Preparation method and application of recombinant varicella zoster virus glycoprotein gE

Abstract

The present invention discloses a preparation method and application of a recombinant varicella-zoster virus glycoprotein gE. By optimizing process parameters in a cell culture process, such as a basal culture medium, a feed culture medium, inoculation density, temperature, time, pH, etc., a cell culture fluid with high protein expression yield and high protein purity is obtained, thereby providing a good basis for the preparation of a varicella-zoster virus vaccine.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a preparation method and application of glycoprotein gE of a recombinant varicella-zoster virus. Background Art

[0002] Varicella zoster virus (VZV) belongs to the genus Herpesvirus, family Herpesviridae. The VZV genome has 71 genes, encoding 67 different proteins, including 6 glycoproteins (gpⅠ~gpⅥ), which are now uniformly named gE, gB, gH, gI, gC and gL. Among them, gE is the most abundant in the viral envelope and is the most important glycoprotein recognized by the host immune system. It is encoded by the orf68 gene and is located in the short region of the vzv genome. It contains 623 amino acids, including 544 in the hydrophilic membrane extracellular region, 17 in the transmembrane region, and 62 in the intracellular region. Studies have shown that the immune response induced by gE as an antigen can protect animals from viral attacks.

[0003] The glycoprotein gE of VZV is mainly expressed in host cells through biological methods, but the current protein yield is not high. It is urgent to explore the optimal preparation process to obtain a higher concentration of gE and thus obtain a vaccine with better immunogenicity. Summary of the invention

[0004] In one aspect of the present invention, a method for culturing cells is provided, wherein the cells express VZV-gE protein, and the method comprises culturing the cells using CD pro, MaxA, MaxC, MaxY, CD fortiCHO or B600S as a basic culture medium.

[0005] Preferably, the basal medium comprises MaxA or CD fortiCHO.

[0006] Preferably, the culture method comprises using MaxFC1 / MaxFB, Nfpro, FeedC or F600aS / F600bS as feed medium;

[0007] More preferably, the feed medium comprises MaxFC1 / MaxFB.

[0008] More preferably, the feed ratio MaxFC1 / MaxFB of the feed medium is (8-12) / 1, which can be any data or range within the above range, such as 8 / 1, 9 / 1, 10 / 1, 11 / 1, 12 / 1. In a specific embodiment, the inoculation density is 10 / 1.

[0009] More preferably, the single MaxFC1 / MaxFB feed concentration is (4%-6%) / (0.4%-0.6%), which can be any data or range within the above range, such as 4% / 0.4%, 5% / 0.5%, 6% / 0.6%. In a specific embodiment, the feed concentration is 4% / 0.4%.

[0010] Preferably, the feed medium includes multiple feedings, for example, feedings can be performed on the 3rd, 5th, 7th, 9th and 11th days respectively.

[0011] Preferably, the culture method further comprises sugar supplementation, wherein the sugar supplementation comprises supplementation when the glucose is <4 g / L, and the sugar is supplemented to a final concentration of 4 to 5 g / L.

[0012] More preferably, the sugar supplementation starts on the 3rd day.

[0013] Preferably, the cell seeding density in the culture method is (0.5-1.5)E6 vc / mL, which can be any data or range within the above range, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5E6 vc / mL. In a specific embodiment, the seeding density is (1.0±0.3)E6 vc / mL.

[0014] Preferably, the initial culture temperature of the culture method is (37±1)°C, such as 36, 37, or 38°C.

[0015] Preferably, the culture method includes cooling the cells in the middle and late stages of cell growth.

[0016] More preferably, the cooling culture comprises culturing at a temperature lowered to 31-35, such as 31, 32, 33, 34, 35°C. In a specific embodiment, the cooling culture temperature is 33°C.

[0017] Preferably, the cooling culture comprises cooling the culture to 33°C on the 6th day.

[0018] Preferably, in the culture method, the pH is 6.80-7.20, such as 6.8, 6.9, 7.0, 7.1, 7.2. In a specific embodiment, the pH is 7.0±0.2.

[0019] Preferably, the culture method further comprises harvesting the supernatant after the culture.

[0020] More preferably, the harvesting step is performed when the cell viability is less than 90%, preferably when the cell viability is less than 85%, less than 80%. Further preferably, the harvesting is performed on day 9-12 of cell culture, for example, day 9, 10, 11, 12. In a specific embodiment, the harvesting is performed on day 10.

[0021] Preferably, the culture method further comprises purifying the supernatant to obtain VZV-gE protein.

[0022] More preferably, the cell comprises an expression vector comprising a nucleic acid encoding the VZV-gE protein.

[0023] Further preferably, the VZV-gE protein is a fusion protein comprising the VZV-gE protein. Further preferably, the fusion protein comprises the VZV-gE protein and the Fc fragment of human IgG4.

[0024] Preferably, the culture method comprises introducing the nucleic acid encoding the above fusion protein into an expression vector, and transfecting the expression vector into cells.

[0025] More preferably, the expression vector is a eukaryotic expression vector.

[0026] In some embodiments, the eukaryotic expression vector is an adenoviral vector.

[0027] In some embodiments, the eukaryotic expression vector is a vector expressed in 293 cells or CHO cells.

[0028] Preferably, the cells are derived from mammalian cells.

[0029] In some embodiments, the mammalian cell is selected from CHO, HEK293, SP2 / 0, BHK, C127, etc.

[0030] In some embodiments, the eukaryotic cell is a CHO cell.

[0031] The second aspect of the present invention provides a supernatant or VZV-gE protein obtained by the above-mentioned culture method.

[0032] The supernatant includes VZV-gE protein.

[0033] The third aspect of the present invention provides a use of the supernatant or VZV-gE protein in preparing a vaccine, wherein the vaccine is used to prevent and / or treat VZV-related diseases or conditions.

[0034] In a fourth aspect, the present invention provides a vaccine, which comprises the supernatant obtained by the above-mentioned culture method or the VZV-gE protein.

[0035] Preferably, the vaccine further comprises one or more adjuvants.

[0036] In a fifth aspect, the present invention provides a method for treating or preventing a disease or condition, the method comprising administering any of the above vaccines to a subject, wherein the disease or condition comprises a disease or condition caused by herpes zoster virus.

[0037] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0038] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0039] As used herein, the term "fusion protein" refers to a natural or synthetic molecule composed of one or more molecules, in which two or more peptide- or protein-based (including glycoprotein) molecules with different specificities are optionally fused together through chemical or amino acid-based linker molecules. The connection can be achieved by CN fusion or NC fusion (in the 5'→3' direction), preferably CN fusion.

[0040] As used herein, the term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from position Cys226, or from the amino acid residue at Pro230 to the carboxyl end of the heavy chain. The C-terminal lysine (residue 447 according to the EU numbering system) in the Fc region can be removed, for example during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody can include an antibody population from which all K447 residues have been removed, an antibody population from which no K447 residues have been removed, and an antibody population with a mixture of antibodies with and without the K447 residue.

[0041] As used herein, the term "disease" or "condition" refers to a state of life or health of a patient or individual that can be treated with the fusion proteins, pharmaceutical compositions or methods provided herein.

[0042] The term "vaccine" is a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or an attenuated virus vaccine or immunogenic composition. In some embodiments, the vaccine or immunogenic composition is a purified fusion protein.

[0043] As used herein, the term "treating" or "treatment" refers to any indicator of successful treatment or improvement of an injury, disease, pathology, or condition, including any objective or subjective parameter, such as elimination; relief; alleviation of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing the rate of degeneration or decline; or making the end point of degeneration less declining; improving the patient's physical or mental health. The treatment or improvement of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric examination, and / or psychiatric assessment. The term "treatment" and its conjugations may include preventing an injury, pathology, condition, or disease. In an embodiment, treatment is prevention. In an embodiment, treatment does not include prevention.

[0044] As used herein (and as is well understood in the art), "treating" or "treatment" also broadly encompasses any method for obtaining a beneficial or desired result (including a clinical result) in a subject's condition. Beneficial or desired clinical results may include, but are not limited to, alleviation or improvement of one or more symptoms or symptoms, alleviation of the extent of the disease, stabilization (i.e., non-exacerbation) of the disease state, prevention of disease transmission or spread, delay or slowing of disease progression, improvement or alleviation of the disease state, reduction of disease recurrence, and alleviation (whether partial or total, and whether detectable or undetectable). In other words, as used herein, "treatment" encompasses any cure, improvement, or prevention of a disease. Treatment may prevent the occurrence of a disease; inhibit the spread of a disease; alleviate the symptoms of a disease, completely or partially remove the underlying cause of a disease, shorten the duration of a disease, or a combination of these things.

[0045] As used herein, "patient" or "subject in need" refers to a living organism that suffers from or is susceptible to a disease or condition that can be treated by administering a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0046] As used herein, the term "administer" means oral administration to a subject, administration in the form of a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or implantation of a slow release device (e.g., a mini-osmotic pump). Administered by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palate, gums, nose, vagina, rectum or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial administration. Other modes of delivery include, but are not limited to, use of liposome formulations, intravenous infusions, transdermal patches, etc. In an embodiment, administration does not include administration of any activating agent other than the narrated activating agent.

[0047] The present invention has the following beneficial effects:

[0048] 1. The present invention screens out a suitable basal medium feed medium. The protein quality obtained by using the basal medium feed medium of the present invention is relatively good, and in the preferred solution, the SEC can be higher than 80%;

[0049] 2. The present invention can ensure the stability of each batch of culture by further adjusting the process conditions such as inoculation density, feed ratio, pH, and cooling culture. Finally, the culture process is confirmed in a 3L reactor. The protein expression amount is as high as 1.5g, and SEC can basically be maintained at more than 80%, and the protein quality is good and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Cell growth curves for culture medium screening;

[0051] Figure 2 Cell growth curves for inoculation density and feeding strategy;

[0052] Figure 3 Cell growth curve diagram for pH and culture temperature control;

[0053] Figure 4 Validation of cell growth curve for 3L reactor cell culture process. DETAILED DESCRIPTION

[0054] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are the terms and routine procedures widely used in the corresponding fields.

[0055] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0056] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0057] Example 1. Cell culture process development

[0058] The host cell is a CHO-K1 cell, and the glycoprotein gE is a fusion protein comprising the glycoprotein E (VZVgE) of varicella-zoster virus (VZV) and the Fc fragment of IgG (see CN116023510A for details, which is incorporated herein by reference as a whole as part of the present application. For example, the fusion protein refers to SEQ ID NO.3 in the document, and the nucleic acid encoding the fusion protein refers to SEQ ID NO.4 in the document).

[0059] Cell culture process development is based on analysis of host cell origin, protein structure, properties and mechanism of action, and evaluates the effects of cell culture process parameters on quality attributes such as protein activity, aggregates and fragments, charge isomers, and major process attributes such as protein yield, cell growth density, cell harvest viability, etc. The main process parameters are determined and targeted research is conducted to ensure the consistency of product quality attributes and major process attributes under fermentation process conditions, and to establish a cell culture process that is efficient, robust and meets the requirements.

[0060] 1.1 Confirmation of production culture medium

[0061] The culture medium provides the necessary substances and living environment for biochemical reactions such as new cell synthesis and cell metabolism, and is the energy source for cell growth and target protein expression. Changes in the culture medium will lead to changes in cell growth and target protein expression and quality. A culture medium suitable for cell growth and expression and with protein quality and delivery time that meet project requirements is selected as the production culture medium.

[0062] The monoclonal clone D-3B7 was selected, and six commercial culture media, CD pro, MaxA, MaxC, MaxY, CD fortiCHO and B600S, which do not contain animal or plant ingredients and have limited chemical components, were used as basal culture media, and four culture media, MaxFC1 / MaxFB, Nfpro, FeedC, F600aS / F600bS, were screened as feed culture media. The culture media used can support high-density cell culture, and have the advantages of increasing yield and shortening the development time of cell culture processes. Because all components of the culture medium are clear, the adverse effects on cell growth and expression caused by differences between culture medium batches are avoided, which is beneficial to the stability of cell growth and expression between protein production batches and ensures product quality. The culture medium screening parameter settings are shown in Table 1.

[0063] Table 1 Culture medium screening process parameter settings

[0064]

[0065]

[0066] The results are as follows Figure 1 As shown in Table 2,

[0067] Table 2 Culture medium screening yield and quality results

[0068]

[0069] The results showed that the protein expression level of the combination of MaxY and MaxFC1 / FB was the highest (SF9 group), and the protein expression level of the combination of MaxC and MaxFC1 / FB was the lowest. The protein quality of the combination of MaxA and MaxFC1 / FB (SF7 group) and the combination of CD fortiCHO and FeedC (SF10 group) was relatively good, with the SEC main peak purity greater than 80%, and the protein expression levels were 0.94g / L and 0.74g / L, respectively. Although the Titer of the SF9 group was 1.66g / L, the purity of the SEC main peak was only 68.7%. Based on the comprehensive experimental results, MaxA and CD fortiCHO were selected as the basic culture medium based on the requirements of protein yield and quality, but CD fortiCHO was an imported culture medium. Based on cost and procurement cycle considerations, MaxA was finally selected as the basic culture medium. MaxFC1 / FB was used as a feed medium for shake flask and reactor process development.

[0070] Example 2: Optimization of inoculation density and feeding strategy

[0071] During the cell culture process, the inoculation density will affect the cell growth density, integral viable cell density (IVCD) and culture time during the entire culture process, and thus affect the protein expression level. The inoculation density needs to be effectively controlled. The addition of feed provides more nutrients for cell growth and maintains cell metabolism. Cells can maintain high-density growth for a long time, thereby increasing IVCD and increasing protein expression. Too high or too low feed amounts will affect cell growth and expression, and will also have a certain impact on protein quality. In order to obtain a high-yield and stable quality cell culture process, it is necessary to optimize the cell inoculation density and feeding strategy to obtain the optimal parameters and operating space. The optimized parameter settings for inoculation density and feeding strategy are shown in Table 3.

[0072] Table 3 Inoculation density and feeding strategy process parameter settings

[0073]

[0074]

[0075] Results Figure 2 and Table 4,

[0076] Table 4 Yield and quality results of inoculation density and feeding strategy

[0077]

[0078] The results showed that different inoculation densities affected cell growth. The higher the cell inoculation density, the faster the cells grew during the logarithmic growth phase, and the higher the cell density during the culture process. When the inoculation density was 0.5E6 vc / ml, increasing the feed ratio slowed down cell growth. When the inoculation density was 1E6 vc / ml or 1.5E6 vc / ml, different feed ratios had little effect on cell growth ( Figure 2 ). Different inoculation densities and feed ratios had certain effects on protein yield and protein quality. The protein expression increased with the increase of inoculation density, and the purity of the SEC main peak increased first and then decreased with the increase of inoculation density; the increase of feed ratio was not conducive to protein expression, and the purity of the SEC main peak also decreased.

[0079] According to the requirements of the project protein yield and key quality attributes, the operating space for setting the inoculation density and feed amount is shown in Table 5.

[0080] Table 5 Operating range of inoculation density and feed ratio

[0081]

[0082] Example 3 Study on Control of Culture pH and Culture Temperature

[0083] Bioreactor culture process parameters include control parameters such as pH, DO, temperature, stirring speed and ventilation, and their effects on cell growth, protein expression and protein quality properties need to be considered comprehensively. pH is one of the important process parameters for cell culture, because it has a certain regulatory effect on the activity of intracellular enzymes, thereby affecting cell growth, expression and product quality such as molecular size isomers, charge isomers, etc. In general, cells can grow and express normally within the pH range of 6.80-7.20. In order to produce products of consistent quality, the operating range of pH needs to be studied.

[0084] Cell culture is generally carried out in a variable temperature culture method. The temperature of the cell exponential growth phase is generally higher than the culture temperature in the middle and late stages. The purpose is to increase the cell growth rate in the growth phase and obtain a sufficient number of cells to provide a basis for later protein expression. The middle and late stages of cell growth are generally the protein expression period. Lowering the culture temperature, controlling the cell growth rate, and extending the cell culture cycle are conducive to increasing the amount of protein expression. The optimization of the culture temperature also has a certain degree of influence on the purity of the target protein, protein glycosylation, and the modification of charge isomers. Given that the optimum temperature for the cell growth phase is 37°C, no optimization is required, so only the temperature parameters and cooling time in the middle and late stages of growth are studied.

[0085] Different harvesting times will have a certain impact on protein yield and quality. It is necessary to study the time of culture harvesting to determine the harvesting time that meets the project quality requirements. The experimental design of the pH and culture temperature control strategy of the 3L reactor is shown in Table 6.

[0086] Table 6 pH and culture temperature control process parameter settings

[0087]

[0088] The results of the pH and temperature process parameters are shown in Figure 3 And Table 7,

[0089] Table 7 Quality results of culture pH and culture temperature control study

[0090]

[0091] The results showed that the cell growth density of the experimental group cooled to 31°C on the 6th day was slightly lower, and the cell growth density of the other experimental groups was basically the same. Different culture processes had little effect on the expression of the target protein. The expression level at harvest was greater than 1g / L, between 1.1 and 1.4g / L, and the SEC main peak purity of the protein expressed by different processes was basically the same. In summary, combined with the results of cell growth, protein expression and quality, the pH of this project was determined to be 7.0±0.2, the initial culture temperature was 37°C, and the temperature was cooled to 33°C on the 6th day.

[0092] The results of the study on different harvesting times of the culture medium of the R04 reactor showed that the expression level increased slightly from the 10th to the 12th day of culture. The purity of the main peak of protein SEC decreased significantly with the extension of culture time. Based on the cell viability, protein yield and quality results, the optimal harvesting time of cell culture in this project was determined to be the 10th day (Table 8).

[0093] Table 8 Quality results of the culture days study

[0094]

[0095] Example 43L reactor culture process confirmation

[0096] The above research determined the process parameters and operating range of the cell culture pilot test, and the 3L reactor was used to confirm the culture process. The 3L reactor culture process parameter settings are shown in Table 9.

[0097] Table 9 Parameter settings for 3L reactor cell culture process confirmation

[0098]

[0099]

[0100] The highest cell growth density in the 3L reactor was 17.4×10 6 vc / mL (see Figure 4 ), the cell viability was about 80% at harvest, and the protein yield was about 1.5g / L. After affinity chromatography of the supernatant, the protein quality results showed that the purity of the SEC main peak was greater than 80%, and the process was relatively robust.

[0101] After affinity chromatography, the results of protein quality analysis of the supernatant (see Table 10) showed that the expression level increased slightly from day 9 to day 11 of culture. With the extension of culture time, the purity of the main peak of protein SEC decreased significantly. Based on the cell viability, protein yield and quality results, it was determined that the optimal harvest time for cell culture in this project was day 10.

[0102] Table 10 Quality results of 3L reactor culture process confirmation study

[0103]

[0104] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cell culture method, characterized in that: The cells express VZV-gE protein, and the cell culture method comprises: The cells are cultured using MaxA as a basal medium and MaxFC1 / MaxFB as a feed medium, wherein the feed volume ratio of the feed medium MaxFC1 / MaxFB is (8-12) / 1; Alternatively, cells were cultured using CD fortiCHO as basal medium and FeedC as feed medium; Feeding was performed on days 3, 5, 7, and 9; The culture method also includes supplementing sugar starting from the third day, wherein the sugar supplementation includes supplementing when the glucose is less than 4 g / L, and supplementing the sugar to a final concentration of 4-5 g / L.

2. The culture method according to claim 1, characterized in that The cell seeding density in the culture method is (0.5-1.5)×10 6 vc / mL.

3. The culture method according to claim 2, characterized in that The cell seeding density in the culture method is (1.0±0.3)×10 6 vc / mL.

4. The culture method according to claim 1, characterized in that In the culture method, the feed ratio MaxFC1 / MaxFB of the feed medium is (4.0±0.5) / (0.4±0.05).

5. The culture method according to any one of claims 1 to 4, characterized in that: The cell includes an expression vector including a nucleic acid encoding a VZV-gE protein.

6. The culture method according to claim 5, characterized in that: The VZV-gE protein is a fusion protein including the VZV-gE protein.

7. The culture method according to any one of claims 1 to 4, characterized in that: The cells are mammalian cells.

8. The culture method according to any one of claims 1 to 4, characterized in that: The initial culture temperature of the culture method is (37±1)°C.

9. The culture method according to claim 8, characterized in that: The culture method comprises lowering the temperature during the middle and late stages of cell growth.

10. The culture method according to claim 9, characterized in that: The temperature reduction culture includes lowering the temperature to (31-35)°C for culture.

11. The culture method according to any one of claims 1 to 4, characterized in that: In the culture method, the pH is 6.80-7.

20.

12. The culture method according to any one of claims 1 to 4, characterized in that: The culturing method further comprises harvesting the supernatant.

13. The culture method according to claim 12, characterized in that: The harvesting step is performed when the cell viability is less than 90%.

14. The culture method according to claim 12, characterized in that: The harvest was performed on day 9-12 of cell culture.

15. The culture method according to any one of claims 1 to 4, characterized in that: The culture method comprises: MaxA was used as the basic medium and MaxFC1 / FB as the feed medium. On days 3, 5, 7, and 9, MaxFC was supplemented with 14% and MaxFB 0.4% according to the working volume; Seeding density: (1.0±0.3)×10 6 vc / mL; Starting from day 3, when glucose ≤ 4 g / L, feed to a final concentration of 4-5 g / L according to the working volume, including 70 g / L glucose in MaxFC1; Culture temperature: 37°C initially, cooled to 33°C on the 6th day; pH 7.0 ± 0.2; Harvest standard: 12th day or survival rate is less than 80%.

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