Perfusion culture method and application of CHO (Chinese hamster ovary) cells

Abstract

The invention discloses a perfusion culture method and application of CHO (Chinese hamster ovary) cells. The perfusion culture method comprises the following steps: inoculating CHO cells, and carrying out cell amplification culture; when the cell density reaches 6 * 10 < 6 > / mL to 10 * 10 < 6 > / mL, perfusion is started, the perfusion rate is 0.5 VVD to 1 VVD, and the perfusion rate is adjusted according to a constant proportion based on the cell density until the cell density reaches 90 * 10 < 6 > / mL to 120 * 10 < 6 > / mL, and the constant proportion is 1 VVD / (33 * 10 < 6 > / mL to 37 * 10 < 6 > / mL); and when the cell density reaches 90 * 10 < 6 > / mL to 120 * 10 < 6 > / mL, cooling and releasing are carried out at the same time, and the cell density is maintained. According to the perfusion culture method, high-density culture of CHO cells is realized, the culture time is greatly prolonged, and the duration for maintaining high cell viability is greatly prolonged.

Description

Technical Field

[0001] This application relates to the field of biopharmaceutical technology, specifically to a perfusion culture method for CHO cells and its application. Background Technology

[0002] Antibody drugs are currently one of the fastest-growing and most advanced and effective classes of drugs. The advent of monoclonal antibody technology has made the research and production of antibody drugs a reality. After more than 30 years of development, therapeutic monoclonal antibody drugs have become one of the most important components of biomedicine, with broad application prospects in disease treatment. They have been successfully used to treat various diseases such as tumors, autoimmune diseases, infectious diseases, and transplant rejection. Since the first therapeutic monoclonal antibody was approved in 1975, more than 100 monoclonal antibodies have been approved for marketing, and the number has been growing rapidly in recent years.

[0003] Most monoclonal antibodies are expressed by mammalian cells, and their research and production are carried out in bioreactors, with the most common working volumes ranging from a few liters to several thousand liters. Taking a 2000L scale as an example, the traditional production process includes: seed resuscitation → shake flask amplification (n~x) → n~2 amplification → n~1 amplification → n production (2000L stirred reactor). The amplification process requires two stages of seed reactor amplification, which increases the design and equipment costs of the workshop and introduces potential operational risks.

[0004] Perfusion culture is a popular method used in recent years for the culture of mammalian cells to produce secretory recombinant therapeutic drugs, such as monoclonal antibodies, chimeric antibodies, and humanized antibodies. Perfusion culture involves adding cells and culture medium together to a reactor, and during cell growth and product formation, continuously removing portions of the conditioned medium while simultaneously injecting fresh medium.

[0005] Due to the complexity of in vitro culture of mammalian cells, problems such as short cell maintenance time, low cell density, and low viability often occur. To solve these problems, there is an urgent need to provide a method for preparing antibodies by perfusion culture of mammalian cells at high cell density. Summary of the Invention

[0006] Therefore, it is necessary to provide a perfusion culture method for CHO cells and its application.

[0007] The first aspect of this application provides a method for perfusion culture of CHO cells, comprising the following steps:

[0008] CHO cells were inoculated and cultured to scale up the cell count.

[0009] When the cell density reaches 6×10 6 Cells / mL ~10×106 Perfusion culture begins after cells reach a density of 100 cells / mL, with a perfusion rate of 0.5 VVD~1 VVD. Once cell growth begins, the perfusion rate is adjusted daily based on cell growth, maintaining a constant ratio based on cell density, until the cell density reaches 90 × 10⁶ cells / mL. 6 Cells / mL ~120×10 6 The perfusion rate was determined by cell density per mL; wherein the perfusion rate was based on a constant ratio adjusted according to cell density of 1 VVD / (33 × 10⁻⁶). 6 37 × 10⁻⁶ cells / mL ~ 37 × 10⁻⁶ 6 (number / mL), the perfusion culture temperature is 36℃~38℃; and,

[0010] When the cell density reaches 90×10 6 Cells / mL ~120×10 6 After reaching a cell density of 100 cells / mL, simultaneous cooling and effluent release were performed to maintain cell density.

[0011] In some implementations, when the cell density reaches 90 × 10 6 Cells / mL ~120×10 6 The temperature after cooling and venting after the flow rate is 31℃~35℃.

[0012] In some implementations, when the cell density reaches 90 × 10 6 Cells / mL ~120×10 6 The volume of fluid released during the step of simultaneously cooling and releasing the fluid after the perfusion volume is 4% to 6% of the perfusion culture volume.

[0013] In some embodiments, the perfusion rate in the perfusion culture is 30 mL / h to 120 mL / h.

[0014] In some embodiments, the perfusion culture method satisfies one or more of the following conditions:

[0015] The glucose concentration in the perfusion culture system is 6 g / L to 8 g / L;

[0016] The dissolved oxygen level in the perfusion culture system is 30%–80%.

[0017] The pH value in the perfusion culture system was 6.8–7.2; and,

[0018] The rotation speed in the perfusion culture system is 200 rpm to 300 rpm.

[0019] In some embodiments, the perfusion culture time is 41 to 43 days.

[0020] In some embodiments, the cell density during the step of seeding CHO cells for cell amplification culture is 0.2 × 10⁻⁶. 6 cells / mL ~0.8×10 6 Cells / mL, and cell amplification culture time is 48h~96h.

[0021] In some embodiments, perfusion culture uses a cell retention system, wherein the pore size of the empty fibers in the cell retention system is 0.1 μm to 2 μm.

[0022] In some embodiments, the CHO cells include at least one of the CHOZN® CHO K1 cell line and the CHOK1SV GS~KO cell line.

[0023] A second aspect of this application provides a method for preparing antibodies, comprising culturing CHO cells containing antibodies expressed using the perfusion culture method described in the first aspect of this application.

[0024] The perfusion culture method provided in this application achieves high-density cell culture by adjusting the perfusion rate according to cell density, reducing temperature, and effluent synergistically, greatly increasing culture time and maintaining high cell viability. Furthermore, this perfusion culture method can culture host CHO cells to high density and obtain high-yield antibodies. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0026] Figure 1 This is a cell density variation curve for different perfusion processes in one embodiment of this application;

[0027] Figure 2 The cell viability variation curves for different perfusion processes in one embodiment of this application are shown.

[0028] Figure 3 This is a comparison curve of the yield of different irrigation processes in one embodiment of this application;

[0029] Figure 4 This is a comparison diagram of cell growth curves between perfusion culture and batch culture in one embodiment of this application;

[0030] Figure 5 This is a comparison chart of the yields of perfusion culture and batch culture in one embodiment of this application. Detailed Implementation

[0031] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0033] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0034] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0035] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0036] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0037] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0038] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0039] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0040] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0041] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0042] Due to the complexity of in vitro culture of mammalian cells, problems such as short cell maintenance time, low density, and low viability often occur.

[0043] Based on this, the embodiments of this application provide at least one method for perfusion culture of CHO cells and its application.

[0044] In this application, unless otherwise specified, "VVD" refers to the ratio of the volume of fresh culture medium added to the culture system each day to the total culture volume.

[0045] In this application, unless otherwise stated, "effervescence" refers to the process of periodically removing a portion of the culture medium from a bioreactor during perfusion culture.

[0046] Unless otherwise stated, "perfusion rate" in this application refers to the volume of fresh culture medium added to the bioreactor per unit time.

[0047] In a first aspect of this application, a perfusion culture method for CHO cells is provided, wherein the perfusion rate is adjusted daily according to cell growth, and the final perfusion rate is determined after the target cell density is reached; simultaneously, cooling and evacuation are performed to control the cells to reach a constant density range, specifically including the following steps:

[0048] S100: Inoculate CHO cells for cell amplification culture;

[0049] S200: When the cell density reaches 6×10⁶ 6 Cells / mL ~10×10 6 Perfusion culture begins after cells reach a density of 100 cells / mL, with a perfusion rate of 0.5 VVD~1 VVD. Once cell growth begins, the perfusion rate is adjusted according to cell growth, maintaining a constant ratio based on cell density, until the cell density reaches 90 × 10⁶ cells / mL. 6 Cells / mL ~120×10 6 The perfusion rate was determined by cell density per mL; the constant ratio of the perfusion rate to the cell density was 1 VVD / (33 × 10⁻⁶). 6 37 × 10⁻⁶ cells / mL ~ 37 × 10⁻⁶ 6 (number / mL), perfusion culture temperature was 36℃~38℃; and,

[0050] S300: When the cell density reaches 90×10 6 Cells / mL ~120×10 6 After reaching a cell density of 100 cells / mL, simultaneous cooling and effluent release were performed to maintain cell density.

[0051] Unless otherwise stated in this application, "1VVD / (33×10 6 37 × 10⁻⁶ cells / mL ~ 37 × 10⁻⁶ 6 "cells / mL" refers to the ratio between cell density and perfusion rate, indicating the increase in perfusion rate required for growth per unit cell density, i.e., the increase in cell density by 33 × 10⁻⁶ cells / mL. 6 cells / mL, 34×10 6 cells / mL, 35×10 6 cells / mL, 36×10 6 cells / mL, 37×10 6For every 1 VVD increase in perfusion rate, the perfusion rate increases by 1 VVD, or any value or range between the two values ​​mentioned above. It is understood that this proportional relationship can be scaled down proportionally, for example, but not limited to, for every 7 × 10⁻⁶ cells / mL growth. 6 The number of cells / mL increased the perfusion rate by 0.2 VVD.

[0052] In some embodiments, in step S100, the density of CHO cells seeded is 0.2 × 10⁻⁶. 6 cells / mL ~0.8×10 6 CHO cell density can be, but is not limited to, 0.2 × 10⁶ cells / mL. Non-limitingly, the seeded CHO cell density can be, but is not limited to, 0.2 × 10⁶ cells / mL. 6 cells / mL, 0.3×10 6 cells / mL, 0.4×10 6 cells / mL, 0.5×10 6 cells / mL, 0.6×10 6 cells / mL, 0.7×10 6 cells / mL, 0.8×10 6 The number of cells / mL or any value or range between the two values ​​mentioned above.

[0053] In some embodiments, in step S100, the cell amplification culture time is 48h to 96h. Non-limitingly, the cell amplification culture time can be, but is not limited to, 48h, 60h, 72h, 84h, 96h, or any value or range between two of the above.

[0054] In some implementations, the scale-up culture process in step S100 is suitable for suspension culture.

[0055] In some embodiments, in step S200, the perfusion culture temperature is 36°C to 38°C. Without limitation, the perfusion culture temperature can be, but is not limited to, 36°C, 37°C, 38°C, or any value or range between two of the above.

[0056] In some embodiments, in step S200, when the cell density reaches 6 × 10⁻⁶ 6 cells / mL, 7×10 6 cells / mL, 8×10 6 cells / mL, 9×10 6 cells / mL, 10×10 6 Perfusion culture begins when the number of cells / mL or any value or range between two of the above values ​​is reached. At this time, the perfusion rate can be, but is not limited to, 0.5 VVD, 0.6 VVD, 0.7 VVD, 0.8 VVD, 0.9 VVD, 1 VVD or any value or range between two of the above values.

[0057] In some embodiments, in step S200, the perfusion rate in the perfusion culture is 30 mL / h to 120 mL / h. Non-limitingly, the perfusion rate in the perfusion culture can be 30 mL / h, 60 mL / h, 90 mL / h, 120 mL / h, or any value or range between two of the above.

[0058] In some embodiments, in step S200, the glucose concentration in the perfusion culture system is 6 g / L to 8 g / L. Without limitation, the glucose concentration in the perfusion culture system can be, but is not limited to, 6 g / L, 7 g / L, 8 g / L, or any value or range between two of the above.

[0059] In some embodiments, in step S200, the dissolved oxygen value in the perfusion culture system is 30% to 80%. Non-limitingly, the dissolved oxygen value in the perfusion culture system can be, but is not limited to, 30%, 40%, 50%, 60%, 70%, 80%, or any value or range between two of the above.

[0060] In some embodiments, in step S200, the pH value of the perfusion culture system is 6.8 to 7.2. Non-limitingly, the pH value of the perfusion culture system can be, but is not limited to, 6.8, 6.9, 7.0, 7.1, 7.2, or any value or range between two of the above.

[0061] In some embodiments, in step S200, the rotation speed in the perfusion culture system is 200 rpm to 300 rpm. Non-limitingly, the rotation speed in the perfusion culture system can be, but is not limited to, 200 rpm, 220 rpm, 240 rpm, 260 rpm, 280 rpm, 300 rpm, or any value or range between two of the above.

[0062] In some embodiments, in step S200, when the cell density reaches 25 × 10⁻⁶ 6 Cells / mL ~30×10 6 The number of bubbles / mL was adjusted from large bubble ventilation to microbubble ventilation.

[0063] In some implementations, the perfusion culture time can be 41 days, 42 days, or 43 days.

[0064] In some embodiments, in step S300, when the cell density reaches 90 × 10⁻⁶ 6 cells / mL, 100×10 6 cells / mL, 110×10 6 cells / mL, 120×10 6 After the number of cells / mL or any value or range between the above two values, cooling and effluent are performed simultaneously.

[0065] In some embodiments, in step S300, when the cell density reaches 90 × 10⁻⁶ 6 Cells / mL ~120×10 6 After the concentration of cells / mL is reduced, the temperature is cooled to 31℃~35℃. Non-limitingly, the temperature after cooling can be, but is not limited to, 31℃, 32℃, 33℃, 34℃, 35℃, or any value or range between two of the above.

[0066] In some embodiments, in step S300, when the cell density reaches 90 × 10⁻⁶ 6 Cells / mL ~120×10 6 After the number of cells / mL is reached, the culture volume is effluent, and the effluent volume is 4% to 6% of the perfusion culture volume. Non-limitingly, the effluent volume can be, but is not limited to, 4%, 5%, 6% of the perfusion culture volume or any value or range between two of the above values.

[0067] In some embodiments, the perfusion culture process is carried out in a bioreactor, which includes, but is not limited to, a disposable stirred bioreactor, a glass stirred bioreactor, and a stainless steel stirred bioreactor.

[0068] Unless otherwise stated, "perfusion culture volume" in this application refers to the working volume of the bioreactor when the perfusion culture system is running stably.

[0069] In some embodiments, the culture medium used for perfusion culture can be any culture medium known in the art that supports CHO cell culture, and can utilize commercially available basal media, such as, but not limited to, EX-CELL® Advanced. TM CHO basal medium and ExpiCHO™ medium.

[0070] In some embodiments, perfusion culture uses a cell retention system, wherein the pore size of the empty fibers in the cell retention system is 0.1 μm to 2 μm. Non-limitingly, the pore size of the empty fibers in the cell retention system can be, but is not limited to, 0.1 μm, 0.5 μm, 1 μm, 1.5 μm, 2 μm, or any value or range between two of the above.

[0071] In some embodiments, the inoculated CHO cells include at least one of the CHOZN® CHO K1 cell line and the CHOK1SV GS-KO cell line.

[0072] In a second aspect of this application, a method for preparing an antibody is provided, comprising culturing CHO cells containing antibody expression using the perfusion culture method described above.

[0073] The following are some examples.

[0074] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0075] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0076] Example 1: Confirmation of process parameters for CHO cell perfusion culture

[0077] The four irrigation processes in Table 1 below were compared to determine the optimal irrigation process:

[0078] Table 1

[0079]

[0080] 1. Irrigation Process 1:

[0081] After resuscitation, CHO cells (original cells derived from Merck's CHOZN) were expanded three times using Corning shake flasks until a density of 6 × 10⁶ cells / year was achieved. 6 Cells / mL ~10×10 6 Once the cell density reaches 400 mL, the cells are inoculated, and Merck Advanced medium is used for cell expansion.

[0082] The cells were inoculated into a fermenter reactor (Applikon my control 3L reactor) and cultured for three days until the cell density reached 8 × 10⁶ cells / day. 6 Perfusion was started after cells / mL (using the Riplegen cell trap), and the perfusion medium was concentrated 1.1× Merck Advanced medium, with an initial perfusion rate of 0.5 VVD.

[0083] As cell density continued to increase, the VVD gradually increased to 3 VVD, and the perfusion rate of this medium was 35 × 10⁻⁶ cells / day per cell density increase. 6 The number of cells / mL increased the perfusion rate by 1 VVD.

[0084] The reactor parameters are as follows: culture temperature 37℃, gradually reduced to 35℃ according to cell density, pH adjustable from 6.5 to 7.5, dissolved oxygen concentration 30% to 80%, stirring speed adjustable from 200 to 300 rpm, glucose content maintained at 6 to 8 g / L per day, and aeration method consisting of large bubbles and microbubbles.

[0085] Cell density reached 120 × 10⁶ on day 11. 6 Cells / mL were removed, the temperature was lowered to 35°C, and the cells were released in a volume of 5% of the culture volume to maintain cell density.

[0086] 2. Irrigation process 2: The basic parameters of irrigation culture are adopted using irrigation process 1, the difference being that only the irrigation rate is changed but no cooling or discharge is performed.

[0087] Simply changing the perfusion rate will lead to a continuous increase in cell density until the reactor reaches its maximum capacity. To maintain cell viability, the amount of perfusion medium used will increase, eventually reaching 6 VVD. Excessive cell density leads to increased medium usage costs, and the continuous increase in medium replacement volume and rapid accumulation of dead cells can cause the hollow fiber tubes to become clogged and stop the culture.

[0088] 3. Irrigation process 3: The basic parameters of irrigation culture are adopted from irrigation process 1, except that the irrigation rate and temperature are changed, but no discharge is performed.

[0089] During the initial stages of this process, cell growth is normal. After cooling, the cell proliferation rate is somewhat inhibited, but growth does not stop. At the end of the culture period, the cell density reaches its peak (the system reaches its maximum peak cell count) and then rapidly declines. Cell viability begins to drop significantly, making it difficult to maintain culture for long periods, resulting in rapid cell death.

[0090] 4. Perfusion Process 4: Perfusion process 4 uses the basic parameters of perfusion culture in perfusion process 1, but only performs discharge to maintain the cell density at 100 × 10⁻⁶ cells / year. 6 The flow rate was maintained at 3 VVD / day from the start of perfusion, without cooling.

[0091] In the early stages of culture, high media exchange volumes allow cells to rapidly proliferate to peak density. However, the amount of antibodies produced during this early cell proliferation phase is very low and is not collected or purified, resulting in a significant waste of culture medium. Increasing the volume of culture by venting during the mid-stage of culture can maintain cell density within a fixed range for continuous culture, but this requires venting at least 15% of the culture volume daily, leading to substantial waste.

[0092] Comparing cell densities in perfusion processes 1, 2, 3, and 4, such as... Figure 1 Only perfusion processes 1 and 4 can be used for long-term culture; compare the cell viability of perfusion processes 1, 2, 3, and 4, such as... Figure 2 Only perfusion processes 1 and 4 can maintain high cell viability for extended periods, allowing for prolonged culture. Comparing protein yields among perfusion processes 1, 2, 3, and 4, for example... Figure 3 Irrigation process 4 resulted in lower yields due to prolonged high flow rates, while irrigation process 1 yielded the highest output. Therefore, irrigation process 1 was selected as the final process.

[0093] Example 2: Comparison of perfusion culture process and batch culture process

[0094] I. Comparison of maximum cell density, viability, and culture period between perfusion culture and batch culture processes

[0095] Experiment 1: Cultured using conventional batch culture processes in this field.

[0096] Experiment 2: Cultivation was carried out using the perfusion process 1 of Example 1.

[0097] Experiment 1 used the traditional batch culture process: the original cells were derived from Merck's CHOZN, and after thawing, they were expanded three times using Corning shake flasks until the density reached 3.5 × 10⁻⁶. 6 cells / mL ~4.5×10 6 Once the cell density reaches 400 mL, inoculation is performed. The reactor culture volume is 1.5 L. Merck Advanced medium is used for cell expansion. The culture temperature is 37℃, D6 temperature is reduced to 35℃, pH is adjustable from 6.5 to 7.5, dissolved oxygen concentration is 30% to 80%, stirring speed is 300 rpm, and aeration is performed using large bubble aeration.

[0098] Experiments 1 and 2 maintained essentially the same parameters during the culture process (such as culture temperature, pH, dissolved oxygen concentration, and stirring speed). Cell density and viability were measured after the culture period, and the results were compared as follows: Figure 4 As shown. The results indicate that the cell density and cell viability of CHO cells cultured using the perfusion process 1 of Example 1 are higher than those obtained using the batch culture process.

[0099] II. Comparison of CHO cell yield between perfusion culture and batch culture

[0100] CHO cells were cultured using the methods described in Experiments 1 and 2 above. The culture media were collected and analyzed using a Roche biochemical analyzer. The results are as follows: Figure 5 As shown, the single-cell yield of CHO cells in perfusion and batch culture remained basically unchanged. Due to the extended culture time, the maximum cell density increased, so the total yield of perfusion was more than 10 times that of batch production.

[0101] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0102] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A method for perfusion culture of CHO cells, characterized in that, Includes the following steps: CHO cells were inoculated and cultured to scale up the cell count. When the cell density reaches 6×10 6 Cells / mL ~10×10 6 Perfusion culture begins after cells reach a density of 100 cells / mL, with a perfusion rate of 0.5 VVD~1 VVD. Once cell growth begins, the perfusion rate is adjusted according to cell growth, maintaining a constant ratio based on cell density, until the cell density reaches 90 × 10⁶ cells / mL. 6 Cells / mL ~120×10 6 The perfusion rate was determined by cell density per mL; wherein the perfusion rate was based on a constant ratio adjusted according to cell density of 1 VVD / (33 × 10⁻⁶). 6 37 × 10⁻⁶ cells / mL ~ 37 × 10⁻⁶ 6 (number / mL), the perfusion culture temperature is 36℃~38℃; and, When the cell density reaches 90×10 6 Cells / mL ~120×10 6 After reaching a cell density of 100 cells / mL, simultaneous cooling and effluent release were performed to maintain cell density.

2. The perfusion culture method for CHO cells as described in claim 1, characterized in that, When the cell density reaches 90×10 6 Cells / mL ~120×10 6 The temperature after cooling and venting is 31℃~35℃ in the step of simultaneously cooling and releasing the sample after being processed at a flow rate of 1 / mL.

3. The perfusion culture method for CHO cells as described in claim 1, characterized in that, When the cell density reaches 90×10 6 Cells / mL ~120×10 6 The volume of fluid released during the step of simultaneously cooling and releasing the fluid after the fluid volume is reduced to 4% to 6% of the perfusion culture volume.

4. The perfusion culture method for CHO cells as described in claim 1, characterized in that, When the cell density reaches 25×10 6 Cells / mL ~30×10 6 After the number of cells / mL was reduced, the ventilation mode was changed from macrobubble ventilation to microbubble ventilation.

5. The perfusion culture method for CHO cells according to any one of claims 1 to 4, characterized in that, The perfusion culture method satisfies one or more of the following conditions: The glucose concentration in the perfusion culture system is 6 g / L to 8 g / L; The dissolved oxygen level in the perfusion culture system is 30%–80%. The pH value in the perfusion culture system is 6.8~7.2; The CO2 concentration in the perfusion culture system is 3%–8%; and, The rotation speed in the perfusion culture system is 200 rpm to 300 rpm.

6. The perfusion culture method for CHO cells according to any one of claims 1 to 4, characterized in that, The perfusion culture time is 41 to 43 days.

7. The perfusion culture method for CHO cells according to any one of claims 1 to 4, characterized in that, In the step of seeding CHO cells for cell amplification culture, the cell density was 0.2 × 10⁻⁶. 6 cells / mL ~0.8×10 6 Cells / mL, and cell amplification culture time is 48h~96h.

8. The perfusion culture method for CHO cells according to any one of claims 1 to 4, characterized in that, The perfusion culture uses a cell retention system, wherein the pore size of the empty fiber of the cell retention system is 0.1 μm to 2 μm.

9. The perfusion culture method for CHO cells according to any one of claims 1 to 4, characterized in that, The CHO cells include at least one of the CHOZN® CHO K1 cell line and the CHOK1SV GS-KO cell line.

10. A method for preparing antibodies, characterized in that, This includes culturing CHO cells containing expressed antibodies using the perfusion culture method for CHO cells as described in any one of claims 1 to 9.

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