Human interferon-beta mutein having double mutation and method for improving safety of human interferon-beta mutein

Abstract

To provide a human interferon-beta mutant having a double mutation and a method for improving the safety of the human interferon-beta mutant.SOLUTION: Provided are a human interferon-beta variant comprising an amino acid sequence in which the 17th amino acid cysteine of human interferon-beta is substituted with serine and the 27th amino acid arginine is substituted with threonine, and a method for improving the stability of a human interferon-beta R27T variant, the method comprising the step of changing the 17th amino acid cysteine of a human interferon-beta R27T variant, in which the 27th amino acid arginine of human interferon-beta is substituted with threonine, to serine.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims priority to Korean Patent Application No. 10-2020-0052513 filed on April 29, 2020, and Korean Patent Application No. 10-2020-0052913 filed on April 29, 2020, the entire specifications of which are incorporated herein by reference.

[0002] The present invention relates to a human interferon-beta variant having a double mutation and a method for improving the safety of a human interferon-beta variant, more specifically to a human interferon-beta variant having an amino acid sequence in which the 17th amino acid, cysteine, of human interferon-beta is replaced with serine and the 27th amino acid, arginine, is replaced with threonine, and a method for improving the safety of a human interferon-beta R27T variant in which the 27th amino acid, arginine, of human interferon-beta is replaced with threonine, comprising a step of replacing the 17th amino acid, cysteine, with serine. [Background technology]

[0003] Interferons (IFNs) are a type of cytokine that exhibit antiviral activity, suppress cell proliferation, and regulate innate immune responses. Among these, interferon-beta (IFN-β) is a globular protein with five alpha helices, measuring 22 kD in size, which becomes 18 kD when the glycosylation chains are removed (Arduini et al., Protein Science 8: pp. 1867-1877, 1999).

[0004] Research into the clinical application of interferon-beta is actively underway, and it is particularly gaining attention as a symptom-relieving, alleviating, or curative agent for multiple sclerosis.

[0005] In addition to multiple sclerosis, interferon beta has been reported to have therapeutic effects on cancer, autoimmune disorders and viral infections, HIV-related diseases, hepatitis C, and rheumatoid arthritis through its various immunological activities, including antiviral activity, cell growth suppression or anti-growth activity, lymphocyte cytotoxicity-increasing activity, immunomodulatory activity, target cell differentiation induction or suppression activity, macrophage activation activity, cytokine production increase activity, cytotoxic T cell effect increase activity, and natural killing cell increase activity.

[0006] There are currently two types of interferon beta used for therapeutic purposes. First, interferon beta-1a is produced from Chinese hamster ovaries (CHO) containing the human interferon beta gene. It is a glycosylated protein consisting of 166 amino acid residues and measuring 22 kDa. Second, interferon beta-1b is produced from Escherichia coli and is a protein consisting of 165 amino acid residues, but lacks sugars, the methionine residue at position 1, and the cysteine ​​residue at position 17 is replaced with serine. Currently available interferon beta-1a products include Rebif and Avonex, while interferon beta-1b products include Betaseron and Extavia.

[0007] Meanwhile, human interferon-beta is also a glycoprotein, and the sugar chain attached to the protein plays an important role in protein activity, so the activity of glycoproteins can be increased by adding sugar chains. That is, protein glycation is known to affect many biochemical properties, such as safety, solubility, intracellular trafficking activity, pharmacokinetics, and antigenicity.

[0008] In response, a case has been reported in which a human interferon-beta variant with increased or improved activity and function was produced by introducing a sugar chain into the glycoprotein human natural interferon-beta (Korean Patent Registration No. 10-0781666). The human interferon-beta variant used in the present invention, R27T, is a recombinant human interferon-beta variant (hereinafter referred to as rhINF-β) designed by replacing arginine (Arg) at position 27 with threonine (Thr) for additional glycosylation at position 25 of interferon-beta 1a. Compared to wild-type interferon-beta 1a (Rebif), it exhibits increased safety, reduced tendency to protein aggregation, and increased half-life. In other words, R27T is a biobetter of rhINF-β produced by additional glycosylation through site-directed mutagenesis.

[0009] One of the major challenges in the development of protein drugs is to provide proteins with sufficient chemical, physical, and biological safety to ensure improved safety during purification and storage. However, achieving high safety remains a difficult task due to the complexity of protein structures, including various levels of intrinsic susceptibility in proteolytic pathways, protein macromolecules, and secondary, tertiary, and quaternary structures.

[0010] More than 30 years have passed since insulin, the first recombinant peptide hormone, was approved and successfully produced in 1982. Numerous successful recombinant protein / peptide drugs have been reported since then. However, the development process of biopharmaceuticals, especially formulation, continues to face challenges due to various factors, such as protein aggregation, physicochemical instability, short half-life, poor solubility, and pharmacokinetic properties.

[0011] In particular, protein aggregation and degradation are major problems that easily occur in almost all biopharmaceutical processes. This is due to the structural and thermodynamic instability of therapeutic proteins in solution during storage. Because therapeutic proteins are sensitive to structural changes due to various factors during purification, processing, and storage, these problems can worsen if proteins are exposed to harsh conditions, such as repeated freezing and thawing or storage in buffers with different pH levels. Furthermore, protein-based biopharmaceuticals are susceptible to physical degradation, such as unfolding, aggregation, and the formation of insoluble particles due to non-native folding. Therefore, it is important to maximize safety by avoiding protein aggregation and physical denaturation.

[0012] In 2004, an interferon-beta mutant protein was developed that altered the glycosylation of interferon (Korean Patent No. 781666), and research is underway to utilize it as a therapeutic agent. Interferon beta is prone to degradation reactions during the purification process, such as cleavage of peptide bonds, deamidation, oxidation of methionine to methionine sulfide, and disulfide exchange (US2012 / 0177603), which can reduce its safety, and these must be taken into consideration.

[0013] Therefore, it is necessary to develop a human interferon-beta variant that exhibits pharmacological effects superior to those of natural interferon-beta, and a method for obtaining this variant in high yield is required. DISCLOSURE OF THE INVENTION technical challenges

[0014] Therefore, the present inventors have endeavored to develop an interferon beta variant with superior pharmacological effects and improved purification efficiency compared to natural interferon beta. As a result, they have found that a human interferon beta variant containing an amino acid sequence in which the 17th amino acid, cysteine, of human interferon beta is replaced with serine and the 27th amino acid, arginine, is replaced with threonine has excellent interferon beta activity and excellent purification efficiency, making it suitable for use in new interferon beta production.

[0015] Furthermore, the inventors have conducted extensive research to develop methods for improving the safety of the R27T mutant, a human interferon beta mutant, more specifically, the purification safety, storage safety, and freezing / thawing safety. As a result, they discovered that this goal could be achieved by replacing the 17th amino acid of the R27T mutant, cysteine, with serine (C17S), thereby completing the present invention.

[0016] Therefore, an object of the present invention is to provide a human interferon-beta mutant having an amino acid sequence in which the 17th amino acid, cysteine, of human interferon-beta is replaced with serine and the 27th amino acid, arginine, is replaced with threonine.

[0017] Another object of the present invention is to provide polynucleotides encoding said human interferon-beta variants.

[0018] It is still another object of the present invention to provide an expression vector which contains the polynucleotide and thereby allows expression of human interferon-beta in animal cells.

[0019] It is still another object of the present invention to provide an animal cell transformed with the vector.

[0020] It is yet another object of the present invention to provide a method for producing a human interferon-beta variant, which comprises the step of culturing said animal cells.

[0021] Another object of the present invention is to provide a pharmaceutical composition containing the human interferon-beta variant as an active ingredient.

[0022] Another object of the present invention is to provide a pharmaceutical composition made from said human interferon-beta variant.

[0023] Another object of the present invention is to provide a pharmaceutical composition essentially comprising said human interferon-beta variant.

[0024] Another object of the present invention is to provide a method for improving the safety of human interferon-beta R27T mutant, which comprises substituting cysteine, the 17th amino acid of human interferon-beta R27T mutant, in which arginine, the 27th amino acid of human interferon-beta, is replaced with threonine, to serine.

[0025] Another object of the present invention is to provide a use of the human interferon beta variant for the manufacture of a preparation having the pharmacological effect of natural human interferon beta against diseases selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infections, HIV-related diseases, and hepatitis C.

[0026] Another object of the present invention is to provide a method for treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infections, HIV-related diseases, and hepatitis C, which has the pharmacological effects of natural human interferon beta and comprises administering to an individual in need thereof an effective amount of a composition comprising said human interferon beta variant.

[0027] To achieve the above-mentioned objectives, the present invention provides a human interferon-beta variant having an amino acid sequence in which the 17th amino acid, cysteine, of human interferon-beta is replaced with serine and the 27th amino acid, arginine, is replaced with threonine.

[0028] In order to achieve another object of the present invention, the present invention provides a polynucleotide encoding said human interferon-beta variant.

[0029] In order to achieve yet another object of the present invention, there is provided an expression vector which contains the polynucleotide and thereby allows expression of human interferon-beta in animal cells.

[0030] To achieve yet another object of the present invention, the present invention provides an animal cell transformed with the vector.

[0031] In order to achieve still another object of the present invention, the present invention provides a method for producing a human interferon-beta variant, which comprises culturing the animal cells.

[0032] To achieve yet another object of the present invention, the present invention provides a pharmaceutical composition containing the human interferon-beta variant as an active ingredient.

[0033] The present invention also provides pharmaceutical compositions made with the human interferon-beta variants.

[0034] The present invention also provides a pharmaceutical composition containing the human interferon-beta variant as an essential active ingredient.

[0035] In order to achieve yet another object of the present invention, an object of the present invention is to provide a method for improving the safety of human interferon-beta R27T mutant, which comprises substituting cysteine, the 17th amino acid of human interferon-beta R27T mutant, in which arginine, the 27th amino acid of human interferon-beta, is replaced with threonine, to serine.

[0036] In order to achieve yet another object of the present invention, the present invention provides a use of the human interferon beta variant for producing a preparation having the pharmacological effect of natural human interferon beta against diseases selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infections, HIV-related diseases, and hepatitis C.

[0037] In order to achieve yet another object of the present invention, the present invention provides a method for treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infections, HIV-related diseases, and hepatitis C, which has the pharmacological effects of natural human interferon beta and comprises administering an effective amount of a composition containing the human interferon beta variant to an individual in need thereof.

[0038] The present invention will be described in detail below. The human interferon beta variant having a double mutation in the present invention is a human interferon beta variant having an amino acid sequence in which the 17th amino acid, cysteine, of human interferon beta is replaced with serine and the 27th amino acid, arginine, is replaced with threonine.

[0039] In the present invention, 'human interferon beta variant' refers to any polypeptide that has all or part of the amino acid sequence derived from human interferon beta, in which the 17th amino acid, cysteine, in natural human interferon beta is replaced with serine and the 27th amino acid, arginine, is replaced with threonine, and that has the activity of human interferon beta.

[0040] In the present invention, "human interferon beta activity" is defined as one or more activities known to be possessed by human interferon beta that are sufficient to identify a polypeptide as human interferon beta. Examples of such activities include the aforementioned activities of alleviating, alleviating, or treating multiple sclerosis, antiviral activity, cell growth suppression activity, antigrowth activity, antiproliferative activity, lymphocyte cytotoxicity-increasing activity, immunomodulatory activity, target cell differentiation induction or inhibition activity, cytokine production-increasing activity, cytotoxic T cell effect-increasing activity, macrophage effect-increasing activity, natural killing cell-increasing activity, cancer prevention or treatment activity, autoimmune disorder prevention or treatment activity, viral infection prevention or treatment activity, HIV-related disease prevention or treatment activity, hepatitis C prevention or treatment activity, and rheumatoid arthritis prevention or treatment activity.

[0041] The most preferred form of the double-mutated human interferon beta variant of the present invention is a polypeptide comprising an amino acid sequence in which the 17th amino acid, cysteine, of natural human interferon beta having the amino acid sequence of SEQ ID NO: 1, is replaced with serine and the 27th amino acid, arginine, is replaced with threonine.

[0042] In the present invention, the mutant in which the 17th amino acid, cysteine, of the natural human interferon-beta is replaced with serine and the 27th amino acid, arginine, is replaced with threonine may contain the amino acid sequence of SEQ ID NO: 3, specifically, may be essentially composed of the amino acid sequence of SEQ ID NO: 3, and more specifically, may be made of the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0043] SEQ ID NO: 1 MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

[0044] SEQ ID NO: 3 MSYNLLGFLQRSSNFQSQKLLWQLNGTLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

[0045] Furthermore, the human interferon beta variant of the present invention may be a human interferon beta variant in which the 17th amino acid, cysteine, of human interferon beta of SEQ ID NO: 1 is replaced with serine and the 27th amino acid, arginine, is replaced with threonine, and which has a sequence homology of 90% or more with wild-type interferon beta of SEQ ID NO: 1 and has the activity of interferon beta.

[0046] As used herein, the term "variant" refers to a protein that differs from the recited sequence by conservative substitutions and / or modifications of one or more amino acids, while maintaining the functions or properties of the protein. A variant differs from an identified sequence by the substitution, addition, or addition of several amino acids. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of a variant may be increased, unchanged, or decreased compared to the native protein. Some variants also include variants in which one or more portions, such as the N-terminal leader sequence or transmembrane domain, have been deleted. Other variants include variants in which portions have been deleted from the N- and / or C-termini of the mature protein. The term "variant" may be expressed in other terms such as "mutation," "modification," "mutated protein," "modified polypeptide," "mutant," "mutein," "divergent," "variant," etc., as long as the term is used in the sense of "mutated," but is not limited thereto. For purposes of the present invention, the variant may be a mutated protein having increased activity compared to a natural wild-type or non-mutated protein, but is not limited thereto.

[0047] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. The variant may, for example, have one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions may generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include glutamic acid and aspartic acid. Among amino acids having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Among the nonpolar amino acids, aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0048] Variants can also include the addition or substitution of additional amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to an N-terminal signal (or leader) sequence of a protein involved in co-translationally or post-translationally protein transfer.

[0049] In one embodiment of the present invention, the human interferon-beta variant may include, but is not limited to, an amino acid sequence having 80% or more homology or identity to the wild-type human interferon-beta protein of SEQ ID NO: 1, in which the 27th amino acid is threonine and the 17th amino acid is serine. Specifically, the variant of the present invention may include a protein having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to the amino acid sequence of SEQ ID NO: 3. It is also clear that proteins having such homology or identity and exhibiting efficacy corresponding to the protein, in which a partial sequence is omitted, modified, replaced, or added beyond the 27th and 17th amino acid positions of the wild-type human interferon-beta protein of SEQ ID NO: 1, are also included within the scope of the present invention.

[0050] As used herein, the term "homology" or "identity" refers to the degree of relatedness between two given amino acid sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0051] Whether any two protein sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, using default parameters as known in the art. Alternatively, the Needleman-Wunsch algorithm, such as that implemented in the Needleman program (version 5.0.0 or later) of the EMBOSS package (including the GCG program package), can be used. For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0052] The present invention also provides polynucleotides encoding the double-mutated human interferon-beta variants of the present invention.

[0053] As used herein, the term "polynucleotide" is defined to include any single-stranded or double-stranded polymer of RNA, DNA, or RNA-DNA.

[0054] Given any amino acid sequence, a person skilled in the art can easily prepare a polynucleotide encoding such an amino acid sequence based on that amino acid sequence, using his or her ordinary skills.

[0055] The present invention also provides an animal cell expression vector comprising the polynucleotide capable of expressing the human interferon-beta variant of the present invention in animal cells.

[0056] The human interferon-beta variant having a double mutation of the present invention contains one or two additional sugar chains compared to the naturally occurring human interferon-beta variant. Considering that such sugar chains are generally formed in animal cells, the animal cell expression vector is specifically (i) a polynucleotide encoding the human interferon beta variant as described above; (ii) a promoter operably linked to the nucleotide sequence of (i) to form an RNA molecule; (iii) a polynucleotide encoding a leader sequence; and (iv) a replication origin; and (v) essentially comprising a 3'-B-decoding site which initiates polyadenylation of the 3'-end of said RNA molecule;

[0057] The promoter refers to a sequence capable of activating transcription, and such sequences are well known in the art. Similarly, the 3'-B-decoding region, which functions to stabilize the leader sequence and mRNA and transport the translated protein to the endoplasmic reticulum where glycosylation occurs, is also well known in the art.

[0058] On the other hand, the expression vector of the present invention may optionally contain a reporter gene (e.g., luciferase and β-glucuronidase) or a selectable marker gene such as an antibiotic resistance gene (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.), and may also optionally contain an enhancer.

[0059] Meanwhile, examples of vectors that can be used as animal cell expression vectors of the present invention include pSV2-neo, pCAGGS, pcDL-SRα296, and pAc373. The vectors exemplified above may optionally contain the promoter, leader, origin of replication, 3'-B-decoding site, reporter gene, selection marker gene, enhancer, etc., as described above.

[0060] The present invention provides animal cells transformed with the expression vector and a method for producing human interferon-beta variants by culturing such animal cells.

[0061] As used herein, "recombinant" refers to the modification of the genotype of a host cell by the introduction of an exogenous polynucleotide (which in this invention means a polynucleotide encoding a double-mutated human interferon-beta variant), and refers to the introduction of the exogenous polynucleotide into the host cell regardless of the method used for the recombination. The exogenous polynucleotide introduced into the host cell may be maintained by being integrated into the genome of the host cell, or may be maintained without being integrated, and the present invention encompasses both.

[0062] Meanwhile, the term "animal cells" as used above includes mammalian cells and insect cells that can be used to produce recombinant proteins. Examples of animal cells that can be used in the present invention include COS cells, CHO cells, C-127 cells, BHK cells, rat HepI cells, rat HepII cells, TCMK cells, human lung cells, human liver tumor cells, HepG2 cells, mouse hepatocytes, DUKX cells, and 293 cells. Examples of insect cells include cultured silkworm cells.

[0063] In another aspect, the present invention relates to a pharmaceutical composition comprising the human interferon-beta variant of the present invention as described above.

[0064] The human interferon-beta contained in the pharmaceutical composition of the present invention has been primarily used as a therapeutic agent for multiple sclerosis, but it has also been reported that it can be used to treat cancer, autoimmune disorders, viral infections, HIV-related diseases, hepatitis C, etc., and its pharmacological effects are continually being reported.

[0065] For this reason, the pharmacological effects of the pharmaceutical composition of the present invention should be understood to include not only the pharmacological effects as a therapeutic agent for multiple sclerosis, but also all other pharmacological effects possessed by human interferon-beta.

[0066] Furthermore, such pharmacological effects must be understood to include not only the pharmacological effects of beta-human interferon that have been known up to now, but also pharmacological effects that will become clear in the future.

[0067] Since the pharmaceutical composition of the present invention is characterized by containing a human interferon-beta variant with increased activity or function obtained by the present invention, it would not be an unduly broadening of the scope of the present invention if the pharmaceutical composition of the present invention includes not only the pharmacological effects of the above drugs that are currently known but also pharmacological effects that will be revealed in the future.

[0068] However, given that human interferon beta variants have still been primarily used as therapeutic agents for multiple sclerosis, and that their therapeutic effects against cancer, autoimmune disorders, viral infections, HIV-related diseases, and hepatitis C have already been demonstrated, it is desirable that the pharmacological effects be as described above.

[0069] Meanwhile, the pharmaceutical composition of the present invention can be administered orally or through many other routes including transdermal, subcutaneous, intravenous or intramuscular.

[0070] In addition, the pharmaceutical compositions of the present invention can be formulated into various dosage forms, and when formulating, they can be formulated using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0071] Furthermore, the daily dosage of the pharmaceutical compositions of the present invention may be administered at a dosage known in the art, generally in the range of 0.01 to 5 mg / kg body weight, and may be administered in a single dose or in divided doses. However, the actual dosage of the pharmaceutical compositions of the present invention is determined in light of many related factors, such as the route of administration, the age, sex, and weight of the patient, and the severity of the patient's condition, and therefore, the dosage should not be construed as limiting the scope of the present invention in any aspect.

[0072] The present invention also provides a method for improving the safety of human interferon-beta R27T mutant, which comprises replacing the 17th amino acid, cysteine, of human interferon-beta R27T mutant, in which the 27th amino acid, arginine, of human interferon-beta is replaced with threonine, with serine.

[0073] In one embodiment of the present invention, the 17th amino acid of human interferon-beta R27T mutant was replaced with serine (C17S) and purified, and then the glycosylation change between mutants with and without the C17S mutation was examined using RP-HPLC. As a result, it was confirmed that the diglycosylation rate after protein purification of the R27T mutant containing C17S was improved compared to the R27T mutant without C17S.

[0074] In another example of the present invention, the 17th amino acid of human interferon-beta R27T mutant was replaced with serine (C17S), and the purified product was then evaluated for storage safety in phosphate buffer and acetate buffer at pH 2.0 to 6.0. As a result, it was confirmed that the protein recovery rate of the R27T mutant containing C17S in each buffer was improved compared to the R27T mutant without C17S.

[0075] In another example of the present invention, the 17th amino acid of human interferon-beta R27T mutant was replaced with serine (C17S), purified, and then repeatedly frozen / thawed to determine the proportion of protein monomers by SEC-HPLC analysis. As a result, it was confirmed that the proportion of monomers of the R27T mutant containing C17S was improved after repeated freezing / thawing compared to the R27T mutant without C17S.

[0076] In the present invention, "human interferon-beta R27T mutant" refers to any polypeptide that has all or part of the amino acid sequence derived from human interferon-beta, in which the 27th amino acid, arginine, in the wild-type human interferon-beta of SEQ ID NO: 1 is replaced with threonine, and has the activity of human interferon-beta.

[0077] In the present invention, the human interferon-beta R27T mutant in which the 27th amino acid of human interferon-beta, arginine, is replaced with threonine may comprise the amino acid sequence of SEQ ID NO: 2, specifically, may be essentially composed of the amino acid sequence of SEQ ID NO: 2, and more specifically, may be made up of the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0078] SEQ ID NO: 2 MSYNLLGFLQRSSNFQCQKLLWQLNGTLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

[0079] Furthermore, the human interferon-beta R27T mutant may include, but is not limited to, an amino acid sequence in which the 27th amino acid of the wild-type human interferon-beta protein of SEQ ID NO: 1 is fixed to threonine and which has 80% or more homology or identity thereto. Specifically, the mutant of the present invention may include a protein having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to the amino acid sequence of SEQ ID NO: 2. It is also clear that proteins having an amino acid sequence in which a partial sequence other than the 27th amino acid position of the wild-type human interferon-beta protein of SEQ ID NO: 1 has been omitted, modified, replaced, or added, as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy to the protein, are also included within the scope of the present invention.

[0080] In the present invention, the step of substituting the 17th amino acid, cysteine, of the human interferon-beta R27T mutant with serine can be carried out by any known method used in the art for introducing amino acid point mutations, without limitation.

[0081] For example, this can be achieved by transforming host cells with a vector containing a polynucleotide encoding a protein in which the 27th amino acid, arginine, of wild-type human interferon-beta of SEQ ID NO: 1 is replaced with threonine and the 17th amino acid, cysteine, is replaced with serine, and then culturing the resulting product in a medium.

[0082] The detailed description of this method for producing a human interferon-beta variant containing R27T and C17S double mutations can be applied as is.

[0083] In the present invention, the polynucleotide encoding the human interferon-beta variant may include, without limitation, a polynucleotide sequence encoding a protein in which the 27th amino acid, arginine, is replaced with threonine and the 17th amino acid, cysteine, is replaced with serine in the wild-type human interferon-beta of SEQ ID NO: 1. Specifically, in the present invention, the coding region of the polynucleotide may be modified in various ways within the scope that does not change the amino acid sequence of the protein, taking into consideration codon degeneracy or codons preferred in the organism in which the protein is to be expressed.

[0084] The human interferon-beta variant produced by the culture may be excreted into the medium or may not yet be able to be excreted and remain intracellularly.

[0085] The method of the present invention may further comprise the step of recovering the human interferon-beta R27T mutant, in which the 17th amino acid, cysteine, is replaced with serine, from the cultured cells or medium.

[0086] The human interferon-beta variant produced in the culturing step can be recovered by collecting the target protein from the culture medium using a suitable method known in the art, depending on the culturing method, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, and the target variant can be recovered from the medium or cells using a suitable method known in the art.

[0087] The recovery step may include a purification step and may be carried out by any suitable method known in the art, such as membrane filtration.

[0088] In one aspect of the present invention, the safety can be selected from the group consisting of purification safety, storage safety, and freezing / thawing safety.

[0089] The term "safe purification" refers to a low rate of protein denaturation that may occur during the purification of a protein recovered from host cells or cell culture medium, and the purification may include, without limitation, conventional methods for purifying proteins in the art, such as salting out (e.g., ammonium sulfate precipitation, phosphoric acid precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.), precipitation, gel filtration, ion exchange, and column chromatography such as reverse-phase column chromatography, preferably column chromatography.

[0090] In one aspect of the present invention, the purification safety may mean that the glycosylation level of the protein is less changed before and after the above-mentioned protein purification process, and specifically, in the case of human interferon-beta R27T mutant, it may mean that the purification rate of diglycosylated protein is improved.

[0091] In another aspect of the present invention, the purification safety can be characterized as a reduction in protein aggregation and degradation during protein concentration and buffer exchange. Generally, proteins or polypeptides can aggregate or degrade during the concentration and buffer exchange steps performed during / after the purification process. However, by substituting serine for cysteine, the 17th amino acid of the human interferon-beta R27T mutant, protein denaturation during the concentration and buffer exchange steps can be reduced.

[0092] Proteins can be concentrated using methods known in the art. Non-limiting exemplary methods that can be used to concentrate proteins include ultrafiltration, tangential flow filtration, centrifugal concentration using membrane concentrators (e.g., cellulose membrane concentrators), precipitation onto a moisture-absorbing material (e.g., a moisture-absorbing polymer), salting out (e.g., using ammonium sulfate), and chromatography (e.g., size exclusion chromatography).

[0093] Ultrafiltration for protein concentration is a separation method in which hydraulic pressure is used to force molecules and solvents across a membrane made of pores with a particle size, or cut-off size. Molecules with larger molecular weights cannot pass through the membrane; only molecules with molecular weights smaller than the membrane's cut-off value can pass through, forming a so-called replenishment solution. The molecules present in the replenishment solution are then concentrated as the solvent flows across the membrane. Ultrafiltration can be used for protein concentration or buffer exchange, or to formulate a target protein into a desired solution or buffer.

[0094] In certain embodiments, concentration of a solution or composition containing a protein of interest can be accomplished by tangential flow filtration (TFF). This method is particularly useful for large-scale concentration, i.e., for concentrating bulk solutions ranging from one liter to several hundred liters. Thus, this method is particularly useful for producing concentrated solutions of a protein of interest on an industrial scale.

[0095] The TFF technique is based on the use of specialized equipment to allow a filtered solution to flow across a semi-permeable membrane, which allows only molecules smaller than the membrane's pores to pass through, forming a filtrate and leaving larger molecules to collect (retentate). Two types of pressure are applied with the TFF process: one to feed the solution into the system and circulate it within the system (inlet pressure), and another pressure applied across the membrane to allow small molecules and solvent to cross the membrane (membrane pressure). Inlet pressures are typically in the 1-3 bar range, e.g., between 1.5-2 bar. Membrane pressures are typically greater than 1 bar.

[0096] When TFF is used to concentrate a composition, the concentrated composition of the target protein can be collected in a buffer. Membranes useful for TFF are typically made of regenerated cellulose or polyethersulfone (PES). The membrane pore size is typically less than 10,000 Mw, e.g., can have a molecular weight cutoff in the range of 10-10,000 Mw.

[0097] In another embodiment, concentration of a composition containing a polypeptide of interest can be accomplished by use of a centrifuge. In this case, the protein of interest is filtered through a membrane by applying centrifugal force to the membrane. Such membranes are often characterized by a molecular weight (Mw) cutoff, i.e., the maximum molecular size of a compound that can pass through the membrane; compounds larger than this molecular size cannot pass through the membrane.

[0098] The membrane may in particular be made of polyethersulfone (PES) or regenerated cellulose. Examples of such suitable commercial filter devices may be, but are not limited to, Centricon Plus-80 or Centricon Plus-15.

[0099] Concentration may generally be carried out at 2000-4500 g, for example between 2500-4000 g, or between 2750-3500 g, or between 3000-3500 g, for example at 3000 g or 3100 g or 3200 g or 3300 g or 3400 g or 3500 g.

[0100] Buffer exchange of the composition containing the concentrated target protein can be performed, for example, by a) diluting the composition containing the concentrated target protein with a buffer or formulation, for example, 5-15 times; b) diluting or concentrating the composition, and after the above steps are performed, the amount of additives contained in the buffer or formulation present in the composition before these steps constitutes, for example, 5 v / v % or less or 1 v / v % or less of the additives of the buffer or formulation present in the composition.

[0101] In the present invention, the storage safety means that the rate of protein denaturation that may occur during the process of storing purified human interferon-beta R27T mutant in a buffer solution or changing the composition of the buffer solution is low.

[0102] In the present invention, the buffer solution may have a pH of 2.0 to 6.0, preferably 2.0 to 5.0, and most preferably 2.0 to 4.0, but is not limited thereto.

[0103] In the present invention, the buffer solution may be selected from the group consisting of acetic acid, phosphoric acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, potassium citrate, potassium metaphosphate, potassium phosphate monobasic, sodium acetate, sodium citrate, sodium lactate solution, dibasic sodium phosphate, monobasic sodium phosphate, bicarbonate, tris(tris(hydroxymethyl)aminomethane), MOPS (3-(N-morpholino)propanesulfonic acid), HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), ACES (2-(2-amino-2-methylpropanol), 2-hydroxy-2-methylpropanol), 2-hydroxy-2-methylpropanol, ... -iodide ethyl)aminoethanesulfonic acid), ADA (N-(2-acetamino)2-iminodiacetic acid), AMPSO (3-(1,1-dimethyl-1,2-hydroxyethylamino-2-propanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, bicine (N,N-bis(2-hydroxyethylglycine), bis-tris(bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), CAPSO (3-(cyclohexylamino) (2-hydroxy-1-propanesulfonic acid), CHES (2-(N-cyclohexylamino)ethanesulfonic acid), DIPSO (3-N,N-bis(2-hydroxyethylamino-2-hydroxy-propanesulfonic acid), HEPPS (N-(2-hydroxyethylpiperazine)-N'-(3-propanesulfonic acid), HEPPSO (N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), triethanolamine, imidazole, glycine, Ethanolamine, phosphate, MOPSO (3-(N-morpholino)-2-hydroxypropanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), POPSO (piperazine-N,N'-bis(2-hydroxypropanesulfonic acid), TAPS (N-trishydroxymethyl)methyl-3-aminopropanesulfonic acid), TAPSO (3-N-tris(hydroxymethyl)methylamino-2-hydroxypropanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid),It may be selected from the group consisting of, but not limited to, tricine (N-tris(hydroxymethyl)methylglycine), 2-amino-2-methyl-1,3-propanediol, and 2-amino-2-methyl-1-propanol.

[0104] In the present invention, the freeze / thaw safety may mean that the possibility of protein denaturation is low when the human interferon-beta R27T mutant stored in a buffer solution is subjected to repeated cycles of freezing and thawing.

[0105] In one aspect of the present invention, the freeze / thaw safety may be characterized as safety after freezing and thawing at -100°C to -10°C, preferably safety after freezing and thawing at -90°C to -30°C, and most preferably safety after freezing and thawing at -80°C to -50°C.

[0106] In another aspect of the present invention, the freeze / thaw safety may be characterized as freeze / thaw safety in an acetate buffer, preferably freeze / thaw safety in an acetate buffer of pH 3.0 to 5.0.

[0107] In another aspect of the present invention, the freeze / thaw safety can be characterized by a reduction in protein aggregation and degradation after three or more freeze / thaw cycles, preferably four or more freeze / thaw cycles, and most preferably four or more freeze / thaw cycles.

[0108] The biological activity of human interferon-beta can change depending on its interaction with the type 1 interferon receptor. The 17th amino acid, which was mutated to improve the safety of the human interferon-beta R27T mutant in the present invention, is located in the binding interface with the type 1 interferon receptor IFNAR2. In particular, the 15th to 23rd amino acid residues are the main receptor binding sites, and mutations in any one or more of these amino acids can alter biological activity. In particular, because free cysteine ​​residues have stronger hydrophobicity than disulfide-bonded cysteine ​​residues, altering the 17th amino acid cysteine ​​can alter activity. As expected, introducing the C17S mutation into the human interferon-beta R27T mutant reduced the hydrophobicity of the R27T mutant, but this did not affect protein activity and improved purification safety, storage safety, and freezing / thawing safety. Furthermore, the introduction of the C17S mutation can induce hydrogen bonding within the protein by the serine residue, thereby improving safety.

[0109] The present invention provides the use of said human interferon beta variant for the manufacture of a preparation having the pharmacological effect of natural human interferon beta against diseases selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infections, HIV-related diseases and hepatitis C.

[0110] The present invention provides a method for treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infections, HIV-related diseases, and hepatitis C, which method comprises administering to an individual in need thereof an effective amount of a composition comprising said human interferon beta variant, which has the pharmacological effect of natural human interferon beta.

[0111] The 'effective amount' of the present invention refers to an amount that, when administered to an individual, shows an effect of improving, treating, preventing, detecting, diagnosing, or suppressing or reducing a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infections, HIV-related diseases, and hepatitis C. The 'individual' may be an animal, preferably a mammal, particularly an animal including a human, or may be a cell, tissue, organ, etc. derived from an animal. The individual may be a patient in need of the effect.

[0112] The term "treatment" as used herein refers to the comprehensive treatment of a disease or symptom of a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infections, HIV-related diseases, and hepatitis C, and may include curing or substantially preventing the disease or improving the condition, and may include, but is not limited to, alleviating, curing, or preventing one or most of the symptoms that began with cancer.

[0113] As used herein, the term 'comprising' is used equivalently to 'including' or 'characterized by' and does not exclude additional components or method steps not specifically mentioned in the compositions or methods of the present invention. The term 'consisting of' means excluding additional elements, steps, or ingredients not otherwise described. The term 'essentially consisting of' means that the scope of a composition or method may include, in addition to the stated materials or steps, materials or steps that do not substantially affect the basic characteristics of the composition or method. Advantageous Effects

[0114] Therefore, the human interferon beta variant with double mutations provided by the present invention has excellent interferon beta activity and significantly improved efficiency in the purification process, making it useful for the production of therapeutic agents.

[0115] Furthermore, according to the method for improving the safety of human interferon-beta R27T mutant of the present invention, the activity of human interferon-beta R27T mutant in which the 27th amino acid of human interferon-beta, arginine, is replaced with threonine is maintained while improving the safety of the protein during the purification process, storage process, and freezing / thawing process, thereby ensuring consistent protein quality during the manufacturing and distribution process. [Brief explanation of the drawings]

[0116] [Figure 1] Figure 1 shows the PCR conditions for the protein expression DNA production experiment. [Figure 2] Figure 2 shows the restriction enzyme treatment and cloning in the protein expression DNA production experiment. [Figure 3] Figure 3 shows the progress of cloning using T4 DNA ligase (NEB) in a protein expression DNA production experiment. [Figure 4] Figure 4 shows the colony PCR conditions for the protein expression DNA production experiment. [Figure 5a] Figures 5a and 5b show cell viability observations after ABN 101(NT) and ABN 101(CS) transduction (ABN 101(NT): R27T mutant interferon-beta, ABN 101(CS): C17S, R27T double mutant interferon-beta). [Figure 5b] Figures 5a and 5b show cell viability observations after ABN 101(NT) and ABN 101(CS) transduction (ABN 101(NT): R27T mutant interferon-beta, ABN 101(CS): C17S, R27T double mutant interferon-beta). [Figure 6a] Figures 6a and 6b show the 50 ml scale fed-batch results for ABN 101(NT) and ABN 101(CS). [Figure 6b]Figures 6a and 6b show the 50 ml scale fed-batch results for ABN 101(NT) and ABN 101(CS). [Figure 7] Figure 7 shows the results for ABN 101(NT) and ABN 101(CS) 1L scale fed-batch. [Figure 8] Figure 8 shows the concentration and buffer exchange in the safety confirmation experiment of the interferon-beta mutant. [Figure 9a] Figures 9a and 9b relate to the interferon-variant RP-HPLC results. [Figure 9b] Figures 9a and 9b relate to the interferon-variant RP-HPLC results. [Figure 10a] 10a to 10d show the change in monomer content during buffer exchange of interferon-mutant. [Figure 10b] 10a to 10d show the change in monomer content during buffer exchange of interferon-mutant. [Figure 10c] 10a to 10d show the change in monomer content during buffer exchange of interferon-mutant. [Figure 10d] 10a to 10d show the change in monomer content during buffer exchange of interferon-mutant. [Figure 11a] Figures 11a to 11f show comparative safety confirmation of interferon-variants in 20 mM Na-Pi buffer free / treated. [Figure 11b] Figures 11a to 11f show comparative safety confirmation of interferon-variants in 20 mM Na-Pi buffer free / treated. [Figure 11c] Figures 11a to 11f show comparative safety confirmation of interferon-variants in 20 mM Na-Pi buffer free / treated. [Figure 11d]Figures 11a to 11f show comparative safety confirmation of interferon-variants in 20 mM Na-Pi buffer free / treated. [Figure 11e] Figures 11a to 11f show comparative safety confirmation of interferon-variants in 20 mM Na-Pi buffer free / treated. [Figure 11f] Figures 11a to 11f show comparative safety confirmation of interferon-variants in 20 mM Na-Pi buffer free / treated. [Figure 12a] 12a to 12c show comparative safety confirmation of interferon-variants in 20 mM Na-OAc buffer free / treated. [Figure 12b] 12a to 12c show comparative safety confirmation of interferon-variants in 20 mM Na-OAc buffer free / treated. [Figure 12c] 12a to 12c show comparative safety confirmation of interferon-variants in 20 mM Na-OAc buffer free / treated. BEST MODE FOR CARRYING OUT THE INVENTION

[0117] The present invention will be described in detail below. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0118] The following experiments were carried out to construct ABN 101(NT), an R27T mutation human interferon beta-1a, and ABN 101(CS), an R27T and C17S double mutation.

[0119] Experimental Method

[0120] 1. Protein Expression DNA Fabrication Primers were designed to add XbaI and PacI enzyme restriction sites to clone ABN 101(NT) and ABN 101(CS) behind the human promoter of the pD2535nt-HDP vector, and the sequences are as follows: Forward: 5'-ggtctagagccaccAtgacca-3' (SEQ ID NO: 4) XbaI Reverse: 5'-cacttagggattaattaatcagttcctcaggtag-3' (SEQ ID NO: 5) PacI The two inserts were amplified using AccuPower PCR PreMix (Bioneer) under the PCR conditions shown in Figure 1. The PCR product obtained through PCR was checked for size on a 0.8% agarose gel and MEGAquick-spin TM Gel extraction was performed using the Plus Total Fragment DNA Purification Kit (Intron). The purified PCR product and pD2535nt-HDP vector were digested with restriction enzymes as shown in Figure 2 below and then cloned. All restriction enzymes and buffers were from Thermo Fisher Scientific. After restriction enzyme digestion, MEGAquick-spin TM We attempted to improve cloning efficiency by obtaining pure DNA fragments using the Plus Total Fragment DNA Purification Kit (Intron). After purification, cloning was performed using T4 DNA ligase (NEB) as shown in Figure 3 below. After ligation, transformation was performed using DH5a Chemically Competent E. coli (Enzynomics). First, DH5a cells were slowly thawed on ice, and then 20 μl of the reaction mixture was added and placed on ice for 30 minutes. Next, heat shock was performed at 42°C for 30 seconds, followed by 2 minutes of ice to stabilize. 400 μl of SOC media (provided by Enzynomics) was added and incubated at 37°C for 1 hour. After healing, the cells were centrifuged at 3000 rpm for 3 minutes and plated on LB medium containing 50 μg of kanamycin. The cells were then grown overnight in a 37°C incubator. Colony PCR was performed to confirm cloning by removing the floating colonies. A single colony, with minimal overlap, was scraped using a 10-point tip and placed in an AccuPower PCR PreMix (Bioneer) tube. PCR was performed using colony PCR primers for pD2535nt-HDP vector provided by Horizon under the conditions shown in Figure 4 below. The PCR products were checked for size on a 0.8% agarose gel to confirm the completion of cloning. The cloned products pD2535nt-HDP::ABN 101(NT) and pD2535nt-HDP::ABN 101(CS) were used to prepare DNA preps for CHO-K1 cell transfection using nucleobond Xtra Maxi Plus (MACHEREY-NAGEL).

[0121] 2. Transduction E. coli harboring the expression plasmid was cultured in LB medium containing 100 μg / mL kanamycin and harvested. DNA was isolated using a QIAGEN Plasmid Midi Prep kit. 50 μg of isolated DNA was added to 30 μl of 10x CutSmart buffer and 2.5 μl of NrU1-HF restriction enzyme to make a final volume of 300 μl. Linearization was then achieved by incubation at 37°C for 2 hours. After 2 hours, 30 μl of a 1 / 10 volume of 3M, pH 5.5 sodium acetate solution and 750 μl of ice-cold ethanol were added, followed by overnight incubation at -80°C. The next day, the ethanol-precipitated DNA was centrifuged and washed with 70% ethanol to ensure high-purity DNA, and its concentration was measured using a Nanodrop™. On the first day for transduction, 3x10 CHO-K1 cells were transfected in CDFortiCHO medium supplemented with 4 mM L-glutamine. 5 The cells were seeded into an E125 shake flask at a concentration of 10 / ml and cultured at 37°C, 5% CO2, and 125 rpm for 24 hours. On the second day, the number of cells seeded on the first day was counted and 5x10 5 The cells were seeded at a concentration of 1x10 / ml and cultured at 37°C, 5% CO2, and 125 rpm for 24 hours. On the third day, the number of seeded cells was measured and 1x10 6 Check if it has reached / ml, and if so, reduce to 1x10 6To prepare for transduction, add 37.5 μg of linearized DNA and 37.5 μl of Freestyle MAX reagent to 600 μl of OptiPRO SFM medium and incubate for 5 minutes at room temperature. Next, transfer the mixture containing DNA to the mixture containing Freestyle MAX reagent, mix, and incubate again at room temperature for 25 minutes. Carefully add the DNA-lipid mixture to the previously seeded CHO-K1 cells. Transduction proceeds by culturing for 48 hours at 37°C, 5% CO2, and 125 rpm. 48 hours after transduction, begin selection of MSX (methionine sulfoximine)-resistant cells. Completely transduced cells are cultured in selection medium supplemented with 25 and 50 μM MSX for approximately 25 days, and monitored every 2-3 days. Monitoring is continued until a viability of 70% or more and a viable cell count of 0.5-1.2 x 10 cells is reached. 6 When the cell density reaches / ml, the cells are pooled and stabilized by suspension culture in a selection medium at 37°C, 5% CO2, and 125 rpm for at least three passages.

[0122] 3.Fed-batch Fed-batch was performed using the pooled cells prepared by the previous transfection. 3x10 cells were cultured in CDFortiCHO medium without MSX. 5 50ml is seeded into an E250 shake flask so that the cell culture medium reaches a concentration of 0.1ml, and cultured at 37°C, 5% CO2, and 125 rpm for approximately 12 days. Cedex Bio analyzes glucose metabolism in the cell culture medium to measure cell viability and viable cell count. Add 5% (V / V) CD Efficient Feed C+ solution on days 3, 5, and 7, and then add 45% Glucose solution on days 4 and 6. After the fed-batch is complete, centrifuge the culture medium and collect the supernatant, which can be stored in the refrigerator or freezer.

[0123] 4.Interferon beta-1a biological activity confirmation (ELISA) The biological activity of the human interferon beta-1a protein expressed in the constructed expressing cell line is analyzed using TORAY's HuIFN-β ELISA KIT. The culture medium secured from the fed-batch is initially diluted 10,000-fold using the diluent provided in the kit, followed by serial dilutions of 2-fold dilutions up to a 1,280,000-fold dilution. The standard substance for measuring biological activity provided in the kit is prepared using diluents ranging from 200 IU / ml to 3.125 IU / ml according to the protocol. The ELISA plate is primed using the wash buffer provided in the kit. After priming, the prepared samples and standard substance are added to the ELISA plate at 100 μl / well, and HRP-conjugated antibody is added to each well at 50 μl / well. The plate is incubated at 27°C for 2 hours. After the reaction is complete, the plate is washed and then 100 μl of color reagent is added per well and incubated at 27°C for 30 minutes to induce the color reaction. Next, 100 μl of stop solution is added per well to terminate the color reaction, and plate detection is performed using a spectrophotometer with a measurement wavelength of 450 nm and a reference wavelength of 620 nm. Based on the absorbance obtained, a standard curve is constructed using a 4-parameter method, and the biological activity of the sample is calculated. The activity of the sample calculated using the standard curve is multiplied by the dilution factor to confirm the biological activity of the original sample.

[0124] 5.Interferon beta-1a expression (concentration) confirmation (ELISA) To confirm the level of human Interferon beta-1a protein expression in the constructed expressing cell line, analysis was performed using TORAY's HuIFN-β ELISA KIT. Similar to the biological activity confirmation method, the culture medium reserved in the fed-batch was initially diluted 10,000-fold using the diluent provided in the kit, followed by serial dilutions of two-fold dilutions up to a 1,280,000-fold dilution. An existing in-house reference standard material was serially diluted two-fold from 2.5 ng / ml to 0.039 ng / ml. The subsequent ELISA experimental process was the same as the biological activity confirmation ELISA method described above. A standard curve was constructed using a four-parameter method based on the absorbance obtained, and the sample Interferon beta-1a expression level was then converted. The expression level calculated using the standard curve was multiplied by the dilution factor to confirm the original sample concentration.

[0125] 6. Purification of Interferon-beta Mutants The cell lines prepared in the above examples were cultured using a cell factory (Nunc, Cat. No. 170069). Each expression cell line was cultured at 5x10 in alpha-MEM medium containing 10% FBS. 4Cells were passaged into cell factories at 1000 cells / ml and cultured at 37°C in 5% CO2 for 72 hours to monitor cell growth. The serum components were removed by washing three times with PBS, and then replaced with serum-free medium (Sigma C8730). After replacing the serum-free medium, the culture medium was harvested every 24 hours and fresh serum-free medium was added. The culture medium was harvested a total of four times and purified. 200 ml of Blue Sepharose resin (Amersham-Pharmacia) was loaded onto an XK50 / 20 column (Amersham-Pharmacia), and 10 column volumes of buffer A (20 mM sodium phosphate, 1 M NaCl, pH 7.4) were allowed to pass through to reach equilibrium. Sterile filtered culture medium was then passed through the equilibrated column at a flow rate of 20 ml / min, and monitored using a UV detector at 280 nm. The column was loaded with buffer B (20 mM sodium phosphate, 1 M NaCl, 30% ethylene glycol, pH 7.4) to wash away unadsorbed components, and then the proteins attached to the resin were extracted with buffer C (20 mM sodium phosphate, 1 M NaCl, 60% ethylene glycol, pH 7.4). The extract was then poured into phosphate buffered saline (PBS), concentrated using a concentrator (Centricon, cut-off 10,000), and poured into phosphate buffered saline (PBS).

[0126] 7. Comparison of diglycosylation purification efficiency of interferon-beta R27T mutant and interferon-beta double mutant (R27T and C17S) Interferon-beta variant (ABN 101(NT)) and interferon-beta double variant (ABN 101(CS)) purified using Blue Sepharose resin were subjected to reverse phase high performance liquid chromatography (RP-HPLC) to measure the content of diglycosylated interferon-beta variants.

[0127] Each interferon mutant was diluted to 0.5 mg / mL and mixed with acetonitrile (ACN) at the initial mobile phase ratio before analysis. RP-HPLC analysis was performed using a YMC-C4 column, and the solvents used were as follows: Mobile phase A was prepared by mixing 0.1% trifluoroacetic acid (TFA) with triple-distilled water, then filtered through a 0.2 μm PVDF filter and degassed for 1 hour. Mobile phase B was prepared by mixing 0.1% TFA with ACN, then filtered through a 0.2 μm PVDF filter and degassed for 1 hour.

[0128] The analytical conditions are shown in the following table. [Table 1] [Table 2]

[0129] 8. Confirmation of the safety of interferon-beta mutants by changing the buffer composition Interferon-beta mutant (ABN 101(NT)) and interferon-beta double mutant (ABN 101(CS)), purified using Blue Sepharose resin, were each subjected to buffer exchange using Centricon. To confirm the extent of quality degradation due to aggregation that occurs during buffer exchange of interferon drugs, each protein released from the Blue Sepharose resin was collected and then concentrated and buffer exchanged using 20 mM sodium phosphate (pH 2.9) buffer at a 7-fold volume ratio using Centricon. After completing buffer exchange using Centricon, the protein was passed through a 0.2 μm PES syringe filter and the protein concentration was measured at a wavelength of 280 nm using a UV spectrophotometer to confirm the recovery rate. To confirm the storage safety of other formulation buffers, the proteins that had been buffer-exchanged with 20 mM phosphate (pH 2.9) were again buffer-exchanged with 20 mM sodium acetate (pH 3.8) using Centricon at the same volume ratio, and the yield was confirmed. This process is shown in Figure 8.

[0130] 9. Freeze / thaw storage safety confirmation by changing buffer composition To confirm freeze / thawing safety, interferon drugs formulated in each buffer were frozen at -70°C for 12 hours or more and thawed at 25°C for 4 hours, with the freeze / thawing cycle repeated three or five times. Size Exclusion High Performance Liquid Chromatography (SEC-HPLC) analysis was then performed to confirm the rate of aggregation, analyzed by high molecular weights (HMWs), and degradation, analyzed by low molecular weights (LMWs). The rate of monomer conversion of the corresponding protein was analyzed to determine the storage safety of the interferon-beta variants. SEC-HPLC was performed using a Tosoh size exclusion column (TSKG2000), and the solvents used were as follows: Mobile phase A was prepared by degassing triple distilled water for 1 hour after filtering through a 0.2 μm PVDF filter, and mobile phase B was prepared by mixing triple distilled water with 150 mM sodium chloride and 100 mM sodium phosphate dibasic dihydrate, titrating the mixture with phosphoric acid to pH 7.0, filtering through a 0.2 μm PVDF filter, and degassing the mixture.

[0131] The analytical conditions are shown in the following table. [Table 3] [Table 4]

[0132] Results and Interpretation 1. Pool development results according to MSX concentration after ABN 101(NT) and ABN 101(CS) gene transduction After transfection of CHO-K1 cells with DNA cloned into the pD2535nt-HDP vector, the cell number and viability were confirmed 48 hours later. Based on this, resistant cell selection was carried out for 25 days using two concentrations of MSX. As a result, ABN 101(NT) acquired resistant cells before ABN 101(CS), and in the case of ABN 101(CS) at 50 μM, the final concentration was 3 x 10 5 The cells obtained were the most resistant, with a viable cell count of 1 / ml and a survival rate of 48%. The low survival rate was attributed to the effect of Interferon beta-1a expression on growth and survival. The results are shown in Figures 5a and 5b below.

[0133] 2. Fed-batch results in the ABN 101(NT) and ABN 101(CS) pool conditions The results of a 50ml small-scale fed-batch using pooled cells are as follows. For both the ABN 101(NT) pool and the ABN 101(CS) pool, viability was maintained longer in the pool with an MSX concentration of 50µM than in the pool with an MSX concentration of 25µM. For both pools, viable cell numbers were maintained at a lower level in the pool with an MSX concentration of 50µM than in the pool with an MSX concentration of 25µM. However, despite the lower viable cell numbers in the pool with an MSX concentration of 50µM than in the pool with an MSX concentration of 25µM, the biological activity of Interferon beta-1a in the culture medium on the final day of the fed-batch was higher, and the biological activity of the ABN 101(CS) pool was more than two-fold higher than that of the ABN 101(NT) pool. The results are shown in Figures 6a and 6b below.

[0134] 3. 1L scale fed-batch results for ABN 101(NT) and ABN 101(CS) pools Based on the results of the previous small-scale fed-batch, a 1L-scale fed-batch was performed using a pool with an MSX concentration of 50uM. The results showed that after 4 days of culture, the biological activity of ABN 101(NT) was 0.69 MIU / ml and that of ABN 101(CS) was 5.99 MIU / ml, demonstrating that the biological activity of ABN 101(CS) was approximately 8.6 times higher. A comparison of the biological activity after 12 days of culture also confirmed that the biological activity of ABN 101(CS) was approximately 4.5 times higher. The results are shown in Figure 7 below.

[0135] 4. Purification results of interferon-beta mutants The purification of the interferon-beta variant showed a higher purification efficiency than existing human interferon-beta. This confirmed that the human interferon-beta variant, which contains an amino acid sequence in which the 17th amino acid, cysteine, is replaced with serine and the 27th amino acid, arginine, is replaced with threonine, has a higher purification efficiency.

[0136] 5. Percentage of Diglycosylation Purification of Interferon-beta R27T Mutant and Interferon-beta Double Mutant (R27T and C17S) The diglycosylation:monoglycosylation content of each interferon-beta variant purified using Blue Sepharose resin was analyzed. As a result, as shown in Table 5 and Figures 9a and 9b below, the diglycosylation content of the ABN 101(CS) double mutant was confirmed to be approximately 10% higher than that of ABN 101(NT). Although there were no significant differences in the biological activity of each substance when compared by ELISA, when the interferon-beta variants were expressed and purified on the same scale, the diglycosylated form of the protein was confirmed to have a higher purification efficiency. [Table 5]

[0137] 6. Safety of Interferon-beta Variants Due to Changes in Buffer Composition To confirm the safety of each buffer composition of interferon-beta variants, we performed SEC-HPLC analysis and protein recovery through buffer exchange using Centricon. After purifying each protein, we exchanged the buffer with 20 mM sodium phosphate (pH 2.9). The recovery rate was highest in ABN 101 (CS), as shown in Table 6 below. [Table 6]

[0138] In addition, to switch each protein exchanged into a different formulation-based buffer, the recovery rate was confirmed by replacing the buffer with 20 mM sodium acetate (pH 3.8) using centricon in the same manner. As a result, as shown in Table 7, even when exchanged into an acetate-based buffer, the recovery rate of ABN 101(CS) was higher than that of ABN 101(NT). This is interpreted as an increase in stability due to the double mutant, resulting from the structural stability of the protein rather than physical factors such as centricon. [Table 7]

[0139] The protein recovery rate and monomer analysis results for each buffer composition change are shown in Table 8 below and Figures 10a to 10d. [Table 8]

[0140] 7. Freeze / thawing safety results due to changes in buffer composition To confirm the freeze / thaw safety of interferon-beta variants according to their buffer composition, the interferon-beta variants in each buffer were subjected to three or five freeze-thaw cycles, and the monomer conversion rate was confirmed through SEC-HPLC analysis. As a result, when the two interferon-beta variants were stored in a 20 mM sodium phosphate (pH 2.9)-based buffer and subjected to repeated freeze-thaw cycles, there was little difference between the two interferon-beta variants (see Tables 9, 10, and Figures 11a to 11f). [Table 9] [Table 10]

[0141] However, after buffer exchange with 20 mM sodium acetate (pH 3.8), freeze-thaw analysis revealed that ABN 101(CS) maintained its monomer content, while ABN 101(NT) exhibited a sharp increase (5-fold increase) in HMWs compared to ABN 101(CS) (see Tables 11, 12, and Figures 12a to 12c). These results suggest that the safety-enhancing effect of the ABN 101(CS) double mutant can compensate for the storage instability of acetate-based interferon-mutant drugs. [Table 11] [Table 12] [Industrial Applicability]

[0142] Therefore, the present invention provides a human interferon-beta variant having an amino acid sequence in which the 27th amino acid, arginine, of human interferon-beta is replaced with threonine and the 17th amino acid, cysteine, is replaced with serine. The present invention provides a human interferon-beta variant with improved purification efficiency, which can be useful in the production of therapeutic agents using the variant and has excellent industrial applicability.

Claims

[Claim 1] A human interferon-beta variant comprising an amino acid sequence in which the 17th amino acid, cysteine, of human interferon-beta is replaced with serine and the 27th amino acid, arginine, is replaced with threonine.

Images