Method for producing varicella-zoster virus surface protein antigen

Abstract

The present invention relates to a method for producing varicella-zoster virus surface protein antigen. The method for producing varicella-zoster virus surface protein antigen according to the present invention is an effective production method capable of obtaining varicella-zoster virus surface protein antigen with high yield and high purity. Therefore, the method is advantageous for producing varicella-zoster virus surface protein antigen to be used as a vaccine composition for preventing or treating varicella or herpes zoster.

Description

Technical Field

[0001] The present invention relates to a method for producing varicella-zoster virus surface protein antigen. Background Art

[0002] Varicella Zoster Virus (VZV) is a virus that mainly causes varicella in children and adolescents. Once infected, VZV remains dormant in the ganglion cells of sensory roots and cranial nerves for several years and is reactivated and causes herpes zoster in adulthood when immunity declines. Varicella is highly contagious; and once infection occurs, it causes a vesicular rash all over the body, accompanied by fever and discomfort. In most normal children, varicella rarely develops into a severe condition and eventually develops into a self-limiting disease. However, many cases where varicella progresses to severe symptoms are known to occur in patients who have received organ transplants or chemotherapy (Adriana Weinberg et al., J Infectious Diseases, 200(7):1068, 2009; Judith Breuer et al., Expert Review of Vaccines, 2017, DOI:10.1080 / 14760584.2017.1394843).

[0003] The initial symptoms of herpes zoster are general pain such as body pain, or severe itching, stinging and burning sensations, accompanied by severe pain like being stabbed by a knife. Herpes zoster is a disease in which blisters appear after several days, the pain increases as the skin lesions develop, and older patients tend to complain of more severe pain. Even when herpes zoster is cured, it may leave neuralgia as a sequela. It is known that in people aged 60 or older, neuralgia can cause them to sleep intermittently, cause them to complain of chronic fatigue, cause them to feel severe pain even with slight contact or friction, or even cause depression, but this neuralgia is relatively rare in adults aged 40 or younger.

[0004] ZOSTAVAX (Merck & Co, Inc.), an attenuated live vaccine produced using the Oka strain, was developed as a prophylactic vaccine against herpes zoster. Due to the fact that the vaccine contains a large amount of virus, the vaccine has been approved and sold in the United States and South Korea under the condition that the vaccine should be used in adults 50 years of age or older rather than children or adolescents. Recently, a vaccine consisting of the viral surface protein (gE) and an adjuvant developed by GlaxoSmithKline Biologicals SA for use in adults 50 years of age or older has been shown to be prophylactically effective in clinical trials (U.S. Patent No. 7,939,084). Summary of the Invention

[0005] Technical Problem

[0006] Accordingly, in studying methods capable of improving the productivity of the varicella-zoster virus (VZV) surface protein (gE) antigen without affecting its immunogenicity, the present inventors have discovered a method for culturing and purifying capable of improving the productivity of the VZV gE antigen, and thus completed the present invention.

[0007] Technical Solution

[0008] In one aspect of the present invention, there is provided a method for producing a varicella-zoster virus (VZV) surface protein (gE) antigen, comprising the steps of: (a) culturing a recombinant cell line producing the VZV gE antigen to obtain a culture solution; and (b) purifying the culture solution.

[0009] The step (a) of obtaining the culture solution may include the steps of: (a-1) seed-culturing the recombinant cell line; and (a-2) performing production culture on the cell line that has been seed-cultured.

[0010] The step (b) of purifying the culture solution may include the steps of: performing anion exchange chromatography; performing hydrophobic interaction chromatography; treating the culture solution with a virus inactivator to inactivate the virus; performing mixed-mode chromatography; and performing concentration and filtration.

[0011] Advantageous Effects

[0012] The method for producing a varicella-zoster virus surface protein antigen according to the present invention is an effective production method capable of obtaining the varicella-zoster virus surface protein antigen with high yield and high purity. Therefore, the method can be used to produce the varicella-zoster virus surface protein antigen for use as a vaccine composition for preventing or treating varicella or herpes zoster. Brief Description of the Drawings

[0013] Figure 1A flowchart showing the process of culturing and purifying to produce VZV gE antigen according to an embodiment of the present invention is shown.

[0014] Figure 2 A schematic diagram showing the process of generating the pMSID2-MGgE vector is shown.

[0015] Figure 3 Results obtained by determining VZV gE productivity according to the type of VZV gE and cell line by Western blot are shown.

[0016] Figure 4 Results obtained by determining VZV gE productivity according to the type of VZV gE and cell line by ELISA are shown.

[0017] Figure 5a Results obtained by determining the viable cell density (VCD) according to the change in culture temperature in the production culture step of the cell line introduced with MGgE are shown.

[0018] Figure 5b Results obtained by determining the productivity according to the change in culture temperature in the production culture step of the cell line introduced with MGgE are shown.

[0019] Figure 6a Results obtained by determining the viable cell density according to the culture temperature in the production culture step of the cell line introduced with MGgE are shown.

[0020] Figure 6b Results obtained by determining the productivity according to the culture temperature in the production culture step of the cell line introduced with MGgE are shown. Detailed Description

[0021] The present invention will be described in detail below.

[0022] In one aspect of the present invention, a method for generating varicella-zoster virus (VZV) surface protein (gE) antigen is provided, which comprises the following steps: (a) culturing a recombinant cell line that produces VZV gE antigen to obtain a culture solution; and (b) purifying the culture solution.

[0023] In the present invention, the varicella-zoster virus surface protein antigen may preferably be one of the varicella-zoster virus surface protein antigens disclosed in International Publication No. WO 2019 / 225962A1. Specifically, the varicella-zoster virus surface protein antigen (MGgE) may be a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:1, and this polypeptide may be encoded by a gene consisting of the nucleotide sequence represented by SEQ ID NO:2.

[0024] The varicella-zoster virus surface protein antigen represented by the amino acid sequence of SEQ ID NO:1 is a varicella-zoster virus surface protein antigen variant derived from the varicella-zoster virus surface protein antigen represented by the amino acid sequence of SEQ ID NO:3, wherein this antigen variant contains a change in which the carboxyl terminus of the amino acid residue at position 537 in the varicella-zoster virus surface protein antigen is truncated.

[0025] The change in which the carboxyl terminus of the amino acid residue at position 537 is truncated means that in the direction from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), the amino acid residues at positions 1 to 537 are retained, and the consecutive amino acid residues from the amino acid residue at position 538 to the carboxyl terminus are truncated.

[0026] The cell line producing the VZV gE antigen may be a cell line transformed with the VZV gE antigen-encoding gene consisting of the nucleotide sequence shown in SEQ ID NO:2. This cell line may have an expression vector containing the VZV gE antigen-encoding gene. In one embodiment of the present invention, the expression vector may be an expression vector including the pMSID2 vector disclosed in Korean Patent No. 1591823. However, for the expression vector, any vector may be used without limitation as long as it is suitable for transfecting the VZV gE antigen into the cell line.

[0027] As used herein, the term "vector" refers to a nucleic acid, which means a nucleotide sequence that can be introduced into a host cell to be recombined and inserted into the genome of the host cell, or a nucleotide sequence that can replicate autonomously as an episome. Suitable expression vectors contain expression regulatory elements such as promoters, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be produced differently according to their purposes. When a gene construct encoding a target protein is administered to an individual, the start codon and stop codon must function in the individual and must be in frame with the coding sequence.

[0028] A host cell or non-human host subject transfected or transformed with a vector according to an embodiment of the present invention can be a host cell or non-human host subject genetically modified by the vector. As used herein, the term "genetically modified" means that the host cell, non-human host subject, precursor or parent contains, in addition to its own genome, a polynucleotide or vector according to an embodiment of the present invention that has entered the host cell, non-human host subject, precursor or parent. Additionally, the polynucleotide or vector according to an embodiment of the present invention can exist as an independent molecule, particularly as a replicable molecule, outside of its genome in the genetically modified host cell or non-human host subject, or can be stably inserted into the genome of the host cell or non-human host subject.

[0029] A host cell according to an embodiment of the present invention is a eukaryotic cell. Eukaryotic cells include fungal cells, plant cells or animal cells. Examples of fungal cells can include yeast, particularly yeast of the genus Saccharomyces sp., and more particularly Saccharomyces cerevisiae. Additionally, examples of animal cells include insect cells or mammalian cells, and specific examples of animal cells include HEK293, 293T, NSO, CHO, MDCK, U2-OS, Hela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A, etc. Additionally, suitable cell lines known in the art can be obtained from cell line depositories such as the American Type Culture Collection (ATCC).

[0030] The VZV gE antigen according to the present invention can be expressed in various types of organisms, such as bacteria, yeast, mammalian cells, plants and transgenic animals. Preferably, mammalian cells can be used in view of the regulations for protein therapeutics and the fact that the produced protein needs to be similar to its natural form. Examples of mammalian cells include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, CapT cells (human amniotic fluid-derived cells), COS cells, etc. According to an embodiment of the present invention, CHO DG44 cells can be used as mammalian cells.

[0031] To introduce the expression vector according to the present invention into the above cell lines, techniques known in the art can be used, examples of which include electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation and calcium chloride (CaCl2) precipitation.

[0032] Specifically, step (a) of obtaining the culture solution may include the following steps: (a-1) seed-culturing the recombinant cell line; and (a-2) production-culturing the cell line that has been seed-cultured.

[0033] As used herein, the term "seed-culture" refers to a culture aimed at obtaining a large number of cell lines. The seed-culture can be carried out under temperature conditions that allow the most active increase in the number of cells. In other words, the seed-culture can be carried out to obtain a certain number of cells for the cell line.

[0034] As used herein, the term "production-culture" refers to the large-scale culture of a cell line aimed at producing a recombinant protein.

[0035] Step (a-1) of carrying out the seed-culture can be such that the cell line producing the VZV gE antigen is cultured by any method selected from subculture, suspension culture, and combinations thereof. For example, the seed-culture of the VZV gE antigen-producing cell line can be carried out by subculture and suspension culture, and its production-culture can be carried out by suspension culture.

[0036] As used herein, the term "subculture" refers to a method of culturing a cell line while transferring the cell line to the same or different fresh medium according to the culture cycle.

[0037] In one embodiment of the present invention, the subculture can be carried out by transferring and inoculating the cell line to the same fresh medium at intervals of 1 to 7 days, 2 to 5 days, or 3 to 4 days. Here, the number of inoculated cells can be, but is not limited to, 2×10 5 cells / mL to 5×10 5 cells / mL, 3×10 5 cells / mL to 4×10 5 cells / mL, or 4×10 5 cells / mL. In addition, the CO2 concentration in the subculture can be, but is not limited to, 4.0% to 6.0%, 4.5% to 5.5%, or 5.0%.

[0038] As used herein, the term "suspension culture" refers to a culture method in which cells are suspended in a culture solution. Suspension culture can be carried out on cells that proliferate in a suspended state even in vivo (such as blood cells or cancer cells in ascites) without shaking or rotation; however, in most cases, suspension culture can be carried out by rotating a stirring paddle (stirring culture), shaking each culture flask (shaking culture), or rotating an incubator (rotary culture).

[0039] In one embodiment of the present invention, the suspension culture can be carried out under stirring speed conditions of 86 rpm to 96 rpm, 88 rpm to 94 rpm, or 90 rpm to 92 rpm; however, the stirring speed is not limited thereto. The number of cells inoculated in the suspension culture can be, but is not limited to, 2×10 5 cells / mL to 5×10 5 cells / mL, 3×10 5 cells / mL to 4×10 5 cells / mL, or 4×10 5 cells / mL. Additionally, the pH can be from pH 6.7 to pH 7.1, or from pH 6.8 to pH 7.0. Additionally, the dissolved oxygen concentration can be, but is not limited to, 10% to 90%, 20% to 80%, or 30% to 60%.

[0040] In the case of producing recombinant proteins by cell culture, particularly animal cell culture, the culture is usually carried out at a temperature suitable for cell growth so that productivity increases with the extension of the culture time, and then the culture is carried out at a temperature lower than the temperature suitable for cell growth so that the cell division cycle stops, and thus the transition to recombinant protein production is achieved (Enhancement of productivity of recombinant α - amidating enzyme by low - temperature culture, Furukawa K et al., Cytotechnol, 1999, Vol.31, 85 to 94; and Enhancing Effect of Low Culture Temperature on Specific Antibody Productivity of Recombinant Chinese Hamster Ovary Cells: Clonal Variation, Yoon S K et al., Biotechnol Prog, 2004, Vol.20, 1683 - 1688). However, the present inventors have determined that in the case of culturing a cell line for producing VZV gE antigen, it is advantageous in terms of yield and purity to carry out the production culture at a temperature lower than the temperature suitable for cell line growth without changing the culture temperature, to carry out the seed culture and / or production culture under constant temperature conditions, or to carry out the seed culture and production culture under constant low - temperature conditions.

[0041] Specifically, the step (a-1) of performing seed culture may include culturing the cell line at a temperature of 34°C to 38°C. Additionally, the step (a-2) of performing production culture may include culturing the cell line at a temperature of 34°C to 35.5°C. For example, the cell line may be subjected to seed culture at a temperature of 36°C to 38°C, 36.5°C to 37.5°C, or 37°C, and subsequently, the cell line that has undergone seed culture may be subjected to production culture at a temperature of 34°C to 35.5°C, 34.5°C to 35.5°C, or 35°C. Additionally, the cell line may be subjected to seed culture at a temperature of 34°C to 35.5°C, 34.5°C to 35.5°C, or 35°C, and subsequently, the cell line that has undergone seed culture may be subjected to production culture at a temperature of 34°C to 35.5°C, 34.5°C to 35.5°C, or 35°C. Additionally, the cell line may be subjected to seed culture and production culture at a constant temperature within the temperature range of 34°C to 35.5°C.

[0042] In one embodiment of the present invention, the production culture may be carried out by suspension culture. Here, the suspension culture may be carried out under stirring speed conditions of 62 rpm to 72 rpm, 64 rpm to 70 rpm, or 66 rpm to 68 rpm; however, the stirring speed is not limited thereto. The number of cells inoculated in the suspension culture may be, but is not limited to, 2×10 5 cells / mL to 5×10 5 cells / mL, 3×10 5 cells / mL to 4×10 5 cells / mL, or 4×10 5 cells / mL. The above cell numbers correspond to the cell density that is optimal for the production culture of the VZVgE antigen. In cases where the cell density is less than 2×10 5 cells / mL, since the number of cells is too small, the amount of protein obtained is small; and in cases where the cell density is greater than 5×10 5 cells / mL, a large amount of host cell protein (HCP), such as cell debris, is also produced, which is problematic because it is difficult to remove the HCP to a concentration that meets the drug standards during the purification process. Additionally, the pH may be pH 6.7 to pH 7.1, or pH 6.8 to pH 7.0. Additionally, the dissolved oxygen concentration may be, but is not limited to, 10% to 90%, 20% to 80%, or 30% to 60%.

[0043] In one embodiment of the present invention, the cultivation can be carried out in a flask or a bioreactor. However, the present invention is not limited thereto. The types of flasks and bioreactors as well as the cultivation conditions can vary within the range that can generally be adjusted by those skilled in the art. Specifically, a wave bioreactor, a stirred tank bioreactor, etc. that can perform suspension culture of animal cells can be used; and the pH of the culture solution can be adjusted to pH 6.8 to pH 7.2 using CO2 or an alkaline solution (such as sodium carbonate and sodium bicarbonate) and used. However, the present invention is not limited thereto.

[0044] For example, the seed culture can be carried out by inoculating a cell line into a shake flask and performing subculture and suspension culture to obtain a certain number of cells; and the production culture can be carried out by inoculating the cell line that has obtained a certain number of cells into a bioreactor and performing suspension culture to obtain a recombinant protein.

[0045] The step (b) of purifying the culture solution may include the following steps: performing anion exchange chromatography; performing hydrophobic interaction chromatography; treating the culture solution with a virus inactivator to inactivate the virus; performing mixed-mode chromatography; and performing concentration and filtration. The chromatography process, the virus inactivation process, and the concentration and filtration process can be carried out in various orders to obtain the best productivity.

[0046] In one embodiment of the present invention, the step (b) of purifying the culture solution may include the following steps: (b-1) performing anion exchange chromatography; (b-2) performing hydrophobic interaction chromatography; (b-3) treating the culture solution with a virus inactivator to inactivate the virus and then performing concentration and diafiltration; (b-4) performing mixed-mode chromatography to obtain an eluate; and (b-5) concentrating and diafiltering the eluate and then performing nanofiltration, and these steps are carried out in a series of orders.

[0047] In addition, before the step (b) of purifying the culture solution, a treatment process can be carried out on the culture solution to remove cell debris therefrom. For example, the purification step can be performed on the culture filtrate obtained by filtering the culture solution. Here, the filter medium for filtration can be a depth filter. In addition, the pore size of the filter medium can be 1 μm or smaller, 0.5 μm or smaller, 0.45 μm or smaller, 0.3 μm or smaller, 0.25 μm or smaller, or 0.2 μm or smaller; and a mixture of filter media with different diameters can be used.

[0048] As used herein, the term "chromatography" refers to the process of separating a target solute, such as a target protein, from other solutes in a mixture under specific buffer conditions, wherein the separation is achieved by percolation of the mixture through an adsorbent that more or less strongly adsorbs or retains solutes due to the properties of the solutes (e.g., pI, hydrophobicity, size, and structure).

[0049] In step (b) of purifying the culture solution, anion exchange chromatography can be used. Anion exchange chromatography can result in the removal of liquid culture medium components and impurities contained in the culture solution or culture filtrate. Resins can be used for anion exchange chromatography, such as DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 (Applied Biosystems), MonoQ, MiniQ, Source 15Q and 3OQ, Q, DEAE, and ANX Sepharose FastFlow, Q Sepharose High Performance, QAE SEPHADEXTM, and Q Sepharose Fast Flow (GE Healthcare), WP PEI, WP DEAM, WP QUAT (J.T. Baker), Hydrocell DEAE, and Hydrocell QA (Biochrom Labs Inc.), UNOsphere Q, Macro-Prep DEAE, and Macro-Prep High Q (Biorad), with Q Sepharose Fast Flow resin being preferred.

[0050] In addition, after equilibrating the column to pH 7.0 ± 0.2, the culture solution to be purified can be loaded at a flow rate of 150 ± 15 cm / hour. Conditions such as resin type, pH, and flow rate are not limited to those described above and can vary within the range that can generally be adjusted by those skilled in the art.

[0051] In addition, for anion exchange chromatography, a washing buffer with a sodium chloride concentration of 150 mM or lower, 0.1 mM to 150 mM, 10 mM to 150 mM, or 100 mM to 150 mM can be used. In addition, an elution buffer with a sodium chloride concentration of 400 mM to 600 mM, 450 mM to 550 mM, or 500 mM can be used. When purification is carried out using washing and elution buffers with sodium chloride concentrations within the above ranges, the impurity content in the culture solution containing VZV gE antigen can be minimized.

[0052] In step (b) of purifying the culture solution, hydrophobic interaction chromatography can be used. Hydrophobic interaction chromatography can be used to remove liquid culture medium components and impurities. Hydrophobic interaction chromatography can be carried out using resins such as Butyl FF, Butyl HP, Octyl FF, Phenyl FF, Phenyl HP, Phenyl FF (high resolution), Phenyl FF (low resolution), Capto Phenyl ImpRes, Capto Phenyl (high resolution), Capto Octyl, Capto Butyl ImpRes, Capto Butyl (GE Healthcare), Super Butyl-550C, Hexyl-650C, Butyl-650C, Phenyl-650C, Butyl 600M, Phenyl-600M, PPG-600M, Butyl-650M, Phenyl-650M, Ether-650M, Butyl-650S, Phenyl-650S, Ether-650S, TSKgel Phenyl-5PW, TSKgel Ether-5PW (Tosoh Bioscience), where Butyl-650M (Tosoh Bioscience) is preferred.

[0053] In addition, after equilibrating the column to pH 7.0 ± 0.2, the culture solution to be purified, such as the eluate from anion exchange chromatography, can be loaded at a flow rate of 100 ± 10 cm / hour. Here, before purification, 1 to 10 M, 2 to 8 M, 3 to 7 M, or 5 M of sodium chloride can be added to the solution to be purified. In addition, a washing buffer containing sodium chloride can be used, and an elution buffer without sodium chloride can be used. Conditions such as resin type, pH, and flow rate are not limited to those described above and can vary within the range that can generally be adjusted by those skilled in the art.

[0054] Step (b) of purifying the culture solution may include a step of treating the culture solution with a virus inactivator to inactivate the virus. The virus inactivation process can result in the inactivation of potential enveloped viruses in the solution containing VZV gE antigen to be purified.

[0055] Specifically, the virus inactivator can be a phosphoric acid solution. For example, the virus inactivator can be added to the eluate from hydrophobic interaction chromatography to adjust the pH of the eluate to pH 2.8 to pH 3.2, pH 2.9 to pH 3.1, or pH 3.0. In the virus inactivation step, when the pH is less than 2.8, the structure of the VZV gE antigen may be denatured; and when the pH value is greater than 3.2, the virus may not be inactivated.

[0056] In step (b) of purifying the culture solution, mixed-mode chromatography can be carried out. Mixed-mode chromatography can result in the removal of dimers and impurities from the solution containing VZV gE antigen. Mixed-mode chromatography can be carried out using a resin such as BAKERBOND ABX TM (J.T. Baker), type I and type II ceramic hydroxyapatite, and fluorinated hydroxyapatite (BioRad), as well as MEP and MBI HyperCel (Pall Corporation), with ceramic hydroxyapatite being preferred.

[0057] In addition, after equilibrating the column to pH 7.0 ± 0.2, the culture solution to be purified, such as a solution obtained by concentrating and filtering the eluate from hydrophobic interaction chromatography, can be loaded at a flow rate of 100 ± 10 cm / hour. In mixed-mode chromatography, the conditions such as, for example, the resin, buffer, and pH are not limited to those described above and can vary within the range that can generally be adjusted by those skilled in the art.

[0058] Step (b) of purifying the culture solution can include steps of concentration and filtration. The concentration and filtration processes can be carried out several times during the purification process of the culture solution and can be carried out between each chromatographic step, between a chromatographic step and a virus inactivation step, or after a chromatographic step or a virus inactivation step.

[0059] For example, before carrying out mixed-mode chromatography, concentration and filtration can be carried out to obtain a solution having conditions suitable for mixed-mode chromatography. For example, the concentration of the VZV gE fraction can be achieved by ultrafiltration and / or diafiltration and can include one or more tangential flow filtration (TFF) steps.

[0060] In addition, after carrying out mixed-mode chromatography, concentration and filtration can be carried out to adjust the VZV gE antigen concentration. Here, diafiltration can be carried out until the pH and conductivity of the filtrate reach the desired values, and then ultrafiltration can be used to adjust the protein concentration.

[0061] In addition, step (b) of purifying the culture solution can include a step of nanofiltration of the eluate from mixed-mode chromatography that has been concentrated and filtered. Here, a filtration system having a nanofiltration membrane can be used for nanofiltration. In order to separate viruses from the solution containing VZV gE antigen, nanofiltration can be carried out using a nanofiltration membrane having a pore size of, for example, 75 nm, less than 50 nm, or less than 15 nm.

[0062] Step (b) of the purified culture solution may further include the step of diluting the filtrate obtained by nanofiltration with a formulated buffer and then filtering. Here, a microfilter with a pore size of 0.05 to 0.8 μm, 0.07 to 0.6 μm, or 0.1 to 0.4 μm can be used for filtration.

[0063] In the case where the filtrate obtained by nanofiltration during step (b) of the purified culture solution is diluted and filtered using a microfilter, the protein can be obtained with a yield of 97% or higher, 98% or higher, 99% or higher, or 99.5% or higher. In addition, the HCP content in the purified culture solution can be about 180 ppm or lower, 170 ppm or lower, 160 ppm or lower, 150 ppm or lower, 100 ppm or lower, 80 ppm or lower, 70 ppm or lower, 60 ppm or lower, 50 ppm or lower, 30 ppm or lower, or 10 ppm or lower.

[0064] Examples

[0065] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

[0066] Example 1. Construction and transformation of an expression vector for varicella-zoster virus surface protein antigen

[0067] The varicella-zoster virus (VZV) surface protein (gE) antigen variant gene (MGgE; SEQ ID NO: 2) disclosed in International Publication No. WO 2019 / 225962 A1 was cloned into the pMSID2 vector disclosed in Korean Patent No. 1591823 to construct the pMSID2-MGgE expression vector ( Figure 2 ). Next, the expression vector was transfected into CHO DG44 (Thermo Fisher Scientific, USA) host cells that had been adapted to a chemically defined medium (CDM4CHO (+0 / 20 nM MTX; methotrexate)) to generate a cell line (MGgE-CHO DG44) that highly expresses the varicella-zoster virus surface protein antigen (MGgE; SEQ ID NO: 1).

[0068] Example 1.1. Determination of protein productivity according to antigen and cell line

[0069] To determine protein productivity according to antigen and cell line type, the pMSID2-MGgE expression vector constructed in Example 1 and the pMSID2-GSKgE expression vector constructed by cloning the gene encoding the truncated gE antigen (GSKgE; SEQ ID NO: 4) into the pMSID2 vector as disclosed in Korean Patent No. 1357204 were transfected into CHO-S host cells that had been adapted to a chemically defined medium (CD forti CHO (+50 nM MTX)) to generate cell lines (MGgE-CHO-S and GSKgE-CHO-S).

[0070] The MGgE-CHO DG44, MGgE-CHO-S, and GSKgE-CHO-S were each cultured in a chemically defined medium for 6 days, and then the expression level of VGV gE was detected by Western blot and enzyme-linked immunosorbent assay (ELISA).

[0071] As a result, referring to Figure 3 and 4 , MGgE showed better productivity than GSKgE in the CHO-S cell line, and MGgE showed much better productivity in the CHO DG44 cell line than in the CHO-S cell line. In particular, MGgE showed the highest productivity in the CHO DG44(S) cell line that had been adapted to the CDM4CHO (+20 nM MTX) medium.

[0072] Example 2. Establishment of a culture method for MGgE production

[0073] Example 2.1. Seed culture to obtain a certain number of cells for the cell line

[0074] The MGgE-CHO DG44 cell line produced in Example 1 was inoculated into a 500 mL flask at 3×10 5 cells / mL or higher to a total of 100 mL and cultured under conditions of 36 °C to 38 °C and 4% to 6% CO2. Then, while increasing the flask volume, the step of inoculating the cell line at 2×10 5 cells / mL to 5×10 5 cells / mL to a total of 200 mL to 800 mL was repeated at intervals of 2 to 4 days to obtain a certain number of cells. The cells obtained in the flask were at 2×10 5 cells / mL to 5×10 5Cells were inoculated into a bioreactor at a concentration of cells / mL and then subjected to suspension culture under the conditions of 36°C to 38°C, pH 6.7 to pH 7.1, 10% to 90% dissolved oxygen, and a stirring speed of 86 rpm to 96 rpm. In this way, a certain number of cells were obtained.

[0075] Example 2.2. Production culture of MGgE antigen

[0076] The MGgE-CHO DG44 cell line, which had obtained a certain number of cells in Example 2.1, was inoculated into a bioreactor at a cell concentration of 2×10 5 cells / mL to 5×10 5 cells / mL and then subjected to suspension culture under the conditions of a temperature of 34°C to 35.5°C, pH 6.7 to pH 7.1, 10% to 90% dissolved oxygen, and a stirring speed of 62 rpm to 72 rpm.

[0077] Example 2.3. Quantification of MGgE antigen

[0078] In the following examples, ELISA was performed to measure the content of MGgE antigen. Specifically, the VZV gE antibody (Cat.#sc-17549, 200 μg / mL) was diluted to a concentration of 1 μg / mL in PBS, and then the resulting solution was added to a 96-well ELISA plate at 100 μL per well. Incubation was carried out overnight at 4°C (coating). The plate was washed 3 times with a washing solution (0.05% Tween 20 / phosphate-buffered saline (PBS)), and then incubated with a PBS solution containing 2% BSA for 1 hour. The ELISA plate was washed again and then incubated for 2 hours in the presence of the diluted sample. Then, the plate was washed again. Each culture solution sample was centrifuged, and then the supernatant was stored at -20°C or lower. The sample was thawed at room temperature and used 1 hour before measurement. The VZV gE antibody (Cat.#sc-56995, 100 μg / mL) was diluted to a concentration of 1 μg / mL, added at 100 μL per well, and incubated at room temperature for 1 hour. The plate was washed. Then, the goat anti-mouse IgG-HRP was diluted to a concentration of 1 / 1000 and then added to it at 100 μL per well. The plate was incubated at room temperature for 1 hour. The plate was washed, and 3,3,5,5'-tetramethylbenzidine (TMB) was added to induce the HRP reaction. Then, 100 μL of 1N H2SO4 was dispensed into each well to terminate the reaction, and the absorbance was measured at 450 nm using a microplate reader. The content of the sample was calculated by substituting the measured absorbance into the standard curve equation and multiplying the resulting value by the dilution factor.

[0079] Experimental Example 1. Determination of Productivity after Change in Culture Temperature

[0080] To determine the effect of a change in culture temperature on the productivity of the MGgE antigen when determining that cells have grown sufficiently during the production culture step, the MGgE-CHO DG44 cell line, which had obtained a certain number of cells in Example 2.1, was inoculated into a bioreactor at a cell concentration of 2×10 5 cells / mL to 5×10 5 cells / mL and subjected to suspension culture. Then, when the cell line reached cell concentrations of approximately 60×10 5 cells / mL, 80×10 5 cells / mL, and 150×10 5 cells / mL, respectively, the culture temperature was lowered to 32°C and production culture was carried out. In addition, the MGgE-CHO DG44 cell line, which had obtained a certain number of cells in Example 2.1, was subjected to production culture under constant temperature conditions of 37°C, and then the productivity of the MGgE antigen was compared (see Table 1, Figures 5A and 5B).

[0081] [Table 1]

[0082]

[0083]

[0084] As a result, referring to Table 1 and Figure 5a and Figure 5b , it can be seen that in the case of a temperature change, the culture duration (during which the cell line survived) increased, while the productivity of the MGgE antigen (q P ) was much lower than that in the case of constant temperature conditions.

[0085] This indicates that during the production culture of the MGgE antigen, the productivity increases when the culture temperature of the MGgE-CHO DG44 cell line is maintained within a certain range without temperature change.

[0086] Experimental Example 2. Determination of MGgE Antigen Productivity According to Production Temperature

[0087] In the production culture step of the MGgE antigen in Example 2.2, the MGgE-CHO DG44 cell line inoculated into a bioreactor at a cell concentration of 2×10 5 cells / mL to 5×10 5 cells / mL was subjected to production culture under constant temperature conditions selected within the range of 33°C to 37°C (Table 2, Figure 6a and Figure 6b ).

[0088] [Table 2]

[0089]

[0090] As a result, referring to Table 2 and Figure 6a and Figure 6b , under low-temperature culture conditions (constant temperature conditions of 34°C to 35.5°C) below 37°C (the suitable temperature for the growth of normal CHO cells), the culture duration and productivity are improved. In addition, under conditions of 33.5°C or lower, cell growth and productivity decline. On the other hand, from the perspective of q P related to the number of cells in the culture, the reason why the q P value calculated at 33°C is quite high is that, although the productivity is very low, the number of cells is very small compared to other batches (see the integrated VCD value). In other words, it can be seen that the low-temperature conditions of 34°C to 35.5°C result in a significant increase in the productivity of the MGgE antigen in the MGgE-CHO DG44 cell line.

[0091] Example 3. Purification of the MGgE antigen

[0092] Example 3.1. Harvesting and clarification

[0093] For the culture solution (MG1120) cultured at 35°C under the conditions used for production culture in Experimental Example 2, in order to remove cell debris in the culture solution, harvesting and clarification were performed using a depth filter (COHC Millipore, USA).

[0094] The pressure conditions during filtration were maintained at 0.9 bar or lower, and the filtrate obtained by removing cell debris was filtered through a 0.45 + 0.2 μm filter (Sartopore II, Sartorius, Germany) to obtain a culture filtrate.

[0095] Example 3.2. Anion exchange chromatography - Comparison of yields according to the sodium chloride concentration in the washing buffer and the elution buffer

[0096] Anion exchange chromatography was performed to remove the liquid culture medium components and impurities contained in the culture filtrate obtained in Example 3.1.

[0097] Specifically, a column was packed with Q Sepharose FF (GE Healthcare, USA) resin and then equilibrated to a pH of 7.0 ± 0.2 using an equilibration buffer. Subsequently, the culture filtrate obtained in Example 3.1 was loaded onto the column at a flow rate of 150 ± 15 cm / hour. Subsequently, the column was washed successively with the equilibration buffer and a wash buffer containing sodium chloride. Then, the MGgE antigen was eluted using an elution buffer containing sodium chloride and collected.

[0098] The yield in the anion exchange chromatography step was compared under the condition of changing the concentration of sodium chloride contained in the wash buffer and the elution buffer.

[0099] Specifically, anion exchange chromatography was performed using a wash buffer containing 150 mM, 200 mM, or 250 mM sodium chloride and an elution buffer containing 500 mM sodium chloride (Table 3).

[0100] [Table 3]

[0101]

[0102] As a result, referring to Table 3, when the concentration of sodium chloride in the wash buffer was 150 mM, the impurity content per MGgE was the lowest.

[0103] Therefore, anion exchange chromatography was performed using a wash buffer containing 150 mM sodium chloride and an elution buffer containing 400 mM, 500 mM, or 600 mM sodium chloride (Table 4).

[0104] [Table 4]

[0105]

[0106] As a result, referring to Table 4, when using an elution buffer containing 500 mM sodium chloride, the impurity content per MGgE was the lowest.

[0107] Therefore, during the purification step of the MGgE antigen, the optimal concentrations of sodium chloride contained in the wash buffer and the elution buffer in the anion exchange chromatography were 150 mM and 500 mM, respectively.

[0108] Example 3.3. Hydrophobic interaction chromatography

[0109] Hydrophobic interaction chromatography was performed on the eluate from the anion exchange chromatography obtained in Example 3.2 to remove the liquid culture components and impurities that were not removed previously.

[0110] Specifically, the column was packed with Toyopearl Butyl-650M (Tosoh) resin and then equilibrated to pH 7.0 ± 0.2 using an equilibration buffer. Then, 5 M sodium chloride was added to the eluate from the anion exchange chromatography eluate obtained in Example 3.2, and the resulting mixture was loaded onto the column at a flow rate of 100 ± 10 cm / hour. Subsequently, the column was washed successively with the equilibration buffer and a wash buffer containing 1 M sodium chloride. Then, the MGgE antigen was eluted using an elution buffer without sodium chloride and collected.

[0111] Example 3.4. Virus Inactivation

[0112] To inactivate potential enveloped viruses in the solution containing the MGgE antigen, a virus inactivation step was carried out in the presence of an added solvent.

[0113] Specifically, phosphoric acid was added to the eluate from the hydrophobic interaction chromatography obtained in Example 3.3 to adjust the pH of the eluate to 3.0 ± 0.2, and the mixture was stirred at 100 ± 20 rpm for 30 minutes at room temperature (control) to inactivate the virus. Then, the pH was adjusted to 6.5 ± 0.5 using disodium phosphate.

[0114] In addition, when the virus inactivation time was changed to 60 minutes, 90 minutes, and 120 minutes, the stability of the MGgE antigen was examined (Table 5).

[0115] [Table 5]

[0116]

[0117] As a result, referring to Table 5, even 120 minutes of inactivation did not cause a significant change in the content and purity of the MGgE antigen. Therefore, under the pH 3.0 condition effective for virus inactivation, MGgE was stable even when the virus inactivation time was 120 minutes.

[0118] Example 3.5. First Concentration and Diafiltration

[0119] The first concentration and diafiltration step was carried out to remove low molecular weight ions from the solution containing the MGgE antigen with added solvent obtained in Example 3.4, and the solution had conditions suitable for the mixed-mode chromatography process.

[0120] Specifically, the solution containing the MGgE antigen with added solvent was ultrafiltered using an ultrafiltration / diafiltration system (Sartocon cassette (50K)), and the filtrate was diafiltered using an equilibration buffer for the mixed-mode chromatography process (the next process) until the filtrate had a pH of 7.2 ± 0.2 and a conductivity of 2.5 mS / cm or lower.

[0121] Example 3.6. Mixed-mode Chromatography

[0122] The dialyzed and / or concentrated solution containing the MGgE antigen obtained in Example 3.5 was subjected to mixed-mode chromatography to remove dimers and impurities therefrom.

[0123] Specifically, the column was packed with ceramic hydroxyapatite (Bio-Rad) resin and then equilibrated to pH 7.2 ± 0.2 using an equilibration buffer. Then, the dialyzed and / or concentrated solution containing the MGgE antigen obtained in Example 3.5 was loaded onto the column such that the flow rate was 100 ± 10 cm / hour, and the unadsorbed solution was collected. Then, the column was washed with the equilibration buffer for collection.

[0124] Example 3.7. Second Concentration and Diafiltration

[0125] A second concentration and diafiltration step was carried out to adjust the protein concentration in the unadsorbed solution obtained from mixed-mode chromatography in Example 3.6 and such that the solution had conditions suitable for the nanofiltration process.

[0126] The unadsorbed solution from mixed-mode chromatography was subjected to an ultrafiltration / diafiltration system (Sartocon cassette (50K)), and the filtrate was diafiltered using the equilibration buffer for the nanofiltration process (which is the next process) until the filtrate had a pH of 7.4 ± 0.2 and a conductivity of 15.5 mS / cm or higher. Then, the resulting product was concentrated by ultrafiltration to a protein concentration of 7.0 ± 0.5 mg / mL.

[0127] Example 3.8. Nanofiltration

[0128] Nanofiltration is a virus removal step where a nanofiltration membrane (Planova 20N, Asahi) is used and the pH is equilibrated to 7.4 ± 0.2 using a formulation buffer.

[0129] Then, the dialyzed and concentrated solution containing the MGgE antigen obtained in Example 3.7 was passed through the nanofiltration membrane under a pressure condition of 1.0 ± 0.2 bar to remove viruses, and then the nanofiltration membrane was washed with the equilibration buffer. The filtrate obtained by nanofiltration and the wash solution were mixed together, and then the protein concentration was measured.

[0130] Example 3.9. Dilution and Filtration

[0131] The filtrate obtained by nanofiltration in Example 3.8 was diluted with a formulation buffer such that the protein concentration was 5.0 ± 0.5 mg / mL, and then filtered using a 0.2 μm filter.

[0132] Then, the obtained solution containing the MGgE antigen was aliquoted and stored at -20 °C or lower temperature.

[0133] Experimental Example 3. Evaluation of the yield of each purification step

[0134] The yield of each step in the purification step was examined by measuring the content of the MGgE antigen in each step of Example 3 (Table 6). Regarding the protein content of MGgE in Table 6, each culture solution marked with * after harvesting, clarification, and anion exchange chromatography steps was measured by ELISA assay; and the culture solution in the subsequent steps was measured by UV.

[0135] [Table 6]

[0136] <110> GREEN CROSS CORPORATION <120> Method for producing varicella-zoster virus surface protein antigen <130> PCC106048GCC <150> KR 10-2020-0085685 <151> 2020-07-10 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 537 <212> PRT <213> Artificial sequence <220> <223> MGgE antigen <400> 1 Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly 1 5 10 15 Ile Ile Thr Gly Thr Leu Arg Ile Thr Asn Pro Val Arg Ala Ser Val 20 25 30 Leu Arg Tyr Asp Asp Phe His Thr Asp Glu Asp Lys Leu Asp Thr Asn 35 40 45 Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp 50 55 60 Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro 65 70 75 80 Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His 85 90 95 Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu 100 105 110 Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp 115 120 125 Asp Thr Gly Ile His Val Ile Pro Thr Leu Asn Gly Asp Asp Arg His 130 135 140 Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly 145 150 155 160 Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val 165 170 175 Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr 180 185 190 Gly Val Arg Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu Thr Cys 195 200 205 Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr 210 215 220 Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr 225 230 235 240 Lys Glu Asp Gln Leu Ala Glu Ile Ser Tyr Arg Phe Gln Gly Lys Lys 245 250 255 Glu Ala Asp Gln Pro Trp Ile Val Val Asn Thr Ser Thr Leu Phe Asp 260 265 270 Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val 275 280 285 Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg 290 295 300 Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys 305 310 315 320 Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro 325 330 335 Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser 340 345 350 Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His 355 360 365 Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp 370 375 380 Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro 385 390 395 400 Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr 405 410 415 Ser Pro His Leu Ala Gln Arg Val Ala Ser Thr Val Tyr Gln Asn Cys 420 425 430 Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser His Met 435 440 445 Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys 450 455 460 Phe Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe Val Val 465 470 475 480 Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val Ser Thr 485 490 495 Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr 500 505 510 Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val 515 520 525 Asn Pro Gly Thr Ser Pro Leu Leu Arg 530 535 <210> 2 <211> 1610 <212> DNA <213> Artificial sequence <220> <223> MGgE antigen <400> 2 atgggaacag tcaacaaacc agtcgtcggc gtgctgatgg gcttcggtat tattacagga 60 actctgagga ttactaaccc cgtgcgcgcc tctgtgctgc ggtacgacga tttccacaca 120 gacgaggata agctggacac caattccgtg tatgagccct actatcactc tgatcacgcc 180 gagagctcct gggtgaaccg gggcgagtct agcaggaagg cttacgacca caacagccct 240 tatatctggc cacggaatga ctacgatggc tttctggaga acgcccacga gcaccacggc 300 gtgtataatc agggcagagg catcgactct ggcgagcggc tgatgcagcc cacccagatg 360 agcgcccagg aggatctggg cgacgataca ggcatccacg tgatccctac cctgaatggc 420 gacgataggc acaagatcgt gaacgtggat cagagacagt acggcgacgt gttcaagggc 480 gatctgaatc ccaagcctca gggccagagg ctgatcgagg tgtccgtgga ggagaaccac 540 cccttcaccc tgagagcccc tatccagcgg atctacggcg tgaggtatac cgagacatgg 600 cccttcaccc tgagagcccc tatccagcgg atctacggcg tgaggtatac cgagacatgg 600 agctttctgc catccctgac atgcaccggc gacgctgctc ctgctatcca gcacatctgc 660 agctttctgc catccctgac atgcaccggc gacgctgctc ctgctatcca gcacatctgc 660 ctgaagcaca ccacatgttt tcaggacgtg gtggtggacg tggattgtgc cgagaataca 720 ctgaagcaca ccacatgttt tcaggacgtg gtggtggacg tggattgtgc cgagaataca 720 aaggaggatc agctggctga gatctcctac cggttccagg gcaagaagga ggccgatcag 780 aaggaggatc agctggctga gatctcctac cggttccagg gcaagaagga ggccgatcag 780 ccttggatcg tggtgaacac ctctacactg tttgacgagc tggagctgga tccccctgag 840 ccttggatcg tggtgaacac ctctacactg tttgacgagc tggagctgga tccccctgag 840 atcgagccag gcgtgctgaa ggtgctgaga accgagaagc agtacctggg cgtgtatatc 900 atcgagccag gcgtgctgaa ggtgctgaga accgagaagc agtacctggg cgtgtatatc 900 tggaacatgc ggggctctga cggcaccagc acatacgcta ccttcctggt cacatggaag 960 tggaacatgc ggggctctga cggcaccagc acatacgcta ccttcctggt cacatggaag 960 ggcgatgaga agacccggaa tccaacacct gctgtgaccc ctcagccaag gggagctgag 1020 ggcgatgaga agacccggaa tccaacacct gctgtgaccc ctcagccaag gggagctgag 1020 tttcacatgt ggaactatca ctcccacgtg ttctctgtgg gcgacacctt tagcctggcc 1080 tttcacatgt ggaactatca ctcccacgtg ttctctgtgg gcgacacctt tagcctggcc 1080 atgcacctgc aatataagat ccacgaggct cctttcgacc tgctgctgga gtggctgtat 1140 atgcacctgc aatataagat ccacgaggct cctttcgacc tgctgctgga gtggctgtat 1140 gtgcccatcg atcctacatg ccagccaatg aggctgtact ccacctgtct gtatcaccca 1200 gtgcccatcg atcctacatg ccagccaatg aggctgtact ccacctgtct gtatcaccca 1200 aatgcccccc aatgcctgag ccacatgaac tccggctgta cctttacaag cccccacctg 1260 aatgcccccc aatgcctgag ccacatgaac tccggctgta cctttacaag cccccacctg 1260 gcccagagag tggcttccac agtgtaccag aactgcgagc acgccgacaa ttacaccgct 1320 tattgtctgg gcatctctca catggagccc agcttcggcc tgatcctgca cgacggcggc 1380 accacactga agtttgtgga tacacccgag tccctgtctg gcctctacgt gttcgtggtg 1440 tacttcaacg gccacgtgga ggccgtggct tatacagtgg tgtctaccgt ggatcacttc 1500 gtgaacgcca tcgaggagag aggatttcca cctaccgctg gacagcctcc agctaccaca 1560 aagcctaagg aaatcacccc tgtcaatcct ggaacttcac ctctgctgcg 1610 <210> 3 <211> 623 <212> PRT <213> Artificial Sequence <220> <223> VZV gE antigen <400> 3 Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly 1 5 10 15 Ile Ile Thr Gly Thr Leu Arg Ile Thr Asn Pro Val Arg Ala Ser Val 20 25 30 Leu Arg Tyr Asp Asp Phe His Thr Asp Glu Asp Lys Leu Asp Thr Asn 35 40 45 Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp 50 55 60 Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro 65 70 75 80 Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His 85 90 95 Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu 100 105 110 Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp 115 120 125 Asp Thr Gly Ile His Val Ile Pro Thr Leu Asn Gly Asp Asp Arg His 130 135 140 Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly 145 150 155 160 Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val 165 170 175 Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr 180 185 190 Gly Val Arg Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu Thr Cys 195 200 205 Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr 210 215 220 Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr 225 230 235 240 Lys Glu Asp Gln Leu Ala Glu Ile Ser Tyr Arg Phe Gln Gly Lys Lys 245 250 255 Glu Ala Asp Gln Pro Trp Ile Val Val Asn Thr Ser Thr Leu Phe Asp 260 265 270 Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val 275 280 285 Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg 290 295 300 Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys 305 310 315 320 Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro 325 330 335 Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser 340 345 350 Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His 355 360 365 Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp 370 375 380 Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro 385 390 395 400 Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr 405 410 415 Ser Pro His Leu Ala Gln Arg Val Ala Ser Thr Val Tyr Gln Asn Cys 420 425 430 Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser His Met 435 440 445 Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys 450 455 460 Phe Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe Val Val 465 470 475 480 Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val Ser Thr 485 490 495 Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr 500 505 510 Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val 515 520 525 Asn Pro Gly Thr Ser Pro Leu Ile Arg Tyr Ala Ala Trp Thr Gly Gly 530 535 540 Leu Ala Ala Val Val Leu Leu Cys Leu Val Ile Phe Leu Ile Cys Thr 545 550 555 560 Ala Lys Arg Met Arg Val Lys Ala Tyr Arg Val Asp Lys Ser Pro Tyr 565 570 575 Asn Gln Ser Met Tyr Tyr Ala Gly Leu Pro Val Asp Asp Phe Glu Asp 580 585 590 Ser Glu Ser Thr Asp Thr Glu Glu Glu Phe Gly Asn Ala Ile Gly Gly 595 600 605 Ser His Gly Gly Ser Ser Tyr Thr Val Tyr Ile Asp Lys Thr Arg 610 615 620 <210> 4 <211> 546 <212> PRT <213> Artificial Sequence <220> <223> GSKgE <400> 4 Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly 1 5 10 15 Ile Ile Thr Gly Thr Leu Arg Ile Thr Asn Pro Val Arg Ala Ser Val 20 25 30 Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn 35 40 45 Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp 50 55 60 Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro 65 70 75 80 Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His 85 90 95 Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu 100 105 110 Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp 115 120 125 Asp Thr Gly Ile His Val Ile Pro Thr Leu Asn Gly Asp Asp Arg His 130 135 140 Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly 145 150 155 160 Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val 165 170 175 Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr 180 185 190 Gly Val Arg Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu Thr Cys 195 200 205 Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr 210 215 220 Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr 225 230 235 240 Lys Glu Asp Gln Leu Ala Glu Ile Ser Tyr Arg Phe Gln Gly Lys Lys 245 250 255 Glu Ala Asp Gln Pro Trp Ile Val Val Asn Thr Ser Thr Leu Phe Asp 260 265 270 Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val 275 280 285 Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg 290 295 300 Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys 305 310 315 320 Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro 325 330 335 Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser 340 345 350 Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His 355 360 365 Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp 370 375 380 Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro 385 390 395 400 Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr 405 410 415 Ser Pro His Leu Ala Gln Arg Val Ala Ser Thr Val Tyr Gln Asn Cys 420 425 430 Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser His Met 435 440 445 Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys 450 455 460 Phe Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe Val Val 465 470 475 480 Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val Ser Thr 485 490 495 Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr 500 505 510 Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val 515 520 525 Asn Pro Gly Thr Ser Pro Leu Ile Arg Tyr Ala Ala Trp Thr Gly Gly 530 535 540 Leu Ala 545

Claims

1. A method for producing varicella-zoster virus (VZV) surface protein (gE) antigen, comprising the following steps: (a) Culturing a recombinant cell line that produces the VZV gE antigen to obtain a culture solution, wherein the recombinant cell line is prepared using Chinese hamster ovary (CHO) cells; and (b) Purifying the culture solution; wherein the step (a) of obtaining the culture solution comprises the following steps: (a-1) Seed-culturing the recombinant cell line; and (a-2) Performing production culture on the cell line that has undergone the seed-culture; wherein the step (a-2) of performing the production culture comprises culturing the cell line at a temperature of 34 °C to 35.5 °C; and wherein the step (b) of purifying the culture solution comprises the following steps: (b-1) Performing anion exchange chromatography; (b-2) Performing hydrophobic interaction chromatography, which comprises adding sodium chloride to the eluate obtained by the anion exchange chromatography, loading it onto a hydrophobic interaction chromatography column, and washing it with a washing buffer containing sodium chloride; (b-3) Treating the culture solution with a virus inactivator to inactivate the virus, and then performing concentration and diafiltration; (b-4) Performing mixed-mode chromatography to obtain an eluate; and (b-5) Concentrating and diafiltering the eluate, and then performing nanofiltration, and these steps are carried out in a series of sequences.

2. The method according to claim 1, wherein the VZV gE antigen is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO:

1.

3. The method according to claim 1, wherein the cell line is transformed with a gene consisting of the nucleotide sequence represented by SEQ ID NO:

2.

4. The method according to claim 1, wherein the step (a-1) of performing the seed-culture is to culture the cell line that produces the VZV gE antigen by any method selected from subculture, suspension culture, and combinations thereof.

5. The method according to claim 1, wherein the step (a-1) of performing the seed-culture comprises culturing the cell line at a temperature of 34 °C to 38 °C.

6. The method according to claim 1, wherein for the anion exchange chromatography, a washing buffer with a sodium chloride concentration of 0.1 mM to 150 mM is used.

7. The method according to claim 1, wherein for the anion exchange chromatography, an elution buffer with a sodium chloride concentration of 400 mM to 600 mM is used.

8. The method according to claim 1, wherein the virus inactivator is a phosphoric acid solution.

9. The method according to claim 1, wherein the virus inactivation step is carried out under the conditions of pH 2.8 to 3.

2.

10. The method according to claim 1, wherein for the nanofiltration step (b-5), a filtration system with a nanofiltration membrane is used.

11. The method according to claim 1, further comprising: After step (b-5), diluting the filtrate obtained by the nanofiltration with a formulation buffer and then performing a filtration step.

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