Pharmaceutical compositions for subcutaneous administration comprising human hyaluronidase PH20 variants and drugs

Abstract

To provide pharmaceutical compositions comprising (a) a drug and (b) a human PH20 variant.SOLUTION: The human PH20 variant contained in the pharmaceutical composition of the invention comprises amino acid substitution at one or more sites selected from alpha helix 8 sequence (S347 to C381), and a linker region (A333 to R346) between alpha helix 7 and alpha helix 8 in the wild-type human PH20 having the amino acid sequence of SEQ ID NO: 1, where the amino acid located at the N terminus or the C terminus is selectively cleaved. The pharmaceutical composition of the invention may further contain a pharmaceutically acceptable additive, in particular a stabilizer. The pharmaceutical composition according to the present disclosure can maximize the therapeutic effect of the drug used in combination therewith, due to the effect of the human PH20 variant.SELECTED DRAWING: Figure 20

Description

Technical Field

[0001] The present invention relates to a variant of human hyaluronidase PH20 with improved enzyme activity and thermal stability, a pharmaceutical composition containing one or more drugs, and a method for treating diseases using the same.

[0002] The pharmaceutical composition according to the present invention can preferably be used for subcutaneous injection applications.

Background Art

[0003] Drugs that require high-volume or large-dose administration, particularly antibody pharmaceuticals, are generally administered by intravenous injection. However, it takes about 90 minutes or more just for the injection, and additional preparation work for intravenous injection is required. Therefore, there are significant inconveniences for both patients and medical staff, and additional costs are incurred. On the other hand, subcutaneous injection has the advantage of being directly administrable, but compared to intravenous injection, the absorption rate is low and absorption is slow. Therefore, when the injection volume is 3 to 5 mL or more, swelling and pain may be induced at the injection site. For this reason, subcutaneous injection of protein therapeutics is usually limited to injection of a small amount of solution within 2 mL. However, if hyaluronidase is administered subcutaneously (subcutaneous administration or subcutaneous injection) together with a therapeutic drug, the hyaluronic acid distributed in the extracellular matrix is hydrolyzed by the action of hyaluronidase, reducing the viscosity of the subcutaneous tissue and increasing the substance permeability. Thus, high-volume and large-dose pharmaceuticals can be easily delivered into the body.

[0004] Humans have six hyaluronidase genes such as 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 cell membrane and acrosome membrane of sperm. HyalPS1 is a pseudogene and is not expressed. PH20 is an enzyme (EC 3.2.1.35) that cleaves the β-1,4 bond between N-acetylglucosamine and glucuronic acid, which are constituent sugars of hyaluronic acid. The optimal pH of human hyaluronidase PH20 is 5.5, but it also shows some activity under pH 7-8 conditions. In contrast, other human hyaluronidases, including Hyal1, have an optimal pH of 3-4 and very weak activity under pH 7-8 conditions. Since the subcutaneous part of humans is slightly neutral at about pH 7.4, among various hyaluronidases, PH20 is widely used clinically. Examples of the clinical use of PH20 include subcutaneous injection of antibody therapeutics, addition of an eye relaxation agent and anesthetic injection during ophthalmic surgery, use for enhancing the accessibility of anticancer therapeutics to tumor cells by hydrolyzing the hyaluronic acid in the extracellular matrix of tumor cells, and use for promoting the reabsorption of excessive body fluids and blood in tissues.

[0005] On the other hand, currently widely used commercially, PH20 is in a form extracted from the testes of cows and sheep. Examples include Amphadase TM (bovine hyaluronidase) and Vitrase TM (ovine hyaluronidase).

[0006] BTH (Bovine testicular hyaluronidase) is a form in which the signal peptide and 56 C-terminal amino acids have been removed from wild-type bovine PH20 during the post-translational modification process. BTH is also a glycoprotein, with mannose accounting for 5% and glucosamine accounting for 2.2% in all components including amino acids (Borders and Raftery, 1968). When animal-derived hyaluronidase is repeatedly administered to the human body in high doses, neutralizing antibodies can be generated, and other animal-derived biological substances contained as impurities along with PH20 may induce allergies. In particular, PH20 extracted from cows has limitations in use due to concerns about mad cow disease. To address such problems, research on recombinant proteins of human PH20 has been conducted.

[0007] Recombinant proteins of human PH20 have been reported to be expressed from yeast (P. pastoris), DS-2 insect cells, animal cells, etc. (Chen et al., 2016, Hofinger et al., 2007). The recombinant PH20 proteins produced from insect cells and yeast have different N-glycosylation patterns from human PH20 during the post-translational modification process.

[0008] Among hyaluronidases, the protein structures of Hyal1 (PDB ID: 2PE4) (Chao et al., 2007) and bee venom hyaluronidase (PDB ID: 1FCQ, 1FCU, 1FCV) have been elucidated. Hyal1 is composed of two domains, a catalytic domain and an EGF-like domain. The catalytic domain forms a (β / α)8 structure in which the alpha helix and beta-strand, which characterize the secondary structure of the protein, are repeated eight times each (Chao et al., 2007). The EGF-like domain is conserved in all mutants spliced such that the C-terminus of Hyal1 is different. The amino acid sequences of Hyal1 and PH20 are 35.1% identical, and the tertiary structure of PH20 has not yet been elucidated.

[0009] According to the research on the structure / function relationship of human PH20, the C-terminal region of PH20 is important for protein expression and enzyme activity. In particular, it has been reported that the termination of the C-terminus at amino acids 477 - 483 is important for enzyme expression and activity (Frost, 2007). The activity of full-length (Full Length) PH20 (amino acids 1 - 509) or a PH20 mutant with the C-terminus truncated after amino acid 467 was only 10% or less of the enzyme activity of a PH20 mutant truncated at any one of amino acids 477 - 483 (Frost, 2007). Halozyme Therapeutics developed rHuPH20 (amino acids 36 - 482), a recombinant protein in which the C-terminus of mature PH20 is truncated at Y482 (Bookbinder et al., 2006; Frost, 2007).

[0010] On the other hand, research is underway to develop various therapeutic pharmaceuticals in the form of subcutaneous injection dosage forms using human PH20, but the problem of the low stability of human PH20 itself remains an issue to be solved.

[0011] Under such a technical background, the inventors of the present invention have filed a patent application (PCT / KR2019 / 009215) after confirming that a human PH20 variant, which contains one or more amino acid substitutions at the alpha helix 8 site (S347 - C381) in the amino acid sequence of wild-type hyaluronidase PH20 and at the linking site between alpha helix 7 and alpha helix 8 (A333 - R346), and in which a part of the amino acids located at the N-terminal and / or C-terminal portions of PH20 is cleaved, has very excellent enzyme activity and thermal stability.

[0012] In addition, the inventors of the present application have found that the variant of PH20 according to the present invention can be applied to a drug, such as a pharmaceutical composition or formulation containing an antibody drug, specifically a high-capacity anti-HER2 antibody or an immune checkpoint antibody. As a result, a pharmaceutical composition and formulation containing a PH20 variant together with a drug such as an anti-HER2 antibody or an immune checkpoint antibody according to the present invention can be used for subcutaneous administration, and it has been confirmed that the activities of drugs such as antibody drugs and the PH20 variant are very stable and long-lasting, thus completing the present invention.

Summary of the Invention

Problems to be Solved by the Invention

[0013] An object of the present invention is to provide a new pharmaceutical composition containing a PH20 variant with improved enzyme activity and thermal stability together with a drug, and particularly a pharmaceutical composition that can be used for subcutaneous administration, in which the thermal stability and activity of the drug and the PH20 variant can be sustained for a long period.

[0014] Another object of the present invention is to provide a method for treating a disease, which comprises administering the pharmaceutical composition according to the present invention to a subject in need of treatment.

Means for Solving the Problems

[0015] To solve the above problems, the present invention provides a pharmaceutical composition comprising (a) a drug and (b) a PH20 variant.

[0016] The PH20 variant contained in the pharmaceutical composition according to the present invention is characterized in that it contains substitution of one or more amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T in wild-type human PH20 having the amino acid sequence of SEQ ID NO: 1, and further contains amino acid substitution at one or more sites selected from the alpha helix 8 sequence (S347 to C381) and / or the linking site (A333 to R346) between alpha helix 7 and alpha helix 8, and is characterized in that a part of the amino acid residues located at the N-terminal and / or C-terminal portions is selectively cleaved.

[0017] In addition, the pharmaceutical composition according to the present invention may further contain one or more selected from pharmaceutically acceptable additives, specifically buffers, stabilizers and surfactants.

[0018] The pharmaceutical composition according to the present invention can be used in the form of an injectable dosage form for subcutaneous administration.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the technical field.

[0021] In one embodiment, the present invention relates to a pharmaceutical composition comprising (a) a drug and (b) a PH20 variant. The pharmaceutical composition according to the present invention can be used for the prevention or treatment of diseases, and is preferably characterized by being used for subcutaneous administration.

[0022] The variant of human PH20 contained in the pharmaceutical composition according to the present invention is the wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), preferably the amino acid sequence of mature wild-type PH20 (among the amino acid sequence of SEQ ID NO: 1, having the sequence consisting of L36 to S490). Among them, the site corresponding to the alpha-helix site and / or its connecting site, preferably the alpha-helix 8 site (S347 to C381) and / or the connecting site between alpha-helix 7 and alpha-helix 8 (A333 to R346), more preferably the amino acid site between T341 and N363, and most preferably the amino acid site corresponding to T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361 or M345 to N363, characterized in that some amino acid residues are substituted.

[0023] In the present invention, "mature wild-type PH20" means a protein consisting of the amino acid residues of L36 to S490 of SEQ ID NO: 1, in which M1 to T35, which is a signal peptide, and A491 to L509, which have no substantial relation to the function of PH20, are deleted in the amino acid sequence of wild-type PH20 of SEQ ID NO: 1.

[0024]

Table 1

[0025] Specifically, the PH20 variant or its fragment contained in the pharmaceutical composition according to the present invention has, in wild-type PH20 having the sequence of SEQ ID NO: 1, one or more mutations selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E, and I361T, preferably including substitution of amino acid residues, and most preferably including substitution of one or more amino acid residues selected from the group consisting of L354I and N356E.

[0026] In the present invention, the "PH20 variant" is not only one that includes mutations of some amino acid residues, preferably substitutions of amino acid residues, in the sequence of wild-type human PH20, but also includes all those in which, together with the substitution of the amino acid residues, deletions of some amino acid residues occur at the N-terminus and / or C-terminus, and is used in the same concept as the expression "PH20 variant or its fragment".

[0027] Based on the experimental results that the inventors of the present invention have previously found that if the amino acid sequences of the alpha-helix 8 region and the connecting region between alpha-helix 7 and alpha-helix 8 of human PH20 are partially substituted with the amino acid sequences of the alpha-helix 8 region and the connecting region between alpha-helix 7 and alpha-helix 8 of Hyal1 with high hydrophilicity, the enzyme activity and the protein aggregation temperature (Tagg.) at neutral pH increase, it has been corrected that a new PH20 variant or its fragment with increased enzyme activity and thermal stability compared to wild-type PH20 can be provided.

[0028] As a result, the PH20 variant contained in the pharmaceutical composition according to the present invention is the wild-type PH20 (having the amino acid sequence of SEQ ID NO: 1), preferably the amino acid sequence of mature wild-type PH20 (having the sequence consisting of L36 to S490 in the amino acid sequence of SEQ ID NO: 1), and has substitution of one or more amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T, preferably substitution of one or more amino acid residues selected from the group consisting of L354I and N356E. It is characterized in that one or more amino acid residues are substituted at a site corresponding to the alpha helix site and / or its connecting site, preferably the alpha helix 8 site (S347 to C381) and / or the connecting site between alpha helix 7 and alpha helix 8 (A333 to R346), more preferably at an amino acid site corresponding to T341 to N363, T341 to I361, L342 to I361, S343 to I361, I344 to I361, M345 to I361 or M345 to N363.

[0029] In particular, the PH20 variant contained in the pharmaceutical composition according to the present invention may have substitution of some amino acid residues in the corresponding site (see Tables 2 and 3) of Hyal1 having the sequence of SEQ ID NO: 51 at the alpha helix 8 site (S347 to C381) and / or the connecting site between alpha helix 7 and alpha helix 8 (A333 to R346) of the wild-type PH20, preferably the mature wild-type PH20, but is not limited thereto.

[0030]

Table 2

[0031]

Table 3

[0032] More specifically, the PH20 variant or its fragment contained in the pharmaceutical composition according to the present invention contains a substitution of amino acid residues of L354I and / or N356E in the amino acid sequence of wild-type PH20, preferably mature wild-type PH20. Furthermore, it preferably contains substitutions of amino acid residues at one or more positions in T341 to N363, particularly at one or more positions selected from the group consisting of T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361 and N363, but is not limited thereto. More preferably, it further comprises substitutions of one or more amino acid residues selected from the group consisting of T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G, but is not limited thereto.

[0033] Preferably, the PH20 variant or its fragment contained in the pharmaceutical composition according to the present invention is characterized by containing substitutions of amino acid residues of M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T. Furthermore, it may be characterized by containing substitutions of one or more amino acid residues selected from the group consisting of T341S, L342W, S343E, I344N and N363G, but is not limited thereto.

[0034] More preferably, the PH20 variant or its fragment contained in the pharmaceutical composition according to the present invention may contain any one substitution selected from the group consisting of the following, but is not limited thereto. (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D and I361T; (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (d) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G; (e) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; and (f) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.

[0035] In the present invention, an expression in which both a one-letter amino acid residue name and a number are described, such as "S347", means the amino acid residue at each position of the amino acid sequence according to SEQ ID NO: 1.

[0036] For example, "S347" means that the amino acid residue at the 347th position in the amino acid sequence of SEQ ID NO: 1 is serine. Also, "S347T" means that the 347th serine in SEQ ID NO: 1 is substituted with threonine.

[0037] The PH20 variant contained in the pharmaceutical composition according to the present invention is interpreted to include variants in which the amino acid residue is conservatively substituted at a specific amino acid residue position.

[0038] As used herein, "conservative substitution" means a modification of a PH20 variant that includes substituting one or more amino acids with amino acids having similar biochemical properties that do not induce loss of the biological or biochemical function of the PH20 variant.

[0039] "Conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are defined in the art and are well known. Those classes include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0040] It is expected that the PH20 variants contained in the pharmaceutical composition according to the present invention can retain their activity even if they have conservative amino acid substitutions.

[0041] In addition, the PH20 variant or a fragment thereof contained in the pharmaceutical composition according to the present invention has substantially the same function and / or effect as the PH20 variant or a fragment thereof according to the present invention, and is also interpreted to include a PH20 variant or a fragment thereof having an amino acid sequence homology of 80% or 85% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99% or more.

[0042] The PH20 variants according to the present invention showed an effect of increased expression in animal cells, increased rate of protein folding and increased thermal stability compared to mature wild-type PH20. Furthermore, despite the increased thermal stability, their enzyme activities were increased or similar compared to mature wild-type PH20.

[0043] On the one hand, it is known that when some of the C-terminal amino acids such as S490 of mature wild-type PH20 are further cleaved, the enzyme activity decreases. However, the PH20 variant according to the present invention has a sequence in which the C-terminal of mature wild-type PH20 is further cleaved. Nevertheless, compared with mature wild-type PH20, it has increased thermal stability and its enzyme activity is increased or similar. Also, even though it has a sequence in which 5 N-terminal amino acids of mature wild-type PH20 are cleaved, it maintains its enzyme activity, indicating that the P41 residue at the N-terminus plays an important role in protein expression and enzyme activity.

[0044] Thus, the PH20 variant contained in the pharmaceutical composition according to the present invention is characterized by substitution of some amino acid residues at the alpha-helix 8 site (S347 - C381) and / or the linking site between alpha-helix 7 and alpha-helix 8 (A333 - R346) of wild-type PH20, and further deletion of some amino acid residues at the C-terminus and / or N-terminus, but is not limited thereto.

[0045] On one aspect, the PH20 variant contained in the pharmaceutical composition according to the present invention has cleavage occurring before an amino acid residue selected from the group consisting of M1 - P42 at the N-terminus of the amino acid sequence of SEQ ID NO:1, preferably before the amino acid residues of L36, N37, F38, R39, A40, P41 or P42, resulting in deletion of some amino acid residues at the N-terminus, and / or after an amino acid residue selected from the group consisting of V455 - W509 at the C-terminus, preferably an amino acid residue selected from the group consisting of V455 - S490, most preferably after the amino acid residues of V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490, resulting in deletion of some amino acid residues at the C-terminus.

[0046] The expression that cleavage occurred before L36, N37, F38, R39, A40, P41 or P42 at the N-terminus means that, in the sequence of SEQ ID NO: 1, up to T35 which is the amino acid residue immediately before M1 - L36, up to L36 which is the amino acid residue immediately before M1 - N37, up to N37 which is the amino acid residue immediately before M1 - F38, up to F38 which is the amino acid residue immediately before M1 - R39, up to R39 which is the amino acid residue immediately before M1 - A40, up to A40 which is the amino acid residue immediately before M1 - P41, up to P41 which is the amino acid residue immediately before M1 - P42 were cleaved and removed. However, the meaning that cleavage occurred before M1 at the N-terminus of SEQ ID NO: 1 means that no cleavage occurred at any N-terminus.

[0047] Also, the expression that cleavage occurred 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 that, in the sequence of SEQ ID NO: 1, cleavage was made and removed from the next amino acid residue after the above V455, C458, D461, C464, I465, D466, A467, F468, K470, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490. For example, the meaning that cleavage occurred after S490 means that cleavage occurred between S490 and A491.

[0048] Preferably, the human PH20 variant contained in the pharmaceutical composition according to the present invention may be selected from the group consisting of the amino acid sequences of SEQ ID NOs: 5 to 50, more preferably, it may have the amino acid sequence of SEQ ID NO: 44, but is not limited thereto. The sequences of the amino acids substituted or cleaved in the PH20 variant prepared in the specific examples according to the present invention are as shown in Table 4.

[0049]

Table 4

[0050] On the other hand, in prior research, it was reported that in the case of wild-type human PH20, the enzyme activity changes according to the cleavage position of the amino acid residue located at the C-terminus. However, in the present invention, a specific alpha helix forming the secondary structure of human PH20 is replaced with the alpha helix of another human hyaluronidase to produce a human PH20 variant with higher stability than wild-type human PH20. These variants are characterized by having enzyme activity above a certain level regardless of the C-terminal cleavage position by showing a different aspect of the interaction formed by the substituted alpha helix domain and other secondary structures of PH20 from that of wild-type PH20.

[0051] In addition, in the present invention, an attempt was made to improve the expression of recombinant PH20 protein by using the signal peptide of another protein that shows a high protein expression level in animal cells instead of the signal peptide specific to human PH20.

[0052] Accordingly, in other aspects, the PH20 variant contained in the pharmaceutical composition according to the present invention is characterized in that, instead of the signal peptide of wild-type PH20 from M1 to T35, it contains a signal peptide derived from human hyaluronidase-1 (Hyal1), human growth hormone or human serum albumin at the N-terminus, and preferably, a signal peptide derived from human growth hormone having the amino acid sequence of MATGSRTSLLLAFGLLCLPWLQEGSA according to SEQ ID NO: 2 as described in Table 5, a signal peptide derived from human serum albumin having the amino acid sequence of MKWVTFISLLFLFSSAYS according to SEQ ID NO: 3, or a signal peptide derived from human Hyal1 having the amino acid sequence of MAAHLLPICALFLTLLDMAQG according to SEQ ID NO: 4, but is not limited thereto.

[0053]

Table 5

[0054] Among the PH20 variants contained in the pharmaceutical composition according to the present invention, the variant with a 6xHis-tag attached to the C-terminus was named HM, and the variant without a 6xHis-tag was named HP. Also, the mature wild-type PH20 (L36 to S490) with a 6xHis-tag attached to the C-terminus was named WT, and the mature wild-type PH20 (L36 to Y482) without a 6xHis-tag and with the C-terminus cleaved after Y482 was named HW2.

[0055] HP46 (Accession No. 44) is a variant of human PH20 in which the amino acid sequence of alpha helix 8 and the amino acid sequence at the connecting site between alpha helix 7 and alpha helix 8 of human PH20 are replaced with those of Hyal1, a human hyaluronidase with a known protein tertiary structure (PDB ID: 2PE4) (Chao et al., 2007), after modeling the protein structure. Specifically, alpha helix 8 is located on the outside in the protein tertiary structure of PH20 and has fewer interactions with adjacent alpha helices or beta-strands compared to other alpha helices. Generally, there is a trade-off relationship between the activity and thermal stability of an enzyme. As the thermal stability of a protein increases, its enzyme activity decreases, and conversely, as the flexibility of the protein structure improves and the enzyme activity increases, the thermal stability tends to decrease. However, the inactivation degree of HP46 measured by the turbidimetric assay method under pH 7.0 conditions was approximately 46 units / μg, which was evaluated to be about twice as high as that of wild-type PH20, approximately 23 units / μg.

[0056] The thermal stability of a protein can be evaluated by the melting temperature (Tm), which is the temperature at which 50% of the protein tertiary structure is denatured, and the aggregation temperature (Tagg), which is the temperature at which aggregation between proteins occurs. Generally, the aggregation temperature of a protein tends to be lower than the melting temperature. Alpha helix 8 of Hyal1 shows higher hydrophilicity compared to alpha helix 8 of PH20. By increasing the surface hydrophilicity of the HP46 protein due to the replaced alpha helix 8 of Hyal1, an effect is induced that delays the aggregation between proteins caused by hydrophobic interactions, resulting in an aggregation temperature of 51°C, which is evaluated to be 4.5°C higher than the aggregation temperature of wild-type PH20, 46.5°C.

[0057] HP46 is obtained by substituting T341 with serine while substituting the amino acids of alpha helix 8 of PH20 and the connecting site between alpha helix 7 and alpha helix 8. When the 341st amino acid residue is threonine, the enzyme activity is similar to that of wild-type PH20, but when it is substituted with serine, it shows the characteristic that the enzyme activity increases by about 2 times. Also, from the substrate gel assay, it was confirmed that hyaluronic acid is hydrolyzed 5 to 6 times more than wild-type PH20. Since the substrate gel assay involves the process of protein denaturation and refolding, it means that the protein tertiary structure refolding and resilience of HP46 are enhanced compared to wild-type PH20.

[0058] The mutant of PH20 in the pharmaceutical composition according to the present invention contains at least 50 units / mL, preferably 100 - 20,000 units / mL, more preferably about 150 - 18,000 units / mL, more preferably 1,000 - 16,000 units / mL, and most preferably 1,500 - 12,000 units / mL.

[0059] Examples of the drugs included in the pharmaceutical composition according to the present invention include protein drugs, antibody drugs, small molecules, aptamers, RNAi, antisense, cell therapeutics such as chimeric antigen receptor (CAR)-T or CAR-natural killer (NK), etc., but are not limited thereto. In addition to currently commercialized drugs, clinically and developmentally ongoing drugs can also be used.

[0060] Preferably, protein drugs or antibody drugs can be used for the drugs.

[0061] The "protein drug" contained in the pharmaceutical composition according to the present invention is a drug composed of amino acids and exhibiting a therapeutic or prophylactic effect on diseases by the activity of the protein, meaning a drug composed of proteins other than antibody drugs, and can be selected from the group consisting of cytokines, therapeutic enzymes, hormones, soluble receptors and their fusion proteins, insulin or its analogues, BMP (Bone Morphogenetic Protein), EPO (erythropoietin), and serum derived proteins, etc., but is not limited thereto.

[0062] The cytokine contained in the pharmaceutical composition according to the present invention can be selected from the group consisting of interferon, interleukin, CSF (colony stimulating factor), TNF (tumor necrosis factor), TGF (tissue growth factor), etc., but is not limited thereto.

[0063] Examples of the therapeutic enzyme include, but are not limited to, beta-glucocerebrosidase and agalsidase beta.

[0064] The soluble receptor contained in the pharmaceutical composition according to the present invention means the extracellular domain of the receptor, and its fusion protein means a protein in which the Fc region of an antibody or the like is fused to the soluble receptor. The soluble receptor is a water-soluble form of the receptor to which a ligand associated with a disease binds, and forms in which an Fc region is fused to a TNF-α soluble receptor (for example, products with the ingredient name Etanercept and similar forms), forms in which an Fc region is fused to a VEGF soluble receptor (products with the ingredient name Aflibercept and similar forms), forms in which an Fc region is fused to CTLA-4 (for example, products with the ingredient names Abatacept or Belatacept and similar forms), forms in which an Fc region is fused to an interleukin 1 soluble receptor (for example, products with the ingredient name Rilonacept and similar forms), forms in which an Fc region is fused to an LFA3 soluble receptor (for example, products with the ingredient name Alefacept and similar forms), etc., but is not limited thereto.

[0065] The hormone contained in the pharmaceutical composition according to the present invention means a hormone or its analog injected from outside the body for the treatment or prevention of diseases caused by hormone deficiency or the like, and includes, but is not limited to, human growth hormone, estrogen, progesterone, etc.

[0066] The plasma-derived proteins contained in the pharmaceutical composition according to the present invention are proteins present in plasma, and include all those extracted from plasma and those produced recombinantly, such as fibrinogen, von Willebrand Factor, albumin, thrombin, FII (Factor II), FV (Factor V), FVII (Factor VII), FVIII (Factor VIII), FIX (Factor IX), FX (Factor X), and FXI (Factor XI), but are not limited thereto.

[0067] The antibody drug contained in the pharmaceutical composition according to the present invention can be a monoclonal antibody drug or a polyclonal antibody drug.

[0068] The monoclonal antibody drug according to the present invention means a protein containing a monoclonal antibody and a monoclonal antibody fragment that can specifically bind to an antigen associated with a specific disease. The monoclonal antibody includes bispecific antibodies, and the protein containing the monoclonal antibody or its fragment is used in the sense of including an ADC (Antibody-drug conjugate).

[0069] Antigens associated with specific diseases include, but are not limited to, 4-1BB, integrin, amyloid beta, angiopoietin (angiopoietin 1 or 2), angiopoietin-like substance 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 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, VEGF receptor, and von Willebrand Factor (VWF).

[0070] Next are proteins including, but not limited to, monoclonal antibodies and monoclonal antibody fragments against the above antigens associated with specific diseases; The antibody against 4-1BB is Utomilumab; The antibodies against integrin are Natalizumab, Etrolizumab, Vedolizumab, Bimagrumab; Antibodies against amyloid beta include bapineuzumab, crenezumab, solanezumab, aducanumab, gantenerumab; Antibodies against angiopoietin include AMG 780 against angiopoietin 1 and 2, MEDI 3617 against angiopoietin 2, nesvacumab, and vanucizumab, a bispecific antibody against angiopoietin 2 and VEGF; Antibodies against angiopoietin - like 3 include evinacumab; Antibodies against B - cell activating factor (BAFF) include tabalumab, lanalumab, belimumab; Antibodies against B7 - H3 include omburtamab; Antibodies against complement 5 include ravulizumab, eculizumab; Antibodies against CCR4 include mogamulizumab; Antibodies against CD3 include otelixizumab, teplizumab, muromonab, tebentafusp, a bispecific antibody against GP100 and CD3, blinatumomab, a bispecific antibody against CD19 and CD3, and REGN1979, a bispecific antibody against CD20 and CD3; Antibodies against CD4 include ibalizumab, zanolimumab; Antibodies against CD6 include itolizumab; Antibodies against CD11a include efalizumab; Antibodies against CD19 include Inebilizumab, Tafasitamab, and the ADC Loncastuximab tesirine; Antibodies against CD20 include Ocrelizumab, Ublituximab, Obinutuzumab, Ofatumumab, Rituximab, Tositumomab, and the ADC Ibritumomab tiuxetan; Antibodies against CD22 include Epratuzumab, the ADC Inotuzumab ozogamicin, and Moxetumomab pasudotox; The ADC against CD30 is Brentuximab vedotin; The ADC against CD33 is Vadastuximab talirine, Gemtuzumab ozogamicin; Antibodies against CD38 include Daratumumab, Isatuximab; The antibody against CD52 is Alemtuxumab; The antibody against CD62 is Crizanlizumab; The ADC against CD79b is Polatuzumab vedotin; The antibody against CD80 is Galiximab; Antibodies against CGRP include Eptinezumab, Fremanezumab, Galcanezumab, Erenumab; Antibodies against Claudin-18 are Zolbetuximab; Antibodies against complement factor D are Lampalizumab; Antibodies against CTLA4 are Tremelimumab, Zalifrelimab, Ipilimumab; The ADC against DLL3 is Rovalpituzumab tesirine; Antibodies against the EGF receptor are Cetuximab, Depatuxizumab, Zalutumumab, Necitumumab, Panitumumab; The bispecific antibody against coagulation factor IX and Factor X, which are hemophilia factors, is Emicizumab; Antibodies against Fc receptors are Nipocalimab, Rozanolixizumab; Antibodies against FGF23 are Burosumab; Antibodies against the folate receptor are Farletuzumab, and the ADC is Mirvetuximab soravtansine; Antibodies against GD2 are Dinutuximab, Naxitamab; Antibodies against GM-CSF are Otilimab; Antibodies against HER2 include Margetuximab, Pertuzumab, Trastuzumab, and ADCs include Trastuzumab deruxtecan, Trastuzumab emtansine, Trastuzumab duocarmazine; Antibodies against HER3 include Patritumab; Antibodies against interferon receptor include Anifrolumab; Antibodies against interferon gamma include Emapalumab; Antibodies against IgE include Ligelizumab, Omalizumab; Antibodies against IGF-1 receptor include Dalotuzumab, Figitumumab, Teprotumumab; Antibodies against interleukin 1 include Gebokizumab, Canakinumab; Antibodies against interleukin 2 receptor include Daclizumab, Basiliximab; Antibodies against interleukin 4 receptor include Dupilumab; Antibodies against interleukin 5 include Mepolizumab, Reslizumab; Antibodies against interleukin 5 receptor include Benralizumab; Antibodies against interleukin 6 include Clazakizumab, Olokizumab, Sirukumab, Siltuximab; Antibodies against interleukin-6 receptor include Sarilumab, Satralizumab, Tocilizumab, REGN88; Antibodies against interleukin-7 include Secukinumab; Antibodies against interleukin-12 / 23 include Ustekinumab, Briakinumab; Antibodies against interleukin-13 include Lebrikizumab, Tralokinumab; Antibodies against interleukin-17A include Ixekizumab, Bimekizumab; Antibodies against interleukin-17 receptor A include Brodalumab; Antibodies against interleukin-23 include Brazikumab, Guselkumab, Risankizumab, Tildrakizumab, Mirikizumab; Antibodies against interleukin-31 receptor include Nemolizumab; Antibodies against interleukin-36 receptor include Spesolimab; Antibodies against LAG3 include Relatlimab; Antibodies against NASP2 include Narsoplimab; Antibodies against NGF include Fasinumab, Tanezumab; Antibodies against PVSK9 include Alirocumab, Evolocumab, Bococizumab; Antibodies against PD-1 include Lambrolizumab, Balstilimab, Camrelizumab, Cemiplimab, Dostarlimab, Progolimab, Sintilimab, Spartalizumab, Tislelizumab, Pembrolizumab, Nivolumab; Antibodies against PD-L1 include Atezolizumab, Avelumab, Envafolimab, Durvalumab and Bintrafusp alpha which is a bispecific antibody of TGF-beta and PD-L1; Antibody against RANK-L is Denosumab; Antibody against SLAMF7 is Elotuzumab; Antibodies against Tissue factor include Concizumab, Marstacimab; Antibodies against TNF, especially TNFα, include Infliximab, Adalimumab, Golimumab, Certolizumab pegol which is an antibody fragment, and Ozoralizumab which is a bispecific antibody of TNF and albumin; Antibodies against VEGF include Brolucizumab, Ranibizumab, Bevacizumab and Faricimab which is a bispecific antibody of VEGF and Ang2; Antibody against VEGF receptor is Ramucirumab; and Antibody against vWF is Caplacizumab

[0071] On the one hand, in about 20 - 25% of breast cancer patients, overexpression of HER2 (Human Epidermal Growth Factor Receptor 2), which promotes cell division, is observed. Breast cancer with HER2 overexpression progresses faster, is more aggressive, and has a lower response to anticancer chemotherapy compared to breast cancer without HER2 overexpression, so the prognosis is poor. Trastuzumab is a monoclonal antibody pharmaceutical targeting HER2. It specifically binds to HER2 on the surface of HER2 overexpressing cancer cells and suppresses the signal transduction of cell replication and proliferation, thereby delaying tumor progression. Trastuzumab was approved as a breast cancer treatment by the US Food and Drug Administration (FDA) in 1998 in the United States and by the Korea Food and Drug Administration (KFDA) in 2003 in Korea. Subsequently, its efficacy has also been recognized in HER2 overexpressing gastric cancer and it is also used as a gastric cancer treatment.

[0072] The Herceptin intravenous injection formulation (trade name Herceptin) of Roche contains 440 mg of Trastuzumab as the main component. The lyophilized Trastuzumab is mixed with physiological saline and injected intravenously. On the other hand, the subcutaneous injection formulation of Trastuzumab (trade name Herceptin SC) is a 5 mL liquid formulation containing 600 mg (120 mg / mL) of Trastuzumab as the main component and, as additives, 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 (g / mL)).

[0073] The expiration date of the Herceptin subcutaneous injection formulation is 21 months. The intravenous injection formulation of Trastuzumab is in lyophilized form and has an expiration date of 30 months, but the subcutaneous injection formulation of Trastuzumab is liquid and has a shorter expiration date of 21 months. The reason is presumably that the stability of one or more of Trastuzumab and recombinant human hyaluronidase PH20 is limited in the liquid formulation.

[0074] In consideration of this, in the present invention, focusing on the fact that the PH20 variant according to the present invention not only has higher enzyme activity compared to human hyaluronidase PH20 wild type and the recombinant human PH20 of Halozyme, but also has the characteristics of a higher measured protein aggregation temperature and improved thermal stability, the object is to set the expiration period of the subcutaneous injection formulation to a long period, preferably 21 months or more.

[0075] In the pharmaceutical composition according to the present invention, the content of the antibody drug can be 5 to 500 mg / mL, preferably 20 to 200 mg / mL, more preferably 100 to 150 mg / mL, and most preferably 120 ± 18 mg / mL, and can be, for example, about 110 mg / mL, about 120 mg / mL or about 130 mg / mL.

[0076] The polyclonal antibody contained in the pharmaceutical composition according to the present invention is preferably, but not limited to, a plasma antibody (serum antibody) extracted from plasma such as immune globulin.

[0077] In the case of low molecular weight compounds, any drug that requires a quick effect for the purpose of prevention or treatment can be used. For example, analgesics of the morphine series can be used (Thomas et al., 2009). Also, when used as a therapeutic agent for tissue necrosis by an anticancer drug, it can be used alone or in combination with drugs of the vinca alkaloid or taxane series, which are antidote drugs (Kreidieh et al., 2016).

[0078] The pharmaceutical composition according to the present invention may further contain one or more selected from the group consisting of a buffer, a stabilizer and a surfactant.

[0079] Any buffer that provides a pH of 4 to 8, preferably 5 to 7, can be used in the composition according to the present invention. Examples of such buffers include 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, BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid), pyrophosphate, and triethanolamine. One or more buffers selected from this group are preferred, and a histidine buffer such as L-histidine / HCl is more preferred, but not limited to this.

[0080] The concentration of the buffer can be 0.001 to 200 mM, preferably 1 to 50 mM, more preferably 5 to 40 mM, and most preferably 10 to 30 mM.

[0081] As the stabilizer in the composition according to the present invention, any substance that is commonly used in the art for the purpose of stabilizing proteins can be used. Preferred examples include one or more selected from the group consisting of carbohydrates, saccharides or their hydrates, sugar alcohols or their hydrates, and amino acids.

[0082] Examples of the carbohydrates, saccharides or sugar alcohols used as the stabilizer include, but are not limited to, trehalose or its hydrate, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomaltol, maltitol, polyglycitol, cyclodextrin, Hydroxypropyl Beta-cyclodextrin, and glucose. One or more selected from the group can be mentioned.

[0083] Examples of the amino acids include, but are not limited to, glutamine, glutamic acid, glycine, lysine, lysyllysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine, and alanine. One or more selected from the group can be mentioned.

[0084] The concentration of the saccharides or sugar alcohols used as the stabilizer in the pharmaceutical composition according to the present invention is 0.001 to 500 mM, preferably 100 to 300 mM, more preferably 150 to 250 mM, and most preferably 180 to 230 mM, and specifically can be about 210 mM.

[0085] In addition, the concentration of the amino acid used as a stabilizer in the pharmaceutical composition according to the present invention is 1 to 100 mM, preferably 3 to 30 mM, more preferably 5 to 25 mM, and most preferably 7 to 20 mM, and specifically may be about 8 to 15 mM.

[0086] The composition according to the present invention may further contain a surfactant.

[0087] Preferably, the surfactant is a nonionic surfactant such as polyoxyethylene-sorbitan fatty acid ester [polysorbate or Tween], polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, such as polyoxyethylene monolauryl ether, alkyl phenyl polyoxyethylene ether [Triton-X], polyoxyethylene-polyoxypropylene copolymer [Poloxamer, Pluronic], and sodium dodecyl sulfate (SDS), but is not limited thereto.

[0088] More preferably, polysorbate can be used. The polysorbate can be polysorbate 20 or polysorbate 80, but is not limited thereto.

[0089] The concentration of the nonionic surfactant in the pharmaceutical composition according to the present invention can be 0.0000001% to 0.5% (w / v), preferably 0.000001% to 0.4% (w / v), more preferably 0.00001% to 0.3% (w / v), and most preferably 0.001% to 0.2% (w / v).

[0090] In one specific example, the pharmaceutical composition according to the present invention can contain 50 to 350 mg / mL of an antibody, such as an anti-HER2 antibody or an immune checkpoint antibody, a histidine buffer providing a pH of 5.5 ± 2.0, 10 to 400 mM of α,α-trehalose, 1 to 50 mM of methionine, and 0.0000001% to 0.5% (w / v) of polysorbate.

[0091] In a more specific embodiment, the pharmaceutical composition according to the present invention comprises an anti-HER2 antibody or immune checkpoint antibody at 120 mg / mL, a 20 mM histidine buffer providing a pH of 5.5 ± 2.0, 210 mM of α,α-trehalose, 10 mM of methionine, and 2,000 units / mL of PH20 variant, and may further contain 0.005-0.1% (w / v) of polysorbate.

[0092] The pharmaceutical composition according to the present invention can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intralung administration, rectal administration, etc., and is preferably administered subcutaneously by subcutaneous injection, and more preferably used in the form of an injection dosage form for subcutaneous injection administration.

[0093] Accordingly, in another aspect, the present invention provides a formulation comprising the pharmaceutical composition according to the present invention, preferably an injection dosage form for subcutaneous administration.

[0094] The injection dosage form for subcutaneous administration can be provided in a ready-to-injection form without an additional dilution process, and can be contained and provided in a pre-filled syringe, a glass ampoule, or a plastic container.

[0095] The present invention also relates to a method for treating a disease using the pharmaceutical composition or formulation according to the present invention.

[0096] There is no particular limitation on the diseases treatable with the pharmaceutical composition or formulation according to the present invention, and there is no limitation as long as it is a disease treatable with a drug used in combination with the PH20 variant according to the present invention.

[0097] Diseases treatable with the pharmaceutical composition or formulation according to the present invention include, but are not limited to, cancer, autoimmune diseases, etc.

[0098] The cancer or carcinoma treatable with the pharmaceutical composition or dosage form according to the present invention is not particularly limited and includes both solid cancers and hematological cancers. Examples of such cancers include skin cancers 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 of the spine, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma, but are not limited thereto. Preferably, the cancer treatable with the pharmaceutical composition or dosage form of the present invention can be selected from the group consisting of gastric cancer, colorectal cancer, breast cancer, lung cancer, and kidney cancer, but is not limited thereto.

[0099] The autoimmune diseases treatable with the pharmaceutical composition or dosage form according to the present invention can be selected from the group consisting of 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.

[0100] In addition, the present invention provides a method for treating a disease, which comprises administering the pharmaceutical composition or dosage form according to the present invention to a subject in need of treatment. Furthermore, the present invention provides the use of the pharmaceutical composition or dosage form according to the present invention for manufacturing a medicament for treating a disease.

[0101] Unless otherwise defined in the technical terms and scientific terms used in the present invention, they have the meanings that are normally understood by those having ordinary knowledge in the technical field to which the present invention pertains. Also, repetitive explanations regarding the same technical configurations and operations as in the prior art are omitted.

[0102] Hereinafter, the present invention will be described in more detail using examples. It will be apparent to those having ordinary knowledge in the technical field that these examples are merely for illustrating the present invention and that the scope of the present invention should not be construed as being limited by these examples.

[0103] Example Example 1. Dosage form development

[0104] As shown in Table 6, four types of subcutaneous injection dosage forms of trastuzumab were manufactured. All of Dosage forms 1 to 4 contain 120 mg / mL of trastuzumab and are composed of 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and PH20 variant. The difference among Dosage forms 1 to 4 is the concentration of the nonionic surfactant, and Dosage form 1 contains 0% polysorbate 20, Dosage form 2 contains 0.005% polysorbate 20, Dosage form 3 contains 0.04% polysorbate 20, and Dosage form 4 contains 0.1% polysorbate 20.

[0105]

Table 6

[0106] Example 2. Measurement using a spectrophotometer

[0107] Dosage forms 1 to 4 were left at 45 °C for 14 days, and the change in protein concentration was analyzed with a spectrophotometer from Beckman. After diluting with distilled water so that the concentration of the sample became 0.4 mg / mL, the absorbance of the protein was measured at 280 nm with the spectrophotometer. There was no significant change in the protein concentration in Dosage forms 1 to 4 in the 14-day 45 °C stress stability test. However, the activity of hyaluronidase showed a phenomenon of rapid decrease at 45 °C, and the enzyme activity was not measured in this example (see Figure 6).

[0108] Example 3. Investigation of the monomer ratio of trastuzumab in each dosage form using size-exclusion chromatography

[0109] For size exclusion chromatography analysis, an HPLC system of Shimadzu Prominence, TSK-gel G3000SWXL (7.8×300 mm, 5 μm) and a TSK guard column (6.0×4.0 mm, 7 μm) were used. As the mobile phase, 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used. An isocratic separation mode with a flow rate of 0.5 mL / min was applied for 35 minutes of analysis. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, 20 μL was injected into the HPLC column, and then the absorbance of the column eluate was recorded at 280 nm. The monomer ratio of trastuzumab was calculated from the HPLC chromatogram and shown in a graph.

[0110] When size exclusion chromatography analysis was performed in the 14-day 45°C stress stability test, Dosage Forms 1 to 4 showed similar patterns of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomers (about 1.5%), and the differences among dosage forms were not significant. Conclusively, as a result of size exclusion chromatography analysis in the 45°C stress stability test, there was no significant difference in the stability profiles among dosage forms due to the concentration of polysorbate 20 (0 - 0.1% (w / v)) (Figure 1).

[0111] Example 4. Measurement of the aggregation temperature of proteins for dosage forms containing trastuzumab and HP46

[0112] Dynamic light scattering (DLS) is used to analyze the denaturation characteristics of proteins due to heat. In this test, it was used for the purpose of calculating the protein aggregation temperature by measuring the size change of protein molecules following temperature changes. For DLS analysis, a Malvern Zetasizer-nano-ZS instrument and a quartz cuvette (ZEN2112) were used. During the analysis process, the temperature was increased from 25 °C to 85 °C at 1 °C intervals. After diluting the sample to 1 mg / mL using each dosage form buffer, 150 μL of the sample was placed in the cuvette for analysis.

[0113] In dosage form 1 without polysorbate 20, the aggregation temperature was 74 °C, and the aggregation temperatures in dosage forms 2 to 4 were 76 °C (Figure 2).

[0114] Example 5. WCX Chromatography Measurement for Dosage Forms Containing Trastuzumab and HP46

[0115] For WCX chromatography analysis, an HPLC system from Shimadzu Prominence, and columns such as TSKgel CM-STAT (4.6×100 mm, 7 μm) and TSKgel guard gel CMSTAT (3.2 mm i.d.×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 for 55 minutes with a linear concentration gradient of 0 - 30% mobile phase B 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 of the column eluate was recorded at 280 nm. The monomer ratio of trastuzumab was calculated from the HPLC chromatogram and shown in a graph.

[0116] When WCX analysis was performed in a 45°C harsh stability test for 14 days, Formulations 1 to 4 showed similar change trends. 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). The differences among the formulations were not significant. Conclusively, in the WCX analysis in the 45°C harsh stability test, the stability of the protein with polysorbate 20 (0 - 0.1%) was similar (Figure 3).

[0117] Example 6. Formulation Development

[0118] As described in Table 7, three types of subcutaneous injection formulations of trastuzumab were manufactured. Formulations 5 to 7 all contained 120 mg / mL of trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and HP46. The difference among Formulations 5 to 7 was the component of Stabilizer 3. Formulation 5 contained 0.04% polysorbate 20, Formulation 6 contained 50 mM Lys-Lys, and Formulation 3 contained glycine.

[0119]

Table 7

[0120] Example 7. Measurement Using a Spectrophotometer

[0121] The formulations of Formulations 5 to 7 were left at 45°C for 14 days, and the change in protein concentration was analyzed with a spectrophotometer from Beckman. After diluting the sample with distilled water so that the concentration was 0.4 mg / mL, the absorbance of the protein was measured at 280 nm with the spectrophotometer. There was no significant change in protein concentration among Formulations 5 to 7 in the 14-day 45°C harsh stability test. However, the activity of hyaluronidase showed a phenomenon of rapid decrease at 45°C, and the enzyme activity was not measured in this example (see Figure 6).

[0122] Example 8. Investigation of the Monomer Ratio of Trastuzumab in Each Formulation Using Size Exclusion Chromatography

[0123] For size exclusion chromatography analysis, an HPLC system from Shimadzu Prominence was used, along with TSK-gel G3000SWXL (7.8×300 mm, 5 μm) as the column and a TSK guard column (6.0×4.0 mm, 7 μm). The mobile phase was 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride. 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, 20 μL of the sample was injected into the HPLC, and the absorbance was measured at 280 nm. The ratio of the monomer of trastuzumab was calculated from the HPLC chromatogram and presented in a graph.

[0124] When size exclusion chromatography analysis was performed in a 14-day stability test at 45 °C, dosage forms 5 to 7 showed similar change trends. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) impurities and a decrease in the monomer (about 1.5%), and the differences among the dosage forms were not significant. Conclusively, in the 45 °C stability test, the protein stability by size exclusion chromatography analysis was similar for the formulations of 0.04% polysorbate 20 with 50 mM Lys-Lys and 50 mM glycine (Figure 4).

[0125] Example 9. WCX Chromatography Analysis of Dosage Forms Containing Trastuzumab and HP46

[0126] For WCX chromatography analysis, an HPLC system from Shimadzu Prominence was used, along with TSKgel CM-STAT (4.6×100 mm, 7 μm) as the column and TSKgel guard gel CM-STAT (3.2 mm i.d.×1.5 cm). 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. A separation mode with a linear concentration gradient of 0 - 30% was applied at a flow rate of 0.8 mL / min for 55 minutes for analysis. 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 the absorbance was recorded at 280 nm. The monomer ratio of trastuzumab was calculated from the HPLC chromatogram and shown in a graph.

[0127] When WCX analysis was performed in a 14-day 45°C stress stability test, formulations 5 - 7 showed similar change trends. Specific changes included an increase in the relative content of acidic variants (about 30% change in 14 days), a decrease in the relative content of the main peak (about 44% change in 14 days), and an increase in the relative content of basic variants (about 15% change in 14 days), and the differences among the formulations were not significant. Conclusively, the protein stability by WCX analysis in the 45°C stress stability test was similar in formulations with 0.04% polysorbate 20, 50 mM lysyl lysine (Lys-Lys), and 50 mM glycine (Figure 5).

[0128] Example 10. Evaluation of the Stability of HP46 at 40°C and 45°C in Subcutaneous Injection Formulations of Trastuzumab and HP46

[0129] To evaluate the stability of HP46 in a subcutaneous injection formulation of trastuzumab, trastuzumab (120 mg / mL) and PH20 (200 units / mL) were mixed. The buffer used here was 20 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, and 0.04% polysorbate 20. The control sample had its enzyme activity measured on day 0, and the test samples had their enzyme activity measured after being left at 40°C or 45°C for 1 day.

[0130] When the activities of hyaluronidase were measured after leaving Herceptin subcutaneous dosage forms, trastuzumab + HW2, and trastuzumab + HP46 at 40 °C for 1 day, they showed activities of 51%, 47%, and 94% respectively, indicating that HP46 has high thermal stability at 40 °C (Figure 6). Also, when the activities of hyaluronidase were measured after leaving Herceptin subcutaneous dosage forms, trastuzumab + HW2, and trastuzumab + HP46 at 45 °C for 1 day, the enzyme activities of Herceptin subcutaneous dosage form and trastuzumab + HW2 disappeared, but 22% of the enzyme activity remained in trastuzumab + HP46 (Figure 6).

[0131] Example 11. Dosage form development

[0132] As described in Table 8, three types of trastuzumab subcutaneous dosage forms were manufactured. Dosage forms 8 to 10 all contain 120 mg / mL of trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and PH20 variant. The difference among dosage forms 8 to 10 is the concentration of nonionic surfactant, including dosage form 8: 0% polysorbate 20, dosage form 9: 0.005% polysorbate 20, and dosage form 10: 0.04% polysorbate 20.

[0133]

Table 8

[0134] Example 12. Measurement using a spectrophotometer

[0135] Dosage forms 8 to 10 were left at 40 °C for 14 days, and the change in protein concentration was analyzed with a Beckman spectrophotometer. After diluting with distilled water so that the concentration of the sample became 0.4 mg / mL, the absorbance of the protein was measured at 280 nm with the spectrophotometer. There was no significant difference in protein concentration among dosage forms 8 to 10 in the 14-day 40 °C stress stability test.

[0136] Example 13. Investigation of the monomer ratio of trastuzumab in each dosage form using size-exclusion chromatography

[0137] For size-exclusion chromatography analysis, an HPLC system from Shimadzu Prominence, TSK-gel G3000SWXL (7.8×300 mm, 5 μm), and a TSK guard column (6.0×4.0 mm, 7 μm) were used. As the mobile phase, 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used. An isocratic separation mode with a flow rate of 0.5 mL / min was applied for 35 minutes of analysis. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, 20 μL of the sample was injected into the HPLC column, and then the absorbance of the column eluate was recorded at 280 nm. The monomer ratio of trastuzumab was calculated from the HPLC chromatogram and shown in a graph.

[0138] When size-exclusion chromatography analysis was performed in the 14-day 40 °C stress stability test, dosage forms 8 - 10 showed similar patterns of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomers (less than about 1.0%), and the differences among the dosage forms were not significant. Conclusively, as a result of size-exclusion chromatography analysis in the 40 °C stress stability test, there were no significant differences in the stability profiles among the dosage forms due to the concentration of polysorbate 20 (0 - 0.04%) (Figure 7).

[0139] Example 14. Measurement of the protein aggregation temperature for dosage forms containing trastuzumab and HP46

[0140] Dynamic light scattering (DLS) is used to analyze the thermal denaturation characteristics of proteins in the field of protein pharmaceuticals. In this test, it was used for the purpose of calculating the protein aggregation temperature by measuring the size change of protein molecules following temperature changes. For DLS analysis, a Malvern Zetasizer-nano-ZS instrument and a quartz cuvette (ZEN2112) were used. During the analysis process, the temperature was increased from 25 °C to 85 °C at 1 °C intervals. The sample was diluted to 1 mg / mL using each dosage form buffer, and then 150 μL of the sample was placed in the cuvette for analysis.

[0141] The aggregation temperature in dosage form 8 without polysorbate 20 was 78.3 °C, in dosage form 9 it was 77.3 °C, and in dosage form 10 it was 77.7 °C. Example 13 showed that there was no change in the protein monomer ratio even without polysorbate 20. Comparing the case without polysorbate 20 and the case with polysorbate 20, it was confirmed that there was no difference in protein aggregation. This result means that minimal polysorbate 20 is not an essential element in the subcutaneous dosage form of trastuzumab (see Figure 8).

[0142] Example 15. WCX Chromatography Analysis of Dosage Forms Containing Trastuzumab and HP46

[0143] For WCX chromatographic analysis, an HPLC system from Shimadzu Prominence was used, along with columns such as TSKgel CM-STAT (4.6×100 mm, 7 μm) and TSKgel guard gel CMSTAT (3.2 mm i.d.×1.5 cm). 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 for 55 minutes with a linear concentration gradient of 0 - 30% mobile phase B 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 of the column eluate was recorded at 280 nm. The monomer ratio of trastuzumab was calculated from the HPLC chromatogram and shown in a graph.

[0144] When WCX analysis was performed in a 14-day 40 °C stress stability test, formulations 8 to 10 showed similar change trends. Specific changes included an increase in the relative content of acidic variants (approximately 10% change over 14 days), a decrease in the relative content of the main peak (approximately 40% change over 14 days), and an increase in the relative content of basic variants (approximately 300% change over 14 days), and the differences among formulations were not significant. Conclusively, in the WCX analysis of the 40 °C stress stability test, the protein stability with polysorbate 20 (0 - 0.04%) was similar (Figure 9).

[0145] Example 16. Measurement of Enzyme Activity for Formulations Containing Trastuzumab and HP46

[0146] The nephelometric analysis method for measuring enzyme activity is a method of measuring, by absorbance, the degree to which hyaluronic acid remaining in the reaction solution binds to acidified albumin (BSA) to form aggregates. When hyaluronic acid is hydrolyzed by PH20, the amount of binding to albumin decreases and the absorbance decreases. Dilute BTH (Sigma) as a standard product to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, 60 unit / mL and prepare in each tube. Dilute the purified PH20 mutant sample 100X, 300X, 600X, 1200X, 2400X with enzyme diluent buffer (20 mM Tris·HCl, pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepare in each tube. Dilute the 3 mg / mL hyaluronic acid solution 10-fold in a new tube so that the concentration becomes 0.3 mg / mL and the volume of each tube becomes 180 μL. Add 60 μL of the sample containing hyaluronidase to the diluted hyaluronic acid solution, mix, and react at 37 °C for 45 minutes. After the reaction is completed, add 50 μL of the enzyme reacted in a 96-well plate and 250 μL of acidic albumin solution to each well, shake for 10 minutes, and then measure the absorbance with a spectrophotometer at 600 nm.

[0147] When activity analysis was performed in a 14-day severe stability test at 40 °C, it was confirmed that the higher the concentration of polysorbate 20, the greater the decrease in activity over time (Figure 10).

[0148] Example 17. Dosage form development

[0149] As described in Table 9, three types of subcutaneous injection dosage forms of trastuzumab were manufactured. Dosage forms 11 to 13 all contain 120 mg / mL of trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and PH20 mutant. The difference in dosage forms 11 to 13 is the concentration of nonionic surfactant, and dosage form 11: 0% polysorbate 80, dosage form 12: 0.005% polysorbate 80, dosage form 13: 0.04% polysorbate 80.

[0150]

Table 9

[0151] When size exclusion chromatography analysis was performed in the 40 °C severe stability test for 14 days, dosage forms 11 to 13 showed similar patterns of change. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in the monomer (less than about 1.0%), and the differences among the dosage forms were not significant. Conclusively, as a result of size exclusion chromatography analysis in the 40 °C severe stability test, there were no significant differences in the stability profiles among the dosage forms due to the polysorbate 80 concentration (0 to 0.04%) (Figure 11).

[0152] Example 18. WCX Chromatography Analysis of Dosage Forms Containing Trastuzumab and HP46

[0153] For WCX chromatography analysis, an HPLC system from Shimadzu Prominence and columns such as TSKgel CM-STAT (4.6 × 100 mm, 7 μm) and TSKgel guard gel CMSTAT (3.2 mm i.d. × 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 for 55 minutes with a linear concentration gradient of 0 to 30% mobile phase B 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 of the column eluate was recorded at 280 nm. The monomer ratio of trastuzumab was calculated from the HPLC chromatogram and shown in a graph.

[0154] When WCX analysis was performed in a 14-day 40°C stress stability test, dosage forms 11 to 13 showed similar change trends. Specific changes included an increase in the relative content of acidic variants (about 10% change in 14 days), a decrease in the relative content of the main peak (about 40% change in 14 days), and an increase in the relative content of basic variants (about 300% change in 14 days). The differences among dosage forms were not significant. Conclusively, in the WCX analysis of the 40°C stress stability test, the protein stability with polysorbate 80 (0 - 0.04%) was similar (Figure 12).

[0155] Example 19. Measurement of Enzyme Activity for Dosage Forms Containing Trastuzumab and HP46

[0156] The nephelometric analysis method for measuring enzyme activity is a method that measures the degree to which hyaluronic acid remaining in the reaction solution binds to acidified albumin (BSA) to form aggregates by absorbance. When hyaluronic acid is hydrolyzed by PH20, the amount of binding to albumin decreases and the absorbance decreases. As standards, BTH (Sigma) is diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, 60 unit / mL and prepared in each tube. The purified protein sample is diluted to 100X, 300X, 600X, 1200X, 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 tube. A 3 mg / mL hyaluronic acid solution is diluted 10-fold in a new tube so that the concentration becomes 0.3 mg / mL and the volume of each tube becomes 180 μL. 60 μL of the sample containing hyaluronidase is added to the diluted hyaluronic acid solution and mixed, and reacted at 37°C for 45 minutes. After the reaction is completed, 50 μL of the reacted enzyme and 250 μL of the acidic albumin solution are added to each well of a 96-well plate, shaken for 10 minutes, and then the absorbance is measured at 600 nm with a spectrophotometer.

[0157] When activity analysis was performed in a 14-day 40°C stress stability test, it was confirmed that the higher the concentration of polysorbate 80, the greater the decrease in activity over time (Figure 13).

[0158] Example 20. Dosage form development

[0159] As described in Table 10, three types of rituximab dosage forms were manufactured. All of Dosage Forms 14 to 16 contain 120 mg / mL of rituximab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and the PH20 variant. The difference among Dosage Forms 14 to 16 is the concentration of the nonionic surfactant, with Dosage Form 1 containing 0% polysorbate 80, Dosage Form 2 containing 0.005% polysorbate 80, and Dosage Form 3 containing 0.06% polysorbate 80.

[0160]

Table 10

[0161] When size exclusion chromatography analysis was performed in the 7-day 40°C stress stability test, Dosage Forms 14 to 16 showed similar patterns 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 (less than about 1.0%), and the differences among the dosage forms were not significant. Conclusively, as a result of size exclusion chromatography analysis in the 40°C stress stability test, there was no significant difference in the stability profiles among the dosage forms due to the polysorbate 80 concentration (0 to 0.06%) (Figure 14).

[0162] Example 21. Measurement of enzyme activity for a dosage form containing rituximab and HP46

[0163] The nephelometric analysis method for measuring enzyme activity is a method of measuring, by absorbance, the degree to which hyaluronic acid remaining in the reaction solution binds to acidified albumin (BSA) to form aggregates. When hyaluronic acid is hydrolyzed by PH20, the amount that binds to albumin decreases and the absorbance decreases. Dilute BTH (Sigma) as a standard product to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, 60 unit / mL and prepare in each tube. Dilute the purified protein sample 100X, 300X, 600X, 1200X, 2400X with enzyme dilution buffer (20 mM Tris·HCl, pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepare in each tube. Dilute the 3 mg / mL hyaluronic acid solution 10-fold in a new tube so that the concentration becomes 0.3 mg / mL and the volume of each tube becomes 180 μL. Add 60 μL of the sample containing hyaluronidase to the diluted hyaluronic acid solution, mix, and react at 37 °C for 45 minutes. When the reaction is complete, add 50 μL of the enzyme reacted in a 96-well plate and 250 μL of the acidic albumin solution to each well, shake for 10 minutes, and then measure the absorbance with a spectrophotometer at 600 nm.

[0164] When performing activity analysis in the 7-day 40 °C severe stability test, it was confirmed that the higher the concentration of polysorbate 80, the greater the decrease in activity over time (Figure 15).

[0165] Example 22. Measurement of enzyme activity in a dosage form of a commercial product without polysorbate

[0166] As described in Table 11, two types of commercial rituximab dosage forms were manufactured. Dosage form 17 is a commercial subcutaneous injection dosage form buffer, and dosage form 18 is a commercial intravenous injection dosage form buffer. Each contains 120, 100 mg / mL of rituximab and the PH20 variant, but unlike the dosage form of the commercial product, it does not contain polysorbate 80.

[0167]

Table 11

[0168] The nephelometric analysis method for measuring enzyme activity is a method of measuring, by absorbance, the degree to which hyaluronic acid remaining in the reaction solution binds to acidified albumin (BSA) to form aggregates. When hyaluronic acid is hydrolyzed by PH20, the amount bound to albumin decreases and the absorbance decreases. Dilute BTH (Sigma) as a standard product to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, 60 units / mL and prepare in each tube. Dilute the purified protein sample 100X, 300X, 600X, 1200X, 2400X with enzyme dilution buffer (20 mM Tris·HCl, pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepare in each tube. Dilute the 3 mg / mL hyaluronic acid solution 10-fold in a new tube so that the concentration becomes 0.3 mg / mL and the volume of each tube becomes 180 μL. Put 60 μL of enzyme into the diluted hyaluronidase solution and mix, and react at 37°C for 45 minutes. After the reaction is completed, put 50 μL of the reacted enzyme and 250 μL of the acidic albumin solution into each well of a 96-well plate, shake for 10 minutes, and then measure the absorbance with a spectrophotometer at 600 nm.

[0169] When activity analysis was performed in a 6-day 40°C severe stability test, it was confirmed that the activity was maintained high even in a dosage form not containing polysorbate 80, and particularly high in Dosage Form 18 (Figure 16).

[0170] Example 23. Dosage Form Development

[0171] Four types of subcutaneous injection formulations of pembrolizumab were manufactured as shown in Table 12. Formulations 19, 20, and 21 all contain 25 mg / mL of pembrolizumab and are composed of 10 mM histidine (pH 5.5), 7% sucrose, 10 mM methionine, and the PH20 variant. The difference among Formulations 19, 20, and 21 lies in the concentration of the non-ionic surfactant, with Formulation 19 containing 0% polysorbate 80, Formulation 20 containing 0.005% polysorbate 80, and Formulation 21 containing 0.02% polysorbate 80. In the case of Formulation 22, it contains 25 mg / mL of pembrolizumab and is composed of 10 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, 0.02% polysorbate 80, and the PH20 variant.

[0172]

Table 12

[0173] Example 24. Measurement Using a Spectrophotometer

[0174] The formulations of Formulations 19, 20, 21, and 22 were left at 40 °C for 7 days, and the change in protein concentration was analyzed using a Beckman spectrophotometer. After diluting with distilled water so that the concentration of the sample became 0.4 mg / mL, the absorbance of the protein was measured at 280 nm using a spectrophotometer.

[0175] There was no significant difference in protein concentration among Formulations 19 to 22 in the 7-day 40 °C stress stability test.

[0176] Example 25. Investigation of the Monomer Ratio of Pembrolizumab in Each Formulation Using Size-Exclusion Chromatography

[0177] For size exclusion chromatography analysis, an HPLC system from Shimadzu Prominence, TSK-gel G3000SWXL (7.8×300 mm, 5 μm), and a TSK guard column (6.0×4.0 mm, 7 μm) were used. As the mobile phase, 0.2 M potassium phosphate (pH 6.2) containing 0.25 M potassium chloride was used. An isocratic separation mode with a flow rate of 0.5 mL / min was applied for 35 minutes of analysis. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, 20 μL of the sample was injected into the HPLC column, and then the absorbance of the column eluate was recorded at 280 nm. The monomer ratio of pembrolizumab was calculated from the HPLC chromatogram and presented in a graph.

[0178] When size exclusion chromatography analysis was performed in the 7-day 40 °C stress stability test, Formulations 19, 20, 21, and 22 showed similar patterns of change. There were no significant differences among the formulations in the patterns of change of high molecular weight (HMW) and low molecular weight (LMW) degradation products. Conclusively, as a result of size exclusion chromatography analysis in the 40 °C stress stability test, there were no significant differences among Formulations 19, 20, 21, and 22, nor were there differences due to the type of sugar (Figure 17). This result was consistent with the results of trastuzumab and rituximab in the above examples.

[0179] Example 26. Measurement of Enzyme Activity for Formulations Containing Pembrolizumab and HP46

[0180] The nephelometric analysis method for measuring enzyme activity measures, by absorbance, the degree to which hyaluronic acid remaining in the reaction solution binds to acidified albumin (BSA) to form aggregates. When hyaluronic acid is hydrolyzed by PH20, the amount that binds to albumin decreases and the absorbance decreases. Dilute BTH (Sigma) as a standard product to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, 60 units / mL and prepare in each tube. Dilute the purified protein sample 100X, 300X, 600X, 1200X, 2400X with enzyme dilution buffer (20 mM Tris·HCl, pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) and prepare in each tube. Dilute the 3 mg / mL hyaluronic acid solution 10-fold in a new tube so that the concentration becomes 0.3 mg / mL and the volume of each tube becomes 180 μL. Put 60 μL of enzyme into the diluted hyaluronidase solution and mix, and react at 37 °C for 45 minutes. When the reaction is complete, put 50 μL of the enzyme reacted in a 96-well plate and 250 μL of the acidic albumin solution into each well, shake for 10 minutes, and then measure the absorbance with a spectrophotometer at 600 nm.

[0181] When performing activity analysis in a 7-day 40 °C severe stability test, it was confirmed that the higher the concentration of polysorbate 80, the slightly larger the decrease in activity over time. When the same polysorbate 80 was included, it was confirmed that the decrease in activity was smaller in the dosage form containing trehalose than in the dosage form containing sucrose. (Figure 18)

[0182] Example 27. pH Activity Profiles of HP46 and Wild-Type HW2

[0183] For the experiment to confirm the pH activity profiles of HP46 and wild-type HW2, the microturbidimetric assay method was used. Hyaluronic acid buffer for dissolving the substrate hyaluronic acid and enzyme buffer for diluting the enzyme were prepared separately for each pH.

[0184] A total of three 96-well plates were prepared for the reaction of the enzyme and the substrate, designated as A, B, and C, and the tests were conducted.

[0185] For the ranges of pH 4.0, 4.5, and 5.0 in the hyaluronic acid buffer, they were prepared using 20 mM acetic acid and 70 mM NaCl. For the ranges of pH 5.5, 6.0, 6.5, 7.0, and 8.0, they 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. Each was diluted with the hyaluronic acid buffer prepared according to different pH values to 500 μL each to obtain concentrations of 0.1, 0.25, 0.45, and 0.7 mg / mL, and 100 μL of each was dispensed into the 96-well plate of Group A. The hyaluronic acid buffer diluted and prepared according to different concentrations was used for the calibration curve for measuring the hyaluronic acid concentration.

[0186] In the enzyme buffer, for the ranges of pH 4.0, 4.5, and 5.0, they were prepared using 20 mM acetic acid, 0.01% (w / v) BSA, and 70 mM NaCl. For the ranges of pH 5.5, 6.0, 6.5, 7.0, and 8.0, they were prepared using 20 mM sodium phosphate, 0.01% (w / v) BSA, and 70 mM NaCl.

[0187] HP46 and wild-type HW2 enzymes were diluted to 10 units / mL with the enzyme buffer prepared according to different pH values and 50 μL of each was dispensed into the 96-well plate of Group B.

[0188] 50 μL of each was transferred from the 96-well plate of Group A to the 96-well plate of Group B and reacted in a 37°C shaking incubator for 45 minutes. 15 minutes before the end of the reaction, 200 μL of the acidic albumin solution was dispensed into the 96-well plate of Group C for preparation. When the enzyme-substrate reaction was completed, 40 μL of each was transferred from the 96-well plate of Group B to the 96-well plate of Group C and reacted for 20 minutes. After 20 minutes had elapsed, the absorbance was measured at 600 nm, and the amount of hyaluronic acid remaining after the enzyme-substrate reaction was calculated to complete the activity profile of the enzyme according to different pH values (Figure 19).

[0189] Example 28. Pharmacokinetic study of Herceptin subcutaneous dosage form and trastuzumab combined with HP46 in Sprague-Dawley rats

[0190] To investigate whether the subcutaneous dosage form of trastuzumab combined with HP46 exhibits pharmacokinetic properties equivalent to those of the subcutaneous dosage form of Herceptin, a study was conducted using 9-week-old Sprague-Dawley rats. The administered amounts of Herceptin and trastuzumab were 18 mg / kg based on the rat body weight, the rHuPH20 contained in the Herceptin subcutaneous dosage form was 100 U, and HP46 was also 100 U. In the pharmacokinetic study, trastuzumab combined with HP46 showed the same area under the curve (AUC) as the Herceptin subcutaneous dosage form (Figure 20).

Industrial Applicability

[0191] The pharmaceutical composition according to the present invention can be used for subcutaneous injection and is very stable, so that the activity of drugs, preferably antibody pharmaceuticals, etc., and PH20 variants can be maintained for a long period. Therefore, in addition to reducing the production cost of the subcutaneous dosage form, it can also contribute to the reduction of medical expenses and is very advantageous in terms of patient convenience.

[0192] References 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. Borders jr., C.L. and Raftery, A. (1968) Purification and Partial Characterization of Testicular Hyaluronidase. J Biol Chem 243, 3756 - 3762 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. 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 Frost, G.I. (2007). Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration. Expert Opin Drug Deliv 4, 427 - 440 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 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. 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) a drug, and (b) a PH20 variant, wherein the PH20 variant comprises substitution of one or more amino acid residues selected from the group consisting of S343E, M345T, K349E, L353A, L354I, N356E and I361T in wild-type PH20 having the sequence of SEQ ID NO:

1. A pharmaceutical composition characterized by this.

2. The pharmaceutical composition according to claim 1, wherein the PH20 variant comprises substitution of one or more amino acid residues selected from the group consisting of L354I and N356E.

3. The pharmaceutical composition according to claim 1, wherein the PH20 variant further comprises substitution of one or more amino acid residues at one or more sites selected from the group consisting of the alpha-helix site of wild-type PH20 of SEQ ID NO: 1 and the sites corresponding to its linking sites.

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

5. The pharmaceutical composition according to claim 4, wherein the sites corresponding to the alpha-helix site of wild-type PH20 of SEQ ID NO: 1 and its linking sites are T341 - N363, T341 - I361, L342 - I361, S343 - I361, I344 - I361, M345 - I361 or M345 - N363.

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

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

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

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

10. The PH20 variant further contains substitutions of one or more amino acid residues selected from the group consisting of T341S, L342W, S343E, I344N, and N363G. The pharmaceutical composition according to claim 9.

11. The PH20 variant contains any one amino acid substitution selected from the group consisting of the following: The pharmaceutical composition according to claim 10. (a) T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (b) L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (c) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; (d) M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, I361T, and N363G; (e) I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T; and (f) S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, L354I, D355K, N356E, E359D, and I361T.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the PH20 variant further has some amino acid residues deleted at one or more of the C-terminal and N-terminal ends.

13. The pharmaceutical composition according to claim 12, wherein the PH20 variant is cleaved before an amino acid residue selected from the group consisting of M1 to P42 at the N-terminal, and some amino acid residues are deleted.

14. The pharmaceutical composition according to claim 13, wherein the PH20 variant is cleaved before the L36, N37, F38, R39, A40, P41 or P42 residue at the N-terminal, and some amino acid residues are deleted.

15. The pharmaceutical composition according to claim 12, wherein the PH20 variant is cleaved after an amino acid residue selected from the group consisting of V455 to L509 at the C-terminal, and some amino acid residues are deleted.

16. The pharmaceutical composition according to claim 15, wherein the PH20 variant is cleaved after an amino acid residue selected from the group consisting of V455 to S490 at the C-terminal, and some amino acid residues are deleted.

17. The pharmaceutical composition according to claim 16, wherein the PH20 variant is cleaved after the V455, C458, D461, C464, I465, D466, A467, F468, K470, P471, P472, M473, E474, T475, E476, P478, I480, Y482, A484, P486, T488 or S490 residue at the C-terminal, and some amino acid residues are deleted.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the PH20 variant further contains a signal peptide derived from human hyaluronidase-1 (Hyal1), human growth hormone or human serum albumin at the N-terminal.

19. The pharmaceutical composition according to any one of claims 1 to 11, wherein the PH20 variant is selected from the group consisting of the amino acid sequences of SEQ ID NO: 5 to SEQ ID NO:

50.

20. The pharmaceutical composition according to claim 19, wherein the PH20 variant has the sequence of SEQ ID NO:

44.

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

22. The pharmaceutical composition according to claim 21, wherein the drug is an antibody, a soluble receptor, or an Fc fusion protein with a soluble receptor.

23. The antibody according to claim 22, which binds to one or more antigens selected from the group consisting of 4-1BB, integrin, amyloid beta, angiopoietin, angiopoietin-like substance 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 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, PCSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, and VWF.

24. The antibodies include Utomilumab, Natalizumab, Etrolizumab, Vedolizumab, Bimagrumab, Bapineuzumab, Crenezumab, Solanezumab, Aducanumab, Gantenerumab, AMG 780, MEDI 3617, Nesvacumab, Vanucizumab, Evinacumab, Tabalumab, Lanalumab, Belimumab, Omburtamab, Ravulizumab, Eculizumab, Mogamulizumab, Otelixizumab, Teplizumab, Muromonab, Tebentafusp, Blinatumomab, REGN1979, Ibalizumab, Zanolizumab, Itolizumab, Efalizumab, Inebilizumab, Tafasitamab, Loncastuximab tesirine, Ocrelizumab, Ublituximab, Obinutuzumab, Ofatumumab, Rituximab, Tositumomab, Ibritumomab tiuxetan, Epratuzumab, Inotuzumab ozogamicin, Moxetumomab pasudotox, Brentuximab vedotin,Vadastuximab talirine, Gemtuzumab ozogamicin, Daratumumab, Isatuximab, Alemtuxumab, Crizanlizumab, Polatuzumab vedotin, Galiximab, Eptinezumab, Fremantuzumab, Galcanezumab, Erenumab, Zolbetuximab, Lampalizumab, Tremelimumab, Zalifrelimab, Ipilimumab, Rovapituizumab tesirine, Cetuximab, Depatuxizumab, Zalutumumab, Necitumumab, Panitumumab, Emicizumab, Nipocalimab, Rozanolixizumab, Burosumab, Farletuzumab, Mirvetuximab soravtansine, Dinutuximab, Naxitamab, Otilimab, Margatuximab, Pertuzumab, Trastuzumab, Trastuzumab deruxtecan, Trastuzumab emtansine, Trastuzumab duocarmazine, Patritumab, Anifrolumab, Emapalumab, Ligezumab,Omalizumab, Dalotuzumab, Figitumumab, Teprotumumab, Gebokizumab, Canakinumab, Daclizumab, Basiliximab, Dupilumab, Mepolizumab, Reslizumab, Benralizumab, Clazakizumab, Olokizumab, Sirukumab, Siltuximab, Sarilumab, Satralizumab, Tocilizumab, REGN88, Secukinumab, Ustekinumab, Briakinumab, Lebrikizumab, Tralokinumab, Ixekizumab, Bimekizumab, Brodalumab, Brazikumab, Guselkumab, Risankizumab, Tildrakizumab, Mirikizumab, Nemolizumab, Spesolimab, Relatlimab, Narsoplimab, Fasinumab, Tanezumab, Alirocumab, Evolocumab, Bocozizumab, Lambrolizumab, Balstilimab, Camrelizumab, Cemiplimab, Dostarlimab, Progolimab, Sintilimab, Spartalizumab,The pharmaceutical composition according to claim 22, characterized in that it is one or more selected from the group consisting of tislelizumab, pembrolizumab, nivolumab, atezolizumab, avelumab, envafolimab, durvalumab, bintrafusp alpha, denosumab, elotuzumab, concizumab, marstacimab, infliximab, adalimumab, golimumab, certolizumab pegol, ozoralizumab, brolizumab, ranibizumab, bevacizumab, faricimab, ramucirumab, and caplacizumab.

25. The soluble receptor contained in the soluble receptor or the Fc fusion protein with the soluble receptor is selected from the group consisting of TNF-α soluble receptor, VEGF soluble receptor, CTLA-4, interleukin 1 soluble receptor, and LFA3 soluble receptor. The pharmaceutical composition according to claim 22, characterized in that.

26. The Fc fusion protein with the soluble receptor is selected from the group consisting of etanercept, aflibercept, abatacept, belatacept, rilonacept, and alefacept. The pharmaceutical composition according to claim 25, characterized in that.

27. The pharmaceutical composition according to claim 1, further comprising one or more selected from the group consisting of a buffer, a stabilizer, and a surfactant.

28. The buffer is one or more buffers selected from the group consisting of malate, formatate, 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, BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid), pyrophosphate, and triethanolamine; The stabilizer is one or more selected from the group consisting of carbohydrates, saccharides or hydrates thereof, sugar alcohols or hydrates thereof, and amino acids; The pharmaceutical composition according to claim 27, wherein the surfactant is one or more nonionic surfactants selected from the group consisting of polyoxyethylene-sorbitan fatty acid, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, polyoxyethylene-polyoxypropylene copolymer, and sodium dodecyl sulfate (SDS).

29. The carbohydrate, saccharide or sugar alcohol is one or more selected from the group consisting of trehalose or its hydrate, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabinitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomaltol, maltitol, polyglycitol, cyclodextrin, hydroxypropyl-β-cyclodextrin and glucose, The amino acid is one or more selected from the group consisting of glutamine, glutamic acid, glycine, lysine, lysyl lysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolysine, histidine and alanine, and the pharmaceutical composition according to claim 28 is characterized in that.

30. The pharmaceutical composition according to claim 27, characterized in that it contains a histidine buffer solution at pH 5.5 ± 2.0, trehalose and methionine.

31. The pharmaceutical composition according to claim 27, characterized in that it contains a histidine buffer solution at pH 5.5 ± 2.0, trehalose, methionine and polysorbate.

32. The pharmaceutical composition according to claim 31, characterized in that it contains a histidine buffer solution at pH 5.5 ± 2.0, 10 to 400 mM of α,α-trehalose, 1 to 50 mM of methionine and 0.0000001% to 0.5% (w / v) of polysorbate.

33. An injectable form for subcutaneous administration containing the pharmaceutical composition according to any one of claims 1 to 32.

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