Culture method of human 293 monoclonal cell for expressing recombinant human serum albumin

Abstract

The invention discloses a culture method of a human 293 monoclonal cell for expressing recombinant human serum albumin. The invention provides a culture method of a human 293 monoclonal cell for expressing recombinant human serum albumin. Monoclonal cells cultured by the first culture medium are fusiform, the cell adherence is tight, the cell boundary is not clear, the cell clone formation rate is high, and the division growth of the monoclonal cells is fast; after the second culture medium is replaced, standing culture is conducted to suspension culture, and finally the recombinant human serum albumin 293 monoclonal cells capable of growing in a suspension mode are obtained. The screening of human 293 monoclonal cells expressing recombinant human serum albumin from single cell cloning to suspension single cell cloning is realized, and meanwhile, a feasible method is provided for screening and culturing the monoclonal cells. Through a batch feeding culture process, the expression quantity of the recombinant human serum albumin can reach 6.3 g / L, and commercial amplification production of the recombinant human serum albumin can be supported.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and particularly to a method for culturing a human 293 monoclonal cell expressing recombinant human serum albumin. Background Art

[0002] Human Serum Albumin (HSA) is the most abundant protein in human plasma and is synthesized in the liver. There are 3.5 - 5.0 g of human serum albumin in every 100 mL of human plasma, accounting for about 40 - 60% of the total plasma protein. Human serum albumin consists of 585 amino acids, contains 17 pairs of disulfide bonds, and is a non-glycosylated globular protein molecule with 3 functional regions and 9 sub-functional regions, with a theoretical molecular weight of 66.5 KD.

[0003] Human serum albumin extracted from the blood of healthy people can be directly injected intravenously into patients. Clinically, it is mainly used for shock caused by blood loss, trauma or burns, cerebral edema and increased intracranial pressure caused by injury, edema or ascites caused by cancer, cirrhosis and kidney diseases, maintenance treatment of advanced cancer patients, prevention and treatment of hypoproteinemia, and neonatal hyperbilirubinemia. It is also widely used in cardiopulmonary bypass, adjuvant treatment of burns, adjuvant treatment of hemodialysis and adult respiratory distress syndrome. It is an essential drug for clinical first aid or critical operations in hospitals and has always occupied a crucial position in the hospital market.

[0004] Currently, HSA used clinically is usually isolated and purified from human plasma, known as pdHAS (Plasma-derived HSA), which is limited by the limited source of human plasma, high production cost, and the risk of carrying viruses in human plasma. In addition, due to various factors such as the restructuring of plasma stations and the strengthening of safety supervision of blood products, the supply of blood products represented by human serum albumin in the domestic market is severely short, and the contradiction between supply and demand is prominent.

[0005] Human serum albumin can not only be used directly by intravenous injection as a drug, but also as an excellent drug carrier, such as albumin-paclitaxel, which can extend the half-life of paclitaxel in the body and improve the therapeutic effect of albumin-paclitaxel. It can also be used as an excipient for biological products such as vaccines, or as a culture medium for cells such as CHO. Producing recombinant human serum albumin by genetic engineering methods has the advantages of rich raw material sources, controllable quality, and scalable production. At the same time, it can completely eliminate the risk of virus contamination, which is of great social and economic significance for alleviating the shortage of domestic human serum albumin supply and eliminating the safety risks of blood-derived products, and has broad development space and market prospects.

[0006] For the production of recombinant human serum albumin (rHSA) by genetic engineering methods, there are currently multiple rHSAs undergoing clinical trials. The rHSA expressed by Tonghua Anruite Biopharmaceuticals using Pichia pastoris is currently in Phase III clinical trials; the rHSA expressed by Beijing Progenesis Biotech using yeast cells is in Phase II clinical trials; the plant-derived recombinant human serum albumin independently developed by Wuhan Heyuan Biotechnology is currently in Phase II / III clinical trials. The rHSA expressed by North China Pharmaceutical using Pichia pastoris has been approved by the NMPA for use as a pharmaceutical excipient, but not for injection drugs. The commonly used expression host platforms for recombinant human serum albumin under clinical research include Saccharomyces cerevisiae expression, rice endosperm cell expression, Escherichia coli expression, Pichia pastoris expression, etc., among which the Pichia pastoris expression system is applied more frequently.

[0007] There is currently no report on the preparation of rHSA protein using human cell lines such as 293 cells. Using suspension 293 cells for the production of recombinant protein drugs has several advantages, including protein modification, being more similar to human proteins, not containing heterologous proteins, and having higher quality; culturing in serum-free medium, without the risk of introducing exogenous viruses; suspension culture, which is conducive to production scale-up. In addition, due to the property of human serum albumin being prone to binding other substances, during the fermentation process of preparing recombinant human serum albumin using yeast and plant expression systems, the protein is easily adsorbed to substances such as heterologous proteins and DNA derived from host cells, and the adsorption amount is much larger than that of human serum albumin from blood sources. This characteristic increases the difficulty of developing the downstream purification process of recombinant expressed human serum albumin.

[0008] The usually administered dose of human serum albumin is in the gram level (5 - 10 g). Detecting trace impurities is a great challenge in the presence of high-concentration proteins. Using the human 293 cell expression system can, to a certain extent, circumvent the immunogenicity-related problems caused by heterologous proteins and DNA impurities.

[0009] Chinese Patent CN118063592A has provided a method for constructing a human serum albumin human-derived recombinant 293 cell (cell pool). However, when using recombinant human-derived 293 cells for large-scale culture, a stable culture process is required, and cell homogeneity must be considered. A cell line with uniform composition and stable growth is a prerequisite for a robust cell culture process. However, limited by the adherent growth characteristics of human 293 cells, it is difficult to amplify from single-cell division into monoclonal cells. Summary of the Invention

[0010] An embodiment of the present invention provides a culture method for human 293 monoclonal cells expressing recombinant human serum albumin to at least solve one of the problems existing in the related art.

[0011] The first object of the present invention is to provide a culture method for amplifying human 293 monoclonal cells expressing recombinant human serum albumin.

[0012] The second object of the present disclosure is to provide a cell culture method for increasing the expression level of recombinant human serum albumin in human 293 monoclonal cells expressing recombinant human serum albumin.

[0013] To achieve this object, the present invention is realized through the following technical solutions.

[0014] The present invention starts from static culture to suspension culture, and finally obtains recombinant human serum albumin 293 monoclonal cells that can grow in suspension. The screening of human 293 monoclonal cells expressing recombinant human serum albumin from single cells to suspension monoclonal cells is realized, and at the same time, a feasible method for monoclonal cell screening and culture is provided.

[0015] The human 293 monoclonal mother cells expressing recombinant human serum albumin in the present invention are derived from Wayne 293 cells transfected with the PLK002-Neo-rHSA-03 vector constructed in Examples 1 to 4 of Patent CN118063592 A.

[0016] Furthermore, single-pressure screening can be performed on Wayne 293 cells transfected with the PLK002-Neo-rHSA-03 vector using the G418 pressure screening medium according to Examples 1 to 5 of Patent CN 118063592 A to obtain cell line M3R as the human 293 monoclonal mother cells expressing recombinant human serum albumin. The G418 pressure screening medium is HyClone CDM4 HEK293 liquid medium supplemented with G418 at a final concentration of 0.2 mg / mL and 4 mM Glutamine. Pressure screening is carried out for 3 to 4 weeks, and half of the medium is replaced every 5 days. Discard the old medium and add new HyClone CDM4 HEK293 liquid medium with a final concentration of 0.2 mg / mL G418. The pressure screening culture conditions are 36.5 °C and 8% (v / v) CO2;

[0017] Alternatively, the culture process of cell line M3R can be further optimized according to Examples 1 to 6 of Patent CN 118063592 A, and the double-pressure screening cell line M3R-F02 (cell line 23S32-C546) can be obtained through fed-batch culture with HyClone CDM4 HEK293 liquid medium, Cell Boost 7a, and Cell Boost 7b feed: according to 0.6×10 6Inoculate the cell line M3R at a density of cells / mL into HyClone CDM4HEK293 liquid medium, culture at a temperature of 37.0 °C, a CO2 concentration of 5.0% (v / v), a rotation speed of 120 rpm, and a humidity of 80%. Record the day of inoculation as D0. Start feeding Cell Boost 7a and Cell Boost 7b on day D3. The feeding method is as follows: on D3, D5, D7, D8, and D9, according to the initial culture volume, the volumes of Cell Boost 7a added are 3% (v / v), 4% (v / v), 5% (v / v), 2% (v / v), and 2% (v / v) in sequence; the volumes of Cell Boost 7b added are 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), 0.2% (v / v), and 0.2% (v / v) in sequence. From D1 to the end of the culture, take cells every day to record the viable cell density and cell viability, and detect the glucose and lactate contents in the culture medium. When the detected value of glucose < 4 g / L, add glucose to 6 g / L.

[0018] The present invention provides a method for culturing a human 293 monoclonal cell expressing recombinant human serum albumin, and the culturing method includes the following steps:

[0019] S1: Plate and culture the revived human 293 monoclonal mother cells expressing recombinant human serum albumin on a cell culture plate with the first medium, 1 cell / well, to obtain monoclonal cells; the first medium includes DMEM / HIGH GLUCOSE Medium or FreeStyle 293 Expression Medium;

[0020] S2: Culture the monoclonal cells in step S1 in a 96-well cell culture plate, a 24-well cell culture plate, a 6-well cell culture plate, and a cell shake tube in sequence with the second medium to obtain expanded monoclonal cells; the second medium includes FreeStyle 293 Expression Medium.

[0021] Further, in step S1, the first medium also contains serum.

[0022] Further, in step S1, the first medium also contains a conditioned medium, and the conditioned medium is the culture supernatant of the revived human 293 monoclonal mother cells expressing recombinant human serum albumin.

[0023] Further, the preparation method of the conditioned medium is: culture the revived human 293 monoclonal mother cells with a cell viability ≥ 90% for 2 - 3 days, and the inoculation density is 0.3 - 0.5×10 6cells / mL to obtain a cell suspension, which was centrifuged, and the supernatant was taken as the conditioned medium.

[0024] Furthermore, the seeding density was 0.3×10 6 cells / mL and cultured for 3 days; the seeding density was 0.5×10 6 cells / mL and cultured for 2 days.

[0025] Furthermore, the medium used was CDM4 HEK293 Medium.

[0026] Further, the culture temperature was 36 - 37 °C, the shaking speed of the shaker culture was 110 - 120 rpm, the CO2 concentration was 5% - 8% v / v, and the humidity was 70% - 80%. Preferably, the culture temperature was 37 °C, the shaking speed of the shaker culture was 120 rpm, the CO2 concentration was 8% v / v, and the humidity was 80%.

[0027] Further, the centrifugation conditions were 20 - 25 °C, (150 - 200)G, and centrifuged for 5 - 10 min. Preferably, it was 25 °C, 200G, and 5 min.

[0028] Further, the first medium was DMEM / HIGH GLUCOSE Medium containing FBS, or FreeStyle 293 Expression Medium containing FBS and conditioned medium.

[0029] Further, the DMEM / HIGH GLUCOSE Medium containing FBS was DMEM / HIGH GLUCOSE Medium containing 5% - 15% v / v FBS, and preferably the FBS concentration was 15% v / v.

[0030] Further, the FreeStyle 293 Expression Medium containing FBS and conditioned medium was FreeStyle 293 Expression Medium containing 5% - 15% v / v FBS and 15% - 20% v / v conditioned medium. Preferably, the FBS concentration was 15% v / v and the conditioned medium concentration was 15% v / v.

[0031] Further, the culture time in step S1 was 14 - 21 days, preferably 14 days.

[0032] Further, the culture temperature in step S1 was 36 - 37 °C, the CO2 concentration was 5% - 8% v / v, and the humidity was 70% - 80%. Preferably, the culture temperature was 36.5 °C, the CO2 concentration was 8% v / v, and the humidity was 80%.

[0033] Further, in step S1, the culture is carried out in a 96-well cell culture plate, and the addition amount of the first culture medium is 120-200 μL, preferably 200 μL.

[0034] Further, in step S1, the human 293 monoclonal mother cells expressing recombinant human serum albumin are cultured with a resuscitation and passage medium, and the resuscitation and passage medium is CDM4 HEK293 Medium containing antibiotics and amino acids.

[0035] Further, the resuscitation and passage medium is CDM4 HEK293 Medium containing puromycin, geneticin and L-glutamine.

[0036] Further, the concentration of puromycin is 0-6 μg / mL, the concentration of geneticin is 0-0.1 mg / mL, and the concentration of L-glutamine is 4-6 mM; preferably, the concentration of puromycin is 2 μg / mL, the concentration of geneticin is 0.1 mg / mL, and the concentration of L-glutamine is 4 mM.

[0037] Further, the preparation of the resuscitated human 293 monoclonal mother cells expressing recombinant human serum albumin includes the following steps:

[0038] Thaw the human 293 monoclonal mother cells expressing recombinant human serum albumin to obtain a cell suspension, add the resuscitation and passage medium, centrifuge to obtain the cells to be resuscitated; culture the cells to be resuscitated with the resuscitation and passage medium, and the cell seeding density is 0.3×10 6 cells / mL - 0.7×10 6 cells / mL, and passage culture is carried out every 24-72 h. After passage culture, the cell density is 1.5×10 6 cells / mL - 3.5×10 6 cells / mL. When inoculating the passage-cultured cells, the cell density is 0.3×10 6 cells / mL - 0.6×10 6 cells / mL, and the cell viability is restored to more than 90%; culture is carried out by shaking in a CO2 shaker, the culture temperature is 36-37 °C, the CO2 concentration is 5%-8% v / v, the humidity is 70%-80%, and the shaking speed of the shaker culture is (110-120) rpm; preferably, the culture temperature is 36.5 °C, the CO2 concentration is 8% v / v, the humidity is 80%, and the shaking speed of the shaker culture is 120 rpm.

[0039] Further, the centrifugation conditions are 20-25 °C, (150-200) G, and centrifugation for 5-10 min; preferably, centrifugation is carried out at 25 °C, 200 G for 5 min.

[0040] Further, step S2 includes the following steps:

[0041] S21: Culture the monoclonal cells described in step S1 in a 96-well cell culture plate with FreeStyle 293 Expression Medium containing serum for 7 - 15 days until the confluence of the monoclonal cells reaches 5% - 10%, and then discard the supernatant.

[0042] S22: Add FreeStyle 293 Expression Medium without serum to each well in step S23 and culture until the confluence rate of the monoclonal cells ≥ 20%.

[0043] S23: Transfer the monoclonal cells with a confluence rate ≥ 20% in step S22 to a 24-well cell culture plate and culture until the confluence rate of the monoclonal cells ≥ 40%.

[0044] S24: Transfer the monoclonal cells with a confluence rate ≥ 40% in step S23 to a 6-well cell culture plate and culture until the confluence rate of the monoclonal cells ≥ 60%.

[0045] S25: Transfer the monoclonal cells with a confluence rate ≥ 60% in step S24 to a cell shake flask and culture until the cell viability of the monoclonal cells ≥ 90%.

[0046] S26: Subculture the monoclonal cells with a cell viability ≥ 90% in step S25.

[0047] The medium used in steps S23, S24, S25 or S26 is FreeStyle 293 Expression Medium containing antibiotics.

[0048] Furthermore, the volume of the discarded supernatant in step S21 is the same as that of the FreeStyle 293 Expression Medium without serum in step S22, and the volume ratio of the FreeStyle 293 Expression Medium containing serum to the discarded supernatant is: FreeStyle 293 Expression Medium containing serum : discarded supernatant = 1 : (0.75 - 1).

[0049] Furthermore, FreeStyle 293 Expression Medium containing serum : discarded supernatant = 1 : 0.75.

[0050] In a specific embodiment of the present invention, in step S21, the volume of FreeStyle 293 Expression Medium containing serum per well is 200 μL, and the volume of the supernatant discarded is 150 μL; in step S22, the volume of FreeStyle 293 Expression Medium without serum per well is 150 μL.

[0051] Furthermore, the culture temperature is 36 - 37 °C, the CO2 concentration is 5% - 8% v / v, and the humidity is 70 - 80%.

[0052] Preferably, the culture temperature is 36.5 °C, the CO2 concentration is 8% v / v, and the humidity is 80%.

[0053] Furthermore, the antibiotics include puromycin and / or geneticin.

[0054] Furthermore, in step S21, the FreeStyle 293 Expression Medium containing serum is FreeStyle 293 Expression Medium containing 5% - 15% v / v FBS; preferably, the FBS concentration is 5% v / v.

[0055] Furthermore, the culture medium used in S23, S24, S25 or S26 is FreeStyle 293 Expression Medium containing puromycin and geneticin.

[0056] Furthermore, the concentration of puromycin is 0 - 6 μg / mL, and the concentration of geneticin is 0 - 0.1 mg / mL; preferably, the concentration of puromycin is 2 μg / mL, and the concentration of geneticin is 0.1 mg / mL.

[0057] Furthermore, the culture time in step S22 is 2 - 6 days, and the culture time in step S23 or S24 is 5 - 7 days.

[0058] Furthermore, in step S23, the volume of the culture medium used in the 24-well cell culture plate is 1 - 1.2 mL, preferably 1 mL.

[0059] Furthermore, in step S24, the volume of the culture medium used in the 6-well cell culture plate is 2 - 3 mL, preferably 2 mL.

[0060] Furthermore, in step S25, the volume of the culture medium used in the cell shake flask is 5 - 10 mL, preferably 7 mL.

[0061] Furthermore, since the monoclonal cells grow adherently, the monoclonal cells need to be pipetted with a pipette tip and then transferred to a cell culture plate of other specifications.

[0062] Furthermore, in step S26, during subculture, the seeding density of monoclonal cells is 0.3 - 0.6×10 6 cells / mL, preferably 0.6×10 6 cells / mL.

[0063] Furthermore, the monoclonal cells after subculture in step S26 are cryopreserved. After centrifuging the monoclonal cells, they are suspended with cryopreservation medium, aliquoted into cryotubes, placed in an -80°C refrigerator overnight, and then transferred to a liquid nitrogen tank for storage; the number of cells in each cryotube is 5E6 - 1E7 cells / mL, preferably 1E7 cells / mL;

[0064] The cryopreservation medium consists of FreeStyle 293 Expression Medium and dimethyl sulfoxide; preferably, the volume ratio of FreeStyle 293 Expression Medium to dimethyl sulfoxide is 90:10 or 92:8, preferably 90:10.

[0065] The present invention also provides a cell culture method for increasing the expression level of recombinant human serum albumin in human 293 monoclonal cells expressing recombinant human serum albumin, comprising the following steps:

[0066] S31: Inoculate the expanded monoclonal cells obtained by culture into HyClone CDM4 HEK293 liquid medium containing amino acids, with the seeding cell density being 0.6×10 6 cells / mL - 1.0×10 6 cells / mL;

[0067] S32: Add feeding medium for fed-batch culture on the 3rd day after inoculation in step S31. The feeding media used are Cell Boost 7a and Cell Boost 7b, and culture for 14 days.

[0068] Furthermore, in step S31, the amino acids include L-glutamine, and the content of L-glutamine is 4 - 6 mM, preferably 4 mM.

[0069] In a specific embodiment of the present invention, in step S31, the seeding cell density is 0.6×10 6 cells / mL.

[0070] Furthermore, in step S32, add feeding medium to the culture medium on the 3rd, 5th, 7th, 8th, 9th, 11th, and 13th days after inoculation,

[0071] On the 3rd day after inoculation, the concentration of Cell Boost 7a added is 3% v / v, and the concentration of Cell Boost 7b added is 0.3% v / v;

[0072] On the 5th day after inoculation, the concentration of Cell Boost 7a added is 5% v / v, and the concentration of Cell Boost 7b added is 0.5% v / v;

[0073] On the 7th day after inoculation, the concentration of Cell Boost 7a added is 5% v / v, and the concentration of Cell Boost 7b added is 0.5% v / v;

[0074] On the 8th day after inoculation, the concentration of Cell Boost 7a added is 2% v / v, and the concentration of Cell Boost 7b added is 0.2% v / v;

[0075] On the 9th day after inoculation, the concentration of Cell Boost 7a added is 2% v / v, and the concentration of Cell Boost 7b added is 0.2% v / v;

[0076] On the 11th day after inoculation, the concentration of Cell Boost 7a added is 2% v / v, and the concentration of Cell Boost 7b added is 0.2% v / v;

[0077] On the 13th day after inoculation, the concentration of Cell Boost 7a added is 2% v / v, and the concentration of Cell Boost 7b added is 0.2% v / v.

[0078] Furthermore, in step S31, ensure that the glucose concentration in the culture medium is not lower than 6 g / L.

[0079] Furthermore, culture with shaking in a CO2 shaker, the culture temperature is 36 - 37 °C, the CO2 concentration is 5% - 8% v / v, the humidity is 70% - 80%, and the shaking speed of the shaker culture is 110 - 120 rpm; preferably, the culture temperature is 36.5 °C, the CO2 concentration is 8% v / v, the humidity is 80%, and the shaking speed of the shaker culture is 120 rpm.

[0080] The embodiments of the present invention have the following beneficial effects:

[0081] (1) The first culture medium used in the present invention is FreeStyle 293 Expression Medium of 15% FBS + DMEM / HIGH GLUCOSE Medium or 15% FBS + 15% Condition Medium. As a monoclonal plating medium, the monoclonal cells cultured have a spindle shape, adhere tightly to the wall, and the boundaries between cells are not clear, but the cell clone formation rate is relatively high, and the monoclonal cells divide and grow relatively fast. After changing to the second culture medium, that is, gradually replacing FreeStyle 293 Expression Medium supplemented with FBS with serum-free FreeStyle 293 Expression Medium, from static culture to suspension culture, finally, recombinant human serum albumin 293 monoclonal cells that can grow in suspension are obtained. The screening of human 293 monoclonal cells expressing recombinant human serum albumin from single cells to suspension-cloned single cells is realized, and at the same time, a feasible method for monoclonal cell screening and culture is provided.

[0082] (2) The monoclonal cell lines obtained in the present invention have a uniform morphology, can be cultured in suspension, and the doubling time is between 24 h and 30 h. Through the Fedbatch batch feeding culture process, the expression level of recombinant human serum albumin can reach 6.3 g / L, which can support the commercial scale-up production of recombinant human serum albumin. Description of the Drawings

[0083] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0084] Figure 1 It is the imaging of cells at different days after monoclonal cell plating culture in a 96-well plate.

[0085] Figure 2 It is the detection result of the expression level of the supernatant obtained by monoclonal cells in the Fedbatch batch feeding culture method.

[0086] Figure 3 It is the SDS-PAGE electrophoresis result of monoclonal cell Fedbatch protein. Detailed Embodiments

[0087] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will elaborate on each implementation manner of the present invention in conjunction with the accompanying drawings. However, those of ordinary skill in the art can understand that in each implementation manner of the present invention, many technical details are provided to help readers better understand the present application. However, even without these technical details and various changes and modifications based on the following implementation manners, the technical solutions claimed in the present application can still be achieved. The division of the following embodiments is for convenience of description and should not constitute any limitation on the specific implementation manner of the present invention. The various embodiments can be combined and cross-referenced with each other on the premise of not being contradictory.

[0088] CDM4 HEK293 Medium: Brand: HyClone; Catalog Number: SH30858.02;

[0089] Puro: Brand: Beyotime; Catalog Number: ST551-50mg;

[0090] G418: Brand: Bioss; Catalog Number: D21054;

[0091] L-Glutamine: Brand: Sigma; Catalog Number: G7513-100mL; L-Glutamine in the following embodiments is simply referred to as Gln;

[0092] DMEM / HIGH GLUCOSE Medium: Brand: Cytiva; Catalog Number: SH30022.01;

[0093] FreeStyle 293 Expression Medium: Brand: Gibco; Catalog Number: 12338-1018;

[0094] Peak expression Medium: Brand: Cytiva; Catalog Number: SH31192.01;

[0095] OPM-293CD05 Medium: Brand: OPMI; Catalog Number: 81075;

[0096] CHO Cloning Medium B: Brand: Quacell; Catalog Number: A11602B;

[0097] CHO Cloning Medium A: Brand: Quacell; Catalog Number: A11602A;

[0098] CHO cloning MaxA: Brand: Maibang Biotech; Catalog Number: MB230706A03T;

[0099] MetaCell TM 293 Cloning Medium: Brand: Sipeng Biotech; Catalog Number: L2006-0010;

[0100] cloning medium: Brand: ExCell; Catalog Number: UE 000-N091;

[0101] HyClone CDM4HEK293 liquid medium: Brand: Cytiva; Catalog Number: SH30858.02;

[0102] Cell Boost 7a: Brand: Hyclone; Catalog Number: SH31026.01;

[0103] Cell Boost 7b: Brand: Hyclone; Catalog Number: SH31027.01CN.

[0104] Example 1 Resuscitation of Human 293 Master Cloning Cells Expressing Recombinant Human Serum Albumin

[0105] 1. Take out a cryopreservation tube of a human 293 master cloning cell line (23S32-C546) expressing recombinant human serum albumin from the liquid nitrogen tank and quickly thaw it in a 37°C constant temperature water bath to obtain a cell suspension.

[0106] 2. Transfer the cell suspension into a 15 mL centrifuge tube containing 7 mL of pre-warmed resuscitation and passage medium, and centrifuge (200G, 5 min, 25°C). Discard the supernatant and retain the cells at the bottom. The resuscitation and passage medium used is: CDM4 HEK293 Medium supplemented with a final concentration of 2 μg / mL Puro (puromycin), 0.1 mg / mL G418 (Geneticin), and 4 mM L-Gln (L-glutamine).

[0107] 3. Resuspend the cells from step 1 with 5 mL of resuscitation and passage medium and place them in a 125 mL cell culture shake flask containing 15 mL of resuscitation and passage medium. The cell density is 0.3×10 6 cells / mL to 0.7×10 6 cells / mL. Incubate with shaking in a CO2 shaker at 36.5°C, 120 rpm, 8% CO2, and 80% humidity. Perform cell passage culture every 24 - 72 h. After passage culture, the cell density is 1.5×10 6 cells / mL to 3.5×10 6 cells / mL. The cell density at the time of inoculation for passage culture cells is 0.3×10 6cells / mL~0.6×10 6 cells / mL, the cell viability was restored to 90% or more, and the revived human 293 master clone cells expressing recombinant human serum albumin were obtained.

[0108] Example 2 Monoclonal Plating Culture

[0109] 1. Use a CountStar cell counter to count the human 293 master clone cells expressing recombinant human serum albumin revived in Example 1. Under the premise that the cell viability > 90% and the doubling returns to normal, subsequent experiments were carried out.

[0110] 2. According to the limited dilution method, add 200 μL of monoclonal cell plating medium to a 96-well plate and preheat it for more than 30 min. The monoclonal cell plating medium is DMEM / HIGH GLUCOSE Medium supplemented with 15% (v / v) FBS (fetal bovine serum) at the final concentration or FreeStyle293 Expression Medium supplemented with 15% (v / v) FBS and 15% (v / v) Condition Medium at the final concentration.

[0111] 3. Plate the qualified revived master clone cells of recombinant human serum albumin 293 in step 1 into the 96-well plate in step 2, 1 cell / well, and place it in a CO2 cell incubator. Incubate statically for 14 days under the conditions of 36.5 °C, 8.0% CO2 and 80% humidity.

[0112] As Figure 1 shown, after culturing for 14 days, most of the cells were in an adherent state, spindle-shaped, and grew in patches.

[0113] Example 3 Effects of Different Monoclonal Cell Plating Media on the Growth of Monoclonal Cells Expressing Recombinant Human Serum Albumin 293

[0114] The monoclonal cell plating media were 10 kinds of media shown in Table 1. After filtering with a 0.22 μm filter or filter cup, they were cultured according to the method of Example 2. Use a CloneSelect Imager cell growth analysis system to photograph the cells cultured by monoclonal plating.

[0115] The preparation method of the Condition Medium used therein is: After the viability of the cell line (23S32-C546) is restored, inoculate the medium CDM4 HEK293 Medium at a density of 0.3×10 6 cells / mL and culture for 3 days, or inoculate at a density of 0.5×10 6Inoculate the medium CDM4 HEK293 Medium at a density of cells / mL, and culture for 2 days in a CO2 shaker at a temperature of 36.5 °C, 120 rpm, 8.0% CO2 and 80% condition, and shake and culture to obtain a cell suspension. Centrifuge the cell suspension (200G, 5 min, 25 °C), and the obtained supernatant is the conditioned medium (Condition Medium).

[0116] Table 1 Monoclonal cell plating medium formulation, plating volume and results

[0117]

[0118]

[0119] The results are shown in Table 1 and Figure 1 As shown, by comprehensively analyzing the CloneSelect Imager cell imaging and clone final formation data, only DMEM / HIGH GLUCOSE Medium supplemented with 15% (v / v) FBS at the final concentration and FreeStyle 293 Expression Medium supplemented with 15% (v / v) FBS and 15% (v / v) Condition Medium at the final concentration can be used as the monoclonal cell plating medium to form single-cell clones.

[0120] As Figure 1 shown, D-1, D-2, D-3, D-4 are images of the growth of the same monoclonal cell on the 0th, 1st, 2nd, and 12th days using DMEM / HIGH GLUCOSE Medium supplemented with 15% (v / v) FBS at the final concentration as the monoclonal cell plating medium; F-1, F-2, F-3, F-4 are images of the growth of the same monoclonal cell on the 0th, 1st, 2nd, and 12th days using FreeStyle 293 Expression Medium supplemented with 15% (v / v) FBS and 15% (v / v) Condition Medium at the final concentration as the monoclonal cell plating medium.

[0121] Example 4 Monoclonal cell expansion culture

[0122] 1. Discard all the supernatant in the 96-well plate cultured for 14 days in Example 2, add 200 μL of FreeStyle 293 Expression Medium supplemented with 5% (v / v) FBS at the final concentration, change the medium and culture for 7 - 15 days, and observe under the microscope. Clones with a confluence of 5% - 10% are formed.

[0123] 2. Discard 150 μL of the supernatant in the 96-well plate after culturing in step 1, and add 150 μL of FreeStyle293 Expression Medium, which is a serum-free medium, and culture for 2 - 6 days until the cell confluence rate ≥ 20%.

[0124] 3. Expand the cells to a 24-well plate: Add 1 mL of pre-warmed FreeStyle 293 Expression Medium supplemented with a final concentration of 2 μg / mL Puro and 0.1 mg / mL G418 to the 24-well plate in advance to obtain a 24-well plate with pre-coated medium; Pipette the cells cultured in step 2 and transfer them to the 24-well plate with pre-coated medium, with a viable cell density of (0.1 - 0.5) cells / mL, and place it in a CO2 cell incubator. Incubate statically at a temperature of 36.5°C, 8.0% CO2, and 80% humidity for 5 - 7 days until the cell confluence rate reaches 40%.

[0125] 4. Expand the cells to a 6-well plate: Add 2 mL of pre-warmed FreeStyle 293 Expression Medium supplemented with a final concentration of 2 μg / mL Puro and 0.1 mg / mL G418 to the 6-well plate in advance to obtain a 6-well plate with pre-coated medium; Pipette the cells cultured in step 3 and transfer them to the 6-well plate with pre-coated medium, with a viable cell density of (0.2 - 0.5) cells / mL, and place it in a CO2 cell incubator. Incubate statically at a temperature of 36.5°C, 8.0% CO2, and 80% humidity for 5 - 7 days until the cell confluence rate reaches 60%. At this time, the cells growing in the 6-well plate are still in an adherent state, but gently pipette the cells, and the cells can easily detach.

[0126] 5. Expand the cells to a 50 mL shake flask culture: Add 7 mL of pre-warmed FreeStyle 293 Expression Medium supplemented with a final concentration of 2 μg / mL Puro and 0.1 mg / mL G418 to the 50 mL shake flask in advance to obtain a shake flask with pre-added medium; Pipette the cells cultured in step 4 and transfer them to the shake flask with pre-added medium, with a viable cell density of (0.2 - 0.5) cells / mL, and place it in a CO2 cell incubator. Incubate with shaking at a temperature of 36.5°C, 220 rpm, 8.0% CO2, and 80% humidity.

[0127] 6. Subculture: Detect that the cell viability after culturing in step 5 has recovered to 90% or above, and according to 0.6×10 6Subculture was carried out in a shake flask of FreeStyle 293 Expression Medium supplemented with Puro at a final concentration of 2 μg / mL and G418 at 0.1 mg / mL at an inoculation density of cells / mL and a culture volume of 15 mL. Subculture was performed every 3 - 4 days to obtain expanded monoclonal cells.

[0128] Example 5 Cryopreservation of Monoclonal Cells

[0129] Cell cryopreservation conditions: The monoclonal cells expanded in Example 4 were counted using a CountStar cell counter, and the monoclonal cells with a viability restored to 90% or more and normal doubling were cryopreserved.

[0130] 1. Prepare the cryopreservation solution: Mix DMSO (dimethyl sulfoxide) and FreeStyle 293 Expression Medium at a volume ratio of DMSO:FreeStyle 293 Expression Medium = 1:9.

[0131] 2. Transfer the cell suspension of monoclonal cells meeting the cryopreservation conditions to a centrifuge tube, centrifuge at 200G at room temperature for 5 min, discard the supernatant, and retain the cells at the bottom.

[0132] 3. Gently pipette the cells centrifuged in step 2 with the cryopreservation solution prepared in step 1 to suspend them. Pipette gently to avoid generating bubbles as much as possible to obtain a cell suspension; Aliquot the cell suspension into labeled cryopreservation tubes, 1 mL per tube, and the number of cells in each cryopreservation tube is 1E7 cells / mL.

[0133] 4. Cryopreserve the cells treated in step 3. Place the cryopreservation tubes in a programmable freezing container, leave them in an -80°C refrigerator overnight, and then transfer them to a liquid nitrogen tank for storage.

[0134] Example 6 Fed-Batch Culture of Monoclonal Cells

[0135] 1. Count the monoclonal cells expanded in Example 4 using a CountStar cell counter. For monoclonal cells with a cell growth viability > 90% and normal doubling, inoculate them at a density of 0.6×10 6 cells / mL into a 125 mL cell culture shake flask filled with basal medium. The name of the shake flask is "clone number + shake flask serial number", for example, "S08 - F01" means "monoclonal No. 08, shake flask No. 1". The basal medium is HyClone CDM4HEK293 liquid medium supplemented with a final concentration of 4 mM L-Gln. The volume of the basal medium is 30 mL per flask. The inoculation day is recorded as D0, and culture is carried out in a CO2 shake incubator at a temperature of 36.5°C, 8.0% CO2, a rotation speed of 120 rpm, and a humidity of 80%.

[0136] 2. Feeding began on the third day after inoculation (D3), with Cell Boost 7a and Cell Boost 7b. The feeding method was as follows: D3, D5, D7, D8, D9, D11 and D13 (3rd, 5th, 7th, 8th, 9th, 11th and 13th days after inoculation). According to the initial culture volume, the volume of Cell Boost 7a added was 3% (v / v), 5% (v / v), 5% (v / v), 2% (v / v), 2% (v / v), 2% (v / v) and 2% (v / v) of Cell Boost 7a; the volume of Cell Boost 7b added was 0.3% (v / v), 0.5% (v / v), 0.5% (v / v), 0.2% (v / v), 0.2% (v / v), 0.2% (v / v) and 0.2% (v / v). The above Cell Boost The volume percentages of 7a and Cell Boost 7a were calculated based on the initial culture volume.

[0137] 3. From D3 to the end of culture, the live cell density and cell viability were detected every day, and the glucose and lactic acid contents in the culture medium were monitored. When the glucose detection value was <4 g / L, glucose was added to 6 g / L (including the glucose contained in the feed). From D10 (the 10th day after inoculation), 1 mL of cell suspension was taken every day and centrifuged (4000 G, 25 min, 4°C) to obtain the supernatant for expression detection. The culture was terminated on D14 (the 14th day after inoculation) or when the cell viability was <60%, the cell suspension was centrifuged (4000 G, 25 min, 4°C), and the supernatant was collected.

[0138] 4. Use ELISA to detect the expression level of supernatant. The test results are shown in Figure 2 The expression of recombinant human serum albumin in human 293 cells can reach 6.3 g / L. SDS-PAGE electrophoresis was performed and the test results are shown in Figure 3 The protein band is single and its size is about 66KD, which is consistent with the theoretical molecular weight of human serum albumin.

[0139] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A culture method for amplifying and expressing human 293 monoclonal cells of recombinant human serum albumin, characterized in that, The cultivation method includes the following steps: S1: Plating and culturing the revived human 293 monoclonal mother cells expressing recombinant human serum albumin on a cell culture plate with a first culture medium, 1 cell / well, to obtain monoclonal cells; the first culture medium includes DMEM / HIGH GLUCOSE Medium or FreeStyle 293 Expression Medium; S2: Culturing the monoclonal cells obtained in step S1 successively in a 96-well cell culture plate, a 24-well cell culture plate, a 6-well cell culture plate, and a cell shake flask with a second culture medium to obtain expanded monoclonal cells; the second culture medium includes FreeStyle 293 Expression Medium.

2. The culturing method according to claim 1, wherein, In step S1, the first culture medium also contains serum.

3. The culturing method according to claim 2, wherein In step S1, the first culture medium also contains a conditioned medium, and the conditioned medium is the culture supernatant of the revived human 293 monoclonal mother cells expressing recombinant human serum albumin.

4. The cultivation method according to claim 3, wherein In step S1, the first culture medium is DMEM / HIGH GLUCOSE Medium containing FBS, or FreeStyle 293 Expression Medium containing FBS and the conditioned medium.

5. The cultivation method according to claim 1, wherein In step S1, the revived human 293 monoclonal cells expressing recombinant human serum albumin are cultured with a revival and passage medium, and the revival and passage medium is CDM4HEK293 Medium containing antibiotics and amino acids.

6. The cultivation method according to claim 1, characterized in that, Step S2 includes the following steps: S21: Culturing the monoclonal cells described in step S1 in a 96-well cell culture plate with FreeStyle 293 Expression Medium containing serum for 7 - 15 days until the confluence of the monoclonal cells reaches 5% - 10%, and discarding the supernatant; S22: Adding FreeStyle 293 Expression Medium without serum to each well in step S23 and culturing until the confluence rate of the monoclonal cells ≥ 20%; S23: Transferring the monoclonal cells with a confluence rate ≥ 20% in step S22 to a 24-well cell culture plate for culture until the confluence rate of the monoclonal cells ≥ 40%; S24: Transferring the monoclonal cells with a confluence rate ≥ 40% in step S23 to a 6-well cell culture plate for culture until the confluence rate of the monoclonal cells ≥ 60%; S25: Transferring the monoclonal cells with a confluence rate ≥ 60% in step S24 to a cell shake flask for culture until the cell viability of the monoclonal cells ≥ 90%; S26: Subculturing the monoclonal cells with a cell viability ≥ 90% in step S25; The culture medium used in steps S23, S24, S25, or S26 is FreeStyle 293 Expression Medium containing antibiotics.

7. The cultivation method according to claim 6, wherein, The volume of the supernatant discarded in step S21 is the same as that of the serum-free FreeStyle 293 Expression Medium in step S22, and the volume ratio of the serum-containing FreeStyle 293 Expression Medium to the discarded supernatant is: serum-containing FreeStyle 293 Expression Medium: discarded supernatant = 1:(0.75 - 1).

8. The cultivation method according to claim 6, characterized in that, The culture temperature is 36 - 37 °C, the CO2 concentration is 5% - 8% v / v, and the humidity is 70 - 80%.

9. The cultivation method according to claim 6, characterized in that, The antibiotics include puromycin and / or geneticin.

10. A cell culture method for increasing the expression level of recombinant human serum albumin in a human 293 monoclonal cell expressing recombinant human serum albumin, characterized in that, It includes the following steps: S31: Inoculate the expanded monoclonal cells obtained by culturing any one of Claims 1 to 9 into HyClone CDM4HEK293 liquid medium containing amino acids, with an inoculated cell density of 0.6×10 6 cells / mL to 1.0×10 6 cells / mL; S32: On the 3rd day after inoculation in step S31, feeding is carried out for fed-batch culture. The feeds used are Cell Boost 7a and Cell Boost 7b, and the culture is carried out for 14 days.

Images