HUMAN INTERFERON-BETA VARIANT WITH DOUBLE MUTATION AND METHOD FOR IMPROVING STABILITY OF HUMAN INTERFERON-BETA VARIANT

Abstract

The present invention relates to a human interferon-beta variant with double mutation and a method for improving stability of a human interferon-beta variant and, more specifically, to a human interferon-beta variant including an amino acid sequence having serine substituted for the 17th amino acid cysteine of human interferon-beta and threonine substituted for the 27th amino acid arginine of the human interferon-beta, and a method for improving stability of human interferon-beta R27T variant, the method comprising a step of substituting serine for the 17th amino acid serine in the human interferon-beta R27T variant in which threonine is substituted for the 27th amino acid arginine of human interferon-beta.

Claims

1. A human interferon-beta variant comprising an amino acid sequence having serine substituted for the 17th amino acid cysteine and threonine substituted for the 27th amino acid arginine of human interferon-beta.

2. A polynucleotide encoding the human interferon-beta variant of claim 1.

3. The polynucleotide of claim 2, wherein the human interferon-beta variant is a human interferon-beta variant comprising an amino acid sequence of SEQ ID NO: 3.

4. An expression vector expressing human interferon-beta in an animal cell comprising the polynucleotide of claim 2.

5. An animal cell transformed with the expression vector of claim 4.

6. A method for preparing a human interferon-beta variant comprising culturing the animal cell of claim 5.

7. A pharmaceutical composition comprising the human interferon-beta variant of claim 1 as an active ingredient, wherein the pharmaceutical composition has a pharmaceutical effect of natural human interferon-beta.

8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition has a pharmaceutical effect of natural human interferon-beta, and the pharmaceutical effect is a pharmaceutical effect of preventing or treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disorder, viral infection, HIV-related diseases, and hepatitis C.

9. A method for improving stability of a human interferon-beta R27T variant, comprising substituting serine for the 17th amino acid cysteine in the human interferon-beta R27T variant in which threonine is substituted for the 27th amino acid arginine of human interferon-beta.

10. The method of claim 9, wherein the human interferon-beta R27T variant consists of an amino acid sequence of SEQ ID NO: 2.

11. The method of claim 9, wherein the stability is selected from the group consisting of purification stability, storage stability and freeze/thawing stability.

12. The method of claim 11, wherein the purification stability improves purification efficiency of 2 glycosylation proteins.

13. The method of claim 11, wherein the purification stability reduces the protein aggregation and degradation during concentration and buffer exchange.

14. The method of claim 11, wherein the storage stability is storage stability in a buffer of pH 2.0 to 6.0.

15. The method of claim 14, wherein the buffer is a buffer 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), 3-(N-morpholino)propanesulfonic acid (MOPS), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(2-amino-2-oxoethyl)aminoethanesulfonic acid (ACES), N-(2-acetamido)2-iminodiacetic acid (ADA), 3-(1,1-dimethyl-1,2-hydroxyethylamino-2-propanesulfonic acid (AMPSO), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), N,N-bis(2-hydroxyethylglycine (Bicine), bis-tris (bis-(2-hydroxyethyl)imino-tris(hydroxymethyl)methane, 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), 3-N,N-bis(2-hydroxyethylamino-2-hydroxy-propanesulfonic acid (DIPSO), N-(2-hydroxyethylpiperazine)-N′-(3-propanesulfonic acid) (HEPPS), N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid (HEPPSO), 2-(N-morpholino)ethanesulfonic acid (MES), triethanolamine, imidazole, glycine, ethanolamine, phosphate, 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES), piperazine-N,N′-bis(2-hydroxypropanesulfonic acid (POPSO), N-trishydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), 3-N-tris(hydroxymethyl)methylamino-2-hydro hydroxy-propanesulfonic acid (TAPSO), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methylglycine (Tricine), 2-amino-2-methyl-1,3-propanediol, and 2-amino-2-methyl-1-propanol.

16. The method of claim 11, wherein the freeze/thawing stability is thawing stability after freezing at −100° C. to −10° C.

17. The method of claim 11, wherein the freeze/thawing stability is freeze/thawing stability in an acetic acid buffer.

18. The method of claim 11, wherein the freeze/thawing stability reduces the protein aggregation and degradation after 3 times or more freeze/thawing cycles.

19. Use of the human interferon-beta variant of claim 1 for preparing an agent having a pharmaceutical effect of natural human interferon-beta on a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infection, HIV-related diseases, and hepatitis C.

20. A method for treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disorders, viral infection, HIV-related diseases, and hepatitis C, comprising administering an effective amount of a composition having a pharmaceutical effect of natural human interferon-beta and comprising the human interferon-beta variant of claim 1 to a subject in need thereof.

21. A human interferon-beta variant having serine substituted for the 17th amino acid cysteine and threonine substituted for the 27th amino acid arginine of human interferon-beta of SEQ ID NO: 1, having at least 90% sequence homology with wild-type interferon beta of SEQ ID NO: 1, and having the activity of interferon beta.

Description

DESCRIPTION OF DRAWINGS

[0119] FIG. 1 illustrates PCR conditions in a protein expression DNA production experiment.

[0120] FIG. 2 illustrates restriction enzyme treatment and cloning in the protein expression DNA production experiment.

[0121] FIG. 3 illustrates the progress of cloning using T4 DNA ligase (NEB) in the protein expression DNA production experiment.

[0122] FIG. 4 illustrates colony PCR conditions in a protein expression DNA production experiment.

[0123] FIGS. 5A and 5B illustrate observation results of cell viability after transduction of ABN 101 (NT) and ABN 101 (CS) (ABN 101 (NT): R27T mutant interferon-beta, ABN 101 (CS): C17S, R27T double mutation interferon-beta).

[0124] FIGS. 6A and 6B illustrate 50 ml scale Fed-batch results of ABN 101 (NT) and ABN 101 (CS).

[0125] FIG. 7 illustrates 1 L scale Fed-batch results of ABN 101 (NT) and ABN 101 (CS).

[0126] FIG. 8 illustrates concentration and buffer exchange in an interferon-beta variant stability confirmation experiment.

[0127] FIGS. 9A and 9B illustrate interferon-variant RP-HPLC results.

[0128] FIGS. 10A to 10D illustrate changes in monomer content during interferon-variant buffer exchange.

[0129] FIGS. 11A to 11F illustrate confirmation of F/T stability comparison of interferon-variants in a 20 mM Na-Pi buffer.

[0130] FIGS. 12A to 12C illustrate confirmation of F/T stability comparison of interferon-variants in a 20 mM Na—OAc buffer.

MODES FOR THE INVENTION

[0131] Hereinafter, the present invention will be described in detail.

[0132] However, the following Examples are just illustrative of the present invention, and the contents of the present invention are not limited to the following Examples.

[0133] The following experiments were performed to prepare ABN 101 (NT), R27T mutation human interferon beta-1a, and ABN 101 (CS), a R27T and C17S double mutation form.

[0134] Experiment Method

[0135] 1. Preparation of Protein Expression DNA

[0136] To clone ABN 101 (NT) and ABN 101 (CS) behind a human promoter of a pD2535nt-HDP vector, primers added with XbaI and PacI enzyme restriction sites were designed, and sequences were as follows.

TABLE-US-00003 Forward: (SEQ ID NO. 4) 5′-ggtctagagccaccAtgacca-3′ XbaI Reverse: (SEQ ID NO. 5) 5′-cacttagggattaattaatcagttcctcaggtag-3′ Pael

[0137] Since two inserts changed cysteine to serine by changing only the 119th DNA sequence, the primers for cloning were used in common. The two inserts were amplified through AccuPower PCR PreMix (Bioneer), and PCR conditions were shown in FIG. 1 below.

[0138] The size of a PCR product obtained through PCR was confirmed through 0.8% agarose gel, and gel extraction was performed using a MEGAquick-Spin™ Plus Total Fragment DNA Purification Kit (Intron). The PCR product and the pD2535nt-HDP vector that had been purified were treated with restriction enzymes as illustrated in FIG. 2 to perform cloning. The restriction enzymes and the buffers thereof were all used with ThermoFisher products.

[0139] After restriction enzyme treatment, it was attempted to increase cloning efficiency by obtaining only pure DNA fragments using the MEGAquick-Spin™ Plus Total Fragment DNA Purification Kit (Intron), and after purification, T4 DNA ligase (NEB) was used as illustrated in FIG. 3 to perform cloning.

[0140] After ligation, transformation was performed using a DH5a Chemically Competent E. coli (Enzynomics) product. First, DH5a cells were slowly thawed in ice, and then 20 μl of a reaction product was fully added and placed on ice for 30 minutes. Then, heat shock was applied at 42° C. for 30 seconds and then stabilized on ice for 2 minutes. 400 μl of SOC media (provided by enzymenomics) were added and shaking-incubated at 37° C. for 1 hour. After healing, cells centrifuged at 3000 rpm for 3 minutes were smeared in an LB medium containing 50 μg kanamycin, and then incubated overnight in a 37° C. incubator. Colonies appearing on a plate were detached and colony PCR was performed to confirm cloning. A single colony without overlapping with other colonies was scraped as much as possible using a 10p tip and then buried in an AccuPower PCR PreMix (Bioneer) tube. PCR was performed with primers for colony PCR of a pD2535nt-HDP vector provided by Horizon, and the conditions were as illustrated in FIG. 4 below.

[0141] The size of the PCR product was confirmed through 0.8% agarose gel to confirm the completion of cloning, and in the cloned pD2535nt-HDP::ABN101 (NT) and pD2535nt-HDP::ABN101(CS), DNA prep for CHO-K1 cell transfection was performed through nucleobond Xtra Maxi Plus (MACHEREY-NAGEL).

[0142] 2. Transduction

[0143] E. coli having an expression plasmid was incubated and harvested in the LB medium containing 100 μg/mL kanamycin, and DNA was isolated using a QIAGEN Plasmid Midi prep kit. 30 μl of a 10× cutsmart buffer and 2.5 μl of an NrU1-HF restriction enzyme were added to 50 μg of the isolated DNA to make a final volume of 300 μl. Then, the isolated DNA was incubated at 37° C. for 2 hours to be linearized. After 2 hours, 30 μl of 1/10 volume of 3 M, pH 5.5 sodium acetate solution and 750 μl of ice-cold ethanol were added and incubated overnight at −80° C. The next day, ethanol-precipitated DNA was centrifuged and washed with 70% ethanol to obtain high-purity DNA, and the concentration was measured using Nanodrop. On the first day for transduction, CHO-K1 cells were seeded in an E125 shake flask to be 3×10.sup.5/ml using a CDFortiCHO culture medium added with L-Glutamine at a concentration of 4 mM and then incubated for 24 hours at 37° C., 5% CO.sub.2, and 125 rpm conditions. On the second day, the number of cells seeded on the first day was measured and the cells were seeded to be 5×10.sup.5/ml and incubated for 24 hours at 37° C., 5% CO.sub.2, and 125 rpm conditions. On the third day, the number of seeded cells was measured to confirm whether the number has reached 1×10.sup.6/ml, and when reached, the cells were finally seeded to be 1×10.sup.6/ml to prepare the transduction. 37.5 μg of linearized DNA and 37.5 μl of a Freestyle MAX reagent were added to 600 μl of an OptiPRO SFM medium, and reacted at room temperature for 5 minutes. Thereafter, the mixture containing DNA was transferred to the mixture containing the Freestyle MAX reagent and mixed, and then reacted at room temperature for 25 minutes. After the reaction, the DNA-lipid mixture was carefully added to the previously seeded CHO-K1 cells. The cells were incubated for 48 hours at 37° C., 5% CO.sub.2, and 125 rpm conditions to perform the transduction. After 48 hours of transduction, the selection of methionine sulfoximine (MSX) resistant cells was started. While the cells were incubated for about 25 days in a selective medium added with 25 or 50 μM of MSX, the fully transfected cells were cultured and monitored once every 2 to 3 days. While monitoring every 2 to 3 days, when the viability reached 70% or more and the number of viable cells reached 0.5 to 1.2×10.sup.6/ml, these cells were selected as a pool, and a suspension culture was 3 or more subcultured on a selective medium at 37° C., 5% CO.sub.2, and 125 rpm conditions to stabilize cells.

[0144] 3. Fed-Batch

[0145] Fed-batch was performed using the cells in the pool state prepared by transduction. 50 ml of the cells were seeded into an E250 shake flask to be 3×10.sup.5/ml using a CDFortiCHO medium without MSX, and incubated for about 12 days at 37° C., 5% CO.sub.2, and 125 rpm conditions. At this time, the glucose metabolites of the cell culture medium were analyzed using cedex bio, and the viability of cells and the number of viable cells were measured. On days 3, 5, and 7, a 5% (V/V) CD Efficient Feed C+ solution was added, and on days 4 and 6, a 45% glucose solution was added. After the Fed-batch was completed, the culture medium was centrifuged, and only a supernatant was taken and stored under a refrigerated or frozen condition.

[0146] 4. Confirmation of Interferon Beta-1a Biological Activity (ELISA)

[0147] How much biological activity of the protein expressed by human Interferon beta-1a was exhibited with the prepared expression cell line was analyzed using a HuIFN-β ELISA KIT of TORAY Co., Ltd. The culture medium secured by Fed-batch was initially diluted 10,000-fold using a diluent in the KIT, and serially diluted two-fold to prepare a 1,280,000-fold diluted sample. A standard material for measuring the biological activity in the KIT was prepared using the diluent from 200 IU/ml to 3.125 IU/ml according to the protocol. An ELISA plate was primed and prepared using a wash buffer in the KIT. After priming, 100 μl/well of the prepared sample and the standard material were added to the ELISA plate, and 50 μl/well of a HRP-conjugated antibody was added to each well. The plate was incubated at 27° C. for 2 hours and reacted. After the reaction was completed, the plate was washed, and then added with a color development reagent at 100 μl/well and incubated at 27° C. for 30 minutes to induce a color development reaction. Thereafter, in order to complete the color reaction, a stop solution was added at 100 μl/well, and plate detection was performed at a wavelength of measurement 450 nm/reference 620 nm using a spectrophotometer device. Based on the absorbance obtained herein, a standard curve was drawn using a 4-parameter method and the biological activity of the sample was converted. The biological activity of the original sample was checked again by multiplying the activity of the sample obtained by conversion from the standard curve by a dilution rate.

[0148] 5. Confirmation of Expression (Concentration) of Interferon Beta-1a (ELISA)

[0149] How much the protein expressed by human Interferon beta-1a was expressed with the prepared expression cell line was analyzed using a HuIFN-β ELISA kit of TORAY Co., Ltd. In the same manner as the biological activity confirmation method, the culture medium secured by Fed-batch was initially diluted 10,000-fold using a diluent in the KIT, and serially diluted two-fold to prepare a 1,280,000-fold diluted sample. The existing reference standard material was prepared by two-fold serial dilution at a concentration from 2.5 ng/ml to 0.039 ng/ml. The subsequent ELISA test process was the same as the biological activity confirmation ELISA test method. Based on the obtained absorbance, a standard curve was drawn using a 4-parameter method and the Interferon beta-1a expression level of the sample was converted. The concentration of the original sample was confirmed again by multiplying the expression level obtained by conversion from the standard curve by the dilution rate.

[0150] 6. Purification of Interferon-Beta Variant

[0151] The cell line prepared in Example above was incubated using a cell factory (Nunc Co., Ltd., Cat No. 170069). Each expression cell line was sub-cultured into the cell factory at 5×10.sup.4 cells/ml with an alpha-MEM medium containing 10% FBS, and incubated at 5% CO.sub.2 and 37° C. for 72 hours to confirm cell growth. After washed three times with PBS, serum components were removed as much as possible and the medium was replaced with a serum-free medium (Sigma C8730). After replaced with the serum-free medium, the culture medium was harvested every 24 hours, and a new serum-free medium was added. The culture medium was harvested for a total of 4 times and purified. After 200 ml of a blue sepharose resin (Amersham-Pharmacia) was filled in an XK50/20 column (Amersham-Pharmacia), 10 C.V. (column volume) of a buffer A (20 mM sodium phosphate, 1 M NaCl, pH 7.4) flowed to reach an equilibrium state. A sterilized and filtered culture solution flowed through a column in an equilibrium state at a flow rate of 20 ml/min, and monitored by connecting a UV detector with a wavelength of 280 nm. A buffer B (20 mM sodium phosphate, 1 M NaCl, 30% Ethylen Glycol, pH 7.4) flowed through the column to wash unadsorbed components, and the protein attached to the resin was eluted with a buffer C (20 mM sodium phosphate, 1 M NaCl, 60% Ethylen Glycol, pH 7.4). The eluate was dialyzed with phosphate buffered saline (PBS), concentrated with a concentrator (Centricon, Cut off 10,000), and dialyzed with phosphate buffered saline (PBS).

[0152] 7. Comparison of 2 Glycosylation Purification Efficiency of Interferon-Beta R27T Variant and Interferon-Beta Double Variant (R27T and C17S)

[0153] In an interferon-beta variant ABN 101 (NT) and an interferon-beta double variant ABN 101 (CS) purified using the blue sepharose resin, the content of a 2 glycosylated interferon-beta variant was measured using Reverse Phase High Performance Liquid Chromatography (RP-HPLC).

[0154] After each interferon variant was diluted to 0.5 mg/mL, acetonitrile (ACN) was mixed at an initial mobile phase ratio and then analyzed. RP-HPLC analysis was performed by a YMC-C4 column, and the solvents used therein were as follows. A mobile phase A was used after mixing 0.1% trifluoroacetic acid (TFA) with tertiary distilled water and then degassing for 1 hour after 0.2 μm PVDF filter. A mobile phase B was used after mixing 0.1% TFA with ACN and then degassing for about 1 hour after 0.2 μm PVDF filter.

[0155] The analysis conditions were indicated in the following Table.

TABLE-US-00004 TABLE 1 Parameter Condition Column TMC Pack C4, 4 × 250 mm, 5 μm Column Temperature 25° C. Flow Rate 1.0 mL/min Autosampler Temperature 4° C. Wavelength 214 nm or 280 nm Injection Volume 50 μl

TABLE-US-00005 TABLE 2 Time (min) Eluent A (%) Eluent B (%) 0.0 70.0 30.0 1.0 70.0 30.0 18.0 45.0 55.0 18.1 0.0 100.0 22.0 0.0 100.0 22.1 70.0 30.0 30.3 70.0 30.0

[0156] 8. Confirmation of Interferon-Beta Variant Stability According to Buffer Composition Change

[0157] In each of an interferon-beta variant ABN 101 (NT) and an interferon-beta double variant ABN 101 (CS) purified using a blue sepharose resin, each buffer was replaced using a centricon. In order to confirm the degree of deterioration in quality due to aggregation occurring during buffer replacement of interferon drugs, each protein eluted from the Blue sepharose resin was obtained, and then concentration and buffer exchange were performed at a volume ratio of about 7 times using a 20 mM sodium phosphate (pH 2.9) buffer in the centricon. After the buffer exchange was completed with the centricon, the protein concentration was measured at a wavelength of 280 nm with a UV spectrophotometer after a 0.2 μm PES syringe filter to confirm the recovery rate.

[0158] In addition, in order to confirm the storage stability with respect to other formulation buffers, the proteins that have been buffer-exchanged with 20 mM phosphate (pH 2.9) were again buffer-exchanged with 20 mM sodium acetate (pH 3.8) using a centricon at the same volume ratio level, respectively. The process was illustrated in FIG. 8.

[0159] 9. Confirmation of Freeze/Thawing Storage Stability According to Buffer Composition Change

[0160] To confirm freeze/thawing stability, for an interferon drug composed of each buffer, a freeze/thawing cycle of freezing at −70° C. for 12 hours or more and thawing at 25° C. for 4 hours was performed 3 or 5 times and then Size Exclusion High Performance Liquid Chromatography (SEC-HPLC) analysis was performed. The storage stability of the interferon-beta variants was measured by confirming a ratio of the aggregation analyzed by a high molecular weight (HMWs) and the degradation analyzed by a low molecular weight (LMWs) to analyze a monomer change rate of the protein. For SEC-HPLC, a size exclusion column (TSKG2000) of Tosoh Co., Ltd. was used, and the solvents used were as follows. A mobile phase A was used by mixing tertiary distilled water and degassing for 1 hour after a 0.2 μm PVDF filter, and a mobile phase B was used by mixing 150 mM sodium chloride and 100 mM sodium phosphate dibasic dihydrate with tertiary distilled water and then titrating to pH 7.0 with phosphoric acid, and degassing after a 0.2 μm PVDF filter.

[0161] The analysis conditions were indicated in the following Table.

TABLE-US-00006 TABLE 3 Parameter Condition Column TSKgel G2000SKxL, 7.8 mm × 300 mm Column Temperature 25° C. Flow Rate 0.5 mL/min Autosampler Temperature 4° C. Wavelength 214 nm or 280 nm Injection Volume Sample: 50 μl GFS: 10 μl

TABLE-US-00007 TABLE 4 Time (min) Eluent A (%) Eluent B (%) 0.0 0.0 100.0 40.0 0.0 100.0

[0162] Results and Interpretation

[0163] 1. Pool Development Result According to MSX Concentration after Gene Transduction of ABN 101 (NT) and ABN 101 (CS)

[0164] After CHO-K1 cells were transduced with DNA cloned into a pD2535nt-HDP vector, the number and viability of the cells were confirmed after 48 hours, and based on the number and viability of the cells, the selection of resistant cells with two concentrations of MSX was performed for 25 days. As a result, ABN 101(NT) obtained resistant cells earlier than ABN 101(CS), and in the case of 50 uM of ABN 101(CS), the final number of viable cells of 3×10.sup.5/ml and the viability of 48% were shown, so that it was confirmed that the resistant cells were slowest obtained. Although the viability was low, it was determined that the growth and viability were affected according to the expression of interferon beta-1a. The results thereof were illustrated in FIGS. 5A and 5B.

[0165] 2. Fed-Batch Results in ABN 101 (NT) and ABN 101 (CS) Pool States

[0166] The results of performing 50 ml small scale Fed-batch with cells in a pool state were as follows. In both the ABN 101 (NT) pool and the ABN 101 (CS) pool, it was confirmed that the viability was maintained longer in the pool with the MSX concentration of 50 μM than 25 μM, and it was confirmed that the number of viable cells was less maintained in the pool with the MSX concentration of 50 μM than 25 μM. However, although the number of viable cells was less maintained in the pool with the MSX concentration of 50 μM than 25 μM, the biological activity of Interferon beta-1a in the culture medium on the last day of Fed-batch was higher and it was confirmed that the biological activity of the ABN 101(CS) pool was at least 2 times higher than that of the ABN 101(NT) pool. The results were illustrated in FIGS. 6A and 6B.

[0167] 3. 1 L Scale Fed-Batch Results of ABN 101 (NT) and ABN 101 (CS) Pools

[0168] Based on the small scale fed-batch results above, 1 L scale fed-batch was performed using a pool with a MSX concentration of 50 μM. As a result, the biological activity on the 4th day of culture was 0.69 MIU/ml for ABN 101(NT) and 5.99 MI/ml for ABN 101(CS), so that it was confirmed that the biological activity of ABN 101(CS) was about 8.6 times higher. When comparing the biological activity of the 12th day of culture, it was confirmed that the biological activity of ABN 101 (CS) was about 4.5 times higher. The results were illustrated in FIG. 7.

[0169] 4. Purification Result of Interferon-Beta Variant

[0170] As a result of the purification of the interferon-beta variant, the purification efficiency was higher than that of conventional human interferon-beta. As a result, it was confirmed that a human interferon-beta variant comprising an amino acid sequence having serine substituted for the 17th amino acid cysteine and threonine substituted for the 27th amino acid arginine had higher purification efficiency.

[0171] 5. Result of 2 Glycosylation Purification Rate of Interferon-Beta R27T Variant and Interferon-Beta Double Variant (R27T and C17S)

[0172] The interferon-beta variants purified using a blue sepharose resin were analyzed with the content of 2 glycosylation:1 glycosylation, respectively. As a result, as shown in Table 5 and FIGS. 9A and 9B below, it was confirmed that the 2 glycosylation content of the ABN 101 (CS) double variant was about 10% higher than that of ABN 101 (NT). There was no significant difference when comparing the biological activities of each material through ELISA, but it was confirmed that the purification efficiency of 2 glycosylated protein was high when the interferon-beta variant was expressed and purified on the same scale.

TABLE-US-00008 TABLE 5 Material 2 glycosylation 1 glycosylation MIU/mg ABN 101(NT) 82.8 17.1 351 ABN 101(CS) 93.1 6.9 360

[0173] 6. Result of Interferon-Beta Variant Stability According to Buffer Composition Change

[0174] In order to confirm the stability of the interferon-beta variant for each buffer composition, protein recovery rate and SEC-HPLC analysis were performed by exchanging each buffer with a centricon. When each protein was purified and then the buffer was exchanged with 20 mM sodium phosphate (pH 2.9), it was confirmed that the recovery rate thereof was high in ABN 101 (CS) as shown in Table 6 below.

TABLE-US-00009 TABLE 6 Initial 20 mM Na-Pi Material Conc.(mg/mL) (mg/mL) Recovery (%) ABN 101(NT) 0.56 (30 mL) 1.1 (8 mL) 52 ABN 101(CS) 0.40 (15 mL) 0.7 (8 mL) 93

[0175] In addition, the recovery rate was confirmed by replacing the buffer with 20 mM sodium acetate (pH 3.8) using a centricon in the same manner to replace each protein exchanged with the corresponding buffer with a buffer based on a different formulation again. As a result, it was confirmed that the recovery rate of ABN 101 (CS) was also higher than that of ABN 101 (NT) when exchanged with an acetate-based buffer as shown in Table 7. This was interpreted as protein structural stability from physical factors such as a centricon by increased stability by the double variant.

TABLE-US-00010 TABLE 7 Initial Conc. 20 mM Na—OAc Material (mg/mL) (mg/mL) Recovery (%) ABN 101(NT) 1.1 (1 mL) 0.86 (1 mL) 78 ABN 101(CS) 1.7 (1.1 mL)  1.7 (1 mL) 91

[0176] The protein recovery rate and the monomer analysis result for each buffer composition change were shown in Table 8 and FIGS. 10A to 10D below.

TABLE-US-00011 TABLE 8 20 mM Na-Pi 20 mM Na—OAc Monomer Material (Monomer %) (Monomer %) Recovery (%) ABN 101(NT) 86.8% 80.4% 92.6 ABN 101(CS) 98.1% 90.2% 91.8

[0177] 7. Result of Freeze/Thawing Stability According to Buffer Composition Change

[0178] In order to confirm the freeze/thawing stability of the interferon-beta variant by buffer composition, the interferon-variant in the form of a substituted buffer was repeatedly freeze-thawed 3 times or 5 times, respectively, and the monomer change rate was confirmed through SEC-HPLC analysis. As a result, when the interferon-variants were stored in a buffer based on 20 mM sodium phosphate (pH 2.9) and subjected to repeated freeze-thawing, a difference between the two interferon-beta variants was not significant (see Table 9, Table 10, and FIGS. 11A to 11F).

TABLE-US-00012 TABLE 9 ABN 101(NT) HMWs (%) Monomers (%) LMWs (%) F/T 1 cy 5 76 19 F/T 3 cy 6 76 18 F/T 5 cy 5 77 18

TABLE-US-00013 TABLE 10 ABN 101(CS) HMWs (%) Monomers (%) LMWs (%) F/T 1 cy 8 89 3 F/T 3 cy 11 86 3 F/T 5 cy 13 84 3

[0179] However, as a result analyzed by exchanging the buffer with 20 mM sodium acetate (pH 3.8) and performing a freeze-thaw test, ABN 101 (CS) maintained the monomer content, but ABN 101 (NT) showed rapidly increased HMWs compared to ABN 101 (CS) (5-fold increased) (see Table 11, Table 12, and FIGS. 12A to 12C). Through these results, it can be expected that the storage instability of the acetate-based interferon-variant drug may be compensated due to the stability-increasing effect of the double variant of ABN 101(CS).

TABLE-US-00014 TABLE 11 ABN 101(NT) (20 mM Na—OAc) HMWs (%) Monomers (%) LMWs (%) Origin 10 80 <10 F/T 3 cy 28 63 <9

TABLE-US-00015 TABLE 12 ABN 101(CS) (20 mM Na—OAc) HMWs (%) Monomers (%) LMWs (%) Origin 9 90 <1 F/T 3 cy 5 94 <1

INDUSTRIAL APPLICABILITY

[0180] Therefore, the present invention provides a human interferon-beta variant comprising an amino acid sequence having threonine substituted for the 27th amino acid arginine and serine substituted for the 17th amino acid cysteine of human interferon-beta. The present invention provides a human interferon-beta variant with improved purification efficiency, which can be usefully used in the production of a therapeutic agent using the same, and thus has excellent industrial applicability.