WO2022262206A1 - Gmp级逆转录病毒载体的纯化方法与应用 - Google Patents

Gmp级逆转录病毒载体的纯化方法与应用 Download PDF

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WO2022262206A1
WO2022262206A1 PCT/CN2021/134470 CN2021134470W WO2022262206A1 WO 2022262206 A1 WO2022262206 A1 WO 2022262206A1 CN 2021134470 W CN2021134470 W CN 2021134470W WO 2022262206 A1 WO2022262206 A1 WO 2022262206A1
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retrovirus
virus
ultrafiltration
microfiltration
membrane
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French (fr)
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朱建高
李佳佳
杨文君
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浙江康佰裕生物科技有限公司
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/00041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/00043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/00051—Methods of production or purification of viral material

Definitions

  • the invention relates to the field of biotechnology, in particular to a purification method and application of a GMP-grade retroviral vector, in particular to a purification method and application of a GMP-grade retroviral vector applicable to chimeric antigen receptor cell therapy.
  • Gene therapy has great application potential in the treatment of many diseases, and is also considered to be the ultimate means of next-generation clinical treatment.
  • Gene therapy refers to the introduction of exogenous DNA fragments into target cells, and targeted intervention on defective and abnormal genes by means of correction, repair, replacement, compensation or silencing, in order to restore normal gene functions and finally achieve the goal of treatment or even complete cure .
  • Retrovirus-derived vectors have become one of the most widely used vectors in gene therapy clinical trials. At present, the preparation process of retroviral vectors still faces great challenges.
  • the clinical application of viral vectors is mainly divided into two types: one is to infect target cells in vitro, and then the target cells with exogenous genes are cultured and expanded in vitro and then reinfused into the human body, which is called ex vivo, such as CAR-T cell therapy ;
  • the other is to directly infect target cells and tissues in vivo with purified viral vectors, which is called in vivo, such as oncolytic virus therapy.
  • the virus activity titer required for clinical practice is generally on the order of 10 7 IP/mL; for the latter, the virus activity titer even requires the order of 10 9 IP/mL.
  • retroviral vector retroviral vector
  • the traditional method of obtaining retrovirus (retroviral vector) liquid by collecting the supernatant of stable producing strains cannot meet this requirement.
  • retroviral vectors With the increasing demand for clinical-grade high-purity retroviruses (retroviral vectors), there is an urgent need to develop new processes for the preparation and purification of retroviruses (retroviral vectors) that are efficient and suitable for large-scale production.
  • retroviruses retroviral vectors
  • the general process for the preparation of retroviruses is to first transfect cell lines expressing retroviral gag, pol and env genes with plasmids containing the target genes, and to obtain stable secretion containing target genes through multiple rounds of subcloning screening. Stable producing strains of recombinant retroviruses. Subsequently, when necessary, the stable producing strains are expanded and cultured, and the supernatant is collected to obtain a crude extract of retrovirus (retroviral vector), which is used in subsequent purification steps. Compared with adenovirus and lentivirus, retrovirus is less stable and more sensitive to shear force.
  • the present invention provides a method for purifying a retrovirus (retroviral vector), said method comprising the steps of:
  • the method also includes the step of filtering and sterilizing the unsterilized virus to obtain a purified retrovirus (retroviral vector).
  • PVDF membrane material filter with a pore size of 0.22 ⁇ m can be used to filter and sterilize the above viruses.
  • the membrane separation pore size (micro-membrane pore size) of the semi-permeable membrane used is 0.45 ⁇ m-0.75 ⁇ m, and/or the membrane material of the semi-permeable membrane used is a modified polymer Ethersulfone.
  • the membrane separation pore size (micro-membrane pore size) may be 0.45 ⁇ m-0.65 ⁇ m, or 0.65 ⁇ m or 0.45 ⁇ m.
  • the flow rate of the cell culture supernatant can be 89mL ⁇ min -1 to 133.5mL ⁇ min -1 , 89mL ⁇ min -1 or 133.5mL ⁇ min -1 .
  • the shear rate of the microfiltration treatment may be 2000s -1 -3000s -1 , 2000s -1 or 3000s -1 .
  • the microfiltration treatment can specifically be any of the following:
  • the membrane separation pore size (micro-membrane pore size) of the semipermeable membrane used is 0.65 ⁇ m
  • the flow rate of the cell culture supernatant is 89 mL ⁇ min -1
  • the shear rate of the microfiltration treatment is 2000 s -1 ;
  • the membrane separation pore size (micro-membrane pore size) of the semipermeable membrane used is 0.65 ⁇ m
  • the flow rate of the cell culture supernatant is 133.5 mL ⁇ min ⁇ 1
  • the shear rate of the microfiltration treatment is 3000 s ⁇ 1 1 ;
  • the membrane separation pore size (micro-membrane pore size) of the semipermeable membrane used is 0.45 ⁇ m
  • the flow rate of the cell culture supernatant is 89 mL ⁇ min ⁇ 1
  • the shear rate of the microfiltration treatment is 2000 s ⁇ 1 .
  • the concentration of nuclease in the reaction system is 1-500 U ⁇ mL -1 , and/or react at 2-8°C for 8-24 hours.
  • the nuclease concentration is 25, 50 or 100 U ⁇ mL ⁇ 1 ;
  • the nuclease treatment time is 8 hours, 16 hours or 24 hours;
  • the nuclease treatment temperature is 4°C or 37°C;
  • the nuclease treatment can specifically be any of the following:
  • the concentration of nuclease in the reaction system is 100U ⁇ mL -1 , and/or react at 4°C for 16 hours;
  • the concentration of nuclease in the reaction system is 100U ⁇ mL -1 , and/or react at 4°C for 24 hours;
  • the concentration of nuclease in the reaction system is 50U ⁇ mL -1 , and/or react at 4°C for 24 hours.
  • the molecular weight cut-off of the ultrafiltration membrane in the ultrafiltration concentration step in step C) is 350KD, 500KD or 750KD, preferably 750KD.
  • step C) carries out ultrafiltration concentration twice respectively as follows:
  • c1) First use a hollow fiber column with an inner diameter of 0.5 mm and a membrane area of 0.16 m 2 ; control the shear rate to 2000 s ⁇ 1 to perform ultrafiltration concentration on the enzyme-digested virus liquid to obtain a 5-fold concentrated primary ultrafiltration concentrated virus liquid;
  • the flow rate of the virus liquid in c1) can be 370mL ⁇ min ⁇ 1 ;
  • c2) Then use a hollow fiber column with an inner diameter of 0.5 mm, a membrane area of 115 cm 2 , and a controlled shear rate of 2000 s -1 to perform ultrafiltration concentration on the primary ultrafiltration concentrated virus liquid obtained in c1) to obtain the volume of c1) virus liquid 5-10 times concentrated virus solution concentrated by secondary ultrafiltration; the flow rate of virus solution in c2) can be 53mL ⁇ min -1 .
  • processing conditions of low-speed centrifugation in step D) are: centrifugal force 4000g-10000g, centrifugation at 4°C for 4-24h; preferably at 4°C, centrifugation at 6000g for 16h;
  • the centrifugal force is 4000-8000g, 4000g, 6000g or 8000g;
  • the centrifugation time is 4-24 hours, 4 hours, 8 hours, 16 hours or 24 hours;
  • the treatment process of the low-speed centrifugation is specifically any of the following:
  • the present invention provides a retrovirus (retroviral vector) obtained by the above method.
  • the present invention provides a product (for example, a medicine or a vaccine) comprising the above-mentioned retrovirus (retroviral vector).
  • the present invention also provides the application of the retrovirus (retroviral vector) in the preparation of gene therapy products and/or cell therapy products and/or immunotherapy products.
  • the gene therapy product is an ex vivo gene therapy product, such as a CAR-T cell therapy product.
  • Retrovirus (retrovirus vector) crude extract is the culture supernatant of a stable strain of retrovirus, which contains a variety of impurities, including host DNA residues (HCD), host protein residues (HCP), bovine serum albumin residues (BSA), etc.
  • HCD host DNA residues
  • HCP host protein residues
  • BSA bovine serum albumin residues
  • HCD is generally not higher than 10ng/100 ⁇ g protein
  • BSA is not higher than 50ng/dose
  • HCP is generally not higher than 0.1% of the total protein content.
  • retrovirus retroviral vector
  • This solution adopts steps such as microfiltration, ultrafiltration, nuclease treatment, and low-speed centrifugation, and has a good removal effect on large fragments, small molecules, and nucleic acid impurities.
  • steps such as microfiltration, ultrafiltration, nuclease treatment, and low-speed centrifugation, and has a good removal effect on large fragments, small molecules, and nucleic acid impurities.
  • the host HCD, HCP, BSA and other impurity components in the retrovirus crude extract can be effectively removed.
  • the process is easy to scale up and meets GMP requirements.
  • BSA ⁇ 200ng/ml, HCP ⁇ 1 ⁇ g/ml, HCD ⁇ 100ng/ml After the purified retrovirus (retroviral vector) feed liquid is tested, BSA ⁇ 200ng/ml, HCP ⁇ 1 ⁇ g/ml, HCD ⁇ 100ng/ml. It has been verified that this virus liquid can be used in the downstream process of ex vivo gene therapy, such as the preparation of CAR-
  • tangential flow refers to the filtration form in which the flow direction of the liquid is perpendicular to the filtration direction.
  • the flow direction of the liquid to be filtered is parallel to the direction of the filter membrane plane, and the liquid will pass through perpendicular to the membrane surface. membrane hole. Tangential flow will generate turbulent flow (secondary flow).
  • the liquid flow Due to turbulent flow, the liquid flow generates shear force on the surface of the filter medium (that is, the surface of the ultrafiltration membrane), which reduces the accumulation of the filter cake layer or gel layer on the membrane surface. , so that the precipitate is stripped from the membrane surface, reducing membrane fouling and ensuring a stable filtration rate.
  • the shear force of tangential flow may damage the envelope structure of retroviruses, so parameters such as flow rate, membrane area, and membrane pore size need to be optimized to control the damage of shear force to retrovirus viability.
  • the present invention adopts mPES material filter membrane with large pore size (molecular mass cut-off 750kDa) and optimizes the shear rate to ensure permeation flux and filtration effect while reducing shear force damage, thereby reducing damage to the envelope structure of retroviruses. destruction, the overall recovery of retroviruses exceeded 80%. 3.
  • the applicant took advantage of the characteristics of large particles and easy sedimentation of retroviruses (retrovirus vectors), and innovatively used a combined process of low-temperature and low-speed centrifugation combined with ultrafiltration to remove more than 90% of BSA and HCP impurities in the supernatant, and at the same time Played the role of concentrated virus fluid. While ensuring the purity of the retrovirus, the scheme can achieve a titer of 10 7 IP/ml in the concentrated virus solution, which meets the requirements of the downstream process.
  • the cell culture supernatant of the recombinant retrovirus (retroviral vector) can be a product that has been obtained, and this embodiment is only an example of the preparation of the cell culture supernatant containing the recombinant retrovirus (retroviral vector) Method: Insert the target gene into the retrovirus expression plasmid to obtain the recombinant retrovirus expression plasmid; transfect the packaging cell line with the recombinant retrovirus expression plasmid, and obtain the recombinant retrovirus (retroviral vector) containing recombinant retrovirus (retroviral vector) after packaging by the packaging cell line Cell culture medium, the cell culture medium containing recombinant retrovirus (retroviral vector) is used to transfect the stable cell line, and after several times of subcloning to obtain the stable cell line of stably packaging retrovirus (retroviral vector) Cultivate the above-mentioned stable transfection cell line, collect the cell supernatant to obtain the cell culture supern
  • the green fluorescent protein EGFP gene as an example, insert the EGFP gene (MK387175.1, SYN 12-AUG-2019) between the XhoI and EcoRI recognition sites of the retroviral expression plasmid (pMSCVneo, Youbao Biology), And keeping other sequences of the plasmid pMSCVneo unchanged, the recombinant retrovirus expression plasmid pMSCVneo-EGFP was obtained.
  • the pMSCVneo-EGFP was packaged according to the following method to obtain a recombinant retrovirus (retroviral vector), which was named EGFP-RV.
  • This crude virus solution was named EGFP-RV-C1.
  • the EGFP-RV-C1 obtained in the previous step was infected with PG13 cells (ATCC, CRL-10686). Two days after infection, EGFP-positive cells were enriched with EGFP antibody (Biolegend) to obtain enriched cells. Take a part of the enriched cells to detect the expression efficiency of EGFP by flow cytometry, take another part of the enriched cells and dilute them into single cells, spread them on a 96-well plate, and use the supernatant on the 5th day after culture in the 96-well plate as the reverse Recording virus (retroviral vector) solution.
  • HT1080 cells ATCC, CCL-121
  • flow cytometry to measure the above-mentioned retrovirus (retroviral vector) fluid titer.
  • the three PG13 cell lines with the highest virus titers in the 96-well plate were screened out and inoculated into the 24-well plate to continue culturing for secondary screening.
  • DMEM complete medium DMEM medium + 10% FBS + 100U/mL penicillin + 10 ⁇ g/mL streptomycin + 2mM Glutamine
  • DMEM complete medium DMEM medium + 10% FBS + 100U/mL penicillin + 10 ⁇ g/mL streptomycin + 2mM Glutamine
  • the cell culture supernatant was collected to obtain a cell culture supernatant containing a retrovirus (retroviral vector), and the supernatant was named EGFP-RV-C2 as a feed solution for subsequent purification.
  • microfiltration clarification and purification system is emptied after the system is balanced, and the cell culture supernatant EGFP-RV-C2 containing retrovirus (retroviral vector) in step 1 is injected into the system to regulate EGFP-RV-
  • the flow rate of C2 is 89mL ⁇ min-1, and the shear rate is 6000s -1 .
  • First close the permeation end of the system let the virus liquid circulate in the system for 2-10 minutes, make the system stable, turn on the switch of the permeation end, start microfiltration, collect the permeate, the permeate is the microfiltration virus liquid, named as EGFP-RV-C3.
  • the enzyme-digested virus liquid obtained in 2.2 is concentrated by ultrafiltration using an ultrafiltration concentration system.
  • the shear rate is 6000s -1 , empty the system after sterilization, and fill the system with sterile distilled water , circulate for 20-30min, until the pH in the purification system is ⁇ 7.0, and the circulation shear rate is 6000s -1 .
  • Collect the retentate press 1:1 (V/V) PBS with the volume of the retentate, turn on the circulation pump to wash for 5 minutes, collect the first wash, and start the cycle with PBS with the volume of the retentate at 1:1 (V/V) The pump circulates the washing for 5 minutes, and collects the second washing liquid.
  • the concentrated solution EGFP-RV-C5 (5) obtained in this step has a volume of 400 mL.
  • a large-volume hollow fiber column (with a membrane area of 0.16m 2 , and the other parameters are the same as above) is added before the above ultrafiltration step to concentrate the starting feed liquid by 5 times.
  • the molecular mass cut-off in the new hollow fiber column is 750KD
  • the inner diameter of the hollow fiber capillary (abbreviated as fiber inner diameter) is 0.5mm
  • the membrane area of the semipermeable membrane is 0.0115m2 .
  • virus suspension EGFP-RV-C7 was filtered and sterilized using a PVDF membrane material filter with a pore size of 0.22 ⁇ m to obtain a purified virus liquid, that is, a GMP-grade retrovirus (retroviral vector), with a volume of 4 mL, named It is EGFP-RV-F and stored at -80°C.
  • the number of HT1080 cells infected with retrovirus per unit volume was indirectly measured, that is, the virus activity titer or the number of infected particles (IP/mL).
  • HT1080 cells in the logarithmic phase of growth were collected, digested with trypsin for 2-3 minutes, and added complete medium to prepare a cell suspension.
  • HT1080 cells in logarithmic growth phase were inoculated at a density of 1.5 ⁇ 10 5 cells/well.
  • the TC6-well plate was shaken up and down, left and right, marked, and placed in a carbon dioxide incubator at 37°C for 20-24 hours.
  • a virus solution containing 8 ⁇ g/mL polybrene was added to each well, and the virus solution and complete medium were diluted in different proportions.
  • the cells were incubated at 37°C in a CO 2 incubator for 1 hour, and shaken once every 15 minutes. After the fourth shaking, 2 mL of complete medium was added, and the culture was continued for 48 hours. Aspirate the virus transduction solution in the wells of the cell plate, and add 2mL PBS to each well to wash the cells. Add 100 ⁇ L of 0.25% trypsin to each well and let stand at room temperature for 1-2 minutes. Digestion was terminated by adding 500 ⁇ L of complete medium to each well. Pipette the cell suspension with a pipette, centrifuge at 1500rpm for 5min, and collect the cell pellet.
  • the cell suspension was inoculated into a 96-well plate at 3 ⁇ 10 4 cells/well, centrifuged at 1500 rpm for 5 min. Wash each well once with 200 ⁇ L FACS buffer, centrifuge at 1500rpm for 5min. According to 1:100 dilution, 20 ⁇ L FITC-labeled EGFP antibody (Biolegend) was added to each well, and incubated in the dark for 10 min. Centrifuge again, and add 200 ⁇ L FACS buffer to each well. Streaming machine testing.
  • IP ⁇ mL -1 (F ⁇ N)/V
  • V volume of virus added in mL.
  • Virus recovery rate harvested virus titer/initial virus titer ⁇ 100%.
  • the host cell residual DNA sample pretreatment kit (magnetic bead method) (Huzhou Shenke, SK030203D100) was used to complete.
  • the qPCR detection of samples is carried out according to the following steps:
  • PG13 cells were collected, and genomic DNA was extracted with a DNA extraction kit (QIAGEN, 80204) as a quantitative reference.
  • a DNA extraction kit QIAGEN, 80204
  • the dilution concentration is 3ng/ ⁇ l, 300pg/ ⁇ L, 30pg/ ⁇ L, 3pg/ ⁇ L, 300fg/ ⁇ L, 30fg/ ⁇ L , 3 fg/ ⁇ L.
  • Set recovery quality control ERC take the prepared sample plus 300pg quantitative reference substance as the ERC, take 100 ⁇ l of the sample to be tested and add it to a 1.5ml clean centrifuge tube. Then add 10 ⁇ L ST2, mix well, and mark as sample ERC.
  • Set up negative quality control NCS Take 100 ⁇ L of sample matrix solution (or DNA dilution) into a 1.5ml clean centrifuge tube, and mark it as negative quality control NCS.
  • Number of reaction wells (standard curve of 6 concentration gradients + 1 no-template control NTC + 1 negative quality control NCS + sample to be tested ⁇ 2) ⁇ 3; calculate the total amount of qPCR MIX required this time according to the number of reaction wells, Prepare qPCR MIX according to the recipe in Table 1.
  • the primer sequences are:
  • Reverse primer 5'-GCCTGGCAAATACAGAAGTGG-3'.
  • Q-PCR probe mix was purchased ready-made commercial master mix (Vazyme, AceQ PCR probe master mix).
  • qPCR reaction put the reagents in Table 2 on ice and mix well, and the total volume after adding the sample is 25 ⁇ L. Establish qPCR template and run parameters. 95°C, 10min, 1cycle; 95°C, 15s, 60°C, 1min, 40cycle; 40°C, 30s, 1cycle.
  • On-machine and result analysis Run the program on the ABI 7500 qPCR instrument.
  • the column of Mean Quantity can read the detection values of the no-template control NTC, negative quality control NCS, sample to be tested, and sample ERC , the unit is pg/10 ⁇ L. Later, the unit can be converted to pg/ ⁇ L or pg/mL in the test report. Calculate the sample recovery rate based on the test results of the sample to be tested and the sample ERC, and the sample recovery rate is required to be between 50% and 150%.
  • the test result of NTC should be Undetermined or Ct value ⁇ 35.
  • the Ct value of NCS should be greater than the lowest concentration Ct value of the curve.
  • the determination method of BSA is: use Bovine Serum Albumin (BSA) ELISA detection kit (Cygnus, F030) to detect the BSA residue in the sample.
  • BSA Bovine Serum Albumin
  • the kit provides an anti-BSA monoclonal antibody-coated plate, and an enzyme-labeled anti-BSA polyclonal antibody is used as a detection antibody to form a double-antibody sandwich ELISA detection kit.
  • According to the supplier's instructions first add the sample or standard to be tested, so that the BSA in it can bind to the antibody immobilized on the plate, after thorough washing, add HRP-labeled anti-BSA polyclonal antibody for incubation.
  • TMB substrate is added, and TMB is converted into blue under the catalysis of HRP, and finally converted into yellow under the action of stop solution.
  • the shade of color is related to the amount of BSA in the sample or standard.
  • the invention adopts the ELISA sandwich method to detect the HCP in the sample.
  • HCP antibody is a polyclonal antibody purified from the serum of rabbits immunized with PG13 cell culture supernatant.
  • the specific detection method is:
  • a. Plate coating use anti-PG13 cell supernatant antibody to coat the surface of the reaction well of the test plate at an optimal concentration. Optimal antibody concentrations were determined by using known concentrations of PG13 cell supernatants to generate a standard curve with the required sensitivity and precision over the required effective concentration range. For PG13 cell supernatant protein, the effective concentration range that this kit can detect is 62.5ng/mL to 4000ng/mL. One of ordinary skill in the art can easily determine whether there is suitable sensitivity and precision within the required range without undue experimentation.
  • washing buffer 0.01mol/L phosphate buffer (0.0027mol/L potassium chloride, 0.137mol/L sodium oxide, pH7.4, containing 0.01% w/v TritonX-100).
  • Plate blocking Add protein and detergent blocking buffer (coating buffer solution containing 1% BSA/0.1% Triton X-100) to the reaction wells. Tablets can be stored in this form.
  • TMBS tetramethylbiphenylsulfonate
  • Nucleases can completely digest RNA and DNA (single-stranded, double-stranded, linear, circular, and supercoiled) with no fewer than 5 phosphate residues, forming 5'-monophosphate ends 3-5 bases in length Oligonucleotides.
  • the invention utilizes the HCD in the nuclease degradation process to make it conveniently removed through subsequent purification methods. However, since nuclease itself is also a protein impurity, it needs to be removed together with other impurities in the subsequent purification process.
  • the present invention detects the residual amount of nuclease in downstream products by purchasing Sino Biological Supernuclease ELISA kit. For specific detection steps, please refer to Sino Biological official website and product manual.
  • step 2 retrovirus (retroviral vector) in Example 1 repeat 3 times to obtain 3 batches of GMP grade retrovirus (retroviral vector) EGFP-RV-F, named respectively
  • the nuclease content, BSA content, HCP content, HCD content and virus titer of batch 1, batch 2 and batch 3 were detected according to the above method.
  • the concentration factor of the feed liquid volume is 500 times.
  • the initial feed titer refers to the titer converted into a volume of 2L feed liquid
  • the titer after purification refers to the titer converted into a volume of 4mL feed solution.
  • Embodiment 2 optimization of retrovirus (retroviral vector) purification process
  • step 2 of Example 1 different flow rates and membrane pore sizes were used to control the shear force to obtain the recovery rate of crude venom microfiltration under different conditions.
  • the cell culture supernatant EGFP-RV-C2 (the titer of the virus is 8.86 ⁇ 10 5 IP ⁇ mL ⁇ min -1 ) was replaced by 133.5mL ⁇ min -1 and 44.5mL ⁇ min -1 in Table 3 from 89mL ⁇ min -1 , and the shear rate was replaced by 1000s -1 in Table 3 from 6000s -1 and 2000s -1 , except that the membrane separation pore size (micro-membrane pore size) was replaced from 0.45 ⁇ m to 0.65 ⁇ m, other operations were the same as step 2.1 of Example 1.
  • nuclease treatment in 2.2 of step 2 of Example 1 With reference to the nuclease treatment in 2.2 of step 2 of Example 1, different nuclease concentrations, treatment times and treatment temperatures were used to obtain the recovery rate of the virus liquid after nuclease treatment under different conditions.
  • the effects of different shear rates and fiber inner diameters on the recovery rate of virus fluid were further explored. Since retroviral (retroviral vector) particles are shear sensitive, the shear rate directly affects viral activity and recovery. In addition, the inner diameter of the hollow fiber tube will directly affect the flux of the feed liquid. For the same volume of feed liquid, the smaller the inner diameter, the smaller the flux and the slower the flow rate.
  • the flow rate of the ultrafiltration virus liquid was controlled at 38, 53, 79, 100, 106, 141, 212, 283 mL ⁇ min -1 , and the shear rate was controlled at 1441, 2000, 3000, 4000s -1 , fiber inner diameter of 0.5, 1.0mm, membrane area of 75, 115cm 2 keeping 2.3 and other treatment processes unchanged, the results obtained are shown in Table 6.
  • test results in Table 6 show that the maximum virus recovery can be obtained when the hollow fiber column with an inner diameter of 0.5 mm and a membrane area of 115 cm 2 is used, the liquid flow rate in the column is 53 mL ⁇ min -1 , and the shear rate is controlled at 2000 s -1 .
  • BSA clearance rate [BSA(EGFP-RV-C5) ⁇ harvested feed volume]/[BSA(EGFP-RV-C4) ⁇ initial feed volume].
  • HCD ⁇ 100ng/ml, and nuclease was lower than the detection limit ( ⁇ 3.15ng/mL), indicating that these two process residue indicators have basically met the requirements.
  • HCP and BSA were still slightly above the norm, with median mean values of 13078 ng/ml and 2056 ng/ml (EGFP-RV-C6), respectively.
  • BSA clearance rate [BSA (EGFP-RV-C7) ⁇ harvested feed volume] / [BSA (EGFP-RV-C6) ⁇ initial feed volume].
  • HCP clearance rate [HCP (EGFP-RV-C7) ⁇ volume of harvested feed solution]/[HCP (EGFP-RV-C6) ⁇ volume of starting feed solution].

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Abstract

公开了纯化逆转录病毒(逆转录病毒载体)的方法，包括以下步骤：A)、对含有逆转录病毒的细胞培养上清液进行微滤处理，以除去细胞培养上清液中的细胞碎片，获得微滤病毒液；B)、对微滤病毒液进行核酸酶处理，以将宿主DNA残留降解为小片段DNA，获得酶消化病毒液；C)、将酶消化病毒液进行超滤，使逆转录病毒留在截留液中，收集截留液，获得超滤病毒液；D)、低速离心处理超滤病毒液，使逆转录病毒沉淀，收集沉淀，得到未经除菌的病毒。该方法获得的逆转录病毒产品在保证逆转录病毒液纯度的同时，浓缩病毒液滴度达到10 7IP/ml，符合下游工艺要求。

Description

GMP级逆转录病毒载体的纯化方法与应用 技术领域
本发明涉及生物技术领域，具体涉及GMP级逆转录病毒载体的纯化方法与应用，特别涉及可应用于嵌合抗原受体细胞治疗的GMP级逆转录病毒载体的纯化方法与应用。
背景技术
基因治疗在多种疾病治疗中具有巨大应用潜力，也被认为是下一代临床治疗的终极手段。基因治疗是指将外源DNA片段导入靶细胞，以纠正、修复、替换、补偿或沉默等方式对缺陷和异常基因进行针对性干预，以期恢复正常的基因功能，最终达到治疗甚至完全治愈的目的。逆转录病毒衍生载体成为基因治疗临床试验中应用最广泛的载体之一。目前，逆转录病毒载体的制备工艺仍面临着巨大挑战。病毒载体的临床应用方式主要分为两种：一种是在体外感染靶细胞，再将导入外源基因的靶细胞体外培养扩增后回输人体，称为ex vivo，例如CAR-T细胞治疗；另一种是直接用纯化的病毒载体感染在体靶细胞和组织，称为in vivo，例如溶瘤病毒疗法。对于前者，临床所需的病毒活性滴度一般在10 7IP/mL数量级；对于后者，病毒活性滴度甚至要求达到10 9IP/mL数量级。而传统的通过收集稳产株上清获取逆转录病毒(逆转录病毒载体)液的方法无法满足这一要求。随着对临床级高纯度逆转录病毒(逆转录病毒载体)的需求日益增加，急需开发高效和适用于规模化生产的逆转录病毒(逆转录病毒载体)制备和纯化的新工艺。
逆转录病毒(逆转录病毒载体)制备的一般流程是，先用包含目的基因的质粒转染表达逆转录病毒gag、pol和env基因的细胞系，通过多轮亚克隆筛选，获得稳定分泌包含目的基因的重组逆转录病毒的稳产株。后续在需要时将稳产株扩大培养，收集上清获得逆转录病毒(逆转录病毒载体)粗提液，用于后续纯化步骤。相比于腺病毒和慢病毒，逆转录病毒的稳定性较差，对于剪切力较为敏感，因此在纯化工艺选择上，对于切向流超滤的工艺控制提出更高要求。另外，传统的超速离心法也不适用于逆转录病毒(逆转录病毒载体)的纯化过程，因为超速离心法需要配备昂贵的离心机，花费大量时间，并且不利于工艺放大。因此，逆转录病毒(逆转录病毒载体)的纯化是目前业界面临的共同难题，尚无适用于临床治疗的GMP(药品生产质量管理规范)级逆转录病毒(逆转录病毒载体)的完整制备和纯化方法。
发明内容
本发明提供纯化逆转录病毒(逆转录病毒载体)的方法，所述方法包括以下步骤：
A)、对含有逆转录病毒(逆转录病毒载体)的细胞培养上清液进行微滤处理，以除去所述细胞培养上清液中的细胞碎片，获得微滤病毒液；
B)、对所述微滤病毒液进行核酸酶处理，以将宿主DNA残留降解为小片段DNA，获得酶消化病毒液；
C)、将所述酶消化病毒液进行超滤，使所述逆转录病毒(逆转录病毒载体)留在截留液中，收集截留液，获得超滤病毒液；
D)、低速离心处理所述超滤病毒液，使所述逆转录病毒(逆转录病毒载体)进行沉淀，收集沉淀，得到未经除菌的病毒。
进一步地，所述方法还包括对所述未经除菌的病毒进行过滤除菌得到纯化的逆转录病毒(逆转录病毒载体)的步骤。
进一步地，可使用0.22μm孔径的PVDF膜材质滤器对上述病毒进行过滤除菌。
进一步地，A)所述微滤处理中，所采用的半透膜的膜分离孔径(微膜孔径)为0.45μm-0.75μm，和/或所采用的半透膜的膜材质为改性聚醚砜。
所述微滤处理中，所述膜分离孔径(微膜孔径)可为0.45μm-0.65μm、或0.65μm或0.45μm。
所述微滤处理中，所述细胞培养上清液的流速可为89mL·min -1-133.5mL·min -1、89mL·min -1或133.5mL·min -1。
所述微滤处理中，所述微滤处理的剪切速率可为2000s -1-3000s -1、2000s -1或3000s -1。
所述微滤处理具体可为下述任一种：
MF1、所采用的半透膜的膜分离孔径(微膜孔径)为0.65μm，所述细胞培养上清液的流速为89mL·min -1，所述微滤处理的剪切速率为2000s -1；
MF2、所采用的半透膜的膜分离孔径(微膜孔径)为0.65μm，所述细胞培养上清液的流速为133.5mL·min -1，所述微滤处理的剪切速率为3000s -1；
MF3、所采用的半透膜的膜分离孔径(微膜孔径)为0.45μm，所述细胞培养上清液的流速为89mL·min -1，所述微滤处理的剪切速率为2000s -1。
进一步地，B)所述核酸酶处理中，反应体系中的核酸酶浓度为1～500U·mL -1，和/或2～8℃反应8-24小时。
述核酸酶处理中，核酸酶浓度为25、50或100U·mL -1；
所述核酸酶处理中，核酸酶的处理时间为8小时，16小时或24小时；
所述核酸酶处理中，核酸酶的处理温度为4℃或37℃；
所述核酸酶处理具体可为下述任一种：
b1)、反应体系中核酸酶的浓度为100U·mL -1，和/或4℃反应16小时；
b2)、反应体系中核酸酶的浓度为100U·mL -1，和/或4℃反应24小时；
b3)、反应体系中核酸酶的浓度为50U·mL -1，和/或4℃反应24小时。
进一步地，步骤C)中超滤浓缩步骤中超滤膜的截留分子量为350KD、500KD或750KD，优选为750KD。
进一步地，步骤C)中分两次进行超滤浓缩分别如下：
c1)首先使用中空纤维柱的内径为0.5mm内径，膜面积0.16m 2；控制剪切速率为2000s -1对酶消化的病毒液进行超滤浓缩获得5倍浓缩的初级超滤浓缩病毒液；c1)中病毒液的流速可为370mL·min -1；
c2)然后使用中空纤维柱的内径为0.5mm、膜面积为115cm 2，控制剪切速率为2000s -1将c1)获得的的初级超滤浓缩病毒液进行超滤浓缩，获得c1)病毒液体积的5-10倍浓缩的二级超滤浓缩病毒液；c2)中病毒液流速可为53mL·min -1。
进一步地，步骤D)中低速离心的处理条件为：离心力4000g～10000g，4℃离心4～24h；优选为在4℃条件下，6000g离心16h；
所述低速离心中，离心力为4000-8000g、4000g、6000g或8000g；
所述低速离心中，所述离心时间为4-24小时、4小时、8小时、16小时或24小时；
所述低速离心的处理工艺具体为如下任一种：
d1)在4℃条件下，6000g离心16h；
d2)在4℃条件下，6000g离心8h。
本发明提供上述方法得到的逆转录病毒(逆转录病毒载体)。
本发明提供一种产品(例如，药物或疫苗)，所述产品含有上述的逆转录病毒(逆转录病毒载体)。
本发明还提供所述逆转录病毒(逆转录病毒载体)在制备基因治疗产品和/或细胞治疗产品和/或免疫治疗产品中的应用。
进一步地，所述基因治疗产品为ex vivo基因治疗产品，例如CAR-T细胞治疗产品。
与现有技术对比，本发明的有益效果如下：
1.采用创新的纯化工艺组合，更有效去除逆转录病毒(逆转录病毒载体)中的杂质成分，提高逆转录病毒(逆转录病毒载体)的纯度。逆转录病毒(逆转录病毒载体)粗提液为逆转录病毒稳产株的培养上清液，其中包含多种杂质，包括宿主DNA残留(HCD)、宿主蛋白残留(HCP)、牛血清白蛋白残留(BSA)等，这些杂质可能在临 床应用过程中被带入人体，对患者带来不可控风险，包括过敏反应、致瘤性等。为此，《中国药典》对于上述各类杂质的残留量上限有着明确规定。例如，对于疫苗类生物制品，HCD一般不高于10ng/100μg蛋白，BSA不高于50ng/剂，HCP一般不高于总蛋白含量的0.1％。而作为ex vivo基因治疗中间产品的逆转录病毒(逆转录病毒载体)，虽然目前的政策法规并无明确规定，但是过高的杂质残留很难在下游工艺中除去。因此，需要在逆转录病毒(逆转录病毒载体)的纯化步骤中尽可能把杂质降到最低水平，避免带入下游工艺。本方案采用微滤，超滤，配合核酸酶处理，以及低速离心等步骤，针对大碎片、小分子以及核酸类杂质具有良好的清除效果。经过上述步骤，可有效去除逆转录病毒粗提液中宿主HCD、HCP、BSA等杂质成分。该工艺容易规模化放大并符合GMP要求。纯化后的逆转录病毒(逆转录病毒载体)料液经检测，BSA<200ng/ml，HCP<1μg/ml，HCD<100ng/ml。经验证，此病毒液可以进入ex vivo基因治疗的下游工艺，例如CAR-T或CAR-NK细胞制备等用途。
2.最大限度保留逆转录病毒(逆转录病毒载体)的生物学活力，提高活性滴度。本方案两次用到了切向流微滤/超滤工艺。所谓的切向流，是指液体流动方向与过滤方向呈垂直方向的过滤形式，切向流过滤时，待过滤的液体的流动方向和过滤膜平面的方向平行，液体就会垂直于膜表面穿过膜孔。切向流会产生湍流(二次流)，由于有了湍流，液体流动在过滤介质表面(即超滤膜表面)产生剪切力，减小了滤饼层或凝胶层在膜表面的堆积，使沉淀从膜表面剥离，降低膜污染，保证稳定的过滤速度。然而，切向流的剪切力可能破坏逆转录病毒的包膜结构，因此需要优化流速、膜面积、膜孔径等参数，以控制剪切力对逆转录病毒活力的损害。本发明通过采用大孔径(截留相对分子质量750kDa)mPES材质滤膜，通过优化剪切速率，保证渗透通量和过滤效果的同时减少剪切力损伤，从而减少对逆转录病毒的包膜结构的破坏，逆转录病毒总体回收率超过80％。3.申请人利用逆转录病毒(逆转录病毒载体)颗粒大，易沉降等特点，创新地使用低温低速离心法配合超滤的组合工艺，去除上清中90％以上的BSA和HCP杂质，同时起到了浓缩病毒液的作用。该方案在保证逆转录病毒纯度的同时，浓缩病毒液滴度达到10 7IP/ml，符合下游工艺要求。
该方案成功避免使用离子交换层析的步骤，因高盐/渗透压可能对包膜的传染性产生不利影响。同时也避免了耗时且昂贵的超速离心步骤，易于规模化放大。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中的实验方法，如无特殊说明，均设三次重复。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、含有重组逆转录病毒(逆转录病毒载体)的细胞上清液的制备及纯化
1、含有重组逆转录病毒(逆转录病毒载体)的细胞培养上清液的制备
重组逆转录病毒(逆转录病毒载体)的细胞培养上清液可以是已经获得的产品，本实施例仅仅是示例性列举含有重组逆转录病毒(逆转录病毒载体)的细胞培养上清液的制备方法：将目的基因插入逆转录病毒表达质粒，得到重组逆转录病毒表达质粒；重组逆转录病毒表达质粒转染包装细胞系，经过包装细胞系包装得到含有重组逆转录病毒(逆转录病毒载体)的细胞培养液，用含有重组逆转录病毒(逆转录病毒载体)的细胞培养液转染稳转细胞系，并经过数次亚克隆得到稳定包装逆转录病毒(逆转录病毒载体)的稳转细胞株；培养上述稳转细胞株，收集细胞上清液即可获得滴度稳定的含有重组逆转录病毒(逆转录病毒载体)的细胞培养上清液。
本实施例中以绿色荧光蛋白EGFP基因为例，将EGFP基因(MK387175.1，SYN 12-AUG-2019)插入逆转录病毒表达质粒(pMSCVneo，优宝生物)的XhoI和EcoRI识别位点间，且保持质粒pMSCVneo的其他序列不变，得到重组逆转录病毒表达质粒pMSCVneo-EGFP。将pMSCVneo-EGFP按照以下方法包装得到重组逆转录病毒(逆转录病毒载体)，命名为EGFP-RV。
S1、包装细胞的培养
每10cm细胞培养皿中添加6×10 6个Phoenix Ecotropic(ECO)细胞(ATCC，CRL-3214，)(小于20代，不过分长满)和10ml的DMEM培养基，充分混匀细胞，37℃培养过夜。
S2、包装细胞的转染
待ECO细胞融合度达到50-60％左右进行转染；在一个管中添加目的质粒pMSCVneo-EGFP 12.5μg，1.25M CaCl 2 250μL，H 2O 1mL，总体积为1.25mL；在另一个管里添加与质粒复合物等体积的2×HBS溶液，将质粒复合物加入2×HBS溶液中，边加质粒复合物边涡旋震荡20s，得到混合物。轻柔地将混合物沿着边加入到ECO细胞培养皿中，37℃培养6h，去除培养基，重新加入预热的新鲜DMEM培养基。
S3、粗病毒液的获得
转染48h后收集上清后，得到病毒液，分装保存于-80℃。此粗病毒液命名为EGFP-RV-C1。
S4、产毒细胞株的建立
将上一步获得的EGFP-RV-C1感染PG13细胞(ATCC，CRL-10686)，感染两天后，用EGFP抗体(Biolegend)对EGFP阳性的细胞进行富集，得到富集后的细胞。取一部分富集后的细胞用流式检测EGFP的表达效率，另取一部分富集后的细胞稀释成单细胞，铺至96孔板，将在96孔板培养后第5天的上清作为逆转录病毒(逆转录病毒载体)液。为测定上述逆转录病毒(逆转录病毒载体)液的病毒滴度，进一步将逆转录病毒(逆转录病毒载体)液感染HT1080细胞(ATCC，CCL-121)，流式细胞术测定上述逆转录病毒(逆转录病毒载体)液的病毒滴度。筛选出96孔板中病毒滴度最高的三个PG13细胞株，接种至24孔板中继续培养，进行二次筛选。继续用培养后第5天的PG13细胞上清作为逆转录病毒液，感染HT1080细胞，流式细胞术测定病毒滴度，选择细胞培养上清病毒滴度最高的PG13细胞作为稳产株，在液氮中长期保存。利用此细胞株在DMEM完全培养基(DMEM培养基+10％FBS+100U/mL青霉素+10μg/mL链霉素+2mM Glutamine)中扩大培养，置于37℃、5％CO 2的恒温培养箱进行培养，收集细胞培养上清液，获得含有逆转录病毒(逆转录病毒载体)的细胞培养上清液，将该上清液命名为EGFP-RV-C2，作为后续纯化的料液。
2、逆转录病毒(逆转录病毒载体)的纯化
2.1、微滤
2.1.1前处理：将微滤澄清纯化系统(瑞普利金，S02-E65U-07-N)按照组装要求进行组装。在该微滤澄清纯化系统中使用膜分离孔径(微膜孔径)为0.65μm、纤维内径为0.75mm，完整无损的中空纤维柱(膜材质为改性聚醚砜mPES)，用硅胶管进行系统连接。用压力保持法检测系统的完整性，并使用0.5M或1M的NaOH溶液循环30-60min进行灭菌处理，剪切速率为2000s -1～8000s -1，灭菌结束后排空系统，用无菌蒸馏水注满系统，循环20-30min，直到纯化系统内PH≤7.0，循环剪切速率为6000/s。
2.1.2再平衡：纯水洗过后排空，注入灭菌处理过的4℃预冷0.1M或0.2M PBS缓冲液，系统循环20min，6000/s。
2.1.3微滤：微滤澄清纯化系统系统平衡后排空，注入步骤1的含有逆转录病毒(逆转录病毒载体)的细胞培养上清液EGFP-RV-C2进入系统，调节EGFP-RV-C2的流速89mL·min-1，使剪切速率为6000s -1。先关闭系统透过端，让病毒液在系统中循环2-10min，使系统稳定，开启透过端开关，开始微滤，收集透过液，该透过液即为微滤病毒液，命名为EGFP-RV-C3。
以上全程在冰盒内操作，保持液体温度为2-8℃。
2.2、核酸酶处理
向2.1获得的微滤病毒液EGFP-RV-C3中加入全能核酸酶(义翘神州)和Mg 2+离子，使核酸酶的含量为100U·mL -1，Mg 2+离子的浓度为2mM，4℃反应24h，得到酶 消化病毒液命名为EGFP-RV-C4。
2.3、超滤浓缩
将2.2获得酶消化病毒液使用超滤浓缩系统，进行超滤浓缩。
2.3.1前处理：将超滤纯化系统(瑞普利金，KrosFlo Reseach 2i)，按照组装要求进行组装。在该超滤纯化系统中使用截留相对分子质量为750KD、中空纤维毛细管的内径(简称纤维内径)为0.5mm、半透膜的膜面积为0.16m 2完整无损的中空纤维柱，用硅胶管进行系统连接。用压力保持法检测系统的完整性，并使用0.5或1M的NaOH溶液循环30-60min进行灭菌处理，剪切速率为6000s -1，灭菌结束后排空系统，用无菌蒸馏水注满系统，循环20-30min，直到纯化系统内PH≤7.0，循环剪切速率为6000s -1。
2.3.2再平衡：纯水洗过后排空，注入灭菌处理过的4℃预冷0.1M或0.2M PBS缓冲液，系统循环20min，6000s -1。
2.3.3病毒液超滤浓缩：取2L步骤2.2的酶消化病毒液EGFP-RV-C4，系统平衡后排空，注入酶消化病毒液EGFP-RV-C4进入系统，调节流速为370mL·min -1，使剪切速率为2000s -1。先关闭系统透过端，让病毒液在系统中循环2-10min，使系统稳定，开启透过端开关，开始超滤。先将病毒液浓缩至1/5，然后连续注入PBS缓冲液进入系统，调节流速使PBS缓冲液注入流速等于透过液流速370mL·min -1，保持病毒液体积不变。收集截留液，按1：1(V/V)截留液体积的PBS，开启循环泵循环洗涤5min，收集第一次洗涤液，按1：1(V/V)截留液体积的PBS，开启循环泵循环洗涤5min，收集第二次洗涤液。按照上述方法循环多次，分别至第3次、第4次、第5次，将所有每次收集的洗涤液混合在一起，即为初级病毒浓缩液即一级超滤病毒液，命名为EGFP-RV-C5(5)。此步骤获得的浓缩液EGFP-RV-C5(5)体积为400mL。
2.3.4病毒液再次超滤浓缩
由于实际的超滤纯化起始料液体积较大，因此还需要额外的超滤浓缩步骤。本发明在上述超滤步骤之前增加了大体积中空纤维柱(膜面积0.16m 2，其余参数同上)对起始料液进行5倍浓缩。更换上述超滤纯化系统的中空纤维柱，该新的中空纤维柱中的截留相对分子质量为750KD、中空纤维毛细管的内径(简称纤维内径)为0.5mm、半透膜的膜面积为0.0115m 2。灭菌、平衡同上，将400mL上述EGFP-RV-C5(5)注入系统，调节流速为53mL·min -1，使剪切速率为2000s -1。先关闭系统透过端，让病毒液在系统中循环2-10min，使系统稳定，开启透过端开关，开始超滤。先将病毒液浓缩至1/10，然后连续注入PBS缓冲液进入系统，调节流速使PBS缓冲液注入流速等于透过液流速53mL·min -1，保持病毒液体积不变。收集截留液，按1：1(V/V)截留液体积的PBS，开启循环泵循环洗涤5min，收集冲洗液，将冲洗液与截留液混合即为二级病毒浓缩液即超滤病毒液，命名为EGFP-RV-C6，体积40mL。
2.4、低速离心及除菌过滤
将2.3获得的40mL超滤病毒液EGFP-RV-C6进一步低速离心处理，使用离心力为6000g，离心16h，离心温度为4℃。离心后弃上清，收集沉淀，该沉淀即为未经除菌的病毒。使用含2％人血白蛋白的PBS重悬该沉淀，重悬体积为离心前的1/10。4℃放置1h用含2％人血白蛋白的PBS复溶，获得病毒重悬液命名为EGFP-RV-C7。
最后，使用0.22μm孔径的PVDF膜材质滤器对病毒重悬液EGFP-RV-C7进行过滤除菌，获得纯化的病毒液，即GMP级逆转录病毒(逆转录病毒载体)，体积为4mL，命名为EGFP-RV-F，放置于-80℃冻存。
3、逆转录病EGFP-RV-F的生物性状检测
3.1、逆转录病毒滴度检测
采用流式法通过检测EGFP表达，间接测定出单位体积逆转录病毒感染的HT1080细胞数，即病毒活性滴度或感染颗粒数(Infectious particles,IP/mL)。
首先，收集处于生长对数期的HT1080细胞，胰酶消化2-3min，加入完全培养基制备成细胞悬液。按照1.5×10 5个/孔的密度接种对数生长期的HT1080细胞。铺细胞后的TC6孔板上下左右摇匀，做好标记，放置于37℃二氧化碳培养箱中培养20～24h。转导时，每孔分别加入含8μg/mL polybrene的病毒液，其中病毒液和完全培养基分别按照不同比例稀释。转导后细胞置于37℃，CO 2培养箱内孵育1h，中间每隔15min摇匀1次，第4次摇匀后加入2mL完全培养基，继续培养48h。吸除细胞板孔内的病毒转导液，每孔加2mL PBS润洗细胞。每孔加入100μL 0.25％胰酶，室温放置1～2min。每孔加入500μL完全培养基，终止消化。用吸头吸液吹打细胞悬液，1500rpm，离心5min，收集细胞沉淀。
将细胞悬液按照3×10 4个细胞/孔接种96孔板，1500rpm，离心5min。每孔用200μL FACS buffer洗1次，1500rpm，离心5min。按照1:100稀释度每孔加入20μL FITC标记的EGFP抗体(Biolegend),避光孵育10min。再次离心，每孔补加200μL FACS buffer。流式上机检测。
按照以下公式计算病毒滴度：
病毒滴度(IP·mL -1)＝(F×N)/V
F：流式检测细胞阳性率(测定值-空白对照CTR值)
N：转染时细胞数(3×10 5)个/孔
V：加入的病毒体积mL。
病毒回收率：收获病毒滴度/初始病毒滴度×100％。
3.2、宿主残留DNA(HCD)检测
样本前处理：采用宿主细胞残留DNA样本前处理试剂盒(磁珠法)(湖州申科， SK030203D100)完成。
样本的qPCR检测按照以下步骤进行：
a.PG13DNA定量参考品和标准曲线的制备：
收集PG13细胞，用DNA提取试剂盒(QIAGEN，80204)提取基因组DNA作为定量参考品。用1×PBS(pH7.4，无Ca和Mg)将DNA定量参考品进行梯度稀释，稀释浓度依次为3ng/μl、300pg/μL、30pg/μL、3pg/μL、300fg/μL，30fg/μL，3fg/μL。
取7支干净的1.5ml离心管，分别标记为ST0，ST1，ST2，ST3，ST4，ST5，ST6。在ST0管中用PBS将DNA定量参考品稀释至3ng/μL，振荡混匀后短时间快速离心10s。在ST1，ST2，ST3，ST4，ST5，ST6管中分别加入90μL PBS。依次做梯度稀释。
设置回收质控ERC：以制备加300pg定量参考品的样本作为ERC，取100μl待测样本加入1.5ml干净的离心管中。再加入10μL ST2，混匀，标记为样本ERC。
设置阴性质控NCS：取100μL样本基质溶液(或DNA稀释液)加入1.5ml干净的离心管中，标记为阴性质控NCS。
b.qPCR反应液(qPCR MIX)的准备
根据所要检测的标准曲线及待测样本数量，计算所需反应孔数，一般做3个重复孔/样。反应孔数＝(6个浓度梯度的标准曲线+1个无模板对照NTC+1个阴性质控NCS+待测样本×2)×3；根据反应孔数计算本次所需的qPCR MIX总量，按照表1配方，配制qPCR MIX。引物序列为：
探针，5’-FAM-AGGGCCCCCAATGGAGGAGCT-TAM-3’；
正向引物，5’-CCCCTTCAGCTCCTTGGGTA-3’；
反向引物，5’-GCCTGGCAAATACAGAAGTGG-3’。
Q-PCR probe mix购买现成的商品化预混液(Vazyme，AceQ PCR probe master mix)。
c.qPCR反应：将表2中各试剂置于冰上混匀，加样后总体积25μL。建立qPCR模板和运行参数。95℃，10min，1cycle；95℃，15s，60℃，1min，40cycle；40℃，30s，1cycle。
d.上机和结果分析：在ABI 7500 qPCR仪上运行程序，在Results的Report面板中，Mean Quantity一栏可读取无模板对照NTC、阴性质控NCS、待测样本、样本ERC的检测值，单位为pg/10μL。后续可在检测报告中将单位换算为pg/μL或pg/mL。根据待测样本和样本ERC的检测结果计算加样回收率，加样回收率要求在50％～150％之间。NTC的检测结果应为Undetermined或Ct值≥35。NCS的Ct值应大于标曲最低浓度Ct值。
表1.qPCR MIX配制表
组分 单孔体积(μL)
qPCR probe mix 12.5
mDNA-F 2
mDNA-R 2
mDNA-P 1
RNAse-free water 补齐至反应体积
Total 20
表2.加样配制表
标准曲线 20μL qPCR MIX+5μL ST1/ST2/ST3/ST4/ST5/ST6
NTC 20μL qPCR MIX+5μL DNA稀释液
阴性对照 20μL qPCR MIX+5μL阴性质控NCS纯化液
待测样本 20μL qPCR MIX+5μL待测样本纯化液
ERC 20μL qPCR MIX+5μL样本ERC纯化液
3.3、牛血清白蛋白(BSA)残留检测
BSA的测定方法为：采用Bovine Serum Albumin(BSA)ELISA检测试剂盒(Cygnus，F030)对样本的BSA残留进行检测。该试剂盒提供了抗BSA单克隆抗体包被板，以酶标记抗BSA多抗为检测抗体，组成双抗体夹心ELISA检测试剂盒。根据供应商的说明书，先加入待测样品或标准品，使其中的BSA与固定在板上的抗体结合，彻底洗涤后加入HRP标记的抗BSA多抗孵育。洗涤后再加入TMB底物，TMB在HRP的催化作用下转化成蓝色，并最终在终止液的作用下转化成黄色。颜色的深浅与样品或标准品中的BSA数量相关。最后，用酶标仪测定各样品孔在450nm处的吸光值，通过BSA标准曲线计算待测样品中的BSA浓度。
3.4、宿主残留蛋白(HCP)检测
本发明采用ELISA夹心法对样本中的HCP进行检测。HCP抗体为用PG13细胞培养上清蛋白免疫家兔后的血清中纯化获得的多克隆抗体。具体的检测方法为：
a.平板包被：用抗PG13细胞上清蛋白抗体，以最佳浓度包被于试验平板的反应孔的表面。最佳抗体浓度是通过用已知浓度的PG13细胞上清蛋白绘制一条标准曲线来确定的，该曲线在所要求的有效浓度范围中具有所要求的灵敏度和精确度。对于PG13细胞上清蛋白，本试剂盒可以检测的有效浓度范围为62.5ng/mL到4000ng/mL。 本领域的普通技术人员，可以方便地确定在所要求范围内是否具有合适灵敏度和精确度，而无须进行多余的实验。
b.平板洗涤：将包被溶液倒去，加入洗涤缓冲液(每孔大约400微升)然后倒去。按要求将这个洗涤循环重复多次。洗涤缓冲液可选为0.01mol/L的磷酸缓冲液(0.0027mol/L氯化钾，0.137mol/L氧化纳，pH7.4，含有0.01％w/v TritonX-100)。
c.平板封闭：将含有蛋白质和去垢剂封闭缓冲液(含1％BSA/0.1％Triton X-100的包被缓冲溶液)加入反应孔。平板可以此形式储存。
d.样品和标准品的加入：平板按上述方式进行洗涤。向平板反应孔内分别加入标准品与待测样本各100μ1，然后每孔分别加入偶联物试剂50μ1，轻轻混合15秒后置37℃孵育60分钟，丢弃反应液，用缓冲液清洗反应板5次，吸干多于水分，向反应孔内加入50μL显色液，置于37℃孵育15分钟，终止反应。将反应板置于酶标仪上读取光密度值。
e.用四甲基联苯肢磺酸盐(TMBS)作为显色底物，读取450nm的吸光度。HCP准确浓度可以通过读取测试样品的吸光度、再参考由HCP标准品所作出的标准曲线来换算得出。
3.5、核酸酶(Supernuclease)残留检测
核酸酶能够完全消化不少于5个磷酸残基的RNA和DNA(单链、双链、线性、圆形和超级螺旋状)，形成长度为3-5个碱基的5'-单磷酸端寡核苷酸。本发明运用核酸酶降解工艺中的HCD，使之方便地通过后续纯化方法去除。然而，由于核酸酶本身也属于蛋白杂质，需要在后续纯化工艺中和其他杂质一起除去。本发明通过购买义翘神州Supernuclease ELISA kit检测下游产品中核酸酶残留量。具体的检测步骤参照义翘神州官网和产品说明书。
按照实施例1中步骤2逆转录病毒(逆转录病毒载体)的纯化中所述方法重复3次得到3个批次的GMP级逆转录病毒(逆转录病毒载体)EGFP-RV-F，分别命名为批次1、批次2和批次3，按照上述方法检测批次1、批次2和批次3的核酸酶含量、BSA含量、HCP含量、HCD含量和病毒滴度。
结果表明经过上述微滤、核酸酶处理、超滤和低速离心步骤之后，料液体积的浓缩倍数500倍。获得GMP级逆转录病毒(逆转录病毒载体)EGFP-RV-F的得率在10-20％之间，活性滴度>1.0E+07，HCD<100ng/ml，核酸酶低于检测限(<3.15ng/mL)，HCP<1μg/mL，BSA<200ng/mL，符合下游生产工艺需求(表9)。
表9逆转录病毒(逆转录病毒载体)最终纯化产物的杂质残留和总体回收率
Figure PCTCN2021134470-appb-000001
注：[1]起始料液滴度指换算成体积为2L料液的滴度；
[2]纯化后滴度指换算成体积为4mL料液的滴度。
实施例2、逆转录病毒(逆转录病毒载体)纯化工艺的优化
2.1、粗毒液微滤工艺优化
参照实施例1的步骤2的2.1微滤，采用不同的流速和膜孔径，控制剪切力，获得不同条件下，粗毒液微滤的回收率。
除将实施例1的步骤2的2.1微滤中的2.1.3的含有逆转录病毒(逆转录病毒载体)的细胞培养上清液EGFP-RV-C2(病毒的滴度为8.86×10 5IP·mL -1)的流速由89mL·min -1分别替换为表3的133.5mL·min -1和44.5mL·min -1，将剪切速率由6000s -1分别替换为表3的1000s -1和2000s -1，将膜分离孔径(微膜孔径)由0.45μm替换为0.65μm外，其它操作均与实施例1的步骤2.1相同。
实验结果见表3。由实验结果可知，流速89mL·min -1剪切力控制在2000s -1，膜孔径0.65μm，可获得逆转录病毒(逆转录病毒载体)的最佳回收率。
表3不同微滤条件对病毒回收率的影响
Figure PCTCN2021134470-appb-000002
Figure PCTCN2021134470-appb-000003
2.2、核酸酶处理工艺优化
参照实施例1的步骤2的2.2的核酸酶处理，采用不同的核酸酶浓度，处理时间和处理温度，获得不同条件下，核酸酶处理后病毒液的回收率。
除将实施例1的步骤2的2.2的核酸酶处理中的核酸酶的含量由100U·mL -1分别替换为表4的50U·mL -1和25U·mL -1，将反应时间由24h分别替换为表4的8h和16h外，其它操作均与实施例1的步骤2的2.2相同。
实验结果见表4。由实验结果可知，酶浓度100U·mL -1，4℃处理24h，可获得最高的HCD清除效率。
表4不同条件下核酸酶处理对HCD的清除作用
Figure PCTCN2021134470-appb-000004
Figure PCTCN2021134470-appb-000005
2.3、超滤工艺优化
不同中空纤维膜截留相对分子质量对病毒液回收率和杂质清除率会产生不同的影响。中空纤维截留相对分子质量越大，杂质清除率越高，但是如果截留相对分子质量超过病毒颗粒的大小，也可能造成部分病毒颗粒的损失，影响回收率。
参照实施例1中2.3超滤工艺的操作流程，采用不同截留相对分子质量的中空纤维柱，获得不同截留相对分子质量的中空纤维膜对病毒回收率及杂质清除率的影响。
将实施例1中2.3中的中空纤维膜的截留相对分子质量替换为750kDa、500kDa、300kDa，保持其他条件不变，得到的结果如表5所示。
表5不同中空纤维膜孔径对病毒回收率和杂质清除率的影响
Figure PCTCN2021134470-appb-000006
注：[1]本项HCD清除率计算是与核酸酶处理前的料液比较，即HCD(EGFP-RV-C6)/HCD(EGFP-RV-C3)×100％。
确定了中空纤维膜截留相对分子质量后，进一步探索不同剪切速率和纤维内径对病毒液回收率的影响。因逆转录病毒(逆转录病毒载体)颗粒对剪切力敏感，因此剪切速率会直接影响病毒活性和回收率。另外，中空纤维管的内径会直接影响料液通量，对于相同体积的料液，内径越小，通量越小，流速越慢。
参照实施例1中2.3超滤的处理工艺，将超滤病毒液的流速控制在38、53、79、100、106、141、212、283mL·min -1，剪切速率控制在1441、2000、3000、4000s -1，纤维内径选择0.5、1.0mm，膜面积选择75、115cm 2保持2.3其他处理工艺不变，得到的结果如表6所示。
表6检测结果表明，用0.5mm内径，膜面积115cm 2的中空纤维柱，柱内液体流 速为53mL·min -1，控制剪切速率为2000s -1时，能获得最大病毒回收率。
表6不同剪切速率和纤维内径对病毒回收率的影响
Figure PCTCN2021134470-appb-000007
注：[1]本项测试通过在确定型号的中空纤维柱中的流速变化来调节剪切速率。
根据超滤的原理，每次循环都能去除一定量的杂质，循环次数越多，杂质清除率越高。但是超滤工艺产生的切向流也会导致一部分的病毒失活，因此保持实施例中2.3超滤工艺的其他步骤不变，测试了不同循环次数对病毒回收率的影响(表7)。检测结果表明，循环超滤5次在最大程度保证回收率的前提下，提高了部分杂质的清除效率。
表7超滤浓缩不同循环次数对病毒回收率和杂质清除率的影响 [1]
Figure PCTCN2021134470-appb-000008
Figure PCTCN2021134470-appb-000009
注：[1]浓缩倍数为2.5倍。
[2]BSA清除率＝[BSA(EGFP-RV-C5)×收获料液体积]/[BSA(EGFP-RV-C4)×起始料液体积]。
2.4、低速离心去除HCP和BSA的工艺优化
经过超滤步骤之后，HCD<100ng/ml，核酸酶低于检测限(<3.15ng/mL)，表明这两项工艺残留指标已基本达到要求。然而，HCP和BSA仍略高于标准，中位数均值分别为13078ng/ml和2056ng/ml(EGFP-RV-C6)。
参照实施例1中2.4低速离心处理工艺步骤，采用不同的离心力和离心时间，保持2.4其他处理工艺不变，获得不同离心力和离心时间的条件下病毒回收率和杂质清除率的结果(表8)。结果表明，在4℃条件下，6000g离心16h后，纯化后的料液中HCP<1μg/mL，BSA<200ng/mL，符合下游生产工艺要求。
表8不同条件低速离心后的病毒回收率和杂质清除率 [1]
Figure PCTCN2021134470-appb-000010
注：
[1]浓缩倍数为5倍。
[2]BSA清除率＝[BSA(EGFP-RV-C7)×收获料液体积]/[BSA(EGFP-RV-C6)×起始料液体积]。[3]HCP清除率＝[HCP(EGFP-RV-C7)×收获料液体积]/[HCP(EGFP-RV-C6)×起始料液体积]。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。

Claims (17)

  1. 纯化逆转录病毒的方法，其特征在于，所述方法包括以下步骤：
    A)、对含有逆转录病毒的细胞培养上清液进行微滤处理，以除去所述细胞培养上清液中的细胞碎片，获得微滤病毒液；
    B)、对所述微滤病毒液进行核酸酶处理，以将宿主DNA残留降解为小片段DNA，获得酶消化病毒液；
    C)、将所述酶消化病毒液进行超滤，使所述逆转录病毒留在截留液中，收集截留液，获得超滤病毒液；
    D)、低速离心处理所述超滤病毒液，使所述逆转录病毒进行沉淀，收集沉淀，得到未经除菌的病毒。
  2. 如权利要求1所述的方法，其特征在于，所述方法还包括对所述未经除菌的病毒进行过滤除菌得到纯化的逆转录病毒的步骤。
  3. 如权利要求1或2所述的方法，其特征在于，所述微滤处理中，所采用的半透膜的膜分离孔径(微膜孔径)为0.45μm-0.75μm，和/或所采用的半透膜的膜材质为改性聚醚砜。
  4. 如权利要求1或2或3所述的方法，其特征在于，所述核酸酶处理中，反应体系中的核酸酶浓度为1U·mL -1～500U·mL -1，和/或2℃～8℃反应8h-24h。
  5. 如权利要求1所述的方法，其特征在于，步骤C)中超滤步骤中，所述超滤的截留相对分子量为350KD-750KD或750KD。
  6. 如权利要求1-5中任一所述的方法，其特征在于，步骤C)中所述超滤包括：
    c1)对所述酶消化病毒液使用截留相对分子质量为750KD、中空纤维毛细管的内径为0.5mm、半透膜的膜面积为0.16m 2的中空纤维柱进行剪切速率为2000s -1的超滤，得到一级病毒超滤液；
    c2)对所述一级病毒超滤液使用截留相对分子质量为750KD、中空纤维毛细管的内径为0.5mm、半透膜的膜面积为0.0115m 2的中空纤维柱进行剪切速率为2000s -1的超滤，得到二级病毒超滤液。
  7. 如权利要求1-6中任一所述的方法，其特征在于，步骤D)中低速离心的处理条件为：离心力4000g～10000g，离心4h～24h。
  8. 如权利要求3所述的方法，其特征在于，所述微滤处理中，所述细胞培养上清液的流速为89mL·min -1-133.5mL·min -1、89mL·min -1或133.5mL·min -1。
  9. 如权利要求3所述的方法，其特征在于，所述微滤处理中，所述微滤处理的剪切速率为2000s -1-3000s -1、2000s -1或3000s -1。
  10. 如权利要求1所述的方法，其特征在于，微滤处理中保持液体的温度在2℃～8℃。
  11. 如权利要求7所述的方法，其特征在于，低速离心的温度是2～8℃。
  12. 如权利要求11所述的方法，其特征在于，低速离心的温度是4℃。
  13. 由权利要求1-12任一项所述方法得到的逆转录病毒。
  14. 如权利要求13所述的逆转录病毒，其特征在于，逆转录病毒的BSA<200ng/ml，HCP<1μg/ml，HCD<100ng/ml。
  15. 一种产品，其特征在于，所述产品含有权利要求13所述的逆转录病毒。
  16. 一种权利要求13所述的逆转录病毒在制备治疗产品中的应用。
  17. 如权利要求16所述的逆转录病毒在制备治疗产品中的应用，其特征在于，所述治疗产品包括基因治疗产品和/或细胞治疗产品和/或免疫治疗产品。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105980571A (zh) * 2013-12-17 2016-09-28 吉尼松公司 用于纯化包膜病毒或病毒载体的方法
CN111876392A (zh) * 2020-06-30 2020-11-03 恒瑞源正（上海）生物科技有限公司 一种大规模快速生产病毒载体的方法
CN111876393A (zh) * 2020-06-30 2020-11-03 恒瑞源正（上海）生物科技有限公司 一种大规模快速生产高纯度高活性慢病毒载体的方法
US20210009966A1 (en) * 2018-03-28 2021-01-14 Shanghai Cellular Biopharmaceutical Group Ltd. Method for large-scale preparation of purified preparation of recombinant lentiviral vector at gmp grade
CN113373120A (zh) * 2021-06-18 2021-09-10 浙江康佰裕生物科技有限公司 Gmp级逆转录病毒载体的纯化方法与应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK1869171T4 (en) * 2005-04-11 2016-01-18 Crucell Holland Bv Virus cleaning using ultrafiltration
CN112143693A (zh) * 2019-06-28 2020-12-29 杭州康万达医药科技有限公司 一种生产病毒的方法及收获液组合物
CN112899242A (zh) * 2021-02-03 2021-06-04 苏州博腾生物制药有限公司 慢病毒纯化工艺

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105980571A (zh) * 2013-12-17 2016-09-28 吉尼松公司 用于纯化包膜病毒或病毒载体的方法
US20210009966A1 (en) * 2018-03-28 2021-01-14 Shanghai Cellular Biopharmaceutical Group Ltd. Method for large-scale preparation of purified preparation of recombinant lentiviral vector at gmp grade
CN111876392A (zh) * 2020-06-30 2020-11-03 恒瑞源正（上海）生物科技有限公司 一种大规模快速生产病毒载体的方法
CN111876393A (zh) * 2020-06-30 2020-11-03 恒瑞源正（上海）生物科技有限公司 一种大规模快速生产高纯度高活性慢病毒载体的方法
CN113373120A (zh) * 2021-06-18 2021-09-10 浙江康佰裕生物科技有限公司 Gmp级逆转录病毒载体的纯化方法与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LOO ET AL.: "Production of High Titer cGMP-Grade SIN Gamma-Retroviral Vectors by Transfection in a Closed System Bioreactor", BLOOD, vol. 112, no. 11, 16 November 2008 (2008-11-16), XP086679747, ISSN: 0006-4971, DOI: 10.1182/blood.V112.11.3539.3539 *
ZENG RONG, LI JIN, ZHOU SHENG, ET AL : "Purification Packaging, Titer and Detection of C-myc Retrovirus Expression Vector", PROGRESS OF ANATOMICAL SCIENCES, vol. 6, no. 3, 20 September 2000 (2000-09-20), XP093015202, ISSN: 1006-2947, DOI: 10.16695/j.cnki.1006-2947.2000.03.018 *

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