A method for maintaining steady state of cell perfusion culture system

Abstract

The present application provides a method for maintaining a steady state of a cell perfusion culture system, and relates to the field of cell culture technology. The method includes: placing cells in a bioreactor and performing a first culture at a temperature of T0; when the density of live cells in the bioreactor reaches the first target value, the temperature of T0 is reduced to the temperature of T1, and a perfusion medium is used for the second culture, and after maintaining for 4-5 days, the temperature of T1 is increased to the temperature of T2, and the third culture is continued, and the second and third cultures are not discharged; or, when the density of live cells in the bioreactor reaches the second target value, the temperature of T0 is reduced to the temperature of T2, and a perfusion medium is used for the fourth culture, and discharge is required in the fourth culture; wherein, T0>T2>T1. The present application adopts the means of two-step cooling, and according to the law of cell first-order kinetic multiplication, a fixed discharge rate is used for discharge, so as to maintain the steady-state growth of the cells.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to a method for maintaining a steady state of a cell perfusion culture system. Background Art

[0002] Since the first abciximab produced using the perfusion process was approved by the US FDA in 1994, the perfusion process has been formally recognized by the biopharmaceutical industry, and the product types include different types of biological products such as antibodies, blood factors, and viral particles. Compared with traditional fed-batch and batch culture, perfusion culture can separate and store the harvested target products in a timely manner, reducing the re-modification of target products such as oxidation and enzymatic degradation outside the cell; the scale of the bioreactor can be reduced to about 1 / 10 of the traditional commercial scale to reduce the cost of early equipment investment.

[0003] Chinese hamster ovary (CHO) cell perfusion culture is a common process for producing protein drugs. In this process, maintaining high cell density and long-term cell viability and density is extremely critical. Therefore, in addition to developing a perfusion culture medium that maintains long-term cell viability and density, the key work in the early stage of process development also requires the development of a suitable process control condition platform.

[0004] Shake tubes are often used as a platform for early perfusion process development due to their high throughput, simple operation, and good gas mass transfer. Using shake tubes for simulated perfusion can initially reflect the cell growth kinetics at different cell densities, as well as the product specific growth rate. However, in the shake tube process, the perfusion medium is usually replaced once a day by centrifugation. However, since the perfusion medium is rich in nutrients and has a very high osmotic pressure, it usually has an adverse effect on cell growth. Therefore, stable control of cell density is crucial in the shake tube process.

[0005] In the perfusion process, a strategy often used is to perform regular cell bleeding to control cell density. The common method of controlling cell density in a shake tube is bleeding once a day to control the upper or lower limit of cell density. However, this control method easily causes large fluctuations in cell density and yield during the steady state, and it is difficult to control the long-term cell viability and density, and it is not possible to achieve the ideal stable cell state. In addition, it is also impossible to accurately control the cell density during the steady state under high density and high specific growth rate conditions, which makes it difficult to reproduce the results evaluated in the shake tube in the reactor. Summary of the invention

[0006] The purpose of the present application is to provide a method for maintaining steady state in a cell perfusion culture system, by adopting a two-step cooling method to allow cells to transition from a growth phase to a steady state phase; at the same time, for conditions of high cell density, according to the first-order kinetic doubling law of cells, discharge is performed at the same discharge rate, thereby maintaining steady-state growth of cells.

[0007] To achieve the above objectives, the technical solution of this application is as follows:

[0008] In a first aspect, the present application provides a method for maintaining a steady state of a cell perfusion culture system, comprising:

[0009] The cells are placed in a bioreactor and cultured at a temperature of T0 for the first time;

[0010] When the viable cell density in the bioreactor reaches a first target value, the T0 temperature is lowered to the T1 temperature, and a second culture is performed using a perfusion medium. After maintaining for 4-5 days, the T1 temperature is raised to the T2 temperature, and a third culture is performed. The second culture and the third culture are not discharged.

[0011] Alternatively, when the viable cell density in the bioreactor reaches the second target value, the T0 temperature is lowered to the T2 temperature, and a fourth culture is performed using a perfusion medium. After maintaining for 1-2 days, the fourth culture begins to be discharged;

[0012] Among them, T0>T2>T1.

[0013] Preferably, the temperature of T0 is 36°C-37°C, the temperature of T2 is 33°C-34°C, and the temperature of T1 is 30°C-32°C.

[0014] Preferably, the first target value is 50×10 6 cells / mL-75×10 6 cells / mL;

[0015] The second target value is 30×10 6 cells / mL-50×10 6 cells / mL.

[0016] Preferably, the first culturing comprises:

[0017] Inoculating the cells in the bioreactor, resuscitating them at the T0 temperature and performing a proliferation treatment using a basal culture medium;

[0018] When the viable cell density after the amplification treatment reaches 10×10 6 cells / mL-20×10 6cells / mL, using the perfusion medium and performing perfusion culture at a fixed perfusion rate every day until the viable cell density in the bioreactor reaches the first target value;

[0019] The fixed perfusion rate is 0.5VVD-1.5VVD.

[0020] Further preferably, when the bioreactor is a shake tube, the cells are inoculated into the shake tube, and the shake tube is placed in an environment at the T0 temperature, a relative humidity of 85%-95%, and a CO2 concentration of 4%-6%, and the shake tube is tilted 15°-20°, and the amplification treatment is performed at a rotation speed of 150rpm-180rpm.

[0021] Preferably, the second culture and the third culture each independently include: using the perfusion medium to perform perfusion at a fixed cell specific perfusion rate every day;

[0022] And / or, the fourth culture comprises: using the perfusion medium, performing perfusion at a fixed cell ratio perfusion rate every day, maintaining for 1-2 days, and then performing discharge at a fixed discharge rate ΔIVCC every day.

[0023] Further preferably, the fixed cell specific perfusion rate is 20pL / (Cells·d)-60pL / (Cells·d);

[0024] The fixed discharge rate ΔIVCC is 40×10 6 cells / mL·day-80×10 6 cells / mL·day.

[0025] Preferably, the cell is selected from the group consisting of:

[0026] CHO, CHO-K1, CHO-S, DXB-11, DG-44, BHK cell lines.

[0027] Preferably, when the cell is selected from the CHO-K1 cell line, it comprises:

[0028] The cells were inoculated in a shake tube and revived at 36.5°C and amplified using a basal medium until the viable cell density in the shake tube reached 10×10 6 After that, the perfusion medium was used at 36.5°C and the perfusion rate was 1VVD per day until the viable cell density in the shake tube reached 50×10 6After the cells / mL were reached, the temperature was lowered to 31°C, and the perfusion treatment was performed every day at a fixed cell ratio perfusion rate of 50 pL / (Cells·d) using the perfusion medium. After maintaining for 4 days, the temperature was raised to 33°C, and the perfusion treatment was continued at the fixed cell ratio perfusion rate every day.

[0029] Preferably, when the cell is selected from the CHO-K1 cell line, it comprises:

[0030] The cells were inoculated in a shake tube and revived at 36.5°C and amplified using a basal medium until the viable cell density in the shake tube reached 10×10 6 After that, the perfusion medium was used at 36.5°C and the perfusion rate was 1VVD per day until the viable cell density in the shake tube reached 30×10 6 After that, the temperature was lowered to 33°C, and the perfusion medium was used to perform perfusion treatment at a fixed cell ratio perfusion rate of 50 pL / (Cells·d) every day. After maintaining for 2 days, discharge was started at the same time, and the fixed discharge rate ΔIVCC was 70×10 6 cells / mL·day.

[0031] Beneficial effects of this application:

[0032] For the traditional one-step cooling method, cells tend to be in a faster growth state after cooling, so that the early density of cells is often greater than the target cell density. The cell density can only be controlled by discharge to reach a stable state, which will lead to waste of culture fluid, and the concentration of nutrients also fluctuates with the fluctuation of cell density, and it is impossible to reach a steady-state concentration close to the theory. The present application adopts a two-step cooling method. After the cells are maintained in a state equivalent to low temperature for a period of perfusion culture, the cells will switch from growth to a steady-state stage, and then the temperature will be increased to accelerate the metabolism of the cells, thereby not affecting the cell division cycle, while maintaining the high secretion state of the cells, so that the cell cycle is maintained in the G1 / S phase, that is, the cell specific growth rate is stably maintained at a low level.

[0033] Alternatively, during the cell growth stage, the present application utilizes the cell growth curve to follow the first-order kinetic growth curve, that is, a geometric increase, and then conceives of releasing cells at high density at the same release rate ΔIVCC, thereby maintaining the long-term steady-state growth law of the cells, stably maintaining the cell density and product yield in the steady-state period, and extending the maintenance time of the perfusion steady state. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0035] Figure 1 It is a line graph of the cell lactic acid production of Comparative Example 1 and Example 1 in Experiment 1 at different culture days;

[0036] Figure 2 It is a line graph of the cell product yields of Comparative Example 1 and Example 1 in Experiment 1 at different culture days;

[0037] Figure 3 It is a line graph of the viable cell density of the cells of Comparative Example 1 and Example 1 in Experiment 1 at different culture days;

[0038] Figure 4 It is a line graph of cell viability of the cells of Comparative Example 1 and Example 1 in Experiment 1 at different culture days;

[0039] Figure 5 It is a line graph of the viable cell density of the cells of Comparative Example 2 and Examples 2-4 in Experiment 2 at different culture days;

[0040] Figure 6 It is a line graph of cell viability of the cells of Comparative Example 2 and Examples 2-4 in Experiment 2 at different culture days;

[0041] Figure 7 It is a line graph of product yield of cells harvested in a single day in Comparative Example 2 and Examples 2-4 in Experiment 2;

[0042] Figure 8 It is a line graph of the cumulative product yield of the cells harvested from Comparative Example 2 and Examples 2-4 in Experiment 2;

[0043] Fig. 9 It is a line chart of the daily percentage of the cell discharge rate of Comparative Example 2 and Examples 2-4 in Experiment 2;

[0044] Fig.10 It is a line graph of the cumulative percentage of cell discharge rate of Comparative Example 2 and Examples 2-4 in Experiment 2. DETAILED DESCRIPTION

[0045] As used herein:

[0046] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus. The conjunction "consisting of" excludes any unspecified element, step, or component.

[0047] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0048] In these examples, parts and percentages are by mass unless otherwise indicated.

[0049] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.

[0050] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0051] In the perfusion culture process, the maintenance of cell density in the steady state stage is directly related to the overall process stability and product quality. Therefore, controlling the steady state of the perfusion culture system mainly means controlling the cell density in it to remain unchanged and maintain a stable temperature.

[0052] Based on this, the present application provides a method for maintaining a steady state of a cell perfusion culture system, comprising: placing cells in a bioreactor and performing a first culture at a temperature of T0; when the density of live cells in the bioreactor reaches a first target value, lowering the temperature of T0 to a temperature of T1, performing a second culture using a perfusion culture medium, maintaining for 4-5 days, and then raising the temperature of T1 to a temperature of T2, continuing a third culture, and the second and third cultures are not discharged. Wherein, T0>T2>T1.

[0053] In one embodiment of the present application, the first culture comprises: inoculating the cells in a bioreactor, resuscitating the cells at a temperature of T0 and performing an amplification process using a basal culture medium;

[0054] The viable cell density after amplification treatment reaches 10×10 6 cells / mL-20×10 6 cells / mL, a perfusion culture medium is used and a fixed perfusion rate is used for perfusion culture every day until the viable cell density in the bioreactor reaches a first target value, wherein the fixed perfusion rate is 0.5VVD-1.5VVD.

[0055] It should be noted that the basal culture medium and the perfusion culture medium may be the same or different depending on the characteristics of each cell product. Generally, the osmotic pressure of the basal culture medium is relatively low, between 280mos-320mos, and is mainly used for seed subculture and batch feeding processes; while the osmotic pressure of the perfusion culture medium is generally 400mos-450mos, which cannot be used for seed subculture expansion and low-density cell growth, and is only used for perfusion processes.

[0056] Further preferably, when the bioreactor is a shake tube, the cells are inoculated into a shake tube (a 50 mL shake tube has a working volume of 10 mL-20 mL), and the shake tube is placed in an environment at a temperature of T0, a relative humidity of 85%-95%, and a CO2 concentration of 4%-6%, and the shake tube is tilted 15°-20° and amplified at a rotation speed of 150 rpm-180 rpm.

[0057] More preferably, the amplification process is performed at a temperature of 36.5° C., a relative humidity of 90%, and a CO 2 concentration of 5%, with the shaking tube tilted at 15° and a rotation speed of 160 rpm.

[0058] In one embodiment of the present application, the temperature of T0 is 36°C-37°C, the temperature of T1 is 30°C-31°C, and the temperature of T2 is 33°C-34°C.

[0059] In one embodiment of the present application, the first target value of the viable cell density in the bioreactor is 50×106 cells / mL-75×10 6 cells / mL.

[0060] In one embodiment of the present application, except for the different culture temperatures, the second culture and the third culture both need to use perfusion culture medium and perform perfusion at a fixed cell ratio perfusion rate every day.

[0061] Among them, the fixed cell specific perfusion rate in the second culture and the third culture can be the same or different, but the fixed cell specific perfusion rate ranges from 20pL / (Cells·d) to 60pL / (Cells·d), for example, it can be 20pL / (Cells·d), 30pL / (Cells·d), 40pL / (Cells·d), 50pL / (Cells·d), 60pL / (Cells·d) or any value between 20pL / (Cells·d) and 60pL / (Cells·d).

[0062] It is understandable that in order to ensure that the cells reach a high cell density, the expanded cells will also be perfused during the logarithmic growth phase. At this time, the cells will be under relatively nutrient-rich conditions, and the factor limiting cell growth is often temperature. Therefore, when the cells reach a certain density, the temperature is lowered to adjust the cells from the growth phase to a stable state.

[0063] The present application first grows the cells at a temperature suitable for cell growth (T0 temperature), then places the cells in a low temperature state (T2 temperature) for a period of time, so that the cells transition from the growth state to the steady state stage, and then accelerates the cell metabolism by raising the temperature (T1 temperature), thereby not affecting the cell division cycle, while maintaining the high secretion state of the cells.

[0064] In one embodiment of the present application, the cultured cells are selected from the group consisting of: CHO, CHO-K1, CHO-S, DXB-11, DG-44, and BHK cell lines.

[0065] When the cell is selected from the CHO-K1 cell line, the method for maintaining a steady state of the cell perfusion culture system specifically comprises:

[0066] S1. The cells were inoculated into a shake tube and revived at 36.5°C, and amplified using basal medium until the viable cell density in the shake tube reached 10×10 6 After 10 cells / mL, the perfusion treatment was continued at 36.5°C with perfusion medium and a perfusion rate of 1 VVD per day;

[0067] S2, wait until the viable cell density in the shaking tube reaches 50×10 6 After the cells / mL was reached, the temperature was lowered to 31°C, and the perfusion treatment was carried out every day at a fixed cell ratio perfusion rate of 50 pL / (Cells·d) using the perfusion medium. After maintaining for 4 days, the temperature was raised to 33°C, and the perfusion treatment was continued at a fixed cell ratio perfusion rate every day.

[0068] In addition, for cells in a state of high cell density, the present application also provides another method for maintaining the steady state of a cell perfusion system, comprising: placing cells in a bioreactor and performing a first culture at a temperature of T0; when the density of live cells in the bioreactor reaches a second target value, lowering the temperature of T0 to a temperature of T2, and performing a fourth culture using a perfusion culture medium, and after maintaining for 1-2 days, the fourth culture begins to be discharged; wherein, T0>T2.

[0069] In this method, after a cooling step, although the cell growth rate slows down relative to the growth rate at the initial culture temperature, the cells after cooling are still in a faster growth state, which often makes the cell density greater than the target cell density. The cell density can only be controlled by bleeding.

[0070] When cells are in a faster growth phase, their growth curves generally follow the first-order kinetics, that is, geometric increase, that is, the cells are in a logarithmic growth phase. Then, according to this growth law, using the same fixed discharge rate ΔIVCC for discharge treatment is more in line with the cell growth law, can steadily maintain the cell density in the steady state period, and can also prolong the perfusion steady-state maintenance time.

[0071] In one embodiment of the present application, the fourth culture includes: first using a perfusion culture medium, perfusing at a fixed cell ratio perfusion rate every day, maintaining for 1-2 days, and then discharging at a fixed discharge rate ΔIVCC every day.

[0072] The fixed cell specific perfusion rate ranged from 20pL / (Cells·d) to 60pL / (Cells·d), and the fixed discharge rate ΔIVCC was 40×10 6 cells / mL·day-80×10 6 cells / mL·day.

[0073] When the cell is selected from the CHO-K1 cell line, the method of using release to maintain steady state specifically includes:

[0074] S1. The cells were inoculated into a shake tube and revived at 36.5°C, and amplified using basal medium until the viable cell density in the shake tube reached 10×106 After 10 cells / mL, the perfusion treatment was continued at 36.5°C with perfusion medium and a perfusion rate of 1 VVD per day;

[0075] S2, wait until the viable cell density in the shaking tube reaches 30×10 6 After the cell count reached 10 cells / mL, the temperature was lowered to 33°C, and perfusion was performed with a fixed cell ratio perfusion rate of 50 pL / (Cells·d) per day using perfusion medium. After 2 days, perfusion was performed every day and the perfusion was started at the same time. The fixed perfusion rate ΔIVCC was 70×10 6 cells / mL·day.

[0076] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0077] Experiment 1: One-step cooling and two-step cooling

[0078] Comparative Example 1

[0079] Cells (cell line: CHO-K1 SV) were cultured at 0.5×10 6 Cells / mL-2.0×10 6 Cells / mL were inoculated into a 50 mL shake tube with a working volume of 10 mL-20 mL.

[0080] Place the shake tube in a shaker at 36.5°C, 90% relative humidity, and 5% CO2 concentration. Set the speed to 160 rpm and tilt the shake tube at 15°. Cultivate until the cell density reaches ≥10×10 on the 4th day. 6 Cells / mL, constant 1VVD perfusion was started and culture was continued until the cell density reached 30×10 on the 6th day. 6 Cells / mL, and then cool the shaking tube to 33°C until the viability of the cultured cells decreases to 80%.

[0081] Example 1

[0082] This example adopts the same method as that of Comparative Example 1 for culturing, except that: when the cell density reaches 30×10 on the 6th day of culturing, 6 Cells / mL, and continue culturing until the 7th day when the cell density reaches 50×10 6Cells / mL, then cooled to 31°C and maintained for 4 days, i.e. after the 11th day of culture, the temperature was adjusted to 33°C until the cell viability decreased to 80%.

[0083] The lactic acid content and product yield, cell density and cell viability of the cells cultured in Comparative Example 1 and Example 1 were tested, respectively. Figure 1 , Figure 2 , Figure 3 , Figure 4 In the figure, the number -1 refers to the result of Comparative Example 1, and the number -2 refers to the result of Example 1.

[0084] from Figure 1-4 It can be seen that in Example 1, two-step cooling is adopted, that is, the culture environment temperature is first adjusted from 36.5°C to 31°C, and then the temperature is increased to 33°C, so that the cell density is maintained at a stable state. After culturing for 20 days, the cell viability is significantly higher than the one-step cooling condition in Comparative Example 1.

[0085] Figure 1 The lactic acid content was monitored from the 9th, 11th, 13th ... days, and the lactic acid production of Comparative Example 1 on the 11th day decreased significantly; and in Example 1, because the two-step cooling condition was used, the temperature was low on the 9th to 11th days, so the expression level of lactic acid was significantly lower than that of Comparative Example 1, but when the temperature was raised to 33°C, there was no significant difference between the expression level and that of Comparative Example 1.

[0086] By comparing the one-step cooling and the two-step cooling in Experiment 1, it is shown that the two-step cooling method in Example 1 can maintain the stability of cell density and achieve steady-state perfusion without additional discharge.

[0087] Experiment 2: Release at high density according to the first-order kinetic growth law

[0088] Comparative Example 2

[0089] Cells (cell line: CHO-K1 ZN) were cultured at 4.0×10 6 Cells / mL-6.0×10 6 Cells / mL were inoculated into a 50 mL shake tube with a working volume of 10 mL-20 mL.

[0090] Place the shake tube in a shaker at 36.5°C, 90% relative humidity, and 5% CO2 concentration. Set the speed to 160 rpm and tilt the shake tube at 15°. When the cell density reaches ≥10×10 6 Cells / mL, constant 1VVD perfusion was started. When the cell density reached 30×10 6Cells / mL, cool to 33°C and continue perfusion culture without draining.

[0091] Example 2

[0092] This example adopts the same method as that of Comparative Example 2 for culturing, except that: when the cell density reaches 30×10 6 Cells / mL, the temperature was lowered to 33°C, and perfusion culture was continued for 2 days, and then the release was started at the same time. The fixed release rate ΔIVCC was 50×10 6 Cells / mL·day. The daily release ratio is based on the following formula: VCD T＝n / VCD T＝n-1 =BP×VCD T＝n+1 / VCD T＝n (Wherein, VCD is the viable cell density, T=n is the culture on the nth day, T=n+1 is the culture on the n+1th day, and BP is the release ratio).

[0093] Example 3

[0094] This example adopts the same method as Example 2 for cultivation, except that after the release begins, the fixed release rate ΔIVCC for daily release is 60×10 6 Cells / mL·day.

[0095] Example 4

[0096] This example adopts the same method as Example 2 for cultivation, except that after the release begins, the fixed release rate ΔIVCC for daily release is 70×10 6 Cells / mL·day.

[0097] The cells cultured in Comparative Example 2 and Examples 2-4 were subjected to a series of tests. The test results are as follows: Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 and Fig.10 In the figure, the number -5 refers to the result of Example 2, the number -6 refers to the result of Example 3, the number -7 refers to the result of Example 4, and the number Control refers to the result of Comparative Example 2.

[0098] from Figure 5-10It can be seen that the cells in Example 2-4 using the release strategy can maintain high density and high vitality for 21 days, while the cell vitality of the cell cultured in Comparative Example 2 without release decreased to less than 90% on the 16th day, and the peak density of the cells was lower than that in Example 2-4. In addition, the daily release rate ΔIVCC in Example 4 was controlled to be 70×10 6 The cells with a yield of 100 cells / ml·day and the yield of cells harvested in a single day were also better than those in Examples 2-3 and Comparative Example 2, and the cumulative yield was also the highest.

[0099] Experiment 2 shows that the cell release strategy in Example 2-4 can maintain high-density cell culture, harvest more target products, and increase the utilization rate of the culture medium for the same number of culture days.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0101] In addition, those skilled in the art will appreciate that, although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, any of the above-mentioned claimed embodiments may be used in any combination. The information disclosed in this background technology section is intended only to deepen the understanding of the overall background technology of the present application and should not be regarded as an admission or in any form of implication that the information constitutes prior art known to those skilled in the art.

Claims

1. A method for maintaining steady state in a cell perfusion culture system, characterized in that: include: The cells are placed in a bioreactor and cultured at a temperature of T0 for the first time; When the viable cell density in the bioreactor reaches a first target value, the T0 temperature is lowered to the T1 temperature, and a second culture is performed using a perfusion medium. After maintaining for 4-5 days, the T1 temperature is raised to the T2 temperature, and a third culture is performed. The second culture and the third culture are not discharged. Wherein, T0>T2>T1; the temperature of T0 is 36℃-37℃, the temperature of T2 is 33℃-34℃, and the temperature of T1 is 30℃-32℃; The group of cells includes CHO-K1 cell line; The first target value is 50×10 6 cells / mL-75×10 6 cells / mL; The first culture comprises: Inoculating the cells in the bioreactor, resuscitating them at the T0 temperature and performing a proliferation treatment using a basal culture medium; When the viable cell density after the amplification treatment reaches 10×10 6 cells / mL-20×10 6 cells / mL, using the perfusion medium and performing perfusion culture at a fixed perfusion rate every day until the viable cell density in the bioreactor reaches the first target value; The fixed perfusion rate is 0.5VVD-1.5VVD; When the bioreactor is a shake tube, the cells are inoculated into the shake tube, the shake tube is placed in an environment at the T0 temperature, the relative humidity is 85%-95%, and the CO2 concentration is 4%-6%, and the shake tube is tilted 15°-20°, and the amplification process is performed at a rotation speed of 150rpm-180rpm; The second culture and the third culture each independently include: using the perfusion medium to perform perfusion at a fixed cell specific perfusion rate every day; The fixed cell specific perfusion rate is 20 pL / (Cells·d)-60 pL / (Cells·d).

2. The method for maintaining steady state of a cell perfusion culture system according to claim 1, characterized in that: When the cell is selected from the CHO-K1 cell line, it comprises: The cells were inoculated in a shake tube and revived at 36.5°C and amplified using a basal medium until the viable cell density in the shake tube reached 10×10 6 After that, the perfusion medium was used at 36.5°C and the perfusion rate was 1VVD per day. When the viable cell density in the shake tube reached 50×10 6 After the cells / mL were obtained, the temperature was lowered to 31°C, and the perfusion treatment was carried out every day using the perfusion medium at a fixed cell ratio perfusion rate of 50pL / (Cells·d). After maintaining for 4 days, the temperature was raised to 33°C, and the perfusion treatment was continued at a fixed cell ratio perfusion rate of 50pL / (Cells·d).

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