Cell culture method based on culture medium optimization and shear force protection

Abstract

The invention relates to the technical field of cell culture, in particular to a cell culture method based on culture medium optimization and shear force protection. The method comprises the following steps: adopting BG01 as a basic culture medium, adding Poloxamer188, and inoculating CHO (Chinese Hamster Ovary) cells; running at a constant temperature of 36.5-37.5 DEG C; carrying out segmented pH control and constant DO setting; switching the annular aerator into a microporous aerator in the fourth to sixth days of culture so as to improve mass transfer and reduce shearing; fF01 supplementary materials are added in batches according to a preset rhythm from the third day to the fifth day, and the total supplementary material amount is 25%-35% of the initial volume; and harvesting when the cell survival rate is reduced to 60-80%. The method inhibits hydrodynamic damage and metabolic fluctuation while ensuring oxygen supply, realizes high titer and stable key quality attribute, has good repeatability and magnification, and is suitable for producing recombinant proteins / antibodies by mammalian cells such as CHO and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, and in particular to a cell culture method based on medium optimization and shear force protection. BACKGROUND

[0002] The use of Chinese hamster ovary (CHO) cells for fed-batch culture is the mainstream technology for producing complex recombinant proteins. Although this process platform is mature, in the process of industrialization, it is always a difficult problem to be solved in the industry to realize the simultaneous optimization of high expression and high quality attributes of the product.

[0003] The existing process first faces the problem of matching the medium system and cell metabolism. If the nutritional ratio of the basic medium and the feed medium is not properly designed, it is easy to cause unbalanced cell metabolism, leading to abnormal accumulation of by-products such as lactic acid and ammonia, and fluctuation of osmotic pressure. This not only limits the high-density growth of cells and the efficient expression of products, but also directly damages the key quality attributes of the product. At the same time, the traditional carbon source supplement strategy mostly uses a fixed mode, lacking a trigger and feedback mechanism based on real-time metabolic state, which easily causes nutrient excess or starvation, further exacerbating metabolic disorders and batch-to-batch differences.

[0004] Secondly, the physical stress in the bioreactor and the complexity of process control constitute another bottleneck. In order to meet the oxygen consumption demand under high cell density, increasing the stirring speed and aeration amount is a common means, but the shear force generated thereby will damage the cells, leading to a decrease in survival rate, product aggregation or degradation. The existing process parameters, such as pH, dissolved oxygen, temperature, and feed height, are highly coupled, and isolated optimization often causes systematic fluctuations, making it difficult to achieve stable process scaling. Although adding shear protectants or using different types of gas distributors is a known means, there is still a lack of clear and scalable engineering strategies for when and how to intervene to achieve the minimization trade-off of oxygen supply and shear.

[0005] In summary, the fundamental problem existing in the field is that the existing cell culture process is disjointed in terms of medium matching, metabolic regulation, physical stress control, and multi-parameter collaborative management. This disconnection leads to poor repeatability, stability, and scalability of the process, and cannot continuously and stably produce products with expected yield and quality when transferring from the laboratory to the production scale. Therefore, there is an urgent technical need and great industrial value to develop a new cell culture process that can systematically solve the above problems and achieve integrated and precise regulation throughout the process. SUMMARY

[0006] To solve the defects of the prior art, the present application provides a cell culture method based on medium optimization and shear force protection, comprising:

[0007] (1) Prepare the basic culture system in the bioreactor: select BG01 medium and add Poloxamer188 to make its concentration in the system 0.5-2.0 g / L; inoculate CHO cells: set the initial inoculation density to 0.5-0.8 x 10 6 cells / mL, and set the working volume to 1000-2000 mL;

[0008] (2) Constant temperature operation: maintain the culture temperature at 36.5-37.5℃;

[0009] (3) Stepwise pH control: maintain the pH at 7.00±0.20 in the pre-culture stage and 6.70-6.90 in the post-culture stage;

[0010] (4) Constant dissolved oxygen control: maintain the dissolved oxygen at 30-50% air saturation by gas composition, aeration rate and stirring cascade adjustment;

[0011] (5) Aeration and stirring setting: air aeration rate is 0.005-0.02 vvm, and stirring speed is set to 250-300 rpm;

[0012] (6) Oxygen transfer strategy: switch from ring aeration to microporous aeration from the 4th day to the 6th day of culture;

[0013] (7) Feeding and carbon source control: from the 3rd day to the 5th day, add FF01 feeding medium every 24±4 hours based on the initial working volume, 3-5% (v / v) at a time, to a total of 25-35% (v / v), or one day before harvest, whichever comes first;

[0014] (8) Terminate the culture and harvest when the cell survival rate decreases to 60-80%.

[0015] Preferably, the amount of Poloxamer188 used in step (1) is 0.8-1.2 g / L.

[0016] Preferably, the initial inoculation density in step (1) is 0.60-0.70 x 10 6 cells / mL.

[0017] Preferably, the culture temperature in step (2) is 36.8-37.2℃.

[0018] Preferably, the dissolved oxygen in step (4) is set to 35-45%.

[0019] Preferably, the air aeration rate in step (5) is 0.008-0.015 vvm.

[0020] Preferably, the stirring speed in step (5) is 260-290 rpm.

[0021] Preferably, the aeration switch point of step (6) is on day 4 or day 5 of the culture.

[0022] Preferably, in step (7), the glucose concentration is monitored during the culture, and when the detected value is lower than the trigger threshold of 2.5-4.0 g / L, it is replenished to the target interval of 5.0-9.0 g / L.

[0023] Preferably, in the carbon source control of step (7), the glucose trigger threshold is 2.8-3.2 g / L, and the replenishment target interval is 6.0-8.0 g / L.

[0024] Preferably, in step (7), the volume of a single replenishment of glucose solution does not exceed 1-3% of the culture volume.

[0025] Preferably, step (5) and step (6) are used in coordination to meet the oxygen demand in the middle and late stages, wherein the DO set point remains unchanged.

[0026] Preferably, in the repeated batch operation of the method, the batch-to-batch coefficient of variation of the peak viable cell density and the final product titer is not more than 10%, preferably not more than 5%.

[0027] Preferably, after purification, the SEC aggregate content of the product is less than 8%, preferably less than 5%.

[0028] The pre-culture section is the period from inoculation to day 4-6 of the culture; the post-culture section is from this time point to the end of the culture.

[0029] The CHO cell is selected from the following group: CHO-S130, CHO-S100, CHO-S136, CHO-S, CHO-K1, CHO-K1SV, CHO-DG44, CHO-DUKX-B11 (or DXB11), a suspension-adapted CHO-K1 derivative strain, a GS-deficient CHO (such as CHO-K1SV GS- / -, CHOZN® GS- / -, etc.) or a derivative strain thereof.

[0030] The BG01 medium, i.e. the Dynamis™ AGT™ medium, can be used for recombinant CHO cell culture in an environment with clear chemical composition, with high batch and fed-batch culture performance and yield; the formula with clear chemical composition, no protein and no animal-derived components can achieve high titer, faster start of process development, and simplify the transfer to scale production (manufacturer: Gibco, catalog number: A26175-01).

[0031] The FF01 feed medium, i.e., BalanCD CHO Feed 4 medium, is a nutrient-rich supplement that can be used in conjunction with any growth medium and is designed to fully exploit the potential of high-performance CHO cell lines to boost the production of recombinant proteins, especially therapeutic antibodies, while ensuring product quality (manufacturer: Fujifilm Irvine, catalog number: 94134).

[0032] The method significantly reduces the local shear caused by large bubble breakage and high rotation speed, and inhibits cell membrane damage and stress response, thereby maintaining a higher and stable cell viability and secretion efficiency, while ensuring sufficient oxygen supply. In the process control dimension, the segmented pH, constant DO, and split feeding, trigger, and carbon source replenishment strategies work together to control the peak values of by-products such as lactic acid and ammonia, reduce the osmotic pressure fluctuations, and make the acid-base and dissolved oxygen curves of the culture medium more consistent with the set band. As a result, the titer plateau is extended, the unit feed yield is improved, the end-point yield is stably improved, and the key quality attributes such as SEC aggregates and RP-HPLC charge / hydrophobic heterogeneity are maintained within the enterprise threshold, and the batch-to-batch consistency is significantly enhanced.

[0033] In terms of repeatability, multiple batches verified under the same process window show that the batch-to-batch variation coefficient CV of cell growth curve, viability decay trend, and end-point titer is maintained at a low level, preferably ≤5%, not higher than 10%, the quality attribute distribution is stable, the deviation is controllable, and the data reproducibility is good. In terms of scale-up, the method uses equal unit volume power as the engineering criterion for cross-device migration, combined with fixed DO set point, segmented pH, standardized feeding rhythm, and consistent aeration switching schedule, to obtain equivalent hydrodynamic and mass transfer environment between reactors of different brands and scales. The process and end-point quality after scale-up are statistically consistent with the small-scale baseline.

[0034] In summary, the method realizes the simultaneous optimization of high titer and high quality without relying on additional metabolic reprogramming or sugar precursor additives, has the engineering characteristics of simplicity, robustness, reproducibility, and scalability, and is suitable for the industrial production of recombinant proteins / antibodies of CHO and other mammalian cells. DETAILED DESCRIPTION

[0035] The endpoints of the ranges and any values disclosed in this specification are not limited to the precise values stated. These ranges and values should be construed and understood to include values adjacent and approximating these ranges and values. For ranges of values, the endpoints of each range are included in the range, and the ranges are inclusive of the individual values.

[0036] The point values can be combined with each other and with individual point values to obtain one or more new numerical value ranges, which should be considered as specifically disclosed in the present application.

[0037] A. Shake flask experiment

[0038] 1. Medium screening

[0039] The cell strain used for medium screening was S130 (PCB), and the scheme is shown in the following table (Table 1-1). The incubation conditions of Group 1 were 37°C, 120 rpm, 5% CO2, and 80% RH; the incubation conditions of Group 2 were 36.5°C, 120 rpm, 5% CO2, and 80% RH. Cell growth and metabolism were detected daily from Day 3, and the glucose concentration was supplemented to 6-8 g / L after the concentration was lower than 3 g / L.

[0040] Table 1-1 Medium screening experiment scheme

[0041] Screening result analysis:

[0042] Table 1-2 Summary of medium screening quality detection data

[0043] According to the quality detection results (Table 1-2), except that Flask-01 and Flask-04 had a shorter shake flask culture period (6 days) and lower expression of target protein, and no corresponding detection of target protein concentration in cell supernatant was performed, and combined with the pCQA evaluation in the early stage of the project, it was determined to select the proportion of peak 1 higher than the proportion of peak 2 as the protein development target of the project. Among all the shake flasks, Flask-03, Flask-05, Flask-06, Flask-07, Flask-08, Flask-09, Flask-12, Flask-14, and Flask-15 had relatively high proportions of peak 1. Considering the accumulation of lactic acid concentration in the later stage and the stability risk assessment of the material source, the shake flask processes finally confirmed by the 2L bioreactor were Flask-06, Flask-07, Flask-08, and Flask-09.

[0044] 2. Culture temperature screening

[0045] The cell strain is S130 (PCB), and the experimental scheme is shown in Table 2-1. The shaking culture conditions are 120 rpm, 8% CO2, and 80% RH. Cell growth and metabolism are detected daily from Day 3, and the glucose concentration is supplemented to 6-8 g / L after feeding when it is lower than 3 g / L.

[0046] Table 2-1 Temperature research experimental scheme

[0047] Screening result analysis:

[0048] Table 2-2 Temperature research expression detection data summary

[0049] Table 2-3 Temperature research quality detection data summary

[0050] According to the expression and quality detection data of the temperature research (Tables 2-2 and 2-3), the control strategy of reducing the culture temperature on Day 5 can maintain the cell viability in the later stage of cell culture, prolong the cell culture time, and improve the final expression. The SEC purity detection results show that the temperature reduction culture process is beneficial to improve the proportion of the main peak, but the proportion of peak 1 in the RP-HPLC detection results of the experimental group is lower than that of the control group. The glycosylation modification changes after temperature reduction culture, and the peak type shifts to peaks 2 and 3 as a whole. The quality results do not meet the expectations, so the cell culture process does not adopt the temperature reduction strategy.

[0051] 3. Additive effect research

[0052] The cell strain is S130 (PCB), and the experimental scheme is shown in Table 2-1. The shaking culture conditions are 120 rpm, 8% CO2, and 80% RH. Cell growth and metabolism are detected daily from Day 3, and the glucose concentration is supplemented to 6-8 g / L after feeding when it is lower than 3 g / L.

[0053] Table 3-1 Additive effect research experimental scheme

[0054] Research result analysis:

[0055] Table 3-2 Additive effect research expression detection data summary

[0056] Table 3-3 Summary of quality detection data of additive effect study

[0057] Based on the expression and quality detection results of all the shake flask with additives (Table 3-2, 3-3), Flask-39 performed the best. Flask-39 used BG01 as basal medium, FG06 as feed medium, and 0.5% Gal + V2, CDFS05 was added at D0. The proportion of peak 1 was greater than peak 2 in the RP-HPLC results, which was contrary to its control Flask-37. Therefore, the effect of adding 0.5% Gal + V2, CDFS05 in larger scale cell culture can be further verified in the subsequent experiments.

[0058] B. 2L bioreactor cultivation process study

[0059] 1. Medium confirmation and pH control strategy study

[0060] The cell strain used in the medium confirmation and pH control strategy study was S130 (PCB), and the scheme is shown in Table (4-1, 4-2). The 2L bioreactor used was from Sartorius. The centrifugal supernatant was sent for SEC / RP-HPLC after one-step purification.

[0061] Table 4-1 Experimental scheme of medium confirmation and pH control strategy study-1

[0062] Table 4-2 Experimental scheme of medium confirmation and pH control strategy study-2

[0063] Analysis of the results of the study:

[0064] Table 4-3 Summary of growth data of medium confirmation and pH control strategy study

[0065] Table 4-4 Summary of expression detection data of medium confirmation and pH control strategy study

[0066] Table 4-5 Summary of expression detection data of medium confirmation and pH control strategy study

[0067] The cell growth and metabolism data and quality results of different medium feeding combinations were analyzed comprehensively (Tables 4-3, 4-4, and 4-5). The cell growth and metabolism were good. The RP-HPLC results met the process development requirements of Vessel02, Vessel03, and Vessel05. The pH control strategy of Vessel05 was different from that of Vessel02 and Vessel03, and the expression level was the lowest. Therefore, the subsequent pH control strategy was determined to be 7.00±0.30 (D0-D5) and 6.80±0.10 (D5-end). The difference between Vessel02 and Vessel03 was that 6 mM glutamine was added to the basic medium of Vessel02, but this had no obvious positive effect on the proportion of peak 1 in the RP-HPLC detection results. Therefore, the medium combination in the subsequent cultivation process was determined to be BG01+FF01.

[0068] 2. Additive effect confirmation

[0069] The cell strain used for additive effect research was S130 (PCB), and the experimental scheme is shown in the following table (Tables 5-1 and 5-2). The control group without additives was Vessel02 and Vessel03 in "1. Medium confirmation and pH control strategy research", and the 2L bioreactor manufacturer was Sartorius. The centrifugal supernatant after chromatography was sent for SEC / RP-HPLC detection.

[0070] Table 5-1 Additive effect confirmation experiment scheme-1

[0071] Table 5-2 Additive effect confirmation experiment scheme-2

[0072] Research result analysis:

[0073] Table 5-3 Additive effect confirmation expression level detection data summary

[0074] Table 5-4 Additive effect confirmation quality detection data summary

[0075] As shown in Table 5-3 and Table 5-4 above, the expression level, SEC, and RP-HPLC results of the two groups of bioreactors, Vessel02 / Vessel06 and Vessel03 / Vessel07, were consistent and had no significant difference. The addition of additives in the 2L reactor had no obvious effect on the improvement of RP peak 1. Although it was found in the shake flask stage that the addition of 0.5% Gal+V2, CDFS05 could significantly improve the proportion of peak 1 in the RP-HPLC detection results, the experimental data of the 2L bioreactor confirmation stage showed that under the culture conditions, the addition of 0.5% Gal+V2, CDFS05 had no obvious positive effect on the improvement of the proportion of peak 1 in the RP-HPLC detection results, therefore, no additives were used in the subsequent culture process of the project.

[0076] 3. Shear force study

[0077] The cell strain used in the shear force study was S130 (PCB), and the experimental scheme is as shown in the following tables (Table 6-1 and Table 6-2). Vessel13 was a duplicate group of Vessel08. Among them, Vessel11 and Vessel12 used bioreactors from Thermo, and the remaining 2L bioreactors used were from Sartorius; the centrifuged supernatant was sent for SEC / RP-HPLC detection after chromatography, and the rotation speed conversion between different reactors followed the principle of equal P / V.

[0078] Table 6-1 Shear force study experimental scheme-1

[0079] Table 6-2 Shear force study experimental scheme-2

[0080] Analysis of research results:

[0081] Table 6-3 Summary of growth data of shear force study

[0082] Table 6-4 Summary of quality detection data of shear force study

[0083] According to the cell growth data and quality detection results (Table 6-3, 6-4), it can be found that under the condition of using micro-sparger aeration strategy, the addition of 1 g / L Poloxamer 188 has no effect on SEC purity and peak type results of reverse phase chromatography detection, but has a positive effect on cell viability. By adding Poloxamer 188 to the culture medium, the cell growth density and cell viability in the late stage of cell culture are higher, and the aeration amount during the culture process is reduced. In contrast, under the condition of not using micro-sparger aeration strategy, whether Poloxamer 188 is added or not has no effect on cell growth. However, the addition of Poloxamer 188 may cause the proportion of peak 1 in RP-HPLC detection results to decrease.

[0084] Poloxamer 188 is widely used as a shear protection agent in cell culture processes to protect cells from turbulent flow in the bioreactor environment. Since the aeration amount increases with the scale-up of the culture scale, the potential risk of increasing aeration amount is to cause the clogging of the tail gas filter and the increase of bag pressure. Therefore, microbubbles are used to control the dissolved oxygen level of the culture system. The addition of Poloxamer 188 can reduce the risk of using microbubbles during process scale-up. Finally, 1 g / L Poloxamer 188 is added to the basal medium for cell culture in this project.

[0085] 4. Process stability confirmation

[0086] Process stability confirmation, see "3. Shear force study" for Vessel08, Vessel09, Vessel13.

[0087] Analysis of research results:

[0088] Table 7-1 Summary of process stability confirmation expression detection data

[0089] Table 7-2 Summary of process stability confirmation quality detection data

[0090] Under the culture process conditions of Vessel08 (Vessel13) or Vessel09, the cell growth and metabolism are good, the expression and quality detection results (Table 7-1, 7-2) are also as expected, and the results of the three bioreactors are basically consistent, proving that the process is relatively stable. Therefore, the process is used as the final process for 2L bioreactor.

[0091] C. Overall research conclusion

[0092] The cell culture development experiment of the project can be divided into two stages. The shake flask stage includes medium screening, culture temperature research and additive effect research. The 2L bioreactor stage includes pH control strategy research, additive effect confirmation, shear force research and process stability confirmation. Finally, a cell culture process with good repeatability, stability and scalability is obtained. The specific process parameters are shown in the following table.

[0093] Table 8 2L bioreactor (Sartorius) culture process

[0094] Example 1:

[0095] The embodiment of the application provides an optimized process for culturing CHO cells to produce recombinant proteins. The process realizes the simultaneous optimization of high expression quantity and high quality attributes of products through systematic medium optimization, precise process control and innovative shear force protection strategy.

[0096] 1. Cell strain and medium preparation

[0097] Cell strain selection and preparation: The engineered Chinese hamster ovary cell CHO-S130 (preservation number: PCB) is selected, which has stable growth characteristics and high efficient target protein expression capacity. Before inoculation, the frozen cells are quickly recovered in a 37℃ water bath, transferred to a shake flask containing BG01 basic medium, and cultured at 37℃, 120rpm, 5% CO2, 80% humidity in a shaking bed. The cell density and survival rate are monitored daily to ensure that the seed liquid is in the logarithmic growth phase and the viability is higher than 95%, providing high quality inoculum for bioreactor inoculation.

[0098] Basic medium preparation: Commercial BG01 medium is used as the basic medium. The medium provides the basic nutrients required for cell growth, including amino acids, vitamins, inorganic salts and carbon sources. In this embodiment, the key improvement is to add Poloxamer 188 with a final concentration of 1.0g / L to the BG01 basic medium. This non-ionic surfactant acts as a shear force protector, which can effectively reduce cell membrane damage caused by stirring vortex and bubble rupture by adsorbing on the cell membrane and gas-liquid interface. The medium is sterile filtered through a 0.22μm filter before use, and samples are taken for sterility test and osmotic pressure test.

[0099] Feed medium and nutrient solution:

[0100] Feed medium: Commercially available FF01 medium was used as feed. This feed concentrate is rich in specific amino acids, vitamins and other key nutrients, and its formulation is highly matched with the BG01 basal medium, aiming to support the nutritional needs of cells and product synthesis during the high-density cultivation phase.

[0101] Glucose stock solution: A glucose solution with a concentration of 400 g / L was prepared as a feed for the main carbon source, and was treated with high-pressure sterilization or sterile filtration before use.

[0102] 2. Bioreactor initialization and inoculation

[0103] Reactor system and preparation: This example used a Sartorius Biostat B-DCU type 2L glass bioreactor system. The reactor and all pipelines and sensors (pH, DO) that contact the culture solution were sterilized with high-pressure steam at 121°C for 30 minutes before use to ensure a sterile state.

[0104] Medium loading and initialization: About 1.2L of the basal medium (BG01 + 1.0 g / L Poloxamer 188) was added to the reactor. Subsequently, the reactor control system was connected, the stirring (initially set to 100 rpm) and temperature control system (37°C) were started, and sterile-filtered air / CO2mixed gas was introduced to equilibrate the medium. At the same time, the pH and DO sensors were calibrated.

[0105] Inoculation operation: After the temperature, pH and DO of the medium were stable, the CHO-S130 seed solution in the logarithmic growth phase was accurately calculated and added to the reactor under a sterile operation table. The initial inoculation density was controlled at 0.65 x 10 6 cells / mL by counting with a cell counter. After inoculation, the working volume of the reactor was accurately supplemented to 1500 mL with fresh basal medium. After inoculation was completed, the stirring speed was immediately increased to the set value, and the 0th day (D0) of the culture was recorded.

[0106] 3. Culture process control

[0107] The culture process was accurately controlled according to the following integrated parameters, and all controls were automatically completed by the reactor control system, and real-time trend data of key parameters were recorded.

[0108] Temperature control: The culture temperature was controlled at 37.0±0.1°C throughout the process. This temperature was determined by shake flask screening (compared with 36.5°C and 37°C) to be most conducive to the growth and product expression of CHO-S130 cells.

[0109] pH control: An optimized two-stage control strategy was used to match the metabolic needs of cells at different growth stages.

[0110] Day 0 to Day 5 (D0-D5): pH is controlled at 7.00 ± 0.20. The higher pH in this stage is beneficial for the rapid proliferation of cells. The precise adjustment is achieved by automatically bubbling CO2 gas and dropping sodium bicarbonate solution with a concentration of 7.5% (w / v).

[0111] Day 5 to the end of the culture (D5-End): The pH set point is adjusted to 6.80 ± 0.10. In the later stage of the culture, a moderate decrease in pH helps to reduce the accumulation of metabolic byproducts such as lactic acid, improve cell viability, and is conducive to maintaining the stability of product proteins and improving quality attributes.

[0112] Dissolved oxygen (DO) control: Through a cascade control strategy, the dissolved oxygen level is strictly controlled at 40% ± 3% air saturation. The control priority is as follows: adjusting the oxygen proportion in the incoming gas (0-100% O2) > adjusting the stirring speed (within the set range) > adjusting the total volume aeration rate. This setting provides a stable and sufficient oxygen supply for cell metabolism.

[0113] Stirring and aeration strategy:

[0114] Stirring: The stirring speed is controlled at a constant 275 rpm. This speed is determined based on the principle of equal unit volume power (P / V), which can control the shear force within an acceptable range while providing sufficient mixing and mass transfer, and forms a synergistic protection with Poloxamer 188.

[0115] Aeration: The total volume aeration rate (Air) is controlled at 0.01 vvm.

[0116] Dynamic oxygen transfer strategy: To solve the contradiction between oxygen supply demand and shear injury at high cell density, the present invention implements a dynamic distributor switching strategy. In the early stage of culture (D0 to D4 / D5), a ring-shaped distributor is used for aeration, which is robust and not easy to block. When the cells enter the late logarithmic growth phase (about the 4th day or the 5th day, judged by the viable cell density > 15 × 10 6 cells / mL), the aeration distributor is switched to a microporous distributor. The microporous distributor can generate a large number of fine bubbles, significantly increasing the gas-liquid mass transfer area, achieving efficient oxygen supply under the same or lower aeration rate, while greatly reducing cell shear injury caused by bubble rupture due to the small size and low rupture energy of the bubbles.

[0117] 4. Feeding and nutrient control strategy

[0118] FF01 feeding strategy: A time-driven and metabolic state-combined feeding method is used. From the 4th day of culture, FF01 feed medium is added daily according to the following precise plan (all percentages are volume percentages relative to the initial culture volume of 1500 mL):

[0119] Day 4 (D4): 4% (i.e. 60 mL) was added.

[0120] Day 5 (D5): 5% (i.e. 75 mL) was added.

[0121] Day 6 (D6): 5% (i.e. 75 mL) was added.

[0122] Day 7 (D7): 5% (i.e. 75 mL) was added.

[0123] Day 8 (D8): 5% (i.e. 75 mL) was added.

[0124] Day 9 (D9): 5% (i.e. 75 mL) was added.

[0125] The total amount of feed was 29% of the initial volume. This strategy ensured a continuous supply of nutrients, avoiding nutrient depletion, and supported high levels of product synthesis.

[0126] Glucose concentration control: Threshold-based feedback control was implemented. From day 3 of cultivation, samples were taken at least once daily and the glucose concentration in the culture broth was monitored using a biochemical analyzer. When a trigger threshold of 3.0 g / L was monitored, 400 g / L of glucose stock solution was immediately added to bring the concentration to the target range of 6.0-8.0 g / L. This "trigger-replenish" logic effectively prevented cell stress and excessive lactate production caused by carbon starvation, while avoiding osmotic shock caused by one-time replenishment of too much. The volume of single replenishment of glucose stock solution did not exceed 2% of the current culture volume.

[0127] 5. Process monitoring and sample analysis

[0128] Daily monitoring: From day 3 after inoculation, samples were taken aseptically daily. An automatic cell counter was used to determine the viable cell density and cell viability. Biochemical analyzers were used to detect the concentrations of key metabolites such as glucose, lactate, glutamine, ammonia, etc.

[0129] Product and quality analysis: Samples were taken regularly during cultivation, and after centrifugation, the supernatant was collected and small-scale purified by protein A affinity chromatography for monitoring the expression titer of the target protein. Harvest batches, host cell proteins.

[0130] Process consistency evaluation: The curves of all key process parameters pH, DO, temperature, as well as the kinetic data of cell growth, metabolism and product expression were recorded and analyzed for evaluating the stability of the process.

[0131] 6. Harvest

[0132] When cell viability dropped to 70%, it was determined as the optimal harvest point. The culture in the reactor was terminated and all the culture broth was transferred to a harvest bag. Subsequently, cells and cell debris were removed by continuous flow centrifugation or depth filtration to obtain a clarified supernatant, which was stored at -80°C or immediately subjected to downstream purification.

[0133] 7. Process performance verification and data

[0134] Three batches of repeated experiments were performed continuously on a 2L Sartorius bioreactor using the complete process parameters described in this example.

[0135] The cell growth curves of the three batches were highly overlapped, with the peak viable cell density stably at 22-24 x 10 6 cells / mL, and the time to reach the peak was consistent. Lactic acid metabolism showed a typical production and consumption pattern, and the peak concentration was controlled at a low level (<2 g / L), indicating healthy metabolism.

[0136] The expression titer of the final target protein was highly consistent between the three batches, and the batch-to-batch coefficient of variation CV was 3.5%, which was significantly better than the industry standard (usually requiring CV <10%).

[0137] Analysis of the purified target protein showed that the size exclusion chromatography (SEC) results showed that the aggregate content was stably between 3.4%, which was lower than the enterprise standard of 5%; the reverse phase high performance liquid chromatography (RP-HPLC) analysis showed that the main peak percentage was stably at 92-94%, and the charge heterogeneity distribution was consistent. This proved that the process not only achieved high yield, but also stably produced products with high quality attributes.

[0138] The process considered the scaling-up requirement from the beginning of the design. The clear shear force control strategy, the stirring setting based on the equal P / V principle, and the standardized pH, DO, and feed control strategy made the process parameters directly related to the physicochemical and biological effects, rather than relying on specific equipment. This laid a solid foundation for smooth scaling-up from 2L laboratory scale to 200L, 2000L, or even larger production scale, ensuring the consistency of process performance after scaling-up.

[0139] The preferred embodiments of the present application are described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and changes without creative labor based on the concept of the present application. Therefore, any technical solutions obtained by logical analysis, reasoning or limited experiments based on the prior art according to the concept of the present application shall be within the protection scope defined by the claims.

Claims

1. A cell culture method based on culture medium optimization and shear force protection, characterized in that, include: (1) Preparation of the basic culture system in the bioreactor: BG01 medium was used, and Poloxamer 188 was added to make the concentration in the system 0.5-2.0 g / L; CHO cells were inoculated: the initial inoculation density was set to 0.5-0.8 × 10⁶ cells / L. 6 cells / mL, and set the working volume to 1000-2000mL; (2) Constant temperature operation: Maintain the culture temperature at 36.5-37.5℃; (3) Segmented pH control: The pH is maintained at 7.00±0.20 in the first stage of culture and at 6.70-6.90 in the second stage of culture; the first stage of culture is the period from inoculation to the 4th-6th day of culture; the second stage of culture is from the time point to the end of culture. (4) Constant dissolved oxygen control: Dissolved oxygen is maintained at 30-50% air saturation by adjusting gas composition, ventilation rate and stirring cascade; (5) Ventilation and stirring settings: air flow rate is 0.005-0.02vvm, stirring speed is set to 250-300rpm; (6) Oxygen transfer strategy: From day 4 to day 6 of culture, the ring aerator was switched to a microporous aerator; (7) Feeding and carbon source control: From day 3 to day 5, FF01 feeding medium was added in batches according to the predetermined rhythm, with the total feeding amount being 25-35% of the initial culture volume; (8) When the cell viability drops to 60-80%, stop the culture and harvest.

2. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, In step (1), the dosage of Poloxamer188 is 0.8-1.2 g / L.

3. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, The initial inoculation density in step (1) is 0.60-0.70 × 10⁻⁶. 6 cells / mL.

4. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, The culture temperature in step (2) is 36.8-37.2℃.

5. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, The dissolved oxygen in step (4) is set to 35-45%.

6. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, The air ventilation rate in step (5) is 0.008-0.015vvm.

7. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, In step (7), glucose concentration is monitored during the culture process. When the detected value is lower than the trigger threshold of 2.5-4.0 g / L, it is replenished to the target range of 5.0-9.0 g / L.

8. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, In step (7), the volume of glucose solution replenished in a single step shall not exceed 1-3% of the culture volume.

9. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, In repeated batch runs, the inter-batch coefficient of variation for peak viable cell density and final product titer does not exceed 10%.

10. The cell culture method based on culture medium optimization and shear force protection as described in claim 1, characterized in that, The CHO cells are selected from one of the following: CHO-S130, CHO-S100, CHO-S136, CHO-S, CHO-K1, CHO-K1SV, CHO-DG44, CHO-DUKX-B11, DXB11, suspension CHO-K1 derivatives, and GS-deficient CHO.

Images