Production method of Oumoprayer's monoclonal antibody stock solution and Oumoprayer's monoclonal antibody stock solution

Abstract

The invention discloses a production method of an emoflaibutin antigen stock solution and the emoflaibutin antigen stock solution, and the production method comprises the following steps: S10, providing cells, and carrying out cell recovery, cell amplification and perfusion culture to obtain a cell culture solution; s20, harvesting and purifying the cell culture fluid to obtain a purified intermediate product; s30, mixing the purified intermediate product with an auxiliary material to obtain the Oumoprayer monoclonal antibody stock solution, the production method of the Oumoprayer monoclonal antibody stock solution adopts a continuous flow process for production, the cell density is high, the cell viability is better maintained, and the production period is long, so that the yield is higher; perfusion culture provides a better microenvironment for protein expression, the expressed target protein can be harvested in time, and the quality is better.

Description

Technical Field

[0001] The present invention relates to the field of cell production, and in particular to a production method of an Omopoulin single antigen liquid and an Omopoulin single antigen liquid. Background Art

[0002] In biopharmaceuticals, "stock solution" (or "API") refers to the active pharmaceutical ingredient obtained after cell culture, harvesting, purification and other steps that has not yet been finalized. The stock solution requires further processing, such as adding excipients, dilution, filling, etc., to prepare it into the final drug form that can be used clinically.

[0003] At present, in the production of stock solution, for example, the production process of Omopoulin monoantigen solution involves complex cell culture and purification processes. Although this type of process is quite mature, there are still some potential disadvantages and problems, the most common of which include the following: high production cost, complex purification steps, complex process, easy mutation, and long-term cell culture process, cells may mutate or change gene expression, leading to unstable product quality. At the same time, the production is limited and the long-term storage of Omopoulin monoantigen solution may be affected by temperature and excipients, and low-temperature storage may be required, increasing the cost and complexity of transportation and storage.

[0004] In general, due to the complexity and high requirements of the production process of Omopubai single antigen liquid, there are still potential challenges in production costs, product consistency, quality control, etc. Summary of the invention

[0005] The main purpose of the present invention is to propose a production method of Omopoulin monoantigen solution and Omopoulin monoantigen solution, aiming to solve the problems in the production process of Omopoulin monoantigen solution in the prior art due to its complexity and high requirements, production cost, product consistency, quality control and other aspects.

[0006] The present invention provides a method for producing an Omopoulin single antigen liquid, and the method for producing an Omopoulin single antigen liquid comprises the following steps:

[0007] S10, providing cells, and performing cell recovery, cell expansion, and perfusion culture on the cells to obtain a cell culture fluid;

[0008] S20, harvesting and purifying the cell culture fluid to obtain a purified intermediate product;

[0009] S30, mixing the purified intermediate product with auxiliary materials to obtain Omopoulin single antigen liquid.

[0010] In some embodiments, in step S10, the cell expansion step comprises:

[0011] S101, providing cells, resuscitating the cells, and obtaining resuscitated cell fluid;

[0012] S102, culturing the resuscitated cell fluid in a CD-CHO subculture medium for N-5 to N-4 generations, performing a first cell expansion, and obtaining a cell fluid after the first cell expansion;

[0013] S103, culturing the cell fluid after the first cell expansion in a CD-CHO subculture medium for N-3 generations, and performing a second cell expansion to obtain a cell fluid after the second cell expansion;

[0014] S104, culturing the cell fluid after the second cell expansion in Actipro subculture medium for N-2 generations, and performing a third cell expansion to obtain a cell fluid after the third cell expansion;

[0015] S105, culturing the cell fluid after the third cell expansion in Actipro subculture medium for N-1 generation, and performing the fourth cell expansion to obtain cell culture fluid.

[0016] In some embodiments, in step S101, the cell density of the cell solution after resuscitation is (0.90-2.80)×10 6 cells / mL, and / or;

[0017] The cell viability of the cell fluid after resuscitation is greater than or equal to 85%.

[0018] In some embodiments, the cell density of the cell solution after the first cell expansion is (1.70-5.00)×10 6 cells / mL, and / or;

[0019] The cell viability of the cell fluid after the first cell expansion is greater than or equal to 90%, and / or;

[0020] The cell density of the cell fluid after the second cell expansion is (1.70-5.00)×10 6 cells / ml, and / or;

[0021] The cell viability of the cell fluid after the second cell expansion is greater than or equal to 90%, and / or;

[0022] The cell density of the cell fluid after the third cell expansion is (1.70-5.00)×10 6 cells / ml, and / or;

[0023] The cell viability of the cell fluid after the third cell expansion is greater than or equal to 90%, and / or;

[0024] The cell density of the cell fluid after the fourth cell expansion was (3.00-6.50)×10 6 cells / ml, and / or;

[0025] After the fourth cell expansion, the cell viability of the cell fluid is greater than or equal to 90%.

[0026] In some embodiments, in step S10, the perfusion culture step includes: selecting cells after cell expansion as initial cells and inoculating them in a culture medium for culture at 36-37° C., and adding different culture media for multiple times during the culture process until the cell density reaches (35.0-50.0)×10 6 cells / ml, lower the temperature to 33-34°C and continue culturing to obtain cell culture fluid.

[0027] In some embodiments, the cells after cell expansion are selected as initial cells, and the cell density of the initial cells is (0.70-1.30)×10 6 cells / ml.

[0028] In some embodiments, in step S20: the harvesting includes ATF harvesting.

[0029] In some embodiments, in step S20, the purification step includes: performing affinity chromatography on the supernatant after harvesting to capture the product to obtain a captured product; and inactivating the captured product and removing impurities multiple times to obtain a purified intermediate product.

[0030] In some embodiments, in step S30, the auxiliary materials are L-histidine, glacial acetic acid, sucrose and polysorbate 80.

[0031] The present invention also provides an Omopubai single antigen liquid, which is produced by any of the above methods for producing the Omopubai single antigen liquid.

[0032] The production method of the Omopoulin single antigen liquid of the present invention is produced through a continuous flow process, with high cell density, better maintenance of cell viability, and a long production cycle, thereby increasing yield; perfusion culture provides a better microenvironment for protein expression, and the expressed target protein can be harvested in a timely manner with better quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0034] Figure 1 This is a schematic diagram of the preparation process of Example 1 provided by the present invention.

[0035] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0036] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0037] At present, in the production of stock solution, for example, the production process of Omopoulin monoantigen solution involves complex cell culture and purification processes. Although this type of process is quite mature, there are still some potential disadvantages and problems, including the following: high production cost, complex purification steps, complex process, easy mutation, and long-term cell culture process, cells may mutate or gene expression changes, leading to unstable product quality. At the same time, the production is limited and the long-term storage of Omopoulin monoantigen solution may be affected by temperature and excipients, and low-temperature storage may be required, increasing the cost and complexity of transportation and storage.

[0038] In general, due to the complexity and high requirements of the production process of Omopubai single antigen liquid, there are still potential challenges in production costs, product consistency, quality control, etc.

[0039] In view of this, the present invention proposes a method for producing an Omopoulin monoantigen solution, aiming to solve the problems in the production process of the Omopoulin monoantigen solution in the prior art due to its complexity and high requirements, production cost, product consistency, quality control and other aspects.

[0040] The present invention provides a method for producing an Omopoulin single antigen solution, comprising the following steps:

[0041] S10, providing cells, and performing cell recovery, cell expansion, and perfusion culture on the cells to obtain a cell culture fluid;

[0042] S20, harvesting and purifying the cell culture fluid to obtain a purified intermediate product;

[0043] S30, mixing the purified intermediate product with auxiliary materials to obtain Omopoulin single antigen liquid.

[0044] The production method of the Omopoulin single antigen liquid of the present invention is produced through a continuous flow process, with high cell density, better maintenance of cell viability, and a long production cycle, thereby higher yield. The perfusion culture provides a better microenvironment for protein expression, and the expressed target protein can be harvested in time with better quality.

[0045] It should be noted that Omopubai monoclonal antibody solution is a monoclonal antibody drug concentrate used for treatment. It is usually a high-concentration protein solution obtained after the monoclonal antibody is purified. Omopubai monoclonal antibody solution is a monoclonal antibody targeting a specific disease. It is highly specific and can recognize and bind to certain targets in the body (such as specific proteins or cells) to achieve a therapeutic effect.

[0046] Further, in step S10, the cells provided in step S10 can be any cell from the WCB cell bank, preferably CHO-K1 cells. In order to obtain a high-yield Omopoulin single antigen solution, the production method of the present invention includes a continuous flow process, especially in the cell culture part and the harvesting step. This process refers to a continuous production process through continuous material input and product output, which is different from the traditional batch process. For example: perfusion culture is a typical continuous flow process, in which the nutrient solution continuously flows into the culture container, waste and metabolic products are removed at the same time, and the cells continue to proliferate, and the product is also continuously produced and harvested.

[0047] Further, in some embodiments of the present invention, in step S10, the cell amplification step includes:

[0048] S101, providing cells, resuscitating the cells, and obtaining resuscitated cell fluid;

[0049] S102, culturing the resuscitated cell fluid in a CD-CHO subculture medium for N-5 to N-4 generations, performing a first cell expansion, and obtaining a cell fluid after the first cell expansion;

[0050] S103, culturing the cell fluid after the first cell expansion in a CD-CHO subculture medium for N-3 generations, and performing a second cell expansion to obtain a cell fluid after the second cell expansion;

[0051] S104, culturing the cell fluid after the second cell expansion in Actipro subculture medium for N-2 generations, and performing a third cell expansion to obtain a cell fluid after the third cell expansion;

[0052] S105, culturing the cell fluid after the third cell expansion in Actipro subculture medium for N-1 generation, and performing the fourth cell expansion to obtain cell culture fluid.

[0053] In this step, the expansion is a step-by-step expansion, and the number of cells is gradually expanded from N-5 to N-1 through multiple generations of subculture, thereby reducing the stress response caused by sudden large-scale expansion. This subculture helps to stabilize the cell growth curve and avoid over-culture or uneven cell populations. At the same time, through the gradual use of different culture media, such as CD-CHO culture medium and Actipro culture medium, this transitional culture medium better prepares for subsequent high-density culture and perfusion culture, optimizes the ability of cells to adapt to environmental changes, thereby improving the efficiency and quality of cell production of target products, and reducing cell stress response and batch differences.

[0054] In some embodiments of the present invention, in step S101, the cell density of the cell fluid after resuscitation is (0.90-2.80)×10 6 cells / mL, and / or;

[0055] The cell viability of the cell fluid after resuscitation is greater than or equal to 85%.

[0056] The cell density of the cell fluid after the first cell expansion is (1.70~5.00)×10 6 cells / mL, and / or;

[0057] The cell viability of the cell fluid after the first cell expansion is greater than or equal to 90%, and / or;

[0058] The cell density of the cell fluid after the second cell expansion is (1.70-5.00)×10 6 cells / ml, and / or;

[0059] The cell viability of the cell fluid after the second cell expansion is greater than or equal to 90%, and / or;

[0060] The cell density of the cell fluid after the third cell expansion is (1.70-5.00)×10 6 cells / ml, and / or;

[0061] The cell viability of the cell fluid after the third cell expansion is greater than or equal to 90%, and / or;

[0062] The cell density of the cell fluid after the fourth cell expansion was (3.00~6.50)×10 6 cells / ml, and / or;

[0063] After the fourth cell expansion, the cell viability of the cell fluid is greater than or equal to 90%.

[0064] It should be noted that high cell viability (≥90%) is the key to ensuring healthy cell growth and efficient production of target products. Low-viability cells can lead to metabolic disorders and product contamination (such as increased cell debris and metabolic waste), which ultimately affects the yield and quality of target proteins (such as monoclonal antibodies). Therefore, maintaining high cell viability is to ensure that cells are in the best production state.

[0065] The present invention gradually increases the cell density to ensure that there are sufficient cells for efficient production in the subsequent high-density culture stage. In particular, in the fourth expansion, the cell density reaches (3.00-6.50)×10 6 cells / ml, indicating that they are fully prepared for subsequent large-scale culture. This step-by-step expansion helps reduce stress responses and ensures that cells remain highly active as the density gradually increases.

[0066] In some embodiments of the present invention, in step S10, the perfusion culture step includes: the perfusion culture step includes: inoculating the cells after cell expansion as initial cells in the culture medium for culture at 36-37°C, and adding different culture media for multiple times during the culture process until the cell density reaches (35.0-50.0)×10 6 cells / ml, lower the temperature to 33-34°C and continue culturing to obtain cell culture fluid.

[0067] Furthermore, the cells after cell expansion are selected as initial cells, and the cell density of the initial cells is (0.70-1.30)×10 6 cells / ml.

[0068] In this step, by gradually expanding the cells and controlling the initial cell density, different culture media were added multiple times to maintain nutrient supply, promote the cell density to reach the target range, and optimize cell metabolism by cooling, thereby extending the culture time and increasing the yield.

[0069] Specifically, the operation steps of S10 include the following:

[0070] 1. Cell recovery: Take a cell from the WCB cell bank, thaw it in a 37.0℃ water bath, and then transfer the seed solution into CD CHO subculture medium for culture. The cell recovery density is (0.20-0.50)×106cells / mL. After 2 days of shaking flask culture, the cell density reaches (0.90-2.80)×106cells / ml and the cell viability is greater than or equal to 85%, then subculture can be carried out.

[0071] 2. Cell expansion (shake flask, N-5 to N-4 generations): The cells were cultured in CD CHO culture medium for N-5 to N-4 generations. When cultured to N-5 generations, the cells were cultured at (0.10-0.40)×10 6 The cells were inoculated at a density of (1.50-5.00)×10 cells / ml and cultured for 3 days. 6 The N-4 generation can be inoculated at a density of (0.10-0.40)×106 cells / ml and cultured for 3 days until the cell density reaches (1.70-5.00)×10 6 cells / mL can be subcultured again. The cell viability during subculture is greater than or equal to 90%.

[0072] 3. Cell expansion (wave reactor, N-3 generation): The cells were cultured in CD CHO culture medium for N-3 generation. When cultured to N-3 generation, (0.10-0.40)×10 6 The cells were inoculated at a density of (1.70-5.00)×10 cells / mL and grown for about 3 days to reach a density of (1.70-5.00)×10 6 When the cell viability is greater than or equal to 90%, the N-2 generation is inoculated.

[0073] 4. Cell expansion (disposable reactor, N-2 generation): The cells were cultured in Actipro subculture medium for N-2 generation. When cultured to N-2 generation, (0.15-0.50)×10 6 The cells were inoculated at a density of (1.70-5.00)×10 cells / mL and grown for about 3 days to reach a density of (1.70-5.00)×10 6 When the cell viability is greater than or equal to 90%, the N-1 generation is inoculated.

[0074] 5. Cell expansion (disposable reactor, N-1 generation): The cells were cultured in Actipro subculture medium for N-1 generation. When cultured to N-1 generation, (0.20-0.50)×10 6 cells / mL. After 3 days of culture, when the cell density increased to (3.00-6.50)×10 6When the cell viability is greater than or equal to 90%, the 1000L reactor can be inoculated for subsequent perfusion culture.

[0075] 6. Disposable bioreactor perfusion culture: initial inoculation density (0.70-1.30) × 10 6 cells / mlThe cells were inoculated in Actipro perfusion medium, and the initial culture temperature was (36.5±0.5)℃. During the culture process, samples were taken every day to detect cell growth and nutrient consumption. Actipro medium was perfused from the first day, CellBoost 7a medium was added from the third day, and Cell Boost 7b medium was added from the fourth day. When the glucose concentration was lower than 2.00g / L, 400g / kg glucose solution was added to increase the glucose concentration to 3.00g / L. The 1000L bioreactor was connected to an ATF device to retain cells in the tank and pump waste liquid and harvest liquid out to the storage bag. The working volume of the bioreactor was maintained at 600±50kg. Alkali solution (1M sodium carbonate solution) and defoaming agent were added according to cell culture requirements. When the cell density reached (35.0-50.0)×10 6 cells / ml, continue to cool down to (33.0±0.5)℃ and continue culturing to obtain cell culture medium.

[0076] In some embodiments of the present invention, in step S20: harvesting includes ATF harvesting.

[0077] It should be noted that ATF is a continuous flow cell retention technology used to separate antibodies from cells and waste in the culture medium. The culture medium continuously flows into the cell retention system, and the product and waste are continuously discharged after separation. The continuous flow culture of the present invention uses the ATF cell retention system, which can achieve continuous high expression harvesting, and the supernatant after harvest can directly enter the downstream purification step without additional treatment.

[0078] In some embodiments of the present invention, in step S20, the purification step includes: performing affinity chromatography on the supernatant after harvesting the cell culture fluid to capture the product to obtain a captured product; and inactivating the captured product and removing impurities multiple times to obtain a purified intermediate product.

[0079] It should be noted that the role of affinity chromatography is to directly capture antibodies, using fillers with Protein A as the ligand, and the collection peak range is 25mAU / mm-main peak-25mAU / mm; the role of low pH virus inactivation and neutralization is to inactivate viruses under low pH conditions, and the steps of multiple impurity removal include intermediate product deep filtration, anion exchange chromatography, cation exchange chromatography, virus removal filtration, ultrafiltration and diafiltration, among which the specific impurities removed are as follows:

[0080] The purpose of deep filtration of intermediate products is to remove impurities;

[0081] Anion exchange chromatography: This step is used to further remove impurities. Anion exchange chromatography is performed using anion fillers. The collection peak range is 50mAU / mm-main peak-50m AU / mm;

[0082] Cation exchange chromatography: This step is used to further remove impurities. Cation exchange chromatography is performed using a cationic filler. The peak range is 25m AU / mm – main peak – 100m AU / mm;

[0083] Virus removal filtration: This step is used to remove viruses.

[0084] Ultrafiltration and diafiltration: This step is to replace the protein into the target buffer.

[0085] Specifically, the operation steps of S20 are:

[0086] 1. ATF harvest: discard the supernatant at the end of ATF from day 1 to day 6, collect the supernatant at the end of ATF from day 7, and purify the collected supernatant every day from day 8. Terminate the culture on day 22 or within 24 hours when the cell viability is less than 55%, whichever comes first.

[0087] 2. Affinity chromatography: Cytiva's MabSelect PrismA filler was used. 50mM Tris-acetate buffer was used as the equilibration and post-loading elution buffer. 30mM sodium acetate-acetic acid was used as the elution buffer for preliminary purification. The collection peak range was 50mAU / mm-main peak-50mAU / mm.

[0088] 3. Low pH virus inactivation and neutralization: Adjust the pH value of the eluate containing the target protein to 3.70-3.80 for low pH virus incubation inactivation, incubate at room temperature for more than or equal to 60 minutes, and adjust the pH to 5.4-5.6 after the low pH incubation.

[0089] 4. Deep filtration of intermediate products: Use Millipore's X0HC membrane package to deep filter the intermediate product in the previous step, and 30mM sodium acetate-acetic acid buffer is used as the eluent.

[0090] 5. Anion exchange chromatography: Anion exchange chromatography was performed using TOSOH's TOYOPEARL GigaCap Q-650M filler. 50 mM sodium acetate-acetic acid was used as the equilibration and post-sampling elution buffer, and the target protein flow-through was collected, with the collection peak range of 100 mAU / mm-main peak-100 m AU / mm.

[0091] 6. Cation exchange chromatography: Cytiva Capto S ImpAct filler was used for cation exchange chromatography. 50 mM sodium acetate-acetic acid buffer was used as the elution buffer, and the target protein elution peak was collected in the range of 50 m AU / mm-main peak-250 m AU / mm.

[0092] 7. Virus removal filtration: After pre-filtration, use Planova Bio EX from Asahi Kasei Company for virus removal filtration. After filtration, rinse and collect the combined filtrate.

[0093] 8. Ultrafiltration and diafiltration: Use Millipore's Pellicon series ultrafiltration membrane packs for concentration and buffer exchange. The protein concentration of the buffer recirculation product reaches 40.0g / L-50.0g / L.

[0094] In some embodiments of the present invention, in step S30, the auxiliary materials are L-histidine, glacial acetic acid, sucrose and polysorbate 80.

[0095] Specifically, the operation steps of S30 are: adding the prepared sucrose mother solution to the intermediate product after ultrafiltration concentration and liquid exchange to make the final concentration of sucrose 8% (w / v); then adding the prepared polysorbate 80 mother solution to make the final concentration of polysorbate 80 0.05% (w / v), then adding the prepared histidine-acetate buffer, and adjusting the target protein concentration to 18.0-22.0 g / L. Finally, a polyvinylidene fluoride (PVDF) material (0.22 μm) filter is used for sterilization filtration to obtain the stock solution.

[0096] The present invention also provides an Omopubai single antigen liquid, which is produced by any of the above methods for producing Omopubai single antigen liquid. Since the Omopubai single antigen liquid is produced by the above method for producing Omopubai single antigen liquid, it also includes the beneficial effects of the above method for producing Omopubai single antigen liquid, which will not be repeated here.

[0097] Experimental materials: Actipro medium was purchased from Hyclone or its specific components; Cell Boost7a medium was purchased from Hyclone or its specific components; Cell Boost 7b medium was purchased from Hyclone.

[0098] Example 1

[0099] The present invention provides a method for producing an Omopoulin single antigen solution, comprising the following steps:

[0100] 1. Cell recovery: Take a cell from the WCB cell bank, thaw it in a 37.0℃ water bath, and then transfer the seed solution into CD CHO subculture medium for culture. The cell recovery density is (0.20-0.50)×106cells / mL. After 2 days of shaking flask culture, the cell density reaches (0.90-2.80)×106cells / ml and the cell viability is greater than or equal to 85%, then subculture can be carried out.

[0101] 2. Cell expansion (shake flask, N-5 to N-4 generations): The cells were cultured in CD CHO culture medium for N-5 to N-4 generations. When cultured to N-5 generations, the cells were cultured at (0.10-0.40)×10 6 The cells were inoculated at a density of (1.50-5.00)×10 cells / ml and cultured for 3 days. 6 The N-4 generation can be inoculated at a density of (0.10-0.40)×106 cells / ml and cultured for 3 days until the cell density reaches (1.70-5.00)×10 6 cells / mL can be subcultured again. The cell viability during subculture is greater than or equal to 90%.

[0102] 3. Cell expansion (wave reactor, N-3 generation): The cells were cultured in CD CHO culture medium for N-3 generation. When cultured to N-3 generation, (0.10-0.40)×10 6 The cells were inoculated at a density of (1.70-5.00)×10 cells / mL and grown for about 3 days to reach a density of (1.70-5.00)×10 6 When the cell viability is greater than or equal to 90%, the N-2 generation is inoculated.

[0103] 4. Cell expansion (disposable reactor, N-2 generation): The cells were cultured in Actipro subculture medium for N-2 generation. When cultured to N-2 generation, (0.15-0.50)×10 6 The cells were inoculated at a density of (1.70-5.00)×10 cells / mL and grown for about 3 days to reach a density of (1.70-5.00)×10 6 When the cell viability is greater than or equal to 90%, the N-1 generation is inoculated.

[0104] 5. Cell expansion (disposable reactor, N-1 generation): The cells were cultured in Actipro subculture medium for N-1 generation. When cultured to N-1 generation, (0.20-0.50)×10 6 cells / mL. After 3 days of culture, when the cell density increased to (3.00-6.50)×10 6When the cell viability is greater than or equal to 90%, the 1000L reactor can be inoculated for subsequent perfusion culture.

[0105] 6. Disposable bioreactor perfusion culture: initial inoculation density (0.70-1.30) × 10 6 cells / mlThe cells were inoculated in Actipro perfusion medium, and the initial culture temperature was (36.5±0.5)℃. During the culture process, samples were taken every day to detect cell growth and nutrient consumption. Actipro medium was perfused from the first day, CellBoost 7a medium was added from the third day, and Cell Boost 7b medium was added from the fourth day. When the glucose concentration was lower than 2.00g / L, 400g / Kg glucose solution was added to increase the glucose concentration to 3.00g / L. The 1000L bioreactor was connected to an ATF device to retain cells in the tank and pump waste liquid and harvest liquid out to the storage bag. The working volume of the bioreactor was maintained at 600±50kg. Alkali solution (1M sodium carbonate solution) and defoaming agent were added according to cell culture requirements. When the cell density reached (35.0-50.0)×10 6 cells / ml, continue to cool down to (33.0±0.5)℃ and continue culturing to obtain cell culture medium.

[0106] 7. ATF harvest: discard the supernatant at the end of ATF from day 1 to day 6, collect the supernatant at the end of ATF from day 7, and purify the collected supernatant every day from day 8. Terminate the culture on day 22 or within 24 hours when the cell viability is less than 55%, whichever comes first.

[0107] 8. Affinity chromatography: Cytiva's MabSelect PrismA filler was used. 50mM Tris-acetate buffer was used as the equilibration and post-loading elution buffer. 30mM sodium acetate-acetic acid was used as the elution buffer for preliminary purification. The collection peak range was 50mAU / mm-main peak-50mAU / mm.

[0108] 9. Low pH virus inactivation and neutralization: Adjust the pH value of the eluate containing the target protein to 3.70-3.80 for low pH virus incubation inactivation, incubate at room temperature for more than or equal to 60 minutes, and adjust the pH to 5.4-5.6 after the low pH incubation.

[0109] 10. Deep filtration of intermediate products: Use Millipore's X0HC membrane package to deep filter the intermediate product in the previous step, and 30mM sodium acetate-acetic acid buffer is used as the eluent.

[0110] 11. Anion exchange chromatography: Anion exchange chromatography was performed using TOYOPEARL GigaCap Q-650M filler from TOSOH. 50 mM sodium acetate-acetic acid was used as the equilibration and post-loading elution buffer, and the target protein flow-through was collected, with the collection peak range being 100 mAU / mm-main peak-100 m AU / mm.

[0111] 12. Cation exchange chromatography: Cytiva Capto S ImpAct filler was used for cation exchange chromatography. 50 mM sodium acetate-acetic acid buffer was used as the elution buffer, and the target protein elution peak was collected in the range of 50 m AU / mm-main peak-250 m AU / mm.

[0112] 13. Virus removal filtration: After pre-filtration, use Planova Bio EX from Asahi Kasei for virus removal filtration. After filtration, rinse. Collect and combine the filtrate.

[0113] 14. Ultrafiltration and diafiltration: Use Millipore's Pellicon series ultrafiltration membrane packs for concentration and buffer exchange. The protein concentration of the buffer recirculation product reaches 40.0g / L-50.0g / L.

[0114] 15. Preparation and packaging of stock solution: Add the prepared sucrose mother solution to the intermediate product after ultrafiltration concentration and liquid exchange, so that the final concentration of sucrose is 8% (w / v); then add the prepared polysorbate 80 mother solution, so that the final concentration of polysorbate 80 is 0.05% (w / v). Then add the prepared histidine-acetate buffer and adjust the target protein concentration to 18.0-22.0 g / L. Finally, use a polyvinylidene fluoride (PVDF) filter (0.22 μm) to sterilize and filter to obtain the stock solution.

[0115] Comparative Example 1

[0116] Different from Example 1, the "continuous flow culture" step used in Example 1 was replaced by "fed batch culture", wherein the specific operation steps of the fed batch culture were as follows: cell recovery and expansion, inoculation into a disposable bioreactor, supplementation of Cell Boost 7a and Cell Boost 7b on days 3, 5, 7, 9, and 11, initial culture temperature 36.5°C, when the cell density reached (10.0-12.0)×10 6 cells / ml, the temperature is lowered to (33.0±0.5)℃ and culture is continued until the 14th day or the viability is below 60%, and the target protein is harvested at one time. The harvested supernatant is subjected to deep filtration, affinity chromatography, low pH virus inactivation, anion chromatography, cation chromatography, virus removal filtration, ultrafiltration and diafiltration, and finally replaced into the auxiliary material prescription to obtain the stock solution.

[0117] Test results:

[0118] The production method of Example 1 was adopted for production, and the expression amount and product plasmid were detected. The detection items included Titer, SEC-LC, CENR, CER and CIEF. Titer (titer) usually indicates the mass of the target protein produced per liter of culture medium, reflecting the concentration and production efficiency of the product; SEC-LC refers to the analysis by size exclusion chromatography (SEC), which determines the polymerization state of the protein and the ratio of its monomers to evaluate the polymerization state and monomer ratio of the protein; CENR (capillary electrophoresis-non-reducing conditions) measures the purity and integrity of the protein without destroying its disulfide bonds; and CER% (capillary electrophoresis-reducing conditions) measures the purity of the protein under a reducing environment (such as after using a reducing agent to destroy the disulfide bonds), both of which are used to evaluate the purity and integrity of the protein under different conditions; CIEF (capillary isoelectric focusing) is a technology used to analyze the isoelectric point (pI) of the protein. The ratio of different charge isomers (isoforms) separated by the electric field shows the charge isomer distribution of the protein, reflecting the possible post-translational modification. The test results are shown in Table 1:

[0119] Table 1

[0120]

[0121] It should be noted that the test results in Table 1 show that the quality of the Omopobai single antigen solution prepared by the present invention is better, because the embodiment pumps fresh culture medium every day, removes the waste culture medium, maintains a better cell state, and produces a better quality of target protein. Among them, the purity of the main peaks of SEC and CIEF is equivalent, the purity of the main peaks of CE NR and CE R is slightly better, and the acidic peak of CIEF is 10.8% vs 20.5%, which is better than batch culture. Moreover, the yield of the embodiment is higher, and the yield is increased by about 8 times, because the continuous flow process has a high cell density and a long production cycle, so the yield is higher.

[0122] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for producing an Omopoulin single antigen solution, characterized in that: The production method of the Omopubai single antigen liquid comprises the following steps: S10, providing cells, and performing cell recovery, cell expansion, and perfusion culture on the cells to obtain a cell culture fluid; S20, harvesting and purifying the cell culture fluid to obtain a purified intermediate product; S30, mixing the purified intermediate product with auxiliary materials to obtain Omopoulin single antigen liquid.

2. The method for producing the single antigen liquid of Omopoulin as claimed in claim 1, characterized in that: In step S10, the cell amplification step includes: S101, providing cells, resuscitating the cells, and obtaining resuscitated cell fluid; S102, culturing the resuscitated cell fluid in a CD-CHO subculture medium for N-5 to N-4 generations, performing a first cell expansion, and obtaining a cell fluid after the first cell expansion; S103, culturing the cell fluid after the first cell expansion in a CD-CHO subculture medium for N-3 generations, and performing a second cell expansion to obtain a cell fluid after the second cell expansion; S104, culturing the cell fluid after the second cell expansion in Actipro subculture medium for N-2 generations, and performing a third cell expansion to obtain a cell fluid after the third cell expansion; S105, culturing the cell fluid after the third cell expansion in Actipro subculture medium for N-1 generation, and performing the fourth cell expansion to obtain cell culture fluid.

3. The method for producing the single antigen liquid of Omopoulin as claimed in claim 2, characterized in that: In step S101, the cell density of the cell solution after resuscitation is (0.90-2.80)×10 6 cells / mL, and / or; The cell viability of the cell fluid after resuscitation is greater than or equal to 85%.

4. The method for producing the single antigen liquid of Omopoulin as claimed in claim 2, characterized in that: The cell density of the cell fluid after the first cell expansion is (1.70~5.00)×10 6 cells / mL, and / or; The cell viability of the cell fluid after the first cell expansion is greater than or equal to 90%, and / or; The cell density of the cell fluid after the second cell expansion is (1.70-5.00)×10 6 cells / ml, and / or; The cell viability of the cell fluid after the second cell expansion is greater than or equal to 90%, and / or; The cell density of the cell fluid after the third cell expansion is (1.70-5.00)×10 6 cells / ml, and / or; The cell viability of the cell fluid after the third cell expansion is greater than or equal to 90%, and / or; The cell density of the cell fluid after the fourth cell expansion was (3.00-6.50)×10 6 cells / ml, and / or; After the fourth cell expansion, the cell viability of the cell fluid is greater than or equal to 90%.

5. The method for producing the Omopoulin single antigen solution according to claim 1, characterized in that: In step S10, the perfusion culture step includes: selecting cells after cell expansion as initial cells and inoculating them in a culture medium for culture at 36-37°C, and adding different culture media for multiple times during the culture process until the cell density reaches (35.0-50.0)×10 6 cells / ml, lower the temperature to 33-34°C and continue culturing to obtain cell culture fluid.

6. The method for producing the Omopoulin single antigen solution according to claim 5, characterized in that: The cells after cell expansion are selected as initial cells, and the cell density of the initial cells is (0.70-1.30)×10 6 cells / ml.

7. The method for producing the Omopoulin single antigen solution according to claim 1, characterized in that: In step S20: the harvesting includes ATF harvesting.

8. The method for producing the Omopoulin single antigen solution according to claim 1, characterized in that: In step S20, the purification step includes: performing affinity chromatography on the harvested supernatant to capture the product to obtain the captured product; and inactivating the captured product and removing impurities multiple times to obtain a purified intermediate product.

9. The method for producing the Omopoulin single antigen solution according to claim 1, characterized in that: In step S30, the auxiliary materials are L-histidine, glacial acetic acid, sucrose and polysorbate 80.

10. An Omopoulin single antigen solution, characterized in that The Omopoulin single antigen liquid is produced by the production method of the Omopoulin single antigen liquid according to any one of claims 1 to 9.

Images