Pharmaceutical composition comprising human hyaluronidase PH20 variant and medicament for subcutaneous injection

Abstract

The present invention relates to a pharmaceutical composition comprising (a) a drug and (b) a human PH20 variant. The human PH20 variant comprised in the pharmaceutical composition according to the present invention comprises amino acid substitutions at one or more positions selected from the alpha helix 8 sequence (S347-C381) and a linking region (A333-R346) between alpha helix 7 and alpha helix 8 on wild-type human pH20 having the amino acid sequence SEQ ID NO: 1, and optionally cleaved at the amino acid located at the N-terminal or C-terminal region. In addition, the pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable additive, in particular a stabilizer. Due to the advantages of the human PH20 variant, the pharmaceutical composition according to the present invention can exhibit a maximum therapeutic effect of a drug used therewith.

Description

[0001] This application is a divisional application of the invention patent application with the application date of March 24, 2020, application number 202080003052.8, and title "Drug composition comprising a human hyaluronidase PH20 variant and a drug for subcutaneous injection". Technical Field

[0002] The present disclosure relates to a drug composition comprising a human hyaluronidase PH20 variant having improved enzyme activity and thermal stability and one or more drugs; and a method of treating a disease using the drug composition.

[0003] The drug composition according to the present disclosure can be preferably used for subcutaneous injection. Background Art

[0004] Drugs (especially antibody drugs, etc.) that should be administered at high doses or multiple doses are usually administered via intravenous injection, and such injection takes about 90 minutes or longer. Intravenous injection is also accompanied by additional preparation procedures, so it is inconvenient for patients, doctors, and medical staff, and additional costs are incurred. In contrast, subcutaneous injection has the advantage of being able to be administered immediately, but compared with intravenous injection, the absorption rate is relatively low, and when the injection volume is 3 - 5 mL or more, it may cause swelling and pain at the injection site due to slow absorption. For this reason, subcutaneous injection of protein therapeutics is usually limited to injection of a small amount of 2 mL or less of solution. However, after hyaluronidase is administered (or subcutaneously injected) together with a therapeutic drug, the hyaluronic acid distributed in the extracellular matrix is hydrolyzed under the action of hyaluronidase, so that the viscosity of the subcutaneous area is reduced and the permeability of substances is increased, and thus high-dose or multiple-dose drugs can be easily delivered into the body.

[0005] There are six types of hyaluronidase genes in humans: Hyal1, Hyal2, Hyal3, Hyal4, HyalPS1, and PH20 / SPAM1. Hyal1 and Hyal2 are expressed in most tissues, and PH20 / SPAM1 (hereinafter referred to as PH20) is expressed in the sperm cell membrane and acrosomal membrane. HyalPS1 is not expressed because it is a pseudogene. PH20 is an enzyme (EC 3.2.1.35) that cleaves the β-1,4 bond between N-acetylglucosamine and glucuronic acid (the sugars that make up hyaluronic acid). The optimal pH of human hyaluronidase PH20 is 5.5, but it also exhibits some activity even at a pH of 7-8, while the optimal pH of other human hyaluronidases (including Hyal1) is 3-4 and their activity is weak at a pH of 7-8. The pH of the human subcutaneous region is approximately 7.4, which is generally neutral. Therefore, among various types of hyaluronidases, PH20 is widely used in clinical applications. Examples of the clinical applications of PH20 include subcutaneous injection of antibody therapeutics, which are used as ocular relaxants and anesthetic additives in ophthalmic surgery; for increasing the access of anticancer therapeutics to tumor cells by hydrolyzing hyaluronic acid in the extracellular matrix of tumor cells; and for promoting the absorption of excessive body fluids and blood in tissues.

[0006] Meanwhile, currently commercially available PH20 is in the form extracted from the testes of cattle or sheep. Examples thereof include (bovine hyaluronidase) and (ovine hyaluronidase).

[0007] Bovine testicular hyaluronidase (BTH) is obtained by removing the signal peptide and 56 amino acids at the C-terminus from bovine wild-type PH20 during the post-translational modification process. BTH is also a glycoprotein and, based on its total amino acid composition, has a mannose content of 5% and a glucosamine content of 2.2% (Borders and Raftery, 1968). When hyaluronidases of animal origin are repeatedly administered to humans at high doses, neutralizing antibodies can be produced, and other animal-derived biomaterials that are present as impurities in addition to PH20 may cause allergic reactions. In particular, the use of PH20 extracted from cattle is restricted due to concerns about mad cow disease. To overcome these problems, research on recombinant human PH20 protein has been carried out.

[0008] The expression of recombinant human PH20 protein in yeast (Pichia pastoris), DS-2 insect cells, animal cells, etc. has been reported (Chen et al., 2016, Hofinger et al., 2007). The recombinant PH20 protein produced in insect cells and yeast is different from human PH20 in terms of the N-glycosylation pattern during the post-translational modification process.

[0009] In hyaluronidases, the protein structures of Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidase (PDB ID: 1FCQ, 1FCU, 1FCV) have been identified. Hyal1 consists of two domains, namely a catalytic domain and an EGF-like domain, and the catalytic domain is in the form of (β / α) 8 where the α-helix and β-strand (which are characteristic of the secondary protein structure) are each repeated eight times (Chao et al., 2007). In variants in which the C-terminus of Hyal1 is alternatively spliced, the EGF-like domain is completely conserved. Hyal1 has 35.1% identity with the amino acid sequence of PH20, and the protein tertiary structure of PH20 has not been found yet.

[0010] In the study of the structure / function relationship of human PH20, it has been found that the C-terminal region of PH20 is important for protein expression and enzyme activity, and specifically, it has been reported that truncating the C-terminus with amino acids 477 - 483 is important for the expression and activity of the enzyme (Frost, 2007). The activity of full-length PH20 (amino acids 1 - 509) or the pH20 variant with a truncated C-terminus at position 467 is only 10% or less of the activity of the pH20 variant with a truncated C-terminus at one site among positions 477 to 483 (Frost, 2007). Halozyme Therapeutics developed rHuPH20 (amino acids 36 - 482), which is a recombinant protein in which the C-terminus of mature PH20 is cleaved at Y482 (Bookbinder et al., 2006; Frost, 2007).

[0011] Meanwhile, although research is underway to develop various therapeutic drugs using the subcutaneous injection form of human PH20, the problem of the low stability of human PH20 itself remains unresolved.

[0012] Against this technical background, the inventors of the present disclosure have confirmed that human PH20 variants (including one or more amino acid residue substitutions in the α-helix 8 region (S347 to C381) and the linker region (A333 to R346) between α-helix 7 and α-helix 8 in the amino acid sequence of wild-type hyaluronidase PH20, and in which some amino acids located at the N-terminus and / or C-terminus of PH20 are cleaved) have high enzyme activity and thermal stability, and thus submitted a patent application (PCT / KR 2019 / 009215) for this.

[0013] The inventors of the present application have also confirmed that the PH20 variants according to the present disclosure can be applied to pharmaceutical compositions or formulations, which contain a drug (such as an antibody drug, especially a high-dose anti-HER2 antibody or an immune checkpoint antibody), and thus the pharmaceutical compositions and formulations according to the present disclosure (containing PH20 variants and drugs, such as anti-HER2 antibodies or immune checkpoint antibodies) can be used for subcutaneous injection, and the activities of the drug (such as an antibody drug) and the PH20 variant are very stable and can be maintained for a long time, thus completing the present disclosure. Summary of the Invention

[0014] Technical Problem

[0015] Therefore, the present disclosure is directed to the above problems, and an object of the present disclosure is to provide a novel pharmaceutical composition, which contains a PH20 variant and a drug with improved enzyme activity and thermal stability, wherein the thermal stability and activity of the drug and the PH20 variant can be maintained for a long time; especially a pharmaceutical composition that can be used for subcutaneous injection.

[0016] Another object of the present disclosure is to provide a method for treating a disease, which includes administering the pharmaceutical composition according to the present disclosure to a subject in need of treatment.

[0017] Technical Solution

[0018] According to the present disclosure, the above and other objects can be achieved by providing a pharmaceutical composition, which contains (a) a drug and (b) a PH20 variant.

[0019] The PH20 variant contained in the pharmaceutical composition according to the present disclosure may contain one or more amino acid residue substitutions selected from S343E, M345T, K349E, L353A, L354I, N356E, and I361T in the wild-type human PH20 having the amino acid sequence of SEQ ID NO: 1; and may further contain one or more amino acid residue substitutions in one or more regions selected from the α-helix 8 region (S347 to C381) and / or the linker region (A333 to R346) between α-helix 7 and α-helix 8, wherein some amino acid residues located at the N-terminus and / or C-terminus are selectively cleaved.

[0020] The pharmaceutical composition according to the present disclosure may further contain one or more selected from pharmaceutically acceptable additives, especially buffers, stabilizers, and surfactants.

[0021] The pharmaceutical composition according to the present disclosure can be used in the form of an injection formulation for subcutaneous injection. Brief Description of the Drawings

[0022] The above and other objects, features and other advantages of the present disclosure will be more clearly understood from the following detailed description in conjunction with the accompanying drawings, in which:

[0023] Figure 1 : A shows the chromatogram of size exclusion chromatography in the stability test of trastuzumab under severe conditions at 45 °C, and B shows the change in the monomer protein purity of trastuzumab according to the formulation in the stability test under severe conditions at 45 °C;

[0024] Figure 2 Shows the results of measuring the protein aggregation temperature of a formulation containing trastuzumab and the novel PH20 variant HP46;

[0025] Figure 3 : A is the weak cation exchange (WCX) chromatogram of trastuzumab in the stability test under severe conditions at 45 °C, B shows the change in the relative amount (%) of acidic variants in the formulation in the stability test under severe conditions at 45 °C, C shows the change in the relative amount (%) of the main peak of the formulation in the stability test under severe conditions at 45 °C, and D shows the change in the relative amount (%) of basic variants in the formulation in the stability test under severe conditions at 45 °C;

[0026] Figure 4 Shows the change in the monomer protein purity of trastuzumab in formulations 5-7 in the stability test under severe conditions at 45 °C;

[0027] Figure 5 : A shows the change in the relative amount (%) of acidic variants in formulations 5-7 in the stability test under severe conditions at 45 °C, B shows the change in the relative amount (%) of the main peak according to formulations 5, 6 and 7 in the stability test under severe conditions at 45 °C, and C shows the change in the relative amount (%) of basic variants according to formulations 5, 6 and 7 in the stability test under severe conditions at 45 °C;

[0028] Figure 6 : A shows the results of measuring the residual enzyme activity of Herceptin subcutaneous injection formulation (Herceptin SC), trastuzumab + wild-type PH20 (HW2) and trastuzumab + PH20 variant HP46 on day 0 and day 1 in the stability test under severe conditions at 40 °C, and B shows the results of measuring the residual enzyme activity of Herceptin subcutaneous injection formulation, trastuzumab + wild-type PH20 (HW2) and trastuzumab + PH20 variant HP46 on day 0 and day 1 in the stability test under severe conditions at 45 °C;

[0029] Figure 7Shows the results of size exclusion chromatography analysis of Formulations 8 - 10 in a stability test lasting 14 days under severe conditions at 40°C;

[0030] Figure 8 : A shows the results of measuring the change in protein particle size of Formulations 8 - 10 using a DLS device, and B shows the results of measuring the protein aggregation temperature;

[0031] Figure 9A Shows the weak cation exchange (WCX) chromatogram of Formulation 8 in a stability test under severe conditions at 40°C, Figure 9B Shows the change (%) in the relative amount of acidic variants in Formulations 8 - 10 in a stability test under severe conditions at 40°C, Figure 9C Shows the change (%) in the relative amount of the main peak of Formulations 8 - 10 in a stability test under severe conditions at 40°C, and Figure 9D Shows the change (%) in the relative amount of basic variants in Formulations 8 - 10 in a stability test under severe conditions at 40°C;

[0032] Figure 10 Shows the change (%) in the relative enzyme activity of Formulations 8 - 10 in a stability test under severe conditions at 40°C;

[0033] Figure 11 Shows the change in trastuzumab monomer purity of Formulations 11 - 13 in a stability test under severe conditions at 40°C,

[0034] Figure 12A Shows the weak cation exchange (WCX) chromatogram of Formulation 11 in a stability test under severe conditions at 40°C, Figure 12B Shows the change (%) in the relative amount of acidic variants in Formulations 11 - 13 in a stability test under severe conditions at 40°C, Figure 12C Shows the change (%) in the relative amount of the main peak of Formulations 11 - 13 in a stability test under severe conditions at 40°C, and Figure 12D Shows the change (%) in the relative amount of basic variants in Formulations 11 - 13 in a stability test under severe conditions at 40°C;

[0035] Figure 13 Shows the change (%) in the relative enzyme activity of Formulations 11 - 13 in a stability test under severe conditions at 40°C;

[0036] Figure 14 Shows the change in rituximab monomer purity of Formulations 14 - 16 in a stability test under severe conditions at 40°C,

[0037] Figure 15Shows the change in relative enzyme activity of Formulations 14 - 16 in a stability test under the severe condition of 40°C;

[0038] Figure 16 Shows the change in relative enzyme activity of Formulations 17 and 18 in a stability test under the severe condition of 40°C;

[0039] Figure 17 Shows the results of size - exclusion chromatography analysis of Formulations 19 - 22 at 40°C;

[0040] Figure 18 Shows the change in relative enzyme activity of Formulations 19 - 22 in a stability test under the severe condition of 40°C;

[0041] Figure 19 Shows the change in enzyme activity caused by the pH change of recombinant human PH20 and HP46; and

[0042] Figure 20 Shows the experimental results of the pharmacokinetics of Herceptin subcutaneous injection product (Herceptin SC) and Herceptin subcutaneous injection biosimilar candidate (trastuzumab + HP46; Herceptin SC BS) in 9 - week - old Sprague - Dawley rats, where Herceptin and Herceptin biosimilar candidate were each injected at 18 mg / kg, and the subcutaneous injection formulation contained 100 units of rHuPH20 and 100 units of HP46 (at pH 5.3). Detailed Description

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the terms used herein are well - known and commonly used in the art.

[0044] Embodiments of the present disclosure relate to a pharmaceutical composition comprising (a) a drug and (b) a PH20 variant, and the pharmaceutical composition according to the present disclosure can be used for the prevention or treatment of diseases, and preferably for subcutaneous injection.

[0045] The human PH20 variant contained in the pharmaceutical composition according to the present disclosure has amino acid residue substitutions in some of the following regions, which correspond to the α-helical regions and / or their linker regions in the amino acid sequence of wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), preferably mature wild-type PH20 (having the sequence consisting of L36 to S490 in the amino acid sequence of SEQ ID NO: 1), preferably α-helix 8 region (S347 to C381) and / or the linker region between α-helix 7 and α-helix 8 (A333 to R346), more preferably the amino acid region from T341 to N363, and most preferably T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361 or M345 to N363.

[0046] In the present disclosure, "mature wild-type PH20" refers to a protein containing the following amino acid residues: L36 to S490 of the amino acid residues of SEQ ID NO: 1, which lacks M1 to T35 that form the signal peptide; and A491 to L509 in the amino acid sequence of wild-type PH20 having the sequence of SEQ ID NO: 1, which is not related to the substantial function of PH20.

[0047] Table 1. Amino acid sequence of wild-type PH20 (SEQ ID NO: 1)

[0048]

[0049] Specifically, the PH20 variant or its fragment contained in the pharmaceutical composition according to the present disclosure contains one or more mutations in wild-type PH20 having the sequence of SEQ ID NO: 1, preferably amino acid residue substitutions selected from S343E, M345T, K349E, L353A, L354I, N356E and I361T, and most preferably one or more amino acid residue substitutions selected from L354I and N356E.

[0050] In the present disclosure, the term "PH20 variant" is intended to include mutations of some amino acid residues in the sequence of wild-type human PH20, preferably amino acid residue substitutions; and deletions of some amino acid residues at the N-terminus and / or C-terminus together with such substitutions of amino acid residues, and is used in substantially the same meaning as the expression "PH20 variant or its fragment".

[0051] The inventors of the present disclosure have confirmed that novel PH20 variants or fragments thereof with improved enzymatic activity and thermal stability compared to wild-type PH20 can be provided based on previous studies with experimental results in which, when the amino acid sequences of the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 of human PH20 are partially replaced with the amino acid sequences of the α-helix 8 region and the linker region between α-helix 7 and α-helix 8 of Hyal1 with high hydrophilicity, the enzymatic activity at neutral pH and the protein aggregation temperature (Tagg) increase.

[0052] Thus, the PH20 variant contained in the pharmaceutical composition according to the present disclosure contains one or more amino acid residue substitutions selected from S343E, M345T, K349E, L353A, L354I, N356E, and I361T in the amino acid sequence of wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), preferably mature wild-type PH20 (having the sequence consisting of L36 to S490 in the amino acid sequence of SEQ ID NO: 1), preferably one or more amino acid residue substitutions selected from L354I and N356E,

[0053] wherein one or more amino acid residues are substituted in the region corresponding to the α-helix region and / or its linker region, preferably in the α-helix 8 region (S347 to C381) and / or in the linker region between α-helix 7 and α-helix 8 (A333 to R346), more preferably in the amino acid region corresponding to T341 to N363, T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361, or M345 to N363.

[0054] In particular, in the PH20 variant contained in the pharmaceutical composition according to the present disclosure, some amino acid residues in the α-helix 8 region (S347 to C381) and / or in the linker region between α-helix 7 and α-helix 8 (A333 to R346) of wild-type PH20, preferably mature wild-type PH20, may be substituted with the amino acid sequences of the corresponding regions of Hyal1 having the sequence SEQ ID NO: 51 (see Tables 2 and 3), but the present disclosure is not limited thereto.

[0055] Table 2. Amino acid sequence of wild-type Hyal1 (SEQ ID NO: 51)

[0056]

[0057] Table 3. Comparison between the α-helices and amino acid sequences of PH20 and Hyal1

[0058]

[0059] More specifically, the PH20 variant or fragment thereof contained in the pharmaceutical composition according to the present disclosure preferably contains amino acid residue substitutions of L354I and / or N356E in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20,

[0060] and preferably further contains amino acid residue substitutions at one or more positions selected from T341 to N363, particularly at one or more positions selected from T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361, and N363, but the present disclosure is not limited thereto, and

[0061] more preferably further contains one or more amino acid residue substitutions selected from T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T, and N363G, but the present disclosure is not limited thereto.

[0062] Preferably, the PH20 variant or fragment thereof contained in the pharmaceutical composition according to the present disclosure may contain amino acid residue substitutions selected from M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T,

[0063] and may further contain one or more amino acid residue substitutions selected from T341S, L342W, S343E, I344N, and N363G, but the present disclosure is not limited thereto.

[0064] More preferably, the PH20 variant or fragment thereof contained in the pharmaceutical composition according to the present disclosure may contain, but is not limited to, any one substitution selected from the following groups:

[0065] (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0066] (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0067] (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0068] (d) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G;

[0069] (e) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; and

[0070] (f) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.

[0071] In the present disclosure, expressions such as "S347" described with a one-letter amino acid residue code together with a number mean the amino acid residue at the corresponding position in the amino acid sequence of SEQ ID NO: 1.

[0072] For example, "S347" means that the amino acid residue at position 347 in the amino acid sequence of SEQ ID NO: 1 is serine. Additionally, "S347T" means that the serine at position 347 in SEQ ID NO: 1 is replaced by threonine.

[0073] The PH20 variants included in the pharmaceutical compositions according to the present disclosure are interpreted to include variants in which the amino acid residues at specific amino acid residue positions are conservatively substituted.

[0074] As used herein, the term "conservative substitution" refers to a modification of a PH20 variant that involves the replacement of one or more amino acids with amino acids having similar biochemical properties, and the latter amino acids do not cause the loss of the biological or biochemical function of the corresponding PH20 variant.

[0075] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined and are well known in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), amino acids having acidic side chains (e.g., aspartic acid and glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0076] It is expected that the PH20 variants included in the pharmaceutical compositions according to the present disclosure will retain their activity despite having conservative amino acid substitutions.

[0077] In addition, the PH20 variants or fragments thereof included in the pharmaceutical compositions according to the present disclosure are interpreted to include PH20 variants or fragments thereof having functions and / or actions that are substantially the same as the functions and / or actions of the PH20 variants or fragments thereof according to the present disclosure, and having at least 80% or 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% amino acid sequence homology with the PH20 variants or fragments thereof according to the present disclosure.

[0078] Compared with mature wild-type PH20, the PH20 variants according to the present disclosure have increased expression levels and increased protein refolding rates in animal cells, and thus have improved thermal stability. In addition, despite the improved thermal stability, the enzymatic activity of the PH20 variants exceeds or is similar to the enzymatic activity of mature wild-type PH20.

[0079] Meanwhile, it is known that when some amino acids (such as S490) at the C-terminus of mature wild-type PH20 are additionally cleaved, the enzymatic activity decreases, but compared with mature wild-type PH20, the PH20 variants according to the present disclosure show improved thermal stability and increased or similar enzymatic activity even when the C-terminus of mature wild-type PH20 has an additionally cleaved sequence. In addition, when up to five amino acid residues are cleaved from the N-terminal amino acids, the PH20 variants maintain their enzymatic activity, indicating that the residues starting from P41 at the N-terminus play an important role in protein expression and enzymatic activity.

[0080] Thus, the PH20 variant comprised in the pharmaceutical composition according to the present disclosure comprises some amino acid residue substitutions in the α-helix 8 region (S347 to C381) of wild-type PH20 and / or in the linker region (A333 to R346) between α-helix 7 and α-helix 8, and further comprises some amino acid residue deletions at the C-terminus and / or N-terminus, but the present disclosure is not limited thereto.

[0081] In one embodiment, the PH20 variant comprised in the pharmaceutical composition according to the present disclosure may comprise some amino acid residue deletions at the N-terminus caused by cleavage before the amino acid residues M1 to P42 at the N-terminus of the amino acid sequence selected from SEQ ID NO: 1, preferably before the amino acid residues L36, N37, F38, R39, A40, P41 or P42; and / or some amino acid residue deletions at the C-terminus caused by cleavage after the amino acid residues V455 to W509 at the C-terminus, preferably after the amino acid residues selected from V455 to S490, and most preferably after the amino acid residues V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490.

[0082] The expression "cleavage before L36, N37, F38, R39, A40, P41 or P42 at the N-terminus" means respectively cleavage and removal of all amino acid residues from M1 to T35 immediately preceding L36, all amino acid residues from M1 to L36 immediately preceding N37, all amino acid residues from M1 to N37 immediately preceding F38, all amino acid residues from M1 to F38 immediately preceding R39, all amino acid residues from M1 to R39 immediately preceding A40, all amino acid residues from M1 to A40 immediately preceding P41, and all amino acid residues from M1 to P41 immediately preceding P42 in the amino acid sequence of SEQ ID NO: 1. The expression "cleavage before M1 at the N-terminus of SEQ ID NO: 1" means that no cleavage occurs at the N-terminus.

[0083] Additionally, the expression "cleavage after V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 at the C-terminus" means cleavage and removal of the amino acid residues after V455, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 in the SEQ ID NO: 1 sequence, respectively. For example, cleavage after S490 means cleavage between S490 and A491.

[0084] Preferably, the α-human PH20 variant contained in the pharmaceutical composition according to the present disclosure may have an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 5 to 50, more preferably having the amino acid sequence of SEQ ID NO: 44, but the present disclosure is not limited thereto. In the PH20 variants constructed in specific embodiments according to the present disclosure, the substituted or cleaved amino acid sequences are shown in Table 4 below.

[0085] Table 4. Amino acid sequences of PH20 variants according to the present disclosure and their substitution / cleavage characteristics

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Meanwhile, previous studies reported that the enzymatic activity of wild-type PH20 varies according to the cleavage position of the amino acid residues located at the C-terminus. However, in the present disclosure, the specific α-helix forming the secondary structure of PH20 is replaced by the α-helices of other human hyaluronidases, thereby constructing PH20 variants with higher stability than wild-type PH20. Among these variants, the interaction between the substituted α-helical domain of PH20 and other secondary structures shows a pattern different from that of wild-type PH20. Therefore, regardless of the cleavage position at the C-terminus, the variants have a certain or higher level of enzymatic activity.

[0103] In addition, in the present disclosure, an attempt was made to increase the expression of recombinant PH20 protein by using the signal peptides of other proteins that exhibit high protein expression levels in animal cells instead of the original signal peptide of human PH20.

[0104] Thus, in another embodiment, the PH20 variant contained in the pharmaceutical composition according to the present disclosure may contain, at its N-terminus, a signal peptide derived from human hyaluronidase-1 (Hyal1), human growth hormone, or human serum albumin instead of the signal peptide of wild-type PH20 consisting of M1 to T35, and preferably may contain a signal peptide derived from human growth hormone having the amino acid sequence MATGSRTSLLLAFGLLCLPWLQEGSA according to SEQ ID NO: 2, a signal peptide derived from human serum albumin having the amino acid sequence MKWVTFISLLFLFSSAYS according to SEQ ID NO: 3, or a signal peptide derived from human Hyal1 having the amino acid sequence MAAHLLPICALFLTLLDMAOG according to SEQ ID NO: 4, but the present disclosure is not limited thereto.

[0105] Table 5. Amino acid sequences of signal peptides of human growth hormone, human serum albumin, or human Hyal1

[0106]

[0107] Among the PH20 variants included in the pharmaceutical composition according to the present disclosure, the variant having a 6xHis tag attached to the C-terminus is referred to as HM, while the variant without the 6xHis tag is referred to as HP. Additionally, the mature wild-type PH20 (L36-S490) having a 6xHis tag attached to its C-terminus is referred to as WT, while the mature wild-type PH20 (L36 to Y482) without the 6xHis tag and with the C-terminus cleaved after Y482 is referred to as HW2.

[0108] HP46 (SEQ ID NO: 44) is a human PH20 variant obtained by the following method: modeling the protein structure using Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) having a known protein tertiary structure, which is a human hyaluronidase, then replacing the amino acid sequence of α-helix 8 and the amino acid sequence of the linker region between α-helix 7 and α-helix 8 with the amino acid sequence of Hyal1, and subjecting the N-terminus to cleavage at F38 and the C-terminus to cleavage after F468. In particular, α-helix 8 is located outside the protein tertiary structure of PH20 and has fewer interactions with adjacent α-helices or β-strands compared to other α-helices. Generally, there is a trade-off relationship between enzyme activity and thermal stability, so the higher the thermal stability of a protein, the lower the enzyme activity, and when the enzyme activity increases due to an increase in the flexibility of the protein structure, the thermal stability tends to decrease. However, the specific activity of HP46 measured by turbidimetry at pH 7.0 is approximately 46 units / μg, which is evaluated to be approximately twice the specific activity of wild-type PH20, which is approximately 23 units / μg.

[0109] The thermal stability of a protein can be evaluated based on the melting temperature Tm (at which 50% of the protein tertiary structure denatures) and the aggregation temperature Tagg (at which aggregation occurs between proteins). Generally, the aggregation temperature of a protein tends to be lower than its melting temperature. The α-helix 8 of Hyal1 is more hydrophilic than the α-helix 8 of PH20. The substituted α-helix 8 of Hyal1 increases the protein surface hydrophilicity of HP46, thereby causing a delayed effect of aggregation between proteins due to hydrophobic interactions. Thus, the aggregation temperature is 51 °C, and it is observed that this is 4.5 °C higher than the aggregation temperature of wild-type PH20 (46.5 °C).

[0110] HP46 is a variant in which the α-helix 8 and the amino acid residues in the linker region between α-helix 7 and α-helix 8 are substituted, and in which T341 is substituted with serine. When the amino acid residue 341 is threonine, the enzyme activity is similar to that of wild-type PH20, but after substitution with serine, the enzyme activity is increased by about 2-fold, and it can be confirmed that even in the substrate gel assay, the hyaluronic acid hydrolyzed by the resulting variant is 5 to 6 times more than that of wild-type PH20. The substrate gel assay involves protein denaturation and refolding processes, which means that the refolding and recovery of the protein tertiary structure of HP46 are enhanced compared to wild-type PH20.

[0111] The amount of the PH20 variant in the pharmaceutical composition according to the present disclosure is at least 50 units / mL, preferably in the range of 100 units / mL to 20,000 units / mL, more preferably in the range of about 150 units / mL to about 18,000 units / mL, still more preferably in the range of 1,000 units / mL to 16,000 units / mL, and most preferably in the range of 1,500 units / mL to 12,000 units / mL.

[0112] Examples of the drugs included in the pharmaceutical composition according to the present disclosure include, but are not limited to, protein drugs, antibody drugs, small molecules, aptamers, RNAi, antisense, and cell therapeutics (such as chimeric antigen receptor (CAR)-T or CAR-natural killer (NK) cells), and not only currently commercially available drugs but also drugs in clinical trials or development can be used.

[0113] As the drug, a protein drug or an antibody drug can be preferably used.

[0114] The "protein drug" included in the pharmaceutical composition according to the present disclosure is a drug composed of amino acids and thus exhibits a therapeutic or prophylactic effect on diseases through the activity of the protein; is a drug composed of proteins other than antibody drugs; and can be selected from cytokines, therapeutic enzymes, hormones, soluble receptors and their fusion proteins, insulin or its analogs, bone morphogenetic protein (BMP), erythropoietin, and serum-derived proteins, but the present disclosure is not limited thereto.

[0115] The cytokines included in the pharmaceutical composition according to the present disclosure can be selected from interferons, interleukins, colony-stimulating factors (CSF), tumor necrosis factor (TNF), and tissue growth factor (TGF), but the present disclosure is not limited thereto.

[0116] The therapeutic enzymes can include, but are not limited to, β-glucocerebrosidase and agalsidase β.

[0117] The soluble receptor contained in the pharmaceutical composition according to the present disclosure is the extracellular domain of the receptor, and its fusion protein is a protein in which the Fc region of an antibody or the like is fused with the soluble receptor. The soluble receptor is a soluble form of the receptor that binds to a disease-related ligand, and examples thereof include a form in which the Fc region is fused with a soluble receptor for TNF-α (for example, a product containing the ingredient etanercept and forms similar thereto), a form in which the Fc region is fused with a soluble receptor for VEGF (a product containing the ingredient alefacept and forms similar thereto), a form in which the Fc region is fused with CTLA-4 (for example, a product containing the ingredient abatacept or belatacept and forms similar thereto), a form in which the Fc region is fused with a soluble receptor for interleukin 1 (for example, a product containing the ingredient rilonacept and forms similar thereto), and a form in which the Fc region is fused with a soluble receptor for LFA3 (for example, a product containing the ingredient alefacept and forms similar thereto), but the present disclosure is not limited thereto.

[0118] The hormone contained in the pharmaceutical composition according to the present disclosure refers to a hormone or its analogue that is injected into the body for treating or preventing diseases caused by hormone deficiency or the like, and examples of the hormone or its analogue include, but are not limited to, human growth hormone, estrogen, and progesterone.

[0119] The serum-derived protein contained in the pharmaceutical composition according to the present disclosure is a protein present in plasma, and includes both proteins extracted from plasma and recombinant proteins produced, and examples thereof may include, but are not limited to, fibrinogen, von Willebrand factor, albumin, thrombin, factor II (FII), factor V (FV), factor VII (FVII), factor IX (FIX), factor X (FX), and factor XI (FXI).

[0120] The antibody drug contained in the pharmaceutical composition according to the present disclosure may be a monoclonal antibody drug or a polyclonal antibody drug.

[0121] The monoclonal antibody drug according to the present disclosure is a protein containing a monoclonal antibody and a monoclonal antibody fragment that can specifically bind to an antigen related to a specific disease. The monoclonal antibody also includes bispecific antibodies, and the protein containing the monoclonal antibody or its fragment conceptually includes antibody-drug conjugates (ADCs).

[0122] Examples of antigens associated with a particular disease include 4-1BB, integrin, amyloid-β, angiopoietin (angiopoietin 1 or 2), angiopoietin-like 3, B-cell activating factor (BAFF), B7-H3, complement 5, CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, claudin-18, complement factor D, CTLA4, DLL3, EGF receptor, factor VIII, Fc receptor, FGF23, folate receptor, GD2, GM-CSF, HER2, HER3, interferon receptor, interferon γ, IgE, IGF-1 receptor, interleukin 1, interleukin 2 receptor, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 7, interleukin 12 / 23, interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin 31 receptor, interleukin 36 receptor, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, VEGF receptor, and von Willebrand factor (vWF), but the present disclosure is not limited thereto.

[0123] The following are, but not limited to, proteins, including monoclonal antibodies or monoclonal antibody fragments directed against antigens associated with a particular disease:

[0124] Umilumab as an anti-4-1BB antibody;

[0125] Natalizumab, etrolizumab, vedolizumab, and bezuclizumab as antibodies against integrin;

[0126] Bapineuzumab, crenezumab, solanezumab, aducanumab, and gantenerumab as antibodies against amyloid-β;

[0127] Antibodies against angiopoietin, such as AMG780 against angiopoietin 1 and 2, MEDI 3617 and nesvacumab against angiopoietin 2, and vanucizumab as a bispecific antibody against angiopoietin 2 and VEGF;

[0128] Evinacumab as an antibody against angiopoietin-like 3;

[0129] Tabalumab, lanalumab, and belimumab, which are antibodies against B-cell activating factor (BAFF);

[0130] Omburtamab, which is an antibody against B7-H3;

[0131] Ravulizumab and eculizumab, which are antibodies against complement 5;

[0132] Mogamulizumab, which is an antibody against CCR4;

[0133] Otelixizumab, teplizumab, and muromonab, which are antibodies against CD3; Tebentafusp, which is a bispecific antibody against GP100 and CD3; Blinatumomab, which is a bispecific antibody against CD19 and CD3; and REGN1979, which is a bispecific antibody against CD20 and CD3;

[0134] Ibalizumab and zanolimumab, which are antibodies against CD4;

[0135] Itolizumab, which is an antibody against CD6;

[0136] Efalizumab, which is an antibody against CD11a;

[0137] Inebilizumab, tafasitamab, and loncastuximab tesirine (the loncastuximab tesirine is an ADC), which are antibodies against CD19;

[0138] Ofatumumab, ublituximab, obinutuzumab, ofatumumab, rituximab, tositumomab, and ibritumomab tiuxetan (the ibritumomab tiuxetan is an ADC), which are antibodies against CD20;

[0139] Epratuzumab, inotuzumab ozogamicin (the inotuzumab ozogamicin is an ADC), and moxetumomab pasudotox as antibodies against CD22;

[0140] Brentuximab vedotin as an ADC against CD30;

[0141] Vadastuximab talirine and gemtuzumab ozogamicin as ADCs against CD33;

[0142] Daratumumab and isatuximab as antibodies against CD38;

[0143] Alemtuzumab as an antibody against CD52;

[0144] Crizanlizumab as an antibody against CD62;

[0145] Polatuzumab vedotin as an ADC against CD79b;

[0146] Galiximab as an antibody against CD80;

[0147] Eptinezumab, fremanezumab, galcanezumab, and erenumab as antibodies against CGRP;

[0148] Zolbetuximab as an antibody against claudin-18;

[0149] Lampalizumab as an antibody against complement factor D;

[0150] Tremelimumab, zalifrelimab, and ipilimumab as antibodies against CTLA4;

[0151] Rovalpituzumab tesirine as an ADC against DLL3;

[0152] Cetuximab, depatuxizumab, zalutumumab, necitumumab, and panitumumab, which are antibodies against the EGF receptor;

[0153] Emicizumab, which is a bispecific antibody against coagulation factor IX and factor X (which are hemophilia factors);

[0154] Nipocalimab and rozanolixizumab, which are antibodies against the Fc receptor;

[0155] Burosumab, which is an antibody against FGF23;

[0156] Farletuzumab, which is an antibody against the folate receptor, and mirvetuximab soravtansine, which is an ADC against the folate receptor;

[0157] Dinutuximab and naxitamab, which are antibodies against GD2;

[0158] Otelixizumab, which is an antibody against GM-CSF;

[0159] Margetuximab, pertuzumab, and trastuzumab, which are antibodies against HER2, and trastuzumab deruxtecan, trastuzumab emtansine, and trastuzumab duocarmazine, which are ADCs against HER2;

[0160] Patritumab, which is an antibody against HER3;

[0161] Anifrolumab, which is an antibody against the interferon receptor;

[0162] Emapalumab, which is an antibody against interferon γ;

[0163] Ligelizumab and omalizumab, which are antibodies against the IgE antibody;

[0164] Dalotuzumab, figitumumab, and teprotumumab, which are antibodies against the IGF-1 receptor;

[0165] Gebokizumab and canakinumab, which are antibodies against interleukin 1;

[0166] Daclizumab and basiliximab, which are antibodies against the interleukin 2 receptor;

[0167] Dupilumab, which is an antibody against the interleukin 4 receptor;

[0168] Mepolizumab and reslizumab, which are antibodies against interleukin 5;

[0169] Benralizumab, which is an antibody against the interleukin 5 receptor;

[0170] Clazakizumab, olokizumab, sirukumab, and siltuximab, which are antibodies against interleukin 6;

[0171] Sarilumab, satralizumab, tocilizumab, and REGN88, which are antibodies against the interleukin 6 receptor;

[0172] Secukinumab, which is an antibody against interleukin 7;

[0173] Ustekinumab and briakinumab, which are antibodies against interleukin 12 / 23;

[0174] Lebrikizumab and tralokinumab, which are antibodies against interleukin 13;

[0175] Ixekizumab and bimekizumab, which are antibodies against interleukin 17A;

[0176] Brodalumab, which is an antibody against the interleukin 17 receptor A;

[0177] Brazikumab, guselkumab, risankizumab, tildrakizumab, and mirikizumab, which are antibodies against interleukin 23;

[0178] Nemolizumab, an antibody against interleukin-31 receptor;

[0179] Spesolimab, an antibody against interleukin-36 receptor;

[0180] Relatlimab, an antibody against LAG3;

[0181] Narsoplimab, an antibody against NASP2;

[0182] Fasinumab and tanezumab, antibodies against NGF;

[0183] Alirocumab, evolocumab and bocozizumab, antibodies against PVSK9;

[0184] Pembrolizumab, balstilimab, camrelizumab, cemiplimab, dostarlimab, prolgolimab, shintilimab, spartalizumab, tislelizumab, pembrolizumab and nivolumab, antibodies against PD-1;

[0185] Atezolizumab, avelumab, envafolimab and durvalumab, antibodies against PD-L1, and bintrafusp α, a bispecific antibody against TGFβ and PD-L1;

[0186] Denosumab, an antibody against RANK-L;

[0187] Elotuzumab, an antibody against SLAMF7;

[0188] Concizumab and marstacimab, antibodies against tissue factor;

[0189] Antibodies against TNF, especially TNFα, including infliximab, adalimumab, golimumab, the antibody fragment certolizumab pegol and ozoralizumab (the ozoralizumab is a bispecific antibody against TNF and albumin);

[0190] Antibodies against VEGF, including brolucizumab, ranibizumab, bevacizumab, and faricimab (the faricimab is a bispecific antibody against VEGF and Ang2);

[0191] Ramucirumab as an antibody against the VEGF receptor; and

[0192] Caplacizumab as an antibody against vWF.

[0193] Meanwhile, overexpression of human epidermal growth factor receptor 2 (HER2) that promotes cell division is observed in approximately 20%-25% of breast cancer patients, and breast cancer with HER2 overexpression progresses rapidly, is invasive, and has a lower response to chemotherapy compared to breast cancer with low HER2 expression, so its prognosis is poor. Trastuzumab is a monoclonal antibody drug targeting HER2, which specifically binds to HER2 on the surface of cancer cells overexpressing HER2 to inhibit the signal transduction of cell replication and proliferation, thereby slowing tumor progression. Trastuzumab was approved by the US Food and Drug Administration (FDA) for the treatment of breast cancer in 1998 and by the Korean Food and Drug Administration (KFDA) for the treatment of breast cancer in 2003. Since then, the efficacy of trastuzumab has also been recognized in gastric cancer with overexpression of HER2, so it has been used as a therapeutic agent for gastric cancer.

[0194] The intravenous injection formulation of Roche's Herceptin (trade name: Herceptin) consists of 440 mg of trastuzumab as the main ingredient, and the lyophilized trastuzumab is mixed with normal saline and injected intravenously. On the other hand, the subcutaneous injection formulation of trastuzumab (trade name: Herceptin SC) is a 5 mL liquid formulation and contains 600 mg (120 mg / mL) of trastuzumab as the main ingredient, and contains 20 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, 0.04% polysorbate 20, and 10,000 units of rHuPH20 (2,000 units / mL, 0.004%, 40 μg / mL) as additives.

[0195] The shelf life of the subcutaneous injection formulation of Herceptin is 21 months. The intravenous injection formulation of trastuzumab is in lyophilized form and has a shelf life of 30 months, but the subcutaneous injection formulation of trastuzumab is in liquid form and has a short shelf life of 21 months. For this reason, it can be estimated that the stability of one or more of trastuzumab and recombinant human hyaluronidase PH20 in the liquid formulation is limited.

[0196] In this case, in the present disclosure, in view of the characteristics of the PH20 variant according to the present disclosure, wherein compared with the wild-type human hyaluronidase PH20 obtained from Halozyme and recombinant human PH20, the PH20 variant not only has increased enzyme activity but also has a measured high protein aggregation temperature, thus exhibiting improved thermal stability, the shelf life of the subcutaneous injection formulation is set to be long term, preferably 21 months or longer.

[0197] The content of the antibody drug in the pharmaceutical composition according to the present disclosure may be in the range of: 5 mg / mL to 500 mg / mL, preferably 20 mg / mL to 200 mg / mL, more preferably 100 mg / mL to 150 mg / mL, and most preferably 120 ± 18 mg / mL, for example, about 110 mg / mL, about 120 mg / mL or about 130 mg / mL.

[0198] The polyclonal antibody contained in the pharmaceutical composition according to the present disclosure is preferably a serum antibody extracted from serum, such as an immunoglobulin, but is not limited thereto.

[0199] In the case of small molecule compounds, any drug that requires a rapid prophylactic or therapeutic effect can be used without limitation. For example, morphine-based painkillers can be used (Thomas et al., 2009). Additionally, when used as a therapeutic agent against tissue necrosis caused by anticancer drugs, the small molecule compound can be used alone or in combination with antidotes such as vinca alkaloids and taxanes (Kreidieh et al., 2016).

[0200] The pharmaceutical composition according to the present disclosure may further comprise one or more selected from buffers, stabilizers and surfactants.

[0201] The buffer contained in the composition according to the present disclosure can be used without limitation as long as it can achieve a pH of 4 to 8, preferably 5 to 7, and the buffer is preferably one or more selected from the following: malate, formate, citrate, acetate, propionate, pyridine, piperazine, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris, bis-Tris, phosphate, ethanolamine, carbonate, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), imidazole, BIS-TRIS propane, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)propanesulfonic acid) (MOPS), N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), pyrophosphate and triethanolamine, more preferably a histidine buffer, such as L-histidine / HCl, but is not limited thereto.

[0202] The concentration of the buffer can be in the following ranges: 0.001 mM to 200 mM, preferably 1 mM to 50 mM, more preferably 5 mM to 40 mM, and most preferably 10 mM to 30 mM.

[0203] The stabilizers in the compositions according to the present disclosure can be used without limitation as long as they are commonly used in the art for the purpose of stabilizing proteins, and preferably, the stabilizers can be one or more selected from the following: carbohydrates, sugars or their hydrates, sugar alcohols or their hydrates, and amino acids.

[0204] The carbohydrates, sugars or sugar alcohols used as stabilizers can be one or more selected from the following: trehalose or its hydrate, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabinitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, heptitol, isomaltulose, maltitol, polydextrose, cyclodextrin, hydroxypropyl cyclodextrin, and glucose, but are not limited thereto.

[0205] The amino acids can be one or more selected from the following: glutamine, glutamate, glycine, lysine, lysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartate, ornithine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine, and alanine, but are not limited thereto.

[0206] The concentration of the sugar or sugar alcohol used as a stabilizer in the pharmaceutical composition according to the present disclosure can be in the following ranges: 0.001 mM to 500 mM, preferably 100 mM to 300 mM, more preferably 150 mM to 250 mM, and most preferably 180 mM to 230 mM, and particularly can be about 210 mM.

[0207] In addition, the concentration of the amino acid used as a stabilizer in the pharmaceutical composition according to the present disclosure can be in the following ranges: 1 mM to 100 mM, preferably 3 mM to 30 mM, more preferably 5 mM to 25 mM, and most preferably 7 mM to 20 mM, and particularly can be in the range of 8 mM to 15 mM.

[0208] The composition according to the present invention may further comprise a surfactant.

[0209] Preferably, the surfactant can be a nonionic surfactant such as polyoxyethylene sorbitan fatty acid esters (polysorbates or Tweens), polyethylene-polypropylene glycols, polyoxyethylene stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ethers [Triton-X], and polyoxyethylene-polypropylene copolymers [poloxamers and pluronics] and sodium dodecyl sulfate (SDS), but not limited thereto.

[0210] More preferably, polysorbate can be used. The polysorbate can be polysorbate 20 or polysorbate 80, but not limited thereto.

[0211] The concentration of the nonionic surfactant in the pharmaceutical composition according to the present disclosure can be in the range of: 0.0000001% (w / v) to 0.5% (w / v), preferably 0.000001% (w / v) to 0.4% (w / v), more preferably 0.00001% (w / v) to 0.3% (w / v), and most preferably 0.001% (w / v) to 0.2% (w / v).

[0212] In one embodiment, the pharmaceutical composition according to the present disclosure can comprise 50 - 350 mg / mL of an antibody (e.g., an anti-HER2 antibody or an immune checkpoint antibody), a histidine buffer (providing a pH of 5.5 ± 2.0), 10 - 400 mM α,α-trehalose, 1 - 50 mM methionine, and 0.0000001% (w / v) to 0.5% (w / v) of polysorbate.

[0213] In a more specific embodiment, the pharmaceutical composition according to the present disclosure can comprise 120 mg / mL of an anti-HER2 antibody or an immune checkpoint antibody, 20 mM histidine buffer (providing a pH of 5.5 ± 2.0), 210 mM α,α-trehalose, 10 mM methionine, and 2,000 units / mL of a PH20 variant, and can further comprise 0.005% (w / v) to 0.1% (w / v) of polysorbate.

[0214] The pharmaceutical composition according to the present disclosure can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intralung administration, rectal administration, etc., and subcutaneous administration is preferably carried out by subcutaneous injection, and more preferably the pharmaceutical composition is used as an injection formulation for subcutaneous injection.

[0215] Therefore, another embodiment of the present disclosure provides a formulation comprising the pharmaceutical composition according to the present disclosure, preferably an injection formulation for subcutaneous injection.

[0216] An injectable formulation for subcutaneous injection can be provided in an immediate injection form without an additional dilution process and can be provided after being contained in a pre-filled syringe, a glass ampoule, or a plastic container.

[0217] This disclosure also relates to a method of treating a disease using a pharmaceutical composition or formulation according to this disclosure.

[0218] There is no particular limitation on the disease that can be treated using the pharmaceutical composition or formulation according to this disclosure, and there is no limitation as long as it is a disease that can be treated with a drug in combination with a PH20 variant according to this disclosure.

[0219] The disease that can be treated using the pharmaceutical composition or formulation according to this disclosure can be cancer or an autoimmune disease, but is not limited thereto.

[0220] There is no particular limitation on the cancer or carcinoma that can be treated with the pharmaceutical composition or formulation according to this disclosure, and it includes solid cancers and blood cancers. Examples of such cancers include skin cancer (such as melanoma), liver cancer, hepatocellular carcinoma, gastric cancer, breast cancer, lung cancer, ovarian cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colorectal cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, kidney cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma, but is not limited thereto. Preferably, the cancer that can be treated with the pharmaceutical composition or formulation according to this disclosure can be selected from gastric cancer, colorectal cancer, breast cancer, lung cancer, and kidney cancer, but is not limited thereto.

[0221] The autoimmune diseases that can be treated with the pharmaceutical composition or formulation according to this disclosure include rheumatoid arthritis, asthma, psoriasis, multiple sclerosis, allergic rhinitis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, type I diabetes, inflammatory bowel disease (IBD), and atopic dermatitis, but are not limited thereto.

[0222] This disclosure also provides a method of treating a disease, which includes administering to a subject in need of treatment a pharmaceutical composition or formulation according to this disclosure, and this disclosure further provides the use of the pharmaceutical composition or formulation according to this disclosure for treating a disease.

[0223] Unless otherwise defined herein, the technical terms and scientific terms used in this disclosure have the meanings commonly understood by those of ordinary skill in the art. Additionally, repetitive descriptions of technical configurations and operations identical to those of the related art will be omitted.

[0224] In the following, the present disclosure will be described in further detail with reference to the following embodiments. These embodiments are provided for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that these embodiments should not be construed as limiting the scope of the present disclosure.

[0225] Example

[0226] Example 1. Formulation Development

[0227] As shown in Table 6, four subcutaneous injection formulations of trastuzumab were prepared. Formulations 1 to 4 generally contained 120 mg / mL trastuzumab and consisted of 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and the PH20 variant. The difference between Formulations 1-4 was the concentration of the non-ionic surfactant, where Formulation 1: 0% polysorbate 20, Formulation 2: 0.005% polysorbate 20, Formulation 3: 0.04% polysorbate 20, and Formulation 4: 0.1% polysorbate 20.

[0228] Table 6. Formulation Composition

[0229]

[0230] Example 2. Measurement Using a Spectrophotometer

[0231] Formulations 1 to 4 were placed at 45 °C for 14 days, and the change in protein concentration was analyzed using a spectrophotometer manufactured by Beckman. Each sample was diluted with distilled water so that the concentration of the sample was 0.4 mg / mL, and then the absorbance of the protein at 280 nm was measured using a spectrophotometer. In the stability test under severe conditions, i.e., at 45 °C for 14 days, the protein concentrations of Formulations 1 to 4 did not change significantly. However, the activity of hyaluronidase decreased rapidly at 45 °C, and thus, the enzyme activity was not measured in this example (see Figure 6 ).

[0232] Example 3. Studying the Monomer Ratio of Trastuzumab in Each Formulation Using Size Exclusion Chromatography

[0233] For size-exclusion chromatography analysis, an HPLC system purchased from Shimadzu Prominence, TSK-gel G3000SWXL (7.8 X 300 mm, 5 μm), and a TSK guard column (6.0 x 4.0 mm, 7 μm) were used. 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. Analysis was carried out by applying an isocratic separation mode at a flow rate of 0.5 mL / min for 35 minutes. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, and after injecting 20 μL into the HPLC column, the absorbance of the column eluate was recorded at 280 nm. The monomer ratio of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0234] When size-exclusion chromatography analysis was carried out in a stability test under severe conditions, i.e., at 45 °C for 14 days, Formulations 1 to 4 showed a similar pattern of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (about 1.5%), and there were no significant differences according to the formulation. In summary, as a result of size-exclusion chromatography analysis in a stability test under severe conditions (i.e., at 45 °C), there were no significant differences in the stability characteristics between the formulations according to the concentration of polysorbate 20 (0 - 0.1% (w / v)) (see Figure 1 ).

[0235] Example 4. Measurement of the protein aggregation temperature of formulations containing trastuzumab and HP46

[0236] Dynamic light scattering (DLS) was used to analyze the denaturation characteristics of proteins attributed to heat. In this experiment, the change in protein molecular size according to temperature change was measured and used to calculate the protein aggregation temperature. For DLS analysis, a Zetasizer-nano-ZS instrument purchased from Malvern and a quartz cuvette (ZEN2112) were used. During the analysis, the temperature was raised from 25 °C to 85 °C at 1 °C intervals, and the sample was diluted to 1 mg / mL with each formulation buffer, and then 150 μL of the sample was added to the cuvette for analysis.

[0237] The aggregation temperature in Formulation 1 without polysorbate 20 was 74 °C, and the aggregation temperatures in Formulations 2 to 4 were 76 °C (see Figure 2 ).

[0238] Example 5. WCX chromatography measurement of formulations containing trastuzumab and HP46

[0239] For WCX chromatographic analysis, an HPLC system commercially available from Shimadzu Prominence was used, and columns such as TSKgel CM-STAT column (4.6 x 100 mm, 7 μm) and TSKgel guard gel CMSTAT (3.2 mm inner diameter x 1.5 cm) were used. Mobile phase A was 10 mM sodium phosphate (pH 7.5), and mobile phase B was 10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. Analysis was carried out at a flow rate of 0.8 mL / min for 55 minutes under a linear concentration gradient of 0 - 30% mobile phase B. The sample was diluted with mobile phase A to a final concentration of 1.0 mg / mL, 80 μL of the sample was injected into the HPLC, and then the absorbance of the column eluate at 280 nm was recorded. The monomer ratio of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0240] When WCX analysis was carried out in a stability test under severe conditions, i.e., at 45°C for 14 days, Formulations 1 to 4 showed a similar pattern of change. Specific changes included an increase in the relative content of acidic variants (a change of approximately 30% over 14 days), a decrease in the relative content of the main peak (a change of approximately 44% over 14 days), and an increase in the relative content of basic variants (a change of approximately 15% over 14 days), and there were no significant differences according to the formulations. In summary, in WCX analysis in a stability test under severe conditions, i.e., at 45°C, the protein stability according to polysorbate 20 (0 - 0.1%) was similar (see Figure 3 ).

[0241] Example 6. Formulation Development

[0242] As described in Table 7, three types of subcutaneous injection formulations of trastuzumab were prepared. Formulations 5 to 7 generally contained 120 mg / mL trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and HP46. The difference between Formulations 5 - 7 was the composition of stabilizer 3: Formulation 5: 0.04% polysorbate 20, Formulation 6: 50 mM Lys-Lys, and Formulation 3: glycine.

[0243] Table 7. Formulation Composition

[0244]

[0245] Example 7. Measurement Using a Spectrophotometer

[0246] Formulations 5 to 7 were placed at 45 °C for 14 days, and the change in protein concentration was analyzed using a spectrophotometer manufactured by Beckman. Each sample was diluted with distilled water so that the concentration of the sample was 0.4 mg / mL, and then the absorbance of the protein at 280 nm was measured using a spectrophotometer. In the stability test under severe conditions, i.e., for 14 days at 45 °C, there was no significant change in the protein concentration of Formulations 5 to 7. However, the activity of hyaluronidase decreased rapidly at 45 °C, and thus, in this example, the enzyme activity was not measured (see Figure 6 ).

[0247] Example 8. Study of the monomer ratio of trastuzumab in each formulation using size exclusion chromatography

[0248] For size exclusion chromatography analysis, an HPLC system available from Shimadzu Prominence was used, and TSK-gel G3000SWXL (7.8 X 300 mm, 5 μm) and a TSK guard column (6.0 x 4.0 mm, 7 μm) were used as columns. 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. An isocratic separation mode was applied at a flow rate of 0.5 mL / min for 35 minutes. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, and after injecting 20 μL of the sample into the HPLC column, the absorbance at 280 nm was measured. The monomer ratio of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0249] When size exclusion chromatography analysis was performed in the stability test under severe conditions, i.e., for 14 days at 45 °C, Formulations 5 to 7 showed a similar pattern of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) impurities and a decrease in monomer content (about 1.5%), and there were no significant differences according to the formulations. In summary, as a result of size exclusion chromatography analysis in the stability test under severe conditions (i.e., at 45 °C), formulations containing 0.04% polysorbate 20, 50 mM Lys-Lys, and 50 mM glycine showed similar protein stability (see Figure 4 ).

[0250] Example 9. WCX chromatography analysis of formulations containing trastuzumab and HP46

[0251] For WCX chromatographic analysis, an HPLC system available from Shimadzu Prominence was used, and columns such as TSKgel CM-STAT (4.6×100 mm, 7 μm) and TSKgel guard gel CM-STAT (3.2 mm inner diameter x 1.5 cm) were used. Mobile phase A was 10 mM sodium phosphate (pH 7.5), and mobile phase B was 10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. Analysis was carried out for 55 minutes using a separation mode with a linear concentration gradient of 0 - 30% at a flow rate of 0.8 mL / min. The sample was diluted with mobile phase A to a final concentration of 1.0 mg / mL, 80 μL of the sample was injected into the HPLC, and then the absorbance at 280 nm was recorded. The monomer ratio of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0252] When WCX analysis was performed during a stability test under severe conditions, i.e., at 45 °C for 14 days, Formulations 5 to 7 showed a similar pattern of change. Specific changes included an increase in the relative content of acidic variants (a change of approximately 30% over 14 days), a decrease in the relative content of the main peak (a change of approximately 44% over 14 days), and an increase in the relative content of basic variants (a change of approximately 15% over 14 days), and there were no significant differences according to the formulations. In summary, as a result of WCX analysis during a stability test under severe conditions (i.e., at 45 °C), the formulations of 0.04% polysorbate 20, 50 mM Lys-Lys, and 50 mM glycine showed similar protein stability (see Figure 5 ).

[0253] Example 10. Evaluation of the stability of HP46 in subcutaneous injection formulations of trastuzumab and HP46 at temperatures of 40 °C and 45 °C

[0254] To evaluate the stability of HP46 in a subcutaneous injection formulation of trastuzumab, trastuzumab (120 mg / mL) and PH20 (2000 units / mL) were mixed. At this time, the buffer used contained 20 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, and 0.04% polysorbate 20. The enzyme activity of the control sample was measured on Day 0, and the experimental samples were placed at 40 °C or 45 °C for 1 day, and then the enzyme activity of each sample was measured.

[0255] The Herceptin subcutaneous injection formulations trastuzumab + HW2 and trastuzumab + HP46 were each placed at 40 °C for 1 day, and then the activity of hyaluronidase was measured. As a result, the activities were 51%, 47%, and 94% respectively in each case, indicating that HP46 has the greatest thermal stability at 40 °C (see Figure 6)。 Additionally, trastuzumab + HW2 and trastuzumab + HP46, the subcutaneous injection formulations of Herceptin, were placed at 45 °C for 1 day, and then the hyaluronidase activity was measured. As a result, the subcutaneous injection formulations of Herceptin and trastuzumab + HW2 had no enzyme activity, but the enzyme activity of trastuzumab + HP46 was retained (see Figure 6 ).

[0256] Example 11. Formulation Development

[0257] As shown in Table 8, three subcutaneous injection formulations of trastuzumab were prepared. Formulations 8 to 10 generally contained 120 mg / mL trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and PH20 variant. The difference between Formulations 8 - 10 was the concentration of the nonionic surfactant, where Formulation 8: 0% polysorbate 20, Formulation 9: 0.005% polysorbate 20, and Formulation 10: 0.04% polysorbate 20.

[0258] Table 8. Formulation Composition

[0259]

[0260] Example 12. Measurement Using a Spectrophotometer

[0261] Formulations 8 to 10 were placed at 40 °C for 14 days, and the change in protein concentration was analyzed using a spectrophotometer manufactured by Beckman. Each sample was diluted with distilled water so that the concentration of the sample was 0.4 mg / mL, and then the absorbance of the protein at 280 nm was measured using a spectrophotometer. In the stability test under severe conditions, that is, at 40 °C for 14 days, the protein concentrations of Formulations 8 to 10 did not change significantly.

[0262] Example 13. Studying the Monomer Ratio of Trastuzumab in Each Formulation Using Size Exclusion Chromatography

[0263] For size exclusion chromatography analysis, an HPLC system available from Shimadzu Prominence was used, and TSK-gel G3000SWXL (7.8X300 mm, 5 μm) and a TSK guard column (6.0x4.0 mm, 7 μm) were used as columns. 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. The analysis was performed by applying an isocratic separation mode at a flow rate of 0.5 mL / min for 35 minutes. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, and after injecting 20 μL of the sample into the HPLC column, the absorbance at 280 nm was measured. The monomer ratio of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0264] When size-exclusion chromatography analysis was performed under severe conditions, i.e., in a stability test at 40 °C for 14 days, Formulations 8 to 10 showed a similar pattern of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (about less than 1.0%), and there were no significant differences according to the formulations. In summary, as a result of size-exclusion chromatography analysis in a stability test under severe conditions (i.e., at 40 °C), there were no significant differences in the stability profiles between the formulations according to the concentration of polysorbate 20 (0 - 0.04%) (see Figure 7 ).

[0265] Example 14. Measurement of the protein aggregation temperature for formulations containing trastuzumab and HP46

[0266] Dynamic light scattering (DLS) was used to analyze the denaturation characteristics of proteins attributable to heat in the field of protein drugs. In this experiment, the change in the size of protein molecules according to temperature change was measured and used to calculate the protein aggregation temperature. For DLS analysis, a Zetasizer-nano-ZS instrument (purchasable from Malvern) and a quartz cuvette (ZEN2112) were used. During the analysis, the temperature was increased from 25 °C to 85 °C at 1 °C intervals, and the samples were diluted to 1 mg / mL using each formulation buffer, and then 150 μL of the sample was added to the cuvette for analysis.

[0267] The aggregation temperature of Formulation 8 without polysorbate 20 was 78.3 °C, that of Formulation 9 was 77.3 °C, and that of Formulation 10 was 77.7 °C. In Example 13, although polysorbate 20 was not contained, the monomer ratio of the protein did not show a change, and as a result of comparing the case without polysorbate 20 with the case containing polysorbate 20, it was confirmed that there was no difference in the aggregation between the proteins. These results indicate that a minimum amount of polysorbate 20 is not necessarily required for subcutaneous injection formulations of trastuzumab (see Figure 8 ).

[0268] Example 15. WCX chromatography analysis of formulations containing trastuzumab and HP46

[0269] For WCX chromatographic analysis, an HPLC system available from Shimadzu Prominence was used, and columns such as a TSKgel CM-STAT column (4.6 x 100 mm, 7 μm) and a TSKgel guard gel CM-STAT (3.2 mm inner diameter x 1.5 cm) were used. Mobile phase A was 10 mM sodium phosphate (pH 7.5), and mobile phase B was 10 mM sodium phosphate containing 0.1 M NaCl (pH 7.2). Analysis was carried out at a flow rate of 0.8 mL / min for 55 minutes under a linear concentration gradient of 0 - 30% mobile phase B. The sample was diluted with mobile phase A to a final concentration of 1.0 mg / mL, 80 μL of the sample was injected into the HPLC, and then the absorbance of the column eluate was recorded at 280 nm. The monomer ratio of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0270] When WCX analysis was carried out in a stability test under severe conditions, i.e., at 40 °C for 14 days, formulations 8 to 10 showed a similar pattern of change. Specific changes included an increase in the relative content of acidic variants (a change of approximately 10% over 14 days), a decrease in the relative content of the main peak (a change of approximately 40% over 14 days), and an increase in the relative content of basic variants (a change of approximately 300% over 14 days), and there were no significant differences according to the formulations. In summary, in WCX analysis in a stability test under severe conditions, i.e., at 40 °C, the protein stability according to polysorbate 20 (0 - 0.04%) was similar (see Figure 9).

[0271] Example 16. Measurement of the enzyme activity of a formulation containing trastuzumab and HP46

[0272] The turbidimetric assay for measuring enzyme activity is a method that measures the degree of aggregate formation of residual hyaluronic acid binding to acidified albumin (BSA) through absorbance. When hyaluronic acid is hydrolyzed by PH20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), a standardized product, was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL and prepared in each test tube. The purified PH20 variant samples were diluted to 100X, 300X, 600X, 1200X, and 2400X with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepared in each test tube. In a fresh test tube, the hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL to make the volume of each test tube 180 μL. 60 μL of the sample containing hyaluronidase was added to the diluted hyaluronic acid solution and mixed with it, and allowed to react at 37 °C for 45 minutes. After the reaction was completed, 50 μL of the reacted enzyme and 250 μL of the acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance at 600 nm was measured using a spectrophotometer.

[0273] As a result of the activity analysis in the stability test under severe conditions, that is, at 40 °C for 14 days, it was confirmed that the higher the concentration of polysorbate 20, the greater the decrease in activity over time (see Figure 10 ).

[0274] Example 17. Formulation Development

[0275] As shown in Table 9, three subcutaneous injection formulations of trastuzumab were prepared. Formulations 11 to 13 generally contained 120 mg / mL trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and PH20 variant. The difference between Formulations 11 - 13 was the concentration of the nonionic surfactant, where Formulation 11: 0% polysorbate 80, Formulation 12: 0.005% polysorbate 80, and Formulation 13: 0.04% polysorbate 80.

[0276] Table 9. Formulation Composition

[0277]

[0278] When size exclusion chromatography analysis was performed under severe conditions, i.e., in a stability test at 40 °C for 14 days, Formulations 11 to 13 showed a similar pattern of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (about less than 1.0%), and there were no significant differences according to the formulations. In summary, as a result of size exclusion chromatography analysis in a stability test under severe conditions (i.e., at 40 °C), there were no significant differences in the stability characteristics between the formulations according to the concentration of polysorbate 80 (0 - 0.04%) (see Figure 11 ).

[0279] Example 18. WCX Chromatography Analysis of Formulations Containing Trastuzumab and HP46

[0280] For WCX chromatography analysis, an HPLC system purchased from Shimadzu Prominence was used, and columns such as a TSKgel CM-STAT column (4.6 x 100 mm, 7 μm) and a TSKgel guard gel CMSTAT (3.2 mm inner diameter x 1.5 cm) were used. Mobile phase A was 10 mM sodium phosphate (pH 7.5), and mobile phase B was 10 mM sodium phosphate (pH 7.2) containing 0.1 M NaCl. Analysis was performed at a flow rate of 0.8 mL / min for 55 minutes under a linear concentration gradient of 0 - 30% mobile phase B. The sample was diluted with mobile phase A to a final concentration of 1.0 mg / mL, 80 μL of the sample was injected into the HPLC, and then the absorbance of the column eluate at 280 nm was recorded. The monomer ratio of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0281] When WCX analysis was performed under severe conditions, i.e., in a stability test at 40 °C for 14 days, Formulations 11 to 13 showed a similar pattern of change. Specific changes included an increase in the relative content of acidic variants (a change of about 10% over 14 days), a decrease in the relative content of the main peak (a change of about 40% over 14 days), and an increase in the relative content of basic variants (a change of about 300% over 14 days), and there were no significant differences according to the formulations. In summary, in WCX analysis in a stability test under severe conditions, i.e., at 40 °C, the protein stability according to polysorbate 80 (0 - 0.04%) was similar (see Figure 12).

[0282] Example 19. Enzyme Activity Measurement of Formulations Containing Trastuzumab and HP46

[0283] The turbidimetric assay for measuring enzyme activity is a method that measures the degree of aggregate formation of residual hyaluronic acid binding to acidified albumin (BSA) by absorbance. When hyaluronic acid is hydrolyzed by PH20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), a standardized product, was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL and prepared in each test tube. The purified protein sample was diluted to 100X, 300X, 600X, 1200X, and 2400X with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepared in each test tube. In a fresh test tube, the hyaluronidase solution at a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL to make the volume of each test tube 180 μL. 60 μL of the sample containing hyaluronidase was added to the diluted hyaluronic acid solution, mixed therewith, and allowed to react at 37 °C for 45 minutes. After the reaction was completed, 50 μL of the reacted enzyme and 250 μL of the acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance at 600 nm was measured using a spectrophotometer.

[0284] As a result of the activity analysis in the stability test under severe conditions, that is, at 40 °C for 14 days continuously, it was confirmed that the higher the concentration of polysorbate 80, the greater the decrease in activity over time (see Figure 13 ).

[0285] Example 20. Formulation Development

[0286] As described in Table 10, three types of rituximab formulations were prepared. Formulations 14 to 16 generally contained 120 mg / mL rituximab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and PH20 variant. The difference between Formulations 14 - 16 was the concentration of the nonionic surfactant: Formulation 14: 0% polysorbate 80, Formulation 15: 0.005% polysorbate 80, and Formulation 16: 0.06% polysorbate 80.

[0287] Table 10. Formulation Composition

[0288]

[0289] When size-exclusion chromatography analysis was performed during a stability test under severe conditions, i.e., at 40 °C for 7 days, Formulations 14 to 16 showed a similar pattern of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (less than about 1.0%), and there were no significant differences according to the formulation. In summary, as a result of size-exclusion chromatography analysis during a stability test under severe conditions (i.e., at 40 °C), there were no significant differences in the stability profiles between the formulations according to the concentration of polysorbate 80 (0 - 0.06%) (see Figure 14 ).

[0290] Example 21. Measurement of the Enzymatic Activity of a Formulation Containing Rituximab and HP46

[0291] The turbidimetric assay used to measure enzymatic activity is a method for measuring the degree of aggregate formation of residual hyaluronic acid binding to acidified albumin (BSA) by absorbance, and when hyaluronic acid is hydrolyzed by PH20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), a standardized product, was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL and prepared in each test tube. The purified protein sample was diluted to 100X, 300X, 600X, 1200X, and 2400X with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepared in each test tube. In a fresh test tube, a hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL to make the volume of each test tube 180 μL. 60 μL of the sample containing hyaluronidase was added to the diluted hyaluronic acid solution, mixed therewith, and allowed to react at 37 °C for 45 minutes. After the reaction was completed, 50 μL of the reacted enzyme and 250 μL of the acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance at 600 nm was measured using a spectrophotometer.

[0292] As a result of activity analysis during a stability test under severe conditions, i.e., at 40 °C for 7 days, it was confirmed that the higher the concentration of polysorbate 80, the greater the decrease in activity over time (see Figure 15 ).

[0293] Example 22. Measurement of the Enzymatic Activity in a Commercially Available Product Formulation without Polysorbate

[0294] As described in Table 11, two types of commercially available rituximab formulations were prepared. Formulation 17 is a commercially available buffer for subcutaneous injection formulations, and Formulation 18 is a commercially available buffer for intravenous injection formulations. Formulations 17 and 18 contain 120 mg / mL and 100 mg / mL of PH20 variant and rituximab, respectively, but unlike the formulations of the commercially available products, they do not contain polysorbate 80.

[0295] Table 11. Formulation composition

[0296]

[0297] The turbidimetric assay for measuring enzyme activity is a method for measuring the degree of aggregate formation of residual hyaluronic acid binding to acidified albumin (BSA) by absorbance, and when hyaluronic acid is hydrolyzed by PH20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), a standardized product, was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL and prepared in each test tube. The purified protein samples were diluted to 100X, 300X, 600X, 1200X, and 2400X with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepared in each test tube. In a fresh test tube, the hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL to make the volume of each test tube 180 μL. 60 μL of the sample containing hyaluronidase was added to the diluted hyaluronic acid solution, mixed therewith, and allowed to react at 37 °C for 45 minutes. After the reaction was completed, 50 μL of the reacted enzyme and 250 μL of the acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance at 600 nm was measured using a spectrophotometer.

[0298] As a result of the activity analysis in the stability test carried out for 6 days under severe conditions (i.e., at 40 °C), it was confirmed that high activity could be maintained even in the formulations without polysorbate 80, and in particular, Formulation 18 maintained high activity (see Figure 16 ).

[0299] Example 23: Formulation development

[0300] As described in Table 12, four types of pembrolizumab formulations were prepared. Formulations 19, 20, and 21 generally contained 25 mg / mL pembrolizumab, 10 mM histidine (pH 5.5), 7% sucrose, 10 mM methionine, and the PH20 variant. The difference between Formulations 19 - 21 was the concentration of the nonionic surfactant: Formulation 19: 0% polysorbate 80, Formulation 20: 0.005% polysorbate 80, and Formulation 21: 0.02% polysorbate 80. Formulation 22 contained 25 mg / mL pembrolizumab and consisted of 10 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, 0.02% polysorbate 80, and the PH20 variant.

[0301] Table 12. Formulation Composition

[0302]

[0303] Example 24. Measurement Using a Spectrophotometer

[0304] Formulations 19, 20, 21, and 22 were placed at 40 °C for 7 days, and the change in protein concentration was analyzed using a spectrophotometer manufactured by Beckman. Each sample was diluted with distilled water such that the concentration of the sample was 0.4 mg / mL, and then the absorbance of the protein at 280 nm was measured using a spectrophotometer.

[0305] In the stability test under severe conditions, i.e., at 40 °C for 7 days, there was no significant change in the protein concentration of Formulations 19 to 22.

[0306] Example 25. Studying the Monomer Ratio of Pembrolizumab in Each Formulation Using Size - Exclusion Chromatography

[0307] For size - exclusion chromatography analysis, an HPLC system available from Shimadzu Prominence was used, and TSK - gel G3000SWXL (7.8 X 300 mm, 5 μm) and a TSK guard column (6.0 x 4.0 mm, 7 μm) were used as columns. 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. The analysis was carried out by applying an isocratic separation mode at a flow rate of 0.5 mL / min for 35 minutes. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, and after injecting 20 μL of the sample into the HPLC column, the absorbance of the column eluate at 280 nm was measured. The monomer ratio of pembrolizumab in the HPLC chromatogram was calculated and plotted.

[0308] When size-exclusion chromatography analysis was performed in a stability test of the formulations for 7 days at a stringent condition, i.e., at 40 °C, 19, 20, 21, and 22 showed similar patterns of change. Depending on the formulations, there were no significant differences in the patterns of change of high molecular weight (HMW) and low molecular weight (LMW) degradation products. In summary, as a result of size-exclusion chromatography analysis in a stability test under stringent conditions (i.e., at 40 °C), none of the formulations 19, 20, 21, and 22 showed any significant differences, and there were no differences depending on the type of sugar (see Figure 17 ). These results are consistent with the cases of trastuzumab and rituximab according to the previous examples.

[0309] Example 26. Measurement of the Enzyme Activity of a Formulation Containing Pembrolizumab and HP46

[0310] The turbidimetry used to measure the enzyme activity is a method of measuring the degree of aggregate formation of residual hyaluronic acid binding to acidified albumin (BSA) by absorbance, and when hyaluronic acid is hydrolyzed by PH20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), a standardized product, was diluted to 1 unit / mL, 2 units / mL, 5 units / mL, 7.5 units / mL, 10 units / mL, 15 units / mL, 20 units / mL, 30 units / mL, 50 units / mL, and 60 units / mL and prepared in each test tube. The purified protein sample was diluted to 100X, 300X, 600X, 1200X, and 2400X with an enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepared in each test tube. In a fresh test tube, a hyaluronidase solution with a concentration of 3 mg / mL was diluted 10-fold to a concentration of 0.3 mg / mL to make the volume of each test tube 180 μL. 60 μL of the enzyme-containing sample was added to the diluted hyaluronic acid solution, mixed therewith, and allowed to react at 37 °C for 45 minutes. After the reaction was completed, 50 μL of the reacted enzyme and 250 μL of the acidic albumin solution were added to each well of a 96-well plate and shaken for 10 minutes, and then the absorbance at 600 nm was measured using a spectrophotometer.

[0311] As a result of the activity analysis in a stability test under stringent conditions, i.e., at 40 °C for 7 days, it was confirmed that as the concentration of polysorbate 80 increased, the decrease in activity over time increased. It was also confirmed that when the same amount of polysorbate 80 was included, the decrease in activity in the formulation containing trehalose was less than that in the formulation containing sucrose (see Figure 18 ).

[0312] Example 27. pH-Activity Curves of HP46 and Wild-Type HW2

[0313] For the experiment to determine the pH-activity curves of HP46 and wild-type HW2, microturbidimetry was used. For each pH, a hyaluronic acid buffer for dissolving hyaluronic acid as the substrate and an enzyme buffer for diluting the enzyme were prepared.

[0314] A total of three 96-well plates were prepared for the reaction between the enzyme and the substrate, named A, B, and C, and the experiment was conducted.

[0315] Hyaluronic acid buffers with pH values of 4.0, 4.5, or 5.0 were prepared using 20 mM acetic acid and 70 mM NaCl, and hyaluronic acid solutions with pH values of 5.5, 6.0, 6.5, 7.0, or 8.0 were prepared using 20 mM sodium phosphate and 70 mM NaCl. 20 mg of hyaluronic acid was dissolved in 10 mL of each prepared hyaluronic acid buffer to prepare the final hyaluronic acid substrate solution, which was then diluted to concentrations of 0.1 mg / mL, 0.25 mg / mL, 0.45 mg / mL, or 0.7 mg / mL with the respective hyaluronic acid buffers prepared according to the pH to prepare 500 μL of the resulting solution, and 100 μL of each solution was dispensed into each well of the 96-well plate named A. The hyaluronic acid buffer diluted and prepared according to the concentration was used as the calibration curve for measuring the hyaluronic acid concentration.

[0316] Enzyme buffers with pH values of 4.0, 4.5, or 5.0 were prepared using 20 mM acetic acid, 0.01% (w / v) BSA, and 70 mM NaCl, and enzyme buffers with pH values of 5.5, 6.0, 6.5, 7.0, or 8.0 were prepared using 20 mM sodium phosphate, 0.01% (w / v) BSA, and 70 mM NaCl.

[0317] The HP46 and wild-type HW2 enzymes were diluted to 10 units / mL with the enzyme buffers prepared according to the pH, and then 50 μL of the resulting solution was dispensed into each well of the 96-well plate named B.

[0318] 50 μL of the sample was transferred from each well of the 96-well plate named A to each well of the 96-well plate named B, and then the reaction was allowed to proceed for 45 minutes in a shaking incubator at 37°C. 15 minutes before the completion of the reaction, 200 μL of acidic albumin solution was dispensed into each well of the 96-well plate named C and prepared, and when the enzyme-substrate reaction was completed, 40 μL of the sample was transferred from each well of the 96-well plate named B to each well of the 96-well plate named C, and then the reaction was allowed to proceed for 20 minutes. After 20 minutes, the absorbance at 600 nm was measured, and the amount of hyaluronic acid remaining after the enzyme-substrate reaction was calculated, and the activity curve of the enzyme according to the pH was completed (seeFigure 19 )。

[0319] Example 28. Pharmacokinetic Testing of Herceptin Subcutaneous Formulation, Trastuzumab, and HP46 in Sprague-Dawley Rats

[0320] To examine whether the subcutaneous formulations of trastuzumab and HP46 exhibit the same pharmacokinetic properties as the Herceptin subcutaneous formulation, experiments were conducted using 9-week-old Sprague-Dawley rats. The administered doses of Herceptin and trastuzumab were 18 mg / kg of rat body weight, the amount of rHuPH20 contained in the Herceptin subcutaneous formulation was 100 U, and the amount of HP46 was also 100 U. In the pharmacokinetic test, trastuzumab and HP46 showed the same area under the curve (AUC) as the Herceptin subcutaneous formulation (see Figure 20 )。

[0321] The present invention also includes the following embodiments:

[0322] 1. A pharmaceutical composition comprising:

[0323] (a) a drug; and

[0324] (b) a PH20 variant,

[0325] wherein the PH20 variant comprises one or more amino acid residue substitutions selected from S343E, M345T, K349E, L353A, L354I, N356E, and I361T in wild-type PH20 having the sequence of SEQ ID NO: 1.

[0326] 2. The pharmaceutical composition according to embodiment 1, wherein the PH20 variant comprises one or more amino acid residue substitutions selected from L354I and N356E.

[0327] 3. The pharmaceutical composition according to embodiment 1, wherein the PH20 variant further comprises one or more amino acid residue substitutions in one or more regions selected from the α-helical region and the linker region corresponding to the wild-type PH20 of SEQ ID NO: 1.

[0328] 4. The pharmaceutical composition according to embodiment 3, wherein the α-helical region of the wild-type PH20 of SEQ ID NO: 1 is the α-helix 8 region (S347 to C381), and the linker region is the linker region between α-helix 7 and α-helix 8 (A333 to R346).

[0329] 5. The pharmaceutical composition according to Embodiment 4, wherein the α-helical region and the region corresponding to its linker region are T341 to N363, T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361 or M345 to N363 of wild-type PH20 of SEQ ID NO: 1.

[0330] 6. The pharmaceutical composition according to Embodiment 4, wherein one or more regions selected from the α-helical 8 region (S347 to C381) of wild-type PH20 of SEQ ID NO: 1 and the linker region (A333 to R346) between α-helical 7 and α-helical 8 are substituted with one or more amino acid residues of the amino acid sequence of the corresponding region of Hyal1.

[0331] 7. The pharmaceutical composition according to Embodiment 1, wherein the PH20 variant contains one or more amino acid residue substitutions of L354I and / or N356E, and further contains one or more amino acid residue substitutions at one or more positions selected from T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361 and N363.

[0332] 8. The pharmaceutical composition according to Embodiment 7, wherein the PH20 variant contains one or more amino acid residue substitutions of L354I and / or N356E, and further contains one or more amino acid residue substitutions selected from T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, 1361T and N363G.

[0333] 9. The pharmaceutical composition according to Embodiment 7, wherein the PH20 variant contains amino acid residue substitutions of M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T.

[0334] 10. The pharmaceutical composition according to Embodiment 9, wherein the PH20 variant further contains one or more amino acid residue substitutions selected from T341S, L342W, S343E, I344N and N363G.

[0335] 11. The pharmaceutical composition according to Embodiment 10, wherein the PH20 variant contains any one amino acid residue substitution selected from the following group:

[0336] (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0337] (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0338] (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T;

[0339] (d) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G;

[0340] (e) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; and

[0341] (f) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.

[0342] 12. The pharmaceutical composition according to any one of embodiments 1 to 11, wherein the PH20 variant further comprises a deletion of one or more amino acid residues in at least one of the C-terminal and the N-terminal.

[0343] 13. The pharmaceutical composition according to embodiment 12, wherein in the PH20 variant, one or more amino acid residues are deleted by cleavage before the amino acid residues selected from M1 to P42 at the N-terminal.

[0344] 14. The pharmaceutical composition according to embodiment 13, wherein in the PH20 variant, one or more amino acid residues are deleted by cleavage before the amino acid residues L36, N37, F38, R39, A40, P41, or P42 at the N-terminal.

[0345] 15. The pharmaceutical composition according to embodiment 12, wherein in the PH20 variant, one or more amino acid residues are deleted by cleavage after an amino acid residue selected from V455 to L509 at the C-terminus.

[0346] 16. The pharmaceutical composition according to embodiment 15, wherein in the PH20 variant, one or more amino acid residues are deleted by cleavage after an amino acid residue selected from V455 to S490 at the C-terminus.

[0347] 17. The pharmaceutical composition according to embodiment 16, wherein in the PH20 variant, one or more amino acid residues are deleted by cleavage after an amino acid residue of V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 at the C-terminus.

[0348] 18. The pharmaceutical composition according to any one of embodiments 1 to 17, wherein the PH20 variant further comprises a signal peptide derived from human hyaluronidase-1 (Hyall), human growth hormone or human serum albumin at the N-terminus.

[0349] 19. The pharmaceutical composition according to any one of embodiments 1 to 11, wherein the PH20 variant has an amino acid sequence selected from amino acid sequences SEQ ID NO: 5 to SEQ ID NO: 50.

[0350] 20. The pharmaceutical composition according to embodiment 19, wherein the PH20 variant has the sequence of SEQ ID NO: 44.

[0351] 21. The pharmaceutical composition according to embodiment 1, wherein the drug is a protein drug, an antibody, a small molecule, an aptamer, RNAi, an antisense or a cell therapy agent.

[0352] 22. The pharmaceutical composition according to embodiment 21, wherein the drug is an antibody, a soluble receptor or an Fc fusion protein thereof.

[0353] 23. The pharmaceutical composition according to Embodiment 22, wherein the antibody binds to one or more antigens selected from the following: 4-1BB, integrin, amyloid beta, angiopoietin, angiopoietin-like 3, B cell activating factor (BAFF), B7-H3, complement 5, CCR4, CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD52, CD62, CD79b, CD80, CGRP, claudin-18, complement factor D, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2, GM-CSF, HER2, HER3, interferon receptor, interferon gamma, IgE, IGF-1 receptor, interleukin 1, interleukin 2 receptor, interleukin 4 receptor, interleukin 5, interleukin 5 receptor, interleukin 6, interleukin 6 receptor, interleukin 7, interleukin 12 / 23, interleukin 13, interleukin 17A, interleukin 17 receptor A, interleukin 31 receptor, interleukin 36 receptor, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, and vWF.

[0354] 24. The pharmaceutical composition according to embodiment 22, wherein the antibody is one or more selected from the following: utomilumab, natalizumab, etrolizumab, vedolizumab, biglumab, bapineuzumab, crenezumab, solanezumab, aducanumab, gantenerumab, AMG 780, MEDI 3617, nescamumab, vanucizumab, eculizumab, molilimumab, ocrelizumab, telizumab, muromonab, tebentafusp, blinatumomab, REGN1979, ibalizumab, zalutumumab, itolizumab, efalizumab, inebilizumab, talquetamab, telisotuzumab, oregovomab, ublituximab, obinutuzumab, ofatumumab, rituximab, tositumomab, iodine-131 tositumomab, ipilimumab, tucotuzumab celmoleukin, gemtuzumab ozogamicin, daratumumab, isatuximab, alemtuzumab, lirilumab, polatuzumab vedotin, galiximab, epratuzumab, fulranumab, guselkumab, lenzilumab, teclistamab, mirikizumab, nemolizumab, pecilizumab, relatlimab, naxitamab, faricimab, tanibirumab, alirocumab, evolocumab, bempedoic acid, palbocizumab, basimglumab, camrelizumab, cemiplimab, dostarlimab, palovarotene, cedazuridine, spartalizumab, tislelizumab, pembrolizumab, nivolumab, atezolizumab, avelumab, envafolimab, durvalumab, bintrafusp alfaα, denosumab, elotuzumab, canakinumab, mavrilimumab, infliximab, adalimumab, golimumab, certolizumab, olokizumab, brodalumab, ranibizumab, bevacizumab, faricimab, ramucirumab, and caplacizumab.

[0355] 25. The pharmaceutical composition according to embodiment 22, wherein the soluble receptor or the soluble receptor comprised in the Fc fusion protein of the soluble receptor is selected from TNF-α soluble receptor, VEGF soluble receptor, CTLA-4, interleukin 1 soluble receptor, and LFA3 soluble receptor.

[0356] 26. The pharmaceutical composition according to embodiment 25, wherein the Fc fusion protein of the soluble receptor is selected from etanercept, aflibercept, abatacept, belatacept, lenaliximab, and alefacept.

[0357] 27. The pharmaceutical composition according to embodiment 1, further comprising one or more selected from buffers, stabilizers, and surfactants.

[0358] 28. The pharmaceutical composition according to embodiment 27, wherein the buffer comprises one or more selected from the following: malate, formate, citrate, acetate, propionate, pyridine, piperazine, cacodylate, succinate, 2-(N-morpholino)ethanesulfonic acid (MES), histidine, Tris, bis-Tris, phosphate, ethanolamine, carbonate, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), imidazole, BIS-TRIS propane, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-(N-morpholino)propanesulfonic acid) (MOPS), N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), pyrophosphate, and triethanolamine;

[0359] The stabilizer comprises one or more selected from the following: carbohydrates, sugars or their hydrates, sugar alcohols or their hydrates, and amino acids; and

[0360] The surfactant comprises one or more nonionic surfactants selected from the following: polyoxyethylene-sorbitan fatty acid esters, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, polyoxyethylene-polyoxypropylene copolymers, and sodium dodecyl sulfate (SDS).

[0361] 29. The pharmaceutical composition according to embodiment 28, wherein the carbohydrate, the sugar or the sugar alcohol comprises one or more selected from the following: trehalose or its hydrate, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, heptitol, isomaltulose, maltitol, polydextrose, cyclodextrin, hydroxypropyl cyclodextrin, and glucose, and

[0362] the amino acid comprises one or more selected from the following: glutamine, glutamic acid, glycine, lysine, lysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine, and alanine.

[0363] 30. The pharmaceutical composition according to embodiment 27, wherein the pharmaceutical composition comprises a histidine buffer providing a pH of 5.5 ± 2.0, trehalose, and methionine.

[0364] 31. The pharmaceutical composition according to embodiment 27, wherein the pharmaceutical composition comprises a histidine buffer providing a pH of 5.5 ± 2.0, trehalose, methionine, and polysorbate.

[0365] 32. The pharmaceutical composition according to embodiment 31, wherein the pharmaceutical composition comprises a histidine buffer providing a pH of 5.5 ± 2.0, 10 - 400 mM α,α-trehalose, 1 - 50 mM methionine, and 0.0000001% (w / v) to 0.5% (w / v) of polysorbate.

[0366] 33. An injection formulation for subcutaneous injection, the injection formulation comprising the pharmaceutical composition according to any one of embodiments 1 to 32.

[0367] Industrial Applicability

[0368] The pharmaceutical composition according to the present disclosure can be used for subcutaneous injection and is also very stable, and the activity of the PH20 variant together with the drug, preferably antibody drugs, etc. can be maintained for a long time. Therefore, the pharmaceutical composition can not only contribute to reducing the production cost of subcutaneous injection formulations, but also reduce the medical cost, and is very effective in terms of patient convenience.

[0369] Although the preferred embodiments of the present disclosure have been disclosed for illustrative purposes, those skilled in the art will understand that various modifications, additions, and substitutions can be made without departing from the scope and spirit of the present invention as disclosed in the appended claims.

[0370] References

[0371] Bookbinder, L.H., Hofer, A., Haller, M.F., Zepeda, M.L., Keller, G.A., Lim, J.E., Edgington, T.S., Shepard, H.M., Patton, J.S., and Frost, G.I. (2006). A recombinant human enzyme for enhanced interstitial transport of therapeutics. J Control Release 114, 230 - 241.

[0372] Borders jr., C.L. and Raftery, A. (1968) Purification and Partial Characterization of Testicular Hyaluronidase. J Biol Chem 243, 3756 - 3762

[0373] Chao, K.L., Muthukumar, L., and Herzberg, O. (2007). Structure of human hyaluronidase - 1, a hyaluronan hydrolyzing enzyme involved in tumor growth and angiogenesis. Biochemistry 46, 6911 - 6920.

[0374] Chen, K.J., Sabrina, S., El - Safory, N.S., Lee, G.C., and Lee, C.K. (2016) Constitutive expression of recombinant human hyaluronidase PH20 by Pichia pastoris. J Biosci Bioeng.122, 673 - 678

[0375] Frost, G.I. (2007). Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin Drug Deliv 4, 427-440

[0376] Hofinger, E.S., Bernhardt, G., and Buschauer, A. (2007) Kinetics of Hyal-1 and PH-20 hyaluronidases: comparison of minimal substrates and analysis of the transglycosylation reaction. Glycobiology 17, 963-971

[0377] Kreidieh, F.Y., Moukadem, H.A., and Saghir, N.S.E. (2016) Overview, prevention and management of chemotherapy extravasation. World J Clin Oncol 7, 87-97.

[0378] Thomas, J.R., Yocum, R.C., Haller, M.F., and Flament J. (2009) The INFUSE-Morphine IIB Study: Use of Recombinant Human Hyaluronidase (rHuPH20) to Enhance the Absorption of Subcutaneous Morphine in Healthy Volunteers. J Pain Symptom Manag 38, 673-682。

Claims

1. A pharmaceutical composition, comprising: (a) a drug; and (b) a PH20 variant, wherein the PH20 variant comprises one or more amino acid residue substitutions selected from S343E, M345T, K349E, L353A, L354I, N356E, and I361T in wild-type PH20 having the sequence of SEQ ID NO:

1.

2. The pharmaceutical composition according to claim 1, wherein the PH20 variant comprises one or more amino acid residue substitutions selected from L354I and N356E.

3. The pharmaceutical composition according to claim 1, wherein the PH20 variant further comprises one or more amino acid residue substitutions in one or more regions selected from the α-helical region and its linker region corresponding to wild-type PH20 of SEQ ID NO:

1.

4. The pharmaceutical composition according to claim 3, wherein the α-helical region of wild-type PH20 of SEQ ID NO:1 is the α-helix 8 region (S347 to C381), and the linker region is the linker region between α-helix 7 and α-helix 8 (A333 to R346).

5. The pharmaceutical composition according to claim 4, wherein the α-helical region and the region corresponding to its linker region are T341 to N363, T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361, or M345 to N363 of wild-type PH20 of SEQ ID NO:

1.

6. The pharmaceutical composition according to claim 4, wherein one or more regions selected from the α-helix 8 region (S347 to C381) of wild-type PH20 of SEQ ID NO:1 and the linker region between α-helix 7 and α-helix 8 (A333 to R346) are substituted with one or more amino acid residues of the amino acid sequence of the corresponding region of Hyal1.

7. The pharmaceutical composition according to claim 1, wherein the PH20 variant comprises one or more amino acid residue substitutions of L354I and / or N356E, and further comprises one or more amino acid residue substitutions at one or more positions selected from T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361, and N363.

8. The pharmaceutical composition according to claim 7, wherein the PH20 variant comprises one or more amino acid residue substitutions of L354I and / or N356E, and further comprises one or more amino acid residue substitutions selected from T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T, and N363G.

9. The pharmaceutical composition according to claim 7, wherein the PH20 variant comprises amino acid residue substitutions of M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.

10. The pharmaceutical composition according to claim 9, wherein the PH20 variant further comprises one or more amino acid residue substitutions selected from T341S, L342W, S343E, I344N, and N363G.

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