A method for cryopreservation culture of CHO cells for expressing dulaglutide

Abstract

The present invention discloses a cryopreservation method for CHO cells expressing dulaglutide, which is a cryopreservation method that prolongs the culture time and maintains a high cell viability rate. This method uses CHO K1SV cells as the culture object, and through optimization experiments on two factors, the cooling temperature and time, it is determined that the cooling time in the first stage is on the 3rd day of culture, and the cells are cooled to 35 °C for culture. The cooling time in the second stage is on the 6th day of culture, and the cells are cooled to 30 °C for culture, and the cooling temperature in the second stage is maintained until the end of the culture. The said method can prolong the culture time, increase the supernatant expression level, maintain a high cell viability rate, reduce the proportion of acidic peaks, and improve the product quality.

Description

Technical Field

[0001] The invention belongs to the technical field of CHO cell culture, and particularly relates to a method for culturing CHO cells for expressing dulaglutide at low temperature. Background Art

[0002] Chinese hamster ovary (CHO) cells are one of the main host expression systems used in the current biopharmaceutical industry, producing products such as monoclonal antibodies, bispecific antibodies, and fusion proteins. While process development has been continuously progressing, cell line engineering and clone selection technologies have greatly increased the yield of recombinant monoclonal antibodies, but how to ensure consistent product quality remains a major challenge.

[0003] Sialylation modification and asparagine deamidation are the main reasons for antibodies to produce acidic substances. There have been relatively many reports in the former relevant literature, and the main reason for its formation lies in the anions existing in sialic acid itself. Asparagine deamidation leads to the appearance of acidic substances due to the introduction of anions, often occurring in the variable region of antibodies, especially in the CDR regions that are exposed and prone to deformation. As a result, aspartic acid or isoaspartic acid will be produced, and the flexibility of the CDR regions will cause mutual influence between different CDRs of antibodies.

[0004] Some literature reports point out that reducing acidic substances in antibody drugs is more important than reducing basic substances. Compared with basic substances, acidic substances will cause a reduction in the in vivo half-life of monoclonal antibody drugs and an acceleration of blood clearance rate, and are adverse to the drug efficacy. For example, the acidic variant of trastuzumab has a lower binding ability to HER2. Basic substances mostly have a positive impact on drug efficacy. For example, in many cases, basic variants show stronger affinity and stronger ADCC effect. However, it is generally considered that protein products with an isoelectric point difference greater than 1 will have significant differences in tissue distribution and pharmacokinetics.

[0005] A large number of literature reports point out that the more suitable temperature for culturing most mammalian cells is generally around 37°C. Too high or too low temperature will inhibit cell growth. However, many experimental studies have shown that reducing the culture temperature can induce the accumulation of G1-phase cells, improve the stability of mRNA, and thus improve the protein expression level. But some studies have also shown that reducing the culture temperature not only cannot improve the product expression level, but also inhibits cell proliferation and reduces the number of viable cells, affecting the accumulation of products and the sialic acid content. Therefore, the influence of temperature on product expression and its quality depends on the cell line and the type of expressed product.

[0006] Currently, most cooling processes are to cool down to 35°C or 33°C. For specific expression hosts, it is still a difficult problem in this field to improve the yield and product quality by controlling the culture conditions. Summary of the Invention

[0007] The present invention discloses a low-temperature culture method for CHO cells used for expressing dulaglutide. Through optimization experimental research on two factors, namely the cooling temperature and time, a culture process of cooling to 35°C on the third day and cooling to 30°C on the sixth day is determined. The method can extend the culture time, increase the supernatant expression level, maintain a high cell viability rate, reduce the proportion of acidic peaks, and improve the product quality.

[0008] A low-temperature culture method for CHO cells used for expressing dulaglutide, which comprises the following steps of inoculation culture and cooling culture:

[0009] The inoculation culture temperature is controlled at 36 - 37°C, the cooling temperature is controlled at 28 - 35°C, the cooling time is controlled on the 2nd - 7th day, and the cooling is carried out in more than two stages. In the first-stage cooling, it is cooled to 35°C for culture, and in the second-stage cooling, it is cooled to 30°C for culture, and the second-stage cooling temperature is maintained until the end of the culture.

[0010] In some embodiments, the first-stage cooling is carried out on the 2nd - 4th day, cooled to 35°C for culture, and the second-stage cooling is carried out on the 5th - 7th day, cooled to 30°C for culture.

[0011] In some embodiments, the first-stage cooling is carried out on the 3rd day, cooled to 35°C for culture, and the second-stage cooling is carried out on the 6th day, cooled to 30°C for culture.

[0012] In some embodiments, the cells are CHO K1 SV cells, with the auxiliary plasmid pOG44, and glutamine needs to be additionally added to the culture medium.

[0013] In some embodiments, the culture method is shake flask culture, the amplitude of the shaker is 25 mm, and the rotation speed is controlled at 140 rpm.

[0014] In some embodiments, the end of the culture means culturing until the 14th day or the cell viability rate drops to 80% to end the culture.

[0015] In some embodiments, the entire culture cycle of the culture method is 14 days.

[0016] In some embodiments, the culture method comprises the following steps:

[0017] 1) The basal medium is SFM4CHO, and the feeding medium is Feed 2;

[0018] 2) After resuscitating the cells in the cell bank and subculturing them for 4 generations, after the cell growth rate is stable, inoculate them in a 125 mL shake flask for culture at a density of 0.5×10 6 cells / mL, and the liquid loading volume is 20 mL;

[0019] 3) Add 4 mM of glutamine during inoculation, and add 2 mM of glutamine on the 2nd and 4th days of culture;

[0020] 4) Take 0.5 mL of samples on the 3rd, 5th, 7th, 9th, 11th, and 13th days of culture to detect cell density, cell viability, glucose, glutamine, and lactate. At the end of the culture, detect the supernatant expression level and the proportion of acid peaks;

[0021] 5) Add the feeding medium on the 3rd, 5th, and 8th days respectively. The feeding medium is Feed2 + N-acetyl-D-glucosamine, and the added volume is 1 / 9 of the initial culture volume;

[0022] 6) Inoculate and culture, and cool down for culture.

[0023] The low-temperature culture method for CHO cells for expressing dulaglutide provided by the present invention can maintain the cell viability above 80% for a long time compared with the non-cooling process. The final supernatant expression level is increased by about 2 times, and the proportion of acid peaks is reduced to about 23%, significantly improving the product yield and quality. Description of the Drawings

[0024] Figure 1 It is the viable cell growth change curve of Example 2.

[0025] Figure 2 It is the cell viability change curve of Example 2.

[0026] Figure 3 It is the lactic acid metabolism change curve of Example 2.

[0027] Figure 4 It is the glutamine metabolism change curve of Example 2.

[0028] Figure 5 It is the glucose consumption change curve of Example 2.

[0029] Figure 6 It is the result of the supernatant expression level of Example 2.

[0030] Figure 7 It is the viable cell growth change curve of Example 3.

[0031] Figure 8 It is the cell viability change curve of Example 3.

[0032] Figure 9 It is the lactic acid metabolism change curve of Example 3.

[0033] Figure 10 It is the glutamine metabolism change curve of Example 3.

[0034] Figure 11Glucose consumption change curve for Example 3.

[0035] Figure 12 Supernatant expression results for Example 3. Detailed implementation manners

[0036] The present invention will be further described in detail below in conjunction with examples, but the implementation manners of the present invention are not limited thereto.

[0037] Construction of CHO engineering cells expressing dulaglutide in Example 1

[0038] Construct its expression plasmid with reference to the dulaglutide sequence described in Patent CN1802386B, co-transfect the auxiliary plasmid pOG44 and the above expression plasmid into FlpIn CHO cells, and the CHO cells are CHO K1 SV, and screen to obtain CHO engineering cells stably expressing dulaglutide.

[0039] Optimization of the cooling culture time of CHO engineering cells in Example 2

[0040] 1. Preparation of culture medium

[0041] (1) SFM4CHO culture medium

[0042] Weigh 19.77 g of SFM4CHO dry powder and place it in a 2 L beaker, add 750 ml of purified water, put in a magnetic stir bar, place the beaker on a magnetic stirrer, turn on the stirrer, and stir for at least 30 minutes until no visible powder remains. Then weigh 1.00 g of poloxamer 188 and 0.585 g of L-glutamine and add them to the beaker, and continue stirring for no less than 20 minutes. Then weigh 2.20 g of sodium bicarbonate and add it to the beaker, and stir for 10 minutes until completely dissolved. Adjust the pH to 6.95 ± 0.05 with dilute hydrochloric acid, and stir for 5 ± 1 minute. Make up the volume to 1 L, stir for 10 ± 1 minute, sterilize by filtration, and store in the dark at 2 - 8 °C.

[0043] (2) BalanCD CHO FEED 2 + N-acetyl-D-glucosamine culture medium

[0044] Weigh 2.605 g of BalanCD CHO FEED 2 dry powder and place it in a 100 ml beaker, add 7.5 ml of purified water, put in a magnetic stir bar, place the beaker on a magnetic stirrer, turn on the stirrer, and stir for at least 30 minutes until no visible powder remains. Then weigh 0.11 g of sodium bicarbonate and add it to the beaker, and stir for 20 minutes until completely dissolved. Adjust the pH to 7.00 - 7.20 with sodium hydroxide, and stir for 10 minutes. Then weigh 1.659 g of N-acetyl-D-glucosamine and add it to the beaker, and stir for 30 minutes. Make up the volume to 50 ml, stir for 10 minutes, sterilize by filtration, and store in the dark at 2 - 8 °C.

[0045] (3) 200 mM L-Glutamine Solution

[0046] Weigh 2.923 g of L-Glutamine powder and place it in a 500 ml beaker. Add 75 ml of purified water, put in a magnetic stir bar, place the beaker on a magnetic stirrer, turn on the stirring, and stir until completely dissolved. Make up the volume to 100 ml, stir for 10 minutes, filter through sterile filtration, and store in the dark at 2 - 8°C.

[0047] 2. Resuscitation and Expansion Culture of CHO Engineered Cells

[0048] Take the CHO engineered cells constructed in Example 1 and perform cell resuscitation and expansion culture according to the experimental conditions shown in Table 1. The resuscitation and expansion culture medium is SFM4CHO.

[0049] Table 1 Conditions for Resuscitation and Expansion Culture of CHO Engineered Cells

[0050]

[0051]

[0052] 3. Cooling Culture of CHO Engineered Cells

[0053] When the cell seeds are expanded to a sufficient number, according to the experimental groups shown in Table 2, start the culture at 36.5°C, without cooling or perform the first-stage cooling on the 2nd, 3rd, and 4th days respectively, and reduce the culture temperature to 35°C; then without cooling or perform the second-stage cooling on the 5th, 6th, and 7th days respectively, and reduce the culture temperature to 32°C until the end of the culture.

[0054] During the culture process, the feeding methods for each experimental group are as follows: Add 4 mM of L-Glutamine solution on the 0th day of inoculation; add 2 mM of L-Glutamine solution on the 2nd and 4th days respectively; add FEED 2 + N-acetyl-D-glucosamine medium solution at 1 / 9 of the initial culture volume on the 3rd, 5th, and 8th days respectively.

[0055] And on the 3rd, 5th, 7th, 9th, 11th, and 13th days, take 0.5 mL of cell culture solution from each shake flask to detect cell density, viability, and the contents of glucose, glutamine, and lactate.

[0056] When the culture reaches the 14th day or the cell viability drops to 80%, end the culture, collect the cell culture solution, detect cell density and viability, then centrifuge at 3000 g for 15 min, collect the supernatant, take 15 ml to detect the contents of glucose, glutamine, lactate, supernatant expression level, and acid-base peak ratio, and store the rest at -20°C / -80°C.

[0057] Table 2 CHO engineering cell cooling culture time conditions

[0058]

[0059]

[0060] Note: R1-2 refers to two parallel experimental groups, R1 and R2, and other experimental groups are similar.

[0061] 4. Detection methods and data processing

[0062] (1) Determination of cell density and viability

[0063] The cell counting method is as follows: 1) Take a clean 1.5mL EP tube and dilute the cells and trypan blue in a certain ratio according to the estimated cell density; 2) After the sample is diluted, take 20μl of the sample and add it to the Countstar counting plate, place the Conutstar counting plate on the instrument stage, and perform counting measurements; 3) Measure 3 fields of view for each sample, and the software system automatically displays the current number of repeated measurements and counting results; 4) Calculate the average value based on the counting results of 2 parallel samples.

[0064] (2) Determination of biochemical parameters

[0065] The counted experimental samples were centrifuged at 10,000 rpm for 5 minutes, and the supernatant was placed in another clean 1.5 mL EP tube and placed in a Cedex Bio biochemical analyzer to detect the lactate, glutamine and glucose content. The average value was calculated based on the test results of the two parallel samples.

[0066] The supernatant expression level and acid-base peak ratio were detected by liquid phase method, and the average value was calculated based on the detection results of two parallel samples.

[0067] 5. Results and Analysis

[0068] from Figure 1 , 2 From Figure 6, we can see that for the first stage cooling time, cooling started on the second day of culture, and the highest cell growth density was slightly lower than that of other experimental groups. Early cooling affected the cell growth rate, resulting in a low final supernatant expression level. Cooling started on the fourth day, which had a certain impact on the final cell viability. Cooling started on the third day was optimal. For the second stage cooling time, cooling to 32°C on the sixth day of culture, the cell density and viability were optimal, and the supernatant expression level was also ideal compared to other experimental groups. Figure 3 , 4As can be seen from 5, the cooling process has a relatively large impact on lactic acid accumulation and consumption. Low temperature can maintain a relatively low lactic acid content in the later stage of cultivation, and cooling to a certain extent reduces the consumption of glucose, but has little impact on glutamine metabolism. In summary, considering the acid-base peak results of different experimental groups in Table 3, selecting the third day of cultivation for the first-stage cooling and the sixth day of cultivation for the second-stage cooling is the optimal cooling timing for the CHO engineering cells.

[0069] Table 3 Acid-base peak results of different experimental groups

[0070] Experimental grouping Main peak (%) Acidic peak (%) Alkaline peak (%) R1-2 29.8 60.5 9.7 R3-4 39.8 46.5 13.7 R5-6 40.1 45.2 14.7 R7-8 41.1 47.1 11.8 R9-10 56.6 39.2 4.2 R11-12 59.5 30.4 10.1 R13-14 47.8 42.6 9.6 R15-16 41.5 45.7 12.8 R17-18 43.5 49.1 7.4 R19-20 42.7 48.3 9.0

[0071] Example 3 Optimization of the cooling culture temperature of CHO engineering cells

[0072] 1. Preparation of the culture medium

[0073] Refer to Example 2 for preparation.

[0074] 2. Resuscitation and expansion culture of CHO engineering cells

[0075] Refer to Example 2 for operation.

[0076] 3. Cooling culture of CHO engineering cells

[0077] When the cell seeds are expanded to a sufficient quantity, according to the experimental grouping shown in Table 4, start the culture at 36.5 °C, without cooling or cooling to 35, 34, 33 °C on the 3rd day of culture respectively, which is the first-stage cooling; then without cooling or cooling to 32, 31, 30 °C on the 6th day of culture respectively, which is the second-stage cooling, and maintain the second-stage cooling temperature until the end of the culture.

[0078] During the culture process, the feeding methods for each experimental group are as follows: add 4 mM L-Glutamine solution on the 0th day of inoculation; add 2 mM L-Glutamine solution on the 2nd day and the 4th day respectively; add FEED 2 + N-acetyl-D-glucosamine culture medium solution at 1 / 9 of the current culture volume on the 3rd day, the 5th day and the 8th day respectively.

[0079] And take 0.5 mL of cell culture solution from each shake flask on the 3rd day, the 5th day, the 7th day, the 9th day, the 11th day and the 13th day respectively to detect the cell density, viability, and the contents of glucose, glutamine and lactic acid.

[0080] When the culture reaches the 14th day or the cell viability drops to 80%, end the culture, collect the cell culture solution, detect the cell density and viability, then centrifuge at 3000 g for 15 min, collect the supernatant, take 15 ml to detect the contents of glucose, glutamine, lactic acid, the supernatant expression level and the acid-base peak ratio, and store the rest at -20 °C / -80 °C.

[0081] Table 4 CHO engineering cell cooling temperature culture conditions

[0082] Experimental grouping Inoculation and culture temperature Cooling temperature in the first stage Cooling temperature in the second stage R21-22 36.5℃ No cooling No cooling R23-24 36.5℃ 35 32 R25-26 36.5℃ 35 31 R27-28 36.5℃ 35 30 R29-30 36.5℃ 34 32 R31-32 36.5℃ 34 31 R33-34 36.5℃ 34 30 R35-36 36.5℃ 33 32 R37-38 36.5℃ 33 31 R39-40 36.5℃ 33 30

[0083] Note: R21-22 refers to the two parallel experimental groups R21 and R22, and the other experimental groups are similar.

[0084] 4. Detection methods and data processing

[0085] Refer to Example 2.

[0086] 5. Results and Analysis

[0087] from Figure 7 , 8 As can be seen from Figures 1 and 12, for the first stage cooling time, the temperature was lowered to 33°C on the third day of culture, and the highest density of cell growth was low. The temperature was too low in the early stage of cell growth, which affected the cell growth rate, resulting in a low final supernatant expression level. Cooling to 34°C also had a certain impact on the cell density and the final supernatant expression level. Cooling to 35°C was the most ideal. For the second stage cooling time, the temperature was lowered to 30°C on the sixth day of culture, and the cell density and viability were optimal, and the supernatant expression level was also ideal compared to other experimental groups. Figure 9 , 10 , 11 It can be seen that the cooling process has a greater impact on lactic acid accumulation and consumption, reducing lactic acid accumulation in the later stage of culture, and cooling reduces glucose consumption to a certain extent, but has little effect on glutamine metabolism. In summary, combined with the acid-base peak results of different experimental groups in Table 5, the culture process of cooling to 35℃ on the third day and cooling to 30℃ on the sixth day, compared with the non-cooling process, the cell viability can be maintained at more than 80%, and the final supernatant expression of low-temperature culture is increased by about 2 times. Compared with the non-cooling process, the final acid peak ratio of low-temperature culture is reduced to about 23%.

[0088] Table 5 Acid-base peak results of different experimental groups

[0089]

[0090]

[0091] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A cryopreservation culture method for CHO cells expressing dulaglutide, characterized in that, It includes the following steps of inoculation culture and cooling culture: The inoculation culture temperature is controlled at 36 - 37°C, and the cooling culture is carried out in two stages: the first-stage cooling is carried out on the 3rd day, cooling to 35°C for culture; the second-stage cooling is carried out on the 6th day, cooling to 30°C for culture; the temperature of the second-stage cooling is maintained until the end of the culture; The cell is CHO K1 SV cell.

2. The cultivation method according to claim 1, characterized in that, The described culture method includes the following steps: 1) The basal medium is SFM4CHO, and the feeding medium is BalanCD CHO Feed 2; 2) After resuscitating the cells in the cell bank, passage them 4 times. After the cell growth rate stabilizes, inoculate them in a 125 mL shake flask for culture at a density of 0.5×10 6 cells / mL, with a liquid loading volume of 20 mL; 3) 4 mM of glutamine is added during inoculation, and 2 mM of glutamine is added on the 2nd and 4th days of culture; 4) 0.5 mL of samples are taken respectively on the 3rd, 5th, 7th, 9th, 11th, and 13th days of culture to detect cell density, cell viability, glucose, glutamine, and lactate, and the supernatant expression level and acid-base peak ratio are detected at the end of the culture; 5) The feeding medium, which is BalanCD CHO Feed2 + N-acetyl-D-glucosamine, is added on the 3rd, 5th, and 8th days respectively, and the added volume is 1 / 9 of the initial culture volume; 6) Inoculation culture and cooling culture.

3. The cultivation method according to claim 1, characterized in that, The described culture method is shake flask culture, the amplitude of the shaker is 25 mm, and the rotation speed is controlled at 140 rpm.

4. The culturing method according to claim 1, characterized in that, The whole culture cycle is 14 days.

5. The cell culture method according to claim 1, characterized in that, The cell is CHO K1 SV cell, the auxiliary plasmid is pOG44, and glutamine needs to be additionally added to the medium.

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