Method for producing human recombinant hyaluronidase

Abstract

The present invention relates to a method for producing hyaluronidase PH20 or a variant thereof and, more specifically, to a method for producing hyaluronidase PH20 or a variant thereof, and a use thereof, hyaluronidase PH20 having a significantly reduced half-life in blood, while maintaining enzyme activity and thermal stability, due to sialidase treatment.

Description

Method for producing human recombinant hyaluronidase

[0001] The present invention relates to a method for producing hyaluronidase PH20 or a variant thereof, and more specifically, to a hyaluronidase that maintains enzyme activity and thermal stability and significantly reduces the blood half-life through sialidase treatment.

[0002] Hyaluronidase is an enzyme that performs important physiological roles within the human body, specifically involved in the breakdown of hyaluronic acid present in subcutaneous tissue. Hyaluronic acid is a major component of the extracellular matrix (ECM) and plays a crucial role in maintaining intercellular space and influencing cell morphology and function. By breaking down hyaluronic acid, hyaluronidase causes transient changes in the structure of the ECM; this increases tissue permeability, thereby playing a vital role in accelerating the diffusion rate of drugs. Through this process, hyaluronidase plays a critical role in drug delivery systems, providing an opportunity to efficiently deliver drugs to tissues such as the skin.

[0003] Hyaluronidase is also useful for relieving subcutaneous pain and edema when used alone. Furthermore, it contributes to expanding the range of anesthetic effects by facilitating the diffusion of local anesthetics. Thanks to these properties, hyaluronidase is utilized in various pharmaceutical formulations, and is particularly effective when mixed with high molecular weight antibody drugs to create subcutaneous injection (SC) formulations. In this case, hyaluronidase enhances tissue permeability through enzymatic activity within the subcutaneous tissue, thereby promoting drug diffusion and helping the drug to be effectively absorbed into the blood vessels beneath the skin.

[0004] While hyaluronidase provides various physiological and medicinal functions, there are still issues to be resolved in current research. In particular, recombinant hyaluronidases such as PH20 primarily focus on the function of breaking down hyaluronic acid in subcutaneous tissue to create space for drug injection. However, if these enzymes maintain their activity even after being absorbed into the bloodstream, they can cause unnecessary toxic reactions and lead to side effects. Therefore, the development of recombinant hyaluronidases that allow for rapid degradation or breakdown of activity in the bloodstream is emerging as a critical task.

[0005] Since the function and safety of hyaluronidase vary significantly depending on the enzyme's structural characteristics, various studies are required to improve them. In particular, the enzyme's N-glycolysis structure significantly influences protein folding, stability, enzymatic activity, and immunogenicity. As N-glycolysis is a critical factor determining the characteristics of recombinant proteins, technologies to regulate it play an essential role in improving the efficacy and safety of these proteins. Therefore, it is important to have technologies that can produce efficient hyaluronidase and develop it into a form suitable for drug delivery systems by controlling the enzyme's N-glycolysis pattern through variations in host cell types and culture conditions.

[0006] Therefore, the development of recombinant hyaluronidase with the characteristics of maintaining enzymatic activity in subcutaneous tissue and rapidly degrading and eliminating it in the bloodstream has become a very important research area in the industry. This expands the range of applications for hyaluronidase and enables the establishment of safer and more effective drug delivery systems.

[0007] The inventors intend to provide a method for producing recombinant hyaluronidase PH20 or a variant thereof, which has a sialic acid content of less than 1%, maintains enzyme activity and thermal stability, and significantly reduces the blood half-life by treating with sialidase.

[0008] The inventors have implemented a method for producing hyaluronidase PH20 and a variant thereof, which maintains enzyme activity and thermal stability while significantly reducing the blood half-life.

[0009] Accordingly, the objective of the present invention is to provide a method for producing hyaluronidase PH20, in which the sialic acid content of the N-sugar chain is less than 1%, and a variant thereof.

[0010] Another objective of the present invention is to provide hyaluronidase PH20 having a sialic acid content of less than 1% in the N-sugar chain and a variant thereof.

[0011] Another objective of the present invention is to provide an injectable composition and a drug delivery material comprising hyaluronidase PH20 and a variant thereof, wherein the sialic acid content of the N-glycan chain is less than 1%.

[0012] The present invention relates to a method for producing hyaluronidase PH20 or a variant thereof, and more specifically, to a method for producing hyaluronidase PH20 or a variant thereof having a sialic acid content of less than 1% in the N-sugar chain and to the use thereof.

[0013] The present invention will be described in more detail below.

[0014]

[0015] One aspect of the present invention is a method for producing hyaluronidase PH20 or a variant thereof.

[0016] The term “hyaluronidase” as used in this specification is one of the enzymes that decomposes hyaluronic acid, and is a substance that decomposes hyaluronic acid to reduce its viscosity and widen the space between tissues.

[0017] The term “PH20” in this specification is an enzyme that cleaves the β-1,4 bond between acetylglucosamine and glucuronic acid of hyaluronic acid. Currently, commercially widely used PH20 is in the form extracted from the testes of cattle or sheep, and includes ampadase (bovine hyaluronidase) and vitrase (sheep hyaluronidase), among others, and rHuPH20, pegylated rHuPH20, etc. are used, but are not limited thereto.

[0018] The term “variant” as used herein refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence of amino acids different from that of the variant prior to modification, while retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant may be increased, unchanged, or decreased compared to the polypeptide prior to modification. Additionally, some variants may include variants in which one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion of the N- and / or C-terminus of a mature protein has been removed. The term "variant" mentioned above may be used interchangeably with terms such as variant, modification, variant polypeptide, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used with the meaning of being mutated.

[0019] Additionally, the variant may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in co-translational or post-translational protein translocation may be conjugated to the N-terminus of the variant. Additionally, the variant may be conjugated with another sequence or linker to enable identification, purification, or synthesis.

[0020] In one embodiment of the present invention, a method for producing hyaluronidase PH20 or a variant thereof may include a acquisition step of acquiring hyaluronidase PH20 or a variant thereof and a processing step of contacting the acquired hyaluronidase PH20 or a variant thereof with sialidase.

[0021] The method for producing hyaluronidase PH20 or a variant thereof according to the present specification is a method of making the sialic acid content of the N-glycan chain of hyaluronidase PH20 or a variant thereof less than 1% through a treatment step of contacting sialydase with hyaluronidase PH20 or a variant thereof.

[0022] Specifically, the method may include a treatment step of contacting hyaluronidase pH 20 or a variant thereof with sialydase at a temperature of 32 to 42°C for 12 to 20 hours at a pH of 5.0 to 6.0, in an amount of 0.1 U (unit) of sialydase per 1 μg of hyaluronidase pH 20 or a variant thereof.

[0023] More specifically, the method may include a treatment step of contacting hyaluronidase PH20 or a variant thereof with sialydase at a temperature of 37°C for 16 hours at a rate of 0.1 U (unit) of sialydase per 1 μg of hyaluronidase PH20 or a variant thereof under conditions of pH 5.5, and may additionally perform a purification step using a HisTrap column to purify the hyaluronidase PH20 or a variant thereof contacted with sialydase (see Example 2).

[0024] Hyaluronidase PH20 treated with sialydase or a variant thereof has a smaller molecular weight than the control group not treated with sialydase (see Example 3 and Fig. 1), has a sialic acid content of less than 1% in the N-sugar chain (see Example 4 and Table 3), and shows no significant difference in enzyme activity compared to hyaluronidase not treated with sialydase (see Example 5).

[0025] Hyaluronidase PH20 or its variants treated with sialydase did not show a significant difference in enzyme activity compared to hyaluronidase not treated with sialydase even after being subjected to harsh conditions of 40°C and 45°C for 24 hours (see Example 6 and Fig. 2). This indicates that there is no change in the thermal stability of hyaluronidase PH20 or its variants even after treatment with sialydase.

[0026] It was confirmed that hyaluronidase PH20 treated with sialydase or a variant thereof has a much shorter half-life compared to hyaluronidase not treated with sialydase (see Example 7 and Fig. 3). This effect of reduced half-life can minimize unpredictable side effects and difficulties in controlling efficacy, such as tissue damage or inflammatory response, excessive pharmacological effects through excessive diffusion of the drug, interactions with other metabolic pathways, or toxicity.

[0027] The term “N-glycan” as used in this specification refers to a structure in which a glycan is bonded to the azimuthal group (-NH2) of an amino acid, and influences the regulation of the structural stability of proteins, protein-protein interactions, cell recognition and immune responses, as well as the activity, stability, and lifespan of proteins.

[0028] The term “sialic acid” as used in this specification refers to a type of sugar molecule, specifically a monosaccharide contained in a sugar chain. Also known as N-acetylneuraminate, it is an important molecule primarily attached to sugar chains on the surface of cells.

[0029] The sialydase according to the present invention may be derived from bacteria.

[0030] Specifically, it may be derived from one or more bacteria selected from the group consisting of Arthrobacter ureafaciens, Vibrio cholera, and Clostridium perfringens.

[0031] More specifically, it may be a sialydase containing the amino acid sequence of SEQ ID NO. 5.

[0032] In some embodiments of the present invention, sialydase may consist of the amino acid sequence of SEQ ID NO. 5.

[0033] In some embodiments of the present invention, sialydase may be functionally equivalent to the amino acid sequence of SEQ ID NO. 5, and may, for example, have sequence similarity of 85%, 90%, 95%, 98%, or 99% or more with the amino acid sequence of SEQ ID NO. 5.

[0034] More specifically, it may be cialidase indicated by sequence number 5, but is not limited thereto.

[0035] The term “sialydase” as used in this specification refers to an enzyme that cleaves sialic acid in glycoproteins or glycolipids containing sialic acid, and is an enzyme that plays an important role in the regulation of intercellular interactions, the regulation of immune responses, or the infection process of pathogens.

[0036] In one embodiment of the present invention, a method for producing hyaluronidase PH20 or a variant thereof may further include a vector introduction step of transforming a host cell with a cloning vector comprising a gene sequence encoding hyaluronidase PH20 or a variant thereof.

[0037] The cloning vector according to the present invention may include the gene sequence of codon-optimized hyaluronidase PH20 or a codon-optimized hyaluronidase PH20 variant in a host cell.

[0038] The term “cloning vector” as used in this specification refers to a DNA molecule used to deliver a specific gene into a cell for replication or expression, primarily used in gene cloning experiments to insert a target gene and for the vector to proliferate or express within a host cell.

[0039] Specific examples may include plasmid vectors, phage vectors, BAC (Bacterial Artificial Chromosome) vectors, YAC (Yeast Artificial Chromosome) vectors, cosmid vectors, viral vectors, and plasmid-phage mixed vectors.

[0040] More specifically, the pCDNA3.1-Zeo plasmid vector can be used.

[0041] The term “host cell” as used in this specification refers to a cell into which a foreign gene, DNA fragment, or virus is introduced to be replicated or expressed, and which serves to accommodate the gene fragment or vector to proliferate it in an internal environment or to induce the expression of a specific protein.

[0042] The host cell in the present invention may be a bacterial cell, an insect cell, a mammalian cell, a yeast cell, etc., but is not limited thereto. More specifically, it may be a CHO (Chinese hamster ovary) cell.

[0043] The term “transformation” as used herein means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed within the host cell. The transformed polynucleotide may include both inserted into and located within the chromosomes of the host cell and extrachromosomally, as long as it can be expressed within the host cell. Additionally, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be introduced into and expressed within the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the elements necessary for self-expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, polynucleotides may be introduced into the host cell in their own form and operably linked to the sequence required for expression in the host cell, but are not limited thereto.

[0044] Any transformation method may be used in the present invention, and it can be easily performed according to conventional methods in the art. Generally, transformation methods include the CaCl2 precipitation method, the Hanahan method which increases efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation method, protoplasmic fusion method, stirring method using silicon carbide fibers, Agrobacterium-mediated transformation method, PEG-mediated transformation method, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods.

[0045] The method for transforming a vector according to the present invention is not limited to the above examples, and transformation or transfection methods commonly used in the art may be used without limitation.

[0046] The term “codon optimization” as used herein refers to altering the codons of a polynucleotide encoding a protein to be preferentially used in a particular organism so that the encoded protein is expressed more efficiently in the organism. Although the genetic code is degenerate in that most amino acids are represented by a few codons referred to as “synonymous” or “synonymous” codons, the codon usage by a particular organism is not random but biased toward specific codon triplets. This bias in codon usage may be higher in relation to certain genes, genes of common function or ancestral origin, highly expressed proteins versus low-copy number proteins, and collective protein-coding regions of the organism’s genome.

[0047] In one embodiment of the present invention, a method for producing hyaluronidase PH20 or a variant thereof may further include a culture step of culturing cells transformed with a cloning vector comprising a gene sequence encoding hyaluronidase PH20 or a variant thereof.

[0048] In the culture step according to the present invention, cell culture can be performed using methods widely known in the industry.

[0049] For example, it can be carried out by batch culture, continuous culture, fed-batch culture, etc.

[0050] The term “batch culture method” as used in this specification refers to a culture method in which culture is continued until all of the initially supplied raw substrate is consumed, and the concentration of the substrate, the concentration of metabolites, the concentration of cells, etc., continuously changes over time.

[0051] The term “continuous culture method” as used in this specification refers to a culture method that maintains a constant state while continuously supplying nutrients to enable cells in culture to grow continuously, and removing waste products or metabolic byproducts.

[0052] The term “batch-fed culture method” as used in this specification refers to a culture method in which a culture medium is supplied intermittently, and in which the amount of substrate supplied can be freely controlled because the substrate in the culture medium is added at an appropriate rate and there is no outflow.

[0053] Specifically, the batch culture method according to the present invention may be a method in which a seed culture immediately prior to the main culture is cultured at 35 to 38°C, followed by inoculation into the main culture and culture at 32 to 37°C. At this time, the cell culture of the main culture may be carried out for 2 to 18 days, specifically for 3 to 16 days, and more specifically for 4 to 14 days, but is not limited thereto. The cell inoculation concentration of the main culture is 1 x 10⁻⁶ 5 cells / mL or more, specifically 2x10 5 cells / mL or more, more specifically 3x10 5 It may be greater than cells / mL, but is not limited thereto (see Example 1).

[0054] The term “main culture” as used in this specification refers to the process of mass-culturing cells to be ultimately used for experiments or production, meaning the transfer of cells proliferated in a seed culture to a larger culture vessel for full-scale cultivation.

[0055] The term “seed culture” as used in this specification refers to the process of pre-proliferating cells to be used in the main culture, meaning culturing cells first in a small flask or culture vessel.

[0056] The term “cell inoculation concentration” as used in this specification refers to the concentration of cells inoculated into the culture medium at the start of culture, indicating how densely the cells are packed. Specifically, it is a value representing the number of cells contained in 1 ml of culture medium, expressed in units of cells / ml (number of cells / milliliter). Cell inoculation concentration is important for optimizing the growth rate and metabolic activity of cells by setting an appropriate concentration according to the type of cell and the purpose of culture.

[0057] The obtaining step according to the present invention is such that the cumulative viable cell density of the cultured cells is 2x10 6 Up to 80x10 6 It may be performed at cells x day / mL. Specifically, 4x10 6 Up to 40x10 6 It can be cells x day / mL, and more specifically, 6x10 6 Up to 20x10 6 It may be performed in the case of cells x day / mL, but is not limited thereto.

[0058] The term “integral viable cell density (IVCD)” as used herein is an indicator representing the cumulative concentration of viable cells during cell culture, and plays an important role in understanding the efficiency of cell culture and the physiological state of the cells.

[0059] Another aspect of the present invention is hyaluronidase PH20 or a variant thereof having a sialic acid content of less than 1% in the N-sugar chain.

[0060] Hyaluronidase PH20 or a variant thereof according to the present invention can be included in various compositions to increase the permeability of subcutaneous tissue to increase the diffusion rate of drugs or to enable absorption into blood vessels inside the skin, and can be used as a drug diffusion agent, and can also be used for the prevention and removal of subcutaneous pain and edema, and for the diffusion of local anesthetics.

[0061] Another aspect of the present invention may be an injectable composition comprising hyaluronidase PH20 or a variant thereof having a sialic acid content of less than 1% of the N-sugar chain.

[0062] More specifically, it may be one or more compositions selected from the group consisting of compositions for subcutaneous injection, compositions for intramuscular injection, and compositions for local anesthetics, but is not limited thereto.

[0063] The term “composition for subcutaneous injection” as used in this specification refers to a component of an injectable preparation for subcutaneous administration. Subcutaneous injection is a method of injecting a drug into the fat layer beneath the skin, allowing the drug to be slowly absorbed to produce a sustained effect. Drugs used for subcutaneous injection may exist in various forms, such as solution, suspension, or emulsion.

[0064] As used herein, the term “composition for intramuscular injection” refers to the compositional components of a drug injected directly into a muscle. Muscle tissue is rich in blood vessels, allowing for rapid absorption of the drug, and some drugs may be designed to provide a sustained-release effect. Intramuscular injections may exist in various forms, such as aqueous solutions, suspensions, and oily formulations.

[0065] The term “local anesthetic” as used in this specification refers to a drug that temporarily blocks or paralyzes sensation by blocking nerve signals in a specific area. Local anesthetics act by blocking the transmission of pain signals through action on nerves and may be used in the form of injections, creams, gels, patches, etc.

[0066] Another aspect of the present invention may be a drug delivery material comprising hyaluronidase PH20 or a variant thereof having a sialic acid content of less than 1% of the N-glycan chain.

[0067] The term “drug delivery material” as used in this specification refers to various physical and chemical systems for effectively delivering a drug to a desired site. Drug delivery materials can help a drug reach a target site within the body and be released continuously and stably. Drug delivery materials may exist in various forms, such as liposomes, nanoparticles, hydrogels, and transdermal drug delivery systems.

[0068] The present invention relates to a method for producing a hyaluronidase having a sialic acid content of less than 1% in the N-sugar chain and a variant thereof. The hyaluronidase or its variant according to the present invention maintains enzyme activity and thermal stability while shortening the blood half-life, thereby reducing concerns about side effects such as toxicity.

[0069] Figure 1 shows the SDS-PAGE results after HisTrap purification of wild-type hyaluronidase PH20.

[0070] Figure 2 shows the results of a comparative analysis of the thermal stability of wild-type hyaluronidase PH20.

[0071] Figure 3 shows the results of a pharmacokinetic study of wild-type hyaluronidase PH20 after intravenous administration in 9-week-old Sprague-Dawley rats.

[0072] A method for producing hyaluronidase PH20 or a variant thereof, comprising a obtaining step of obtaining hyaluronidase PH20 or a variant thereof and a processing step of contacting the obtained hyaluronidase PH20 or a variant thereof with sialidase.

[0073] The present invention will be explained in more detail below through the following examples. However, these examples are merely illustrative of the invention, and the scope of the invention is not limited by these examples.

[0074]

[0075] Example 1. Preparation of Hyaluronidase PH20

[0076]

[0077] 1-1. Creation of Cloning Vector

[0078] To produce hyaluronidase, gene synthesis was performed by optimizing codons in GeneScript so that the wild-type PH20 gene sequence could be efficiently expressed in CHO (Chinese hamster ovary) cells from F38 to N483.

[0079] The PH20 gene was amplified using polymerase chain reaction (PCR) and inserted into the HindIII and XhoI restriction enzyme sites of the pCDNA3.1-Zeo vector. ExpiCHO-S TM For expression in (ThermoFisher, Product No. A29127) cells, human serum albumin was used as the signal peptide instead of the intrinsic signal peptide of PH20, and a 10XHis-tag was inserted at the 3'-terminus for protein purification using a HisTrap column. The primer sequences used for gene amplification are shown in Table 1 below.

[0080] Sequence number name sequence (5`- 3`) 1PH20 ForGGCTAGCGTTTAAACTTAAGCTT2PH20 RevGCCCTCTAGACTCGAGTTAGTGGTGGTGGTGGTGGTGGTGGTGGTGGTGGTTGTAGAAGATCTGTGGCTCTT3Hsa PH20 ForTTCAGCTCTGCTTACTCTTTCAGAGCTCCTCCTGTGATA4Hsa PH20 RevAGAGTAAGCAGAGCTGAAC

[0081] 1-2. Protein Expression

[0082] After gene transformation into CHO cells using the ExpiFectamine CHO reagent, 25–30 ml of cells were cultured for 4–8 days in a 37°C shaker with approximately 80% humidity and 5–8% CO2. The expressed proteins were identified using SDS-PAGE and Western blot, and then filtered through a 0.22 µm filter for purification.

[0083]

[0084] 1-3. Protein Purification

[0085] Purification was performed using a HisTrap HP column. Prior to use, the column was equilibrated using binding buffer (500 mM NaCl, 20 mM sodium phosphate, pH 7.4). The prepared culture medium was flowed through the prepared column to bind the target protein, followed by a wash with binding buffer. Subsequently, elution buffer (500 mM NaCl, 20 mM sodium phosphate, 0.5 M imidazole, pH 7.4) was used to elute the bound target protein, and the target protein was selected using a linear gradient method. All processes were carried out at a flow rate of 1–5 ml / min. The selected target protein was filtered through a 0.2 µm filter, and its activity was measured. Fast Protein Liquid Chromatography (FPLC) was performed using a Cytiva AKTA pure instrument.

[0086]

[0087] Example 2. Sialidase treatment

[0088] Sialidase A derived from Arthrobacter ureafaciens was added to pH 20 culture medium expressed in ExpiCHO-S cells or purified samples at a concentration of 0.1 U per 1 µg of protein in reaction buffer (5 mM CaCl2, 50 mM sodium acetate, pH 5.5) and reacted at 37°C for 16 hours to remove sialic acid from pH 20. Subsequently, purification was performed using a HisTrap column to analyze pH 20 enzyme activity, thermal stability, and sialic acid content. The amino acid sequence of the sialicase used is shown in Table 2 below.

[0089] Sequence number name Sequence (N`- C`)5Sia AMAPTPPNSPTLPPGSFSETNLAADRTAANFFYRIPALTYLGNDVVLAAWDGRPGSAADAPNPNSIVQRRSTDGGKTWGPVQVIAAGHVADASGPRYGYSDPSYIYDAEANKVFAFFVYSKDQGFGGSQFGNDDADRNVISSAVIESSDAGVTWSQPRLITSVTKPGTSKTNPAAGDVRSNFASSGEGIQLKYGPHKGRLIQQYAGDVRQADGSNKIQAYSVYSDDHGVTWHKGANVGDRMDENKTVELSDGRVLLNSRDNANRGYRKVAVSTDGGATYGPVSQDTELPDPANNGAIARMFPNAAQGSADAKKLIFTNANSKTGRENVSARVSCDDGETWPGVRTIRSGFSAYSTVTRLADGKFGVLYEGNYTDNMPFATFDDAWLNYVCAPLAVPAVNIAPSATQEVPVTVTNQEATTLSGATATVYTPSGWSATTVPVPDVAPGASVTVTVALTAPADASGPRSLNAAFTTADGR VSQFTFTATTPVAPQVGLTIGSAPARDVAANPYQAGDVLGYTLNVKSTANVAANSVPLTGTFDSGFLPPAAPNCRYNNLAAGASYNCTTAKHTITAADMERGYFVPEATFSITSTTTPSLTKTVQFTGAAVALRDGLISADISGARTDVGRDLATRPYAAGELVPYAFTVKNTSPFVEQVVPTAGNFSPFLPAGAGNCRYLSLPAGQSYECATPRHAVTAEEVEQGFFVPDTTWEVSAAGQSTRTYRINGGEVDLLVRDAALSATVVAEWKDADGDRFASAGDPVTFTYTVGNAGNVALTGLEAPDAGISLPFLAPGDTATATREHVLTAADVAGGSLAASAFEATARANGSKEVTATAEGQPLELKVQPAQPSKEPELTVQDLEDQTPPFDLGTAFKYRTGQKVSLAGLEYGQWYYVYLNKTGYRLGWMFPTTGDTVEFILPPEVRNGRDDVVVLDKDGRRVSFDRLQVTPKGEKI

[0090] Example 3. SDS-PAGE analysis of purified hyaluronidase PH20

[0091] After purifying the wild-type pH 20 sample and the wild-type pH 20 sample with terminal sialic acid removed by treatment with sialydase A using a HisTrap column, the SDS-PAGE results showed that only one identical band was detected, and it was confirmed that no impurities were detected with a purity of over 98% (Fig. 1). Additionally, the positional difference resulting from the difference in molecular weight between the wild-type pH 20 sample and the wild-type pH 20 sample with terminal sialic acid removed by treatment with sialydase A was confirmed.

[0092]

[0093] Example 4. Analysis of sialic acid content in hyaluronidase pH 20

[0094] The purified sialydase obtained in Example 2 was analyzed using an instrument (Sialic acid Assay kit, sigma-Aldrich, cat no. mak314) to analyze the sialic acid content released by hydrolysis from PH20 treated with the sialydase and PH20 not treated. This sialic acid analysis is performed by the enhanced Warren method in which sialic acid reacts with thiobarbituric acid to form a pink product by oxidizing to form formylpyruvic acid, and the value analyzed by fluorescence measurement (λex=555nm, λem=585nm) is proportional to the sialic acid concentration of the sample.

[0095]

[0096] Example 5. Analysis of Enzyme Activity of Hyaluronidase PH20

[0097] The enzymatic activity of hyaluronidase PH20 was measured using the following Morgan-Elson method.

[0098] The Morgan-Elson method is a colorimetric analysis method that quantifies the red substance produced by the reaction of the reducing end of N-acetyl-D-glucosamine (GlcNAc), which is generated when hyaluronic acid is hydrolyzed by hyaluronidase, with para-dimethylaminobenzaldehyde (DMAB), which is Ehrlich's reagent, by 545 nm absorption. N-acetyl-D-glucosamine (GlcNAc, Sigma) was diluted to 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 20 mM in dilution buffer solution (0.1 M sodium phosphate, 0.1 M NaCl, 1.3 mg / ml BSA, pH 6.0). Each test tube was treated with tetraborate, heated at 100°C for 5 minutes, and then DMAB was added to induce a color reaction at 37°C for 20 minutes. After the reaction, the absorbance was measured at 545 nm to construct a standard reaction curve for GlcNAc. 5 μl of diluted hyaluronidase solution was added to 125 μl of dilution buffer solution (1.5 mg / ml HA, 0.1 M sodium phosphate, 0.1 M NaCl, 1.5 mM saccharin acid 1,4-lactone, pH 6.0) to form the substrate hyaluronic acid, and the mixture was reacted at 37°C for 45 minutes. The enzyme reaction was then terminated by heating at 100°C for 5 minutes. After adding tetraborate and heating at 100°C for 5 minutes, DMAB was added to allow the color development reaction to proceed at 37°C for 20 minutes. After the reaction, the absorbance was measured at 545 nm, and the enzyme activity was determined using the standard reaction curve of GlcNAc above.

[0099] Table 3 presents the results of analyzing the sialic acid content and enzyme activity for wild-type pH 20 samples and wild-type pH 20 samples with terminal sialic acid removed by treatment with sialydase A. These results showed that the sialic acid content of the N-glycosylate chain had no correlation with enzyme activity.

[0100] The sialic acid content in Table 3 below is a value obtained by hydrolyzing hyaluronidase pH 20 or a variant thereof with a weak acid solution to separate sialic acid, then determining the concentration of N-acetylneuraminic acid using an analytical method such as liquid chromatography or the Warren method, and converting it into a ratio of mol / mol.

[0101] Protein Sialitric Acid Content Morgan-Elson Analysis (OD=545nm) GlNAc(mM) Wild Type PH20 (Batch 1) 7.91% 1.049 19.99 Wild Type PH20 (Batch 1)+ Sialidase A 0.27% 1.081 20.63 Wild Type PH20 (Batch 2) 7.4% 0.998 19.02 Wild Type PH20 (Batch 2)+ Sialidase A 0.46% 1.002 19.11

[0102] Example 6. Analysis of the thermal stability of hyaluronidase at pH 20

[0103] Experiments to investigate the thermal stability of wild-type human hyaluronidase pH 20 were conducted as follows. The hyaluronidase formulation was prepared by dissolving wild-type pH 20 at a concentration of 0.5 mg / ml in a PBS (phosphate buffer saline) pH 7.0 solution, which is most similar to the human body condition, and changes in enzyme activity were checked after 24 hours under harsh conditions at 40°C and 45°C.

[0104] Figure 2 presents the results of analyzing enzyme activity after 24 hours at harsh conditions of 40°C and 45°C for wild-type pH 20 samples and wild-type pH 20 samples treated with sialydase A to remove terminal sialic acid. The activity of pH 20 was measured by the Morgan-Elson method, and it was confirmed that the enzyme activity between the two samples was almost identical.

[0105]

[0106] Example 7. Pharmacokinetic analysis of hyaluronidase PH20 in Sprague-Dawley rats

[0107] To perform pharmacokinetic analysis according to sialic acid content, wild-type PH20 samples with sialic acid contents of 7.91% and 0.27% were intravenously injected into rats at a concentration of 21 µg / kg, respectively. For the pharmacokinetic analysis of PH20, blood samples were collected at 2, 5, 10, 15, 30, and 60 minutes after administration of the substance and analyzed using the following methods.

[0108] The experiment was conducted using the Human Hyaluronidase PH-20 (SPAM1) ELISA kit. Standards and analytical samples were prepared prior to the experiment. The standards were prepared by diluting them 1 / 2 with the sample diluent provided in the kit. Subsequently, after washing with wash buffer, HRP-avidin diluted 1:100 was added, and the reaction was carried out at 37°C for 1 hour. After the reaction was complete, the plates were washed with wash buffer, TMB solution was added, and the reaction was carried out in a dark room at 37°C for 30 minutes.

[0109] Finally, once it was confirmed that the sample entered the standard, a stop solution was added and the reaction was terminated. The results were verified by measuring the absorbance of the reaction plate at 450 nm / 570 nm.

[0110] The results of pharmacokinetic analysis performed on samples not treated with sialydase A and samples treated with sialydase A with a sialic acid content of less than 1% are presented in Figure 3. It was confirmed that the blood concentration of pH 20 decreased significantly upon treatment with sialydase A.

[0111] The present invention relates to a method for producing hyaluronidase PH20 or a variant thereof, and more specifically, to a hyaluronidase that maintains enzyme activity and thermal stability and significantly reduces the blood half-life through sialidase treatment.

Claims

1. A method for producing hyaluronidase PH20 or a variant thereof, wherein the production method A acquisition step of acquiring hyaluronidase PH20 or a variant thereof; and A treatment step of contacting the obtained hyaluronidase PH20 or a variant thereof with sialidase; A method for producing hyaluronidase PH20 or a variant thereof, comprising 2. A method for producing hyaluronidase PH20 or a variant thereof, wherein the processing step is to contact sialydase such that the sialic acid content of the N-sugar chain of hyaluronidase PH20 or a variant thereof is less than 1%.

3. A method for producing hyaluronidase PH20 or a variant thereof, wherein the sialydase is derived from bacteria, in accordance with claim 1.

4. A method for producing hyaluronidase PH20 or a variant thereof, wherein, in paragraph 3, the bacteria are derived from one or more bacteria selected from the group consisting of Arthrobacter ureafaciens, Vibrio cholera, and Clostridium perfringens.

5. A method for producing hyaluronidase PH20 or a variant thereof, wherein, in claim 1, the sialydase comprises the amino acid sequence of SEQ ID NO.

5.

6. A method for producing hyaluronidase PH20 or a variant thereof, wherein the treatment step of claim 1 involves contacting sialydase under conditions of pH 5.0 to 6.

0.

7. A method for producing hyaluronidase PH20 or a variant thereof, wherein the production method according to claim 1 further comprises a purification step of purifying hyaluronidase PH20 or a variant thereof that has been contacted with sialydase.

8. A method for producing hyaluronidase PH20 or a variant thereof, wherein the production method according to claim 1 further comprises a vector introduction step of transforming a host cell with a cloning vector comprising a gene sequence encoding hyaluronidase PH20 or a variant thereof.

9. A method for producing hyaluronidase PH20 or a variant thereof, wherein, in claim 8, the host cell is a CHO (Chinese hamster ovary) cell.

10. A method for producing hyaluronidase PH20 or a variant thereof, wherein the cloning vector comprises a gene sequence of codon-optimized hyaluronidase PH20 or a codon-optimized hyaluronidase PH20 variant in the host cell.

11. A method for producing hyaluronidase PH20 or a variant thereof, wherein the production method further comprises a culture step of culturing cells transformed with a cloning vector comprising a gene sequence encoding hyaluronidase PH20 or a variant thereof.

12. Hyaluronidase PH20 or a variant thereof having a sialic acid content of less than 1% in the N-glycan chain.

13. An injectable composition comprising hyaluronidase PH20 or a variant thereof having an N-glycan sialic acid content of less than 1%.

14. A drug delivery material comprising hyaluronidase PH20 or a variant thereof having an N-glycan sialic acid content of less than 1%.

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