Soluble hyaluronidase and production thereof

Abstract

A method for producing a hyaluronidase polypeptide may include: a) introducing a nucleic acid molecule operably linked to a promotor into a eukaryotic cell, the nucleic acid molecule encoding a hyaluronidase polypeptide and including a stop codon; (b) culturing the cells in a growth medium having copper sulfate, L-glutamine, and L-cysteine sulfate under conditions whereby an encoded hyaluronidase polypeptide is produced by expression of the introduced nucleic acid molecule and secreted by the cell, the growth medium being devoid of sodium butyrate and insulin; and (c) recovering the expressed polypeptide.

Description

[0001] SOLUBLE HYALURONIDASE AND PRODUCTION THEREOF FIELD OF THE INVENTION

[0002] The inventions herein relate to recombinant human proteins, in particular, recombinant soluble hyaluronidases, methods for the production of these proteins, and uses thereof.

[0003] INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

[0004] An electronic version of the Sequence Listing is filed herewith, the contents of which are incorporated by reference in their entirety. The electronic file was created on December 1, 2025, is 124,000 bytes in size, and is titled 063995-5121-WO.

[0005] BACKGROUND

[0006] Hyaluronidases are a family of enzymes that degrade hyaluronic acid (also known as hyaluronan or hyaluronate), an essential component of the extracellular matrix and a major constituent of the interstitial barrier. By catalyzing the hydrolysis of hyaluronic acid, hyaluronidase lowers the viscosity of hyaluronic acid, thereby increasing tissue permeability. As such, hyaluronidases have been used, for example, as a spreading or dispersing agent in conjunction with other agents, drugs and proteins to enhance their dispersion and delivery. Hyaluronidases also have other therapeutic and cosmetic uses. Because of the increasing use of hyaluronidases for therapeutic and cosmetic uses, there is a need for large-scale quantities of purified hyaluronidase.

[0007] Hyaluronidase compositions and methods for producing hyaluronidase compositions have been described in the art. For example, the Applicant’s own work as set out, e.g., in US 2004 / 0268425 Al describes early foundation work on a hyaluronidase expression construct, in particular, the achievement of a soluble recombinant human hyaluronidase product by introducing C-terminal truncations to remove the glycosylphosphatidylinositol (GPI) anchor and modifying the signal peptide. In terms of the production process, US 2004 / 0268425 Al only contains one small-scale proof-of-concept study demonstrating that the engineered protein of US 2004 / 0268425 Al can be produced in a recombinant expression system and is soluble and enzymatically active. This early version of the Applicant’s own, Generation 1 or “Genl” hyaluronidase production process as described in US 2004 / 0268425 Al was not suitable for large scale commercial applications. Later work by the Applicant included development of a larger scale Genl hyaluronidase production process based on the work set out in, e.g., US 2004 / 0268425 Al, which was initially used for commercial applications.

[0008] DBl / 164647321.1 1 The Applicant’s own further work as set out, e.g., in WO 2009 / 111066 Al describes a subsequently developed, large-scale hyaluronidase production process that is referred to as the Generation 2 or “Gen2” hyaluronidase production process. This Gen2 hyaluronidase production process differs from the Genl hyaluronidase production process in several steps, which were all specifically altered to increase the relative content of active enzyme in the resulting composition.

[0009] TECHNICAL PROBLEM AND SOLUTION

[0010] Although certain hyaluronidase compositions and methods for producing hyaluronidase compositions have been described, a need remains in the art to provide further improved hyaluronidase compositions and methods for producing hyaluronidase compositions. This is important, for example, because of the increasing amounts of different uses of hyaluronidase, including therapeutic and cosmetic uses. In particular, the methods disclosed herein provide an improved hyaluronidase composition and an improved hyaluronidase production process. The hyaluronidase production process provided herein (also referred to as the Gen3 hyaluronidase production process) is improved in that, for example, it achieves significantly higher yields of rHuPH20 through optimization of bioreactor conditions that enhance cellular health and productivity. The optimized culture conditions also directly result in an improved hyaluronidase (rHuPH20 composition). For example, the optimized culture environment as provided in the Gen3 hyaluronidase production process reduces cellular stress, resulting in decreased release of intracellular hydrolytic enzymes that would otherwise degrade rHuPH20. Consequently, the rHuPH20 final product exhibits improved structural integrity and maturity, as evidenced by, for example, extended C-terminal sequences (a higher overall content of longer polypeptide species), a glycosylation profile with longer, more mature glycan chains, and improved structural integrity of the polypeptide species (e.g., as evidenced by reduced hydrolytic fragmentation, measurable as a lower amount of “hydrolyzed” rHuPH20, as defined herein). Furthermore, the optimized culture conditions also result in a reduced amount of contaminating species such as CHO host cell proteins and DNA, throughout production and in the final hyaluronidase composition. Being able to produce higher quantities of hyaluronidase in the same culture volume reduces the cost of production. Furthermore, providing a hyaluronidase composition with improved structural characteristics and reduced contamination, for example, reduces the risk of adverse reactions upon administration to subjects. Thus, the invention as set out herein solves the problem of providing improved hyaluronidase compositions.

[0011] DBl / 164647321.1 2 SUMMARY

[0012] Among the objects herein, it is an object to provide methods for the production and purification of hyaluronidases. Provided herein are methods for the production and purification of soluble hyaluronidases. In particular, provided herein are methods for the production and purification of soluble recombinant human PH20 (rHuPH20). It is a further object to provide hyaluronidase polypeptides obtainable by the methods described herein. The hyaluronidase polypeptides obtainable by the methods described herein are a mixture of hyaluronidase polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8. The methods and steps described herein are amendable for scale-up or scale-down, as would be apparent to one of skill in the art. It is a further object to provide a composition comprising a mixture of hyaluronidase polypeptides, said mixture comprising the hyaluronidase polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the hyaluronidase polypeptides in said mixture of hyaluronidase polypeptides consist of the sequence set forth in SEQ ID NO: 5. In this composition, preferably at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc. It is a further object to provide a harvest cell culture fluid comprising a mixture of hyaluronidase polypeptides, said mixture of hyaluronidase polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein the enzymatic activity of hyaluronidase is at least 25,000 U / mL, preferably at least 30,000 U / mL.

[0013] In a first aspect of the invention, there is provided a hyaluronidase polypeptide produced by a method comprising (a) introducing nucleic acid molecule operably linked to a promotor into a eukaryotic cell, the nucleic acid molecule encoding a hyaluronidase polypeptide and including a stop codon; (b) culturing the cell in a growth medium having copper sulfate, L-glutamine, and L-cysteine sulfate under conditions whereby an encoded hyaluronidase polypeptide is produced by expression of the introduced nucleic acid molecule and secreted by the cell, the growth medium being devoid of sodium butyrate and insulin; and (c) recovering the expressed polypeptide, wherein the growth culture has a hyaluronidase activity of at least about 25,000 U / ml.

[0014] The hyaluronidase polypeptide is neutral active and contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide. The hyaluronidase polypeptide may consist of the sequence of amino acids set forth as amino acids 1-477 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 39), ammo acids 1-478 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 40), amino acids 1-479 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 41), amino acids 1- 480 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 42), amino acids 1-481 set forth in SEQ ID

[0015] DBl / 164647321.1 3 NO: 1 (i.e., SEQ ID NO: 43), amino acids 1-482 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 3) or amino acids 1-483 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 44), or consists of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as amino acids 1-477 of SEQ ID NO: 1 (i.e., SEQ ID NO: 39), amino acids 1-478 of SEQ ID NO: 1 (i.e., SEQ ID NO: 40), ammo acids 1-479 of SEQ ID NO: 1 (i.e., SEQ ID NO: 41), ammo acids 1-480 of SEQ ID NO: 1 (i.e., SEQ ID NO: 42), ammo acids 1-481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 43) or ammo acids 1-482 of SEQ ID NO: 1 (i.e., SEQ ID NO: 3) or amino acids 1-483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 44). The hyaluronidase polypeptide may consist of the sequence of amino acids set forth as amino acids 36-477 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 9), ammo acids 36-478 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 8), ammo acids 36-479 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 7), amino acids 36-480 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 6), ammo acids 36-481 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 5), amino acids 36-482 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 4), or amino acids 36-483 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 46) or contains amino acid substitutions in the sequence of amino acids set forth as amino acids 36-477 of SEQ ID NO: 1 (i.e., SEQ ID NO: 9), ammo acids 36-478 of SEQ ID NO: 1 (i.e., SEQ ID NO: 8), ammo acids 36-479 of SEQ ID NO: 1 (i.e., SEQ ID NO: 7), ammo acids 36-480 of SEQ ID NO: 1 (i.e., SEQ ID NO: 6), ammo acids 36-481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5), ammo acids 36-482 of SEQ ID NO: 1 (i.e., SEQ ID NO: 4), or ammo acids 36-483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 46), whereby the amino-acid substituted hyaluronidase glycoprotein consists of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as amino acids 36-477 of SEQ ID NO: 1 (i.e., SEQ ID NO: 9), ammo acids 36-478 of SEQ ID NO: 1 (i.e., SEQ ID NO: 8), ammo acids 36-479 of SEQ ID NO: 1 (i.e., SEQ ID NO: 7), ammo acids 36-480 of SEQ ID NO: 1 (i.e., SEQ ID NO: 6), ammo acids 36-481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5), ammo acids 36-482 of SEQ ID NO: 1 (i.e., SEQ ID NO: 4), or ammo acids 36-483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 46).

[0016] The hyaluronidase polypeptide may further include a polymeric molecule selected from the group consisting of polyalkylene oxides (PAO), PEG-glycidyl ethers (Epox-PEG), PEG- oxycarbonylimidazole (CDI-PEG), branched polyethelene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans, heparin, homologous albumin, celluloses, hydrolysates of chitosan, starches, glycogen, agaroses and derivatives thereof, guar

[0017] DBl / 164647321.1 4 gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, and biopolymers.

[0018] In a second aspect of the invention, there is provided a method for producing a hyaluronidase polypeptide, comprising: (a) introducing a nucleic acid molecule operably linked to a promotor into a eukaryotic cell, the nucleic acid molecule encoding a hyaluronidase polypeptide and including a stop codon; (b) culturing the cells in a growth medium having copper sulfate, L-glutamine, and L-cysteine sulfate under conditions whereby an encoded hyaluronidase polypeptide is produced by expression of the introduced nucleic acid molecule and secreted by the cell, the growth medium being devoid of sodium butyrate and insulin; and (c) recovering the expressed polypeptide.

[0019] The method may further include modifying the recovered polypeptide with a polymeric molecule, wherein the polymeric molecule is selected from among polyalkylene oxides (PAO), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethelene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans, heparin, homologous albumin, celluloses, hydrolysates of chitosan, starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, and bio-polymers.

[0020] In the method, recovering, harvesting, purifying, processing, or isolating the expressed polypeptide may further include (i) transferring the cell culture fluid (CCF) from (b) through a positively charged media to separate cells, cell debris, and host cell impurities from the expressed polypeptide and (ii) chasing the transferred CCF through the charged media with at least one buffer. A positively charged media may include a quaternary ammonium charged synthetic fiber matrix, such as 3M™ Harvest RC.

[0021] The polypeptide may be further purified, processed, or isolated from the harvest cell culture (HCCF) mixture by (i) loading onto a chromatography media; (ii) washing the chromatography media with at least one wash solution; and (iii) eluting the chromatography media with an elution buffer to obtain the purified polypeptide. The chromatography media may include a cibacron dye ligand. The at least one wash solution may include sodium phosphate, ammonium sulfate, Tris, sodium chloride, urea or a combination thereof.

[0022] The chromatography media may include a second process step comprising hydrophobic interaction chromatography (HIC) media, a third process step comprising affinity interaction

[0023] DBl / 164647321.1 5 chromatorgraphy, and / or a fourth process step comprising of mixed-mode or ion exchange chromatography (IEX).

[0024] According to a third aspect of the invention, there is provided a recombinant antihyaluronidase monoclonal antibody, comprising a heavy chain variable region comprising or consisting of one of (i) amino acid residues 19-136 of SEQ ID NO: 73 (i.e., SEQ ID NO: 73 without the signal peptide), (ii) amino acid residues 20-137 of SEQ ID NO: 74 (i.e., SEQ ID NO: 74 without the signal peptide) or (iii) amino acid residues 20-135 of SEQ ID NO: 75 (i.e., SEQ ID NO: 75 without the signal peptide); and a light chain variable region comprising or consisting of one of (iv) amino acid residues 21-131 of SEQ ID NO: 76 (i.e., SEQ ID NO: 76 without the signal peptide), (v) amino acid residues 21-127 of SEQ ID NO: 77 (i.e., SEQ ID NO: 77 without the signal peptide), or (vi) amino acid residues 16-121 of SEQ ID NO: 78 (i.e., SEQ ID NO: 78 without the signal peptide). The antibody may include a light chain region including a constant region comprising a mouse Ig-kappa light chain comprising SEQ ID NO: 66. The antibody may include a heavy chain region including a constant region comprising a mouse IgGl heavy chain comprising SEQ ID NO: 61.

[0025] In a fourth aspect of the invention, there is provided hyaluronidase polypeptides obtainable by the methods described herein. In particular, there is provided a composition comprising a mixture of polypeptides, said mixture comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein the composition is obtainable by the methods described herein, in particular the methods according to the first and / or second aspect of the invention described above.

[0026] In a fifth aspect of the invention, there is provided a composition comprising a mixture of hyaluronidase polypeptides, said mixture comprising the polypeptides set forth in SEQ ID NO: 4-8. In this composition, preferably at least 60% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5. In this composition, preferably at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc.

[0027] In a sixth aspect of the invention, there is provided a harvest cell culture fluid comprising a mixture of hyaluronidase polypeptides, said mixture of hyaluronidase polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein the enzymatic activity of hyaluronidase is at least 25,000 U / mL, preferably at least 30,000 U / mL. In some preferred embodiments, the enzymatic activity of hyaluronidase in the harvest cell culture fluid is 40,000 - 50,000 U / mL.

[0028] FIGURES

[0029] DBl / 164647321.1 6 Figure 1: Flow chart of exemplary Gen3 hyaluronidase production process according to the present invention.

[0030] Figure 2A-B: (A) Enzyme activity in of soluble rHuPH20 in the HCCF in units of enzyme activity per mL of HCCF (U / mL): comparative data showing improvements for Gen3 rHuPH20 in comparison to Gen2 rHuPH20 and Genl rHuPH20. (B) Process yield of soluble rHuPH20 shown in g of produced rHuPH20 per 100 L of culture: comparative data showing improvements for Gen3 rHuPH20 in comparison to Gen2 rHuPH20 and Genl rHuPH20.

[0031] Figure 3A-B: (A) CHO Host Cell Proteins (HCP) in final soluble rHuPH20 drug product: comparative data showing improvement in the Gen3 rHuPH20 final drug product in comparison to the Gen2 rHuPH20 final drug product. (B) CHO HCP level (ng / mg) throughout processing representative batches of Gen2 and Gen3 rHuPH20 (Q Seph = Q Sepharose purification step; Phenyl = Phenyl sepharose purification step; APB = aminophenyl boronate purification step; CHT = ceramic hydroxyapatite purification step; Virosart = viral filtration step ; CBHS = beaded crosslinked agarose purification step comprising Cibacron Blue is Capto™Blue (high sub); Benzyl Ultra = beaded benzyl-substituted crosslinked poly(styrene-divinylbenzene) purification step using is POROS™ Benzyl Ultra (BU))

[0032] Figure 4: Levels of % hydrolyzed rHuPH20 in rHuPH20 final drug product expressed as % of total rHuPH20 as defined herein: comparative data showing improvements for Gen3 rHuPH20 in comparison to Gen2 rHuPH20 and Genl rHuPH20.

[0033] Figure 5A-B: (A) Clearance of DNA from the production steps of the process according to W02009 / 111066 (upper line) and the process according to the present invention (lower line) as determined by PicoGreen. (B) Enlarged version of Figure 5 A.

[0034] Figure 6A: Sequence determination of heavy chain variable regions for 3E8, 1H3 and 10A4 clones and murine IgGl heavy chain constant region. Sequence determination of heavy chain variable region contains partial sequence of heavy chain constant region (shown in blue). Figure 6B: Sequence determination of light chain variable regions for 3E8, 1H3 and 10A4 clones and murine IgGl light chain constant region. Sequence determination of light chain variable region contains at least partial sequence of light chain constant region (shown in blue). Figure 7A-7C: Sequences for alignment of heavy and light chain for the anti-rHuPH20 antibodies designated 3E8, 1H3 and 10A4. (A) Heavy and light chain sequences of the anti- rHuPH20 antibodies designated 3E8, 1H3 and 10A4 (black = signal peptides; red = variable regions; blue = constant regions); (B) Sequence alignment of heavy chains for the anti-rHuPH20 antibodies designated 3E8, 1H3 and 10A4 (black = signal peptides; red = variable regions;

[0035] DBl / 164647321.1 7 purple = constant region CHI ; brown = constant region hinge region; green = constant region CH2; blue = constant region CH3); (C) Sequence alignment of light chains for the anti- rHuPH20 antibodies designated 3E8, 1H3 and 10A4 (black = signal peptides; red = variable regions; blue = constant regions)

[0036] Figure 8A-B: (A) Structure of N-glycan species (NeuAc)2(Gal- GlcNAc)2(Man)3(GlcNAc)2Fuc; (B) Box plots illustrating a statistical analysis of abundance of (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc glycan species expressed as % of total N-linked glycans (designated “Mean (%Peak 1)” in the Figure): comparative data showing improvements for Gen3 rHuPH20 (designated “HZ203” in this Figure) in comparison to Gen2 rHuPH20 (designated “HZ202” in this Figure) and Genl rHuPH20 (designated “HZ201” in this Figure). Figure 9A-B: C-terminal heterogeneity: (A) Bar graphs showing abundances of each of SEQ ID NO: 4-8 in rHuPH20 compositions, with abundances of individual polypeptides in rHuPH20 compositions determined by LC-MS analysis based on intact mass: comparative data showing differences between Gen2 (n = 10) and Gen3 (n = 5) rHuPH20 compositions, means and standard deviations shown in tables below each graph. (B) Box plots illustrating a statistical analysis of abundance of SEQ ID NO: 5 (i.e., the 1-446 polypeptide, thus designated “C-term (1-446) %” in this Figure) in rHuPH20 compositions determined by LC-MS analysis based on intact mass: comparative data showing increase in SEQ ID NO: 5 abundance from Genl rHuPH20 to Gen2 rHuPH20 and further increase to Gen3 rHuPH20, expressed as % of total of SEQ ID NO: 4+5+6+7+8.

[0037] DETAILED DESCRIPTION

[0038] A. DEFINITIONS

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety. In the event that there is a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0040] DBl / 164647321.1 8 As used herein, hyaluronidase refers to an enzyme that degrades hyaluronic acid. Hyaluronidases include bacterial hyaluronidases (EC 4.2.99.1), hyaluronidases from leeches, other parasites, crustaceans (EC 3.2.1.36), and mammalian-type hyaluronidases (EC 3.2.1.35). Hyaluronidases also include any of non-human origin including, but not limited to, murine, canine, feline, leporine, avian, bovine, ovine, porcine, equine, piscine, ranine, bacterial, and any from leeches, other parasites, and crustaceans. Exemplary non-human hyaluronidases include, hyaluronidases from cows (SEQ ID NO: 10), yellow jacket wasp (SEQ ID NO: 11 and 12), honey bee (SEQ ID NO: 13), white-face hornet (SEQ ID NO: 14), paper wasp (SEQ ID NO: 15), mouse (SEQ ID NO: 16-18, 29), pig (SEQ ID NO:19-20), rat (SEQ ID NO:21-23, 28), rabbit (SEQ ID NO:24), sheep (SEQ ID NO:25), orangutan (SEQ ID NO:26), cynomolgus monkey (SEQ ID NO:27), guinea pig (SEQ ID NO:30), Staphylococcus aureus (SEQ ID NO:31), Streptococcus pyogenes (SEQ ID NO:32), and Clostridium perfringens (SEQ ID NO:33). Exemplary human hyaluronidases include HYAL1 (SEQ ID NO:34), HYAL2 (SEQ ID NO:35), HYAL3 (SEQ ID NO:36), HYAL4 (SEQ ID NO:37), and PH20 (SEQ ID NO: 1). Also included amongst hyaluronidases are soluble human PH20 and soluble rHuPH20.

[0041] Reference to hyaluronidases includes precursor hyaluronidase polypeptides and mature hyaluronidase polypeptides (such as those in which a signal sequence has been removed), truncated forms thereof that have activity, and includes allelic variants and species variants, variants encoded by splice variants, and other variants, including polypeptides that have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the precursor polypeptides set forth in SEQ ID NO: 1 and 10-37, or the mature form thereof. For example, reference to hyaluronidase also includes the human PH20 precursor polypeptide variants set forth in. SEQ ID NO:48-49. Hyaluronidases also include those that contain chemical or posttranslational modifications and those that do not contain chemical or posttranslational modifications. Such modifications include, but are not limited to, pegylation, albumination, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art.

[0042] As used herein, soluble human PH20 or sHuPH20 include mature polypeptides or recombinant polypeptides lacking all or a portion of the glycosylphosphatidyl inositol (GPI) attachment site at the C-terminus such that upon expression, the polypeptides are soluble. Exemplary sHuPH20 polypeptides include mature polypeptides having an amino acid sequence set forth in any one of SEQ ID NO:4-9 and 45-46. The precursor polypeptides for such exemplary sHuPH20 polypeptides include a signal sequence. Exemplary of the precursors are

[0043] DBl / 164647321.1 9 those set forth in SEQ ID NO:3 and 38-44, each of which contains a 35 amino acid signal sequence at amino acid positions 1-35. Soluble HuPH20 polypeptides also include those degraded during or after the production and purification methods described herein.

[0044] As used herein, soluble rHuPH20 refers to a soluble form of human PH20 that is recombinantly expressed in Chinese Hamster Ovary (CHO) cells. Soluble rHuPH20 is encoded by nucleic acid that includes the signal sequence and is set forth in SEQ ID NO:47. Also included are DNA molecules that are allelic variants thereof and other soluble variants. The nucleic acid encoding soluble rHuPH20 is expressed in CHO cells which secrete the mature polypeptide. As produced in the culture medium there is heterogeneity at the C-terminus so that the product includes a mixture of species that can include one or more of SEQ ID NOS. 4-8 in various abundance. Corresponding allelic variants and other variants also are included, including those corresponding to the precursor human PH20 polypeptides set forth in SEQ ID NO:48-49. Other variants can have 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NOS. 4-8 as long they retain a hyaluronidase activity and are soluble.

[0045] The expressions “heterogeneity at the C-terminus” and “C-terminal heterogeneity” are used interchangeably herein and refer to a situation where one nucleic acid encoding soluble rHuPH20 as defined herein when expressed in CHO cells as defined herein, such as 2B2 cells as defined herein, produces a mixture of polypeptides having different lengths, specifically having different C-terminal ends. In particular, the soluble rHuPH20 compositions described herein comprise a mixture of the polypeptides as set forth in SEQ ID NO: 4-8. SEQ ID NO: 4-8 are sequentially shortened at the C-terminus by one amino acid, with SEQ ID NO: 4 being the longest polypeptide, SEQ ID NO: 5 being one amino acid shorter at the C-terminus than SEQ ID NO: 4, SEQ ID NO: 6 being one amino acid shorter at the C-terminus than SEQ ID NO: 5, SEQ ID NO: 7 being one amino acid shorter at the C-terminus than SEQ ID NO: 6, and SEQ ID NO: 8 being one amino acid shorter at the C-terminus than SEQ ID NO: 7. The expressions “heterogeneity at the C-terminus” and “C-terminal heterogeneity” define that hyaluronidase polypeptides with such different C-terminal ends are present in the same composition, such that the composition has heterogeneity at the C-terminus or C-terminal heterogeneity.

[0046] The sequences of the polypeptides as set forth in SEQ ID NO: 4-8 are provided herein below:

[0047] DBl / 164647321.1 10

[0048] DB1 / 164647321.1 11

[0049] The abundances provided for “SEQ ID NO: 4” herein correspond to the total abundance of detected “1-447” and “1-447S” polypeptides together. Both of these polypeptide species have the same amino acid sequence as indicated above. The “1-447S” polypeptide merely differs from the “1-447” polypeptide in that the “1-447S” polypeptide carries a sulfation at Tyr447. The “1- 447” polypeptide and “1-447S” polypeptide can be detected separately when a mass spectrometry assay of high sensitivity is used.

[0050] The expression “at least [x]% of the polypeptides in the mixture of polypeptides consists of the sequence set forth in SEQ ID NO: [x]” as used herein defines the relative abundances of

[0051] DBl / 164647321.1 12 individual polypeptides in a mixture of polypeptides. For example, when the mixture of polypeptides comprises the polypeptides set forth in SEQ ID NO: 4-8, the sum of the abundances of SEQ ID NO: 4-8, (i.e., SEQ ID NO: 4 + SEQ ID NO: 5 + SEQ ID NO: 6 + SEQ ID NO: 7 + SEQ ID NO: 8) is 100%. Abundances of individual polypeptides in this mixture are expressed relative thereto.

[0052] The abundances of individual polypeptides in rHuPH20 compositions comprising a mixture of SEQ ID NO: 4-8 can be measured by different methods, and using different methods can produce slightly different results.

[0053] For example, one method for determining the abundances of individual polypeptides in rHuPH20 compositions comprising a mixture of SEQ ID NO: 4-8 is analysis based on peptide fragments. In this method, a sample containing the mixture of polypeptides is digested with endoproteinase Asp-N, which specifically cleaves peptide bonds N-terminally at aspartic and cysteic acid. This releases the C-terminal portion of the soluble rHuPH20 at the aspartic acid at position 431 of SEQ ID NO: 4. The resulting C-terminal fragments can then be separated and characterized to determine the sequence and abundance of each individual polypeptide in the mixture. This method has been used to determine the abundances of individual polypeptides in rHuPH20 compositions, for example, as described in the applicant’s own earlier work on Gen2 hyaluronidase compositions as described in WO 2009 / 111066.

[0054] Alternatively, another method for determining the abundances of individual polypeptides in rHuPH20 compositions comprising a mixture of SEQ ID NO: 4-8 is Liquid chromatographymass spectrometry (LC-MS) analysis based on intact mass. In this method, a sample comprising the mixture of intact polypeptides consisting of the entire sequences of SEQ ID NO: 4-8 (i.e., polypeptides of SEQ ID NO: 4-8 that have not been digested) is first denatured and deglycoslyated to remove all N- and O-linked glycans (e.g., by incubation with O-glycosidase, sialidase and N-gycanase at 37°C overnight), before being injected in size-exclusion ultra performance liquid chromatography (SE-UPLC), wherein detection is carried our using both UV 280 nm and electrospray ionization mass spectrometry (ESI-MS). This method of LC-MS analysis based on intact mass of rHuPH20 polypeptides is the method used to generate the data shown herein. The presence of C-terminally heterogenous polypeptides (i.e., of all of SEQ ID NO: 4-8) in the mixture of hyaluronidase polypeptides and the relative abundances of each of these polypeptides (i.e., of each of SEQ ID NO: 4-8) make up a structural fingerprint that is characteristic for the hyaluronidase compositions described herein.

[0055] As used herein, the term “hydrolyzed species” or “hydrolyzed polypeptide (species)” or

[0056] DBl / 164647321.1 13 “hydrolyzed” refers to the amount of hyaluronidase that has been hydrolyzed between residues 311 and 312 (with reference to the residue numbering of SEQ ID NO: 4, i.e., between Arginine 311 and Serine 312). Hyaluronidase has a natural protease cleavage site on the C-terminal side of arginine 311 (with reference to the residue numbering of SEQ ID NO: 4). The more active proteases are present in a given batch, the more cleavage at this site will be observed. This type of protein degradation is thus an indicator of quality of the hyaluronidase provided herein. The content of hydrolyzed hyaluronidase impurity can be calculated by using Reversed Phase High Performance Liquid Chromatography (RP-HPLC) to determine the % area under the respective peaks in the elution profile, i.e.: hydrolyzed (% area) = (area under hydrolyzed peak) / (area under monomer peak + area under hydrolyzed peak + area under oxidized peaks)) x 100

[0057] As used herein, "soluble rHuPH20-expressing cells" refers to any CHO cell that expresses soluble rHuPH20. Exemplary soluble rHuPH20-expressing cells include 2B2 and 3D35M cells, as further defined herein. Soluble rHuPH20-expressing cells are CHO cells into which nucleic acid that contains the sequence set forth in SEQ ID NO: 51 has been introduced.

[0058] As used herein, the terms “cell line derived from” or “derived cell line”, should be understood as a cell line derived from a cell line provided herein, such as the 2B2 cells described herein, by means of, e.g., genetic engineering, radiation and / or chemical treatment, and / or selection, adaptation, screening, etc. It is preferred that the derived cell line is a functionally equivalent mutant, e.g., a cell line that has substantially the same, or improved, properties with respect to expressing rHuPH20 as the parent cell line. Such a derived cell line is a part of the present invention. Especially, the term “derived cell line” refers to a cell line obtained by subjecting 2B2 cells to any conventionally used adaptation method for increasing expression of a transformed protein. For example, by increasing the concentration of methotrexate in the culture media, the hyaluronidase-expressing 2B2 cells are forced to produce more dihydrofolate reductase to remain viable. This can be affected by, for example, gene amplification or rearrangement of the integrated DNA to a more stable and productive arrangement. Thus, forcing an increase in the production of dihydrofolate reductase also can result in an increase in the production of srHuPH20. Desirable derived cell lines can be selected as those cell lines that still have the capacity to produce soluble rHuPH20, preferably an improved capacity to produce soluble rHuPH20. In one aspect, such a cell line derived from 2B2 cells can be obtained by gradually adapting 2B2 cells to grow in cell culture medium containing more than 20 pM methotrexate by gradually increasing the amount of methotrexate in the cell culture medium

[0059] DBl / 164647321.1 14 above 20 pM methotrexate when passaging the cells. In one aspect, such a cell line that is derived from 2B2 cells in this manner is selected based on a higher level of enzymatic activity of hyaluronidase measured in the cell culture fluid. This enzymatic activity of hyaluronidase can be determined by using the turbidimetric- based assay described herein (see e.g., Example 14 and the section “Monitoring soluble rHuPH20 production”).

[0060] A mutant may have been subjected to several mutagenesis treatments (a single treatment should be understood one adaptation step followed by a screening / selection step), but it is presently preferred that no more than 20, no more than 10, or no more than 5, treatments are carried out. In a presently preferred derived cell line, less than 1%, or less than 0.1%, less than 0.01%, less than 0.001% or even less than 0.0001% of the nucleotides in the genome have been changed (such as by replacement, insertion, deletion or a combination thereof) compared to the parent cell line.

[0061] As used herein, “CHO Host Cell Proteins” or “CHO HCP” refers to proteins that are secreted and released from lysed cells during the production process of rHuPH20 and accumulate extracellularly during the cultures of recombinant CHO cells. If these HCPs are not effectively removed during purification, this can have a negative impact on product quality. Thus, a quality attribute of rHuPH20 compositions is the amount of residual HCP in the final composition (drug substance). The lower the amount of residual HCP in the rHuPH20 composition, the higher the quality of the rHuPH20 composition.

[0062] As used herein, the terms “rDNA” and “residual DNA” and “DNA” in the context of contaminants are used interchangeably and refer to DNA that is released from lysed host cells during the rHuPH20 production process and accumulates in the cell culture medium. The lower the amount of residual DNA in the rHuPH20 composition, the higher the quality of the rHuPH20 composition.

[0063] As used herein, “(NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc)” refers to the N-linked glycan species consisting of units N-Acteyl-Neuraminic Acids (NeuAc), two units Galactose (Gal), two units N-Acetylglucosamine (GlcNAc), three units Mannose (Man), two further units GlcNAc and one unit Fucose (Fuc). This glycan structure is illustrated in Figure 8A.

[0064] As used herein, hyaluronidase activity refers to any activity exhibited by a hyaluronidase polypeptide. Such activities can be tested in vitro and / or in vivo and include, but are not limited to, enzymatic activity, such as to effect cleavage of hyaluronic acid, ability to act as a dispersing or spreading agent and antigenicity.

[0065] As used herein, enzymatic activity refers to the activity of a hyaluronidase, as assessed in

[0066] DBl / 164647321.1 15 in vitro enzymatic assays, to cleave a substrate, such as hyaluronic acid. In vitro assays to determine the enzymatic activity of hyaluronidases, such as soluble rHuPH20, are known in the art and described herein. Exemplary assays include turbidimetric-based assay described below (see e.g., Example 14 and the section “Monitoring soluble rHuPH20 production”) that measures cleavage of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when the uncleaved hyaluronic acid binds with serum albumin. Enzymatic activity values as measured by, e.g., the turbidimetric-based assay described below are provided as units of enzymatic activity per ml, also abbreviated as “units / mL”, “units / ml”, “U / mL” or “U / ml” herein.

[0067] As used herein, specific activity with reference to soluble rHuPH20 is the enzyme activity relative to the amount of soluble rHuPH20 reference standard. Specific activity is calculated by dividing the enzymatic activity (units / mL) by the protein concentration (mg / mL).

[0068] As used herein, "exhibits at least one activity" or "retains at least one activity" refers to the activity exhibited by a variant soluble rHuPH20 as compared to any soluble rHuPH20 set forth in SEQ ID NO:4-8 under the same conditions. Typically, a variant soluble rHuPH20 that retains or exhibits at least one activity of a soluble rHuPH20 set forth in SEQ ID NO:4-8 retains at or about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more of the activity of a soluble rHuPH20 set forth in SEQ ID NO:4-8. Exemplary activities include, but are not limited to, hyaluronidase activity and enzymatic activity.

[0069] As used herein, beaded crosslinked agarose column chromatography refers to chromatography using a column packed with beaded crosslinked agarose. Exemplary of beaded crosslinked agarose is beaded crosslinked agarose comprising Cibacron Blue (also sometimes referred to as Cibacron Blue F3G-A, 3G-A or 3G). A commercially available example of a beaded crosslinked agarose comprising Cibacron Blue is Capto™Blue (high sub) (HS) (CBHS). Capto™ Blue HS is an affinity chromatography resin designed for capture of a variety of molecules, including enzymes, comprising a Cibacron Blue ligand bound to a rigid agarose matrix.

[0070] As used herein, beaded crosslinked poly(styrene-divinylbenzene) column chromatography refers to chromatography using a column packed with beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality, such as beaded benzyl-substituted crosslinked poly(styrene-divinylbenzene). Exemplary of beaded benzyl-

[0071] DBl / 164647321.1 16 substituted crosslinked poly(styrene-divinylbenzene) is POROS™ Benzyl Ultra (BU). POROS™ Benzyl Ultra resin is a hydrophobic interaction chromatography (HIC) resin utilizing crosslinked poly(styrene-divinylbenzene) POROS™ base beads manufactured by Thermo Fisher with a highly hydrophobic aromatic benzyl ligand.

[0072] As used herein, aminophenylboronate (APB) column chromatography refers to chromatography using a column packed with beaded aminophenylboronate substituted crosslinked agarose. Exemplary of beaded aminophenylboronate substituted crosslinked agarose is Aminophenylboronate Agarose 6XL. Aminophenylboronate Agarose 6XL resin is a mixedmode chromatography resin utilizing cross-linked agarose base beads with a m- aminophenylboronic acid ligand manufactured by Astrea Bioseparations.

[0073] As used herein, the term “cell culture fluid” or “CCF” refers to the culture fluid before harvest of the cells from the bioreactor and before separation of the cell culture medium from the cells, cell debris and other aggregates.

[0074] As used herein, the terms “harvested cell culture fluid” or “harvest cell culture fluid” (HCCF) are used interchangeably and refer to the fluid obtained following harvest of the cells from the bioreactor and separation of the cell culture medium from the cells, cell debris, and other aggregates. In some instances herein, the HCCF is also referred to as the “clarified harvest”. The cell culture that is harvested from the bioreactor can be filtered to clarify the culture, removing the cells, cell debris and other aggregates to leave the harvested cell culture fluid. In other words, the HCCF is defined as the fluid that has been separated from cells, cell debris and aggregates. The HCCF is obtained from the CCF with minimal change to the volume of the fluid in comparison to the CCF. More specifically, the volume of fluid between the CCF and the HCCF may increase by up to about 15% due to the nature of the clarification step. As used herein, cell density refers to the number of cells in a given volume of medium. As used herein, cell culture or culture refers to a cell population that is suspended in a medium under conditions suitable to maintain viability of the cells or grow the cells.

[0075] As used herein, medium, cell medium or cell culture medium refers to a solution containing nutrients sufficient to promote the growth of cells in a culture. Typically, these solutions contain essential and non-essential amino acids, vitamins, energy sources, lipids and / or trace elements. The medium also can contain additional supplements, such as hormones, growth factors and growth inhibitors.

[0076] As used herein, the residues of naturally occurring amino acids are the residues of those 20 a-amino acids found in nature which are incorporated into protein by the specific recognition

[0077] DBl / 164647321.1 17 of the charged tRNA molecule with its cognate mRNA codon in humans.

[0078] As used herein, "in amounts sufficient to increase" when referring to a substance increasing parameters such as cell growth rate, peak cell density, protein synthesis or cell cycle arrest refers to the amount of a substance that effects an increase in one of these parameters compared to that observed in the absence of the substance. The parameters can be assessed in the presence and absence of a substance, and the amount of substance that increases the parameter (such as cell growth rate, peak cell density, protein synthesis or cell cycle arrest) compared in the absence of the substance can be determined. The growth rate, peak cell density, protein synthesis or cell cycle arrest in the presence of the substance can be increased by at or about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to the growth rate, peak cell density, protein synthesis or cell cycle arrest in the absence of the substance. As used herein, nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double-stranded. When referring to probes or primers, which are optionally labeled, such as with a detectable label, such as a fluorescent or radiolabel, single-stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or of low copy number (typically less than 5, generally less than 3) for probing or priming a library. Generally, a probe or primer contains at least 14, 16, or 30 contiguous nucleotides of sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids long.

[0079] As used herein, a peptide refers to a polypeptide that is from 2 to 40 amino acids in length. As used herein, the amino acids which occur in the various sequences of amino acids provided herein are identified according to their known three-letter or one-letter abbreviations (Table 1). The nucleotides which occur in the various nucleic acid fragments are designated with the standard single-letter designations used routinely in the art.

[0080] As used herein, an "amino acid" is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids.

[0081] For purposes herein, amino acids include the twenty naturally-occurring amino acids, non-natural amino acids, and amino acid analogs (i.e., amino acids wherein the carbon has a side chain).

[0082] As used herein, “amino acid residue” refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described

[0083] DBl / 164647321.1 18 herein are presumed to be in the “L” isomeric form. Residues in the “D” isomeric form, which are so designated, can be substituted for any L-amino acid residue as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243: 3552-3559 (1969), and adopted 37 C.F.R., §§ 1.821-1.822, abbreviations for amino acid residues are shown in Table 1.

[0084] Table 1 : Table of Correspondence It should be noted that all amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxylterminus. In addition, the phrase "amino acid residue" is broadly defined to include the amino acids listed in the Table of Correspondence (Table 1) and non-natural amino acids, modified, and unusual amino acids, such as those referred to in 37 C.F.R. §§ 1.821-1.822, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues, to an amino-terminal group such as NH2 or to a carboxyl-terminal group such as COOH.

[0085] DBl / 164647321.1 19 As used herein, "naturally occurring amino acids" refer to the 20 L-amino acids that occur in polypeptides.

[0086] As used herein, "non- natural amino acid" refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or analogs of amino acids other than the 20 naturally-occurring amino acids and include, but are not limited to, the D-isostereomers of amino acids. Exemplary nonnatural amino acids are described herein and are known to those of skill in the art.

[0087] As used herein, a DNA construct is a single or double stranded, linear or circular DNA molecule that contains segments of DNA combined and juxtaposed in a manner not found in nature. DNA constructs exist as a result of human manipulation and include clones and other copies of manipulated molecules.

[0088] As used herein, “similarity” between two proteins or nucleic acids refers to the relatedness between the sequence of amino acids of the proteins or the nucleotide sequences of the nucleic acids. Similarity can be based on the degree of identity and / or homology of sequences of residues and the residues contained therein. Methods for assessing the degree of similarity between proteins or nucleic acids are known to those of skill in the art. For example, in one method of assessing sequence similarity, two amino acid or nucleotide sequences are aligned in a manner that yields a maximal level of identity between the sequences. "Identity" refers to the extent to which the amino acid or nucleotide sequences are invariant. Alignment of amino acid sequences, and to some extent nucleotide sequences, also can take into account conservative differences and / or frequent substitutions in amino acids (or nucleotides). Conservative differences are those that preserve the physico-chemical properties of the residues involved. Alignments can be global (alignment of the compared sequences over the entire length of the sequences and including all residues) or local (the alignment of a portion of the sequences that includes only the most similar region or regions).

[0089] “Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part i, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G, Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there exists a number of

[0090] DBl / 164647321.1 20 methods to measure identity between two polynucleotide or polypeptides, the term "identity" is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 48: 1073 (1988)).

[0091] As used herein, homologous (with respect to nucleic acid and / or amino acid sequences) means about greater than or equal to 25% sequence homology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence homology; the precise percentage can be specified if necessary. For purposes herein the terms "homology" and "identity" are often used interchangeably, unless otherwise indicated. In general, for determination of the percentage homology or identity, sequences are aligned so that the highest order match is obtained (see, e.g.: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G, Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) SIAM J Applied Math 48: 1073). By sequence homology, the number of conserved amino acids is determined by standard alignment algorithms programs and can be used with default gap penalties established by each supplier.

[0092] Substantially homologous nucleic acid molecules would hybridize typically at moderate stringency or at high stringency all along the length of the nucleic acid of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the hybridizing nucleic acid molecule.

[0093] Whether any two molecules have nucleotide sequences or amino acid sequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" or "homologous" can be determined using known computer algorithms such as the "FASTA" program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et al, .1 Molec Biol 215:403 (1990)); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo etal. (1988) SIAM J Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, WI) and the University of Wisconsin Genetics Computer Group (UWG) "Gap" program (Madison WI). Percent homology or identity of proteins and / or nucleic acid molecules can be determined, for example, by comparing sequence information using a GAP computer program

[0094] DBl / 164647321.1 21 (e.g., Needleman etal. (1970) J. MoL Biol. 48:443, as revised by Smith and Waterman (1981) Adv. ApplMath. 2:482). Briefly, the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids), which are similar, divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a unary, comparison matrix (containing a value of 1 for identities and 0 for nonidentities) and the weighted comparison matrix of Gribskov etal. (1986) Nucl. Acids Res. 14:6745, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.

[0095] Therefore, as used herein, the term “identity” or “homology” represents a comparison between a test and a reference polypeptide or polynucleotide. As used herein, the term at least “90% identical to” refers to percent identities from 90 to 99.99 relative to the reference nucleic acid or amino acid sequence of the polypeptide. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 100 amino acids are compared. No more than 10% (i.e., 10 out of 100) of the amino acids in the test polypeptide differs from that of the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of a polypeptide or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g. 10 / 100 amino acid difference (approximately 90% identity). Differences are defined as nucleic acid or amino acid substitutions, insertions or deletions. At the level of homologies or identities above about 85- 90%, the result should be independent of the program and gap parameters set; such high levels of identity can be assessed readily, often by manual alignment without relying on software.

[0096] As used herein, an aligned sequence refers to the use of homology (similarity and / or identity) to align corresponding positions in a sequence of nucleotides or amino acids. Typically, two or more sequences that are related by 50% or more identity are aligned. An aligned set of sequences refers to 2 or more sequences that are aligned at corresponding positions and can include aligning sequences derived from RNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

[0097] As used herein, "primer" refers to a nucleic acid molecule that can act as a point of initiation of template- directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and a polymerization agent, such as DNA polymerase, RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.

[0098] DBl / 164647321.1 22 It will be appreciated that a certain nucleic acid molecules can serve as a "probe" and as a "primer." A primer, however, has a 3' hydroxyl group for extension. A primer can be used in a variety of methods, including, for example, polymerase chain reaction (PCR), reversetranscriptase (RT)-PCR, RNA PCR, LCR, multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3' and 5' RACE, in situ PCR, ligation-mediated PCR and other amplification protocols.

[0099] As used herein, an allelic variant or allelic variation references any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation and can result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequence. The term "allelic variant" also is used herein to denote a protein encoded by an allelic variant of a gene. Typically, the reference form of the gene encodes a wildtype form and / or predominant form of a polypeptide from a population or single reference member of a species. Typically, allelic variants, which include variants between and among species typically have at least 80%, 90%, or greater amino acid identity with a wildtype and / or predominant form from the same species; the degree of identity depends upon the gene and whether comparison is interspecies or intraspecies. Generally, intraspecies allelic variants have at least about 80%, 85%, 90% or 95% identity or greater with a wildtype and / or predominant form, including 96%, 97%, 98%, 99% or greater identity with a wildtype and / or predominant form of a polypeptide. Reference to an allelic variant herein generally refers to variations in proteins among members of the same species.

[0100] As used herein, "allele," which is used interchangeably herein with "allelic variant" refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for that gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene also can be a form of a gene containing a mutation.

[0101] As used herein, species variants refer to variants in polypeptides among different species, including different mammalian species, such as mouse and human. As used herein, a splice variant refers to a variant produced by differential processing of a primary transcript of genomic DNA that results in more than one type of mRNA.

[0102] As used herein, modification is in reference to modification of a sequence of amino acids

[0103] DBl / 164647321.1 23 of a polypeptide or a sequence of nucleotides in a nucleic acid molecule and includes deletions, insertions, and replacements of amino acids and nucleotides, respectively. Methods of modifying a polypeptide are routine to those of skill in the art, such as by using recombinant DNA methodologies.

[0104] As used herein, the term promoter means a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding region of genes.

[0105] As used herein, isolated or purified polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis, and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance.

[0106] Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound.

[0107] The term substantially free of cellular material includes preparations of proteins in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly-produced. In one embodiment, the term substantially free of cellular material includes preparations of enzyme proteins having less that about 30% (by dry weight) of nonenzyme proteins (also referred to herein as a contaminating protein), generally less than about 20% of non-enzyme proteins or 10% of non-enzyme proteins or less that about 5% of nonenzyme proteins. When the enzyme protein is recombinantly produced, it also is substantially free of culture medium, i.e., culture medium represents less than about or at 20%, 10% or 5% of the volume of the enzyme protein preparation.

[0108] As used herein, the term substantially free of chemical precursors or other chemicals includes preparations of enzyme proteins in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. The term includes preparations of enzyme proteins having less than about 30% (by dry weight) 20%, 10%, 5% or

[0109] DBl / 164647321.1 24 less of chemical precursors or non-enzyme chemicals or components.

[0110] As used herein, synthetic, with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods.

[0111] As used herein, an expression vector includes vectors capable of expressing DNA that is operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.

[0112] As used herein, vector also includes “virus vectors” or “viral vectors.” Viral vectors are engineered viruses that are operatively linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0113] As used herein, the term assessing is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a protease, or a domain thereof, present in the sample, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the activity. Assessment can be direct or indirect and the chemical species actually detected need not of course be the proteolysis product itself but can for example be a derivative thereof or some further substance. For example, detection of a cleavage product of a complement protein, such as by SDS-PAGE and protein staining with Coomassie blue. As used herein, a composition refers to any mixture. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof. As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof.

[0114] As used herein, the term “treatment” or “treating” refers to alleviating the specified condition, eliminating or reducing the symptoms of the condition, slowing or eliminating the progression, invasion, or spread of the condition and reducing or delaying the reoccurrence of

[0115] DBl / 164647321.1 25 the condition in a previously afflicted subject.

[0116] As used herein, the term “prevention” or “preventing” refers to precluding developing a disease, disorder, or condition or reducing the risk of developing the disease, disorder, or condition or reducing the symptoms thereof.

[0117] As used herein, a pharmaceutically effective agent, includes any therapeutic agent or bioactive agents, including, but not limited to, for example, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs and therapeutic proteins.

[0118] As used herein, a patient refers to a human subject.

[0119] As used herein, an effective amount is the quantity of a therapeutic agent necessary for preventing, curing, ameliorating, arresting or partially arresting a symptom of a disease or disorder.

[0120] As used herein, animal includes any animal, such as, but are not limited to primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs and other animals. Non-human animals exclude humans as the contemplated animal. The hyaluronidases provided herein are from any source, animal, plant, prokaryotic and fungal. Most enzymes are of animal origin, including mammalian origin.

[0121] As used herein, a control refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be from a normal volunteer not affected with the condition of interest. A control also can be an internal control.

[0122] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a compound, comprising “an extracellular domain” includes compounds with one or a plurality of extracellular domains.

[0123] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 mM” means “about 5 mM” and also “5 mM.”

[0124] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11 :1726).

[0125] B. Overview

[0126] DBl / 164647321.1 26 Provided herein are methods for the large-scale production of soluble hyaluronidases, such as soluble human hyaluronidases, including soluble human PH20 (sHuPH20), such as, for example, soluble rHuPH20. Also provided herein are soluble hyaluronidases, such as soluble human hyaluronidases, including soluble human PH20 (sHuPH20), such as, for example, soluble rHuPH20, obtainable by the methods described herein. These soluble hyaluronidases that are obtainable by the methods described herein are a mixture of hyaluronidase polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8. Also provided herein is a composition comprising a mixture of polypeptides, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5. In this composition, preferably at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc. Also provided herein is a harvest cell culture fluid comprising a mixture of polypeptides, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein the enzymatic activity of hyaluronidase in the harvest cell culture fluid is at least 25,000 U / mL, preferably at least 30,000 U / mL. In some embodiments, the enzymatic activity of hyaluronidase in the harvest cell culture fluid is 40,000- 50,000 U / mL. The methods typically utilize bioreactors to culture cells that produce the soluble hyaluronidase, such as CHO cells (e.g. DG44 CHO cells). Exemplary of such cells are 2B2 cells, which produce soluble rHuPH20. The volume of cell culture in the bioreactor can range from 1 L to 5000 L or more, but typically is or is about 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000 or 4000 liters. Prior to inoculation of the bioreactor, the cells are expanded through a series of increasing cell culture volumes to generate the required number of cells for seeding of the bioreactor. Typically, the cell culture in the bioreactor is seeded with 105to 106cells / mL but can be seeded with more or less. The cells are then incubated in the bioreactor for 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more days.

[0127] During this incubation, feed media is added to the cell culture to supply additional nutrients and supplements. Feed media is preferably added to the cell culture at a frequency to ensure non-depletion of essential components for building cell mass and recombinant protein, e.g., daily. Exemplary supplements or nutrients that can be included in the feed media include, but are not limited to, glucose, glutamine or glutamine-substitute, such as L-alanyl-L-glutamine, asparagine, insulin, sodium butyrate, vitamins, minerals, copper sulfate, L-Cysteine Sulfate, and feed media that have been designed to replenish depleted nutrients in CHO cell cultures during fed-batch production, thereby promoting cell growth and productivity. Examples of such feed DBl / 164647321.1 27 media for fed-batch cultures of CHO cells that are commercially available include, but are not limited to, Ex-Cell® Advanced CHO Feed 1 and Cellvento® 4Feed. One of skill in the art knows, from their common general knowledge, how to identify and obtain suitable feed media for fed- batch cultures of CHO cells that promote cell growth and productivity. Such media can comprise, e.g., amino acids including the essential amino acids, vitamins, salts, and trace elements. The type and amount of supplement added can influence cell growth and protein production. For example, insulin and glutamine or glutamine-substitute can be incorporated into the first feed media added to the cell culture to increase cell growth and peak cell density. Subsequent feed media can be designed to promote protein production more than cell growth. Following protein production in the bioreactor the cell culture fluid (CCF) contains the soluble hyaluronidase that has been secreted into the cell culture medium, and further contains cells, cell debris and other aggregates. The CCF can be removed from the bioreactor and processed to obtain the harvest cell culture fluid (HCCF) by separating the cell culture medium comprising the soluble hyaluronidase from the cells, cell debris and other aggregates. This step of separating can be carried out, e.g., by using a filtration step on the CCF to obtain the HCCF. After the HCCF has been obtained, the solution comprising the soluble hyaluronidase, such as soluble rHuPH20can be subjected to a further step of concentrating prior to initiation of the purification process, if required. In some implementations, the concentration and diafiltration step may be omitted from the process. The soluble hyaluronidase is then purified from the protein solution using a series of purification steps. Exemplary of purification methods that are used for the methods herein may combine anion-exchange fiber filtration, membrane filtration, ion-exchange chromatography, mixed-mode chromatography, hydrophobic interaction chromatography, affinity chromatography, and ultrafiltration and diafiltration in any order. The purified protein is then processed through a final filtration step and filled into final container closure.

[0128] Utilizing the methods described herein, between about 0.5-50 grams of soluble hyaluronidases, such as soluble rHuPH20, is produced per 100 L of cell culture. In some examples, the amount of soluble rHuPH20 produced per 100 L of culture is or is about 1, 2, 3, 4, 5, 10, 15, 20, 30, or 40 grams or more. In a preferred embodiment, the amount of soluble rHuPH20 produced per 100 L of culture is at least 20 grams per 100 L of culture, more preferably at least 25 grams per 100 L of culture. In some examples, the enzymatic activity of the cell culture fluid after harvest clarification and prior to concentration and purification (i.e., the enzymatic activity of the HCCF) can be greater than or about 25,000 units / mL, such as 30,000, 32,000, 34,000, 36,000, 38,000, 40,000, 42,000, 44,000, 46,000, 48,000, 50,000, 52,000, 54,000,

[0129] DBl / 164647321.1 28 56,000, 58,000 or 60,000 units / mL. In a preferred embodiment, the enzymatic activity of the HCCF is 30,000-60,000 units / mL. In a more preferred embodiment, the enzymatic activity of the HCCF is 40,000-50,000 units / mL. In some examples, the process yield of soluble hyaluronidase following purification can range from between or between about 10% to 50% of the amount produced before purification. For example, the process yield following purification can be or be about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 55%, 60%, 65%, 70%, 75, 80%, or more of the amount produced before purification. Generally, the specific activity of soluble rHuPH20 produced using the methods herein is at least or about 80,000, 100,000, 120,000, 140,000, 160,000, or 180,000 units / mg.

[0130] Typically, the soluble rHuPH20 is harvested from the production phase by processing through a cell removal and clarification chromatographic media having a quaternary ammonium (Q) functionalized polypropylene fiber, combined with a 0.2 pm poly ethersulfone membrane. Exemplary examples are provided below. In other words, the HCCF is defined as the fluid that has been separated from cells, cell debris and aggregates. The HCCF is obtained from the CCF with minimal change to the volume of the fluid in comparison to the CCF. More specifically, the volume of fluid between the CCF and the HCCF may increase by up to about 15% due to the nature of the clarification step. The resulting harvest cell culture fluid (HCCF) can then be further purified by sequential purification using chromatography methods, such as a chromatography method using beaded crosslinked agarose resin comprising Cibacron Blue (affinity chromatography; a commercially available example of a beaded crosslinked agarose resin comprising Cibacron Blue is Capto™ Blue HS), a chromatography method using beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality (hydrophobic interaction chromatography; a commercially available example of a beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality is POROS™ Benzyl Ultra), a chromatography method using beaded aminophenylboronate substituted crosslinked agarose resin (mixed-mode chromatography; a commercially available example of a beaded aminophenylboronate substituted crosslinked agarose resin is Aminophenylboronate Agarose 6XL), and, if required, a chromatography method using mixedmode ion exchange (IEX) resin. Each of these purification steps exhibit different binding properties with regards to hyaluronidase, such that the affinity chromatography step (e.g. using beaded crosslinked agarose resin comprising Cibacron Blue, such as chromatography using a Capto™ Blue HS column) is a bind and elute capture step (i.e. soluble rHuPH20 is bound to the resin through hydrophobic and electrostatic interactions while some other proteins and media

[0131] DBl / 164647321.1 29 components flow through), the hydrophobic interaction chromatography step (e.g. using beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality, such as chromatography using a POROS™ Benzyl Ultra column) is a flow through step (i.e. soluble rHuPH20 flows through the column while some other proteins are bound via hydrophobic interactions), the mixed-mode chromatography step (e.g. using beaded aminophenylboronate substituted crosslinked agarose resin, such as chromatography using an Aminophenylboronate Agarose 6XL column) is a bind and elute polishing step (i.e. soluble rHuPH20 is bound to the resin through hydrophobic interactions during loading and through electrostatic interactions during wash steps) , the mixed-mode ion exchange chromatography step (IEX) in bind and elute (i.e. soluble rHuPH20 is bound to the resin through electrostatic or metal ion affinity interactions during wash steps) or flowthrough mode (i.e. soluble rHuPH20 flows through the resin while undesired impurities are bound to the resin through electrostatic or metal ion affinity interactions) to further purify the soluble rHuPH20 from residual host cell proteins, residual DNA, potential viruses, and product related variants remaining in solution.

[0132] Prior to use, the columns are typically sanitized and then equilibrated. Sanitization can be affected by any method known in the art, including, but not limited to, sanitization with 0.5 M to 1.0 M NaOH. Equilibration can be affected by the addition of an appropriate buffer to the column, such as a buffer similar to or the same as the buffer used to subsequently wash the column or the buffer in which the protein is contained in prior to loading. One of skill in the art can readily determine buffers suitable for use in equilibrating each column. Exemplary buffers are provided below. Between each chromatography step, the eluted protein can be filtered, such as through a 0.22 pm sterilizing grade filter, to remove any contaminating microorganism or large aggregates. In some examples, the filtered eluate is stored, such as in sterile storage vessels or bags, prior to use in the next step. Following column chromatography, the purified hyaluronidase can subsequently be subjected to a virus inactivation step and a virus filtration removal step, followed by protein concentration and buffer exchange for final formulation. Exemplary purification methods are described in more detail below.

[0133] In the composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprises the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5, optionally wherein 60-80% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5, optionally wherein 60-70% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5. In one embodiment

[0134] DBl / 164647321.1 30 of said composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprises the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 61%, at least 62%, at least 63%, at least 64%, at least 65% or at least 66% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5. In a preferred embodiment of said composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprises the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 63% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5, optionally wherein 63-80% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5, optionally wherein 63-70% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5.

[0135] In the composition comprising a mixture of polypeptides provided herein, preferably at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc, optionally wherein 6-12% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are (NeuAc)2(Gal- GlcNAc)2(Man)3(GlcNAc)2Fuc.

[0136] In the composition comprising a mixture of polypeptides provided herein, the amount of residual CHO Host Cell Proteins (HCP) is reduced in comparison to compositions previously described in the art. The data provided in the examples demonstrates that the Gen3 rHuPH20 provided herein, including the final product, i.e., the composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8 provided herein, contains lower levels of HCP than rHuPH20 compositions described in the prior art, in particular lower than the Gen2 hyaluronidase compositions described in WO 2009 / 111066. In one embodiment, the HCCF provided herein contains less than 1 mg / ml of CHO HCP, preferably less than 0.5 mg / ml of CHO HCP. In another embodiment, the composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, contains less than 250 ng HCP per mg rHuPH20 polypeptide, preferably less than 200 ng HCP per mg rHuPH20 polypeptide, more preferably less than 150 ng HCP per mg rHuPH20 polypeptide, even more preferably less than 100 ng HCP per mg rHuPH20 polypeptide.

[0137] In the composition comprising a mixture of polypeptides provided herein, the amount of contaminating DNA or residual DNA is substantially reduced in comparison to compositions previously described in the art. The data provided in the examples demonstrates that the Gen3 rHuPH20 provided herein, including the final product, i.e., the composition comprising a mixture DB1 / 164647321.1 31 of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8 provided herein, contains lower levels of DNA than rHuPH20 compositions described in the prior art, in particular lower than the Gen2 hyaluronidase compositions described in WO 2009 / 111066. In one embodiment, the HCCF provided herein contains less than 1000 ng DNA per mg of rHuPH20, preferably less than 800 ng DNA per mg of rHuPH20.

[0138] In the composition comprising a mixture of polypeptides provided herein, the amount of hydrolyzed species, i.e., the amount of rHuPH20 that has been hydrolyzed at the natural protease cleavage site between Arginine 311 and Serine 312 (with reference to the residue numbering of SEQ ID NO: 4) is substantially reduced in comparison to compositions previously described in the art. The amount of hydrolyzed rHuPH20 as defined herein is expressed as a % of total rHuPH20 in a given sample. The data provided in the examples demonstrates that the Gen3 rHuPH20 provided herein, including the final product, i.e., the composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8 provided herein, contains much lower levels of hydrolyzed rHuPH20 than rHuPH20 compositions described in the prior art, in particular lower than the Gen2 hyaluronidase compositions described in WO 2009 / 111066 and also lower than the Genl hyaluronidase compositions based on what has been described in US 2004 / 0268425 Al. In one embodiment, final drug product, i.e., the composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, contains less than 0.5% hydrolyzed species, preferably less than 0.4% hydrolyzed species. In other words, in one embodiment, the present invention provides a composition comprising a mixture of rHuPH20 polypeptides, said mixture of polypeptides comprising the rHuPH20 polypeptides set forth in SEQ ID NO: 4-8, wherein less than 0.5%, preferably less than 0.4% of the rHuPH20 polypeptides have been hydrolyzed between Arginine 311 and Serine 312.

[0139] C. HYALURONIDASES

[0140] Hyaluronidases are a family of enzymes that degrade hyaluronic acid (the substrate, also known as hyaluronan or hyaluronate), an essential component of the extracellular matrix and a major constituent of the interstitial barrier. By catalyzing the hydrolysis of hyaluronic acid, hyaluronidase lowers the viscosity of hyaluronic acid, thereby increasing tissue permeability. As such, hyaluronidases have been used, for example, as a spreading or dispersing agent in conjunction with other agents, drugs and proteins to enhance their dispersion and delivery. In addition, hyaluronidases have also been used for the treatment of multiple diseases due to their enzymatic action and the role that their substrate, hyaluronic acid, has in diseases such as cancer.

[0141] DBl / 164647321.1 32 In such treatment, hyaluronidase can either be used as a sole active ingredient or in combination with further active ingredients- Hyaluronidase is known to have synergistic effects together with multiple other active ingredients that goes beyond the additive effects of hyaluronidase and said further active ingredients.

[0142] 1. Structure and function of hyaluronidases

[0143] There are three general classes of hyaluronidases; mammalian hyaluronidase, bacterial hyaluronidase and hyaluronidase from leeches, other parasites and crustaceans. Mammalian-type hyaluronidases (EC 3.2.1.35) are r - / i- / V-acetyl-hexosaminidases that hydrolyze the / il ^4 glycosidic bond of hyaluronic acid into various oligosaccharide lengths such as tetrasaccharides and hexasaccharides. They have both hydrolytic and transglycosidase activities and can degrade hyaluronic acid and chondroitin sulfates, such as C4-S and C6-S. Hyaluronidases of this type include, but are not limited to, hyaluronidases from cows (SEQ ID NO: 10), yellow jacket wasp (SEQ ID NO: 11 and 12), honey bee (SEQ ID NO: 13), white-face hornet (SEQ ID NO: 14), paper wasp (SEQ ID NO:15), mouse (SEQ ID NO:16-18, 29), pig (SEQ ID NO: 19-20), rat (SEQ ID NO:21-23, 228), rabbit (SEQ ID NO:24), sheep (SEQ ID NO:25), orangutan (SEQ ID NO:26), cynomolgus monkey (SEQ ID NO:27), guinea pig (SEQ ID NO: 30) and human hyaluronidases.

[0144] There are six hyaluronidase-like genes in the human genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20 / SPAM1. HYALP1 is a pseudogene, and HYAL3 (SEQ ID NO: 36) has not been shown to possess enzyme activity toward any known substrates. HYAL4 (precursor polypeptide set forth in SEQ ID NO: 37) is a chondroitinase and exhibits little activity towards hyaluronic acid. HYAL1 (precursor polypeptide set forth in SEQ ID NO: 34) is the prototypical acid-active enzyme and PH20 (precursor polypeptide set forth in SEQ ID NO: 1) is the prototypical neutral-active enzyme. Acid-active hyaluronidases, such as HYAL1 and HYAL2 (precursor polypeptide set forth in SEQ ID NO:35) generally lack catalytic activity at neutral pH (i.e. pH 7). For example, HYAL1 has little catalytic activity in vitro over pH 4.5 (Frost et al. (1997) Anal Biochemistry 251:263-269). HYAL2 is an acid-active enzyme with a very low specific activity in vitro. The hyaluronidase-like enzymes also can be characterized by those which are generally locked to the plasma membrane via a glycosylphosphatidyl inositol anchor such as human HYAL2 and human PH20 (Danilkovitch-Miagkova, et al. (2003) Proc Natl Acad Sci USA. 100(8):4580-5), and those which are generally soluble such as human HYAL1 (Frost et al, (1997) Biochem Biophys Res Commun. 236(1): 10-5). By catalyzing the hydrolysis of hyaluronic acid, a major constituent of the interstitial barrier, hyaluronidase lowers the viscosity

[0145] DBl / 164647321.1 33 of hyaluronic acid, thereby increasing tissue permeability. It also has been shown to exhibit anticancer and anti-carcinogenic activities.

[0146] N-linked glycosylation of some hyaluronidases can be very important for their catalytic activity and stability. While altering the type of glycan modifying a glycoprotein can have dramatic effects on a protein's antigenicity, structural folding, solubility, and stability, many enzymes are not thought to require glycosylation for optimal enzyme activity. Hyaluronidases are, therefore, unique in this regard, in that removal of N-linked glycosylation can result in near complete inactivation of the hyaluronidase activity. For such hyaluronidases, the presence of N- linked glycans is critical for enzyme expression.

[0147] There are seven potential N-linked glycosylation sites at N82, N166, N235, N254, N368, N393, N490 of human PH20 exemplified in SEQ ID NO: 1. Disulfide bonds form between the cysteine residues C60 and C351 and between C224 and C238 to form the core hyaluronidase domain. However, additional cysteines are required in the carboxy terminus for neutral enzyme catalytic activity such that amino acids 36 to 464 of SEQ ID NO: 1 contains the minimally active human PH20 hyaluronidase domain. Thus, N-linked glycosylation site N490 is not required for proper hyaluronidase activity.

[0148] N-linked oligosaccharides fall into several major types (oligomannose, complex, hybrid, sulfated), all of which have (Man) 3-GlcNAc-GlcNAc-cores attached via the amide nitrogen of Asn residues that fall within- Asn-Xaa-Thr / Ser-sequences (where Xaa is not Pro). Glycosylation at an-Asn-Xaa-Cys-site has been reported for coagulation protein C. In some instances, the hyaluronidase can contain both N-glycosidic and O-glycosidic linkages. For example, rHuPH20 (as produced in the methods described herein) has O-linked oligosaccharides as well as N-linked oligosaccharides.

[0149] The methods described herein provide a process for the production and purification of large quantities of a soluble preparation of human PH20 hyaluronidase preparation.

[0150] 2. PH20

[0151] Human PH20 (also known as sperm surface protein PH20), as noted above, is the prototypical neutral-active enzyme that is generally locked to the plasma membrane via a glycosylphosphatidyl inositol (GPI) anchor. It is naturally involved in sperm-egg adhesion and aids penetration by sperm of the layer of cumulus cells by digesting hyaluronic acid. The PH20 mRNA transcript is normally translated to generate a 509 amino acid precursor protein containing a 35 amino acid signal sequence at the N-terminus (amino acid residue positions 1-

[0152] DBl / 164647321.1 34 35). The mature PH20 polypeptide is, therefore, a 474 amino acid polypeptide with an amino acid sequence set forth in SEQ ID NO:2.

[0153] Soluble forms of human PH20 (sHuPH20) can be produced and purified using the methods described herein. The generation of srHuPH20 are described in related U.S. Patent Application Nos. 10 / 795,095, 11 / 065,716 and 11 / 238,171 (also referred to in these applications as sHASEGP or rHuPH20). The soluble forms are produced by expressing nucleic acid encoding C-terminal truncations of the mature PH20 polypeptide that lack the GPI-attachment sites. Soluble forms of human PH20 include soluble rHuPH20, which is produced and purified using the methods provided herein.

[0154] 3. Therapeutic uses of hyaluronidases

[0155] Various forms of hyaluronidases have been prepared and approved for therapeutic use in humans. For example, animal-derived hyaluronidase preparations include Vitrase® (ISTA Pharmaceuticals), a purified ovine testicular hyaluronidase, and Amphadase® (Amphastar Pharmaceuticals), a bovine testicular hyaluronidase. Hylenex® (Halozyme Therapeutics) is a human recombinant hyaluronidase produced by genetically engineered Chinese Hamster Ovary (CHO) cells containing nucleic acid encoding for soluble rHuPH20. Approved therapeutic uses for hyaluronidase include use as an adjuvant to increase the absorption and dispersion of other injected drugs, for hypodermoclysis (subcutaneous fluid administration), and as an adjunct in subcutaneous urography for improving resorption of radiopaque agents. In addition to these indications, hyaluronidases, including sHuPH20, can be used as a therapeutic or cosmetic agent for the treatment of additional diseases and conditions either alone or in combination with further active ingredients.

[0156] As noted above, hyaluronidase is a spreading or diffusing substance which modifies the permeability of connective tissue through the hydrolysis of hyaluronic acid, a polysaccharide found in the intercellular ground substance of connective tissue, and of certain specialized tissues, such as the umbilical cord and vitreous humor. When no spreading factor is present, materials injected subcutaneously, such as drugs, proteins, peptides and nucleic acid, spread very slowly. Co-inj ection with hyaluronidase, however, can cause rapid spreading. The rate of diffusion is proportional to the amount of enzyme, and the extent of diffusion is proportional to the volume of solution. Absorption and dispersion of injected drugs and agents can be enhanced by adding 10-100,000 units hyaluronidase to the injection solution. In some examples, 150 U hyaluronidase is added. Hyaluronidases have multiple uses, including and in addition to their use as a spreading agent. Hyaluronidase is commonly used, for example, for peribulbar block in local

[0157] DBl / 164647321.1 35 anesthesia prior ophthalmic surgery. The presence of the enzyme prevents the need for additional blocks and speeds the time to the onset of akinesia (loss of eye movement). Peribulbar and subTenon's block are the most common applications of hyaluronidase for ophthalmic procedures. Hyaluronidase also can promote akinesia in cosmetic surgery, such as blepharoplasties and face lifts. Exemplary therapeutic and cosmetic uses for hyaluronidase are described below.

[0158] Provided herein are all therapeutic and other uses of hyaluronidases as set out herein, in particular all therapeutic and other uses described below of the composition comprising a mixture of hyaluronidase polypeptides, said mixture of hyaluronidase polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, as disclosed herein. In a preferred embodiment, a composition comprising a mixture of polypeptides, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60%, preferably at least 63%, of the polypeptides in said mixture of polypeptides consists of the sequence set forth in SEQ ID NO: 5, is for use as a medicament, such as for use as a spreading agent, for use in hypodermoclysis, for use in the treatment or prevention of vitrectomy and ophthalmic disorders and conditions, for use in gene therapy, for use in cosmetic applications, for use in organ transplantation, for use in cancer treatment, for use in treatment of glycosaminoglycan accumulation in the brain, for use in treatment of glycosaminoglycan accumulation in cardiovascular disease, for use in the treatment or prevention of pulmonary disease, and / or for any other use set out in more detail below. a. Use as a spreading agent

[0159] Hyaluronidases, such as soluble rHuPH20 produced using the methods described herein, can be used to promote or enhance the delivery agents and molecules to any of a variety of mammalian tissues in vivo. It can be used to facilitate the diffusion and, therefore, promote the delivery, of small molecule pharmacologic agents as well as larger molecule pharmacologic agents, such as proteins, nucleic acids and ribonucleic acids, and macromolecular compositions than can contain a combination of components including, but not limited to, nucleic acids, proteins, carbohydrates, lipids, lipid-based molecules and drugs. For example, molecules and macromolecular complexes ranging from about 10 nm to about 500 nm in diameter, can exhibit dramatic improvements in delivery through interstitial spaces when the interstitial space has been previously, or is coincidently, exposed to hyaluronidase (see e.g. U.S. Patent Application Nos. 10 / 795,095, 11 / 065,716 and 11 / 238,171).

[0160] Examples of pharmaceutical, therapeutic and cosmetic agents and molecules that can be administered with hyaluronidase include, but are not limited to, anesthetics; anti-metabolites, anti-neoplastics and other anti-cancer agents; anti-virals; anti-infectives, including anti-bacterials

[0161] DBl / 164647321.1 36 and other antibiotics, anti-fungals and other anti-infectives; immunomodulatory agents; steroidal and non-steroidal anti-inflammatories; beta blockers; sympathomimetics; ducosanoids, prostaglandins and prostaglandin analogs; miotics, cholinergics and anti-cholinesterases; anti- allergenics and decongestants; hormonal agents; growth factors; immunosuppressants; vaccines and toxoids; immune sera; antibodies; and any combination thereof. In one example, soluble rHuPH20 is administered with a cathepsin, such as cathepsin L. b. Use in hypodermoclysis

[0162] Hypodermoclysis, the infusion of fluids and electrolytes into the hypodermis of the skin, is a useful and simple hydration technique suitable for mildly to moderately dehydrated adult patients, especially the elderly. Although considered safe and effective, the most frequent adverse effect is mild subcutaneous edema that can be treated by local massage or systemic diuretics. Approximately 3 L can be given in a 24-hour period at two separate sites. Common infusion sites include the chest, abdomen, thighs and upper arms. Solutions used in hypodermoclysis include, for example, normal saline, half-normal saline, glucose with saline and 5% glucose. Potassium chloride also can be added to the solution. The addition of hyaluronidase to the solution can enhance fluid absorption and increase the overall rate of administration. c. Use in vitrectomy and ophthalmic disorders and conditions

[0163] Hyaluronidase can be used to minimize the detachment or tearing of the retina during vitrectomy. This could cause, for example, the vitreous body to become uncoupled or "disinserted" from the retina, prior to removal of the vitreous body. Such disinsertion or uncoupling of the vitreous body can minimize the likelihood that further tearing or detachment of the retina will occur as the vitreous body is removed.

[0164] Hyaluronidase can be used for various ophthalmic applications, including the vitrectomy adjunct application described in U.S. Pat. No. 5,292,509. The use of a highly purified hyaluronidase, such as, for example, soluble rHuPH20 produced and purified by the methods described herein, is preferable for intraocular procedures to minimize immunogenicity and toxicity. In some examples, a PEGylated hyaluronidase can be used to prolong residence within the vitreous and prevent localized uptake.

[0165] Hyaluronidases can be used to treat and / or prevent ophthalmic disorders by, for example, preventing neovascularization and increasing the rate of clearance from the vitreous of materials toxic to the retina. Hyaluronidase can be administered in an amount effective to liquefy the vitreous humor of the eye without causing toxic damage to the eye. Liquefaction of the vitreous humor increases the rate of liquid exchange from the vitreal chamber. This increase in exchange

[0166] DBl / 164647321.1 37 removes the contaminating materials whose presence can cause ophthalmologic and retinal damage.

[0167] Hyaluronidase also can be used to reduce postoperative pressure. Hyaluronic acid has been used in the eye primarily as a spacer during cataract and intraocular lens surgical procedures. It also is used in other ocular surgical procedures such as glaucoma, vitreous and retina surgery and in corneal transplantation. A common side effect occurring in postoperative cataract patients is a significant early, and occasionally prolonged, rise in intraocular pressure. Such a condition is sometimes serious, especially in patients with glaucomatous optic disc changes. Hyaluronidase can be co-administered with hyaluronic acid to the eye prior to surgery to reduce postoperative pressure in the eye. The hyaluronidase is administered in an amount effective to reduce the intraocular pressure to pre-operative levels by breaking down the hyaluronic acid without decreasing its effectiveness during surgery nor causing side effects in the patient (U.S. Patent No. 6,745,776).

[0168] Hyaluronidase also can be administered to patients with glaucoma to remove glycosaminoglycans from the trabecular meshwork and reduce intraocular pressure, and can be applied to the vitreous to promote the resolution of vitreous hemorrhages (i.e. extravasation of blood into the vitreous), which can occur in connection with conditions such as diabetic retinopathy, retinal neovascularization, retinal vein occlusion, posterior vitreous detachment, retinal tears, ocular traumas and the like. The presence of vitreous hemorrhages, which are typically slow to resolve, can delay, complicate or prevent procedures that require the retina to be visualized through the vitreous for diagnosis and / or for treatment procedures such as laser photocoagulation and the like which are often primary treatments for conditions such as proliferative diabetic retinopathy. d. Use in gene therapy

[0169] The efficacy of most gene delivery vehicles in vivo does not correspond to the efficacy found observed in vitro. Glycosaminoglycans can hinder the transfer and diffusion of DNA and viral vectors into many cell types. The levels of such extracellular matrix material can hinder the process considerably. Administration of hyaluronidase can open channels in the extracellular matrix, thus enhancing delivery of gene therapy. For example, hyaluronidase can be administered with collagenase to facilitate transduction of DNA in vivo (Dubensky et al. (1984) Proc Natl Acad Sci USA 81(23):7529-33). Hyaluronidase also can enhance gene therapy using adeno-associated virus (Favre et al, (2000) Gene Therapy 7(16): 1417-20). The channels opened following administration of hyaluronidase are of a size that typically enhance diffusion of

[0170] DBl / 164647321.1 38 smaller molecules such as retroviruses, adenoviruses, adeno-associated viruses and DNA complexes (as well as other therapeutic and pharmacological agents of interest). The pores are not so large, however, as to promote the dislocation and movement of cells.

[0171] In some examples, viruses can be engineered to express hyaluronidase to facilitate their replication and spread within a target tissue. The target tissue can be, for example, a cancerous tissue whereby the virus is capable of selective replication within the tumor. The virus also can be a non-lytic virus wherein the virus selectively replicates under a tissue specific promoter. As the viruses replicate, the co-expression of hyaluronidase with viral genes can facilitate the spread of the virus in vivo. e. Cosmetic uses

[0172] Hyaluronidases can be administered to remove glycosaminoglycans involved in the accumulation of cellulite and to promote lymphatic flow. In some examples, human hyaluronidases, such as for example, soluble rHuPH20, are used for the treatment of cellulite. The hyaluronidase can be administered through repeated subcutaneous injections, through transdermal delivery in the form of ointments or creams or through the use of injectable slow release formulations to promote the continual degradation of glycosaminoglycans and prevent their return.

[0173] Hyaluronidase also can be used to treat conditions such as “pigskin” edema or "orange peel" edema. Hyaluronidases can affect depolymerization of the long mucopolysaccharide chains that can accumulate in the dermis and which are responsible for the retention of bound water and of the slowing, by capillary compression, of the diffusion of organic liquids, which eliminate metabolic wastes. Such retention of water and wastes associated with fat overloading of the lipocytes, constitutes classical "pigskin" edema or "orange peel" edema. Depolymerization can cut the long chains of mucopolysaccharides into shorter chains, resulting in the elimination of the bound water and wastes and restoration of the venous and lymphatic circulation, culminating in the disappearance of local edema. f. Use in organ transplantation

[0174] The content of hyaluronic acid in an organ can increase with inflammation. An increased concentration of hyaluronic acid has been observed in tissue from different organs characterized by inflammatory-immunological injury such as alveolitis (Nettelbladt et al. (1991) Am. Rev. Resp. Dis. 139: 759-762) and myocardial infarction (Waldenstrom et al. (1991) J. Clin. Invest. 88(5): 1622-1628). Other examples include allograft rejection after a renal (Hallgren et al. (1990) J. Exp. Med. 171: 2063-2076; Wells et al. (1990) Transplantation 50: 240-243), small bowel

[0175] DBl / 164647321.1 39 (Wallander et al. (1993) Transplant. Int. 6: 133-137) or cardiac (Hallgren et al. (1990), J Clin Invest 185:668-673) transplantation; or a myocardial inflammation of viral origin (Waldenstrom et al. (1993) Eur. J. Clin. Invest. 23: 277-282). The occurrence of interstitial edemas in connection with the grafting of an organ constitutes a severe problem in the field of transplantation surgery. Grafts with interstitial edemas can swell to such a degree that the function is temporarily lost. In some instances, the swelling can cause disruption of the kidney, resulting in a massive hemorrhage. Hyaluronidases can be used to degrade accumulated glycosaminoglycans in an organ transplant. Removal of such glycosaminoglycans promotes removal of water from the graft and thus enhances organ function. g. Use in cancer treatment

[0176] Hyaluronidase has direct anticarcinogenic effects. Hyaluronidase prevents growth of tumors transplanted into mice (De Maeyer et al., (1992) Int. J. Cancer 51:657-660) and inhibits tumor formation upon exposure to carcinogens (Pawlowski et al. (1979) Int. J. Cancer 23:105- 109). Hyaluronidase is effective as the sole therapeutic agent in the treatment of brain cancer (gliomas) (WO 198802261). In addition to these effects, hyaluronidases also can be used to enhance penetration of chemotherapeutic agents into solid tumors. They can be injected intratumorally with anti-cancer agents or intravenously for disseminated cancers or hard to reach tumors. The anticancer agent can be a chemotherapeutic, an antibody, a peptide, or a gene therapy vector, virus or DNA. Additionally, hyaluronidase can be used to recruit tumor cells into the cycling pool for sensitization in previously chemorefractory tumors that have acquired multiple drug resistance (St Croix et al., (1998) Cancer Lett September 131(1): 35-44). Hyaluronidases, such as, for example soluble rHuPH20, also can enhance delivery of biologies such as monoclonal antibodies, cytokines and other drugs to tumors that accumulate glycosaminoglycans.

[0177] Hyaluronidases can also be used to increase the sensitivity of tumors that are resistant to conventional chemotherapy. For example, hyaluronidase, such as soluble rHuPH20, can be administered to a patient having a tumor associated with a HYAL1 defect in an amount effective to increase diffusion around the tumor site (e.g., to facilitate circulation and / or concentrations of chemotherapeutic agents in and around the tumor site), inhibit tumor cell motility, such as by hyaluronic acid degradation, and / or to lower the tumor cell apoptosis threshold. This can bring the tumor cell(s) to a state of anoikis, which renders the tumor cell more susceptible to the action of chemotherapeutic agents. Administration of hyaluronidase can induce responsiveness of previously chemotherapy-resistant tumors of the pancreas, stomach, colon, ovaries, and breast

[0178] DBl / 164647321.1 40 (Baumgartner et al. (1988) Reg. Cancer Treat. 1:55-58; Zanker et al. (1986) Proc. Amer. Assoc. Cancer Res. 27:390).

[0179] Hyaluronidases can be used as anti-cancer therapeutics either alone or in combination with other known anti-cancer agents. Such combinations of hyaluronidases and other anti-cancer agents can lead to synergistic effects.

[0180] In one example, hyaluronidases are used in the treatment of metastatic and non-metastatic cancers, including those that have decreased endogenous hyaluronidase activity relative to non- cancerous cells. Hyaluronidases can be used as a chemotherapeutic agent alone or in combination with other chemotherapeutics. Exemplary cancers include, but are not limited to, small lung cell carcinoma, squamous lung cell carcinoma, and cancers of the breast, ovaries, head and neck, or any other cancer associated with depressed levels of hyaluronidase activity or decreased hyaluronic acid catabolism. h. Use in treatment of glycosaminoglycan accumulation in the brain

[0181] Hyaluronic acid levels are elevated in a number of cerebrospinal pathologic conditions. Levels of cerebrospinal hyaluronic acid are normally less than 200 pg / L in adults (Laurent et al. (1996) Acta Neurol Scand September 94(3): 194-206) but can elevate to levels of over 8000 pg / L in diseases such as meningitis, spinal stenosis, head injury and cerebral infarction.

[0182] Hyaluronidases, such as, for example, soluble rHuPH20, can be utilized to degrade critically elevated levels of substrate.

[0183] The lack of effective lymphatics in the brain also can lead to life threatening edema following head trauma. Hyaluronic acid accumulation is a result of increased synthesis by hyaluronic acid synthases and decreased degradation. Accumulation of hyaluronic acid can initially serve the beneficial purpose of increasing water content in the damaged tissue to facilitate leukocyte extravasation, but continued accumulation can be lethal. Administration of hyaluronidase, such as intrathecally or intravenously, to a patient suffering from head trauma can serve to remove tissue hyaluronic acid accumulation and the water associated with it.

[0184] Hyaluronidases also can be used in the treatment of edema associated with brain tumors, particularly that associated with glioblastoma multiform. The edema associated with brain tumors results from the accumulation of hyaluronic acid in the non-cancerous portions of the brain adjacent the tumor. Administration of hyaluronidase to the sites of hyaluronic acid accumulation (e.g., by intravenous injection or via a shunt) can relieve the edema associated with such malignancies by degrading the excess hyaluronic acid at these sites.

[0185] DBl / 164647321.1 41 i. Use in treatment of glycosaminoglycan accumulation in cardiovascular disease

[0186] Hyaluronidase can be used in the treatment of some cardiovascular disease.

[0187] Administration of hyaluronidase in animal models following experimental myocardial infarct can reduce infarct size (Maclean, et al (1976) Science 194(4261): 199-200). One proposed mechanism by which this can occur is by reducing hyaluronic acid accumulation that occurs following ischemia reperfusion. Reduction of infarct size is believed to occur from increased lymph drainage and increased tissue oxygenation and reduction of myocardial water content.

[0188] Hyaluronidases also can be used to limit coronary plaques from arteriosclerosis. Such plaques accumulate glycosaminoglycans and mediate macrophage and foam cell adhesion (Kolodgie et al. (2002) Arterioscler Thromb Vase Biol. 22(10): 1642-8). j. Use in pulmonary disease

[0189] Levels of hyaluronic acid in broncheoalveolar lavages (BAL) from normal individuals are generally below 15 ng / ml. However, hyaluronic acid levels in BAL rise dramatically in conditions of respiratory distress (Bjermer et al. (1987) Br Med J (Clin Res Ed) 295(6602): 803- 6). The increased hyaluronic acid in the lung can prevent oxygen diffusion and gas exchange as well as activating neutrophil and macrophage responses. Purified preparations of soluble rHuPH20, such as those produced using the methods provided herein, can be delivered by either pulmonary or intravenous delivery to patients presenting with such conditions to reduce hyaluronan levels. Hyaluronidases also can be administered to patients suffering from other pulmonary complications that are associated with elevated glycosaminoglycans or to enhance the delivery of other co delivered molecules to the lung. k. Other uses

[0190] In further examples of its therapeutic use, hyaluronidase can be used for such purposes as an antidote to local necrosis from paravenous injection of necrotic substances such as vinka alkaloids (Few et al. (1987) Amer. J. Matern. Child Nurs. 12, 23-26), treatment of ganglion cysts (Paul et al. (1997) J Hand Surg. 22 (2): 219-21) and treatment of tissue necrosis due to venous insufficiency (Elder et al. (1980) Lancet 648-649). Hyaluronidases also can be used to treat ganglion cysts (also known as a wrist cyst, Bible cyst, or dorsal tendon cyst), which are the most common soft tissue mass of the hand and are fluid filled sacs that can be felt below the skin.

[0191] Hyaluronidases can be used in the treatment of spinal cord injury by degrading chondroitin sulfate proteoglycans (CSPGs). Following spinal cord injury, glial scars containing CSPGs are produced by astrocytes. CSPGs play a crucial role in the inhibition of axon growth. In

[0192] DBl / 164647321.1 42 addition, the expression of CSPG has been shown to increase following injury of the central nervous system (CNS). Hyaluronidases also can be utilized for the treatment of herniated disks in a process known as chemonucleolysis. Chondroitinase ABC, an enzyme cleaving similar substrates as hyaluronidase, can induce the reduction of intradiscal pressure in the lumbar spine. There are three types of disk injuries. A protruded disk is one that is intact but bulging. In an extruded disk, the fibrous wrapper has torn and the NP has oozed out, but is still connected to the disk. In a sequestered disk, a fragment of the NP has broken loose from the disk and is free in the spinal canal. Chemonucleolysis is typically effective on protruded and extruded disks, but not on sequestered disk injuries.

[0193] Hyaluronidase can be used for example in the treatment of chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, myositis, systemic sclerosis and the like, either alone or in combination with for example Efgartigimod, with which it can have synergistic effects.

[0194] Hyaluronidase can be used for example in the treatment of advanced renal cell carcinoma, melanoma, and the like, either alone or in combination with for example Nivolumab and / or Relatimab, with which it can have synergistic effects.

[0195] Hyaluronidase can be used for example in treatment or pre-exposure prophylaxis of HIV, either alone or in combination with for example Cabotegravir, with which it can have synergistic effects.

[0196] Hyaluronidase can be used for example in the treatment of Relapsing and Progressive Multiple Sclerosis, either alone or in combination with for example Ocrelizumab, with which it can have synergistic effects.

[0197] Hyaluronidase can be used for example in the treatment of Non-Hodgkin's Lymphoma (NHL), either alone or in combination with for example Rituximab, with which it can have synergistic effects.

[0198] Hyaluronidase can be used for example in the treatment of early-stage or metastatic breast cancer that is HER2-positive, either alone or in combination with for example Trastuzumab and / or Pertuzumab, with which it can have synergistic effects.

[0199] Hyaluronidase can be used for example in cancer immunotherapy, either alone or in combination with for example Atezolizumab, with which it can have synergistic effects.

[0200] Hyaluronidase can be used for example in the treatment of Non-Small Cell Lung Cancer, either alone or in combination with for example Tiragolumab and / or Atezolizumab, with which it can have synergistic effects.

[0201] DBl / 164647321.1 43 Hyaluronidase can be used for example in the treatment of multiple myeloma, either alone or in combination with for example Daratumumab, with which it can have synergistic effects.

[0202] Hyaluronidase can be used for example in the treatment of advanced or metastatic nonsmall cell lung cancer (NSCLC), either alone or in combination with for example Amivantamab, with which it can have synergistic effects.

[0203] Hyaluronidase can be used in combination with for example Rilipivirine, with which it can have synergistic effects.

[0204] Hyaluronidase can be used for example in the treatment of Alzheimer's Disease, either alone or in combination with for example Sabirnetug, with which it can have synergistic effects.

[0205] Hyaluronidase can furthermore be used with any combination of the above compounds.

[0206] Hyaluronidase can be used in combination with a compound such as an antibody or a fragment thereof, a bispecific antibody, a nanobody, a small molecule drug, or a FC receptor antagonist, with which it can have synergistic effects.

[0207] Hyaluronidase can be used either alone or in combination with other active ingredients with which they can have synergistic effects for the treatment or prevention of or use in: Advanced renal cell carcinoma (RCC), Alzheimer's Disease, AMR, Amyotrophic Lateral Sclerosis, BP, breast cancer such as HER2 positive breast cancer, cancer immunotherapy, Gastroesophageal Junction Cancer, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), Classical Hodgkin Lymphoma, Colorectal Cancer, Congenital Myasthenia, Esophageal Adenocarcinoma, Esophageal Cancer, Gastric Cancer, Hemophilia A, Hepatocellular Carcinoma, HIV PrEP and HIV Treatment, Malignant Pleural Mesothelioma, Melanoma, multifocal motor neuropathy (MMN), Multiple Myeloma, myasthenia gravis, myositis, Nephropathy, Non-Hodgkin's Lymphoma (NHL), NonSmall Cell Lung Cancer, NSCLC, Ocular MG, pelvic inflammatory disease, PFS BE, PID for example pediatric PID, RCC, Relapsing and Progressive Multiple Sclerosis, Sjogren’s, Spinal Muscular Atrophy, Squamous Cell Carcinoma of Head / Neck, Systemic Sclerosis, TED, and Urothelial Carcinoma.

[0208] D. Soluble hyaluronidases

[0209] Soluble hyaluronidases include any that, upon expression, are secreted from a cell and exist in soluble form. Such soluble hyaluronidases include, for example, but are not limited to, bacterial soluble hyaluronidases, non-human soluble hyaluronidases, such as bovine PH20 and ovine PH20, human soluble PH20, and variants thereof. Generally soluble forms of PH20 are

[0210] DBl / 164647321.1 44 produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity, since glycosylation is important for the catalytic activity and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g., DG44 CHO cells). In the present invention, the CHO cells that are preferably used for expression of soluble hyaluronidases are 2B2 cells, as further defined herein. rHuPH20 refers to the composition produced upon expression in a cell, such as CHO cell, of nucleic acid encoding residues 36-482 of SEQ ID NO: 3, generally linked to the native or a heterologous signal sequence (residues 1-35 of SEQ ID NO: 3). rHuPH20 is produced by expression of a nucleic acid molecule, such as encoding amino acids 1 -482 (set forth in SEQ ID NO: 3) in a mammalian cell. Translational processing removes the 35 amino acid signal sequence.

[0211] Compositions comprising soluble rHuPH20 as provided herein exhibit certain structural characteristics, such as a specific C-terminal heterogeneity pattern and / or abundance of particular glycans and / or a particular abundance of “hydrolyzed” polypeptide species. Without wishing to be bound by theory, these structural characteristics can be considered to be indicators of product quality, as further explained below.

[0212] In one aspect, compositions comprising soluble rHuPH20 as provided herein have a particular pattern of C-terminal heterogeneity, i.e., a particular pattern of the relative abundances of C-terminally truncated polypeptides within the composition. This pattern of C-terminal heterogeneity amounts to a structural “fingerprint” that defines the composition disclosed herein and distinguishes it from, e.g., other compositions described in the prior art (e.g., compositions disclosed in earlier work from the Applicant, in particular Genl hyaluronidase compositions as disclosed in US 2004 / 0268425 Al and Gen2 hyaluronidase compositions as disclosed in WO 2009 / 111066 Al). More specifically, as produced in the culture medium, there is heterogeneity at the C-terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more polypeptides and some shorter polypeptides, in various abundance. In a preferred embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 5). The hyaluronidase compositions provided herein have a higher proportion of longer polypeptide species (specifically, of SEQ ID NO: 4 and 5 combined, and in particular of SEQ ID NO: 5) than hyaluronidase compositions disclosed in the state of the art (e.g., in Genl hyaluronidase compositions as disclosed in US 2004 / 0268425 Al

[0213] DBl / 164647321.1 45 and Gen2 hyaluronidase compositions as disclosed in WO 2009 / 111066 Al). The hyaluronidase compositions provided herein are more homogenous than hyaluronidase compositions disclosed in the state of the art (e.g., in Genl hyaluronidase compositions as disclosed in US 2004 / 0268425 Al and in Gen2 hyaluronidase compositions as disclosed in WO 2009 / 111066 Al).

[0214] In another aspect, compositions comprising soluble rHuPH20 as provided herein have a particular glycosylation pattern, such as a particular abundance of mature N-linked glycans, such as a particular abundance of the N-linked glycan (NeuAc)2(Gal- GlcNAc)2(Man)3(GlcNAc)2Fuc. rHuPH20 and forms of soluble hyaluronidase are produced in cells, such as CHO cells, for example DG44 CHO cells, preferably 2B2 cells, that facilitate N- glycosylation. PH20 is a glycoprotein, and as known in the art, requires glycosylation to retain activity. See, e.g., U.S. Patent Nos. 8,927,249 and 9,284,543 (and PCT Publication No. WO 2010 / 077297), which describe the effects of glycosylation and partial glycosylation and elimination of glycosylation on the activity of soluble forms of PH20. These patents and publications also describe and exemplify soluble C-terminally truncated forms of PH20. In one embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are:

[0215] (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc.

[0216] The hyaluronidase compositions provided herein have a higher proportion of the mature N-linked glycan (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc than hyaluronidase compositions disclosed in the state of the art (e.g., in Genl hyaluronidase compositions as disclosed in US 2004 / 0268425 Al and Gen2 hyaluronidase compositions as disclosed in WO 2009 / 111066 Al).

[0217] Another quality characteristic of compositions comprising soluble rHuPH20 as provided herein is a reduction in protein degradation. This can be detected, e.g., as a reduction in the amount of hydrolyzed species. Correspondingly, in another aspect, the compositions comprising soluble rHuPH20 as provided herein contain a particularly low proportion of “hydrolyzed” rHuPH20. In one embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein less than 0.5%, preferably less than 0.4% of the rHuPH20 polypeptides have been hydrolyzed between Arginine 311 and Serine 312.

[0218] The rHuPH20 compositions provided herein have a lower proportion of this hydrolyzed species than hyaluronidase compositions disclosed in the state of the art (e.g., in Genl DBl / 164647321.1 46 hyaluronidase compositions as disclosed in US 2004 / 0268425 Al and Gen2 hyaluronidase compositions as disclosed in WO 2009 / 111066 Al).

[0219] In a preferred aspect, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are:

[0220] (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc.

[0221] In a preferred aspect, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 5, and wherein less than 0.5%, preferably less than 0.4% of the rHuPH20 polypeptides have been hydrolyzed between Arginine 311 and Serine 312.

[0222] In a preferred aspect, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are:

[0223] (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc, and wherein less than 0.5%, preferably less than 0.4% of the rHuPH20 polypeptides have been hydrolyzed between Arginine 311 and Serine 312.

[0224] In a more preferred aspect, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 6% of the total N- linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are:

[0225] (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc, and wherein less than 0.5%, preferably less than 0.4% of the rHuPH20 polypeptides have been hydrolyzed between Arginine 311 and Serine 312.

[0226] Without wishing to be bound by theory, all three of these structural characteristics (i.e., a higher abundance of longer hyaluronidase polypeptides, a higher abundance of longer, mature glycans, and a lower abundance of “hydrolyzed” species) are thought to reflect a more mature protein form. This is believed to result from the soluble rHuPH20-expressing cells being in a more optimal state with reduced cell stress during production, which in turn may lead to fewer degrading enzymes being released from dying cells. Such enzymes could otherwise shorten the

[0227] DBl / 164647321.1 47 rHuPH20 polypeptides and glycans and / or hydrolyzed the rHuPH20 polypeptides. While other factors could contribute to these features, all three characteristics are considered indicators of an improved hyaluronidase composition.

[0228] Another quality characteristic of compositions comprising soluble rHuPH20 as provided herein is a reduction in the presence of contaminants, such as CHO Host Cell Proteins (HCP) and DNA. Without wishing to be bound by theory, lower levels of such contaminants throughout the production process and in the final product are thought to be an indicator of an improved production process, wherein the cells are in a more optimal state and suffer from less cell death, thus reducing the amount of intracellular contaminants that are secreted into the culture medium due to cell lysis. In one embodiment, the HCCF provided herein contains less than 1 mg / ml of CHO HCP, preferably less than 0.5 mg / ml of CHO HCP. In another embodiment, the composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, contains less than 250 ng HCP per mg rHuPH20 polypeptide, preferably less than 200 ng HCP per mg rHuPH20 polypeptide, more preferably less than 150 ng HCP per mg rHuPH20 polypeptide, even more preferably less than 100 ng HCP per mg rHuPH20 polypeptide. In another embodiment, the HCCF provided herein contains less than 1000 ng DNA per mg of rHuPH20, preferably less than 800 ng DNA per mg of rHuPH20. The data provided in the examples demonstrates that the Gen3 rHuPH20 provided herein, including the final product, i.e., the composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8 provided herein, contains lower levels of HCP than rHuPH20 compositions described in the prior art, in particular lower than the Gen2 hyaluronidase compositions described in WO 2009 / 111066. In one embodiment, the HCCF provided herein contains less than 1 mg / ml of CHO HCP, preferably less than 0.5 mg / ml of CHO HCP. In another embodiment, the composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, contains less than 250 ng HCP per mg rHuPH20 polypeptide, preferably less than 200 ng HCP per mg rHuPH20 polypeptide, more preferably less than 150 ng HCP per mg rHuPH20 polypeptide, even more preferably less than 100 ng HCP per mg rHuPH20 polypeptide.

[0229] 1. Forms of Soluble Human PH20

[0230] Soluble hyaluronidases include bovine and ovine PH20, and recombinant and humanized forms thereof. Human PH20 in nature includes a GPI anchor and exists linked to sperm cells; it is not soluble. C -terminally -truncated forms thereof are soluble. Soluble forms of recombinant human PH20 have been produced and can be used in the compositions, combinations and

[0231] DBl / 164647321.1 48 methods described herein. Descriptions of and production of such soluble forms of PH20 are described, for example, in U.S. Patent Nos. 7,767,429; 8,202,517; 8,431,380; 8,431,124; 8,450,470; 8,765,685; 8,772,246; 7,871,607; 7,846,431; 7,829,081; 8,105,586; 8,187,855; 8,257,699; 8,580,252; 9,677,061; and 9,677,062, each incorporated by reference herein. The soluble hyaluronidases, thus include forms of human PH20, which are neutral active hyaluronidases and which require glycosylation for activity.

[0232] SEQ ID NO: 1 sets forth the sequence of the precursor polypeptides; the mature PH20 polypeptide (residues 36-509); soluble forms also include those with amino acid truncations at the N-terminal, such as deletions of the first one, two, three, or fours residues, such that the resulting polypeptides have an N-terminus, for example, at residue 36, 37, 38, 39, or 40, and a C- terminus at a residue from 465 to 500, and variants thereof, including, but not limited to, variants discussed below, variants known in the art, and allelic variants.

[0233] Hyaluronidases for use in the compositions, combinations, and methods herein are soluble neutral active hyaluronidases. Exemplary thereof are the soluble C-terminally truncated forms of mature human PH20. Soluble forms that have hyaluronidase activity, include but are not limited to, those that are truncated at residues from 465 to 500 of SEQ ID NO: 1 , and that are, upon expression, secreted. Exemplary thereof are polypeptides that have sequence: 36-465 of SEQ ID NO: 1, 36-466 of SEQ ID NO: 1, 36-467 of SEQ ID NO: 1, 36-468 of SEQ ID NO: 1, 36-469 of SEQ ID NO: 1, 35-470 of SEQ ID NO: 1, 36-471 of SEQ ID NO: 1, 36-472 of SEQ ID NO: 1, 36-474 of SEQ ID NO: 1, 36-475 of SEQ ID NO: 1, 36-476 of SEQ ID NO: 1, 35-477 of SEQ ID NO: 1, 36-478 of SEQ ID NO: 1 (i.e., SEQ ID NO: 8), 36-479 of SEQ ID NO: 1 (i.e., SEQ ID NO: 7), 36-480 of SEQ ID NO: 1 (i.e., SEQ ID NO: 6), 36-481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5), 36-482 of SEQ ID NO: 1 (i.e., SEQ ID NO: 4), 36-483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 46), 35-484 of SEQ ID NO: 1, 36-485 of SEQ ID NO: 1, 36-486 of SEQ ID NO: 1, 36-487 of SEQ ID NO: 1, 36-488 of SEQ ID NO: 1, 36-489 of SEQ ID NO: 1, 36-490 of SEQ ID NO: 1, 35-491 of SEQ ID NO: 1, 36-492 of SEQ ID NO: 1, 36-493 of SEQ ID NO: 1, 36-494 of SEQ ID NO: 1, 36-495 of SEQ ID NO: 1, 36-496 of SEQ ID NO: 1, 36-497 of SEQ ID NO: 1, 35-498 of SEQ ID NO: 1, 36-499 of SEQ ID NO: 1, and 36-500 of SEQ ID NO: 1, as well as N- terminally truncated forms of each of the preceding that lack two to five residues at the N- terminus, such as for example 37-368 of SEQ ID NO: 1, 38-468 of SEQ ID NO: 1, and any others that exhibit hyaluronidase activity at neutral pH, such as pH in the range of 7.0-7.4.

[0234] Thus, such soluble forms include truncated forms of the mature form of human PH20 lacking all or a portion of the C-terminal GPI anchor, so long as the hyaluronidase is soluble and

[0235] DBl / 164647321.1 49 retains hyaluronidase activity. Soluble forms are secreted upon expression in mammalian cells, and are encoded with a signal sequence, such are residues 1-35 of SEQ ID NO. 1 or a heterologous signal sequence that is cleaved by the cell to effect secretion. Soluble forms are forms that, when expressed in a cell, lack the signal peptide. Also included among soluble hyaluronidases are variants of the soluble PH20 polypeptides that exhibit hyaluronidase activity. Variants include polypeptides having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the PH20 polypeptides 36-465 of SEQ ID NO: 1, 36-466 of SEQ ID NO: 1, 36-467 of SEQ ID NO: 1, 36-468 of SEQ ID NO: 1, 36-469 of SEQ ID NO: 1, 35-470 of SEQ ID NO: 1, 36-471 of SEQ ID NO: 1, 36-472 of SEQ ID NO: 1, 36-474 of SEQ ID NO: 1, 36-475 of SEQ ID NO: 1, 36-476 of SEQ ID NO: 1, 35-477 of SEQ ID NO: 1, 36-478 of SEQ ID NO: 1 (i.e., SEQ ID NO: 8), 36-479 of SEQ ID NO: 1 (i.e., SEQ ID NO: 7), 36-480 of SEQ ID NO: 1 (i.e., SEQ ID NO: 6), 36-481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5), 36-482 of SEQ ID NO: 1 (i.e., SEQ ID NO: 4), 36-483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 46), 35-484 of SEQ ID NO: 1, 36-485 of SEQ ID NO: 1, 36-486 of SEQ ID NO: 1, 36-487 of SEQ ID NO: 1, 36-488 of SEQ ID NO: 1, 36-489 of SEQ ID NO: 1, 36-490 of SEQ ID NO: 1, 35-491 of SEQ ID NO: 1, 36-492 of SEQ ID NO: 1, 36-493 of SEQ ID NO: 1, 36-494 of SEQ ID NO: 1, 36-495 of SEQ ID NO: 1, 36-496 of SEQ ID NO: 1, 36-497 of SEQ ID NO: 1, 35-498 of SEQ ID NO: 1, 36-499 of SEQ ID NO: 1, and 36-500 of SEQ ID NO: 1. Ammo acid variants include conservative and non-conservative insertions, or deletions, or replacements, and include the modifications, singly or combinations of the modifications detailed, for example, in U.S. Patent No. 11,041,149 and International PCT publication No. WO 2013 / 102144. U.S. Patent No. 11,041,149 and International PCT publication No. WO 2013 / 102144 describe a systematic analysis and results identifying the effects of amino acid modifications at each residue in PH20 to thereby provide a structure / function map of PH20; a skilled person can identify replacement residues and consequent alterations in properties and activities, such as for effecting increases in enzymatic activity, stability in denaturing conditions, and also residues whose replacement or deletion decreases or eliminates enzymatic activity.

[0236] It is understood that residues that are important or otherwise required for the activity of a hyaluronidase, such as any described above or known to those of skill in the art, are generally invariant and, except for possible conservative amino acid substitutions, cannot be changed. These include, for example, active site residues. For example, amino acid residues 111, 113 and 176 (corresponding to residues in the mature PH20 polypeptide) of a human PH20 polypeptide, or soluble form thereof, are generally invariant and are not altered. Other residues that confer

[0237] DBl / 164647321.1 50 glycosylation and formation of disulfide bonds required for proper folding also can be invariant.

[0238] The soluble human PH20 hyaluronidase is GPI-anchored and is rendered soluble by truncation at the C-terminus by removal of all or a part of the GPI anchor. Such truncation can remove all of the GPI anchor attachment sequence or can remove only some of the GPI anchor attachment sequence. The resulting polypeptide, however, is soluble. In instances where the soluble hyaluronidase retains a portion of the GPI anchor attachment signal sequence, 1, 2, 3, 4, 5, 6, 7 or more amino acid residues in the GPI anchor attachment signal sequence can be retained, provided the polypeptide is soluble. Polypeptides containing one or more amino acids of the GPI anchor are termed extended soluble hyaluronidases. One of skill in the art can determine whether a polypeptide is GPI-anchored using methods well known in the art. Such methods include, but are not limited to, using known algorithms to predict the presence and location of the GPI anchor attachment signal sequence and co-site, and performing solubility analyses before and after digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) or D (PI-PLD).

[0239] Extended soluble hyaluronidases, which terminate for example, at residues 495, 496, 497, 498, 499, and 500, with reference to SEQ ID NO: 1 can be produced by making C-terminal truncations to any naturally GPI-anchored hyaluronidase such that the resulting polypeptide is soluble and contains one or more amino acid residues from the GPI anchor attachment signal sequence (see, e.g., U.S. Patent No. 8,927,249). These include hyaluronidases that are neutral active, soluble, contain amino acid substitutions, and have at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% or more sequence identity to amino acid sequences terminating at residues 495, 496, 497, 498, 499, and 500, with reference to SEQ ID NO:1.

[0240] Typically, for use in the compositions, combinations and methods herein, a soluble human hyaluronidase, such as a soluble human PH20, is used, such as a PH20 and variants having, for example, at least 91% or 95% or 98% sequence identity thereto, including those with 1 to 5 N-terminal residues deleted. Hyaluronidases used in the regimens, combinations, compositions, and methods herein can be recombinantly produced or can be purified or partially purified from natural sources, such as, for example, from testes extracts. Methods for production of recombinant proteins, including recombinant hyaluronidases, are well known in the art.

[0241] Recombinant soluble forms of human PH20 have been generated and can be used in the compositions, combinations and methods provided herein. For example, with reference to SEQ ID NO: 1, which sets forth the sequence of full length precursor PH20, which includes a signal sequence (residues 1-35), soluble forms include, but are not limited to, C-terminal truncated

[0242] DBl / 164647321.1 51 polypeptides of human PH20 set forth in SEQ ID NO: 1 having a C-terminal amino acid residue 467 of the sequence of amino acids set forth in SEQ ID NO: 1, 468 of the sequence of amino acids set forth in SEQ ID NO: 1, 469 of the sequence of amino acids set forth in SEQ ID NO: 1, 470 of the sequence of amino acids set forth in SEQ ID NO: 1, 471 of the sequence of amino acids set forth in SEQ ID NO: 1, 472 of the sequence of amino acids set forth in SEQ ID NO: 1, 473 of the sequence of amino acids set forth in SEQ ID NO: 1, 474 of the sequence of amino acids set forth in SEQ ID NO: 1, 475 of the sequence of amino acids set forth in SEQ ID NO: 1, 476 of the sequence of amino acids set forth in SEQ ID NO: 1, 477 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 39), 478 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 40), 479 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 41), 480 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 42), 481 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 43), 482 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 3), 483 of the sequence of amino acids set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 44), 484 of the sequence of amino acids set forth in SEQ ID NO: 1, 485 of the sequence of amino acids set forth in SEQ ID NO: 1, 486 of the sequence of amino acids set forth in SEQ ID NO: 1, 487 of the sequence of amino acids set forth in SEQ ID NO: 1, 488 of the sequence of amino acids set forth in SEQ ID NO: 1, 489 of the sequence of amino acids set forth in SEQ ID NO: 1, 490 of the sequence of amino acids set forth in SEQ ID NO: 1, 491 of the sequence of amino acids set forth in SEQ ID NO: 1, 492 of the sequence of amino acids set forth in SEQ ID NO: 1, 493 of the sequence of amino acids set forth in SEQ ID NO: 1, 494 of the sequence of amino acids set forth in SEQ ID NO: 1, 495 of the sequence of amino acids set forth in SEQ ID NO: 1, 496 of the sequence of amino acids set forth in SEQ ID NO: 1, 497 of the sequence of amino acids set forth in SEQ ID NO: 1, 498 of the sequence of amino acids set forth in SEQ ID NO: 1, 499 of the sequence of amino acids set forth in SEQ ID NO: 1 or 500 of the sequence of amino acids set forth in SEQ ID NO: 1, or polypeptides that exhibit at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity thereto, when aligned with the unmodified sequence of the soluble PH20, have activity at neutral pH, and are soluble (secreted into the medium when expressed in a mammalian cell). Soluble forms of human PH20 generally include those that contain amino acids 36-464 set forth in SEQ ID NO: 1 and terminate at any of residues, 465-500, and optionally include a 1-3 amino acid deletion at the N-terminus (i.e., lack residues 36, 36-37, or 36-38 of SEQ ID NO:1). For example, when expressed in mammalian cells, the 35 amino acid N-terminal signal sequence (residues 1-35 of SEQ ID NO: 1) is cleaved

[0243] DBl / 164647321.1 52 during processing, and a soluble form of the protein is secreted.

[0244] Thus, the mature soluble polypeptides include those that contain amino acids 36 to 467 of SEQ ID NO: 1, 468 of SEQ ID NO: 1, 469 of SEQ ID NO: 1, 470 of SEQ ID NO: 1, 471 of SEQ ID NO: 1, 472 of SEQ ID NO: 1, 473 of SEQ ID NO: 1, 474 of SEQ ID NO: 1, 475 of SEQ ID NO: 1, 476 of SEQ ID NO: 1, 477 of SEQ ID NO: 1 (i.e., SEQ ID NO: 9), 478 of SEQ ID NO: 1 (i.e., SEQ ID NO: 8), 479 of SEQ ID NO: 1 (i.e., SEQ ID NO: 7), 480 of SEQ ID NO: 1 (i.e., SEQ ID NO: 6), 481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5), 482 of SEQ ID NO: 1 (i.e., SEQ ID NO: 4), 483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 46), and up to and including 500 of SEQ ID NO: 1. Exemplary of soluble hyaluronidases are soluble human PH20 polypeptides that are 442 (i.e., SEQ ID NO: 9), 443 (i.e., SEQ ID NO: 8), 444 (i.e., SEQ ID NO: 7), 445 (i.e., SEQ ID NO: 6), 446 (i.e., SEQ ID NO: 5) or 447 (i.e., SEQ ID NO: 4) amino acids in length, such as set forth those set forth above, and variants thereof that have, for example, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and retains hyaluronidase activity. The generation of such soluble forms of recombinant human PH20 are described, for example, in U.S. Patent Nos. 7,767,429; 8,202,517; 8,431,380; 8,431,124; 8,450,470; 8,765,685; 8,772,246; 7,871,607; 7,846,431; 7,829,081; 8,105,586; 8,187,855; 8,257,699; 8,580,252; 9,677,061; and 9,677,062.

[0245] Generally soluble forms of PH20 are produced using protein expression systems that facilitate correct N-glycosylation to ensure the polypeptide retains activity, since glycosylation is important for the catalytic activity and stability of hyaluronidases. Such cells include, for example Chinese Hamster Ovary (CHO) cells (e.g., DG44 CHO cells).

[0246] The composition that is recombinantly produced from mammalian cells, such as CHO cells, has been referred to rHuPH20. Preferably, rHuPH20 as provided herein is produced in 2B2 cells, as further defined herein. The rHuPH20 composition refers to the composition produced upon expression in a cell, such as CHO cell, preferably 2B2 cells as defined herein, of nucleic acid encoding residues 36-482 of SEQ ID NO: 1 (i.e., SEQ ID NO: 4), generally linked to the native (residues 1-35 of SEQ ID NO: 1; residues 1-482 of SEQ ID NO: 1 are set forth in SEQ ID NO: 3) or a heterologous signal sequence. rHuPH20 is produced by expression of a nucleic acid molecule, such as encoding amino acids 1-482 (set forth in SEQ ID NO: 1; residues 1-482 of SEQ ID NO: 1 are set forth in SEQ ID NO: 3) or 36 to 482 (residues 36-482 of SEQ ID NO: 1 are set forth in SEQ ID NO: 4) with a heterologous signal sequence. Post translational processing removes the 35 amino acid signal sequence, resulting in polypeptide or a mixture of polypeptides, including those set forth in SEQ ID NO: 4-8. As produced in the culture medium

[0247] DBl / 164647321.1 53 there is heterogeneity at the C-terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more of SEQ ID NO: 4-8 in various abundance. More specifically, as produced in the culture medium, there is heterogeneity at the C-terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more polypeptides and some shorter polypeptides, in various abundance. In a preferred embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 5).

[0248] Generally, the soluble hyaluronidases (rHuPH20) are produced in cells that facilitate correct N-glycosylation to retain activity, such as CHO cells (e.g., DG44 CHO cells), preferably 2B2 cells as defined herein. Human soluble PH20 hyaluronidase requires glycosylation for activity. When produced recombinantly from a vector encoding residues 36-582, the most abundant species is the 446 amino acid polypeptides corresponding to residues 36-481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 5). The particular distribution of resulting polypeptides can depend upon the particular method of production. An exemplary method for production of high levels of PH20 is detailed, for example in U.S. Patent Nos. 8,187,855 and 8,343,487.

[0249] 2. C-terminal heterogeneity rHuPH20 as provided herein is a composition comprising a mixture of polypeptides, specifically a mixture of hyaluronidase polypeptides set forth in SEQ ID NO: 4-8. More specifically, as produced in the culture medium, there is heterogeneity at the C-terminus such that the product, designated rHuPH20, includes a mixture of species that can include any one or more polypeptides and some shorter polypeptides, in various abundance. In a preferred embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 5. In a more preferred embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 5.

[0250] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably

[0251] DBl / 164647321.1 54 wherein less than 10% of the polypeptides in said mixture of polypeptides (i.e., less than 10% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7, preferably wherein 9% or less of the polypeptides in said mixture of polypeptides (i.e., 9% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7.

[0252] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein less than 3% of the polypeptides in said mixture of polypeptides (i.e., less than 3% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8, preferably wherein 2% or less of the polypeptides in said mixture of polypeptides (i.e., 2% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8.

[0253] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 11% of the polypeptides in said mixture of polypeptides (i.e., at least 11% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 4.

[0254] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein 8% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6, preferably 7% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6.

[0255] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein less than 10% of the polypeptides in said mixture of polypeptides (i.e., less than 10% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7, preferably

[0256] DBl / 164647321.1 55 wherein 9% or less of the polypeptides in said mixture of polypeptides (i.e., 9% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7. In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein less than 3% of the polypeptides in said mixture of polypeptides (i.e., less than 3% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8, preferably wherein 2% or less of the polypeptides in said mixture of polypeptides (i.e., 2% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8.

[0257] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 11% of the polypeptides in said mixture of polypeptides (i.e., at least 11% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 4, and wherein 8% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6, preferably 7% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6.

[0258] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 11% of the polypeptides in said mixture of polypeptides (i.e., at least 11% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 4, and wherein less than 10% of the polypeptides in said mixture of polypeptides (i.e., less than 10% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7, preferably wherein 9% or less of the polypeptides in said mixture of polypeptides (i.e., 9% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7.

[0259] DBl / 164647321.1 56 In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 11% of the polypeptides in said mixture of polypeptides (i.e., at least 11% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 4, wherein less than 3% of the polypeptides in said mixture of polypeptides (i.e., less than 3% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8, preferably wherein 2% or less of the polypeptides in said mixture of polypeptides (i.e., 2% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8.

[0260] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 11% of the polypeptides in said mixture of polypeptides (i.e., at least 11% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 4, and wherein 8% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6, preferably 7% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6, and wherein less than 10% of the polypeptides in said mixture of polypeptides (i.e., less than 10% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7, preferably wherein 9% or less of the polypeptides in said mixture of polypeptides (i.e., 9% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7.

[0261] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 11% of the polypeptides in said mixture of polypeptides (i.e., at least 11% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 4, and wherein less than 10% of the polypeptides in said mixture of polypeptides (i.e., less than 10% of SEQ ID NO:

[0262] DBl / 164647321.1 57 4-8) consist of the sequence set forth in SEQ ID NO: 7, preferably wherein 9% or less of the polypeptides in said mixture of polypeptides (i.e., 9% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7, and wherein less than 3% of the polypeptides in said mixture of polypeptides (i.e., less than 3% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8, preferably wherein 2% or less of the polypeptides in said mixture of polypeptides (i.e., 2% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8.

[0263] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 11% of the polypeptides in said mixture of polypeptides (i.e., at least 11% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 4, and wherein 8% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6, preferably 7% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6, and wherein less than 3% of the polypeptides in said mixture of polypeptides (i.e., less than 3% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8, preferably wherein 2% or less of the polypeptides in said mixture of polypeptides (i.e., 2% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8.

[0264] In another embodiment, rHuPH20 as provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, preferably wherein at least 60% of the polypeptides in said mixture of polypeptides (i.e., at least 60% of SEQ ID NO: 4-8), more preferably at least 63% of the polypeptides in said mixture of polypeptides (i.e., at least 63% of SEQ ID NO: 4-8), consist of the sequence set forth in SEQ ID NO: 5, and wherein at least 11% of the polypeptides in said mixture of polypeptides (i.e., at least 11% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 4, and wherein 8% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6, preferably 7% or less of the polypeptides in said mixture of polypeptides (i.e., 8% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 6, and wherein less than 10% of the polypeptides in said mixture of polypeptides (i.e., less than 10% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID

[0265] DBl / 164647321.1 58 NO: 7, preferably wherein 9% or less of the polypeptides in said mixture of polypeptides (i.e., 9% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 7, and wherein less than 3% of the polypeptides in said mixture of polypeptides (i.e., less than 3% of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8, preferably wherein 2% or less of the polypeptides in said mixture of polypeptides (i.e., 2% or less of SEQ ID NO: 4-8) consist of the sequence set forth in SEQ ID NO: 8.

[0266] 3. Glycosylation of hyaluronidases

[0267] Glycosylation, including N- and O-linked glycosylation, of some hyaluronidases, including the soluble PH20 hyaluronidases, can be important for their catalytic activity and stability. For some hyaluronidases, removal of N-linked glycosylation can result in near complete inactivation of the hyaluronidase activity. For such hyaluronidases, the presence of N- linked glycans can be important for generating an active enzyme.

[0268] N-linked oligosaccharides fall into several primary types (oligomannose, complex, hybrid, sulfated), all of which have (Man) 3-GlcNAc-GlcNAc- cores attached via the amide nitrogen of Asn residues that fall within -Asn-Xaa-Thr / Ser-sequences (where Xaa is not Pro). Glycosylation at an -Asn-Xaa-Cys-site has been reported for coagulation protein C.

[0269] N-linked glycans can be separated and individually determined by methods such as hydrophilic interaction liquid chromatography (HILIC) separation with fluorescence detection of N-hydroxysuccinimide (NHS) ester labeled glycans, as further defined in the section “Monitoring and Assays” herein.

[0270] In one embodiment, the rHuPH20 composition provided herein is a composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4- 8 are:

[0271] (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc.

[0272] In this embodiment, the “% of the total N-linked glycans on the polypeptides” can be measured by HILIC separation with fluorescence detection of NHS ester labeled glycans, as further defined in the section “Monitoring and Assays” herein.

[0273] In some instances, a hyaluronidase, such as a PH20 hyaluronidase, can contain N- glycosidic and O-glycosidic linkages. For example, PH20 has O-linked oligosaccharides as well as N-linked oligosaccharides. There are six potential N-linked glycosylation sites at N82, N166, N235, N254, N368, N393 and one O-linked glycosylation site at T440 of human PH20 exemplified in SEQ ID NO: 1.

[0274] DBl / 164647321.1 59 As already defined herein, structural characteristics of the rHuPH20 provided herein include both C-terminal heterogeneity, i.e., the various abundances of SEQ ID NO: 4, 5, 6, 7 and / or 8 as defined herein, as well as the abundance of specific glycans, in particular the N- linked glycan (NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc. Any combinations of abundances of SEQ ID NO: 4, 5, 6, 7 and / or 8 as defined herein and abundances of (NeuAc)2(Gal- GlcNAc)2(Man)3(GlcNAc)2Fuc are thus also disclosed together, since they both relate to the compositions provided herein.

[0275] 4. Variants

[0276] As discussed above, variants of PH20 are known to those of skill in the art, or readily can be prepared in view of the skill and knowledge in the art. Variants include those with amino acid replacements, insertions, and deletions. Variants of the soluble PH20 polypeptides that have altered properties, such as increased stability and / or activity, have been produced. U.S. Patent No. 9,447,401 and family members U.S. Patent Nos. 10,865,400, 11,041,149 and 11,066,656 describe and provide a structure / function map of human PH20 detailing the effects of amino acid replacements at every residue in the catalytic domain of PH20. These patents provide about 7000 examples in which the effects of replacing each amino acid with 15 other amino acids on activity and stability were identified and described. By virtue of those patents, and earlier publications / patents, describing virtually all variants of soluble PH20 polypeptides are known in the art. A skilled person readily can prepare soluble hyaluronidases and variants thereof and know the properties of the resulting hyaluronidase.

[0277] Other variants also are known to those of skill in the art, and can be used in the combinations, regimens, and methods described herein. For example, see, International PCT Publication Nos. W02020022791 and W02020197230, which are incorporated by reference, and which describe modified PH20 polypeptides. These polypeptides, which include variants of the PH20 polypeptides that generally span residues 38-468, and include replacements, insertions, and deletions. The variants include for example one or more amino acid residues changes S343E, I344N, M345T, M348K, K349E, L353A, L354I, N356E, and I361T (with reference to SEQ ID NO: 1 ), and others, including about 15 amino acid variations, and truncations at the N-terminus and C-terminus. Variants that contain such modifications and others are set forth in SEQ ID NO: 52-107 of International PCT publication No. W02020022791. International PCT Publication No. WO2021150079 provides variant PH20 polypeptides described as having increased stability relative to unmodified PH20, such as those in rHuPH20. These variant polypeptides have been shown to have PH20 activity and are described as having use for subcutaneous co-administration

[0278] DBl / 164647321.1 60 with other agents.

[0279] E. Soluble rHuPH20-expressing cells

[0280] The methods described herein can be used to generate and purify large quantities of soluble rHuPH20. Soluble rHuPH20 is expressed in CHO cells that are grown in large-scale cell culture. Expression is affected using an expression vector that contains the nucleotide sequence encoding the sequence of amino acids set forth in SEQ ID NO: 3 (corresponding to amino acids 1 to 482 of the precursor human PH20 polypeptide set forth in SEQ ID NO:1). Following translation, the 35 amino acid signal sequence is cleaved and soluble rHuPH20 is secreted into the medium. The vector also contains an IRES downstream of the soluble rHuPH20 encoding region, a mouse dihydrofolate reductase gene and the SV40 pA sequence. The expression vector was introduced into DG44 cells, which are dihydrofolate reductase deficient (dhfr-) that have been adapted to grow in suspension culture in a chemically defined, animal product-free medium. The resulting soluble rHuPH20-expressing cells include cells designated 3D35M, 2B2, 3E10B, 1B3, 5C1, 1G11 and 2G10 cells. In a preferred embodiment, the soluble rHuPH20- expressing cells are 2B2 cells as further defined herein.

[0281] Other cells can be used to produce hyaluronidases similar to rHuPH20. Generally, protein expression systems suitable for the introduction of O-linked and / or N-linked glycosylation residues on hyaluronidases are used. Such cells include, for example, yeast cells, fungal cells, plant cells, insect cells and mammalian cells. Many cell lines are available for mammalian expression including mouse, rat human, monkey, chicken, and hamster cells. Exemplary cell lines include but are not limited to CHO (including DG44 cells and CHO-S cells), Balb / 3T3, HeLa, MT2, mouse NSO (nonsecreting) and other myeloma cell lines, hybridoma and heterohybridoma cell lines, lymphocytes, fibroblasts, Sp2 / 0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines also are available adapted to serum-free media, which facilitates purification of secreted proteins from the cell culture media.

[0282] 1. 3D35M cells

[0283] Exemplary of soluble rHuPH20-expressing cells are 3D35M cells, described in Example 1, below, and U.S. Patent Publication Nos. 20040268425, 20050260186 and 20060104968. 3D35M cells are dihydrofolate reductase deficient (dhfr-) DG44 CHO cells that express soluble rHuPH20. The cells were transformed with an HZ24 expression vector having the nucleotide sequence set forth in SEQ ID NO: 50. This vector contains a CMV promoter driving expression of nucleic acid encoding a 482 amino acid (SEQ ID NO:3) polypeptide that corresponds to amino acid positions 1 to 482 of the full length human PH20 set forth in SEQ ID NO: 1. This

[0284] DBl / 164647321.1 61 includes a 35 amino acid N-terminal signal sequence. The vector also contains an internal ribosome entry site (IRES) after the PH20-encoding sequence, followed by a mouse dihydrofolate reductase gene and the SV40 polyadenylation sequence. Following translation, the 482 amino acid polypeptide is processed to remove the 35 amino acid signal sequence, resulting in the secretion of soluble rHuPH20.

[0285] Soluble rHuPH20 produced from 3D35M cells by the methods herein is a mixture of species that can include one or more of the polypeptides having sequences set forth in SEQ ID NO:4-8.

[0286] The 3D35M cells can be grown in cell culture medium with or without methotrexate. Additional supplements, such as glutamine, also can be added. In some examples, the cells are grown in cell culture medium containing, for example, 50 nM, 100 nM, 500 nM, 1 pM, or 2 pM methotrexate and lacking hypoxanthine and thymidine. In one example, 3D35M cells are cultured at 37°C in 5-7% CO2 in culture medium (such as CD CHO Medium, Invitrogen) without hypoxanthine and thymidine and with 100 nM methotrexate and glutamine or a glutamine substitute, such as L-alanyl-L-glutamine, a stabilized, dipeptide form of L-glutamine. Other cell culture media appropriate for CHO cells can be used to culture 3D35M cells including, but not limited to, Dulbecco's modified Eagle's medium (DMEM), Eagle’s Minimum essential medium (EMEM), Iscove’s modified Eagle's medium (IMEM), F12 and RPMI. 3D35M cells grown under such conditions in shaking flasks can produce in excess of 1000 units / mL hyaluronidase activity. When cultured in a bioreactor, 3D35M cells can produce soluble rHuPH20 with enzymatic activity in excess of 2000 units / mL.

[0287] 2. 2B2 cells

[0288] Exemplary of soluble rHuPH20-expressing cells for production of rHuPH20 in the methods provided herein are 2B2 cells described herein. Preferably, 2B2 cells are used for production of rHuPH20 as provided herein. 2B2 cells are dihydrofolate reductase deficient (dhfr- ) DG44 CHO cells that express soluble rHuPH20. More specifically, 2B2 cells were generated by adapting 3D35M cells to higher methotrexate levels (i.e. 20 pM) and selecting clones that grew in the higher methotrexate concentration. This adaptation increased the hyaluronidase activity produced by the cells. DG44 cells are dihydrofolate reductase-deficient (dhfr-) and, therefore, cannot make nucleosides. The expression vector present in 3D35M and 2B2 cells contains, in addition to the PH20 gene, the coding sequence for mouse dihydrofolate reductase. Methotrexate is a strong competitive dihydrofolate reductase inhibitor. Therefore, by increasing the concentration of methotrexate in the culture media, the hyaluronidase-expressing cells are

[0289] DBl / 164647321.1 62 forced to produce more mouse dihydrofolate reductase to remain viable. This can be affected by, for example, gene amplification or rearrangement of the integrated DNA to a more stable and productive arrangement. Thus, forcing an increase in the production of mouse dihydrofolate reductase also can result in an increase in the production of soluble rHuPH20. A comparison of enzymatic activity of soluble rHuPH20 produced by 2B2 cells and 3D35M cells demonstrated that activity was typically between 80% and 100% higher in 2B2 cells compared to 3D35M cells.

[0290] 2B2 cells were selected from amongst the cell clones that were isolated following selection with 20 pM methotrexate as the cell line that produced soluble rHuPH20 having the greatest enzymatic activity. In one example, southern blot analysis of Spe I-, Xba I- and BamH I / Hind Ill-digested genomic 2B2 cell DNA using a probe specific for the nucleic acid region encoding soluble rHuPH20 revealed the following restriction digest profile: one major hybridizing band of -7.7 kb and four minor hybridizing bands (-13.9, -6.6, -5.7 and -4.6 kb) with DNA digested with Spe I; one major hybridizing band of -5.0 kb and two minor hybridizing bands (-13.9 and -6.5 kb) with DNA digested with Xba I; and one single hybridizing band of -1.4 kb observed using 2B2 DNA digested with BamH I / Hind III.

[0291] 2B2 cells can be grown in cell culture medium with or without methotrexate. Additional supplements, such as L-cysteine, glutamine, insulin and yeast extract also can be added. In some examples, the cells are grown in cell culture medium that does not include insulin or yeast extract. In some examples, the cells are grown in cell culture medium containing, for example, 50 nM, 100 nM, 500 nM, 1 pM, 2 pM, 5 pM, 10 pM, 20 pM or more methotrexate and lacking hypoxanthine and thymidine. In one example, 2B2 cells are cultured at 37 °C in 5-7% CO2 in culture medium (such as CD CHO Medium, Invitrogen) without hypoxanthine and thymidine and with 20 pM methotrexate and glutamine or L-alanyl-L-glutamine, a stabilized, dipeptide form of L-glutamine. Other cell culture media appropriate for CHO cells can be used to culture 2B2 cells, including, but not limited to, Dulbecco's modified Eagle's medium (DMEM), Eagle’s Minimum essential medium (EMEM), Iscove’s modified Eagle's medium (IMEM), F12 and RPMI. 2B2 cells grown under such conditions in shaking flasks can produce high hyaluronidase activity. By adding anti-foaming agents and / or copper sulfate to the cell culture medium, oxidation of the hyaluronidase in the cell culture medium can be controlled or eliminated to further increase the enzymatic activity of hyaluronidase. When cultured in a bioreactor under such conditions, 2B2 cells can produce soluble rHuPH20 having enzymatic activity in excess of 25,000 units / mL hyaluronidase activity.

[0292] DBl / 164647321.1 63 2B2 cells can be stored frozen, such as at -20 °C, -70 °C or -80 °C, preferably below -60 °C, or maintained in culture at, for example, 37 °C. Frozen 2B2 cells can be thawed using routine methods for thawing CHO cells that are well-known in the art, and can subsequently be tested for viability using methods for viability testing of CHO cells that are well-known in the art. Briefly, a frozen vial of 2B2 cells can be removed from liquid nitrogen storage and gently thawed in pre- warmed growth medium, before adding the cells to a culture flask and maintaining in culture at, for example, 37 °C. Viability can be determined, e.g., using a trypan blue viability assay to distinguish live from dead cells based on membrane integrity. Automatic cell counters (hemocytometers) can be used, or the cells can be counted manually. Soluble rHuPH20 produced from 2B2 cells by the methods herein is a mixture of species of polypeptides having sequences set forth in SEQ ID NO:4-8. As noted for soluble rHuPH20 produced from 3D35M cells, the heterogeneity in the soluble rHuPH20 preparation from 2B2 cells is likely a result of C-terminal cleavage by peptidases present during the production and purification methods provided herein. In particular, without wishing to be bound by theory, it is thought that intracellular peptidases may be released into the culture medium as cell death occurs during culture, and these released peptidases may then negatively affect the structural integrity of the hyaluronidase protein product in the cell culture medium (i.e., structural integrity as evidenced, e.g., by C-terminal heterogeneity, abundance of longer, more mature glycans and abundance of “hydrolyzed” species).

[0293] 2B2 cells have been deposited at the American Type Culture Collection (ATCC, 10801 University Blvd, Manassas, VA 20110 USA) by Halozyme, Inc. on November 13, 2025. The identification reference for 2B2 cells given by the depositor is HZ24-2B2. The deposit accession number for 2B2 cells given by the ATCC is PTA-127953. The deposit was made at the ATCC (10801 University Blvd, Manassas, VA 20110 USA) according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The terms “2B2 cells” and “HZ24-2B2 cell line” are used interchangeably herein. The Applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted. Also disclosed herein are cell lines that are derived from 2B2 cells that still have the capacity to produce soluble rHuPH20. In one aspect, such a cell line derived from 2B2 cells can be obtained by gradually adapting 2B2 cells to grow in cell culture medium containing more than 20 pM methotrexate by gradually increasing the amount of methotrexate in the cell culture medium

[0294] DBl / 164647321.1 64 above 20 pM methotrexate when passaging the cells. In one aspect, such a cell line that is derived from 2B2 cells in this manner is selected based on a higher level of enzymatic activity of hyaluronidase measured in the cell culture fluid. This enzymatic activity of hyaluronidase can be determined by using the turbidimetric- based assay described herein (see, e.g., Example 14 and the section “Monitoring soluble rHuPH20 production”). For example, this assay includes incubating soluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period of 10 minutes, precipitating undigested sodium hyaluronate with addition of acidified serum albumin, measuring turbidity of resulting sample at 640 nm after a 20 minute development period, and comparing with a calibration curve generated with dilutions of a soluble rHuPH20 assay working reference standard.

[0295] F. Cell culture expansion

[0296] The methods described herein can be scaled up or down by one of skill in the art. Further, modifications to, for example, cell media, incubation times, or feeding protocols may be made. One of skill in the art can empirically determine the appropriate conditions for protein production for any given bioreactor and cell type.

[0297] The methods described herein employ bioreactors to grow large volumes of cell culture to produce large quantities of soluble rHuPH20. The soluble rHuPH20-expressing cells, such as 2B2 cells, are initially expanded from an original inoculum, such as an aliquot of cells from a working cell bank (WCB) or master cell bank (MCB), to a larger volume prior to culture in the bioreactor for the production phase. The final culture volume in the expansion phase is directly proportional to the volume of the bioreactor used in the following production phase.

[0298] The soluble rHuPH20-expressing cells are expanded through a series of cultures, each increasing in volume from the previous one, and each being used as the inoculum for the subsequent culture. Exemplary of such cells are 2B2 cells. Preferably the rHuPH20-expressing cells are 2B2 cells. The original inoculum is typically one in which the purity and identity of the cells and the cell number are defined. These cells can be stored frozen, such as at -20 °C, -70 °C or -80 °C, preferably below -60 °C, or maintained in culture at, for example, 37 °C. In some instances, the original inoculum is a MCB or WCB aliquot that has been stored frozen. In some examples, the target split ratio range for the first passage out of thaw are 1 :2 to 1:8 may be used, preferably a target split ratio range of 1 :4 to 1:8 may be used. The target split ratio range for all subsequent passages in unbaffled shake flasks may be between 1 : 5 to 1: 11. When the cells reach the desired density, such as, for example, greater than 0.8 x 106cells / mL (e.g. between 0.8 x 106cells / mL and 2.5 x 106cells / mL), the cell culture is used to inoculate a larger bioreactor

[0299] DBl / 164647321.1 65 containing a volume of fresh cell culture media. Cells can be inoculated into this expanded culture at a density of 0.2 x 106to 0.5 x 106cells / mL, such as 0.4 x 106cells / mL.

[0300] In one embodiment, the inoculum may be thawed in a 37 °C water bath and maintained in an unbaffled shake flask. The inoculum may be maintained in an unbaffled shake flask. The basal media may be CD CHO Medium (e.g., Gibco 12490025) with 8 mM L-Glutamine (e.g., Gibco 21051040) and 20 pM Methotrexate (e.g., Millipore Sigma M7824). The shake flask may have a percentage of CO2 is between 4% and 11%, typically between 6.0% and 9.0%, such as between 6% and 8%, and the target CO2 concentration may be 7.0. Typically, the temperature is between 35 °C and 39 °C, typically between 36 °C and 38 °C, and the target temperature may be 37 °C. For example, 2B2 and 3D35M cells can be grown in a 36 °C humidified incubator with 7% CO2. The culture can be agitated, such as at 90-140 rpm, during this process, preferably between 120-140 rpm, and the target agitation may be at 130 rpm. The shake flasks may have volume of about 25 mL, 50 mL, 75 mL, 100 mL, 125 mL, 150 mb, 175 mb, or 200 mb or more. This cell culture can then be used to expand the cell culture prior to being used in a production bioreactor.

[0301] In one embodiment, the inoculum may be in a series of unbaffled shake flasks, with transfers based on target viable cell concentration (VCC). The basal media may be CD CHO Medium (e.g., Gibco 12490025) with 8 mM L-Glutamine (e.g., Gibco 21051040) and 20 pM Methotrexate (e.g., Millipore Sigma M7824). The rocking bioreactors may have a percentage of CO2 is between 4% and 11%, typically between 6.0% and 9.0%, such as between 6% and 8%, and the target CO2 concentration may be 7.0. Typically, the temperature is between 35 °C and 39 °C, typically between 36 °C and 38 °C, and the target temperature may be 37 °C. The culture can be agitated, such as at 90-140 rpm, during this process, preferably between 120-140rpm, and the target agitation may be at 130 rpm. Target split ratio range for the first passage out of thaw are preferably, 1 :4 to 1:8 but a split ratio as low as 1 :2 may be used. Target split ratio range for all subsequent passages in unbaffled shake flasks are preferably 1 : 5 to 1: 11.

[0302] The inoculum may then be further expanded in a series of rocking bioreactors, with transfers based on target viable cell concentration (VCC). The basal media may be CD CHO Medium (e.g., Gibco 12490025) with 8 mM L-Glutamine (e.g., Gibco 21051040) and 20 pM Methotrexate (e.g., Millipore Sigma M7824). Target split ratio range for all rocking bioreactor expansion steps are preferably between 1 : 5 and 1 :11.

[0303] As used herein, the term “basal media” refers to the culture media used during initial scale-up (expansion) of the cells. As described above, a basal media for the described expansion

[0304] DBl / 164647321.1 66 steps can comprise, e.g., CD CGO AGT™ media (Invitrogen), supplemented with 8 mM glutamine and 20 pM methotrexate. Exemplary alternative basal media include CD CHO media (Invitrogen), or reconstituted powdered CD CGO AGT™ media (Invitrogen), supplemented with 8 mM glutamine or L-alanyl-L-glutamine and 20 pM methotrexate or 8 mM glutamine or L- alanyl-L-glutamine and 100 mM methotrexate.

[0305] In one embodiment, a final step of expansion may be using a N-l bioreactor which can be used to grow 2B2 cells to the optimum VCC, viability, and working volume to seed a production bioreactor. In some examples, the inoculum bioreactor culture may be maintained in CD CHO Medium (e.g., Gibco 12490025) with 8 mM L-Glutamine (e.g., Gibco 21051040) without Methotrexate. In some examples, antifoam, such as Ex-Cell® Antifoam is added as needed. Typically, the temperature is between 35°C and 39°C, typically between 36°C and 38°C, and the target temperature may be 37°C. A transfer VCC may be achieved about day 3-5 and be about 3.5 x 106 cells / mL to 4.5 x 106cells / mL, and the target may be 4 x 106cells / mL. The target split ratio for such an inoculum bioreactor (N-l) expansion step is preferably between 1 :4 and 1: 10.

[0306] This process, like any of the processes described herein, also can be scaled-up by one of skill in the art for introduction of the cells into a bioreactor with a culture volume larger than 300 L. For example, the process can be scaled up for introduction of the cells into a bioreactor with a 2000 L culture volume. Thus, in one example of the methods provided herein, following thawing, the cells are serially expanded through a 125 mL shaker flask (working volume of 20- 30 mL, such as 25 mL), a 250 mL shaker flask (working volume of 45-55 mL, such as 50 mL), a 1 L shaker flask (working volume of 190-210 mL, such as 200 mL), two 2 L shaker flasks (working volume of 350-450 mL per flask, such as 400 mL per flask), six 2 L shaker flasks (working volume of 350-450 mL per flask, such as 400 mL per flask), a 25 L wave bioreactor (working volume of 14-16 L, such as 15 L), a 100 L wave bioreactor (working volume of 75-85 L, such as 80 L), and a 600 L seed bioreactor (working volume of 440-520 L, such as 480 L).

[0307] G. Protein production

[0308] Following cell expansion, the soluble rHuPH20-expressing cells are transferred to a production bioreactor for the production phase, during which large quantities of soluble rHuPH20 are secreted into the cell media. This phase typically is designed such that the cells growth is maximized in the first half of the bioreactor run, and soluble rHuPH20 production is maximized in the second half of the bioreactor run. The cells are provided with feed media at particular time points throughout the production to regulate this process. The term “feed media” as used herein refers to media containing specific supplements to enable sustaining of the

[0309] DBl / 164647321.1 67 culture. The bioreactor is typically monitored to ensure optimal environmental conditions, e.g., temperature, dissolved oxygen, pH, osmolarity, are maintained throughout the process. Bioreactors of different sizes and designs can be utilized in the methods herein. Bioreactors with working volumes of between 1 L and 6000 L or more can be used in the methods herein. In some examples, a 5 L, 36 L, 125 L, 400 L or 3500 L bioreactor is used in the methods herein to culture cells in volumes of approximately 4 L, 23 L, 100 L, 300 L and 2500 L, respectively. Bioreactors may be sterilized prior to the addition of cell culture media or cells. The bioreactor can be supplied pre-sterilized in a single use format by either gamma ray irradiation or X-ray irradiation. Sterilization of stainless steel bioreactors can be affected by autoclaving or otherwise treating with steam for some bioreactors, or by treatment with a sterilizing solution, such as dilute sodium hydroxide, dilute nitric acid or sodium hypochlorite. In some examples, the bioreactor is sterilized by steam at 121 °C, 20 PSI for 30 minutes. Following sterilization, cell culture media can be added to the bioreactor and then assessed for contamination, such as by microbial contamination, after a period of time to ensure that the sterilization process was effective.

[0310] The cell culture from the expansion phase, described above, is added to the sterilized bioreactor containing fresh cell culture media. Generally, the soluble rHuPH20-expressing cells are inoculated into the fresh cell culture media at a cell density of 104to 107cells / mL, such as 105to 106cells / mL. For example, cells can be inoculated at a density of 1 x 104, 5 * 104, 1 x105, 5xl05, I xlO6, 4xl06, or I xlO7cells / mL. In one example, soluble rHuPH20-expressing cells are inoculated at a cell density of 4 x 105cells / mL. The total cell count following inoculation can be, therefore, between 107and 1014, depending on the size of the bioreactor and the cell density. For example, a cell culture volume of 100 L can have a cell density following inoculation of approximately 109, 1010, 1011or 1012cells. In another example, a cell culture volume of 2500 L can have a cell density following inoculation of approximately 1010, 1011, 1012or 1013cells.

[0311] The volumes of inoculating cell culture and fresh culture media used are dependent upon the size of the bioreactor and the cell density of the inoculum. For example, approximately 30 L of soluble rHuPH20-expressing cells, such as 2B2 cells, can be added to a 400 L bioreactor containing 230 L fresh cell culture media, for a total volume of approximately 260 L and an inoculation cell density of 4 x 105cells / mL (total cell count of approximately 1011cells). This can be scaled up or down as necessary, depending on the bioreactor. For example, approximately 20 L of soluble rHuPH20-expressing cells, such as 3D35M cells, can be added to a 125 L bioreactor containing 65 L fresh cell culture media, for a total volume of approximately 85 L and

[0312] DBl / 164647321.1 68 an inoculation cell density of 4 x 105cells / mL (total cell count of approximately 3.4 x IO10cells). In another example, for production in a 3500 L bioreactor, 2B2 cells are added to fresh cell culture media for a total cell culture volume of 1900-2300 L, such as 2100 L.

[0313] The fresh cell culture media contains the appropriate supplements to provide the necessary nutrients to the cells to promote cell growth.

[0314] In some instances, the basal medium contains sufficient glucose that no further glucose needs to be added. In other instances, glucose is added to the media later in the production process, such as in subsequent feed media. In some examples, glucose is added to the cell culture medium periodically such as, for example, once daily. For example, a bolus of glucose at 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM or 20 mM or, alternatively, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 15 g / Lor 20 g / Lto achieve a glucose concentration range of about 2 g / L to about 8 g / L of production culture can be added to the cell culture medium every 24 hours.

[0315] Glutamine or glutamine-substitutes, such as L-alanyl-L-glutamine, and / or feed media that have been designed to replenish depleted nutrients in CHO cell cultures during fed-batch production can support cell cycle progression and also enhance cell growth. Examples of such feed media for fed-batch cultures of CHO cells that are commercially available include, but are not limited to, Ex-Cell® Advanced CHO Feed 1 and Cellvento® 4Feed. One of skill in the art knows, from their common general knowledge, how to identify and obtain suitable feed media for fed-batch cultures of CHO cells that promote cell growth and productivity. Such media can comprise, e.g., amino acids including the essential amino acids, vitamins, salts, and trace elements. One of skill in the art can empirically determine the amount and quality of the nutrients that can be supplemented to the basal medium. In some examples, these supplements can be added to the basal cell culture medium at 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM or 20 mM. For example, basal cell culture medium supplemented with 8 mM L-alanyl-L-glutamine can be used as the fresh cell culture media into which the soluble rHuPH20-expressing cells are inoculated. Similar approaches can additionally or alternatively be taken with asparagine. Additional supplements, such as antibiotics, anti- fungals, indicators, salts, vitamins, amino acids, minerals, fatty acids, and growth factors also can be added. In one embodiment, the basal medium for production culture is CD CHO Medium (e.g., Gibco 12490025) with 8 mM L Glutamine (e.g., Gibco 21051040) without Methotrexate.

[0316] The individual parameters of the bioreactor can be set to maintain optimal conditions throughout the protein production process. The specific parameters that can be set depend on the

[0317] DBl / 164647321.1 69 bioreactor used, and can include, but are not limited to, temperature, pH, dissolved oxygen, impeller speed, vessel pressure, air sparge and air overlay. In one example, the conditions of a 2000 L bioreactor containing an initial culture volume of approximately 1500 L are set to; temperature: 36 °C (or between 34 °C and 38 °C, with a shift to 33 °C (or 32 °C to 34 °C), in one preferred embodiment, during culturing a temperature of the growth medium is maintained to be 36 °C for first 9 days and changed to 33 °C at day 9; impeller rotation: 20 W / m3; vessel pressure: 5 psi (or 3-7 psi); air sparge: 12 L / minute (or 11-13 L / minute); dissolved oxygen: 25% (or 20% to 30%); pH controlled at: 6.9 (or pH 6.5-7.3) One of skill in the art can empirically determine the appropriate conditions for growth of a particular soluble rHuPH20-expressing cell in a particular bioreactor.

[0318] The soluble rHuPH20-expressing cells are typically cultured in the bioreactor for between 10 and 25 days. In some examples, the soluble rHuPH20-expressing cells are cultured in the bioreactor for 12, 13, 14, 15 or 16 days before harvesting. In other examples, the cells are harvested when the viability % falls to a particular level, such as, for example, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 70 %, 80 % or 90 %. In one example, the cells are harvested within 24 hours of the viability % dropping below 80 %.

[0319] During the bioreactor culture, the cells can be grown as batch cultures, in which the culture is grown to completion without the addition of further nutrients. In other examples, the cells are grown as fed-batch cultures and provided with a series of feed media at particular time points to supplement nutrients and glucose throughout. In some instances, the nutrients provided in the cell culture medium into which the cells were inoculated have depleted by days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more post-inoculation. Thus, providing additional nutrients or supplements can produce higher yields of protein than batch cultures. In one example, cells are provided with feed media daily. Preferably, this may be on days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more. Alternatively, feed media may be added daily between days 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 21 days, or more, preferably 2 to 16 days. In another example, cells are provided on days

[0320] 6, 9 and 11 post-inoculation. In further example, cells are provided with feed media on days 7, 9 and 11 post-inoculation. In a yet further example, cells are provided with feed media on days 5,

[0321] 7, 9 and 11 post-inoculation. In yet another example, cells are provided with feed media daily after 2 days. In one example, glucose (e.g., Gibco 15023) is fed daily starting on day 2, as needed, back to a target concentration of 5.0 g / L. Bolus Copper Sulfate (100 mM stock solution, e.g., Millipore Sigma C8027) is added on day 9 to a target concentration of 100 pM CuSC of

[0322] DBl / 164647321.1 70 the current working volume. Following the bolus Copper Sulfate addition on day 9, a temperature shift from 36.0 °C to 33 °C is performed. In one embodiment, pH may be controlled using carbon dioxide and sodium bicarbonate. The volume of feed media added to the bioreactor culture per feed can range between, for example, 0.5 % and 20%, such as 1-20%, 2-15%, 3-10% or 4-5% of the cell culture volume. In some instances, the feed media is added at a volume per feed equivalent to 1% of the cell culture volume. Total feed media added to the reactor can range from, for example, 5% to 50%.

[0323] The addition of various supplements to the feed media also can be used to regulate the growth and / or cell cycle of the cells, cell viability, and / or cell productivity. Exemplary nutrients and supplements that can be included in the feed media include, but are not limited to, glutamine or glutamine-substitute, such as L-alanyl-L-glutamine, 1-cysteine s-sulfate sodium salt sesquihydrate, glucose, and feed media that have been designed to replenish depleted nutrients in CHO cell cultures during fed-batch production. Examples of such feed media for fed-batch cultures of CHO cells that are commercially available include, but are not limited to, Ex-Cell® Advanced CHO Feed 1 and Cellvento® 4Feed. One of skill in the art knows, from their common general knowledge, how to identify and obtain suitable feed media for fed-batch cultures of CHO cells that promote cell growth and productivity. Such media can comprise, e.g., amino acids including the essential amino acids, vitamins, salts, and trace elements. Furthermore, the feed media also can be concentrated, thus providing additional nutrients, such as amino acids, which in some embodiments may be essential amino acids, that may have been depleted during cell culture. The feed media can be 2*, 3*, 4*, 5*, 6* or more concentrated. In other examples, the feed media is less concentrated, or the same concentration as the cell culture media in the bioreactor.

[0324] The supplements included in the feed media can be used to regulate cell growth and protein production. For example, the first feed media added to the cell culture can include nutrients that enhance cell cycle progression, cell growth and peak cell density. Subsequent feed media can promote cell growth arrest and / or protein synthesis. The amount of each supplement in each feed media can vary, such as by increasing or decreasing from one feed media to the next or can be the same from one feed media to the next. In some examples, the amount of a supplement is increased from one feed media to the next, such as by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or more. In other examples, the amount of a supplement is decreased from one feed media to the next, such as by 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In one example, a supplement is omitted from a

[0325] DBl / 164647321.1 71 feed media. In other examples, the amount of a supplement in the feed media stays the same. One of skill in the art can empirically determine the optimum amount of each supplement for each feed media to promote the desired amount of cell growth and protein production.

[0326] Exemplary supplements or nutrients that can be included in the feed media include, but are not limited to, glucose, 1-cysteine s-sulfate sodium salt sesquihydrate, glutamine or glutamine-substitute, such as L-alanyl-L-glutamine, and feed media that have been designed to replenish depleted nutrients in CHO cell cultures during fed-batch production. Examples of such feed media for fed-batch cultures of CHO cells that are commercially available include, but are not limited to, Ex-Cell® Advanced CHO Feed 1 and Cellvento® 4Feed. The type and amount of supplement added can influence cell growth and protein production. For example, glutamine or glutamine-substitute can be incorporated into the first feed media added to the cell culture to increase cell growth and peak cell density. Subsequent feed media can be designed to promote protein production more than cell growth. Supplements such as glutamine, glutamine-substitute, or L-alanyl-L-glutamine can be excluded or reduced in amount. In addition, supplements that enhance cell cycle arrest and, therefore, increased protein production, also can be included.

[0327] Glutamine or glutamine-substitute, such as L-alanyl-L-glutamine, Glucose, Cysteine Sulfate, Copper (II) Sulfate Pentahydrate, feed media that have been designed to replenish depleted nutrients in CHO cell cultures during fed-batch production, also can be added to the feed media. Examples of feed media that have been designed to replenish depleted nutrients in CHO cell cultures during fed-batch production that are commercially available include, but are not limited to, Ex-Cell® Advanced CHO Feed 1 and Cellvento® 4Feed. In some instances, the amount of glutamine or glutamine-substitute added to the first feed media is more than the amount of glutamine or glutamine-substitute added to subsequent feed media. In particular examples, the amount of glutamine or glutamine-substitute added to each subsequent feed media is reduced compared to the amount added in the prior feed media. The optimal amount added to each feed media can be determine empirically by one of skill in the art, and can include, for example, concentrations of glutamine or glutamine-substitute at or about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM or more.

[0328] In one embodiment, the feed media is feed media comprising amino acids including the essential amino acids, vitamins, salts and trace elements, and further comprises Cysteine Sulfate. For example, the feed media comprises a 50 / 50 blend of Ex-Cell® Advanced CHO Feed 1 (Millipore Sigma 24368C) and Cellvento® 4Feed (Millipore Sigma 103796) with L-Cysteine Sulfate (e.g., Millipore Sigma 137116). In other embodiments, the feed media is feed media

[0329] DBl / 164647321.1 72 comprising amino acids including the essential amino acids, vitamins, salts and trace elements, and further comprises 0.5 M L-Cysteine Sulfate, 40% Glucose, and 100 mM CuSO4. For example, the feed media comprises Ex-Cell® Advanced CHO Feed 1, Cellvento® 4Feed + 20mM L-Cysteine Sulfate, 40% Glucose, and 100 mM CuSO4. For example, the feed medium additive formulation may be 34.10 g / kg Ex-Cell® Feed 1 powder, 130.35 g / kg Cellvento® 4Feed powder, 40 ml / kg 0.5 M L-Cysteine Sulfate, 400 g / mL Glucose, powder, and 25 g / kg Copper (II) Sulfate Pentahydrate.

[0330] Preferred amino acid concentrations for the base media and feed media are set out in Table 2.

[0331] Table 2: Exemplary Base media and feed media amino acid concentrations

[0332] *using 14% total feed based on w / w or v / v

[0333] ** calculated as individual amino acid LB divided by total amino acids UB ***calculated as individual amino acid UB divided by total amino acids LB LB = lower bound;

[0334] DB1 / 164647321.1 73

[0335] UB = upper bound

[0336] In some embodiments, the initial cell culture medium contains at least 40 mM in amino acids or greater than or equal to 40% of the total amino acids supplied, and total amino acids provided are at least 100 mM.

[0337] In some instances, the concentration of various amino acids may be varied. For example, the total amount of asparagine and aspartate supplied are at least 10 mM combined, preferably 13 mM combined, or the total amount of cystine and cysteine supplied are at least 1 mM combined, or the total amount of asparagine, aspartate, glutamine, glutamate, and glutamax are at least 20%, preferably 25%, of the total amino acids supplied, or the total amount of serine is at least 11%, preferably 16%, of the total amino acids supplied.

[0338] In some instances, the expressed hyaluronidase polypeptide is neutral active and contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide. For example, the expressed hyaluronidase polypeptide may consist of the sequence of amino acids set forth as amino acids 1-477 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 39), amino acids 1-478 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 40), amino acids 1-479 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 41), ammo acids 1-480 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 42), amino acids 1-481 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 43), amino acids 1- 482 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 3) or amino acids 1-483 set forth in SEQ ID NO: 1 (i.e., SEQ ID NO: 44) or consist of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as amino acids 1 -477 of SEQ ID NO: 1 (i.e., SEQ ID NO: 39), ammo acids 1-478 of SEQ ID NO: 1 (i.e., SEQ ID NO: 40), ammo acids 1-479 of SEQ ID NO: 1 (i.e., SEQ ID NO: 41), ammo acids 1-480 of SEQ ID NO: 1 (i.e., SEQ ID NO: 42), ammo acids 1-481 of SEQ ID NO: 1 (i.e., SEQ ID NO: 43) or ammo acids 1-482 of SEQ ID NO: 1 (i.e., SEQ ID NO: 3) or ammo acids 1-483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 44).

[0339] In some instances, the expressed hyaluronidase polypeptide contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide, and the hyaluronidase polypeptide is soluble and catalytically active. For example, the polypeptide may (i) consists of a contiguous sequence of amino acids contained within SEQ ID NO: 1, whereby the polypeptide comprises amino acids 36-464 of SEQ ID NO:1 and terminates at a

[0340] DBl / 164647321.1 74 residue selected from among 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO:1; or (ii) a polypeptide that contains amino acid substitutions in the sequence of amino acids of the polypeptide of (i), whereby the amino acid-substituted polypeptide consists of a sequence of amino acids that has at least about 91% amino acid sequence identity with the polypeptide of (i). In some examples, the hyaluronidase polypeptide consists of a sequence of amino acids that has at least 98% amino acid sequence identity with the sequence of amino acids set forth as amino acid residues 36-483 of SEQ ID NO: 1 (i.e., SEQ ID NO: 46).

[0341] In some instances, the expressed hyaluronidase polypeptide contains a contiguous sequence of amino acid residues of SEQ ID NO: 1, but it is C-terminally truncated so that it does not contain all or a portion of the GPI anchor, whereby the polypeptide is soluble and neutral active.

[0342] In a preferred embodiment, the expressed hyaluronidase polypeptide comprises a mixture of polypeptides that comprises the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides consists of the sequence set forth in SEQ ID NO: 5, more preferably wherein at least 63% of the polypeptides in said mixture of polypeptides consists of the sequence set forth in SEQ ID NO: 5.

[0343] The production bioreactor is typically harvested either on Day 15 to 17, preferably Day 16 or within 24 hours of receiving a viability measurement < 80%, whichever comes first.

[0344] H. Purification

[0345] The soluble rHuPH20 is purified from the host cell protein solution using a series of purification steps. Many purification techniques are known in the art and can be utilized in the methods herein. Such methods can include, but are not limited to, chromatographic and non- chromatographic methods such as anion-exchange chromatography (AEX), mixed-mode chromatography (MMC), size-exclusion chromatography (SEC), affinity chromatography (AC), charged depth filtration, high performance liquid chromatography (HPLC), reversed phase chromatography (RPC) and hydrophobic interaction chromatography (HIC), and gel filtration methods, or any combination thereof.

[0346] 1. Harvest Clarification

[0347] The cells are harvested from the bioreactor and processed through a cell removal and clarification system to separate the cell culture fluid containing the hyaluronidase from cells and cell debris, e.g., by using a depth filter, resulting in the HCCF. One example of such a system is a synthetic fibrous anion exchange (AEX) chromatographic media, e.g., having a quaternary ammonium (Q) functionality, combined with a 0.2 pm poly ethersulfone membrane to separate

[0348] DBl / 164647321.1 75 the cells, cell debris, and host cell impurities from the harvest fluid containing the soluble rHuPH20. Cells and cell debris are retained by the filter based on size and host cell impurities are bound based on charge, while soluble rHuPH20 flows through the media.

[0349] An exemplary functionalized harvest clarification media useful for depth filtration that contains charged based separation capabilities includes, but is not limited to, Harvest RC, available from 3M™.

[0350] The HCCF is defined as the fluid that has been separated from cells, cell debris and aggregates. The HCCF is obtained from the CCF with minimal change to the volume of the fluid in comparison to the CCF. More specifically, the volume of fluid between the CCF and the HCCF may increase by up to about 15% due to the nature of the clarification step.

[0351] Previous purification methods such as the Applicant’s own earlier Gen2 method as described in W02009 / 111066 use a further Harvest Clarification Tangential Flow Filtration Step. In the present invention, this step has been removed which prevents volumetric loss of protein that occurs when concentrating, recovering, and filtering the rHuPH20 containing solution, further increasing overall process yield of rHuPH20.

[0352] 2. Affinity Chromatography

[0353] After harvest clarification, the resulting harvest cell culture fluid (HCCF) containing hyaluronidase is further processed through a series of chromatography steps. One example of such a step is affinity chromatography, e.g., having Cibacron Blue ligand containing sulfonic groups that can take part in ionic exchange interactions and other parts of the ligand that can take part in hydrophobic interactions. The harvest cell culture fluid (HCCF) is passed through such an affinity column. One example of a suitable affinity resin is a resin comprising Cibacron Blue (also sometimes referred to as Cibacron Blue F3G-A, 3G-A or 3G), such as Capto™ Blue (HS), available from Cytiva.

[0354] Resins comprising Cibacron Blue, such as Capto™ Blue HS, can be engineered for higher flow rates, greater mechanical stability, and better resolution than other resins such as resins comprising crosslinked 6% agarose beads with quaternary ammonium (Q) strong anion exchange groups (such as the exemplary commercially available Q Sepharose Fast Flow), which leads to faster processing times and improved binding capacity at higher flow rates. Beaded crosslinked agarose resin comprising Cibacron Blue (such as the exemplary commercially available Capto™ Blue HS) preferentially binds rHuPH20 primarily through hydrophobic interactions between exposed hydrophobic regions on rHuPH20 and the hydrophobic dye ligand (Cibacron Blue F3GA) on the resin. This leads to a more selective

[0355] DBl / 164647321.1 76 binding of rHuPH20 compared to previous purification methods that only uses generic electrostatic interactions that binds both rHuPH20 and other host cell proteins. This leads to increased product quality for beaded crosslinked agarose resin comprising Cibacron Blue (such as the exemplary commercially available Capto™ Blue HS) compared to resins comprising crosslinked 6% agarose beads with quaternary ammonium (Q) strong anion exchange groups (such as the exemplary commercially available Q Sepharose Fast Flow).

[0356] The bound soluble rHuPH20 can then be washed and eluted using appropriate buffers. The dimensions of the column used are typically dependent on the volume of the HCCF and / or mass of rHuPH20. For example, in one embodiment, HCCF from a culture of hyaluronidaseexpressing cells in a 1000 L bioreactor culture can be loaded onto a column that is 20 cm high, 80 cm in diameter and contains 100 L resin. In another embodiment, HCCF from a culture of hyaluronidase-expressing cells in a 2000 L bioreactor culture can be loaded onto a column that is 20 cm high, 100 cm in diameter and contains 157 L resin. In a yet further embodiment, HCCF from a culture of hyaluronidase-expressing cells in a 300 L bioreactor culture can be loaded onto a column that is 29 cm high, 20 cm in diameter and contains 9 L resin. This can be scaled up or down as necessary, depending on the volume of the HCCF and the expected amount of protein. For example, HCCF in a 2000 L bioreactor culture can be loaded onto a Capto™ Blue HS column that is 20 cm high, 100 cm in diameter and contains 157 L resin. Typically, the flow rate for the affinity column is 270-330 cm / hr, preferably 300 cm / hr.

[0357] After loading the HCCF, the affinity column is washed. A suitable buffer for washing such columns containing bound soluble rHuPH20 may have a pH in a range from about 6to about 9. The column can be washed with one or more types of buffer in any order. The buffer(s) may typically have a buffer conductivity of about 12 mS / cm (or about 5 mS / cm to about 37 mS / cm). For example, in one embodiment, the column may be washed with at least one wash solution comprises sodium phosphate, ammonium sulfate, Tris, sodium chloride, urea, or a combination thereof. In another embodiment, the washing buffer for at least one of the washing steps may additionally include urea, arginine, guanidine, sodium chloride, caprylate or a combination thereof.

[0358] In one embodiment, the column may be washed with 50 mM Na^PO-i Sodium Phosphate, 70 mM NaCl, pH 6.8. In another embodiment, the buffer(s) are selected to reduce specific host cell proteins. For example, the buffer(s) may include 200 mM Ammonium Sulfate, pH 7.0 and / or 100 mM Tris, pH 8.0, and / or , and / or 50mM Sodium Phosphate, IM urea, lOOmM Sodium Chloride, pH 6.8. In one embodiment, a first wash can be performed using a buffer comprising

[0359] DBl / 164647321.1 77 50 mM Sodium Phosphate, 70 mM NaCl, pH 6.8, followed by a second wash buffer comprising 20 mM Sodium Phosphate, 200 mM Ammonium Sulfate, pH 7.0, and followed by a third wash comprising 100 mM Tris, pH 8.0. In another embodiment, a first wash can be performed using a buffer comprising 50 mM Sodium Phosphate, 70 mM NaCl, pH 6.8, followed by a second wash buffer comprising 50mM Sodium Phosphate, IM urea, lOOmM Sodium Chloride, pH 6.8, and followed by a third wash comprising 100 mM Tris, pH 8.0

[0360] The at least one wash solution may comprise a pH in a range from about 5 to about 9, with a first washing buffer preferably with a pH range about 6.4 to 7.2, a second washing buffer preferably with a pH range of about 6.6 to 7.4, and a third washing buffer preferably with a pH range of about 7.6 to 8.4.

[0361] Typically, washing is affected by passing through 1, 2, 3, 4, 5, 6, 7, 8, 9 or more column volumes of buffer. In some examples, 5 column volumes of buffer is used to wash the column.

[0362] The remaining bound soluble rHuPH20 is then eluted using a buffer with a higher salt concentration. The elution buffer may comprise sodium phosphate, sodium chloride, arginine, or a combination thereof, such as for example, 50 mM Sodium Phosphate, 400 mM Sodium Chloride, pH 6.8. As another example, the buffer may include 20 mM Sodium Phosphate, 250 mM Sodium Chloride, 250 mM Arginine, pH 8.0. The elution buffer may have a pH in a range from about 7 to 9, preferably about 7.6 to 8.4 and the washing step may be carried out at a flow rate of about 200 cm / hr to about 400 cm / hr. In some examples, the absorbance at A280 is monitored to determine when to collect the eluate, as any absorbance during this process generally indicates the presence of soluble rHuPH20. Thus, in one example, the eluate is collected when the absorbance begins reading increases to 0.5 OD and ends collection when the absorbance decreases to 0.5 OD. In other examples, the eluate collection begins when the absorbance increases to 0.025 OD and ends collection when the absorbance decreases to 0.5 OD. Typically, the eluate is filtered through an appropriate filter, such as a 0.22 pm filter, before being stored, such as in a sterile storage bag. The protein eluate from the column purification using beaded crosslinked agarose resin comprising Cibacron Blue (such as the exemplary commercially available Capto™ Blue HS resin) also is supplemented with sodium sulfate and acetic acid. These can be supplemented to the protein pool to final concentrations of, for example, about 0.25 M sodium sulfate and 15 mM acetic acid, pH 7.0.

[0363] 3. Hydrophobic Interaction Chromatography

[0364] Following purification through the affinity column, the protein solution can be subjected to hydrophobic interaction chromatography using a beaded crosslinked poly(styrene-

[0365] DBl / 164647321.1 78 divinylbenzene) column with hydrophobic ligand functionality, such as a POROS™ Benzyl Ultra column, in which the soluble rHuPH20 flows through the column while other contaminating host cell proteins are captured.

[0366] Beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality, such as POROS™ Benzyl Ultra, has significantly higher hydrophobicity than other resins used for such an intermediate step, for example resins composed of cross-linked 6% agarose beads modified with standard aromatic phenyl groups via uncharged, chemically-stable ether linkages, such as Phenyl Sepharose Fast Flow, and binds rHuPH20 much stronger, allowing for higher binding capacities. Thus, the column comprising the beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality, such as POROS™ Benzyl Ultra column, is engineered for higher flow rates, greater mechanical stability, and better resolution which leads to faster processing times and improved binding capacity at higher flow rates.

[0367] The column used in the methods herein can range in size, depending on the volume and amount of protein being purified though it. Exemplary sizes include columns that are 20 cm high, 45 cm in diameter with 31.8 L resin for use in the purification of soluble rHuPH20 from cells grown in a 2000 L bioreactor culture. One of skill in the art can scale up or down as appropriate.

[0368] The sanitized beaded crosslinked poly(styrene-divinylbenzene) column, such as a POROS™ Benzyl Ultra column, can be equilibrated prior to loading of the protein with an appropriate buffer such as, for example, 20 mM sodium phosphate, 0.4 M sodium sulfate, pH 7.0. In some examples, the absorbance at A280 is monitored during loading to determine when to collect the flow through, as any absorbance during this process generally indicates the presence of soluble rHuPH20. Thus, in one example, the flow through is collected when the absorbance reading increases to 0.5 OD. In other examples, the flow through collection begins when the absorbance increases to 0.05 OD. Following loading and flow through start collection of the protein, the column can be washed with one or more types of buffer in any order. The buffer(s) may typically have a buffer conductivity of about 50 mS / cm (or about 40 mS / cm to about 60 mS / cm). For example, the column may be washed with a wash solution comprising from about 5 mM to 50 mM sodium phosphate, 0.2 mM to 0.6 mM Sodium Sulfate or a combination thereof, preferably 20 mM Sodium Phosphate, 0.4 M Sodium Sulfate, pH 7.0. Preferably, the washing solution comprises 20 mM Sodium Phosphate, 0.4 M Sodium Sulfate, pH 7.0. During the wash step, the flow through collection ends, in one example, when the absorbance reading decreases to

[0369] DBl / 164647321.1 79 0.5 OD. The washing step may be carried out at a flow rate of about 100 cm / hr to about 400 cm / hr and the wash solution may have a pH in a range from about 5.0 to about 8.5. The washing step may be repeated at least one, two, three, four or five times.

[0370] Typically, the flow through is filtered through an appropriate filter, such as a 0.22 pm filter, before being stored, such as in a sterile storage bag. The protein flow through from the column purification using beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality (such as the exemplary commercially available POROS™ Benzyl Ultra resin), also is supplemented with sodium sulfate. This can be supplemented to the protein pool to final concentrations of, for example, about 0.5 M sodium sulfate, pH 7.0.

[0371] 4. Mixed Mode Chromatography

[0372] Following hydrophobic interaction chromatography, the pH and conductivity adjusted protein solution can be loaded onto a beaded cross linked agarose column with aminophenylboronate ligand functionality (APB column), such as an Aminophenylboronate Agarose 6XL column, for further purification. Aminophenylboronate ligand-mediated chromatography differs from many other ligands used for mixed mode chromatography. Whereas most ligands bind to a particular binding site on a protein by a mixture of noncovalent interactions, aminophenylboronate interacts predominantly by forming a temporary covalent bond with 1,2-cis-diol groups. These 1,2-cis-diol groups are present in glycans and thus highly glycosylated proteins like rHuPH20 bind well to the APB column. The aminophenylboronate ligand will bind to any molecule containing the appropriate group, including soluble rHuPH20, which is highly glycosylated.

[0373] The APB column used in the methods herein can range in size, depending on the volume and amount of protein being purified though it. Exemplary sizes include columns that are 25 cm high, 100 cm in diameter with 235.5 L resin for use in the purification of hyaluronidase from cells grown in a 2000 L bioreactor culture. One of skill in the art can scale up or down as appropriate. Buffers suitable for equilibrating the APB column include, for example, buffers 20 mM sodium phosphate, 650 mM sodium sulfate, pH 7.0.

[0374] Following loading of the conductivity adjusted HIC column- purified protein onto the APB column, the column is washed with suitable wash buffers. The wash solution may comprise sodium phosphate, sodium sulfate, glycine, sodium chloride, or a combination thereof. Exemplary wash buffers include, but are not limited to, from about 10 mM to 1000 mM sodium phosphate, from about 30 mM to 200 mM sodium chloride, from about 400 mM to 1000 mM sodium sulfate, from about 20 mM to 200 mM glycine, or a combination thereof. In one

[0375] DBl / 164647321.1 80 example, the APB column with the bound hyaluronidase is washed first with 20 mM sodium phosphate, 650 mM sodium sulfate, pH 7.0, then with 80 mM glycine, 850 mM sodium phosphate, pH 9.0 and finally with 80 mM glycine, 100 mM NaCl, pH 10.0. The wash solution may have a pH in a range from about 5 to about 1, with a first washing buffer preferably with a pH range about 6.6 to 7.4, a second washing buffer preferably with a pH range of about 8.6 to 9.4, and a third washing buffer preferably with a pH range of about 9.6 to 10.4.

[0376] The bound hyaluronidase can then be eluted, such as with 50 mM HEPES, 100 mM NaCl, pH 6.9. One of skill in the art can modify one or more of the buffers to similarly effect purification. Typically, the eluate is filtered through an appropriate filter, such as a 0.22 pm filter, before being stored, such as in a sterile storage bag.

[0377] Previous purification methods such as the Applicant’s own Gen2 method as described in W02009 / 111066 uses a further chromatography step (preferably using Ceramic Hydroxyapatite). In the present invention, this step can be removed. Removal of this step can prevent additional rHuPH20 loss while maintaining effective impurity and viral clearance Roughly 10-15% of rHuPH20 is unrecoverable across the step in the process of separating rHuPH20 from impurities. With the optional removal of this step from the process of the present invention, the rHuPH20 loss can be eliminated, further increasing overall process yield of rHuPH20.

[0378] 5. Mixed Mode Ion Exchange (IEX) Chromatography (if required)

[0379] Following mixed mode chromatography, the protein solution can be loaded onto a column containing an additional mixed mode resin, such as Ceramic Hydroxyapatite, Capto Adhere Impress, or other mixed mode chromatography for further purification.

[0380] The Mixed Mode column used in the methods herein can range in size, depending on the volume and amount of protein being purified though it. One of skill in the art can scale up or down as appropriate.

[0381] The mixed-mode chromatography step in bind and elute (i.e. soluble rHuPH20 is bound to the resin through hydrophobic, electrostatic, or metal ion affinity interactions, or combination thereof during wash steps) or flowthrough mode (i.e. soluble rHuPH20 flows through the resin while undesired impurities are bound to the resin through hydrophobic, electrostatic, or metal ion affinity interactions, or a combination thereof).

[0382] 6. Virus Inactivation and Removal, Protein Concentration, and Buffer Exchange

[0383] The soluble rHuPH20 obtained following column chromatography is subjected to virus inactivation and virus removal steps to ensure it is free from potential contamination and suitable

[0384] DBl / 164647321.1 81 for therapeutic use. The solution can also be subjected to post-purification steps that serve to formulate the protein in the desired buffer at the desired concentration.

[0385] The virus inactivation is performed at a low pH such as, for example, 4.0 (or from about 3.8 to about 4.2). In some instances, during this process, stabilizers may be added to protect the protein at the low pH. One example of a stabilizer is L-arginine, which serves to stabilize and protect soluble rHuPH20 from degradation at low pH. Without wishing to be bound by theory, L-arginine is believed to augment the inactivation of the virus by disrupting viral envelopes, and thus, allows for the use of relatively higher pH values such as 4.0 relative to detergent-based viral inactivation methods that use surfactants such as Triton. One of skill in the art can empirically determine a suitable concentration of L-arginine to be used during the viral inactivation process.

[0386] Viral removal is typically affected with the use of a filter that allows only the soluble protein to pass through while retaining any viruses (and other contaminants that are equal in size or larger than viruses). Such filters are available commercially, and any can be used in the methods herein. Pore sizes of filters useful for viral removal include, but are not limited to, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 75 nm and 100 nm. In one example, the purified hyaluronidase is filtered through a filter containing 20 nm pores. The protein can be transferred through the filter by, for example, peristaltic pump or by use of a pressure tank.

[0387] In one example, the Sartorius Virosart CPV filter is used to filter viruses based on size. This filter may be used in conjunction with the Sartorius Virosart Max pre- filter which provides removal of potential foulants of the Sartorius Virosart CPV virus filter.

[0388] Following viral removal, the soluble rHuPH20 can be concentrated and subjected to buffer exchange. In one example, the process may include an ultrafiltration (UF) or diafiltration step (DF) to concentrate and buffer exchange the virus filtration (VF) pool prior to the final bulk filtration step and filling. Any method of protein concentration known in the art can be utilized. Exemplary of such methods include concentration using tangential flow filtration (TFF) systems with molecular weight cut off (MWCO) filters. For example, the purified hyaluronidase can be passed through a series of 30 kDa MWCO regenerated cellulose filters to concentrate the protein 50-fold.

[0389] In one example, ultrafiltration-diafiltration may be performed using a 30kD Millipore Pellicon 3 Ultracel with regenerated cellulose, D screen membrane. The soluble rHuPH20 can be concentrated by 2*, 3*, 4*, 5*, 6*, 7*, 8*, 9*, 10*, 1 l x, 12*, 13 x or more. In some examples, the protein is concentrated approximately 50 / . This can result in, for example, a concentration of

[0390] DBl / 164647321.1 82 between 0.1 g / L and 50 g / L. In other examples, the purified hyaluronidase is concentrated to approximately 25 mg / mL. This pool may then be diafiltered with a minimum of 8 diavolumes of formulation buffer. A buffer exchange is generally performed following protein concentration to formulate the protein in the desired buffer for subsequent use, for example, as a therapeutic. One of skill in the art can empirically determine an appropriate buffer. Exemplary of suitable buffers are saline buffers, including, but not limited to, 10 mM HEPES, 130 mM NaCl, pH 7.0 and 10 mM Histidine, 130 mM NaCl, pH 6.5. Another exemplary suitable buffer is 20 mM Histidine, 130 mM NaCl, 0.05 % PS80, pH 6.5. After a low dP recirculation, the diafiltrated pool maybe recovered via air / buffer displacement and a final dilution to 1 Og / L performed with a formulation buffer. For large-scale production of hyaluronidase, such as, for example, 2000 L cultures, filters with surface areas of between 0.5 and 5 square meters are typically employed for this purpose. In some examples, filters with a surface area of 1.14 square meters or 2.28 square meters are used.

[0391] A buffer exchange may be performed following protein concentration to formulate the protein in the desired buffer for subsequent use, for example, as a therapeutic. One of skill in the art can empirically determine an appropriate buffer. Exemplary of suitable buffers are saline buffers, including, but not limited to, 10 mM HEPES, 130 mM NaCl, pH 7.0 and 10 mM Histidine, 130 mM NaCl, pH 6.5. Another exemplary suitable buffer is 20 mM Histidine, 130 mM NaCl, 0.05 PS80, pH 6.5. The purified hyaluronidase can, in some examples, be passed through another filter, such as a 0.22 pm capsule filter, before being stored in a sterile environment.

[0392] I. Filling

[0393] The methods described herein for the production and purification of soluble rHuPH20 also can include a filling step, in which the purified protein is aseptically filled into smaller containers for long-term storage and use. The soluble rHuPH20 can be filled into the containers as a liquid formulation, or as a powder, such as following lyophilization. For large-scale production, automated filling systems that include, for example, pumps to transfer the protein to the containers and weighing stations to measure the fill volume are typically used and are widely available. Manual or a combination of automated and manual filling of containers also can be performed, however. Suitable containers include, but are not limited to, glass or plastic vials, blister packs, bottles, tubes, inhalers, pumps, bags, syringes, bottles, or any other suitable container. Suitable closures or caps also can be used to seal the container. The filling process can include first passing the soluble rHuPH20 through a filter prior to filling to remove microbial contaminant and larger aggregates or sediment. For example, the protein can be filtered through

[0394] DBl / 164647321.1 83 a 0.22 pm sterilizing grade filter before being aliquoted into suitable containers. One of skill in the art can determine the appropriate fill volume and can include, for example, volumes ranging from 0.1 mL to 100 mL. In some examples, vials are aseptically filled with 1 mL, 5 mL, or 100 mb soluble rHuPH20.

[0395] In one example, the bulk filtration step includes filtering the UF / DF pool through a presterilized 0.2 pm sterilizing grade filter into final product storage containers. An exemplary filter which can be used in this step is the Millipore Durapore, Hydrophilic PVDF Filter, 0.22 pm.

[0396] Following capping or closure of the containers, the containers can be stored at an appropriate temperature. In some examples, the containers are flash frozen and stored at between -15 °C and -35 °C. In other examples, the containers are refrigerated, such as at between 3 °C and 15 °C. In another example, the final product is stored at 4 °C prior to freezing at -80 °C, and final storage at -20 ± 5 °C. Typically, long-term storage of liquids is at lower temperatures to minimize degradation. Soluble rHuPH20 in powder form can be stored for long periods at room temperature without significant degradation.

[0397] Examples of appropriate storage containers are the Thermo Scientific Nalgene PC Platinum Certified Clean Biotainer Bottles such as the 5 mL Biotainer: Catalog #CE-N3500-05 and 125 mL Biotainer : Catalog #CE-N3030-42.

[0398] J. Monitoring and Assays

[0399] The methods described herein can be monitored at one or more steps, measuring one or more conditions, parameters or products at each point. This can ensure that optimal conditions are maintained throughout, and also can be used to assess efficiency and productivity of the process. Monitoring can occur, for example, one or more times during the cell expansion phase, protein production phase (i.e. in the bioreactor), and / or the protein purification stage, as well as any time between, before or after, such as during concentration / buffer exchange procedures or filling. Monitoring can include, but is not limited to, measuring pH, temperature, volumes, contamination, purity, protein concentration, enzyme activity, cell viability, and cell number. In addition to monitoring conditions, parameters or products throughout the process, the purified soluble rHuPH20 produced as the end product also can be assessed and characterized with respect to, for example, protein concentration, enzyme activity, impurities, contamination, osmolarity, degradation, post-translational modifications, and monosaccharide content.

[0400] 1. Monitoring the conditions

[0401] The conditions during one or more of the steps in the methods provided herein can be monitored to ensure optimal conditions are maintained throughout the process. If the monitoring

[0402] DBl / 164647321.1 84 demonstrates that the conditions are not within an optimal range, then the conditions can be altered. Conditions that can be monitored vary for each process. For example, during the cell culture phases (i.e. cell expansion and protein production in the bioreactor), conditions to be monitored include, but are not limited to, temperature, cell culture pH, cell culture nutrients (e.g. glucose), CO2 levels, and O2 levels. Typically, the conditions are monitored automatically using in-built systems in, for example the incubator or bioreactor.

[0403] During the protein purification stage, conditions that can be monitored include, but are not limited to, pH, conductivity, and flow rate. These conditions can be monitored before, during and / or after one or more column chromatography steps. For example, the buffers used to equilibrate, wash or elute the column can be monitored. This can be performed before the buffer is loaded or after the buffer has run through the column.

[0404] 2. Monitoring soluble rHuPH20 production

[0405] Soluble rHuPH20 production, and parameters associated with soluble rHuPH20 production, also can be monitored throughout the process. These include, but are not limited to, cell number, cell viability, contamination, protein concentration, enzyme activity, purity, osmolarity, post-translational modifications. Any method to assess these parameters can be used. For example, mammalian cell viability can be assessed by taking a small aliquot of the cell culture and staining with trypan blue, which permeates only damaged cell membranes, thus staining only dead cells. The cells can be visualized under a microscope and counted using, for example, a hemocytometer. Other methods include assessing cell viability by measuring metabolic activity. For example, an aliquot of the cell culture can be incubated with a tetrazolium salt (e.g. MTT, XTT or WST-1) that is cleaved into a colored formazan product by metabolically active cells.

[0406] Soluble rHuPH20 concentration in a particular sample can be assessed by methods well- known in the art, including but not limited to, enzyme-linked immunosorbent assays (ELISA); SDS-PAGE; Bradford, Lowry, and / or BCA methods; UV absorbance, and other quantifiable protein labeling methods, such as, but not limited to, immunological, radioactive and fluorescent methods and related methods. Additionally, the presence and extent of degradation can be measured by standard techniques such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting of electrophoresed hyaluronidase-containing samples and chromatography, such as, for example, RP-HPLC. The purity of a hyaluronidasecontaining sample can be assessed by, for example, SDS-PAGE, RP-HPLC, size-exclusion chromatography, ion-exchange chromatography, for example anion or cationic-exchange

[0407] DBl / 164647321.1 85 chromatography and isoelectric focusing (IEF). Soluble rHuPH20-containing samples, such as samples containing purified hyaluronidase, can be further characterized by assessing the sialic acid and monosaccharide content. This can be accomplished by, for example, hydrolyzing the sample with 40% trifluoroacetic acid, fluorescently labeling the released monosaccharides and separating them using RP-HPLC.

[0408] Soluble rHuPH20 produced and purified using the methods provided herein also can be assessed for the presence of post-translational modifications. Such assays are known in the art and include assays to measure glycosylation (such as HILIC separation with fluorescence detection of NHS ester labeled glycans), hydroxylation, and carboxylation. In an exemplary assay for glycosylation, carbohydrate analysis can be performed, for example, with SDS-PAGE analysis of soluble rHuPH20 exposed to hydrazinolysis or endoglycosidase treatment. Hydrazinolysis releases N- and O-linked glycans from glycoproteins by incubation with anhydrous hydrazine, while endoglycosidase release involves PNGase F, which releases most N- glycans from glycoproteins. Hydrazinolysis or endoglycosidase treatment of soluble rHuPH20 polypeptides generates a reducing terminus that can be tagged with a fluorophore or chromophore label. Labeled soluble rHuPH20 polypeptides can be analyzed by fluorophore- assisted carbohydrate electrophoresis (FACE). The fluorescent tag for glycans also can be used for monosaccharide analysis, profiling or fingerprinting of complex glycosylation patterns by HPLC. Exemplary HPLC methods include hydrophilic interaction chromatography, electronic interaction, ion-exchange, hydrophobic interaction, and size-exclusion chromatography. Exemplary glycan probes include, but are not limited to, 3-(acetylamino)-6-aminoacridine (AA- Ac) and 2-aminobenzoic acid (2-AA). Carbohydrate moieties can also be detected through use of specific antibodies that recognize the glycosylated hyaluronidase polypeptide. An exemplary assay to measure b-hydroxylation comprises reverse phase HPLC analysis of soluble rHuPH20 polypeptides that have been subjected to alkaline hydrolysis (Przysiecki et al. (1987) PNAS 84:7856-7860). Carboxylation and g-carboxylation of hyaluronidase polypeptides can be assessed using mass spectrometry with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis, as described in the art see, e.g. Harvey et al. J Biol Chem 278:8363- 8369, Maum et al. Prot Sci 14:1171-1180).

[0409] N-linked glycans profiles on rHuPH20 can be generated and the abundance of individual glycan species can be quantified. For example N-linked glycans can be separated from hyaluronidase and from each other, and subsequently individually determined / quantified from an oligosaccharide profile generated by methods such as hydrophilic interaction liquid

[0410] DBl / 164647321.1 86 chromatography (HILIC) separation with fluorescence detection of N-hy dr oxy succinimide (NHS) ester labeled glycans. Briefly, HILIC separation with fluorescence detection of N- hydroxysuccinimide (NHS) ester labeled glycans can be carried out by the following method: (i) obtaining a sample comprising a protein of interest, such as soluble rHuPH20; (ii) denaturing the sample comprising the protein of interest, such as soluble rHuPH20; (iii) deglycosylating the sample comprising the protein of interest, such as soluble rHuPH20, using PNGaseF, thereby releasing all N-linked glycans as glycosamine, which contains an active primary amine group; (iv) reacting the active primary amine group on the released N-linked glycans (glycosamines) with a fluorescently tagged N-Hydroxysuccinimide (NHS) ester to form a stable conjugate; (v) subjecting the sample to HILIC to separate the fluorescently labelled N-glycan polar molecules. HILIC is a type of high performance liquid chromatography (HPLC), wherein separation is based on the combined effect of hydrophilic partitioning, hydrogen bonding, electrostatic interaction, or van der Waals interaction. By this method, a profile of N-linked glycans can be obtained that allows determination of relative abundances of individual glycan species in % of total N-linked glycans based on the area under peak in the profile.

[0411] The enzymatic activity of soluble rHuPH20 in a sample can be assessed at any point during the methods described herein. The sample may be, for example, a sample of the HCCF. In one example, activity is measured using a turbidimetric based assay. This is based on the formation of an insoluble precipitate when hyaluronic acid binds with serum albumin. The activity is measured by incubating soluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period of time (e.g., 10 minutes) and then precipitating the undigested sodium hyaluronate with the addition of acidified serum albumin. The turbidity of the resulting sample is measured at 640 nm after an additional (e.g., 20 minute) development period. The decrease in turbidity resulting from enzyme activity on the sodium hyaluronate substrate is a measure of the soluble rHuPH20 enzymatic activity. A unit of hyaluronidase activity can then be determined by comparing the turbidity measured at 640 nm with a calibration curve generated with dilutions of a soluble rHuPH20 assay working reference standard. One of skill in the art knows how to obtain such a calibration curve from their common general knowledge. In another example, enzymatic activity is measured using a microtiter assay in which residual biotinylated hyaluronic acid is measured following incubation with the sample containing soluble rHuPH20 (see e.g. Frost and Stern (1997) Anal. Biochem. 251:263-269, U.S. Patent Publication No. 20050260186). The free carboxyl groups on the glucuronic acid residues of hyaluronic acid are biotinylated, and the biotinylated hyaluronic acid substrate is covalently coupled to a microtiter plate. Following

[0412] DBl / 164647321.1 87 incubation with the sample containing soluble rHuPH20, the residual biotinylated hyaluronic acid substrate is detected using an avidin-peroxidase reaction and compared to that obtained following reaction with hyaluronidase standards of known activity. Other assays to measure enzymatic activity also are known in the art and can be used in the methods herein (see e.g. Delpech et al., (1995) Anal. Biochem. 229:35-41; Takahashi et al., (2003) Anal. Biochem. 322:257-263).

[0413] The presence of any contamination also can be monitored. Contamination can include, but is not limited to, microbial contamination (e.g. viruses, bacteria and mycoplasma), microbial products contamination (e.g. endotoxin), or other process-related impurities. Any suitable method or assay can be used. For example, viruses and bacteria can be cultured using methods well known in the art to determine whether they are present or not in a sample, and if so, in what quantities. Microscopy also can be used to detect microbial contamination. For example, a sample can be assessed for the presence of viruses or bacteria using transmission electron microscopy (TEM). Mycoplasma detection can be affected using, for example, biochemical or molecular techniques, including, but not limited to, PCR to amplify mycoplasma-specific nucleic acid, biochemical tests to detect mycoplasmal enzymes and cell-based fluorescence to detect mycoplasmal antigens or nucleic acid.

[0414] The presence of microbial products, such as bacterial endotoxins, also can be monitored. An example of a suitable assay for detecting the presence of endotoxin is the Limulus Amebocyte Lysate (LAL) assay. Two types of LAL assays can be used: gel clot and photometric (chromogenic and turbidimetric). LAL is an aqueous extract of blood cells (amebocytes) from the horseshoe crab. Endotoxin triggers a cascade of enzymatic reactions, which result in activated clotting enzyme. In the presence of bacterial endotoxins, at an elevated temperature, the LAL reagent will clot after addition of reagent. The formation of the gel clot is proportional to the concentration of the endotoxin. In the kinetic assay, the proenzyme in the LAL is activated when in contact with endotoxins produced by gram negative bacteria. The rate of activation is directly proportional to the concentration of the endotoxin present. The level of activation can be measured through a subsequent substrate reaction which is measured spectrophotometrically.

[0415] C-terminal heterogeneity of soluble rHuPH20 can be assessed by methods well-known in the art. For example, one method for determining the abundances of individual polypeptides in rHuPH20 compositions comprising a mixture of SEQ ID NO: 4-8 is analysis based on peptide fragments. In this method, a sample containing the mixture of polypeptides is digested with endoproteinase Asp-N, which specifically cleaves peptide bonds N-terminally at aspartic and

[0416] DBl / 164647321.1 88 cysteic acid. This releases the C-terminal portion of the soluble rHuPH20 at the aspartic acid at position 431 of SEQ ID NO: 4. The resulting C-terminal fragments can then be separated and characterized to determine the sequence and abundance of each individual polypeptide in the mixture. This method has been used to determine the abundances of individual polypeptides in rHuPH20 compositions, for example, as described in the applicant’s own earlier work on Gen2 hyaluronidase compositions as described in WO 2009 / 111066.

[0417] Alternatively, another method for determining the abundances of individual polypeptides in rHuPH20 compositions comprising a mixture of SEQ ID NO: 4-8 is Liquid chromatographymass spectrometry (LC-MS) analysis based on intact mass. In this method, a sample comprising the mixture of intact polypeptides consisting of the entire sequences of SEQ ID NO: 4-8 (i.e., polypeptides of SEQ ID NO: 4-8 that have not been digested) is first denatured and deglycoslyated to remove all N- and O-linked glycans (e.g., by incubation with O-glycosidase, sialidase and N-gycanase at 37°C overnight), before being injected in size-exclusion ultra performance liquid chromatography (SE-UPLC), wherein detection is carried our using both UV 280 nm and electrospray ionization mass spectrometry (ESI-MS). This method of LC-MS analysis based on intact mass of rHuPH20 polypeptides is the method used to generate the data shown herein. The presence of C-terminally heterogenous polypeptides (i.e., of all of SEQ ID NO: 4-8) in the mixture of hyaluronidase polypeptides and the relative abundances of each of these polypeptides (i.e., of each of SEQ ID NO: 4-8) make up a structural fingerprint that is characteristic for the hyaluronidase compositions described herein. Exemplary SE-UPLC parameters for LC-MS intact protein analysis may be:

[0418] UPLC Instrument Waters Acquity UPLC I Class

[0419] MS Instrument Waters XEVO G2 QTOF Mass Spectrometer

[0420] UPLC Column ACQUITY UPLC Protein BEH SEC Column, 200A, 1.7 pm, 4.6 mm x 150 mm

[0421] Autosampler Temperature 5°C

[0422] Column Temperature 40°C

[0423] Mobile Phase Mobile phase A (MPA): 0.2% formic acid in water Mobile Phase B (MPB): 0.2% formic acid in acetonitrile UV Detection 280 nm

[0424] MS Detection ESI-MS positive ion mode Flow Rate 0.3 mL / min Injection Volume 15 pL

[0425] DBl / 164647321.1 89 Run Time 10 min

[0426] Gradient Isocratic (50% MPA / 50% MPB)

[0427] Mass spectrometric data were collected on a Waters Xevo G2 QTOF mass spectrometer using electrospray ionization (ESI) in positive ion mode with real-time lockmass correction. Data was macquired from m / z 1000-3000 in MS mode at both low collision energy (4 V) and high collision energy modes (ramped from 15 to 45 V). The sample cone voltage, the capillary voltage, and the extraction cone voltage were set at 30 V, 2 kV, and 4 V respectively. The source temperature and the desolvation temperature were set at 150°C and 450°C respectively. Argon was used for the collision gas. The real-time lockmass correction was performed by monitoring the molecular ion, [M+H]+ of Leucine Enkephalin at m / z 556.2771 in positive MS mode. Prior to analysis, the mass spectrometer was calibrated using a 5th order fit on fragment ions of sodium iodide (Nal) covering an m / z range from 50 to 2000.

[0428] The skilled person knows, from their common general knowledge, how to perform techniques such as SE-UPLC to detect proteins of different lengths individually in a manner that allows a direct comparison of different compositions using techniques that are known in the art.

[0429] Since the rHuPH20 provided herein is produced in CHO cells, CHO Host Cell Proteins (HCP) are secreted and released from lysed cells during the production process and accumulate extracellularly during the cultures of recombinant CHO cells. If these HCPs are not effectively removed during purification, this can have a negative impact on product quality. Thus, a quality attribute of rHuPH20 compositions is the amount of residual HCP in the final composition (drug substance). The lower the amount of residual HCP in the rHuPH20 composition, the higher the quality of the rHuPH20 composition. CHO Host Cell Proteins (HCP) can be measured in the final rHuPH20 drug substance, e.g., using methods that are well-known in the art, such as ELISA. For example, in one exemplary method, HCP can be measured using the Cygnus Technologies’ 3rdGeneration CHO Host Cell Proteins kit. Capture antibodies against the proteins of interest, CHO HCPs, are bound to each well of a 96-well plate. A labelled detection antibody solution, anti-CHO HCP-HRP, is then added along with the standards and test articles. If any CHO HCPs are present, they bind to the capture and detection antibodies. After incubation, any unbound material is removed by a washing step. A substrate is then added, 3,3’,5,5’-tetramethylbenzindine (TMB), which produces a blue color when oxidized by the horseradish peroxidase conjugate (HRP) bound to the detection antibody. The TMB reaction is then stopped by the addition of acid which turns the solution from blue to yellow. The intensity of the colour can then be quantified in a plate reader. The concentration of CHO HCP is

[0430] DBl / 164647321.1 90 calculated relative to a reference standard curve in the assay, unknowns are quantified using curve-fitting software.

[0431] The data provided in the examples demonstrates that the Gen3 rHuPH20 provided herein, including the final product, i.e., the composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8 provided herein, contains lower levels of HCP than rHuPH20 compositions described in the prior art, in particular lower than the Gen2 hyaluronidase compositions described in WO 2009 / 111066. In one embodiment, the HCCF provided herein contains less than 1 mg / ml of CHO HCP, preferably less than 0.5 mg / ml of CHO HCP. In another embodiment, the composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, contains less than 250 ng HCP per mg rHuPH20 polypeptide, preferably less than 200 ng HCP per mg rHuPH20 polypeptide, more preferably less than 150 ng HCP per mg rHuPH20 polypeptide, even more preferably less than 100 ng HCP per mg rHuPH20 polypeptide.

[0432] Since the rHuPH20 provided herein is produced in cells, DNA can be released from lysed cells during the production process and accumulate extracellularly during the cultures of recombinant CHO cells. If this DNA is not effectively removed during purification, this can have a negative impact on product quality. Thus, a quality attribute of rHuPH20 compositions is the amount of residual DNA in the final composition (drug substance). The lower the amount of residual DNA in the rHuPH20 composition, the higher the quality of the rHuPH20 composition. Residual DNA can be measured in any sample throughout and at the end of processing, including the CCF, HCCF, eluate from any purification column and the final product. Measurement can be performed, e.g., using the PicoGreen Methodology that is available, e.g., as commercial kits that use an ultra-sensitive fluorescent nucleic acid stain for quantifying double-stranded DNA in solutions. The Quant-iT PicoGreen dsDNA reagent is an asymmetrical cyanine dye. Free dye does not fluoresce, but upon binding to dsDNA it exhibits a fluorescence enhancement. PicoGreen is more sensitive than UV absorbance methods, and highly selective for dsDNA over ssDNA and RNA. The concentration of dsDNA concentration is calculated relative to a reference standard curve in the assay, unknows can be quantified using curve fitting software.

[0433] The data provided in the examples demonstrates that the Gen3 rHuPH20 provided herein, including the final product, i.e., the composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8 provided herein, contains lower levels of DNA than rHuPH20 compositions described in the prior art, in particular lower than the Gen2 hyaluronidase compositions described in WO 2009 / 111066. In one embodiment, the HCCF

[0434] DBl / 164647321.1 91 provided herein contains less than 1000 ng DNA per mg of rHuPH20, preferably less than 800 ng DNA per mg of rHuPH20.

[0435] Another structural quality attribute of rHuPH20 that can be measured is the amount of degraded protein material. This can be measured, for example, by quantifying the amount of hyaluronidase that has been hydrolyzed between residues 311 and 312 (also referred to as “hydrolyzed” species). Hyaluronidase has a natural protease cleavage site on the C-terminal side of arginine 311. The more active proteases are present in a given batch, the more cleavage at this site will be observed. This type of protein degradation is thus an indicator of quality of the hyaluronidase provided herein. The presence of this hydrolyzation impurity can be measured, e.g., by using reverse phase high performance liquid chromatography (RP-HPLC) to detect oxidized, main peak and hydrolyzed species. This technique identifies hydrolyzed species, because the retention time of the hydrolyzed peak on the HPLC elution profile is shifted relative to the main peak for the intact hyaluronidase. The content of hydrolyzed hyaluronidase impurity was calculated by using RP-HPLC to determine the % area under the respective peaks in the elution profile, i.e.: hydrolyzed (% area) = (area under hydrolyzed peak) / (area under monomer peak + area under hydrolyzed peak+ area under oxidized peaks)) x 100 The data provided in the examples demonstrates that the Gen3 rHuPH20 provided herein, including the final product, i.e., the composition comprising a mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8 provided herein, contains much lower levels of “hydrolyzed” species than rHuPH20 compositions described in the prior art, in particular lower than the Gen2 hyaluronidase compositions described in WO 2009 / 111066 and also lower than the Genl hyaluronidase compositions described in US 2004 / 0268425 Al. In one embodiment, final drug product, i.e., the composition comprising a mixture of polypeptides provided herein, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, contains less than 0.5% hydrolyzed species, preferably less than 0.4% hydrolyzed species. In other words, in one embodiment, the present invention provides a composition comprising a mixture of rHuPH20 polypeptides, said mixture of polypeptides comprising the rHuPH20 polypeptides set forth in SEQ ID NO: 4-8, wherein less than 0.5%, preferably less than 0.4% of the rHuPH20 polypeptides have been hydrolyzed between Arginine 311 and Serine 312. K. Modifications to rHuPH20 and Compositions having rHuPH20

[0436] The purified rHuPH20 obtained from the methods disclosed herein can be suitably modified for particular therapeutic uses. Thus, in some instances, the purified rHuPH20 may be

[0437] DBl / 164647321.1 92 modified to include a linker and a targeting agent, wherein the linker is attached to the targeting agent and wherein the linker is attached directly or indirectly to the recovered polypeptide. Such modified rHuPH20, for example, can be used for targeting delivery thereof to particular types of cells so as to improve efficacy of the therapeutic use. One of skill in the art would be able to empirically determine a suitable method for addition for such linker and target molecules to rHuPH20.

[0438] In other instances, the modified rHuPH20 may include a polymeric molecule designed to protect and / or stabilize the hyaluronidase polypeptide, e.g., in the blood stream or for improving its shelf life. For example, the polymeric molecule may be selected from polyalkylene oxides (PAO), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethelene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans, heparin, homologous albumin, celluloses, hydrolysates of chitosan, starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, and bio-polymers. One of skill in the art would be able to empirically determine a suitable method for modifying rHuPH20 to include such polymeric molecule(s).

[0439] In some instances, the purified rHuPH20 may include post-translational modifications such as, for example, glycosylation, sialylation, albumination, famesylation, carboxylation, hydroxylation, and phosphorylation. One of skill in the art would be able to empirically determine a suitable method for such post-translational modification of rHuPH20.

[0440] In some instances, the purified rHuPH20 obtained using methods disclosed herein may be formulated into a suitable pharmaceutical composition. The pharmaceutical composition may include a therapeutically active agent such as, for example, a polypeptide, a protein, a nucleic acid, a drug, a small molecule, or an organic molecule. In some examples, the therapeutically active agent is selected from among a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonacidal agent, an anti-Parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, an antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dilator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent,

[0441] DBl / 164647321.1 93 an electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a psychic energizer agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, an urinary agent, a vaginal agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, and a sleep inducer.

[0442] In some examples, the therapeutically active agent is an antibody. In some examples, the therapeutically active agent is Factor VIII polypeptide.

[0443] L. Affinity purification using anti-rHuPH20 antibody

[0444] Also provided herein are anti-rHuPH20 antibodies. In particular, provided herein are three anti-rHuPH20 antibodies designated “3E8”, “1H3” and “10A4” with the sequences as set forth in Figures 6 and 7. The sequences of each of the 3E8 anti-rHuPH20 antibody, the 10A4 anti-rHuPH20 antibody and the 1H3 anti-rHuPH20 antibody are provided below.

[0445] DBl / 164647321.1 94

[0446] DB1 / 164647321.1 95

[0447] DB1 / 164647321.1 96

[0448] The anti-rHuPH20 antibody can be any one or more antibodies selected from the group consisting of the 3E8 anti-rHuPH20 antibody, the 10A4 anti-rHuPH20 antibody and / or the 1H3 anti-rHuPH20 antibody. In a preferred embodiment, the anti-rHuPH20 antibody is the 1H3 anti- rHuPH20 antibody. The signal peptides are cleaved from the sequences shown above during posttranslational processing and do not form part of the final antibody structure.

[0449] In one embodiment, the anti-rHuPH20 antibody is the 38E antibody, i.e., an antibody that binds to rHuPH20 having the sequence as set forth in any of SEQ ID NO: 4-8, such as the sequence as set forth in SEQ ID NO: 4, wherein said antibody comprises a light chain sequence as set forth in amino acid residues 21-238 of SEQ ID NO: 68 (i.e., SEQ ID NO: 68 without the signal peptide) or a light chain sequence that has at least 95% sequence identity to this sequence and / or a heavy chain sequence as set forth in amino acid residues 19-460 of SEQ ID NO: 67 (i.e., SEQ ID NO: 67 without the signal peptide) or a heavy chain sequence that has at least 95% sequence identity to this sequence. In one embodiment, the anti-rHuPH20 antibody is an antibody that binds to rHuPH20 having the sequence as set forth in any of SEQ ID NO: 4-8, such as the sequence as set forth in SEQ ID NO: 4, wherein said antibody comprises a light chain

[0450] DB1 / 164647321.1 97 variable region with the sequence as set forth in amino acid residues 21-131 of SEQ ID NO: 76 (i.e., SEQ ID NO: 76 without the signal peptide) or a light chain variable region that has at least 95% sequence identity to this sequence and / or a heavy chain variable region with the sequence as set forth in amino acid residues 19-136 of SEQ ID NO: 73 (i.e., SEQ ID NO: 73 without the signal peptide) or a heavy chain variable region that has at least 95% sequence identity to this sequence.

[0451] In one embodiment, the anti-rHuPH20 antibody is the 10A4 antibody, i.e., an antibody that binds to rHuPH20 having the sequence as set forth in any of SEQ ID NO: 4-8, such as the sequence as set forth in SEQ ID NO: 4, wherein said antibody comprises a light chain sequence as set forth in amino acid residues 16-228 of SEQ ID NO: 72 (i.e., SEQ ID NO: 72 without the signal peptide) or a light chain sequence that has at least 95% sequence identity to this sequence and / or a heavy chain sequence as set forth in amino acid residues 20-459 of SEQ ID NO: 71 (i.e., SEQ ID NO: 71 without the signal peptide) or a heavy chain sequence that has at least 95% sequence identity to this sequence. In one embodiment, the anti-rHuPH20 antibody is an antibody that binds to rHuPH20 having the sequence as set forth in any of SEQ ID NO: 4-8, such as the sequence as set forth in SEQ ID NO: 4, wherein said antibody comprises a light chain variable region with the sequence as set forth in amino acid residues 16-121 of SEQ ID NO: 78 (i.e., SEQ ID NO: 78 without the signal peptide) or a light chain variable region that has at least 95% sequence identity to this sequence and / or a heavy chain variable region with the sequence as set forth in amino acid residues 20-135 of SEQ ID NO: 75 (i.e., SEQ ID NO: 75 without the signal peptide) or a heavy chain variable region that has at least 95% sequence identity to this sequence.

[0452] In a preferred embodiment, the anti-rHuPH20 antibody is the 1H3 antibody, i.e. an antibody that binds to rHuPH20 having the sequence as set forth in any of SEQ ID NO: 4-8, such as the sequence as set forth in SEQ ID NO: 4, wherein said antibody comprises a light chain sequence as set forth in amino acid residues 21-234 of SEQ ID NO: 70 (i.e., SEQ ID NO: 70 without the signal peptide) or a light chain sequence that has at least 95% sequence identity to this sequence and / or a heavy chain sequence as set forth in amino acid residues 20-461 of SEQ ID NO: 69 (i.e., SEQ ID NO: 69 without the signal peptide) or a heavy chain sequence that has at least 95% sequence identity to this sequence. In a more preferred embodiment, the anti-rHuPH20 antibody is an antibody that binds to rHuPH20 having the sequence as set forth in any of SEQ ID NO: 4-8, such as the sequence as set forth in SEQ ID NO: 4, wherein said antibody comprises a light chain variable region with the sequence as set forth in amino acid residues 21-127 of SEQ

[0453] DBl / 164647321.1 98 ID NO: 77 (i.e., SEQ ID NO: 77 without the signal peptide) or a light chain variable region that has at least 95% sequence identity to this sequence and / or a heavy chain variable region with the sequence as set forth in amino acid residues 20-137 of SEQ ID NO: 74 (i.e., SEQ ID NO: 74 without the signal peptide) or a heavy chain variable region that has at least 95% sequence identity to this sequence.

[0454] The skilled person knows how to prepare antibodies with known amino acid sequences using their common general knowledge. For example, the skilled person can determine DNA sequences encoding the antibody heavy chains and light chains based on amino acid sequences using their common general knowledge of the genetic code. The skilled person can then assemble and synthesize the DNA sequences (e.g., using de novo DNA synthesis) encoding heavy chains and light chains, and clone them into a suitable mammalian expression vector, such as pcDNA3.4. The expression vector comprising the DNA sequences encoding the heavy chains and light chains of a given antibody can then be transfected into suitable mammalian expression cells, such as CHO cells, and expressed and purified using techniques that are well-known in the art, such as expression in CHO cells and purification by Protein G affinity chromatography.

[0455] Also provided herein is an anti-rHuPH20 affinity resin that is generated by conjugating the above-mentioned anti-rHuPH20 recombinant monoclonal antibodies purified from CHO cells to a chromatography resin, such as to a cyanogen bromide (CnBr)-activated size exclusion chromatography resin (e.g., CNBr-activated Sepharose 4 Fast Flow is a commercially available example of such a resin that can be used for conjugating the above-mentioned anti-rHuPH20 recombinant monoclonal antibodies). In one embodiment, the anti-rHuPH20 affinity resin is generated by conjugating the 3E8 anti-rHuPH20 recombinant monoclonal antibodies as defined herein to a chromatography resin, such as to a cyanogen bromide (CnBr) -activated size exclusion chromatography resin. In another embodiment, the anti-rHuPH20 affinity resin is generated by conjugating the 10A4 anti-rHuPH20 recombinant monoclonal antibodies as defined herein to a chromatography resin, such as to a cyanogen bromide (CnBr)-activated size exclusion chromatography resin. In a preferred embodiment, the anti-rHuPH20 affinity resin is generated by conjugating the 1H3 anti-rHuPH20 recombinant monoclonal antibodies as defined herein to a chromatography resin, such as to a cyanogen bromide (CnBr)-activated size exclusion chromatography resin. The anti-rHuPH20 affinity resin that is generated by conjugating the above-mentioned anti-rHuPH20 recombinant monoclonal antibodies purified from CHO cells to a chromatography resin is also referred to herein as “beads”. These beads can be used to capture rHuPH20, followed by one or more wash steps and elution, e.g., using a low pH buffer (e.g., pH

[0456] DBl / 164647321.1 99 lower than pH 4, preferably lower than pH 3, such as pH 2.7). Purified rHuPH20 samples may be further concentrated, filtered, and assessed for product purity using RP-HPLC analysis. More specifically, these beads comprising the anti-rHuPH20 1H3 affinity resin can be added to any in- process or end-of-process samples containing rHuPH20. The capturing step is performed by incubating the sample with the beads for 2 hours. The beads are then washed and eluted using a low pH buffer to provide a purified sample.

[0457] Also provided herein is a method of analysing a sample comprising rHuPH20, the method comprising a step of incubating said sample with the anti-rHuPH20 affinity resin that is generated by conjugating the above-mentioned anti-rHuPH20 recombinant monoclonal antibodies purified from CHO cells to a chromatography resin, and further comprising one or more wash steps and an elution step, e.g., elution using a low pH buffer (e.g., pH lower than pH 4, preferably lower than pH 3, such as pH 2.7). The sample comprising rHuPH20 may be any sample, including any in-process or end-of process sample from the rHuPH20 production process, such as the CCF (including any samples of the culture fluid taken from the bioreactor during protein expression), the HCCF, the load, wash or elution samples from the affinity chromatography step, the load, wash or elution samples from the hydrophobic interaction chromatography step, the load, wash or elution samples from the mixed-mode chromatography Aminophenylboronate step, the load wash or elution samples from the mixed-mode ion exchange chromatography step, any sample from the viral inactivation step, any sample from the ultrafiltration / diafiltration step, or the final drug product. The anti-rHuPH20 affinity resin in this method may comprise the 3E8 anti-rHuPH20 recombinant monoclonal antibody as defined herein, the 10A4 anti-rHuPH20 recombinant monoclonal antibody as defined herein, and / or the 1H3 anti-rHuPH20 recombinant monoclonal antibody as defined herein. In a preferred embodiment, the anti-rHuPH20 affinity resin in this method comprises the 1H3 anti-rHuPH20 recombinant monoclonal antibody as defined herein.

[0458] Also provided herein are uses of the above-mentioned anti-rHuPH20 recombinant monoclonal antibodies purified from CHO cells in monitoring rHuPH20 during a production process. In these uses, the above-mentioned anti-rHuPH20 recombinant monoclonal antibodies purified from CHO cells may be conjugated to a chromatography resin to generate an anti- rHuPH20 affinity resin. This anti-rHuPH20 affinity resin can be incubated with any in-process or end-of process sample comprising rHuPH20 to separate the rHuPH20 prior to subjecting the purified rHuPH20 to any analysis techniques, such as the monitoring techniques described herein.

[0459] DBl / 164647321.1 100 This method of analysing a sample comprising rHuPH20 comprising a step of incubating said sample with the anti-rHuPH20 affinity resin and / or these uses of the anti-rHuPH20 recombinant monoclonal antibodies in monitoring rHuPH20 during a production process provide the benefit of being able to analyse rHuPH20 not only at the end of the rHuPH20 production process, but also throughout the process, at any in-process step. This enables identification, for example of certain impurities and / or monitoring of certain structural quality attributes (e.g., C- terminal heterogeneity, glycosylation profile, and / or abundance of “hydrolyzed” species) throughout the rHuPH20 production process. This method thus enables detection of possible issues with a batch early on during production, such that a decision can be made to either stop or continue producing a batch. Overall, this provides an additional benefit to hyaluronidase production, because overall production costs can be dramatically reduced when it is possible to identify issues with a batch early on during production, and a batch with issues can thus be abandoned.

[0460] Examples

[0461] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. A flow chart of an exemplary process according to the present invention is shown in Figure 1.

[0462] Exemplary steps for the production and purification of hyaluronidases are described. The skilled person will be aware that certain steps may be optional depending on need.

[0463] Example 1 - Cell Line and Molecule Information

[0464] The cell line and molecule information for the process described in the following examples is outlined in Table 3.

[0465] Table 3 : Cell Line and Molecule Information

[0466] Generation of the HZ24-2B2 cell line is set out in Applicant’s own patent application W02009 / 111066, which is incorporated by reference in its entirety. Furthermore, the HZ24-2B2 cell line, also referred to herein as “2B2 cells”, has been deposited at the American Type Culture

[0467] DBl / 164647321.1 101 Collection (ATCC, 10801 University Blvd, Manassas, VA 20110 USA) by Halozyme, Inc. on . The identification reference for 2B2 cells given by the depositor is HZ24-2B2. The deposit accession number for 2B2 cells given by the ATCC is . The deposit was made at the ATCC (10801 University Blvd, Manassas, VA 20110 USA) according to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0468] Example 2 - Step 1 - Vial Thaw and Inoculum Expansion

[0469] Vial thaw and seed train expansion operations can be used to grow HZ24-2B2 cells to the optimum viable cell concentration (VCC), viability, and working volume to seed the N-l bioreactor.

[0470] Summary of Exemplary Process

[0471] Vial thaw through rocking bioreactor cultures may be maintained in CD CHO Medium (e.g., Gibco 12490025) with 8 mM L-Glutamine (e.g., Gibco 21051040) and 20 pM Methotrexate (Millipore Sigma M7824), prepared with additives as shown in Table 4.

[0472] One (1) rHuPH20 Working Cell Bank (WCB) vial is thawed in a pre- warmed water bath with water for injection (WFI) and maintained in an unbaffled shake flask with vented cap according to Table 5. The inoculum is then expanded in a series of unbaffled shake flasks according to Table 6, with transfers based on target viable cell concentration (VCC). Target split ratio range for the first passage out of thaw are 1 :4 to 1:8; however, a split ratio as low as 1 :2 may be used. Target split ratio range for all subsequent passages in unbaffled shake flasks are 1 :5 to 1: 11.

[0473] The inoculum is then expanded in a series of rocking bioreactors according to Table 7, with transfers based on target viable cell concentration (VCC). Target split ratio range for all rocking bioreactor expansion steps are between 1 : 5 and 1:11.

[0474] Table 4: Exemplary Basal Medium Additives for Vial Thaw and Rocking Bioreactor Expansion

[0475] Cultures

[0476] Per kg of post-Q.S. preparation medium weight.

[0477] Table 5: Exemplary Vial Thaw Culture Operating Parameters

[0478] DBl / 164647321.1 102

[0479] *A 1:2 split is performed at 120 hours or when the culture reaches 0.8xl06vc / mL, whichever comes first.

[0480] Table 6: Exemplary Shake Flask Expansion Culture Parameters Table 7: Exemplary Rocking Bioreactor Operating Conditions

[0481] Example 3 - Step 2 - Inoculum Bioreactor (N-l) Expansion

[0482] N-l bioreactor expansion can be used to grow HZ24-2B2 cells to the optimum VCC, viability, and working volume to seed the production bioreactor. Summary of Exemplary Process

[0483] The inoculum is further expanded in a stirred tank bioreactor according to Table 9 to obtain sufficient cell mass to inoculate a production bioreactor. The inoculum bioreactor culture may be maintained in CD CHO Medium (e.g., Gibco 12490025) with 8 mM L-Glutamine (e.g., Gibco 21051040) without Methotrexate, prepared with additives as shown in Table 8. Antifoam, such as Ex-Cell® Antifoam, is added as needed.

[0484] DBl / 164647321.1 103 Air and oxygen gases are used to control dissolved oxygen (DO). pH is only controlled at the upper limit with carbon dioxide. The target split ratio for the inoculum bioreactor (N-l) expansion step is between 1:4 and 1:10.

[0485] Table 8: Exemplary Basal Medium Additives for the Inoculum Bioreactor (N-l) Expansion

[0486] Cultures

[0487] *Per kg of post-Q.S. preparation medium weight.

[0488] Table 9: Exemplary Inoculum Bioreactor (N-l) Operating Conditions

[0489] Example 4 - Step 3 - Production Bioreactor

[0490] A production bioreactor can be used as the final vessel for production of rHuPH20. Summary of Exemplary Process

[0491] The basal medium for production culture is CD CHO Medium (e.g., Gibco 12490025) with 8 mM L-Glutamine (e.g., Gibco 21051040) without Methotrexate, as shown in Table 10. The production bioreactor is fed 1% w / w of the working volume daily on Day 2 through Day 15. The feed strategy consists of two feed media to replenish depleted nutrients in CHO cell cultures during fed-batch production, comprising, e.g., the essential amino acids, vitamins, salts and trace elements, and further comprising L-Cysteine Sulfate. For example, two feed media can be fed as a 50 / 50 blend of Ex-Cell® Advanced CHO Feed 1 (Millipore Sigma 24368C) and Cellvento® 4Feed (Millipore Sigma 103796) with 20 mM L-Cysteine Sulfate (Millipore Sigma 137116), as shown in Table 11. Antifoam, such as Ex-Cell® Antifoam (Millipore Sigma 59920C) is added as needed. Glucose (Gibco 15023) is fed daily starting on Day 2, as needed, back to a target concentration of 5.0 g / L. Bolus Copper Sulfate (100 mM stock solution, Millipore Sigma C8027) is added on Day 9 to a target concentration of 100 pM CuSO4 of the current working volume. Following the bolus Copper Sulfate addition on Day 9, a temperature shift from 36.0 °C to 33 °C is performed.

[0492] DB1 / 164647321.1 104 pH is controlled using carbon dioxide and sodium bicarbonate. pCCh is maintained below 100 mmHg by adjusting air sparge, as needed. Air is sparged through a drilled hole sparer. Oxygen and carbon dioxide are sparged through a frit. The production bioreactor operating conditions are shown in Table 13, and the operational schedule (feed additions, set- point shifts) are shown in Table 14.

[0493] The production bioreactor is harvested either on Day 16 or within 24 hours of receiving a viability measurement < 80%, whichever comes first.

[0494] Table 10: Exemplary Basal Medium Additives for Production Culture

[0495] *Per kg of post-Q.S. preparation medium weight. Table 11 : Exemplary Feed Medium Additive Formulations for Production Culture

[0496] *Per kg of post-Q.S. preparation medium weight.

[0497] Table 12: Exemplary Production Bioreactor Operating Parameters

[0498] DBl / 164647321.1 105 Table 13: Exemplary Production Culture: Exemplary Scheduled Operations

[0499] DBl / 164647321.1 106

[0500] Example 5 - Step 4 - Harvest via Depth Filtration

[0501] A harvest step can be performed to separate CHO cells, associated cell debris and other insoluble components from the product-containing supernatant. Summary of Exemplary Process

[0502] The cell culture harvest process clarifies the cell culture fluid (CCF) through a series of depth filters (using positively charged media comprising a quaternary ammonium charged synthetic fiber matrix, such as 3M™ Harvest RC) and sterile filters before it enters the Harvested Cell Culture Fluid (HCCF) tank, which is then held at 20°C for subsequent processing. Prior to harvest operations, the production bioreactor is chilled to 20°C and held at 25% DO throughout the harvest.

[0503] DB1 / 164647321.1 107 Table 14: Exemplary Primary Recovery Operation Summary

[0504] Table 15: Exemplary Harvest Filter Information

[0505] Example 6 - Step 5 -Affinity Chromatography

[0506] Affinity chromatography can be performed to capture and purify the rHuPH20 enzyme product from HCCF components including cell culture media components, host cell protein (HCP), DNA, and potential virus and endotoxin.

[0507] DB1 / 164647321.1 108 Summary of Exemplary Process

[0508] The affinity chromatography step uses beaded crosslinked agarose resin comprising Cibacron Blue, such as the commercially available Cytiva Capto™ Blue HS resin, and is the first chromatography step in the downstream process that purifies rHuPH20 from the HCCF. The resin binds rHuPH20 while the majority of HCP, host cell DNA and other HCCF components flow through to waste. The resin is equilibrated, loaded, washed, eluted, and sanitized. The volume of HCCF to be loaded for each cycle is calculated based on the rHuPH20 titer with the maximum load of 5 g-rHuPH20 / L-resin target loading density per cycle. During elution, the pool is sterile filtered into a product storage vessel and held for up to 24 hours at 15-25 °C, or up to 72 hours at 2-8 °C.

[0509] Table 16: Exemplary Affinity Chromatography Column Information

[0510] Table 17: Exemplary Affinity Chromatography Column Operations

[0511] DBl / 164647321.1 109

[0512] DB1 / 164647321.1 110 Table 18: Exemplary Hydrophobic Interaction Chromatography Load Conditioning

[0513] Example 7 - Step 6 - Hydrophobic Interaction Chromatography

[0514] An intermediate chromatography step using Hydrophobic Interaction Chromatography (HIC) can be performed to reduce residual Cibacron Blue ligand, HCP, DNA, and potential virus. Summary of Exemplary Process

[0515] Hydrophobic interaction chromatography utilizes beaded crosslinked poly(styrene- divinylbenzene) resin with hydrophobic ligand functionality, such as Thermo Scientific POROS™ Benzyl Ultra resin, and is the intermediate purification step in the rHuPH20 downstream process and is operated in a flowthrough mode. During column operations, the resin is equilibrated, loaded and washed at low conductivity. The pool from the affinity chromatography step using beaded crosslinked agarose resin comprising Cibacron Blue (e.g., in this exemplary process, Capto™ Blue HS pool) is adjusted with a stock solution to match equilibration buffer conductivity of 50 mS / cm. rHuPH20 is selectively recovered from the resin during flow-through, while impurities remain bound. During flow through collection, the pool is sterile filtered into a product storage vessel and held for up to 24 hours at 15-25 °C, or up to 72 hours at 2-8 °C.

[0516] Table 19: Exemplary Hydrophobic Interaction Chromatography Column Information

[0517] DBl / 164647321.1 111 Table 20: Exemplary Hydrophobic Interaction Chromatography Column Operations

[0518] DBl / 164647321.1 112

[0519] Table 21: Exemplary Hydrophobic Interaction Chromatography Pool Conditioning

[0520] Example 8 - Step 7 - Mixed-Mode Chromatography A polishing chromatography step using Aminophenylboronate (APB) mixed-mode chromatography can be performed to further reduce HCP, DNA and potential viruses and endotoxin.

[0521] Summary of Exemplary Process

[0522] Mixed-mode chromatography utilizes beaded aminophenylboronate substituted crosslinked agarose resin (APB resin), such as Astrea Bioseparations Aminophenylboronate A6XL resin, in bind and elute mode and is the final polishing step in the rHuPH20 downstream process. APB resin utilizes both hydrophobic interactions and diol affinity properties to bind rHuPH20 while less hydrophobic and non-glycosylated impurities, such as HCP, DNA, and potential viruses flow through. The pool from the hydrophobic interaction chromatography step using beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality (e.g., in this example, POROS™ Benzyl Ultra HIC pool) is adjusted with a stock solution to match equilibration buffer conductivity of 70 mS / cm. The resin is equilibrated, loaded, washed, eluted, and sanitized. During elution, the pool is sterile filtered into a product storage vessel and held for up to 24 hours at 15-25 °C or up to 72 hours at 2-8 °C.

[0523] Table 22: Exemplary Mixed-Mode Chromatography Column Information

[0524] DB1 / 164647321.1 H 3

[0525] Table 23: Exemplary Mixed-Mode Chromatography Column Operations

[0526] DBl / 164647321.1 114

[0527] Example 9 - Step 8 - Mixed-Mode Ion Exchange Chromatography

[0528] A polishing chromatography step using mixed-mode chromatography can be performed to further reduce HCP, DNA and potential viruses and endotoxin.

[0529] DBl / 164647321.1 115 Example 10 - Step 8 - Viral Inactivation

[0530] A low pH hold step can be used to inactivate any potential enveloped viruses that may be present in the feed stream and is included in the process to ensure product safety.

[0531] Summary of Exemplary Process

[0532] The mixed-mode chromatography pool is adjusted to a target concentration of 0.8 M Arginine and pH 4.0 ± 0.1 with a 2.0 M Arginine, 100 mM Citrate, pH 3.8 viral inactivation solution and held for a target of 60 - 90 minutes to allow for viral inactivation, then immediately followed by a pH adjustment to pH 7.0 using 2.0 M Tris Base.

[0533] Table 24: Exemplary Low pH Viral Inactivation Pool Adjustment

[0534] Example 11 - Step 9 - Viral Filtration

[0535] A virus retentive filtration step can be included in the process as an orthogonal virus clearance step to ensure viral safety through mechanical retention and reduction of potential viruses in the product pool.

[0536] Summary of Exemplary Process

[0537] The Sartorius Virosart CPV filter is a normal flow virus retentive filter capable of reducing potential virus contaminants from the Viral Inactivated pool based on size. This filtration ensures adequate viral reduction capacity in the rHuPH20 process. The Sartorius Virosart Max pre-filter provides removal of potential foulants of the Sartorius Virosart CPV virus filter. Both filters are designed for single use and are flushed and equilibrated independently with APB elution buffer (50 mM HEPES, 100 mM NaCl, pH 6.9) prior to use. They are then configured in-line for product filtration of the Neutralized Viral Inactivated (NVI) pool performed at constant pressure of 30 psid, DB1 / 164647321.1 H6 either by pump or source tank pressure. The Sartorius Virosart CPV is integrity tested pre- and post-use.

[0538] Table 25: Exemplary Viral Filtration Filter Summary

[0539] Table 26: Exemplary Viral Filtration Operation Summary

[0540] DBl / 164647321.1 117

[0541] Example 12 - Step 10 - Ultrafiltration / Diafiltration Operations

[0542] An Ultrafiltration / Diafiltration step can be used to concentrate and buffer exchanges the virus filtration (VF) pool. Summary of Exemplary Process

[0543] Ultrafiltration-Diafiltration is a tangential flow filtration process used to both concentrate and buffer exchange rHuPH20. rHuPH20 is retained by a membrane filter while smaller species, specifically buffer excipients and water molecules, pass through the pores and are removed. For example, a 30kD Millipore Pellicon 3 Ultracel with regenerated cellulose, D screen membrane can be used for ultrafiltration-diafiltration. The VF pool is initially concentrated to 15 g / L during UF1. The UF1 pool is then diafiltered with a minimum of 8 diavolumes of formulation buffer. After a low dP recirculation, the DF pool is recovered via air / buffer displacement and a final dilution to lOg / L is performed with formulation buffer. Table 27: Exemplary UF / DF Filter Information

[0544] Table 28: Exemplary UF / DF Cassette Installation Operation Summary

[0545] DBl / 164647321.1 118

[0546] Table 29: Exemplary Ultrafiltration and Diafiltration Main Operations Summary

[0547] DB1 / 164647321.1 119

[0548]

[0549] DB1 / 164647321.1 120

[0550] Example 13 - Step 11 - Bulk Filtration Operations

[0551] During the bulk filtration process, the Diluted UF / DF Pool can be sterile filtered into the final product storage container. Summary of Exemplary Process

[0552] The Diluted UF / DF pool is filtered through a pre-sterilized 0.2 pm sterilizing grade filter into the final product storage containers and stored at 4 °C prior to freezing at -80 °C, and final storage at -20 ± 5 °C.

[0553] Table 30: Exemplary Bulk Filtration Step Information

[0554] DBl / 164647321.1 121 Table 31: Exemplary Bulk Filtration Operations Summary

[0555] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. In some embodiments, the Long-Term Storage Temperature is < -20 °C or less; or < -65 °C; or about -80 °C.

[0556] Example 14: Affinity Purification Method

[0557] Anti-rHuPH20 1H3 affinity resin is generated by conjugating anti-rHuPH20 recombinant monoclonal antibody 1H3 purified from CHO cells to CNBr-activated Sepharose 4 Fast Flow and are subsequently referred to as “beads” throughout this method. Once coupled with 1H3, the affinity resin is added to in-process samples containing rHuPH20. During a 2-hour incubation period, rHuPH20 is captured by the beads. The beads are then washed and eluted using a low pH buffer to provide a purified sample. Purified rHuPH20 samples are further concentrated, filtered, and assessed for product purity using RP-HPLC analysis. Purification Buffer A (Wash Buffer) is lx Tris-buffered saline (TBS) 20 mM L-

[0558] DBl / 164647321.1 122 Methionine pH 7.4. Purification Buffer B (Elution Buffer) is 0.1 M Glycine 20 mM L- Methionine pH 2.7. Purification Buffer C (Neutralization Buffer) is 0.3 M Tris-HCl 20 mM L- Methionine pH 8.0.

[0559] Efficient isolation of rHuPH20 is dependent on bead concentration, rHuPH20 concentration, pH, rHuPH20-lH3 affinity, and incubation time. This procedure is based on 0.5 mL of beads when the volume of beads to the volume of Purification Buffer A is 1:2 in a settled state. For example, if the bead volume is 10 mL in a 50 mL conical tube when beads are allowed to settle for > 3 min, then the Purification Buffer A layer is up to the 20 mL mark. In this example, 0.5 mL (when beads + Purification Buffer A is mixed to a homogenous mixture) is equal to 0.5 mL of beads. For binding of rHuPH20 to 1H3 affinity resin, beads are stored in a 50 mL conical tube. Invert this 50 mL conical test tube until beads are thoroughly mixed with buffer. Transfer 0.5 mL of beads to a labelled 2 mL LoBind Eppendorf tube. Add 1 mL of in-process rHuPH20 sample to a labelled Eppendorf tube that has 0.5 mL of beads. Incubate at 2-8 °C with tilting and rotation for 2 hours to capture rHuPH20. Ensure to limit exposure to light during incubation.

[0560] For isolation of rHuPH20, a poly prep chromatography column (1 column per 1 testing sample) with manifold stopcock to glass manifold vacuum is used. Upon completion of 2 hour incubation, load material from the incubation to the poly prep chromatography column with stopcock open. Wash beads 3 times using ~1 mL of Purification Buffer A using disposable transfer pipette tip. Allow Buffer A to flow through (gravity filtration) and go into waste with the stopcock lock open. At the end of last wash, apply vacuum to ensure all residual Purification Buffer A is extracted out of the beads. Don’t apply excessive vacuum to avoid over-drying of the beads.

[0561] For elution of rHuPH20, turn stopcock sideways to close and carefully lift the glass block lid and set aside. Install the test tube rack inside the vacuum manifold with labelled and uncapped 15 mL centrifugal unit with 10K filter device. Place the lid back and make sure corresponding centrifugal unit is placed under the matching column. At the start of stopwatch (t = 00:00), add 1 mL of Purification Buffer B to chromatography column. Pipette up and down three times. At t = 02:00, turn open the stopcock and let Purification Buffer B flow through (gravity filtration). Apply vacuum at the end to extract all eluate from beads. Lift glass block lid, unload test tube rack and proceed with centrifugation and filtration steps.

[0562] For centrifugation and filtration of eluate, cap 15 mL centrifugal unit with 10K filter device containing the eluate and place in the centrifuge. Concentrate eluate until total volume

[0563] DBl / 164647321.1 123 reaches ~230 pL at 4,000 rpm for 5 minute increments at room temperature. Once volume reaches -230 pL, remove centrifugal unit from centrifuge and transfer the concentrated eluate in 10K filter device to a labelled microcentrifuge 0.22 pm filter unit. Load filter unit in microcentrifuge and allow eluate to filter through by quick spin down at 5,000 rpm for 10 seconds.

[0564] Example 15: Determination of enzymatic activity of soluble rHuPH20

[0565] Enzymatic activity of soluble rHuPH20 in samples such as cell cultures, the CCF or the HCCF, purification fractions and purified solutions was determined using a turbidimetric assay, which is based on the formation of an insoluble precipitate when hyaluronic acid binds with serum albumin. The activity is measured by incubating soluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period of time (10 minutes) and then precipitating the undigested sodium hyaluronate with the addition of acidified serum albumin. The turbidity of the resulting sample is measured at 640 nm after a 30 minute development period. The decrease in turbidity resulting from enzyme activity on the sodium hyaluronate substrate is a measure of the soluble rHuPH20 enzymatic activity. The method is run using a calibration curve generated with dilutions of a soluble rHuPH20 assay working reference standard, and sample activity measurements are made relative to this calibration curve.

[0566] Dilutions of the sample were prepared in Enzyme Diluent Solutions. The Enzyme Diluent Solution was prepared by dissolving 33.0 ± 0.05 mg of hydrolyzed gelatin in 25.0 mL of the 50 mM PIPES Reaction Buffer (140 mM NaCl, 50 mM PIPES, pH 5.5) and 25.0 mL of SWFI, and diluting 0.2 mL of 25% Buminate solution into the mixture and vortexing for 30 seconds. This was performed within 2 hours of use and stored on ice until needed. The samples were diluted to an estimated 1-2 U / mL. Generally, the maximum dilution per step did not exceed 1:100 and the initial sample size for the first dilution was not less than 20 pL.

[0567] Dilutions of known soluble rHuPH20 with a concentration of 2.5 U / mL were prepared in Enzyme Diluent Solution to generate a standard curve and added to the Optilux plate in triplicate. The dilutions included 0 U / mL, 0.25 U / mL, 0.5 U / mL, 1.0 U / mL, 1.5 U / mL, 2.0 U / mL, and 2.5 U / mL. “Reagent blank” wells that contained 60 pL of Enzyme Diluent Solution were included in the plate as a negative control. The plate was then covered and warmed on a heat block for 5 minutes at 37 °C. The cover was removed and the plate was shaken for 10 seconds. After shaking, the plate was returned to the plate to the heat block and the MULTIDROP 384 Liquid Handling Device was primed with the warm 0.25 mg / mL sodium hyaluronate solution (prepared by dissolving 100 mg of sodium hyaluronate (LifeCore

[0568] DBl / 164647321.1 124 Biomedical) in 20.0 mL of SWFI. This was mixed by gently rotating and / or rocking at 2-8 °C for 2-4 hours, or until completely dissolved). The reaction plate was transferred to the MULTIDROP 384 and the reaction was initiated by pressing the start key to dispense 30 pL sodium hyaluronate into each well. The plate was then removed from the MULTIDROP 384 and shaken for 10 seconds before being transferred to a heat block with the plate cover replaced. The plate was incubated at 37 °C for 10 minutes.

[0569] The MULTIDROP 384 was prepared to stop the reaction by priming the machine with Serum Working Solution and changing the volume setting to 240 pL. (25 mL of Serum Stock Solution [1 volume of Horse Serum (Sigma) was diluted with 9 volumes of 500 mM Acetate Buffer Solution and the pH was adjusted to 3.1 with hydrochloric acid] in 75 mL of 500 mM Acetate Buffer Solution). The plate was removed from the heat block and placed onto the MULTIDROP 384 and 240 pL of serum Working Solutions was dispensed into the wells. The plate was removed and shaken on a plate reader for 10 seconds. After a further 15 minutes, the turbidity of the samples was measured at 640 nm and the enzyme activity (in U / mL) of each sample was determined by fitting to the standard curve.

[0570] Specific activity (Units / mg) was calculated by dividing the enzyme activity (U / ml) by the protein concentration (mg / mL).

[0571] The data shown in Figure 2 A demonstrates that the HCCF produced by the Gen3 rHuPH20 production process exhibits significantly higher enzymatic activity than the HCCF produced be the Gen2 or Genl rHuPH20 production processes.

[0572] Example 16: ELISA for Measurement of CHO Host Cell Proteins (HCP) in rHuPH20 Drug Substance

[0573] HCP can be measured using the Cygnus Technologies’ 3rdGeneration CHO Host Cell Proteins kit. Capture antibodies against the proteins of interest, CHO HCPs, are bound to each well of a 96-well plate. A labelled detection antibody solution, anti-CHO HCP-HRP, is then added along with the standards and test articles. If any CHO HCPs are present, they bind to the capture and detection antibodies.

[0574] After incubation, any unbound material is removed by a washing step. A substrate is then added, 3,3’,5,5’-tetramethylbenzindine (TMB), which produces a blue color when oxidized by the horseradish peroxidase conjugate (HRP) bound to the detection antibody. The TMB reaction is then stopped by the addition of acid which turns the solution from blue to yellow. The intensity of the color can then be quantitated in a plate reader. The concentration of CHO HCP is

[0575] DBl / 164647321.1 125 calculated relative to a reference standard curve in the assay, unknowns are quantified using curve-fitting software.

[0576] The concentration of HCP was determined for the process described in W02009 / 111066 and for the process of the present invention. Table 32: Comparison of concentration of HCP from an exemplary process as described in

[0577] W02009 / 111066 (Gen2) and an exemplary process according to the present invention (Gen3)

[0578] The fold removal of the present invention is therefore 4.3 times that of the process described in W02009 / 111066.

[0579] Figure 3A and 3B contain further data illustrating the HCP content of Gen2 rHuPH20 and Gen3 rHuPH20. More specifically, Figure 3 A illustrates a statistical analysis of HCP content per mg of rHuPH20 in representative compositions from several Gen2 (n = 10) and Gen3 (n = 6)

[0580] DBl / 164647321.1 126 batches. Figure 3B shows data on the HCP content per mg of rHuPH20 throughout several representative Gen2 (n = 2) and Gen3 (n = 6) rHuPH20 production processes.

[0581] Example 17: Content, Oxidation, and Hydrolyzation

[0582] The purpose of this analytical test method is two-fold. The first part involves the isolation and purification of rHuPH20 bioreactor and in-process samples from process related impurities and cell culture media using the 1H3 (a recombinant murine IgG kappa anti-rHuPH20 monoclonal antibody described in W02009 / 111066) coupled Q Sepharose affinity resin. The second part involves either quantification of purified rHuPH20 content (mg / mL) or purity of oxidation and hydrolyzation by Reversed Phase High Performance Liquid Chromatography (RP- HPLC).

[0583] Summary of method(s)

[0584] Anti-rHuPH20 1H3 affinity resin is generated by conjugating anti-rHuPH20 recombinant monoclonal antibody 1H3 purified from Chinese Hamster Ovary (CHO) cells to CNBr-activated Sepharose 4 Fast Flow and are subsequently referred to as “beads” throughout this method.

[0585] Once coupled with 1H3, the affinity resin is added to in-process samples containing rHuPH20. During a 2-hour incubation period, rHuPH20 is captured by the beads. The beads are then washed and eluted using a low pH buffer to yield a purified sample. Purified rHuPH20 samples are assessed for product quantity using RP-HPLC.

[0586] RP-HPLC is a chromatographic technique that separates molecules based on their relative hydrophobicity. Reversed Phase separation is achieved by binding hydrophobic molecules to the resin in an aqueous (hydrophilic) environment and gradually reducing the polarity of the mobile phase by adding a water-miscible organic solvent such as acetonitrile and gradually increasing the percentage of organic solvent over time. This percent solvent change over time process is called a gradient elution. The more hydrophilic components will elute under conditions of relatively low organic solvent and the more hydrophobic compounds will elute under conditions of relatively high organic solvent. Ion-pairing (IP) reagents, such as trifluoroacetic acid (TFA), are used as buffer additives in order to modify the charge groups on sample molecules. These reagents have two functionalities, hydrophobicity and charge, allowing them to bind hydrophobically to the stationary phase and ionically to the molecule charge groups. They also serve the purpose of blocking residual silanols (hydroxyl derivatives of silanes which may cause an ion-pairing effect).

[0587] To measure content and to correct for sample dilution during affinity purification and

[0588] DBl / 164647321.1 127 elution, Bovine Serum Albumin (BSA) is added to the elution buffer (low pH) for use as an internal standard. BSA has no affinity to the beads, co-elutes with rHuPH20 during elution but is completely resolved during RP-HPLC analysis. The ratio of BSA signal in the elution buffer (unadulterated) to the BSA signal in the rHuPH20 affinity purified samples is used to correct for sample dilution during the purification. The amount of rHuPH20 (mg / mL) is extrapolated from a calibration curve obtained by plotting rHuPH20 peak area as a function of rHuPH20 concentration. The predicted sample concentration is then multiplied by the BSA ratio correction...

Claims

CLAIMS1. A hyaluronidase polypeptide produced by a method comprising:(a) introducing nucleic acid molecule operably linked to a promotor into a eukaryotic cell, the nucleic acid molecule encoding a hyaluronidase polypeptide and including a stop codon;(b) culturing the cell in a growth medium having copper sulfate and L-cysteine sulfate under conditions whereby an encoded hyaluronidase polypeptide is produced by expression of the introduced nucleic acid molecule and secreted by the cell, the growth medium being devoid of sodium butyrate and insulin; and(c) recovering the expressed polypeptide, wherein the growth medium has a hyaluronidase activity of at least about 25,000 U / ml.

2. The hyaluronidase polypeptide of claim 1, wherein the growth medium comprises a basal medium and a concentrated feed medium.

3. The hyaluronidase polypeptide of claim 2, wherein the basal medium comprises CD CHO and L-Glutamine.

4. The hyaluronidase polypeptide of claim 2, wherein the concentrated feed medium comprises essential amino acids, vitamins, salts and trace elements, and further comprises copper sulfate, glucose, and L-cysteine sulfate.

5. The hyaluronidase polypeptide of claim 2, wherein the concentrated feed medium comprises essential amino acids, vitamins, salts and trace elements, and further comprises L-Cysteine Sulfate.

6. The hyaluronidase polypeptide of claim 1, that: is neutral active; and contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide.

7. The hyaluronidase polypeptide of claim 6, consisting of the sequence of amino acids set forth as ammo acids 1-477, 1-478, 1-479, 1-480, 1-481, 1-482 or 1-483 set forth in SEQ ID NO: 1 or consisting of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as amino acids 1-477, 1-478, 1-479, 1-480, 1-481 or 1 -482 or 1 -483 of SEQ ID NO : 1.DBl / 164647321.

18. The hyaluronidase polypeptide of claim 6, consisting of the sequence of amino acids set forth as ammo acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 set forth in SEQ ID NO: 1 or containing amino acid substitutions in the sequence of amino acids set forth as ammo acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO: 1, whereby the amino-acid substituted hyaluronidase polypeptide consists of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO: 1.

9. The hyaluronidase polypeptide of claim 1, that: contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide; is catalytically active; and is soluble.

10. The hyaluronidase polypeptide of claim 9, selected from: i) a polypeptide that consists of a contiguous sequence of amino acids contained within SEQID NO: 1, whereby the polypeptide comprises amino acids 36-464 of SEQ ID NO: 1 and terminates at a residue selected from among 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO: 1; or ii) a polypeptide that contains amino acid substitutions in the sequence of amino acids of the polypeptide of i), whereby the amino acid- substituted polypeptide consists of a sequence of amino acids that has at least about 91% amino acid sequence identity with the polypeptide of i).

11. The hyaluronidase polypeptide of claim 9, consisting of a sequence of amino acids that has at least 98% amino acid sequence identity with the sequence of amino acids set forth as amino acid residues 36-483 of SEQ ID NO: 1.

12. The hyaluronidase polypeptide of claim 1, that contains a contiguous sequence of amino acid residues of SEQ ID NO: 1, but it is C-terminally truncated so that it does not contain all or a portion of GPI anchor, whereby the polypeptide is soluble and neutral active.

13. The hyaluronidase polypeptide of claim 1, further comprising a linker and a targeting agent, wherein the linker is attached to the targeting agent and wherein the linker is attached directly or indirectly to the recovered polypeptide.DB1 / 164647321.

114. The hyaluronidase polypeptide of claim 1, further comprising a polymeric molecule selected from the group consisting of polyalkylene oxides (PAO), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethelene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans, heparin, homologous albumin, celluloses, hydrolysates of chitosan, starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, and bio-polymers.

15. The hyaluronidase polypeptide of claim 1, wherein a unit of hyaluronidase activity is determined by incubating soluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period of 10 minutes, precipitating undigested sodium hyaluronate with addition of acidified serum albumin, measuring turbidity of resulting sample at 640 nm after a 20 minute development period, and comparing with a calibration curve generated with dilutions of a soluble rHuPH20 assay working reference standard.

16. A pharmaceutical composition comprising a hyaluronidase polypeptide of claim 1, and Factor VIII polypeptide.

17. A method for producing a hyaluronidase polypeptide, comprising:(a) introducing a nucleic acid molecule operably linked to a promotor into a eukaryotic cell, the nucleic acid molecule encoding a hyaluronidase polypeptide and including a stop codon;(b) culturing the cells in a growth medium having copper sulfate, and L-cysteine sulfate under conditions whereby an encoded hyaluronidase polypeptide is produced by expression of the introduced nucleic acid molecule and secreted by the cell, the growth medium being devoid of sodium butyrate and insulin; and(c) recovering the expressed polypeptide.

18. The method of claim 17, wherein the growth medium comprises a basal medium and a concentrated feed medium.

19. The method of claim 18, wherein the basal medium comprises CD CHO and L-Glutamine.

20. The method of claim 18, wherein the concentrated feed medium comprises essential amino acids, vitamins, salts and trace elements, and further comprises copper sulfate, glucose, and L-cysteine sulfate.DB1 / 164647321.

121. The method of claim 20, wherein the concentrated feed medium comprises essential amino acids, vitamins, salts and trace elements, and further comprises L-Cysteine Sulfate.

22. The method of claim 17, wherein the concentrated feed medium is added to the culture daily starting on day 2 and daily thereafter.

23. The method of claim 17, wherein amount of the concentrated feed medium is 1% of the bioreactor volume per day.

24. The method of claim 17, wherein the hyaluronidase polypeptide encoded by the nucleic acid molecule is selected from among: a polypeptide that consists of a sequence of amino acids selected from among amino acid residues 1-477, 1-478, 1-479, 1-480, 1-481, 1-482, 1-483, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO: 1; and a polypeptide that consists of a sequence of amino acids that contains amino acid substitutions in the sequence of amino acids set forth as 1-477, 1-478, 1-479, 1-480, 1-481, 1-482, 1-483, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1, whereby the amino acid-substituted hyaluronidase polypeptide consists of a sequence of amino acids that has at least 95% amino acid sequence identity to a sequence of amino acids set forth as amino acid residues 1-477, 1-478, 1-479, 1-480, 1-481, 1-482, 1-483, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO: 1.

25. The method of claim 24, wherein the encoded hyaluronidase polypeptide consists of a sequence of amino acids that contain amino acid substitutions in the sequence of amino acids set forth as 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1, whereby the amino acid- substituted hyaluronidase polypeptide consists of a sequence of amino acids that has at least 95% amino acid sequence identity to a sequence of amino acids set forth as amino acid residues 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1.

26. The method of claim 17, wherein the nucleic acid encodes amino acids 36-477, 36-478, 36- 479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO: 1.

27. The method of claim 17, wherein the nucleic acid comprises the sequence of nucleotides set forth in SEQ ID NO:47.DB1 / 164647321.

128. The method of claim 17, wherein the nucleic acid comprises the sequence of nucleotides set forth as nucleotides 106-1446 of SEQ ID NO:47 or degenerates thereof or the sequence of nucleotides set forth in SEQ ID NO:47.

29. The method of claim 17, wherein the encoded hyaluronidase polypeptide is selected from among: a polypeptide that consists of a sequence of amino acids selected from among amino acid residues 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1; and a polypeptide that consists of a sequence of amino acids that contain amino acid substitutions in the sequence of amino acids set forth as 36-477, 36-478, 36-479, 36-480, 36-481, 36- 482 or 36-483 of SEQ ID NO: 1, whereby the amino acid- substituted hyaluronidase polypeptide consists of a sequence of amino acids that has at least 95% amino acid sequence identity to a sequence of amino acids set forth as amino acid residues 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1; and the nucleic acid molecule comprises a sequence of nucleotides encoding a signal sequence for secretion of the encoded polypeptide.

30. The method of claim 29, wherein the encoded signal sequence for secretion is an IgG kappa chain leader peptide.

31. The method of claim 30, wherein the IgG kappa chain leader peptide is encoded by the sequence of nucleotides 1 to 105 as set forth in SEQ ID NO: 47.

32. The method of claim 17, wherein the recovered polypeptide contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide.

33. The method of claim 17, wherein the recovered polypeptide: is neutral active; contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide; and consists of the sequence of amino acids set forth as amino acids 1-477, 1-478, 1-479, 1-480, 1-481, 1-482 or 1-483 set forth in SEQ ID NO: 1 or consists of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as ammo acids 1-477, 1-478, 1-479, 1-480, 1-481 or 1-482 or 1-483 of SEQ ID NO: 1.DB1 / 164647321.

134. The method of claim 17, wherein the recovered polypeptide: is soluble; is neutral active; contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptide; and consists of the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36- 480, 36-481, 36-482, or 36-483 set forth in SEQ ID NO:1 or contains amino acid substitutions in the sequence of amino acids set forth as amino acids 36-477, 36-478, 36- 479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO: 1, whereby the amino-acid substituted hyaluronidase polypeptide consists of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as ammo acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO:1.

35. The method of any of claims 17-34, whereby the method results in recovered polypeptide that is substantially free of at least one impurity.

36. The method of any of claims 17-35, wherein the eukaryotic cell is a Chinese hamster ovary (CHO) cell.

37. The method of any of claims 17-36, wherein during culturing, pH of the growth medium is maintained to be in a range from 6 to 8, preferably 6.6 to 7.2.

38. The method of any of claims 17-37, wherein the culturing is performed for a period in a range from 14 days to 21 days.

39. The method of claim 17-38, wherein temperature of the growth medium is greater than or equal to 36 degrees C, and then subsequently changed to a temperature less than or equal to 35 degrees C, preferably less than or equal to 33 degrees C.

40. The method of claim 39, wherein during culturing a temperature of the growth medium is maintained at greater than or equal to 36 °C for first 9 days and changed to less than or equal to 33 °C at day 9.

41. The method of claim 17-40, wherein initial cell culture medium contains least 40 mM in amino acids or greater than or equal to 40% of total amino acids supplied, and total amino acids provided are at least 100 mM.DB1 / 164647321.

142. The method of claim 17-41, wherein the total amount of asparagine and aspartate supplied are at least 10 mM combined, preferably 13 mM combined.

43. The method of claim 17-42, wherein the total amount of cystine and cysteine supplied are at least 1 mM combined.

44. The method of claim 17-43, wherein the total amount of asparagine, aspartate, glutamine, glutamate, and glutamax are at least 20%, preferably 25%, of the total amino acids supplied.

45. The method of claim 17-44, wherein the total amount of serine is at least 11%, preferably 16%, of the total amino acids supplied.

46. The method of claims 17-45, wherein prior to recovering the expressed polypeptide, the cultured eukaryotic cells have a cell viability greater than about 80%.

47. The method of claim 46, wherein prior to recovering the expressed polypeptide, growth culture has a hyaluronidase activity is at least about 25,000 U / ml, preferably at least about 28,000 U / ml.

48. The method of claim 47, wherein a unit of hyaluronidase activity is determined by incubating soluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period of 10 minutes, precipitating undigested sodium hyaluronate with addition of acidified serum albumin, measuring turbidity of resulting sample at 640 nm after a 20 minute development period, and comparing with a calibration curve generated with dilutions of a soluble rHuPH20 assay working reference standard.

49. The method of claim 17, further comprising preparing a pharmaceutical composition comprising the recovered polypeptide with a Factor VIII polypeptide.

50. The method of claim 17, the recovered polypeptide: contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide; is catalytically active; is soluble, and is selected from: i) a polypeptide that consists of a contiguous sequence of amino acids contained within SEQ ID NO: 1, whereby the polypeptide comprises amino acids 36-464 of SEQ ID NO: 1 and terminates at a residue selected from among 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO: 1; orDB1 / 164647321.1ii) a polypeptide that contains amino acid substitutions in the sequence of amino acids of the polypeptide of i), whereby the amino acid- substituted polypeptide consists of a sequence of amino acids that has at least about 91% amino acid sequence identity with the polypeptide of i).

51. The method of claim 17, wherein the recovered polypeptide: contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide; is catalytically active; is soluble; and consists of a sequence of amino acids that has at least 98% amino acid sequence identity with the sequence of amino acids set forth as amino acid residues 36-483 of SEQ ID NO:1.

52. The method of claim 17, further comprising: conjugating the recovered polypeptide with a linker and a targeting agent, wherein the linker is attached to the targeting agent and wherein the linker is attached directly or indirectly to the recovered polypeptide.

53. The method of claim 17, wherein the recovered polypeptide contains a contiguous sequence of amino acid residues of SEQ ID NO:1, but it is C-terminally truncated so that it does not contain all or a portion of GPI anchor, whereby the polypeptide is soluble and neutral active.

54. The method of claim 17, further comprising modifying the recovered polypeptide with a polymeric molecule, wherein the polymeric molecule is selected from among polyalkylene oxides (PAO), PEG-glycidyl ethers (Epox-PEG), PEG-oxy carbonylimidazole (CDI-PEG), branched polyethelene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans, heparin, homologous albumin, celluloses, hydrolysates of chitosan, starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, and biopolymers.

55. The method of any of claims 17-54, wherein recovering, harvesting, purifying, processing, or isolating the expressed polypeptide comprises (i) transferring cell culture fluid (CCF)from (b) through a positively charged media to separate cells, cell debris, and host cell impuritiesDB1 / 164647321.1from the expressed polypeptide and (ii) chasing the transferred CCF through the charged media with at least one buffer.

56. The method of claim 55, wherein a positively charged media comprises a quaternary ammonium charged synthetic fiber matrix.

57. The method of claim 55, wherein the at least one buffer comprises about 10 mM to 100 mM sodium phosphate, 30 mM to 150 mM sodium chloride, or a combination thereof, preferably 50 mM Sodium Phosphate, 70 mM NaCl, pH 6.8.

58. The method of claim 55 wherein the at least one buffer comprises a pH in a range from about 6 to 8, preferably about 6.4 to 7.2.

59. The method of claim 55, wherein the polypeptide is further purified, processed, or isolated from harvest cell culture (HCCF) mixture by (i) loading onto a chromatography media; (ii) washing the chromatography media with at least one wash solution; and (iii) eluting the chromatography media with an elution buffer to obtain purified polypeptide.

60. The method of claim 59, wherein the chromatography media comprises a cibacron dye ligand.

61. The method of claim 60, wherein the cibacron dye ligand media comprises Cibacron Blue.

62. The method of claim 59, wherein the at least one wash solution comprises sodium phosphate, ammonium sulfate, Tris, sodium chloride, urea or a combination thereof.

63. The method of claim 62, wherein the at least one wash solution comprises from about 10 mM to 100 mM sodium phosphate, from about 30 mM to 150 mM sodium chloride, from about 100 mM to 300 mM ammonium sulfate, from about 50 mM to 200 mM Tris, from about 0.2 M to 1.2 M urea, or a combination thereof, preferably, a first washing buffer comprising 50 mM Sodium Phosphate, 70 mM NaCl, pH 6.8, a second washing buffer comprising 20 mM Sodium Phosphate, 200 mM Ammonium Sulfate, pH 7.0, or a second wash buffer comprising 50 mM Sodium Phosphate, IM Urea, 100 mM Sodium Chloride, pH 6.8, and a third washing buffer comprising 100 mM Tris, pH 8.0.

64. The method of claim 59, wherein the at least one wash solution comprises a pH in a range from about 5 to about 9, with a first washing buffer preferably with a pH range about 6.4 to 7.2, a second washing buffer preferably with a pH range of about 6.6 to 7.4, and a third washing buffer preferably with a pH range of about 7.6 to 8.4.DB1 / 164647321.

165. The method of claim 17, wherein an elution buffer comprises sodium phosphate, sodium chloride, arginine, or a combination thereof66. The method of claim 65, wherein an elution buffer comprises about from about 5 mM to 50 mM sodium phosphate, from about 50 mM to 400 mM sodium chloride, from about 100 mM to 400 mM arginine, or a combination thereof, preferably 20 mM Sodium Phosphate, 250 mM NaCl, 250 mM Arginine, pH 8.

67. The method of claim 59, wherein an elution buffer has a pH in a range from about 7 to 9, preferably about 7.6 to 8.4.

68. The method of claim 59, wherein the washing step is carried out at a flow rate of about 200 cm / hr to about 400 cm / hr.

69. The method of claim 59, wherein the washing step is repeated at least one, two, three, four or five times.

70. The method of claim 59, wherein the chromatography media comprises a second process step comprising hydrophobic interaction chromatography (HIC) media.

71. The method of claim 70, wherein the HIC media comprises a beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality.

72. The method of claim 59, wherein at least one wash solution comprises sodium phosphate, sodium sulfate, or a combination thereof.

73. The method of claim 72, wherein the at least one wash solution comprises from about 5 mM to 50 mM sodium phosphate, 0.2 mM to 0.6 mM Sodium Sulfate or a combination thereof, preferably 20 mM Sodium Phosphate, 0.4 M Sodium Sulfate, pH 7.0.

74. The method of claim 59, wherein the at least one wash solution has a pH in a range from about 5.0 to about 8.5.

75. The method of claim 59, wherein the washing step is carried out at a flow rate of about 100 cm / hr to about 400 cm / hr.

76. The method of claim 59, wherein the washing step is repeated at least one, two, three, four or five times.

77. The method of claim 59, wherein the chromatography media comprises a third process step comprising aminophenylboronate ligand media.

78. The method of claim 77, wherein the aminophenylboronate ligand media comprises beaded aminophenylboronate substituted crosslinked agarose resin.DB1 / 164647321.

179. The method of claim 59, wherein the at least one wash solution comprises sodium phosphate, sodium sulfate, glycine, sodium chloride, or a combination thereof.

80. The method of claim 79, wherein the at least one wash solution comprises from about 10 mM to 1000 mM sodium phosphate, from about 30 mM to 200 mM sodium chloride, from about 400 mM to 1000 mM sodium sulfate, from about 20 mM to 200 mM glycine, or a combination thereof, preferably a first wash buffer comprising 20 mM Sodium Phosphate, 650 mM Sodium Sulfate, pH 7.0, a second wash buffer comprising 80 mM Glycine, 850 mM Sodium Sulfate, pH 9.0, and a third wash buffer comprising 80 mM Glycine, 100 mM Sodium Chloride, pH 10.0.

81. The method of claim 59, wherein the at least one wash solution has a pH in a range from about 5 to about 1, with a first washing buffer preferably with a pH range about 6.6 to 7.4, a second washing buffer preferably with a pH range of about 8.6 to 9.4, and a third washing buffer preferably with a pH range of about 9.6 to 10.

482. The method of claim 59, wherein an elution buffer comprises HEPES, sodium chloride, or a combination thereof.

83. The method of claim 65, wherein an elution buffer comprises from about 10 mM to 100 mM HEPES, from about 10 mM to 200 mM sodium chloride, or a combination thereof, preferably 50 mM HEPES, 100 mM Sodium Chloride, pH 6.9.

84. The method of claim 59, wherein an elution buffer has a pH in a range from about 5 to 9, preferably about 6.5 to 7.3.

85. The method of claim 59, wherein the washing step is carried out at a flow rate of about 50 cm / hr to about 400 cm / hr.

86. The method of claim 59, wherein the washing step is repeated at least one, two, three, four, five, or six times.

87. The method of any of claims 17-63, wherein introducing nucleic acid molecule comprises culturing the eukaryotic cell with a vector comprising the nucleic acid molecule.

88. The method of claim 87, wherein the vector is a Pichia vector, an E. coli vector, or a viral vector.

89. The method of claim 87, wherein the vector is a viral vector.

90. The method of claim 55, wherein recovering the expressed polypeptide further comprises viral inactivation in presence of L-arginine.DB1 / 164647321.

191. The method of claim 90, wherein the viral inactivation comprises applying a buffer solution comprising L-arginine, wherein the buffer solution has a pH in a range from 3.5 to 4.3.

92. The method of claim 91 wherein the buffer solution comprises from about 500 mM to 1400 mM L-arginine, preferably 800 mM Arginine, pH 4.0.

93. The method of claim 92, wherein the viral inactivation comprises holding the buffer conditioned protein solution for a duration from about 30 to 120 minutes.

94. The method of any of claims 55-68, further comprising depth filtration, membrane filtration, a cation exchange chromatography, a mixed-mode chromatography, ultrafiltration, diafiltration, or combination thereof.

95. The method of any of claims 55-69, further comprising analyzing at least one impurity.

96. A recombinant anti-hyaluronidase monoclonal antibody, comprising: a variable region comprising or consisting of one of (i) amino acid residues 21-131 of SEQ ID NO: 76, (ii) amino acid residues 21-127 of SEQ ID NO: 77 or (iii) amino acid residues 16-121 of SEQ ID NO: 78; and a variable region comprising or consisting of one of (iv) amino acid residues 19-136 of SEQ ID NO: 73, (v) amino acid residues 20-137 of SEQ ID NO: 74, or (vi) amino acid residues 20-135 of SEQ ID NO: 75.

97. The recombinant anti-hyaluronidase monoclonal antibody of claim 96, wherein the antibody comprises a light chain region including a constant region comprising or consisting of a mouse Ig-kappa light chain comprising SEQ ID NO: 66.

98. The recombinant anti-hyaluronidase monoclonal antibody of claim 96, wherein the antibody comprises a heavy chain region including a constant region comprising or consisting of a mouse IgGl heavy chain comprising SEQ ID NO: 61.

99. The method of claim 59, wherein the chromatography comprises Sepharose bound to the recombinant anti-hyaluronidase monoclonal antibody of any of claims 96-98.

100. A composition comprising a mixture of polypeptides, said mixture comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein the composition is obtainable by the following method:(a) introducing a nucleic acid molecule operably linked to a promotor into a eukaryotic cell, the nucleic acid molecule encoding a hyaluronidase polypeptide and including a stop codon;DB1 / 164647321.1(b) culturing the cell in a cell culture comprising growth medium having copper sulfate and L-cysteine sulfate under conditions whereby hyaluronidase polypeptide is produced by expression of the introduced nucleic acid molecule and secreted by the cell, the growth medium being devoid of sodium butyrate and insulin; and(c) recovering the expressed polypeptide, wherein the expressed polypeptide is a mixture of hyaluronidase polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8.

101. The composition of claim 100, wherein in said method the growth medium comprises a basal medium and a concentrated feed medium.

102. The composition of claim 101, wherein in said method the basal medium comprises CD CHO and L-Glutamine.

103. The composition of claim 101, wherein in said method the concentrated feed medium comprises amino acids, vitamins, salts and trace elements, and further comprises copper sulfate, glucose, and L-cysteine sulfate.

104. The composition of claim 101, wherein in said method the concentrated feed medium comprises amino acids, vitamins, salts and trace elements, and further comprises glucose and L-cysteine sulfate.

105. The composition of any of claims 101-104, wherein in said method the concentrated feed medium is added to the culture daily starting on day 2 and daily thereafter.

106. The composition of any of claims 101-105, wherein in said method the concentrated feed medium is added to the culture at a volume of 1% of bioreactor volume per day.

107. The composition of any of claims 100-106, wherein in said method the eukaryotic cell is a Chinese hamster ovary (CHO) cell.

108. The composition of any of claims 100-107, wherein in said method during culturing, pH of the growth medium is maintained to be in a range from 6 to 8, preferably 6.6 to 7.2.

109. The composition of any of claims 100-108, wherein in said method the culturing is performed for a period in a range from 14 days to 21 days.

110. The composition of any of claims 100-109, wherein in said method during culturing temperature of the growth medium is greater than or equal to 36 °C, and then subsequently changed to a temperature less than or equal to 35 °C, preferably less than or equal to 33 °C.DB1 / 164647321.1111. The composition of any of claims 100- 110, wherein in said method during culturing the temperature of the growth medium is maintained at greater than or equal to 36 °C for the first 9 days and changed to less than or equal to 33 °C on day 9.

112. The composition of any of claims 101-111, wherein in said method the basal medium contains at least 40 mM in amino acids or greater than or equal to 40% of total amino acids supplied, and the total amino acids supplied are at least 100 mM.

113. The composition of any of claims 100-112, wherein in said method total amount of asparagine and aspartate supplied are at least 10 mM combined, preferably 13 mM combined.

114. The composition of any of claims 100-113, wherein in said method total amount of cystine and cysteine supplied are at least 1 mM combined.

115. The composition of any of claims 100-114, wherein in said method total amount of asparagine, aspartate, glutamine, glutamate, and glutamax are at least 20%, preferably 25%, of total amino acids supplied.

116. The composition of any of claims 100-115, wherein in said method total amount of serine is at least 11%, preferably 16%, of total amino acids supplied.

117. The composition of any of claims 100-116, wherein in said method prior to recovering the expressed polypeptide, the eukaryotic cells in the cell culture have a cell viability greater than about 80%.

118. The composition of any of claims 100-117, wherein in said method prior to recovering the expressed polypeptide the cell culture comprising the expressed polypeptide has a hyaluronidase activity that is at least 25,000 units / ml, preferably at least 28,000 units / ml.

119. The composition of any of claims 100-118, wherein in said method recovering the expressed polypeptide comprises harvesting, purifying, processing, and / or isolating the expressed polypeptide, wherein this recovering comprises (i) transferring cell culture fluid through a positively charged medium to separate cells, cell debris, and host cell impurities from the expressed polypeptide and (ii) chasing the transferred cell culture fluid through the positively charged medium with at least one buffer, thereby obtaining a harvest cell culture fluid.

120. The composition of claim 119, wherein in said method the positively charged medium comprises a quaternary ammonium charged synthetic fiber matrix.DB1 / 164647321.1121. The composition of any of claims 119-120, wherein in said method the at least one buffer comprises about 10 mM to 100 mM sodium phosphate, 30 mM to 150 mM sodium chloride, or a combination thereof, preferably 50 mM Sodium Phosphate, 70 mM NaCl, pH 6.8.

122. The composition of any of claims 119-121, wherein in said method the at least one buffer has a pH in a range from about 6 to 8, preferably about 6.4 to 7.2.

123. The composition of any of claims 119-122, wherein in said method the expressed polypeptide is further purified, processed, and / or isolated from the harvest cell culture fluid by (i) loading onto chromatography medium; (ii) washing the chromatography medium with at least one wash solution; and (iii) eluting from the chromatography medium with an elution buffer to obtain purified polypeptide.

124. The composition of claim 123, wherein in said method the chromatography medium comprises a cibacron dye ligand.

125. The composition of claim 124, wherein in said method the cibacron dye ligand medium comprises Cibacron Blue.

126. The composition of any of claims 123-125, wherein in said method the at least one wash solution comprises sodium phosphate, ammonium sulfate, Tris, sodium chloride, urea, or a combination thereof.

127. The composition of any of claims 123-126, wherein in said method the at least one wash solution comprises from about 10 mM to 100 mM sodium phosphate, from about 30 mM to 150 mM sodium chloride, from about 100 mM to 300 mM ammonium sulfate, from about 50 mM to 200 mM Tris, from about 0.2 M to 1.2 M urea, or a combination thereof, preferably, a first washing buffer comprising 50 mM Sodium Phosphate, 70 mM NaCl, pH 6.8, a second washing buffer comprising 20 mM Sodium Phosphate, 200 mM Ammonium Sulfate, pH 7.0, or 50mM Sodium Phosphate, IM Urea, lOOmM Sodium Chloride, pH 6.8, and a third washing buffer comprising 100 mM Tris, pH 8.0.

128. The composition of any of claims 123-127, wherein in said method the at least one wash solution has a pH in a range from about 5 to about 9, with a first washing buffer preferably with a pH range about 6.4 to 7.2, a second washing buffer preferably with a pH range of about 6.6 to 7.4, and a third washing buffer preferably with a pH range of about 7.6 to 8.4.

129. The composition of any of claims 123-128, wherein in said method the elution buffer comprises sodium phosphate, sodium chloride, arginine, or a combination thereof.DB1 / 164647321.1130. The composition of any of claims 123-129, wherein in said method the elution buffer comprises about from about 5 mM to 50 mM sodium phosphate, from about 50 mM to 400 mM sodium chloride, from about 100 mM to 400 mM arginine, or a combination thereof, preferably 20 mM Sodium Phosphate, 250 mM NaCl, 250 mM Arginine, pH 8.

131. The composition of any of claims 123-130, wherein in said method the elution buffer has a pH in a range from about 7 to 9, preferably about 7.6 to 8.4.

132. The composition of any of claims 123-131, wherein in said method the step of washing the chromatography medium is carried out at a flow rate of about 200 cm / hr to about 400 cm / hr.

133. The composition of any of claims 123-132, wherein in said method the step of washing the chromatography medium is repeated at least one, two, three, four or five times.

134. The composition of any of claims 123-133, wherein in said method the expressed polypeptide is further purified, processed, and / or isolated by (i) loading onto a second chromatography medium; (ii) washing the second chromatography medium with at least one wash solution; and (iii) eluting from the second chromatography medium with an elution buffer to obtain purified polypeptide, wherein the second chromatography medium comprises hydrophobic interaction chromatography (HIC) medium.

135. The composition of claim 134, wherein in said method the HIC medium comprises a beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality.

136. The composition of any of claims 134-135, wherein in said method the at least one wash solution comprises sodium phosphate, sodium sulfate, or a combination thereof.

137. The composition of any of claims 134-136, wherein in said method the at least one wash solution comprises from about 5 mM to 50 mM sodium phosphate, 0.2 mM to 0.6 mM Sodium Sulfate or a combination thereof, preferably 20 mM Sodium Phosphate, 0.4 M Sodium Sulfate, pH 7.0.

138. The composition of any of claims 134-137, wherein in said method the at least one wash solution has a pH in a range from about 5.0 to about 8.5.

139. The composition of any of claims 134-138, wherein in said method the step of washing the second chromatography medium is carried out at a flow rate of about 100 cm / hr to about 400 cm / hr.DB1 / 164647321.1140. The composition of any of claims 134-139, wherein in said method the step of washing the second chromatography medium is repeated at least one, two, three, four or five times.

141. The composition of any of claims 123-140, wherein in said method the expressed polypeptide is further purified, processed and / or isolated by (i) loading onto a third chromatography medium; (ii) washing the third chromatography medium with at least one wash solution; and (iii) eluting from the third chromatography medium with an elution buffer to obtain purified polypeptide, wherein the third chromatography medium comprises aminophenylboronate ligand medium.

142. The composition of claim 141, wherein in said method the aminophenylboronate ligand medium comprises beaded aminophenylboronate substituted crosslinked agarose resin.

143. The composition of any of claims 141-142, wherein in said method the at least one wash solution comprises sodium phosphate, sodium sulfate, glycine, sodium chloride, or a combination thereof.

144. The composition of any of claims 141-143, wherein in said method the at least one wash solution comprises from about 10 mM to 1000 mM sodium phosphate, from about 30 mM to 200 mM sodium chloride, from about 400 mM to 1000 mM sodium sulfate, from about 20 mM to 200 mM glycine, or a combination thereof, preferably a first wash buffer comprising 20 mM Sodium Phosphate, 650 mM Sodium Sulfate, pH 7.0, a second wash buffer comprising 80 mM Glycine, 850 mM Sodium Sulfate, pH 9.0, and a third wash buffer comprising 80 mM Glycine, 100 mM Sodium Chloride, pH 10.0.

145. The composition of any of claims 141-144, wherein in said method the at least one wash solution has a pH in a range from about 5 to about 1, with a first washing buffer preferably with a pH range about 6.6 to 7.4, a second washing buffer preferably with a pH range of about 8.6 to 9.4, and a third washing buffer preferably with a pH range of about 9.6 to 10.4.

146. The composition of any of claims 141-145, wherein in said method the elution buffer comprises HEPES, sodium chloride, or a combination thereof.

147. The composition of any of claims 141-146, wherein in said method the elution buffer comprises from about 10 mM to 100 mM HEPES, from about 10 mM to 200 mM sodium chloride, or a combination thereof, preferably 50 mM HEPES, 100 mM Sodium Chloride, pH 6.9.DB1 / 164647321.1148. The composition of any of claims 141-147, wherein in said method the elution buffer has a pH in a range from about 5 to 9, preferably about 6.5 to 7.3.

149. The composition of any of claims 141-148, wherein in said method the step of washing the third chromatography medium is carried out at a flow rate of about 50 cm / hr to about 400 cm / hr.

150. The composition of any of claims 141-149, wherein in said method the step of washing the third chromatography medium is repeated at least one, two, three, four, five, or six times.

151. The composition of any of claims 100-150, wherein in said method introducing the nucleic acid molecule into the eukaryotic cell comprises culturing the eukaryotic cell with a vector comprising the nucleic acid molecule.

152. The composition of claim 151, wherein in said method the vector is a Pichia vector, an E. coli vector, or a viral vector.

153. The composition of claim 151, wherein in said method the vector is a viral vector.

154. The composition of any of claims 100-153, wherein in said method recovering the expressed polypeptide further comprises viral inactivation in presence of L-arginine.

155. The composition of claim 154, wherein in said method the viral inactivation comprises applying a buffer solution comprising L-arginine, wherein the buffer solution has a pH in a range from 3.5 to 4.3.

156. The composition of claim 155, wherein in said method the buffer solution comprises from about 500 mM to 1400 mM L-arginine, preferably 800 mM Arginine, pH 4.0.

157. The composition of any of claims 155-156, wherein in said method the viral inactivation comprises holding the buffer solution in the presence of the protein solution for a duration from about 30 to 120 minutes.

158. The composition of any of claims 100-157, wherein the method further comprises depth filtration, membrane filtration, a cation exchange chromatography, a mixed-mode chromatography, ultrafiltration, diafiltration, or combination thereof.

159. The composition of any of claims 100-158, wherein the method further comprises analyzing at least one impurity.

160. The composition of any of claims 100-159, wherein the polypeptides in the mixture of polypeptides: are neutral active; andDB1 / 164647321.1contain at least one sugar moiety that is covalently attached to an asparagine (N) residue of the polypeptides.

161. The composition of any of claims 100-160, wherein the polypeptides in the mixture of polypeptides: contain at least one sugar moiety that is covalently attached to an asparagine residue of the polypeptides; are catalytically active; and are soluble.

162. The composition of any of claims 100-161, wherein the polypeptides in the mixture of polypeptides contain a contiguous sequence of amino acid residues of SEQ ID NO: 1, but the polypeptides are C-terminally truncated so that they do not contain all or a portion of GPI anchor, whereby the polypeptides are soluble and neutral active.

163. The composition of any of claims 100- 162, wherein the polypeptides in the mixture of polypeptides further comprise a linker and a targeting agent, wherein the linker is attached to the targeting agent and wherein the linker is attached directly or indirectly to the recovered polypeptides.

164. The composition of any of claims 100-163, wherein the polypeptides in the mixture of polypeptides further comprise a polymeric molecule selected from the group consisting of polyalkylene oxides (PAO), PEG-glycidyl ethers (Epox-PEG), PEG-oxycarbonylimidazole (CDI-PEG), branched polyethelene glycols (PEGs), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D,L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydride, dextrans, heparin, homologous albumin, celluloses, hydrolysates of chitosan, starches, glycogen, agaroses and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, and bio-polymers.

165. The composition of any of claims 100-164, wherein a unit of hyaluronidase activity is determined by incubating soluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period of 10 minutes, precipitating undigested sodium hyaluronate with addition of acidified serum albumin, measuring turbidity of resulting sample at 640 nm after a 20 minute development period, and comparing with a calibration curve generated with dilutions of a soluble rHuPH20 assay working reference standard.DB1 / 164647321.1166. A pharmaceutical composition comprising the mixture of polypeptides obtainable by the method of any of claims 100-165, and Factor VIII polypeptide.

167. A composition comprising a mixture of polypeptides, said mixture comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein the composition is obtainable by the following method:(a) introducing a nucleic acid molecule operably linked to a promotor into a eukaryotic cell, the nucleic acid molecule encoding a hyaluronidase polypeptide and including a stop codon;(b) inoculating a basal medium in a bioreactor with an inoculum of the eukaryotic cells that encode the hyaluronidase polypeptide to produce a cell culture, wherein: the bioreactor contains at least 100 liters of cell culture;IO10- 1011cells are inoculated per 100 liters cell culture; and the cells are cultured at a set temperature;(c) on days 2 to 8 of the cell culture, feeding the cells daily with a concentrated feed medium comprising amino acids, vitamins, salts and trace elements with glucose and L-cysteine added to the concentrated feed medium as needed, wherein the concentrated feed medium is added to the culture at a volume of 0.5% to 20% of cell culture volume;(d) on day 9 of the cell culture, feeding the cells with a concentrated feed medium comprising amino acids, vitamins, salts and trace elements, and adding a copper sulfate bolus, wherein the concentrated feed medium is added to the culture at a volume of 0.5% to 20% of the cell culture volume; and lowering the temperature compared to the temperature in step (c) to a temperature sufficient to increase cell cycle arrest, increase cell viability and stabilize the expressed hyaluronidase polypeptide;(e) on day 10 and subsequent days of the cell culture, feeding the cells daily with a concentrated feed medium comprising amino acids, vitamins, salts and trace elements, and adding glucose and L-cysteine to the cell culture as needed, wherein the concentrated feed medium is added to the culture at a volume of 0.5% to 20% of the cell culture volume;(f) continuing to culture the cells until within 24 hours of the viability % dropping below 80 %;(g) obtaining harvest cell culture fluid; and(h) purifying the hyaluronidase polypeptide from the harvest cell culture fluid,DB1 / 164647321.1wherein the hyaluronidase polypeptide is a mixture of hyaluronidase polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8.

168. The composition of claim 167, wherein the hyaluronidase polypeptide is soluble recombinant human PH20 (rHuPH20).

169. The composition of any of claims 167-168, wherein in said method the temperature in step (c) is greater than or equal to 36 °C.

170. The composition of any of claims 167-169, wherein in said method the temperature in step (d) is less than or equal to 35 °C, preferably less than or equal to 33 °C.

171. The composition of any of claims 167-170, wherein in step (g) of said method (i) the cell culture fluid is transferred through a positively charged medium to separate cells, cell debris, and host cell impurities from the expressed polypeptide and (ii) the cell culture fluid is chased through the positively charged medium with at least one buffer, thereby obtaining the harvest cell culture fluid.

172. The composition of claim 171, wherein in said method a positively charged medium comprises a quaternary ammonium charged synthetic fiber matrix.

173. The composition of any of claims 167-172, wherein in said method purifying the expressed hyaluronidase polypeptide from the harvest cell culture fluid is effected by column chromatography.

174. The composition of claim 173, wherein in said method the column chromatography comprises affinity chromatography, hydrophobic interaction chromatography and mixed-mode chromatography.

175. The composition of any of claims 173-174, wherein in said method the column chromatography comprises using beaded crosslinked agarose resin comprising Cibacron Blue for affinity chromatography, beaded crosslinked poly(styrene-divinylbenzene) resin with hydrophobic ligand functionality for hydrophobic interaction chromatography, and beaded aminophenylboronate substituted crosslinked agarose resin for mixed-mode chromatography.

176. Composition of any of claims 167-175, wherein in said method the concentrated feed medium is added to the cell culture at a volume of 1% of the cell culture volume177. Composition of any of claims 167-176, wherein in said method:DB1 / 164647321.1in step b) the basal medium comprises CD CHO and L-Glutamine; in step c) glucose is added as 40% glucose to the cell culture as needed, and L-cysteine added as 0.5 M L-cysteine sulfate to the cell culture at a volume of 1% of the cell culture volume; in step d) the copper sulfate is added as a 100 mM copper sulfate (CuSCU) bolus, wherein the concentrated feed medium is added to the cell culture at a volume of 1% of the cell culture volume; and in step e) the concentrated feed medium is added to the cell culture at a volume of 1% of the cell culture volume.

178. Composition of any of claims 167-177, wherein at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or 40 grams or more grams of hyaluronidase polypeptide is produced per 100 L of cell culture.

179. Composition of any of claims 167-178, wherein enzymatic activity of the harvest cell culture fluid comprising the hyaluronidase polypeptide prior to purification is greater than 25,000 units / mL, such as 30,000, 32,000, 34,000, 36,000, 38,000, 40,000, 42,000, 44,000, 46,000, 48,000, 50,000, 52,000, 54,000, 56,000, 58,000 or 60,000 units / mL.

180. Composition of any of claims 167-179, wherein specific activity of the hyaluronidase polypeptide is at least 80,000, 100,000, 120,000, 140,000, 160,000, or 180,000 units / mg.

181. Composition of any of claims 167-180, wherein the volume of cell culture in the bioreactor is 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000 or 4000 liters.

182. Composition of any of claims 167-181, wherein the cells that encode the hyaluronidase polypeptide are DG44 CHO cells.

183. Composition of any of claims 167-182, wherein the hyaluronidase polypeptide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 47.

184. A composition comprising a mixture of polypeptides, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 60% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5, optionally wherein 60-80% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5.DB1 / 164647321.1185. The composition of claim 184, wherein 60-70% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5.

186. The composition of claim 184, wherein at least 63% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5, optionally wherein 63-80% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5.

187. The composition of claim 186, wherein 63-70% of the polypeptides in said mixture of polypeptides consist of the sequence set forth in SEQ ID NO: 5.

188. A composition according to any of claims 184-187, for use as a medicament.

189. A composition comprising a mixture of polypeptides, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein at least 6% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are:(NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc, optionally wherein 6-12% of the total N-linked glycans on the polypeptides set forth in SEQ ID NO: 4-8 are:(NeuAc)2(Gal-GlcNAc)2(Man)3(GlcNAc)2Fuc.

190. A composition according to claim 189, for use as a medicament.

191. A harvest cell culture fluid comprising a mixture of polypeptides, said mixture of polypeptides comprising the polypeptides set forth in SEQ ID NO: 4-8, wherein the enzymatic activity of hyaluronidase is at least 25,000 units / mL, such as 30,000, 32,000, 34,000, 36,000, 38,000, 40,000, 42,000, 44,000, 46,000, 48,000, 50,000, 52,000, 54,000, 56,000, 58,000 or 60,000 units / mL.

192. The harvest cell culture fluid of claim 191, wherein the enzymatic activity of hyaluronidase is 30,000-60,000 units / mL.

193. The harvest cell culture fluid of any of claims 191-192, wherein the enzymatic activity of hyaluronidase is 30,000-50,000 units / mL.

194. The harvest cell culture fluid of any of claims 191-193, wherein the enzymatic activity of hyaluronidase is 40,000-50,000 units / mL.

195. A cell line deposited at American Type Culture Collection (ATCC, 10801University Blvd, Manassas, VA 20110 USA) on with the depositor identification reference HZ24-2B2 and the ATCC accession numberDB1 / 164647321.1196. A cell line that is derived from the deposited cell line of claim 195, wherein said cell line that is derived from said deposited cell line still has the capacity to produce soluble rHuPH20.

197. A method for obtaining a cell line derived from the deposited cell line of claim 196, comprising a step of gradually adapting said deposited cell line to grow in cell culture medium containing more than 20 pM methotrexate, wherein said deposited cell line is gradually adapted to grow in cell culture medium containing more than 20 pM methotrexate by gradually increasing the amount of methotrexate in the cell culture medium above 20 pM methotrexate when passaging the cells, optionally wherein the method further comprises a step of selecting said cell line derived from said deposited cell line based on a higher level of enzymatic activity of hyaluronidase measured in the cell culture fluid.

198. The harvest cell culture fluid of any of claims 191-194 or the method according to claim 197, wherein a unit of enzymatic activity of hyaluronidase is determined by incubating soluble rHuPH20 with sodium hyaluronate (hyaluronic acid) for a set period of 10 minutes, precipitating undigested sodium hyaluronate with addition of acidified serum albumin, measuring turbidity of resulting sample at 640 nm after a 20 minute development period, and comparing with a calibration curve generated with dilutions of a soluble rHuPH20 assay working reference standard.

199. A cell line obtainable by the method of claim 196 or claim 197.DB1 / 164647321.1

Images