CN111849870B - High-density culture process of serum-free Vero cell fixed bed bioreactor - Google Patents
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Abstract
The invention discloses a high-density culture process of a serum-free Vero cell fixed bed bioreactor, belonging to the field of cell culture. The culture process comprises the following steps: 1) inoculating Vero cells cultured by a serum-free culture medium into a fixed bed bioreactor until the solution is clear; 2) and (3) setting the following parameters for culturing under a non-perfusion condition within 12-24 hours after inoculation: the temperature is 37.0 +/-0.2 ℃, the pH is 7.20 +/-0.1, the dissolved oxygen is 60 +/-20%, and the rotating speed is 90-110 rpm; 3) and starting perfusion culture 12-24 hours after inoculation. The method can be used for culturing high-density Vero cells, does not involve the use of bovine serum and animal pancreatin, and greatly improves the safety of biological products obtained from the Vero cells.
Description
Technical Field
The present invention belongs to the field of cell culture.
Background
The culture of the cells is an indispensable precondition for viral vaccines and other most biological products, and the high-density and high-quality cells can greatly improve the product yield.
Vero cells are African green monkey (Cercopticitech aethiops) kidney cells, cell lines were established by Yasumura and Kawakita of the university of Qianya, Japan, 3 months and 27 days 1962. The Vero cell line was brought to the united states' laboratories of tropical virology (laboratory of tropical virology), National Institute of Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH) by doctor b.simulu.s.1964 at 6, 15 th month, when the passage level of the cells was 93 rd generation. At the passage level of 113 th generation, the cells were again submitted to the American Type Culture Collection (ATCC), cultured at ATCC for passage to 121 th generation, and pooled, the cell pool being designated ATCC CCL-81 TM.
Serum-free Vero cells (Vero-SF-ACF) are obtained by acclimatizing ATCC from CCL-81TM (Lot58954145) in 2011 in serum-free and animal-origin-free culture medium, and the ATCC is numbered as CCL-81.5.
At present, no serum-free cell (Vero-SF-ACF) culture mode for large-scale production through serum-free and animal-derived domestication is reported. Reports on small-scale culture experiments mainly used cell factories, spinner flasks or microsphere bioreactors. The advantages and disadvantages of each culture method are shown in Table 1.
TABLE 1 advantages and disadvantages of cell factories, spinner flasks or microspherical bioreactors
The summary table shows: the bottle transfer or cell factory is not suitable for large-scale production due to the defects of low production efficiency, easy pollution and the like.
And the quality of the non-static cultured cells of the microsphere bioreactor is easily influenced greatly, so that the requirement on the product quality or the difficulty of a downstream treatment process is improved.
A fixed-bed bioreactor is a device for immobilizing cultured cells, the cells can be attached to a carrier immobilized therein, and a culture solution in the reactor is fluidized. The fixed bed bioreactor can reduce the shearing force of culture and has high cell culture efficiency, wherein the density of Vero cells can reach 1.0 × 107cells/ml (Yang Yi, Ru Dongyu, Guo Xixia, Caiguan, etc. the basket bioreactor is used to prepare rabies vaccine for Vero cell man [ J)]Journal of biological sciences of china, 5 months 2011, volume 25, stage 5 ], but still has room for improvement.
Disclosure of Invention
The invention aims to solve the problems that: the characteristic of static culture of a fixed bed bioreactor is applied, and a perfusion mode is used to provide a large-scale, high-quality and high-density culture process of serum-free Vero cells (Vero-SF-ACF).
The technical scheme of the invention is as follows:
a high-density culture process of a serum-free Vero cell fixed bed bioreactor comprises the following steps:
1) inoculating Vero cells cultured by a serum-free culture medium into a fixed bed bioreactor until the solution is clear;
2) and (3) setting the following parameters for culturing under a non-perfusion condition within 12-24 hours after inoculation: the temperature is 37.0 +/-0.2 ℃, the pH is 7.20 +/-0.1, the dissolved oxygen is 60 +/-20%, and the rotating speed is 90-110 rpm;
3) starting perfusion culture 12-24 hours after inoculation;
the serum-free culture medium with the following formula is used in each step: 15.0-17.6 g/L, 2-4 mmol/L of glutamine-alanine dipeptide, 0.5-2.0 g/L of fructose and 0.5-2.0 g/L of Tricine;
during perfusion, the concentration of glucose in the culture medium is ensured to be not lower than 1.0g/L by controlling the perfusion speed of the culture medium.
The Vero cells are serum-free Vero cells with the serial number of CCL-81.5 of American Standard culture Collection (ATCC) by the culture process.
The culture process is characterized in that the inoculation density of the step 1) is 5-8 multiplied by 105cells/ml。
As in the culture process, the perfusion volume in the step 3) is 1.0-2.0 times of the culture volume per day.
The invention has the following beneficial effects:
1) the culture process of the invention can greatly improve the cell density to 3 multiplied by 107cells/ml, higher than 1.0 × 10 of the prior literature7cells/ml (Yang Yi, Ru Dongyu, Guo Xixia, Caiguan, etc. the basket bioreactor is used to prepare rabies vaccine for Vero cell man [ J)]Journal of biological products, 2011 5 months, volume 25, phase 5).
2) The culture process of the invention does not need serum and passage does not relate to animal pancreatin, thus greatly reducing the pollution of exogenous factors.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
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FIG. 1: the structure of the fixed bed bioreactor is sketch map.
FIG. 2: cell growth trend graph.
Detailed Description
EXAMPLE 1 high Density culture in a serum-free Vero cell fixed bed bioreactor
1. Production cell
The production cells are Vero cells Vero-SF-ACF adapted to serum-free culture, purchased from American Type Culture Collection (ATCC) and numbered as CCL-81.5.
2. Method of producing a composite material
1) The sheet-shaped carrier (as in the purple region of FIG. 1) was packed at 70g/L in the carrier-packed region of the fixed-bed bioreactor.
2) Placing the working cell bank serum-free Vero cells into a T75 square bottle, culturing with a serum-free culture medium, digesting the cells with recombinant cell digestive enzyme (instead of animal-derived pancreatin) when growing into a compact monolayer, expanding and passaging according to a ratio of 1:6, digesting the cells with the recombinant cell digestive enzyme, and inoculating into a fixed bed bioreactor, wherein the cell density in the reactor is 6 x 10 during inoculation5cells/ml。
3) And (3) completely entering the cell suspension into the reactor for 60min, completing cell adsorption, and setting cell culture parameters after the solution becomes clear: the temperature is 37.0 ℃, the pH value is 7.20, the dissolved oxygen content is 60 percent, and the rotating speed is 100 rpm; no perfusion is performed.
4) Starting perfusion 18 hours after the cell inoculation is finished, wherein the perfusion volume is 1.5 times of the culture volume every day, namely, 1.5 times of the culture volume of culture medium is input into the bioreactor every day, the equal volume of the cultured solution is output, and the culture parameters in the step 3) are maintained during the perfusion culture period
The serum-free culture medium with the following formula is used in each step:
during perfusion, the concentration of glucose in the culture medium is ensured to be not lower than 1.0 g/L.
3. Results
The cell density of serum-free Vero cells (Vero-SF-ACF) in a fixed bed bioreactor can reach 3.0 multiplied by 107The cell/ml is higher than the prior literature report by more than 100 percent, and the cell growth trend graph is shown in figure 2.
EXAMPLE 2 high Density culture in a serum-free Vero cell fixed bed bioreactor
1. Production cell
The production cells are Vero cells Vero-SF-ACF adapted to serum-free culture, purchased from American Type Culture Collection (ATCC) and numbered as CCL-81.5.
2. Method of producing a composite material
1) The sheet-shaped carrier (as in the purple region of FIG. 1) was packed at 60g/L in the carrier-packed region of the fixed-bed bioreactor.
2) Placing the working cell bank serum-free Vero cells into a T75 square bottle, culturing with a serum-free culture medium, digesting the cells with recombinant cell digestive enzyme (instead of animal-derived pancreatin) when growing into a compact monolayer, expanding and passaging according to a ratio of 1:3, digesting the cells with the recombinant cell digestive enzyme, and inoculating into a fixed bed bioreactor, wherein the cell density in the reactor is 5 x 10 during inoculation5cells/ml。
3) And (3) completely entering the cell suspension into the reactor for 60min, completing cell adsorption, and setting cell culture parameters after the solution becomes clear: the temperature is 36.8 ℃, the pH value is 7.3, the dissolved oxygen content is 40 percent, and the rotating speed is 90 rpm; no perfusion is performed.
4) Starting perfusion 12 hours after the cell inoculation is finished, wherein the perfusion volume is 1.0 time of the culture volume every day, namely, 1.0 time of the culture volume of culture medium is input into the bioreactor every day, the equal volume of the cultured solution is output, and the culture parameters in the step 3) are maintained during the perfusion culture period
The serum-free culture medium with the following formula is used in each step:
during perfusion, the concentration of glucose in the culture medium is ensured to be not lower than 1.0 g/L.
3. Results
The cell density of serum-free Vero cells (Vero-SF-ACF) in a fixed bed bioreactor can reach 2.5 multiplied by 107cells/ml, which is more than 100% higher than the prior literature report.
EXAMPLE 3 high Density culture in a serum-free Vero cell fixed bed bioreactor
1. Production cell
The production cells are Vero cells Vero-SF-ACF adapted to serum-free culture, purchased from American Type Culture Collection (ATCC) and numbered as CCL-81.5.
2. Method of producing a composite material
1) The sheet-shaped carrier (as in the purple region of FIG. 1) was packed at 70g/L in the carrier-packed region of the fixed-bed bioreactor.
2) Placing the working cell bank serum-free Vero cells into a T75 square bottle, culturing with a serum-free culture medium, digesting the cells with recombinant cell digestive enzyme (instead of animal-derived pancreatin) when growing into a compact monolayer, expanding and passaging according to a ratio of 1:6, digesting the cells with the recombinant cell digestive enzyme, and inoculating into a fixed bed bioreactor, wherein the cell density in the reactor is 8 x 10 during inoculation5cells/ml。
3) And (3) completely entering the cell suspension into the reactor for 60min, completing cell adsorption, and setting cell culture parameters after the solution becomes clear: the temperature is 37.2 ℃, the pH value is 7.3, the dissolved oxygen content is 80 percent, and the rotating speed is 110 rpm; no perfusion is performed.
4) Starting perfusion 24 hours after the cell inoculation is finished, wherein the perfusion volume is 2.0 times of the culture volume every day, namely, 2.0 times of culture volume of culture medium is input into the bioreactor every day, equal volume of solution after culture is output, and the culture parameters in the step 3) are maintained during the perfusion culture period
The serum-free culture medium with the following formula is used in each step:
during perfusion, the concentration of glucose in the culture medium is ensured to be not lower than 1.0 g/L.
3. Results
The cell density of serum-free Vero cells (Vero-SF-ACF) in a fixed bed bioreactor can reach 2.8 multiplied by 107cells/ml, which is more than 100% higher than the prior literature report.
The following is further illustrated in the form of experimental examples.
Experimental example 1 high-density culture of rabies vaccine in serum-free Vero cell fixed bed bioreactor
1. Production cell
The production cells are Vero cells Vero-SF-ACF adapted to serum-free culture, purchased from American Type Culture Collection (ATCC) and numbered as CCL-81.5.
2. Poison seed
The production seed virus is rabies virus fixed virus rPV-2061 strain, introduced from the U.S. CDC.
3. Vaccine preparation
(1) Cell culture: the same as in example 1.
(2) Inoculating and culturing viruses: when the cell density in the bioreactor reaches 0.7X 107Inoculating rabies virus (rPV-2061 strain) at cell/ml, and the MOI is 0.005; 6 to 8 culturesh, starting perfusion of the cell maintenance solution.
The maintenance liquid formula comprises:
the culture parameters are as follows: perfusion culture is carried out at 34 ℃, pH7.50, dissolved oxygen of 60 percent and rotating speed of 100 rpm.
(3) And (3) toxin collection: the virus was harvested for 15 consecutive days (day 3 to day 17 after seed virus). Single virus harvest (virus titer is 6.5-7.5 lgLD)50Per ml), coarse-filtered with a 0.65 μm filter and concentrated 20-30 times with a 300kD membrane.
(4) Inactivation: adding beta-propiolactone into the concentrated virus harvest liquid according to the proportion of 1:4000, and inactivating the rabies virus at 4 ℃ for 24 hours; the mixture was left at 37 ℃ for 2 hours to hydrolyze beta-propiolactone.
(5) And (3) purification: 140U/ml nuclease (non-restriction endonuclease) was added and the digestion was carried out at 37 ℃ for 24 hours. Sepharose 4FF was selected as the packing for molecular sieve chromatography purification with phosphate buffer pH 7.6 as the mobile phase and the loading volume was not more than 5% of the column volume.
(6) Preparing stock solution: after purification, human serum albumin with the final concentration of 3 percent (m/v) and maltose with the final concentration of 5 percent (m/v) are added as stabilizing agents to prepare stock solution.
(7) Preparing a preparation: adding a freeze-drying protective agent (phosphate buffer solution containing human serum albumin with the final concentration of 3% (m/v) and maltose with the final concentration of 5% (m/v)) into the stock solution to obtain a semi-finished product with the antigen content of 30 IU/dose, wherein each dose is 1 mL; and (4) freeze-drying the semi-finished product to prepare the vaccine preparation.
4. Comparison of vaccine production efficiency
A commercially available rabies vaccine (from manufacturer A, B, C, D) was tested and compared to the product prepared by the procedure of this example. The detection method comprises the following steps:
4.1. potency assay
Rabies vaccine titer determination (NIH method) for human is carried out according to the general rules 3503 of the 2015 edition of pharmacopoeia of the people's republic of China:
(1) dilution reference vaccine and test article
The reference vaccine and the test article are respectively diluted into 1: 25. 1: 125. 1: 625 (re-dilution after reconstitution of the lyophilized preparation).
(2) Priming mice
The test article and the reference vaccine with different dilutions are used for immunizing 16 mice with 12-14 g respectively, and 0.5ml is injected into the abdominal cavity of each mouse.
(3) Two-free mouse
Mice were immunized again 7 days after priming using the same protocol.
(4) CVS challenge mice
(5) Calculation of attack virus
Assuming that 31LD is contained per 0.03ml50The viral load of (a) is subjected to intracerebral challenge, the viral titer of the CVS used being C (lgLD)500.03ml), lg31 ≈ 1.50, the CVS is diluted 10C-1.50Doubling is that every 0.03ml contains 31LD50Viral amount of viral suspension.
(6) Injection mouse
On day 14 after priming, the pre-titrated CVS was diluted to 5-100 LD per 0.03ml with virus diluent50A suspension of virus amounts as challenge virus. Performing intracerebral attack with 0.03ml of challenge virus per mouse; simultaneously diluting the challenge virus to 100、10-1、10-2And 10-3Performing virulence titration, wherein each dilution is not less than 8 mice, placing the cage filled with the mice on a cage frame after injection, observing for 14 days day by day, recording death conditions, and counting the number of the mice which die and show typical rabies brain symptoms after 5 days.
(7) Titer P formula:
dT is the dosage of 1 patient to be tested, ml; dS is the 1-time human dose, ml, of the reference vaccine; d is the titer of the reference vaccine, IU/ml.
4.2. Heat stability test
After standing at 37 ℃ for 28 days, the titer was measured.
Detection of Vero cell protein residue
The residual amount of Vero Cell Proteins was determined by the method (pharmacopoeia of the people's republic of China, 2020 edition, enzyme-linked immunosorbent assay) using Cygnus kit "Vero Cell hosts Cell Proteins".
Vero cell DNA residue detection
(1) Pharmacopoeia method
The culture cell supernatant was not less than 1ml and was subjected to the determination of the residual amount of exogenous DNA (rule 3407) in the pharmacopoeia of the people's republic of China (2015 edition), DNA probe hybridization.
(2) qPCR method (incorporated into the pharmacopoeia 2020 edition)
The test is carried out in 2 steps, wherein the first step is to extract host cell DNA, and the second step is to carry out fluorescence quantitative PCR on the DNA. Taking 100 mul of culture cell supernatant, adding lysis solution to crack and release host cell DNA; adsorbing the released DNA by using magnetic beads, keeping the magnetic beads, and removing a lysate; washing the magnetic beads; and eluting the host cell DNA adsorbed on the magnetic beads by using an eluent, transferring the eluent, and performing fluorescent quantitative PCR. Negative control, blank extraction control, extraction recovery control and standard curve are arranged in the whole test to ensure the accuracy and reliability of the result.
In addition, the inventor also obtained the single-tank yield information by inquiring the manufacturer A, B, C, D and checking the quantity of the product on the market in each batch through the official website of the Chinese food and drug testing research institute.
The results are as follows:
the single pot yields and titers are shown in table 2. The number of preparations produced by the culture process is far higher than that of the manufacturer A, C, D, the volume of the incubator of the manufacturer B is 8 times that of the invention, but the single-pot yield is less than 2 times that of the invention. The potency of the preparation obtained by the invention is much higher than that of the manufacturer A, B, C.
TABLE 2 comparison of production efficiencies
Note: ND, not detected.
Therefore, by means of the cell high-density culture process, the produced vaccine preparation has large quantity, the titer of unit preparation is high, and the overall production efficiency is far higher than the common level in the field.
In conclusion, the cell high-density culture process can improve the cell density and is beneficial to improving the output of related biological products; on the other hand, the pollution of exogenous factors can be reduced (animal-derived pancreatin is not used in cell passage, bovine serum albumin is not used in a culture medium, and Vero protein residue can be reduced by a culture mode), and the safety of related biological products is improved.
Claims (3)
1. A high-density culture process of a serum-free Vero cell fixed bed bioreactor is characterized by comprising the following steps:
1) inoculating Vero cells cultured by a serum-free culture medium into a fixed bed bioreactor until the solution is clear;
2) and (3) setting the following parameters for culturing under a non-perfusion condition within 12-24 hours after inoculation: the temperature is 37.0 +/-0.2 ℃, the pH is 7.20 +/-0.1, the dissolved oxygen is 60 +/-20%, and the rotating speed is 90-110 rpm;
3) starting perfusion culture 12-24 hours after inoculation;
the serum-free culture medium with the following formula is used in each step: 15.0-17.6 g/L of VP-SFM, 2-4 mmol/L of glutamine dipeptide, 0.5-2.0 g/L of fructose and 0.5-2.0 g/L of Tricine;
during perfusion, the concentration of glucose in the culture medium is ensured to be not lower than 1.0g/L by controlling the perfusion speed of the culture medium;
the perfusion volume of the step 3) is 1.0-2.0 times of the culture volume per day.
2. The process of claim 1, wherein: the Vero cells are serum-free Vero cells with the serial number of CCL-81.5 of American Standard culture Collection (ATCC).
3. The process of claim 1 or 2, wherein: the inoculation density of the step 1) is 5-8 multiplied by 105cells /ml。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107532150A (en) * | 2015-02-13 | 2018-01-02 | 武田疫苗股份有限公司 | The viral method of vaccine is prepared for generation |
| US20200216820A1 (en) * | 2019-01-08 | 2020-07-09 | Ge Healthcare Bio-Sciences Ab | Method for virus propagation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107532150A (en) * | 2015-02-13 | 2018-01-02 | 武田疫苗股份有限公司 | The viral method of vaccine is prepared for generation |
| US20200216820A1 (en) * | 2019-01-08 | 2020-07-09 | Ge Healthcare Bio-Sciences Ab | Method for virus propagation |
Non-Patent Citations (2)
| Title |
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| 应用篮式生物反应器制备Vero细胞人用狂犬病疫苗;杨屹 等;《中国生物制品学杂志》;20110531;第24卷(第5期);第584页左栏第3段 * |
| 无血清培养Vero细胞及其传代稳定性分析;张香玲 等;《中国生物制品学杂志》;20170131;第30卷(第1期);第68页摘要结论部分，第69页左栏第1-3段 * |
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