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

Abstract

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

Description

[0001] This application is a divisional application of the invention patent application filed on March 24, 2020, with application number 202080003052.8 and entitled "Pharmaceutical composition comprising human hyaluronidase PH20 variant and medicament for subcutaneous injection". Technical Field

[0002] This disclosure relates to a pharmaceutical composition comprising a human hyaluronidase PH20 variant having enhanced enzyme activity and thermal stability and one or more drugs; and a method of treating a disease using said pharmaceutical composition.

[0003] The pharmaceutical compositions according to this disclosure are preferably intended for subcutaneous injection. Background Technology

[0004] Medications requiring high or multiple doses (especially antibody drugs) are typically administered intravenously, a process that takes approximately 90 minutes or longer and involves additional preparation procedures, making it inconvenient and costly for patients, doctors, and healthcare professionals. In contrast, subcutaneous injection offers the advantage of immediate administration, but absorption is relatively lower compared to intravenous injection, and at doses of 3-5 mL or more, it can cause swelling and pain at the injection site due to slow absorption. For this reason, subcutaneous injections of protein therapies are usually limited to small doses of 2 mL or less. However, when hyaluronidase is administered subcutaneously (or via subcutaneous injection) along with the therapeutic drug, hyaluronic acid distributed in the extracellular matrix is ​​hydrolyzed by hyaluronidase, reducing viscosity and increasing permeability in the subcutaneous region, thus allowing for the easy delivery of high or multiple doses of the drug into the body.

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

[0006] Meanwhile, currently available commercial PH20 is in the form extracted from bovine or sheep testes. Examples include... (bovine hyaluronidase) and (Sheep hyaluronidase).

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

[0008] Recombinant human PH20 protein has been reported to be expressed in yeast (Pichia pastoris), DS-2 insect cells, and animal cells (Chen et al., 2016; Hofinger et al., 2007). The recombinant PH20 protein produced in insect cells and yeast differs from human PH20 in its N-glycosylation pattern during post-translational modification.

[0009] 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 identified. Hyal1 consists of two domains: a catalytic domain and an EGF-like domain. The catalytic domain is in the form of (β / α)8, where the α-helix and β-chain (characteristic of secondary protein structures) are each repeated eight times (Chao et al., 2007). The EGF-like domain is completely conserved in variants where the C-terminus of Hyal1 is spliced ​​differently. Hyal1 shares 35.1% amino acid sequence identity with PH20, and the tertiary structure of PH20 has not yet been identified.

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

[0011] Meanwhile, although research is underway to develop various therapeutics using human PH20 via subcutaneous injection, the issue of low stability of human PH20 itself remains unresolved.

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

[0013] The inventors of this application have also confirmed that the PH20 variant according to this disclosure can be applied to pharmaceutical compositions or preparations containing drugs (e.g., antibody drugs, particularly high-dose anti-HER2 antibodies or immune checkpoint antibodies), and therefore the pharmaceutical compositions and preparations according to this disclosure (containing the PH20 variant and drugs, such as anti-HER2 antibodies or immune checkpoint antibodies) can be used for subcutaneous injection, and the activity of the drugs (such as antibody drugs) and the PH20 variant is very stable and can be maintained for a long time, thus completing this disclosure. Summary of the Invention

[0014] Technical issues

[0015] Therefore, this disclosure is made in response to the above-mentioned problems, and one object of this disclosure is to provide a novel pharmaceutical composition comprising a PH20 variant having enhanced enzyme activity and thermal stability, and a drug, wherein the thermal stability and activity of the drug and the PH20 variant can be maintained for a long time; particularly a pharmaceutical composition suitable for subcutaneous injection.

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

[0017] Technical solutions

[0018] According to this disclosure, the above and other objectives can be achieved by providing a pharmaceutical composition comprising (a) a drug and (b) a PH20 variant.

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

[0020] The pharmaceutical compositions according to this disclosure may further comprise one or more of pharmaceutically acceptable additives, particularly buffers, stabilizers and surfactants.

[0021] The pharmaceutical compositions according to this disclosure can be used in the form of injectable preparations for subcutaneous injection. Attached Figure Description

[0022] The above and other objects, features and advantages of this disclosure will become clearer from the following detailed description taken in conjunction with the accompanying drawings, in which:

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

[0024] Figure 2 Results of measuring protein aggregation temperature in formulations containing trastuzumab and the novel PH20 variant HP46 are shown.

[0025] Figure 3 A is a weak cation exchange (WCX) chromatogram of trastuzumab in a stability test under harsh conditions at 45°C; B shows the relative change (%) of the acid variant in the formulation in a stability test under harsh conditions at 45°C; C shows the relative change (%) of the main peak in the formulation in a stability test under harsh conditions at 45°C; and D shows the relative change (%) of the basic variant in the formulation in a stability test under harsh conditions at 45°C.

[0026] Figure 4 The changes in the purity of trastuzumab monomer protein in formulations 5-7 are shown in a stability test under harsh conditions of 45°C.

[0027] Figure 5 A shows the change (%) of the relative amount of acidic variants in formulations 5-7 during stability testing under harsh conditions at 45°C; B shows the change (%) of the relative amount of the main peaks of formulations 5, 6 and 7 during stability testing under harsh conditions at 45°C; and C shows the change (%) of the relative amount of basic variants of formulations 5, 6 and 7 during stability testing under harsh conditions at 45°C.

[0028] Figure 6 A shows the results of residual enzyme activity measured on days 0 and 1 in a stability test under harsh conditions of 40°C for Herceptin subcutaneous injection (Herceptin SC), trastuzumab + wild-type PH20 (HW2), and trastuzumab + PH20 variant HP46, and B shows the results of residual enzyme activity measured on days 0 and 1 in a stability test under harsh conditions of 45°C for Herceptin subcutaneous injection, trastuzumab + wild-type PH20 (HW2), and trastuzumab + PH20 variant HP46.

[0029] Figure 7Size exclusion chromatography analysis results of formulations 8-10 were shown during a stability test conducted under harsh conditions at 40°C for 14 days.

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

[0031] Figure 9A The weak cation exchange (WCX) chromatogram of formulation 8 is shown in the stability test under harsh conditions of 40°C. Figure 9B The changes (%) in the relative amounts of acidic variants in formulations 8-10 are shown in stability tests conducted under harsh conditions at 40°C. Figure 9C The relative change (%) of the main peak of formulations 8-10 in stability tests under harsh conditions of 40°C is shown, and Figure 9D The relative changes (%) of basic variants in formulations 8-10 are shown in stability tests conducted under harsh conditions at 40°C.

[0032] Figure 10 The changes (%) in relative enzyme activity of formulations 8-10 during stability testing under harsh conditions at 40°C are shown.

[0033] Figure 11 The changes in the purity of trastuzumab monomers in formulations 11-13 are shown in stability tests under harsh conditions of 40°C.

[0034] Figure 12A The weak cation exchange (WCX) chromatogram of formulation 11 is shown in the stability test under harsh conditions of 40°C. Figure 12B The changes (%) in the relative amounts of acidic variants in formulations 11-13 during stability testing under harsh conditions at 40°C are shown. Figure 12C The relative changes (%) of the main peaks of formulations 11-13 in stability tests under harsh conditions of 40°C are shown, and Figure 12D The relative changes (%) of basic variants in formulations 11-13 are shown in stability tests under harsh conditions at 40°C.

[0035] Figure 13 The changes (%) in relative enzyme activity of formulations 11-13 under harsh conditions of 40°C are shown.

[0036] Figure 14 The changes in rituximab monomer purity in formulations 14-16 are shown in stability tests conducted under harsh conditions at 40°C.

[0037] Figure 15The changes in relative enzyme activity of formulations 14-16 under harsh conditions of 40°C in stability tests are shown.

[0038] Figure 16 The changes in relative enzyme activity of formulations 17 and 18 are shown in stability tests under harsh conditions of 40°C.

[0039] Figure 17 The results of size exclusion chromatography analysis of formulations 19-22 at 40°C are shown.

[0040] Figure 18 The changes in relative enzyme activity of formulations 19-22 under harsh conditions of 40°C in stability tests are shown.

[0041] Figure 19 The changes in enzyme activity caused by pH variations in recombinant human PH20 and HP46 are shown; and

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

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

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

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

[0046] In this disclosure, “mature wild-type PH20” means a protein containing the following amino acid residues: amino acid residues L36 to S490 of SEQ ID NO:1, which lack M1 to T35 for forming a signal peptide; and amino acid residues A491 to L509 of the amino acid sequence of wild-type PH20 having the sequence of SEQ ID NO:1, which are not related to the substantive function of PH20.

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

[0048]

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

[0050] In this disclosure, the term "PH20 variant" is intended to include mutations, preferred substitutions of amino acid residues in the sequence of wild-type human PH20; and deletions of some amino acid residues at the N-terminus and / or C-terminus along with such substitutions of amino acid residues, and is used in a substantially the same sense as the expression "PH20 variant or fragment thereof".

[0051] The inventors of this disclosure have demonstrated that novel PH20 variants or fragments thereof with enhanced enzyme activity and thermostability compared to wild-type PH20 can be provided based on previous research and experimental results in which the enzyme activity and protein aggregation temperature (Tagg) at neutral pH are increased when the amino acid sequence of the α-helix 8 region and the linker region between the α-helix 7 and α-helix 8 of human PH20 is partially replaced by the amino acid sequence of the α-helix 8 region and the linker region between the α-helix 7 and α-helix 8 of the highly hydrophilic Hyal1.

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

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

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

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

[0056]

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

[0058]

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

[0060] Furthermore, it is preferred that amino acid residue substitutions be further included at one or more positions selected from T341 to N363, particularly at one or more positions selected from T341, L342, S343, I344, M345, S347, M348, K349, L352, L353, D355, E359, I361, and N363, but this disclosure is not limited thereto, and

[0061] More preferably, it further comprises one or more amino acid residues selected from T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T and N363G, but this disclosure is not limited thereto.

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

[0063] It may further include one or more amino acid residues selected from T341S, L342W, S343E, I344N and N363G as substitutions, but this disclosure is not limited thereto.

[0064] More preferably, the PH20 variant or fragment thereof contained in the pharmaceutical composition according to this disclosure may contain, but is not limited to, any substitution selected from the group consisting of:

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

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

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

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

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

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

[0071] In this disclosure, expressions such as “S347”, which are described by a one-letter amino acid residue code along with a number, refer to the amino acid residue at the corresponding position in the amino acid sequence of SEQ ID NO:1.

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

[0073] According to this disclosure, the PH20 variant contained in the pharmaceutical composition is interpreted as including a variant in which an amino acid residue at a specific amino acid residue position is conservatively substituted.

[0074] As used herein, the term “conservative substitution” refers to a modification of the PH20 variant in which one or more amino acids are replaced by amino acids with similar biochemical properties, the latter of which do not cause a loss of the biological or biochemical function of the corresponding PH20 variant.

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

[0076] It is expected that the PH20 variant contained in the pharmaceutical composition according to this disclosure will retain its activity despite having conserved amino acid substitutions.

[0077] Furthermore, the PH20 variant or fragment thereof contained in the pharmaceutical composition according to this disclosure is interpreted as including the PH20 variant or fragment thereof having substantially the same function and / or effect as the PH20 variant or fragment thereof according to this disclosure, and having at least 80% or 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99% amino acid sequence homology with the PH20 variant or fragment thereof according to this disclosure.

[0078] Compared to mature wild-type PH20, the PH20 variant according to this disclosure exhibits increased expression levels and increased protein refolding rate in animal cells, resulting in improved thermal stability. Furthermore, despite the improved thermal stability, the PH20 variant exhibits enzymatic activity exceeding or similar to that of mature wild-type PH20.

[0079] Furthermore, it is known that enzyme activity decreases when certain amino acids (such as S490) at the C-terminus of mature wild-type PH20 are additionally cleaved. However, the PH20 variant according to this disclosure exhibits improved thermostability and increased or similar enzyme activity compared to mature wild-type PH20, even though the C-terminus of mature wild-type PH20 has an additionally cleaved sequence. Additionally, the PH20 variant maintains its enzyme activity when cleaved from up to five amino acid residues from the N-terminus, suggesting that residues starting at P41 from the N-terminus play an important role in protein expression and enzyme activity.

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

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

[0082] The statement "cleavage before L36, N37, F38, R39, A40, P41, or P42 at the N-terminus" means cleaving and removing all amino acid residues immediately preceding L36 (M1 to T35), immediately preceding N37 (M1 to L36), immediately preceding F38 (M1 to N37), immediately preceding R39 (M1 to F38), immediately preceding A40 (M1 to R39), immediately preceding P41 (M1 to A40), and immediately preceding P42 (M1 to P41). The statement "cleavage before M1 at the N-terminus of SEQ ID NO:1" means no cleavage occurs at the N-terminus.

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

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

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

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Meanwhile, previous studies have reported that the enzyme activity of wild-type PH20 varies depending on the cleavage position of the amino acid residues located at the C-terminus. However, in this disclosure, the specific α-helix forming the secondary structure of PH20 is replaced by the α-helix of other human hyaluronidases, thereby constructing PH20 variants with higher stability than wild-type PH20. In these variants, the interaction between the substituted α-helix domain of PH20 and other secondary structures exhibits a different pattern than that of wild-type PH20, thus the variants possess certain or higher levels of enzyme activity regardless of the cleavage position at the C-terminus.

[0103] In addition, this disclosure attempts to increase the expression of recombinant PH20 protein by replacing the original signal peptide of human PH20 with the signal peptide of other proteins that exhibit high protein expression levels in animal cells.

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

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

[0106]

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

[0108] HP46 (SEQ ID NO:44) is a human PH20 variant obtained by modeling the protein structure using Hyal1 (PDB ID: 2PE4) (Chao et al., 2007), which has a known protein tertiary structure and is human hyaluronidase. The amino acid sequence of α-helix 8 and the linker region between α-helix 7 and α-helix 8 is then substituted with the amino acid sequence of Hyal1, and the N-terminus is cleaved at F38 and the C-terminus is cleaved after F468. Specifically, α-helix 8 is located outside the PH20 protein tertiary structure and interacts less with adjacent α-helices or β chains compared to other α-helices. Generally, there is a trade-off between enzyme activity and thermostability; therefore, higher protein thermostability corresponds to lower enzyme activity, and when enzyme activity increases due to increased protein structural flexibility, thermostability tends to decrease. However, the specific activity of HP46, measured by turbidity determination at pH 7.0, was approximately 46 units / μg, which was evaluated as approximately twice the specific activity of wild-type PH20, which had a specific activity of approximately 23 units / μg.

[0109] The thermal stability of proteins can be evaluated based on the melting temperature Tm (at which 50% of the protein's tertiary structure denatures) and the aggregation temperature Tagg (at which proteins aggregate). Generally, the aggregation temperature of proteins tends to be lower than their melting temperature. The α-helix 8 of Hyal1 has greater hydrophilicity than that of PH20. The substituted α-helix 8 of Hyal1 increases the surface hydrophilicity of HP46, thereby causing a delayed effect on protein aggregation due to hydrophobic interactions, resulting in an aggregation temperature of 51 °C, which is observed to be 4.5 °C higher than that of wild-type PH20 (46.5 °C).

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

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

[0112] Examples of pharmaceuticals included in the pharmaceutical compositions according to this disclosure include, but are not limited to, protein drugs, antibody drugs, small molecules, aptamers, RNAi, antisenses, and cell therapy agents (such as chimeric antigen receptor (CAR)-T or CAR-natural killer (NK) cells), and may include not only currently commercially available drugs, but also drugs in clinical trials or under development.

[0113] As a drug, protein drugs or antibody drugs are preferred.

[0114] The “protein drug” included in the pharmaceutical composition according to this disclosure is a drug composed of amino acids and thus exhibiting a therapeutic or preventive effect on disease through the activity of proteins; a drug composed of proteins other than antibody drugs; and may be selected from cytokines, therapeutic enzymes, hormones, soluble receptors and their fusion proteins, insulin or analogues thereof, bone morphogenetic protein (BMP), erythropoietin and serum-derived proteins, but this disclosure is not limited thereto.

[0115] The cytokines contained in the pharmaceutical compositions according to this disclosure may be selected from interferon, interleukin, colony-stimulating factor (CSF), tumor necrosis factor (TNF) and tissue growth factor (TGF), but this disclosure is not limited thereto.

[0116] Therapeutic enzymes may include, but are not limited to, β-glucocerebrosidase and agalsidase β.

[0117] The soluble receptor included in the pharmaceutical composition according to this disclosure is the extracellular domain of a receptor, and its fusion protein is a protein in which the Fc region of an antibody is fused with the soluble receptor. A soluble receptor is a soluble form of a disease-associated ligand-binding receptor, and examples include forms in which the Fc region is fused with a TNF-α soluble receptor (e.g., products containing the ingredient etanercept and similar forms), forms in which the Fc region is fused with a VEGF soluble receptor (e.g., products containing the ingredient alefacept and similar forms), forms in which the Fc region is fused with a CTLA-4 receptor (e.g., products containing the ingredients abatacept or beraccept and similar forms), forms in which the Fc region is fused with an interleukin-1 soluble receptor (e.g., products containing the ingredient linascept and similar forms), and forms in which the Fc region is fused with an LFA3 soluble receptor (e.g., products containing the ingredient alefacept and similar forms), but this disclosure is not limited thereto.

[0118] The hormones contained in the pharmaceutical compositions according to this disclosure refer to hormones or analogues thereof injected into the body for the treatment or prevention of diseases caused by hormone deficiency, and examples of said hormones or analogues include, but are not limited to, human growth hormone, estrogen, and progesterone.

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

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

[0121] According to this disclosure, a monoclonal antibody drug comprises a monoclonal antibody and a protein containing a monoclonal antibody fragment capable of specifically binding to an antigen associated with a specific disease. Monoclonal antibodies also include bispecific antibodies, and proteins containing monoclonal antibodies or fragments thereof conceptually include antibody-drug conjugates (ADCs).

[0122] Examples of antigens associated with specific diseases include 4-1BB, integrin, β-amyloid, angiopoietin (angiopoietin 1 or 2), angiopoietin analogue 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, tight junction protein-18, complement factor D, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2, and GM. -CSF, HER2, HER3, interferon receptor, interferon-γ, IgE, IGF-1 receptor, interleukin-1, interleukin-2 receptor, interleukin-4 receptor, interleukin-5, interleukin-5 receptor, interleukin-6, interleukin-6 receptor, interleukin-7, interleukin-12 / 23, interleukin-13, interleukin-17A, interleukin-17 receptor A, interleukin-31 receptor, interleukin-36 receptor, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, VEGF receptor and von Willebrand factor (vWF), but this disclosure is not limited thereto.

[0123] The following are, but are not limited to, proteins, including monoclonal antibodies or monoclonal antibody fragments targeting antigens associated with specific diseases:

[0124] Utomilumab is an anti-4-1BB antibody.

[0125] Natalizumab, etorizumab, vedolizumab, and bigromab are antibodies targeting integrin.

[0126] Bapineuzumab, crenezumab, solanezumab, aducanumab, and gantenezumab are antibodies targeting β-amyloid protein.

[0127] Antibodies against angiopoietin, such as AMG780 against angiopoietin 1 and 2, MEDI 3617 and Nescalumab against angiopoietin 2, and vancomycin as a bispecific antibody against angiopoietin 2 and VEGF.

[0128] Eviacumab is an antibody targeting angiopoietin analogue 3.

[0129] Tabalumab, lanalumab, and belimumab are antibodies targeting B cell activating factor (BAFF).

[0130] omburtamab is an antibody targeting B7-H3.

[0131] Ravulizumab and eculizumab are antibodies targeting complement 5.

[0132] Mogamulizumab is an antibody targeting CCR4.

[0133] Osteizumab, teplizumab, and muromonab are antibodies against CD3; tebentafusp is a bispecific antibody against GP100 and CD3; blinatumomab is a bispecific antibody against CD19 and CD3; and REGN1979 is a bispecific antibody against CD20 and CD3.

[0134] Ibacillinumab and zanolimumab are antibodies targeting CD4.

[0135] Itrazumab is an antibody targeting CD6.

[0136] efalizumab is an antibody targeting CD11a.

[0137] Inbilizumab, tafasitamab, and loncastuximab tesirine are antibodies against CD19 (the loncastuximab is an ADC).

[0138] Oribitusumab, ublituximab, obbituzumab, ofamumab, rituximab, tosimomab, and ibritumomab tiuxetan are antibodies against CD20 (the latter being an ADC).

[0139] Epratuzumab, inotuzumabozogamicin (the inotuzumab being an ADC), and moxetumomab pasudotox are antibodies against CD22.

[0140] Brentuximab vedotin is an ADC targeting CD30.

[0141] Tadalirine (vadastuximab talirine) and gemtuzumab (gemtuzumab ozogamicin) are ADCs targeting CD33.

[0142] Daratumumab and isatuximab are antibodies targeting CD38.

[0143] alemtuzumab is an antibody targeting CD52;

[0144] Crizanlizumab is an antibody targeting CD62.

[0145] Porcuzumab vedotin is an ADC targeting CD79b.

[0146] Galiximab is an antibody targeting CD80;

[0147] Eptinezumab, fremanezumab, galcanezumab, and erenumab are antibodies against CGRP.

[0148] Zoloximab is an antibody targeting tight junction protein-18;

[0149] Lanpamumab is an antibody targeting complement factor D.

[0150] Trimemomab, zalifrelimab, and ipilimumab are antibodies targeting CTLA4.

[0151] Rovapituzumab tesirine is an ADC targeting DLL3;

[0152] Cetuximab, depatuximab, zarumumab, necitumumab, and panitumumab are antibodies targeting the EGF receptor.

[0153] emicizumab is a bispecific antibody targeting coagulation factor IX and factor X (which are hemophilia factors).

[0154] Nipocalimab and rozanolixizumab are antibodies targeting the Fc receptor.

[0155] Burosumab is an antibody targeting FGF23.

[0156] Farletuzumab, an antibody targeting the folate receptor, and mirvetuximab soravtansine, an ADC targeting the folate receptor.

[0157] Dinutuximab and naxitamab are antibodies targeting GD2.

[0158] Otelimab is an antibody targeting GM-CSF;

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

[0160] Patritumab is an antibody targeting HER3.

[0161] Anifrolumab is an antibody that targets the interferon receptor.

[0162] Emapalumab is an antibody targeting interferon-gamma.

[0163] Ligozumab and omalizumab are antibodies targeting IgE.

[0164] Dalotuzumab, figitumumab, and teprotumumab are antibodies targeting the IGF-1 receptor.

[0165] Gebokizumab and canakinumab are antibodies against interleukin-1.

[0166] Dacizumab and baliximab are antibodies targeting the interleukin-2 receptor;

[0167] Dupilumab is an antibody targeting the interleukin-4 receptor.

[0168] Mepozumab and Rosumab are antibodies targeting interleukin-5;

[0169] Benalizumab is an antibody targeting the interleukin-5 receptor.

[0170] Clagizumab, ologiizumab, slucurumab, and stoximab are antibodies targeting interleukin-6.

[0171] Sarralizumab, satralizumab, tocilizumab, and REGN88 are antibodies targeting the interleukin-6 receptor.

[0172] Secukinumab is an antibody targeting interleukin-7;

[0173] Utecumab and briakinumab are antibodies targeting interleukin-12 / 23;

[0174] Lebrikizumab and tralokinumab are antibodies targeting interleukin-13.

[0175] Ixekizumab and bimekizumab are antibodies targeting interleukin-17A.

[0176] Brodalumab is an antibody targeting interleukin-17 receptor A.

[0177] Brazikumab, guselkumab, risankizumab, tildrakizumab, and mirikizumab are antibodies targeting interleukin-23.

[0178] Nemozumab is an antibody targeting the interleukin 31 receptor;

[0179] Pesolimab is an antibody targeting the interleukin-36 receptor.

[0180] Relatlimab is an antibody targeting LAG3.

[0181] Narsoplimab is an antibody targeting NSP2.

[0182] Faximab and tanezumab are antibodies against NGF.

[0183] Alikumab, evokumab, and bococizumab are antibodies targeting PVSK9.

[0184] Antibodies targeting PD-1 include lambrolizumab, balstilimab, camrelizumab, cemiplimab, dostarlimab, prolgolimab, shintilimab, spartalizumab, tislelizumab, pembrolizumab, and nivolumab.

[0185] Atezolizumab, avelumumab, envafolimab, and duvamarumab are antibodies against PD-L1, and bintrafuspα is a bispecific antibody against TGFβ and PD-L1.

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

[0187] Elozizumab, an antibody targeting SLAMF7;

[0188] Concizumab and marstacimab are antibodies against tissue factor.

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

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

[0191] Ramucirumab, an antibody targeting the VEGF receptor; and

[0192] Caplacizumab is an antibody targeting vWF.

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

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

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

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

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

[0198] The polyclonal antibodies contained in the pharmaceutical compositions according to this disclosure are preferably serum antibodies extracted from serum, such as immunoglobulins, but not limited thereto.

[0199] In the case of small molecule compounds, any drug requiring rapid preventative or therapeutic effects can be used without restriction. For example, morphine-based analgesics can be used (Thomas et al., 2009). Additionally, when used as a therapeutic agent for tissue necrosis caused by anticancer drugs, small molecule compounds can be used alone or in combination with antidotes such as vinca alkaloids and taxanes (Kreidieh et al., 2016).

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

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

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

[0203] Stabilizers in the compositions according to this disclosure may be used without limitation, provided that they are generally used in the art for the purpose of stabilizing proteins, and preferably, the stabilizers may be, for example, one or more selected from: carbohydrates, sugars or their hydrates, sugar alcohols or their hydrates, and amino acids.

[0204] The carbohydrate, sugar, or sugar alcohol used as a stabilizer may be selected from one or more of the following: trehalose or its hydrate, sucrose, saccharin, glycerol, erythritol, threitol, xylitol, arabinitol, ribitol, mannitol, sorbitol, galactitol, fucitol, idutol, inositol, heptaheptanol, isomaltitol, maltitol, polyglucitol, cyclodextrin, hydroxypropyl cyclodextrin, and glucose, but is not limited thereto.

[0205] Amino acids may be selected from one or more of the following: glutamine, glutamic acid, glycine, lysine, lysine, leucine, methionine, valine, serine, selenomethionine, citrulline, arginine, asparagine, aspartic acid, ornithine, isoleucine, taurine, theanine, threonine, tryptophan, tyrosine, phenylalanine, proline, pyrrolidone, histidine, and alanine, but are not limited thereto.

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

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

[0208] The compositions according to the invention may further contain surfactants.

[0209] Preferably, the surfactant can be 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, alkylphenyl polyoxyethylene ether [Triton-X], and polyoxyethylene-polyoxypropylene copolymer [poloxam and Plonic] and sodium dodecyl sulfate (SDS), but is not limited thereto.

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

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

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

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

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

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

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

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

[0218] There are no particular limitations on the diseases that can be treated with the pharmaceutical compositions or preparations according to this disclosure, and there are no limitations on them as long as they are diseases that can be treated with a combination of a drug with a PH20 variant according to this disclosure.

[0219] The diseases that can be treated with the pharmaceutical compositions or preparations according to this disclosure may be cancer or autoimmune diseases, but are not limited thereto.

[0220] There are no particular limitations on the cancers or carcinomas that can be treated with the pharmaceutical compositions or formulations according to this disclosure, and these include solid cancers and hematologic cancers. Examples of such cancers include, but are not limited to, 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, ureteral cancer, osteosarcoma, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma. Preferably, the cancers that can be treated with the pharmaceutical compositions or formulations according to this disclosure may be selected from, but are not limited to, gastric cancer, colorectal cancer, breast cancer, lung cancer, and kidney cancer.

[0221] Autoimmune diseases that can be treated with the pharmaceutical compositions or formulations disclosed herein include, but are not limited to, 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.

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

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

[0224] The present disclosure will be described in further detail below with reference to the following embodiments. These embodiments are provided for illustrative purposes only, and it will be clear to those skilled in the art that they should not be construed as limiting the scope of the present disclosure.

[0225] Example

[0226] Example 1. Formulation Development

[0227] As shown in Table 6, four trastuzumab subcutaneous injection formulations were prepared. Formulations 1 through 4 typically contain 120 mg / mL trastuzumab and consist of 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and a PH20 variant. The difference between formulations 1-4 is the concentration of nonionic surfactant, where formulation 1: 0% polysorbate 20, formulation 2: 0.005% polysorbate 20, formulation 3: 0.04% polysorbate 20, and formulation 4: 0.1% polysorbate 20.

[0228] Table 6. Composition of the preparation

[0229]

[0230] Example 2. Measurement using a spectrophotometer

[0231] Preparations 1 to 4 were incubated at 45°C for 14 days, and changes in protein concentration were analyzed using a Beckman spectrophotometer. Each sample was diluted with distilled water to a concentration of 0.4 mg / mL, and the absorbance of the protein at 280 nm was measured using a spectrophotometer. Under the harsh conditions of 14 days at 45°C, the protein concentration of preparations 1 to 4 did not change significantly. However, hyaluronidase activity decreased rapidly at 45°C; therefore, enzyme activity was not measured in this embodiment (see [link to documentation]). Figure 6 ).

[0232] Example 3. Size exclusion chromatography was used to study the monomer ratio of trastuzumab in each formulation.

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

[0234] Size exclusion chromatography (SGC) analysis during a stability test at harsh conditions (45°C) for 14 days showed similar patterns of change in formulations 1 through 4. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (approximately 1.5%), with no significant differences between formulations. In summary, as a result of size exclusion chromatography analysis during a stability test at harsh conditions (i.e., 45°C), there were no significant differences in stability characteristics between formulations based on the concentration of polysorbate 20 (0–0.1% (w / v)) (see [link to relevant documentation]). Figure 1 ).

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

[0236] Dynamic light scattering (DLS) was used to analyze the denaturing properties of proteins attributed to heat. In this experiment, changes in protein molecule size with temperature variations were measured and used to calculate protein aggregation temperatures. For DLS analysis, a Zetasizer-nano-ZS instrument and quartz cuvettes (ZEN2112), available from Malvern, were used. During the analysis, the temperature was increased from 25°C to 85°C at 1°C intervals, and the sample was diluted to 1 mg / mL using each prepared buffer before adding 150 μL of sample to the cuvette for analysis.

[0237] The polymerization temperature in formulation 1, which does not contain polysorbate 20, is 74°C, and the polymerization temperature in formulations 2 to 4 is 76°C (see [link to formulation 1]). Figure 2 ).

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

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

[0240] When WCX analysis was performed under harsh conditions, i.e., a 14-day stability test at 45°C, formulations 1 through 4 showed similar patterns of change. Specific changes included an increase in the relative content of the acidic variant (approximately 30% change after 14 days), a decrease in the relative content of the main peak (approximately 44% change after 14 days), and an increase in the relative content of the basic variant (approximately 15% change after 14 days), with no significant differences observed across formulations. In summary, the protein stability of polysorbate 20 (0–0.1%) was similar under harsh conditions, i.e., a 45°C stability test, according to WCX analysis (see [link to WCX analysis]). Figure 3 ).

[0241] Example 6. Formulation Development

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

[0243] Table 7. Composition of the preparation

[0244]

[0245] Example 7. Measurement using a spectrophotometer

[0246] Preparations 5 to 7 were incubated at 45°C for 14 days, and changes in protein concentration were analyzed using a Beckman spectrophotometer. Each sample was diluted with distilled water to a concentration of 0.4 mg / mL, and the absorbance of the protein at 280 nm was measured using a spectrophotometer. In the stability test under harsh conditions, i.e., continuous 14 days at 45°C, the protein concentration of preparations 5 to 7 did not change significantly. However, hyaluronidase activity decreased rapidly at 45°C; therefore, enzyme activity was not measured in this embodiment (see [link to documentation]). Figure 6 ).

[0247] Example 8. The monomer ratio of trastuzumab in each formulation was studied using size exclusion chromatography.

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

[0249] Size exclusion chromatography (SGC) analysis of formulations 5 through 7 showed similar patterns of change during a stability test conducted under harsh conditions (i.e., at 45°C) for 14 days. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) impurities and a decrease in monomer content (approximately 1.5%), and no significant differences were observed between formulations. In summary, as a result of size exclusion chromatography analysis during a stability test under harsh conditions (i.e., at 45°C), formulations of 0.04% polysorbate 20, 50 mM Lys-Lys, and 50 mM glycine showed similar protein stability (see [link to relevant documentation]). Figure 4 ).

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

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

[0252] When WCX analysis was performed under harsh conditions (i.e., 45°C) for 14 days of stability testing, formulations 5 through 7 showed similar patterns of change. Specific changes included an increase in the relative content of the acidic variant (approximately 30% change over 14 days), a decrease in the relative content of the main peak (approximately 44% change over 14 days), and an increase in the relative content of the basic variant (approximately 15% change over 14 days), with no significant differences observed across formulations. In summary, as a result of WCX analysis under harsh conditions (i.e., 45°C), formulations of 0.04% polysorbate 20, 50 mM Lys-Lys, and 50 mM glycine showed similar protein stability (see [link to relevant documentation]). Figure 5 ).

[0253] Example 10. Stability of HP46 was evaluated in subcutaneous injection formulations of trastuzumab and HP46 at temperatures of 40°C and 45°C.

[0254] To assess the stability of HP46 in the trastuzumab subcutaneous injection formulation, trastuzumab (120 mg / mL) and pH20 (2000 units / mL) were mixed. The buffer used contained 20 mM histidine (pH 5.5), 210 mM trehalose, 10 mM methionine, and 0.04% polysorbate 20. Enzyme activity in the control samples was measured on day 0, and the experimental samples were incubated at 40°C or 45°C for 1 day, after which enzyme activity was measured for each sample.

[0255] Herceptin subcutaneous injection formulations of trastuzumab + HW2 and trastuzumab + HP46 were each incubated at 40°C for 1 day, and hyaluronidase activity was then measured. The results showed 51%, 47%, and 94% activity in each case, respectively, indicating that HP46 exhibited the greatest thermostability at 40°C (see [link to product description]). Figure 6Additionally, the Herceptin subcutaneous formulations trastuzumab + HW2 and trastuzumab + HP46 were incubated at 45°C for one day, and then hyaluronidase activity was measured. As a result, the Herceptin subcutaneous formulation and trastuzumab + HW2 showed no enzyme activity, but trastuzumab + HP46 retained its enzyme activity (see [link to original text]). Figure 6 ).

[0256] Example 11. Formulation Development

[0257] As shown in Table 8, three trastuzumab subcutaneous injection formulations were prepared. Formulations 8 to 10 typically contain 120 mg / mL trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and a pH 20 variant. The difference between formulations 8 and 10 is the concentration of nonionic surfactant, with formulation 8 containing 0% polysorbate 20, formulation 9 containing 0.005% polysorbate 20, and formulation 10 containing 0.04% polysorbate 20.

[0258] Table 8. Composition of the preparation

[0259]

[0260] Example 12. Measurement using a spectrophotometer

[0261] Preparations 8 to 10 were incubated at 40°C for 14 days, and changes in protein concentration were analyzed using a Beckman spectrophotometer. Each sample was diluted with distilled water to a concentration of 0.4 mg / mL, and the absorbance of the protein at 280 nm was measured using a spectrophotometer. Under the harsh conditions of 14 days at 40°C, the protein concentration of preparations 8 to 10 showed no significant change.

[0262] Example 13. The monomer ratio of trastuzumab in each formulation was studied using size exclusion chromatography.

[0263] For size exclusion chromatography analysis, an HPLC system available from Shimadzu Prominence was used, with a TSK-gel G3000SWXL (7.8 x 300 mm, 5 μm) and a TSK guard column (6.0 x 4.0 mm, 7 μm) as the column. A 0.2 M potassium phosphate solution (pH 6.2) containing 0.25 M potassium chloride was used as the mobile phase. Analysis was performed in isocratic separation mode at a flow rate of 0.5 mL / min for 35 minutes. The sample was diluted with the analytical solvent to a final concentration of 10 mg / mL, and the absorbance was measured at 280 nm after injecting 20 μL of sample into the HPLC column. The monomer ratio of trastuzumab in the HPLC chromatogram was calculated and plotted.

[0264] Size exclusion chromatography (SGC) analysis during a stability test at harsh conditions (40°C) for 14 days showed similar patterns of change in formulations 8 through 10. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (approximately less than 1.0%), and no significant differences were observed between formulations. In summary, as a result of size exclusion chromatography analysis during a stability test at harsh conditions (40°C), there were no significant differences in the overall stability profile between formulations based on the concentration of polysorbate 20 (0–0.04%) (see [link to relevant documentation]). Figure 7 ).

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

[0266] Dynamic light scattering (DLS) was used to analyze the denaturation properties of proteins attributed to heat in the field of protein pharmaceuticals. In this experiment, the change in protein molecule size with temperature variations was measured and used to calculate the protein aggregation temperature. For DLS analysis, a Zetasizer-nano-ZS instrument and quartz cuvettes (ZEN2112), available from Malvern, were used. During the analysis, the temperature was increased from 25°C to 85°C at 1°C intervals, and the sample was diluted to 1 mg / mL using each prepared buffer before adding 150 μL of sample to the cuvette for analysis.

[0267] The aggregation temperature of formulation 8 (without polysorbate 20) was 78.3°C, that of formulation 9 was 77.3°C, and that of formulation 10 was 77.7°C. In Example 13, although polysorbate 20 was absent, the monomer ratio of the protein did not show any change, and as a comparison between the cases without and with polysorbate 20, no difference in protein aggregation was confirmed. These results indicate that subcutaneous injection formulations of trastuzumab do not necessarily require a minimum amount of polysorbate 20 (see [link to relevant documentation]). Figure 8 ).

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

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

[0270] When WCX analysis was performed under harsh conditions (40°C) for 14 days, formulations 8 through 10 showed similar patterns of change. Specific changes included an increase in the relative content of the acidic variant (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 the basic variant (approximately 300% change over 14 days), with no significant differences observed across formulations. In summary, the protein stability of polysorbate 20 (0–0.04%) was similar under harsh conditions (40°C) in WCX analysis (see Figure 9).

[0271] Example 16. Enzyme activity measurement of formulations containing trastuzumab and HP46

[0272] The turbidity assay used to measure enzyme activity is a method that measures the degree of aggregate formation by binding residual hyaluronic acid to acidified albumin (BSA) via absorbance. When hyaluronic acid is hydrolyzed by pH 20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), as a standardized product, was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units / mL and prepared in each tube. Purified pH 20 variant samples were diluted with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X and prepared in each tube. In fresh test tubes, dilute the 3 mg / mL hyaluronidase solution 10-fold to a concentration of 0.3 mg / mL, making each tube 180 μL. Add 60 μL of the sample containing hyaluronidase to the diluted hyaluronic acid solution and mix, allowing the reaction to proceed at 37°C for 45 minutes. After the reaction is complete, add 50 μL of the reacted enzyme and 250 μL of acidic albumin solution to each well of a 96-well plate and shake for 10 minutes. Then, measure the absorbance at 600 nm using a spectrophotometer.

[0273] The results of activity analysis, conducted under harsh conditions (40°C for 14 days), confirmed that higher concentrations of polysorbate 20 correlated with a greater decrease in activity over time (see [link to relevant documentation]). Figure 10 ).

[0274] Example 17. Formulation Development

[0275] As shown in Table 9, three trastuzumab subcutaneous injection formulations were prepared. Formulations 11 to 13 typically contain 120 mg / mL trastuzumab, 20 mM histidine / histidine-HCl (pH 5.5), 210 mM trehalose, 10 mM methionine, and a pH20 variant. The difference between formulations 11-13 is the concentration of nonionic surfactant, with formulation 11 containing 0% polysorbate 80, formulation 12 containing 0.005% polysorbate 80, and formulation 13 containing 0.04% polysorbate 80.

[0276] Table 9. Composition of the preparation

[0277]

[0278] Size exclusion chromatography (SGC) analysis during a stability test at harsh conditions (40°C) for 14 days showed similar patterns of change in formulations 11 through 13. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (approximately less than 1.0%), and no significant differences were observed between formulations. In summary, as a result of size exclusion chromatography analysis during a stability test at harsh conditions (40°C), there were no significant differences in stability characteristics between formulations based on the concentration of polysorbate 80 (0–0.04%) (see [link to relevant documentation]). Figure 11 ).

[0279] Example 18. WCX chromatographic analysis of a formulation containing trastuzumab and HP46.

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

[0281] When WCX analysis was performed under harsh conditions, i.e., a 14-day stability test at 40°C, formulations 11 through 13 showed similar patterns of change. Specific changes included an increase in the relative content of the acidic variant (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 the basic variant (approximately 300% change over 14 days), with no significant differences observed across formulations. In summary, the protein stability of polysorbate 80 (0–0.04%) was similar under harsh conditions, i.e., a 14-day stability test at 40°C, according to WCX analysis (see Figure 12).

[0282] Example 19. Enzyme activity measurement of formulations containing trastuzumab and HP46

[0283] The turbidity assay used to measure enzyme activity is a method that measures the degree of aggregate formation by binding residual hyaluronic acid to acidified albumin (BSA) via absorbance. When hyaluronic acid is hydrolyzed at pH 20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), as a standardized product, was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units / mL and prepared in each tube. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X and prepared in each tube. In fresh test tubes, dilute the 3 mg / mL hyaluronidase solution 10-fold to a concentration of 0.3 mg / mL, making each tube 180 μL. Add 60 μL of the sample containing hyaluronidase to the diluted hyaluronic acid solution, mix, and allow to react at 37°C for 45 minutes. After the reaction is complete, add 50 μL of the reacted enzyme and 250 μL of acidic albumin solution to each well of a 96-well plate and shake for 10 minutes. Then, measure the absorbance at 600 nm using a spectrophotometer.

[0284] The results of activity analysis, conducted under harsh conditions (40°C for 14 days), confirmed that higher concentrations of polysorbate 80 correlated with a greater decrease in activity over time (see [link to relevant documentation]). Figure 13 ).

[0285] Example 20. Formulation Development

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

[0287] Table 10. Composition of the preparation

[0288]

[0289] Size exclusion chromatography (SGC) analysis during a stability test at harsh conditions (40°C for 7 days) showed similar patterns of change in formulations 14 through 16. The main changes were an increase in high molecular weight (HMW) and low molecular weight (LMW) degradation products and a decrease in monomer content (less than approximately 1.0%), with no significant differences between formulations. In summary, as a result of size exclusion chromatography analysis during a stability test at harsh conditions (40°C), there were no significant differences in the overall stability profile between formulations based on the concentration of polysorbate 80 (0–0.06%) (see [link to relevant documentation]). Figure 14 ).

[0290] Example 21. Measurement of enzyme activity in formulations containing rituximab and HP46

[0291] The turbidity assay used to measure enzyme activity is a method that measures the degree of aggregate formation by binding residual hyaluronic acid to acidified albumin (BSA) via absorbance. When hyaluronic acid is hydrolyzed at pH 20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), as a standardized product, was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units / mL and prepared in each tube. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X and prepared in each tube. In fresh test tubes, dilute the 3 mg / mL hyaluronidase solution 10-fold to a concentration of 0.3 mg / mL, making each tube 180 μL. Add 60 μL of the sample containing hyaluronidase to the diluted hyaluronic acid solution, mix, and allow to react at 37°C for 45 minutes. After the reaction is complete, add 50 μL of the reacted enzyme and 250 μL of acidic albumin solution to each well of a 96-well plate and shake for 10 minutes. Then, measure the absorbance at 600 nm using a spectrophotometer.

[0292] The results of activity analysis, conducted under harsh conditions (7 days at 40°C), confirmed that higher concentrations of polysorbate 80 correlated with a greater decrease in activity over time (see [link to relevant documentation]). Figure 15 ).

[0293] Example 22. Measurement of enzyme activity in formulations containing commercially available polysorbate-free products.

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

[0295] Table 11. Composition of the preparation

[0296]

[0297] The turbidity assay used to measure enzyme activity is a method that measures the degree of aggregate formation by binding residual hyaluronic acid to acidified albumin (BSA) via absorbance. When hyaluronic acid is hydrolyzed at pH 20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), as a standardized product, was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units / mL and prepared in each tube. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X and prepared in each tube. In fresh test tubes, dilute the 3 mg / mL hyaluronidase solution 10-fold to a concentration of 0.3 mg / mL, making each tube 180 μL. Add 60 μL of the sample containing hyaluronidase to the diluted hyaluronic acid solution, mix, and allow to react at 37°C for 45 minutes. After the reaction is complete, add 50 μL of the reacted enzyme and 250 μL of acidic albumin solution to each well of a 96-well plate and shake for 10 minutes. Then, measure the absorbance at 600 nm using a spectrophotometer.

[0298] As a result of activity analysis conducted under harsh conditions (i.e., at 40°C) for 6 consecutive days, it was confirmed that high activity was maintained even in formulations without polysorbate 80, and in particular, formulation 18 maintained high activity (see [link to relevant documentation]). Figure 16 ).

[0299] Example 23: Formulation Development

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

[0301] Table 12. Composition of the preparation

[0302]

[0303] Example 24. Measurement using a spectrophotometer

[0304] Preparations 19, 20, 21, and 22 were incubated at 40°C for 7 days, and changes in protein concentration were analyzed using a Beckman spectrophotometer. Each sample was diluted with distilled water to a concentration of 0.4 mg / mL, and the absorbance of the protein at 280 nm was measured using a spectrophotometer.

[0305] In a stability test conducted under harsh conditions, namely at 40°C for 7 consecutive days, the protein concentration of formulations 19 to 22 did not change significantly.

[0306] Example 25. Size exclusion chromatography was used to study the monomer ratio of pembrolizumab in each formulation.

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

[0308] Size exclusion chromatography (SGC) analysis of formulations under harsh conditions (40°C) for 7 days showed similar patterns of change in formulations 19, 20, 21, and 22. There were no significant differences in the patterns of change for high molecular weight (HMW) and low molecular weight (LMW) degradation products, depending on the formulation. In summary, as results of size exclusion chromatography analysis under harsh conditions (40°C), formulations 19, 20, 21, and 22 showed no significant differences, and there were no differences based on sugar type (see [link to relevant documentation]). Figure 17 These results are consistent with those of trastuzumab and rituximab according to previous embodiments.

[0309] Example 26. Measurement of enzyme activity in formulations containing pembrolizumab and HP46

[0310] The turbidity assay used to measure enzyme activity is a method that measures the degree of aggregate formation by binding residual hyaluronic acid to acidified albumin (BSA) via absorbance. When hyaluronic acid is hydrolyzed at pH 20, the degree of binding to albumin decreases, resulting in a decrease in absorbance. BTH (Sigma), as a standardized product, was diluted to 1, 2, 5, 7.5, 10, 15, 20, 30, 50, and 60 units / mL and prepared in each tube. Purified protein samples were diluted with enzyme dilution buffer (20 mM Tris·HCl pH 7.0, 77 mM NaCl, 0.01% (w / v) bovine serum albumin) to 100X, 300X, 600X, 1200X, and 2400X and prepared in each tube. In fresh test tubes, dilute the 3 mg / mL hyaluronidase solution 10-fold to a concentration of 0.3 mg / mL, making each tube 180 μL. Add 60 μL of the enzyme-containing sample to the diluted hyaluronic acid solution, mix, and allow to react at 37°C for 45 minutes. After the reaction is complete, add 50 μL of the reacted enzyme and 250 μL of acidic albumin solution to each well of a 96-well plate and shake for 10 minutes. Then, measure the absorbance at 600 nm using a spectrophotometer.

[0311] The results of activity analysis, conducted under stringent conditions (7 days at 40°C), confirmed that the decrease in activity over time increased with increasing polysorbate 80 concentration. It was also confirmed that, when containing the same amount of polysorbate 80, the decrease in activity in trehalose-containing formulations was less than that in sucrose-containing formulations (see [link to relevant documentation]). Figure 18 ).

[0312] Example 27. pH-activity curves of HP46 and wild-type HW2

[0313] For experiments conducted to determine the pH-activity profiles of HP46 and wild-type HW2, microturbidity assays were used. For each pH, ​​hyaluronic acid buffer was prepared to dissolve hyaluronic acid as the substrate, and enzyme buffer was prepared to dilute the enzyme.

[0314] Three 96-well plates were prepared for the enzyme-substrate reaction and named A, B, and C, and experiments were conducted.

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

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

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

[0318] 50 μL of sample was transferred from each well of a 96-well plate designated A to each well of a 96-well plate designated B, and the reaction was allowed to proceed in a 37°C shaking incubator for 45 minutes. Fifteen minutes before the reaction was complete, 200 μL of acidic albumin solution was dispensed into each well of a 96-well plate designated C for preparation. When the enzyme substrate reaction was complete, 40 μL of sample was transferred from each well of the 96-well plate designated B to each well of the 96-well plate designated C, and the reaction was allowed to proceed for 20 minutes. After 20 minutes, absorbance was measured at 600 nm, and the amount of residual hyaluronic acid after the enzyme substrate reaction was calculated. An enzyme activity curve based on pH was then constructed (see [link to relevant documentation]). Figure 19 ).

[0319] Example 28. Pharmacokinetic studies of Herceptin subcutaneous formulation, trastuzumab, and HP46 in Sprague-Dawley rats.

[0320] To examine whether the subcutaneous formulations of trastuzumab and HP46 exhibited the same pharmacokinetic properties as the subcutaneous formulation of Herceptin, 9-week-old Sprague-Dawley rats were used. Herceptin and trastuzumab were administered at a dose of 18 mg / kg rat body weight. The subcutaneous formulation of Herceptin contained 100 U of rHuPH20, and the amount of HP46 was also 100 U. In pharmacokinetic tests, trastuzumab and HP46 showed the same area under the curve (AUC) as the subcutaneous formulation of Herceptin (see [link to pharmacokinetic study]). Figure 20 ).

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

[0322] 1. A pharmaceutical composition comprising:

[0323] (a) medicine; and

[0324] (b) PH20 variant,

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

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

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

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

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

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

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

[0332] 8. The pharmaceutical composition according to embodiment 7, wherein the PH20 variant comprises one or more amino acid residues substituted with L354I and / or N356E, and further comprises one or more amino acid residues substituted with T341S, L342W, S343E, I344N, M345T, S347T, M348K, K349E, L352Q, L353A, D355K, E359D, I361T, and N363G.

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

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

[0335] 11. The pharmaceutical composition according to embodiment 10, wherein the PH20 variant comprises any amino acid residue substitution selected from the group consisting of:

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

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

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

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

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

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

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

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

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

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

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

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

[0348] 18. The pharmaceutical composition according to any one of embodiments 1 to 17, wherein the PH20 variant further comprises a signal peptide derived from human hyaluronidase-1 (Hyal1), human growth hormone, or human serum albumin at the N-terminus.

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

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

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

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

[0353] 23. The pharmaceutical composition according to embodiment 22, wherein the antibody binds to one or more antigens selected from: 4-1BB, integrin, β-amyloid, angiopoietin, angiopoietin analogue 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, tight junction protein-18, complement factor D, CTLA4, DLL3, EGF receptor, hemophilia factor, Fc receptor, FGF23, folate receptor, GD2, GM-CSF, HER2, HER3, interferon receptor, interferon-γ, IgE, IGF-1 receptor, interleukin-1, interleukin-2 receptor, interleukin-4 receptor, interleukin-5, interleukin-5 receptor, interleukin-6, interleukin-6 receptor, interleukin-7, interleukin-12 / 23, interleukin-13, interleukin-17A, interleukin-17 receptor A, interleukin-31 receptor, interleukin-36 receptor, LAG3, LFA3, NGF, PVSK9, PD-1, PD-L1, RANK-L, SLAMF7, tissue factor, TNF, VEGF, and vWF.

[0354] 24. The pharmaceutical composition according to embodiment 22, wherein the antibody is selected from one or more of the following: utorumab, natezumab, ertrolizumab, vedolizumab, bigramab, bavizumab, kranezimab, sorazizumab, aducacanibab, gantezumab, AMG 780, MEDI3617, Nescalumab, Vanguzumab, Ivamarumab, Tabelucizumab, Lanarumab, Belimumab, Oboterumab, Rifalizumab, Ikulizumab, Molizumab, Oxizumab, Teflizumab, Morozumab, Tebentafusp, Bonatumab, REGN1979, Ipalzumab, Zhamumab, Itrazumab, Efalizumab, Inbilzumab, Tafacitazumab, Tironutuzumab, Ogrizumab, Utoxizumab, Obituzumab, Ofalimumab, Rituximab, Tosimomab, Tefimumab, Ipatizumab, Ointituzumab, Parcitumomab, Bentuzumab, Taliban-Vardatuxizumab, Ozolmicin- Gelatinumab, Daremaxillab, Ixatuximab, Alenumab, Lizalizumab, Polotuximab, Galiliximab, Epranetumab, Furenalzumab, Gacalizumab, Ernomarab, Zoloxicam, Lanpamab, Trimelimumab, Zeflizumab, Ipilimumab, Rovatropinumab, Cetuximab, Datoximab, Zalumumab, Nexituximab, Panitumab, Emetuzumab, Nipocalizumab, Loliximab, Broxamarab, Faruzumab, Socin-Mituximab, Datoximab, Naxituximab, Otetil, Magutuximab, Pertuximab, Trastuzumab, Trastuzumab-Drunotecan, Trastuzumab-Metuximab, Trastuzumab-Metuximab Ducarmazine, Pertrastuzumab, Anifrucizumab, Epatavazumab, Ligozulumab, Omalizumab, Darozizumab, Fentuximab, Titrumizumab, Gevocil, Connorumab, Dactocil, Baliximab, Dupilumab, Meperizumab, Rosumarab, Benalizumab, Clagizumab, Olojizumab, Selukumab, Seluximab, Saliruzumab, Shatraziluzumab, Tocilizumab, REGN88, Secukinumab, Utekelumab, Brenuzumab, Lebirexumab, Traroluzumab, Ikkizumab, Bimezumab, Brodatumab, Brazkumab, Gusekumab, Lisangazolizumab, Titraziluzumab, Mijizumab, Nemozumab, Pe Solimumab, rilatumab, nasolpirumab, fasilimumab, tanizumab, alicurumab, evokumab, bercosizumab, pembrolizumab, basililimumab, camrelizumab, cimipril, dotalimumab, palolilimumab, sildenafil, spartazumab, tislelilimumab, pembrolizumab, nivolumab, atezolilimumab, averulimumab, envorizumab, duvalimumab, bintrafuspα, dinosumab, elozima, mastalimumab, infliximab, adalimumab, golimumab, cytorelin, orizumab, broxol, lanonilimumab, bevacizumab, farecilimumab, ramoruzumab, and capsulbizumab.

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

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

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

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

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

[0360] The surfactant includes one or more nonionic surfactants selected from the following: polyoxyethylene-sorbitan fatty acid ester, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, polyoxyethylene-polyoxypropylene copolymer, and sodium dodecyl sulfate (SDS).

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

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

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

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

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

[0366] 33. An injectable preparation for subcutaneous injection, said injectable preparation comprising a pharmaceutical composition according to any one of embodiments 1 to 32.

[0367] Industrial applicability

[0368] The pharmaceutical composition according to this disclosure can be used for subcutaneous injection and is very stable, and the activity of the PH20 variant, along with the drug, preferably an antibody drug, can be maintained for a long time. Therefore, the pharmaceutical composition can not only help reduce the production cost of subcutaneous injection preparations, but also reduce medical costs, and is very effective in terms of patient convenience.

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

[0370] References

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

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

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

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

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

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

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

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

Claims

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

1.

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

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

1.

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

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

1.

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

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

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

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

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

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