CN111394390A - Method for batch production of recombinant adenovirus for novel coronavirus gene vaccine - Google Patents
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Abstract
The invention discloses a method for batch production of recombinant adenovirus for novel coronavirus gene vaccine, which comprises the following steps: expanding cells; the cells obtained by amplification are inoculated into a packed bed bioreactor for culture; infecting cells in the packed bed bioreactor with virus, and harvesting culture solution containing recombinant adenovirus; filtering and concentrating the collection liquid of the recombinant adenovirus; adding broad-spectrum nuclease into the recombinant adenovirus collecting solution for nuclease treatment; and separating and purifying the nuclease-treated recombinant adenovirus collecting solution by using anion exchange column chromatography to obtain the purified recombinant adenovirus. The infection and the amplification of the recombinant adenovirus are carried out by adopting a packed bed bioreactor, the efficiency is high, the packed bed bioreactor is more suitable for the requirement of a GMP production environment, and the full-automatic column chromatography separation and purification is adopted, so that the economic and effective large-scale industrialized GMP production of recombinant adenovirus preparation products is realized, and the packed bed bioreactor is used for preventing the infection and the infection of novel coronavirus.
Description
Technical Field
The invention belongs to the technical field of biological pharmaceutical technology, relates to a method for batch production of recombinant adenovirus for novel coronavirus gene vaccine, is applied to the batch production process of GMP (good manufacturing practice) of recombinant adenovirus gene therapy products, and particularly relates to the batch production process of GMP (good manufacturing practice) of recombinant adenovirus gene vaccine for novel coronavirus.
Background
After decades of research and development, the field of gene therapy has been developed and product breakthrough is realized, the recombinant adenovirus p53 gene therapy product of Shenzhen Sauruo gene technology company in China, the Glybera recombinant adeno-associated virus (AAV) product of UniQure company in the Netherlands and the L uxturna recombinant adeno-associated virus (AAV) product of Spark Therapeutics in America are approved by the government medical regulatory agency and are on the market successively.
The burst of the novel coronavirus (COVID-19) brings huge crisis to social public health, the infectivity is very strong, and the novel coronavirus brings huge social health and economic influences to human beings and is highly valued by people. There is currently no effective therapeutic and prophylactic vaccine. For the health of people's public, the safety of the country and society, there is an urgent need to develop vaccine products that can prevent the infection of novel coronavirus.
The commonly used development methods of viral vaccines include 1) inactivated viral vaccines, 2) inactivated viral vaccines, 3) subunit vaccines, 4) V L P (virus like particle) vaccines, 5) RNA vaccines, and 6) recombinant viral vector vaccines, the high-tech recombinant viral vector gene vaccine is the latest technology for vaccine development, the vaccine is safe and effective, and the development time is short.
After decades of research and development, the field of gene therapy products based on recombinant adenovirus vectors has been developed for a long time. The traditional preparation method of gene therapy products in laboratories can not meet the requirements of GMP large-scale industrialization of gene therapy products. By means of years of research and development experience and investment, Shenzhen Saururo gene technology company in China develops a set of brand-new GMP batch production recombinant adenovirus gene therapy products and processes, and realizes product breakthrough. The industrialization of the recombinant adenovirus p53 product is successfully realized today, and the product is approved and marketed by the government medical regulatory agency in 2004. On the basis of the production method and process of the now-living recombinant adenovirus p53 product, Shenzhen Shennosheng gene technology company in China developed a set of brand-new production method and process of recombinant adenovirus vector gene vaccine products, which are used for GMP large-scale production of vaccine products and can be widely used for preventing infection and infection of novel coronavirus.
Disclosure of Invention
The invention aims to provide a method for producing recombinant adenovirus on a large scale for novel coronavirus gene vaccine, a brand-new GMP large-scale batch production method for recombinant adenovirus, and successfully realizes the industrialization of recombinant adenovirus vector gene vaccine products.
The technical scheme of the invention is to provide a method for producing recombinant adenovirus in a large scale for a novel coronavirus gene vaccine, which comprises the following steps:
s1, taking cells of a working cell bank of the recombinant adenovirus packaging cells, thawing the cells, and inoculating the cells into a cell culture container for cell amplification;
s2, inoculating the cells obtained by amplification in the cell culture container into a packed bed bioreactor for culture;
s3, taking the working virus library virus of the recombinant adenovirus to infect cells in the packed bed bioreactor, releasing the recombinant adenovirus into the culture solution, and harvesting the culture solution containing the recombinant adenovirus;
s4, filtering and concentrating the culture solution containing the recombinant adenovirus, and diluting the recombinant adenovirus collection solution with a buffer solution;
s5, adding broad-spectrum nuclease into the recombinant adenovirus collecting solution for nuclease treatment, and degrading free nucleic acid molecules;
s6, separating and purifying nuclease-treated recombinant adenovirus collecting liquid by anion exchange column chromatography to obtain purified recombinant adenovirus;
all the operation steps of the technical scheme are carried out in a GMP clean room, the packed bed bioreactor is adopted to carry out three-dimensional high-cell-concentration culture and adherent growth of the SBN-1293 clone cells for the infection and the amplification of the recombinant adenovirus, the preparation efficiency is high, the requirements of the GMP production environment are met, the full-automatic column chromatography separation and purification are adopted, the more economical and effective large-scale industrialized GMP production of the recombinant adenovirus product is realized, and the recombinant adenovirus product is used for the clinical treatment of cancer patients.
In an example of the above technical solution, the step S1, the seeding the cells into the cell culture container after the cells are thawed, and performing cell expansion includes:
seeding in cell culture container with density of 0.5 × 104To 5x104Per centimeter2Culturing at 36-37 deg.C and 37 deg.C in 5% carbon dioxide incubator until the cell density reaches above 90%, removing cell wall with trypsin-EDTA, harvesting, and repeating cell expansion of the harvested cells to reach required amount.
In an example of the above technical solution, in step S1, the cells in the working cell bank are producer cells of recombinant adenovirus, and are clonal cells with adherent growth characteristics.
Further, the clone cell is SBN-1293 clone cell.
In an example of the above technical solution, in the step S2, the packed bed bioreactor adopts a filler composed of a polyester net and a polypropylene support frame, which can achieve three-dimensional high cell concentration, DMEM + 10% FBS is used as a cell culture solution, the pH of the culture solution is 6.5-8.2, the dissolved oxygen value is 15% -75%, the temperature is 36-37 ℃, and the stirring speed is 50-200 rpm.
In one example of the technical scheme, during the culture in the packed bed bioreactor, culture solution is adopted for perfusion, nutrients are supplemented, harmful byproducts of cell metabolism are eliminated, and the glucose concentration of the culture solution is kept to be not lower than 0.5 g/L.
In one example of the above technical solution, in the step S3, the virus multiplicity of infection is controlled to be 10-100 vp/cell.
In one example of the foregoing technical solution, in the step S4, the filtering and concentrating includes:
the harvested culture broth containing the recombinant adenovirus was filtered using a 0.65 μm tangential flow microfiltration system to remove large impurities, and the filtered harvest was concentrated using another 300KD MWCO tangential flow ultrafiltration system.
In an example of the foregoing technical solution, in step S6, the step of separating and purifying by anion exchange column chromatography is a full-automatic purification system controlled by Unicorn software, and the steps include:
sterilizing the purification system and the column filled with anion exchange resin with 1.0N NaOH solution;
the treated column was equilibrated with 20mM Tris, pH8.0 buffer A;
after balancing, putting the recombinant adenovirus collection liquid on a column, and washing the column by using a buffer solution A with 5-8 times of column volume until the UV absorption value reaches the bottom line;
eluting the recombinant adenovirus adsorbed on the column with a linear NaCl gradient from 30 column volumes of buffer A to 20mM Tris, pH8.0,2M NaCl in buffer B;
the Unicorn software determined the appearance of recombinant adenovirus peaks during elution by automatic monitoring of the UV absorbance at 260nm, with the purified recombinant adenovirus peaks being accepted when the UV absorbance at 260nm increased to 0.1AU and being stopped when the UV absorbance at 260nm decreased to 0.2 AU.
In an example of the above technical solution, the method further comprises step S7, converting the purified recombinant adenovirus into a solution for preparing a recombinant adenovirus gene vaccine product for the novel coronavirus.
In the step S7, the purified recombinant adenovirus is further concentrated by a small 300KD MWCO tangential flow ultrafiltration system to reach the index of the virus titer of the recombinant adenovirus biological preparation, and the purified recombinant adenovirus is converted into a preparation solution by percolation with the preparation solution to prepare and obtain the recombinant adenovirus gene vaccine;
drawings
FIG. 1 is a flow chart of a method for preparing a recombinant adenovirus biological agent of the present invention.
FIG. 2 is a schematic diagram showing an example of a packed bed bioreactor in the embodiment of the present invention.
FIG. 3 is a high definition structural image of the packing employed in a packed bed bioreactor in an example of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1: development of process for producing recombinant adenovirus vector gene vaccine product
This example illustrates the production of recombinant adenoviral vector gene vaccine for the prevention of novel coronavirus, which specifically includes the following steps, all of which are performed in a GMP clean room.
First, cell expansion
Taking 1 cell of recombinant adenovirus working cell bank, wherein the cell contains recombinant adenovirus vector gene for preventing novel coronavirus, and the cell amount is about 5x106To 1x107After the cells were thawed, the cells were inoculated into a T-flask for cell culture at an inoculation density of about 0.5X104To 5x104Per centimeter2. Culturing at 37 deg.C in 5% carbon dioxide incubator until the cell density reaches about 90%, and removing the cell wall with trypsin-EDTA for harvesting.
The harvested cells were seeded into more T-flasks to expand the cells, with the seeding density still maintained at 0.5X104To 5x104Per centimeter2The cell expansion procedure can be repeated until the cell mass reaches 1 × 109Can be used for inoculating a bioreactor.
Secondly, inoculating cells in the bioreactor
A packed bed bioreactor (such as CelliGen plus type bioreactor, New Brunswick scientific, Co. Inc. USA) is used for large-scale cell culture, and FIG. 2 shows the structure and working principle of the packed bed bioreactor, the structure comprises a reactor cavity 1, a heat-preservation water jacket 2 is arranged outside the reactor cavity, circulating water 21 in the heat-preservation water jacket enters from a water inlet 3 at the bottom and is discharged from a water outlet 4, a stainless steel fiber bed basket 5 is arranged in the reactor cavity, a filler 6 is arranged in the basket, a rotary stirring paddle 8 on a stirring device 7 provides circulating flow of culture solution in the reactor cavity, and a culture solution feeding port 9 and a collecting pipe 10, a buffer solution collecting pipe 11 and an air inlet 12 are arranged at the upper part of the reactor cavity.
About 250 grams of Fibra-Cel Disks (New Brunswick Scientific, Co.Inc.) packing was packed into the stainless steel basket of the cleaned packed bed bioreactor. Then 10 liters of PBS was added to the bioreactor. After connecting the required rubber tubing and the bioprobe, the bioreactor was sterilized in a sterilization cabinet at 121 ℃ for 45 minutes.
The adopted fibre-Cel Disks filler is composed of a polyester net and a polypropylene support frame, a high-definition structural image of the fibre-Cel Disks is shown in figure 3, and cells grow on the surface and in the inner gaps of the fibre-Cel Disks, so that high cell concentration can be achieved.
The PBS solution was removed from the sterilized bioreactor, 5 liters of DMEM + 10% FBS cell culture medium was added, and cells harvested from the T-flask were inoculated into the bioreactor for culture at about 1X109To 1x1010. The pH and Dissolved Oxygen (DO) of the culture solution are automatically controlled by a bioreactor controller through a pH and dissolved oxygen probe, the pH is controlled to be 6.5-8.2, and the DO is controlled to be 15% -75%. The temperature of the culture solution is automatically controlled between 36 ℃ and 37 ℃ by circulating water in the water jacket, the stirring of the culture solution is provided by a rotary stirring paddle of the bioreactor, and the stirring speed is controlled between 50 rpm and 200 rpm.
On the 3 rd day after cell inoculation, culture solution perfusion is started, nutrients are continuously provided, harmful byproducts of cell metabolism are continuously removed, the perfusion amount of the culture solution is adjusted to keep the glucose concentration of the culture solution in the bioreactor not lower than 0.5 g/L, and recombinant adenovirus infection can be carried out after the culture solution grows for 5 days.
Third, recombinant adenovirus infection and amplification
The method comprises the steps of taking a working virus bank virus of the recombinant adenovirus to infect cells in a bioreactor, controlling the virus infection complex number to be between 10 and 100 vp/cell, then culturing for 7 days, releasing the recombinant adenovirus into a culture solution after the virus is infected for 3 to 5 days, starting to harvest the culture solution containing the recombinant adenovirus at the moment until the culture process is finished, harvesting a virus collecting solution 35L with the virus amount of 1.6x1015vp, temporarily storing the culture solution containing the recombinant adenovirus harvested every day in a refrigeration house at 4 ℃, and finally collecting, processing and purifying.
Fourth, recombinant adenovirus culture solution is filtered and concentrated
Firstly, filtering the collected recombinant adenovirus culture solution by using a tangential flow microfiltration system with the diameter of 0.65 mu m to remove large impurities such as cell debris, and then concentrating the filtered collection solution by 10-50 times by using another tangential flow ultrafiltration system with 300KD MWCO to obtain a 3L collection solution with the total virus content of 1X1015 vp.
Further performing diafiltration treatment on the concentrated collection, and converting the collection into another buffer solution to facilitate subsequent nuclease treatment and ion exchange column chromatography separation and purification, wherein the diafiltration coefficient is between 3 and 10. The tangential flow ultrafiltration concentration and diafiltration process pressure parameters are controlled between 5-20psi and to increase the efficiency of nuclease treatment, the concentrated and diafiltered harvest is diluted with buffer prior to nuclease addition.
Fifthly, nuclease treatment
Uses a broad-spectrum nuclease Benzonase which is widely used in the production process of biological productsTM. Mixing BenzonaseTMThe free nucleic acid molecules are degraded by addition to the concentrated and diafiltered pool diluted with buffer, at a dose of between 10 and 20 u/ml, and the degradation treatment is carried out in a water bath at 37 ℃ for 1 hour.
Sixthly, full-automatic ion exchange column chromatography separation and purification
Separating and purifying the Benzonase by anion exchange column chromatography at room temperatureTMAnd (3) purifying the treated recombinant adenovirus collection liquid by adopting a full-automatic purification system controlled by Unicorn software.
The anion exchange resin is loaded in a column of BPG300, the loading volume is about 2.5 liters, and the anion exchange resin can adopt Sepharose Q X L or Source Q resin.
Before using the column, the column was tested for acceptable performance by HETP (height Equivalent to thermal plate), and before the column was loaded, the purification system and the column containing the anion exchange resin were first sterilized with 1.0N NaOH solution.
The treated column was first equilibrated with buffer A (20mM Tris, pH8.0), after which the column was equilibrated, and the recombinant adenovirus pool was started to be applied to the column, which was then washed with 5 column volumes of buffer A until the UV absorbance reached the bottom.
The recombinant adenovirus adsorbed on the column was then eluted with a linear NaCl gradient from 30 column volumes of A buffer to B buffer (20mM Tris, pH8.0,2M NaCl).
The Unicorn software determined the appearance of recombinant adenovirus peaks during elution by automatic monitoring of UV absorbance at 260 nm. When the UV absorption value of 260nm is increased to 0.1AU, the purified adenovirus group peak is automatically received into a sterile bottle, and when the UV absorption value of 260nm is decreased to 0.2AU, the receiving is stopped, and the virus liquid volume is 600ml, the virus concentration is 60X1010vp/ml, the virus purity is 95%, and the total virus amount is 6X1014 vp.
The column and purification system are then washed sequentially with alkaline, buffer, and acid.
Seventhly, preparing, subpackaging and freeze-drying the product
The recombinant adenovirus product was further diafiltered (10mM Tris-HC L +1mM MgCl2 pH8.0, containing 6% mannitol, 5% sucrose and 0.5% human serum albumin) with a diafiltration coefficient between 10 and 15 to ensure complete conversion of the purified recombinant adenovirus into the product formulation and to ensure product stability.
Filtering the recombined adenovirus with a 0.22-micron sterile filter to produce sterile bulk liquid medicine of the product, sampling 24ml of the sterile bulk liquid medicine, performing quality inspection on items such as sterility, bacterial endotoxin, virus concentration and the like, temporarily storing the sterile bulk liquid medicine in a freezing box at the temperature of minus 80 ℃, and bottling the qualified sterile bulk liquid medicine in recombined adenovirus medicine preparations.
A batch of sterile bulk drug solution was thawed at room temperature overnight. Filtering with 0.22 μm sterile filter to obtain sterile liquid medicine, sampling, inspecting, bottling with automatic filling machine, and adding lyophilized bottle stopper.
Filling the canned bottles into a freeze dryer in a sterile environment, and controlling the freeze drying process by adopting a program, wherein the process comprises the following steps:
1. setting the temperature of the freeze-drying rack at-45 ℃, and freezing the product in the bottle for 2 hours.
2. Setting the temperature of the freeze-drying rack at-45 ℃, starting a vacuum pump, setting the vacuum at 400mT, and keeping the time for 5 hours.
3. The temperature of the freeze-drying rack is set at-35C, the vacuum is set at 200mT, and the time is 13 hours.
4. The freeze-drying rack temperature was set at-22C and the vacuum was set at 100mT for 15 hours.
5. The temperature of the freeze-drying rack is set at-10 ℃, the vacuum is set at 100mT, and the time is 5 hours.
6. The freeze-drying rack temperature was set at 10C and the vacuum was set at 100mT for 4 hours.
7. The stopper was pressed under vacuum to seal the product in the bottle.
8. And (5) finishing the freeze-drying process, taking out the freeze-dried product, and capping.
After the freeze drying is finished, sampling the recombinant adenovirus anticancer injection product for quality inspection and detection. The detection items comprise: asepsis, appearance (light white liquid without visible foreign matters), endotoxin less than 10 EU/count, mycoplasma negative by adopting culture method and fluorescent staining method, virus concentration 1X 1012 vp/count, virus purity more than or equal to 95%, virus activity more than or equal to 3.3X 1010 IU/count, specific activity more than or equal to 3.3%, biological activity cancer cell TCID 50: 100-500 MOI, replication active virus less than 1RCA/3X 1010vp, residual cell protein less than or equal to 100 ng/count, residual bovine serum albumin less than or equal to 50 ng/count, residual cell DNA less than or equal to 10 ng/count, residual BenzonaseTMLess than or equal to 1 ng/branch without AAV pollutionAbnormal toxicity, insoluble particles, osmolality, etc. Qualified products are approved for clinical use by the quality control department.
Claims (9)
1. A method for the batch production of recombinant adenovirus for the gene vaccine of new type coronavirus,
the method comprises the following steps:
s1, taking cells of a working cell bank of the recombinant adenovirus packaging cells, thawing the cells, and inoculating the cells into a cell culture container for cell amplification;
s2, inoculating the cells obtained by amplification in the cell culture container into a packed bed bioreactor for culture;
s3, taking the working virus library virus of the recombinant adenovirus to infect cells in the packed bed bioreactor, releasing the recombinant adenovirus into the culture solution, and harvesting the culture solution containing the recombinant adenovirus;
s4, filtering and concentrating the culture solution containing the recombinant adenovirus, and diluting the recombinant adenovirus collection solution with a buffer solution;
s5, adding broad-spectrum nuclease into the recombinant adenovirus collecting solution for nuclease treatment, and degrading free nucleic acid molecules;
s6, separating and purifying nuclease-treated recombinant adenovirus collecting liquid by anion exchange column chromatography to obtain purified recombinant adenovirus.
2. The method for mass production of recombinant adenovirus for novel coronavirus gene vaccine according to claim 1,
in step S1, the step of inoculating the thawed cells into a cell culture container for cell expansion comprises:
seeding in cell culture container with density of 0.5 × 104To 5x104Per centimeter2Culturing at 36-37 deg.C and 37 deg.C in 5% carbon dioxide incubator until the cell density reaches above 90%, removing cell wall with trypsin-EDTA, harvesting, and repeating cell expansion of the harvested cells to reach required amount.
3. The method for mass production of recombinant adenovirus for novel coronavirus gene vaccine according to claim 1 or 2,
in step S1, the cells in the working cell bank are producer cells of recombinant adenovirus, and are clonal cells with adherent growth characteristics.
4. The method for mass production of recombinant adenovirus for novel coronavirus gene vaccine according to claim 3,
the clone cell is SBN-1293 clone cell.
5. The method for mass production of recombinant adenovirus for novel coronavirus gene vaccine according to claim 1,
in the step S2, the packed bed bioreactor adopts a filler which can realize three-dimensional high cell concentration and is composed of a polyester net and a polypropylene support frame, DMEM and 10% FBS are adopted as cell culture solution, the pH value of the culture solution is 6.5-8.2, the dissolved oxygen value is 15% -75%, the temperature is 36-37 ℃, and the stirring speed is 50-200 rpm.
6. The method for mass production of recombinant adenovirus for novel coronavirus gene vaccine according to claim 5,
in the process of culturing in the packed bed bioreactor, culture solution is adopted for perfusion, nutrients are supplemented, harmful byproducts of cell metabolism are eliminated, and the glucose concentration of the culture solution is kept to be not lower than 0.5 g/L.
7. The method for mass production of recombinant adenovirus for novel coronavirus gene vaccine according to claim 1,
in the step S3, the virus infection complex number is controlled to be 10-100 vp/cell.
8. The method for mass production of recombinant adenovirus for novel coronavirus gene vaccine according to claim 1,
in step S4, the filtering and concentrating includes:
the harvested culture broth containing the recombinant adenovirus was filtered using a 0.65 μm tangential flow microfiltration system to remove large impurities, and the filtered harvest was concentrated using another 300KD MWCO tangential flow ultrafiltration system.
9. The method for mass production of recombinant adenovirus for novel coronavirus gene vaccine according to claim 1,
in step S6, the step of separating and purifying by anion exchange column chromatography is a full-automatic purification system controlled by Unicorn software, and includes the steps of:
sterilizing the purification system and the column filled with anion exchange resin with 1.0N NaOH solution;
the treated column was equilibrated with 20mM Tris, pH8.0 buffer A;
after balancing, putting the recombinant adenovirus collection liquid on a column, and washing the column by using a buffer solution A with 5-8 times of column volume until the UV absorption value reaches the bottom line;
eluting the recombinant adenovirus adsorbed on the column with a linear NaCl gradient from 30 column volumes of buffer A to 20mM Tris, pH8.0,2M NaCl in buffer B;
the Unicorn software determined the appearance of recombinant adenovirus peaks during elution by automatic monitoring of the UV absorbance at 260nm, with the purified recombinant adenovirus peaks being accepted when the UV absorbance at 260nm increased to 0.1AU and being stopped when the UV absorbance at 260nm decreased to 0.2 AU.
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