High density continuous cell culture method and its applications

Abstract

The present invention discloses a high-density continuous inoculation cell culture method and its applications. The method includes the steps of (1) providing a cell culture and subjecting the cell culture to resuscitation, expansion culture in shake flasks, and expansion culture in swing reaction bags; (2) transferring the resuscitated and expanded cells to a final-stage cell expansion tank and continuing the expansion culture; (3) inoculating the cells in the final-stage cell expansion tank into a culture fermenter using high-density continuous inoculation and conducting fermentation culture; and (4) harvesting the target product. The high-density continuous inoculation cell culture method of the present invention can reduce the cell passage time per batch and improve the production efficiency of cells and their expressed products.

Description

[Technical field]

[0001] Priority and Related Applications This application claims priority to Chinese Patent Application No. 201910641684.6, entitled “High-density continuous inoculation cell culture method and its application,” filed on July 16, 2019, the entire contents of which, including the appendix, are incorporated herein by reference.

[0002] The present invention relates to the technical field of mammalian cell culture, specifically to a high-density continuous seeding cell culture method and its applications. [Background technology]

[0003] After decades of development, cell culture technology has become a mature technique for producing many important proteins, especially complex macromolecular proteins, which are often used for the prevention and treatment of serious human diseases, such as cancer, viral infections, genetic defects and other chronic diseases.

[0004] At present, there are three main in vitro cell culture modes: batch culture, fed-batch culture, and continuous culture (perfusion). Among them, batch culture is a culture method in which a certain amount of culture medium is put into a closed reactor, and then a microbial strain is inoculated and cultured. Fed-batch culture is a culture method in which a certain amount of culture medium is first put into a reactor, cells are inoculated under appropriate conditions, and the cells are cultured so that they grow continuously and products are formed continuously. In this process, nutrients are constantly consumed, and new nutrients are constantly replenished into the system so that the cells can further grow and metabolize until the product is taken out after the entire culture is completed. Fed-batch culture is characterized by being able to adjust the concentration of nutrients in the culture environment, and by adding fresh culture medium during the culture process, the reaction volume of the entire process changes. Compared with batch culture and fed-batch culture, continuous culture (also called open culture, perfusion culture) has been attracting more and more attention and becoming more popular due to its unique advantages such as sustainability in production, high cell density obtained by culture, and good uniformity of the final product.

[0005] Continuous culture has recently become the recommended culture method for culturing mammalian cells to produce secreted recombinant therapeutics, such as genetically engineered antibodies, including monoclonal antibodies, chimeric antibodies, and humanized antibodies. Continuous culture is a method in which cells are placed in a reactor together with a culture medium, and then during the process of cell growth and product formation, a portion of the conditioned medium is continuously removed while fresh medium is continuously perfused. Its greatest advantages are: (1) the cells can exist in a nutrient-rich culture environment with a low concentration of harmful metabolic wastes; and (2) the cell density is high, typically 10 7 ~10 8 (3) the density of cells cultured by this method is high and their survival time is long, resulting in a high recovery rate of the target product; and (4) the residence time of the target product in the culture fermenter is short, allowing it to be immediately recovered and stored at low temperature, which helps maintain the activity of the target product.

[0006] Continuous and stepwise cultivation using shake flasks and reactors is a typical cell amplification process. When the cell culture volume and cell density reach the desired level, the cells are inoculated into a culture fermenter for cultivation and expression. In the conventional process (see Figure 1), the cells generally require about 19 days (D0-D19) from the start of resuscitation to inoculation into the culture fermenter, and the production time is about 15 days (i.e., the production time in the culture fermenter is about 15 days), so the total cell culture time is about 34 days. Prior to producing a large amount of the target protein, the cells need to reach a suitable density in the culture fermenter for about 7 days. Therefore, the first 7 days are called the growth period and the last 8 days are called the expression period. Inoculating the culture fermenter with an inoculation density 10 times higher than the normal inoculation density can significantly shorten the growth period and allow the cells to reach the yield of the previous 15 days in a shorter time, and this technique is called high density inoculation. However, currently, high-density seeding technology has not been widely applied in the biopharmaceutical field, and the most important reason is that the traditional end-stage cell amplification tank (also called N-1 seed tank) cannot provide such a large amount of cells.

[0007] CN108641960A discloses a multi-purpose bioreactor including a culture tank, an agitation mechanism, an aeration mechanism, a feeding mechanism, a harvesting device and an exhaust device, which can meet the needs of various cells by replacing the relevant module assemblies in the bioreactor according to the cultured cells, and can be widely applied to large-scale high-density cell culture and expression of target products in the biopharmaceutical industry.CN108949558A discloses a drum-type hollow fiber reactor used for high-density culture of plant cells, which has excellent mixing properties, low shear force and energy saving during cell culture, and the reactor can greatly reduce the tight aggregation of cells and improve their dissolved oxygen and improve the mass transfer permeability of the membrane by driving the fluid flow in the chamber by the rotation of the drum, which is conducive to cell growth and metabolism and is favorable for high-density cell culture. CN109576212A discloses a method for culturing seed cells in high-density inoculation culture, which increases the density of viable cells by fed-batch culture after N-1 seed culture and subsequent amplification culture. In the fed-batch culture method described in this document, the total amount of material is constantly increasing, the metabolic products and target proteins expressed by cells are constantly accumulating, and the cell debris is also constantly increasing, making it difficult to maintain the optimal state of the cell culture environment, and therefore it is difficult to improve the density of the seed medium.

[0008] In large-scale production conditions (usually larger than 1000L), a certain amount of cells is required as the starting "seed" in the culture fermenter, and when the cell culture volume and cell density reach a certain standard, the cells are transferred to the bioreactor to continue growing and express the product. However, this part of the cells must be progressively amplified in multiple steps to reach the required density requirement. In the conventional process, the amplification starts with cell resuscitation, and then the cells are amplified by continuous subculture, and the culture devices selected include shake flasks, WAVE wave bioreactors, and stirred reactors, etc. When the conventional process is selected for cell culture, the cell amplification step is time-consuming and inefficient, and as the future market demand for bioproducts increases, it is urgent to establish a new amplification process that is efficient and time-saving. Summary of the Invention [Problem to be solved by the invention]

[0009] Regarding the problems existing in the prior art, the present application provides a high-density continuous inoculation cell culture method and its application, which can reduce the cell passage time per batch and improve the production efficiency of cells and their expression products. [Means for solving the problem]

[0010] In view of the problems existing in the prior art described above, the present inventors have conducted extensive research and repeated testing, and have optimized the cell amplification process to shorten the passage time per batch of cells, thereby completing the present invention. That is, the present invention is as follows.

[0011] The first aspect of the present invention is (1) providing a cell culture, and subjecting the cell culture to resuscitation, amplification culture using a shake flask, and amplification culture using a swing reaction bag; (2) transferring the resuscitated and amplified cells to a final stage cell amplification tank and continuing the amplification culture; (3) inoculating the cells in the final cell amplification tank into a culture fermentation tank by a high-density continuous inoculation method, and carrying out fermentation culture; (4) harvesting the target product. A method for cell culture with high density continuous seeding is provided.

[0012] In the step (2), a filtration device is provided in the cell amplification tank in the final stage, and the cell density in the cell amplification tank in the final stage is reduced to 10 7 The non-cellular material in the medium can be replaced with fresh medium such that the number of cells / mL is greater than 1, preferably the filtration device is an ATF, Spin filter or TFF.

[0013] Furthermore, in step (3), the high-density continuous inoculation is 10 7 In a specific embodiment of the present invention, in step (3), the high-density continuous inoculation is to inoculate the cells into the culture fermenter at a density of more than 10 cells / mL. 7 The method involves inoculating the culture fermenter at a density greater than 10000 cells / mL, replenishing the final stage cell amplification tank with the same volume of fresh medium after inoculation is completed so that another new culture fermenter can be continued the day after inoculation, and repeating the above-mentioned operations of inoculating the culture fermenter and replenishing the final stage cell amplification tank with fresh medium.

[0014] Specifically, the subculture process in the initial stage of the high-density continuous inoculation cell culture method according to the present invention is consistent with the conventional method. However, after culturing the cells in the N-1 seed tank for 3 days, at most half of the volume of the cell culture solution is taken and inoculated into the culture fermentation tank, and the same volume of fresh medium is supplemented. Since the doubling time of CHO cells is about 24 hours, according to the method of the present invention, by the next day, seed cells with a density close to that for inoculating the new culture fermentation tank can be obtained, and so on. According to the maximum culture days (B) defined by the cell stability data, the N-1 seed tank can achieve continuous culture and continuous inoculation within a certain period (<B), and can greatly save time and cost. The high-density continuous inoculation cell culture method according to the present invention may first increase the seed density of the N-1 seed tank by means of a perfusion culture process and then inoculate.

[0015] The second aspect of the present invention is an improvement of the high-density continuous inoculation cell culture method of the first aspect, that is, based on the first aspect, the cell culture described in step (1) is resuscitated in two batches, amplified by a shaking flask, and amplified by a swing reaction bag, and then another final-stage cell amplification tank is added in step (2).

[0016] Specifically, in order to further solve the defect that the maximum culture days of the N-1 seed tank in the technical solution according to the first aspect of the present invention are limited due to insufficient cell stability, after one batch of cells is resuscitated for C days (C is smaller than the maximum days defined by the subculture stability), the second batch of cells is resuscitated, and if the cells are cultured in the N-1 seed tank for C days by the culture method according to the first aspect of the present invention, they can be taken out of the tank. At this time, the preparation of the second batch of seeds in the N-1 seed tank is completed, and the inoculation of the culture fermentation tank can be continued. The culture method may first increase the seed density of the N-1 seed tank by means of a perfusion culture process and then inoculate.

[0017] The high-density continuous inoculation cell culture methods according to the first and second aspects of the present invention are characterized in that the volume of the final-stage cell amplification tank described in step (2) is 2L to 1000L.

[0018] In a specific embodiment, the heating type of the reactor tank body used in the 2L-10L stage of the high-density continuous inoculation cell culture method according to the present invention is heating by a glass tank heating pad, and the bottom vent end is an annular vent or a direct microvent; in the 250L and 1000L stages, heating by a water-filled jacket of the stainless steel tank body is used, and the culture liquid is filled into a disposable culture bag without contacting the stainless steel tank. The reactor tanks of each specification are all commercially available third-party products. When the high-density continuous inoculation cell culture method according to the present invention is applied to actual production in a factory, a culture vessel with a volume of 1000L or more may be used, such as a disposable reaction bag with a volume of 1000L or a stainless steel reactor with a volume of 3000L or more.

[0019] A third aspect of the present invention provides an application of said high density continuous seeding cell culture method in mammalian cell culture.

[0020] Wherein, the mammalian cell is selected from the group consisting of CHO, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, T cell line, B cell line, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, HT1080, L929, hybridoma and cancer cell line.Preferably, the mammalian cell is CHO-S cell line of Chinese hamster ovary cells or CHO-K1 cell of GS-deficient expression system.

[0021] In a specific embodiment, said mammalian cell comprises a nucleotide sequence encoding a heterologous protein, preferably said heterologous protein is an antibody, more preferably said antibody is a monoclonal antibody or a bispecific antibody. Effect of the Invention

[0022] As can be seen from the test results of the present invention, compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0023] 1. Based on the conventional process, the present invention improves the cell amplification method in the cell amplification tank, and in particular optimizes the cell amplification method in the final stage cell amplification tank (i.e., N-1 seed tank). By using continuous culture technology, the N-1 seed tank is made dense enough to obtain sufficient cells, and the culture fermenter is inoculated at high density, thereby shortening the production cycle and reducing costs. Conventional cell inoculation has a low density (usually 10 6 Approximately 7 days are required after inoculation to reach the highest density and enter the high yield stage. 7 With this (>3.5 cells / mL) technology, cells can reach high yield density faster, significantly shortening production time and improving production efficiency.

[0024] 2. In the present invention, the N-1 seed tank can continue to inoculate the culture fermenter the next day by replenishing the N-1 seed tank with fresh medium and continuing the culture. The second culture fermenter only has a cell amplification time of one day, which greatly shortens the cell culture cycle compared with the conventional cell culture method (which requires about 19 days from seed resuscitation to inoculation into the culture fermenter). According to the method of the present invention, the N-1 seed tank can be used to inoculate six culture fermenters for six consecutive days, and under the condition that the total yield is the same, a total of 90 days of seed amplification time and 42 days of production time can be saved. In addition, one N-1 seed tank can be used to inoculate the culture fermenter for several consecutive days, so theoretically, the method of the present invention can achieve a production efficiency of one batch per day, and finally achieve a harvest of one batch per day. Therefore, the production efficiency is greatly improved and the cost is reduced.

[0025] 3. The present invention applies a continuous culture method to cell expansion in an N-1 seed tank, and non-cellular materials in the continuous culture environment, such as metabolites, cell debris, and produced proteins, are constantly replaced with fresh medium through a filtration device (e.g., ATF, spin filter, TFF, etc.), thereby maintaining an optimal cell culture environment and improving the density of the seed medium.

[0026] 4. By adding one N-1 seed tank, the high-density continuous inoculation cell culture method of the present invention is also applicable to the cultivation of less stable host cells and the expression of target proteins.

[0027] 5. The reactor tanks of various specifications (2L to 1000L) used in the high-density continuous inoculation cell culture method of the present invention are all commercially available third-party products, and the culture method can be easily carried out in laboratories or production workshops.

[0028] In order that the above and other objects, features and advantages of the present invention may be more clearly and easily understood, preferred embodiments of the present invention will be described in detail below in conjunction with the drawings. [Brief description of the drawings]

[0029] [Figure 1] This is the conventional process for cell culture. [Figure 2-1] FIG. 2-1 is a schematic diagram of the structure of an INGLP bispecific antibody, and FIG. 2-2 is a specific structural diagram of an INGLP bispecific antibody. [Figure 2-2] Same as above. [Diagram 3] FIG. 1 shows graphs of cell viability (VIB) and viable cell density (VCD) over time for seed amplification of CHO cells capable of stably expressing the ADI-31853 monoclonal antibody in a 2L Applikon reactor using an ATF perfusion device. [Figure 4] 1 shows a graph of viable CHO cell density over time stably expressing the ADI-31853 monoclonal antibody, for high density inoculation (20×106 cells / mL, abbreviated as "inoculation 20") and for regular fed-batch inoculation. [Diagram 5] A graph showing the expression level over time of CHO cells expressing the ADI-31853 monoclonal antibody in high-density inoculation (20 x 106 cells / mL, abbreviated as "inoculation 20") and inoculation by conventional fed-batch culture is shown. [Figure 6] FIG. 1 shows graphs of cell viability (VIB) and viable cell density (VCD) over time for seed amplification of CHO seed cells capable of stably expressing INGLP bispecific antibodies in 10 L culture bags using a Sartorius RM20 reactor. [Figure 7] FIG. 1 shows a graph of viable CHO cell density over time stably expressing INGLP bispecific antibody for two different high density inoculations (10×106 cells / mL and 20×106 cells / mL, abbreviated as "10 inoculation" and "20 inoculation", respectively) and for regular fed-batch inoculation. [Figure 8] A graph showing the expression level over time of CHO cells expressing INGLP bispecific antibody with two different high density inoculations (10 x 106 cells / mL and 20 x 106 cells / mL, abbreviated as "10 inoculation" and "20 inoculation", respectively) and with conventional fed-batch inoculation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The technical solution of the present invention will be further described below by specific embodiments. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Moreover, any chapter titles used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter described.

[0031] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. More specifically, as used herein and in the appended claims, the singular forms "one," "one," and "the" include plural referents unless otherwise indicated by context. In this application, the use of "or" means "and / or" unless otherwise indicated. Furthermore, the use of the term "comprises" and other forms (such as "includes," "containing," etc.) is not limiting. Furthermore, the ranges provided in the specification and appended claims include all values ​​between the endpoints and the breakpoints.

[0032] definition "About", "approximately": As used herein, the terms "about" and "approximately", when used with one or more cell culture conditions, refer to a set of values ​​similar to a reference value designated for said culture conditions. In some embodiments, the term "about" refers to a set of values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value designated for said culture conditions.

[0033] As used herein, the term "next day" refers to any time between 24 hours and 48 hours after inoculation.

[0034] "Medium" refers to the liquid environment in direct contact with the cells and dependent on them for survival; it is the collection of various nutrients and buffer systems.

[0035] mammalian cells As used herein, "mammalian cells" refers to cell lines of mammalian origin for the production and preparation of biological drugs, selected and adapted for pharmaceutical industry production according to production conditions, and whose post-translational modifications of expressed proteins are beneficial for maintaining the biological activity, stability, and antigenicity of the proteins. Many cell lines are available from commercial sources, such as the American Type Culture Collection (ATCC). Non-limiting examples of mammalian cells that can be used according to the invention include the group consisting of CHO, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, T cell lines, B cell lines, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, HT1080, L929, hybridoma and cancer cell lines. In at least one embodiment, the mammalian cells are Chinese Hamster Ovary cells CHO-S cell line or CHO-K1 cells of the GS-deficient expression system.

[0036] Heterologous Proteins The term "heterologous protein" as used herein refers to a gene encoding a target protein other than a host cell within the scope of genetic engineering, and a DNA recombinant technology is used to effectively amplify and express the host cell to produce a protein of practical value. The heterologous protein is known in the art and can be obtained from commercial sources or by methods known in the art. In at least one embodiment, the heterologous protein is an Fc fusion protein, an antibody, or an enzyme, and optionally, the antibody is a monoclonal antibody or a bispecific antibody, and the monoclonal antibody may be any monoclonal antibody suitable for heterologous expression by CHO-K1 cells, including, but not limited to, IgG, IgA monoclonal antibodies. The term "bispecific antibody" as used herein refers to an antibody having two antigen-binding sites, each of which binds to a different epitope of the same antigen or a different epitope of different antigens. In one embodiment, the "bispecific antibody" has binding specificity for a first antigen and a second antigen.

[0037] In at least one embodiment, the enzyme refers to a medicinal enzyme, which is an enzyme for use in therapeutic drugs, including, but not limited to, glucosidase, lipase, and the like.

[0038] The host cell used in the embodiment of the present invention is CHO-K1 cell, and the nucleic acid molecule encoding ADI-31853 monoclonal antibody and INGLP bispecific antibody (BsAb) is introduced into the host cell by conventional molecular biology means, and a stable expression cell line is screened. The experimental equipment and experimental reagents used in this embodiment can be obtained from the market, and the specific information is as follows:

[0039] Laboratory equipment [Table 1]

[0040] Experimental Reagents [Table 2]

[0041] Among them, the amino acid sequences of the heavy chain (HC) and light chain (LC) of the ADI-31853 monoclonal antibody (also called anti-LAG3 antibody) are SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0042] The structural diagram of the INGLP bispecific antibody (also called anti-LAG3 / PDL1 antibody) is shown in Figure 2-1, in which the antigen A is LAG-3 and the antigen B is PD-L1, and the anti-LAG-3 monoclonal antibody is connected to the heavy chain variable domain (VHH) of the anti-PD-L1 single domain antibody (sdAb) via a flexible linker (with the sequence GGGGSGGGGS) to form a bispecific antibody (each of which contains peptide chain #1 and peptide chain #2), which is symmetrical on the left and right, and the specific structure of the antibody is shown in Figure 2-2. The amino acid sequences of peptide chain #1 and peptide chain #2 are SEQ ID NO:3 and SEQ ID NO:4, respectively. EXAMPLES

[0043] Example 1 Application of high density seeding cell culture method in the production of ADI-31853 monoclonal antibody

[0044] The antibody was produced by the following method using CHO-K1 cells stably expressing ADI-31853 monoclonal antibody as the test material, and the upstream process of producing this antibody is seed resuscitation, amplification by shake flask, amplification by swing reaction bag, production by N-1 seed tank and production by culture fermenter. Take the production process in a 2L bioreactor as an example.

[0045] First, the seed cells were thawed in a water bath at 37°C and then expanded in a shake flask containing CD CHO medium. The seed resuscitation culture conditions were 36.5°C, 130 rpm, 6% CO2 The shake flask amplification step was performed at 0.5 × 10 6 The cells were seeded at 100 cells / mL and cultured at 36.5°C, 130 rpm, and 6% CO 2 The cells were amplified stepwise every 3 days until the 12th day.

[0046] Through the above procedure, approximately 1.0 × 10 CHO seed cells capable of stably expressing the ADI-31853 monoclonal antibody were obtained. 6 The cells were inoculated into a 2L Ezcontrol reactor from Applikon equipped with an ATF perfusion device (cells were collected at an aperture width of 30 kD) at a cell density of 1.2 L / mL, the culture volume was 1.2 L, and the medium was Dynamis mixed with 5% Feed C+ / 483 medium. The graphs of cell viability (VIB) and viable cell density (VCD) over time in the N-1 seed tank of the CHO seed cells are shown in Figure 3.

[0047] When cultured in the N-1 seed tank on day 8, the viable cell density was 60 × 10 6 When the N-1 seed culture vessel reaches more than 20 × 10 cells / mL, take out sufficient seed cell solution (less than half the volume of the N-1 seed culture vessel) and culture 20 × 10 cells / mL in a 2 L reactor. 6 The cells were inoculated at a density of 10 ...

[0048] To further illustrate the benefits of the high-density inoculation cell culture method described in this invention, the following experiment was also carried out. 6 High density of 1×10 cells / mL and 1×10 cells / mL are used in conventional fed-batch culture. 6 The cells were inoculated at a density of 10000000 cells / mL, and the effects of different initial inoculation densities on cell viability and viable cell density were compared. See Figure 4 for specific test results. Under the above conditions of different inoculation densities, the time-dependent trend of protein expression levels in CHO seed cells was measured. See Figure 5 for specific test results.

[0049] As can be seen from the results in Figures 4 and 5, (1) the ATF perfusion device can enable the CHO seed cells stably expressing the ADI-31853 monoclonal antibody to reach a high cell density, and the cell condition can be well maintained (see Figure 4); (2) after selecting high-density inoculation fed-batch, the expression level of 15 days by normal fed-batch culture was achieved on the 7th to 8th day, and the culture period was shortened (see Figure 5).

[0050] Example 2 Application of high density continuous seeding cell culture method in the production of INGLP bispecific antibodies

[0051] Using CHO-K1 cells stably expressing INGLP bispecific antibody as the test material, the antibody was produced by the following method, and the upstream process of the antibody production includes seed resuscitation, amplification in shake flasks, amplification in swing reaction bags, production in N-1 seed tanks, and production in culture fermenters. Take the production process in a 2L bioreactor as an example.

[0052] First, the seed cells were thawed in a water bath at 37°C and then expanded in a shake flask containing CD CHO medium. The culture conditions for seed resuscitation were 36.5°C, 130 rpm, 6% CO 2 The shake flask amplification step was performed at 0.5 × 10 6 The cells were seeded at 100 cells / mL and cultured at 36.5°C, 130 rpm, and 6% CO 2The cells were amplified stepwise every 3 days until the 12th day.

[0053] A culture bag indicated as 10 L was loaded in the Sartorius RM20 reactor, and a filter membrane (0.2 μm) was provided at the bottom of the culture bag to filter the culture solution and collect the cells. The medium was Dynamis mixed with 5% Feed C+ / 483 culture solution. The density of CHO seed cells to be inoculated into the N-1 seed tank was approximately 80 × 10 6 The CHO seed cells were cultured to a concentration of 1000 cells / mL. A graph of the cell viability (VIB) and viable cell density (VCD) over time in the N-1 seed tank is shown in FIG.

[0054] As can be seen from the viable cell density curve in Figure 6, on the 8th day of cell culture, the viable cell density in the N-1 seed tank was 79.37 × 10 6 At this time, 5 L of cell culture medium was taken out from the N-1 seed tank and inoculated into the culture fermenter, and after inoculation was completed, 5 L of fresh medium was replenished into the N-1 seed tank to continue cell amplification culture. On the 9th day of cell amplification culture, the viable cell density in the N-1 seed tank was 85.68 × 10 6 Once the cell / mL is reached, another new culture fermentor can be inoculated.

[0055] To further illustrate the merits of the cell culture method of high-density continuous seeding described in the present invention, the following test was also performed. 6 When cultured to cells / mL, each was 10 × 10 6 cells / mL and 20×10 6 Two 2 L reactors of Sartorius BiostatA with a culture volume of 1.2 L were inoculated at a cell density of 100 cells / mL, the initial culture temperature was 36.5 °C, and the cell density was approximately 25 × 10 6When it reached cells / mL, the temperature was decreased to 33.0 °C. Feeding method: Five days before D5, it was matched with the past fed-batch feeding amount by the integral of viable cell density over time (IVCC), and fed every other day. After D5, about 2.8% (v / v) of the feed material was added every other day until the yield became equivalent to the harvest in the control fed-batch culture (i.e., the normal fed-batch culture mode).

[0056] Into a 2 L reactor of Sartorius Biostat A, 10×10 6 cells / mL and 20×10 6 cells / mL of high density and 1×10 6 cells / mL of density used for normal fed-batch culture were inoculated, and the effects on the cell viability and viable cell density at different initial inoculation densities were compared. For specific test results, refer to Figure 7. Under the above conditions of different inoculation densities, the time-dependent trend of the protein expression level of CHO seed cells was measured. For specific test results, refer to Figure 8.

[0057] As can be seen from the results of Figure 7 and Figure 8, (1) By perfusion culture for collecting cells with RM20, CHO cells stably expressing INGLP bispecific antibody can reach a high cell density, and the cell state can be well maintained (refer to Figure 7). (2) After selecting high-density inoculation fed-batch of 20×10 6 cells / mL and 10×10 6 cells / mL, the expression levels achieved by normal fed-batch culture for 15 days were reached on the 8th day and the 10th day respectively, and the culture cycle was shortened (refer to Figure 8).

[0058] The specific embodiments of the present invention have been described in detail above, but they are merely exemplary, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications or substitutions made to the embodiments are included within the scope of the present invention. Therefore, any equivalent conversions or modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention.

Claims

1. (1) providing a cell culture, and subjecting the cell culture to resuscitation, expansion culture in a shake flask, and then expansion culture in a swing reaction bag; (2) transferring the resuscitated and amplified cells to a final stage cell amplification tank and continuing the amplification culture; (3) inoculating the cells in the final cell amplification tank into a culture fermentation tank by a high-density continuous inoculation method, and carrying out fermentation culture; (4) harvesting the target product, In step (2), a filtration device is provided in the final stage cell amplification tank, and the filtration device is used to reduce the cell density in the final stage cell amplification tank to 10 ７ cells / mL > 10 ８ Non-cellular material in the medium can be exchanged with fresh medium to provide less than cells / mL, and the filtration device is an alternating tangential flow filtration (ATF), a spin filter, or a tangential flow filtration (TFF), In step (3), the high-density continuous inoculation is performed by inoculating the cell culture solution in the final stage cell amplification tank with a volume of less than half the volume of the cell culture solution in the final stage cell amplification tank. ７ cells / mL > 10 ８ The cell culture medium in the final stage cell amplification tank is inoculated at a density of less than 10 cells / mL. After the inoculation is completed, the cell culture medium in the final stage cell amplification tank is inoculated with 10 ７ cells / mL > 10 ８ The final stage cell amplification tank is replenished with fresh medium in an amount equal to or less than half the volume of the cell culture medium in the final stage cell amplification tank so that another new culture fermenter can be inoculated at a density smaller than cells / mL, and the above-mentioned operations of inoculating the culture fermenter and replenishing the final stage cell amplification tank with fresh medium are repeated. Cell culture method.

2. The cell culture described in step (1) is subjected to resuscitation, amplification culture in a shake flask and then amplification culture in a swing reaction bag in two batches, and another final stage cell amplification tank is added in step (2); 2. The method of claim 1 .

3. The volume of the final stage cell amplification tank described in step (2) is 2 L to 1000 L; 3. The method according to claim 1 or 2.

4. Use of the method according to any one of claims 1 to 3 in the cultivation of mammalian cells.

5. The mammalian cell is selected from the group consisting of CHO, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, T cell line, B cell line, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, HT1080, L929, hybridoma and cancer cell line; 5. The use according to claim 4.

6. The mammalian cell is a CHO-S or CHO-K1 cell.

6. Use according to claim 4 or 5.

7. The use according to any one of claims 4 to 6, wherein the mammalian cell comprises a nucleotide sequence encoding a heterologous protein.

8. The use according to claim 7, wherein the heterologous protein is an antibody.

9. The antibody is a monoclonal antibody or a bispecific antibody.

9. The use according to claim 8.

Images