Lentiviral vector purification method and application thereof

Abstract

The invention provides a lentiviral vector purification method and application thereof. The lentiviral vector obtained by the method is high in purity and low in impurity content, and very high transfection efficiency can be obtained. Besides, the volatility among all batches of lentiviral vectors produced by the method is extremely small, the lentiviral vectors are generally stable in the aspects of yield and recovery rate, and all batches of lentiviral vectors can meet the requirement of virus injection level in the aspect of impurity level.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, and particularly relates to a lentivirus vector purification method and application thereof. BACKGROUND

[0002] Due to the higher efficiency of virus infection than that of transfection method for delivering nucleic acid, virus vector becomes a high-efficiency method for delivering nucleic acid into cells. Commonly used virus vectors include adenovirus vector, adeno-associated virus vector, retrovirus vector and lentivirus vector, etc. Lentivirus vector (LV) is a kind of vector derived from retrovirus, which is a replication-defective lentivirus modified from human immunodeficiency virus (HIV) vector system, and contains genetic information required for packaging, transfection and stable integration. While the pathogenic factors of lentivirus are removed, the ability of its genome to integrate into the host genome is retained, and under the action of reverse transcriptase, the exogenous gene fragment can be randomly and stably integrated into the host cell genome to realize the persistent and stable expression of the target gene.

[0003] The establishment of HIV-type lentivirus vector system has experienced a gradual improvement process to improve the biological safety of the vector. Lentivirus vector system is mainly composed of lentivirus transfer vector, packaging vector and cell membrane vector, and is generally composed of three plasmids (second-generation lentivirus packaging system) or four plasmids (third-generation lentivirus packaging system, improved on the basis of the second-generation system). The cells used for packaging lentivirus are generally 293T cells, and currently, the third-generation vector system which is relatively safe is widely used. At present, lentivirus vector system has been widely used in gene expression regulation such as overexpression, interference and gene knockout, and its application in the field of cell and gene therapy is also becoming more and more extensive, especially in CAR-T therapy.

[0004] Gene therapy and cell therapy not only have requirements for the titer of lentivirus, but also have very high requirements for the purity of lentivirus. According to the requirements of the Drug Review Center of the Drug Administration on the issuance of “Technical Guidelines for the Pharmaceutical Research and Evaluation of In Vitro Gene Modification System (Trial)”, there are clear requirements for the impurities of lentivirus, especially BSA and nucleic acid fragments. The upstream process of lentivirus is difficult to produce differentiation, and the biggest challenge lies in the downstream purification process of lentivirus. Generally, large cells, cell fragments and part of impurities are removed by clarification (centrifugation or filtration), then the virus is concentrated by ultrafiltration system, the solution is replaced to remove part of impurities, and finally the lentivirus vector is further purified by two-layer chromatography and ultrafiltration liquid exchange technology. Although the existing lentivirus production process can generally meet the requirements of large-scale and high-titer, the lentivirus vector prepared by the process may have problems such as low purity and process-related impurity residues, which cannot be directly used for human body or has a great influence on the downstream process.

[0005] Therefore, there is an urgent need in the art to construct a new method for purifying lentiviral vectors to improve purity and remove impurities. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a novel method for purifying lentiviral vectors to achieve the purpose of improving purity and removing impurities.

[0007] In the present invention, a method for purifying a lentiviral vector is provided, comprising the steps of:

[0008] (a) providing a cell transfected with a four-plasmid system, wherein the four-plasmid system comprises a packaging plasmid 1, a packaging plasmid 2, an envelope plasmid, and a shuttle plasmid, wherein the mass ratio of the four plasmids in the system is 1-4:1-4:1:1-10, preferably, 1-3:1-3:1:2-5;

[0009] (b) culturing the cells in a first culture medium containing 1-5% (volume ratio) (preferably, 2-5%) FBS to obtain a culture solution containing the lentiviral vector;

[0010] (c) filtering the culture solution obtained in step (b);

[0011] (d) concentrating, replacing, and removing impurities from the culture solution obtained in step (c);

[0012] (e) enzymatically hydrolyzing the culture solution obtained in step (d) with a nuclease;

[0013] (f) performing composite chromatography and ion exchange chromatography on the culture solution obtained in step (e);

[0014] (g) finally concentrating the culture medium obtained in step (f) and replacing it with a lentivirus preservation medium containing 0.1-4% (preferably 0.2-2%, more preferably 0.4-2%) human serum albumin by volume to obtain a purified lentiviral vector.

[0015] In another preferred embodiment, the four-plasmid system includes packaging plasmid 1pMDLg / pRRE (Kan+), packaging plasmid 2pRSV-Rev (Kan+); envelope plasmid pCMV-VSV-G (Kan+), and shuttle plasmid pCDH-EF1-TX103CAR (Kan+).

[0016] In another preferred embodiment, the method further comprises the steps of sterilization filtration and subpackaging for storage.

[0017] In another preferred embodiment, a capsule filter is used to remove cell debris and impurities.

[0018] In another preferred embodiment, the first culture medium comprises DMEM medium.

[0019] In another preferred embodiment, the cell comprises a mammalian cell.

[0020] In another preferred embodiment, the cell comprises a human kidney epithelial cell.

[0021] In another preferred embodiment, the cell comprises a 293T cell.

[0022] In another preferred embodiment, the cell comprises a human kidney epithelial cell 293T or a cell derived therefrom.

[0023] In another preferred embodiment, the cell is transfected at a pH of 6.8-7.2 (preferably, 7.0-7.2, more preferably, 7.05-7.15, more preferably, 7.12±0.03).

[0024] In another preferred embodiment, the culturing time in step (b) is 24h.

[0025] In another preferred embodiment, a capsule filter is used for filtration in step (c).

[0026] In another preferred embodiment, a hollow fiber column is used for concentration and removal of impurities in step (d).

[0027] In another preferred embodiment, the flow rate of the hollow fiber column is 4.89-6.52 L / min.

[0028] In another preferred embodiment, the shear force of the hollow fiber column is 1450 -sec -1850 -sec .

[0029] In another preferred embodiment, the concentration of the nuclease in step (e) is 100-2000 U / mL, preferably, 100-1500 U / mL, preferably, 100-800 U / mL, preferably, 100-600 U / mL, preferably, 200-400 U / mL, more preferably, 300±3 U / mL.

[0030] In another preferred embodiment, an anion exchange column is used for ion exchange chromatography in step (f).

[0031] In another preferred embodiment, the elution NaCl concentration used when performing ion exchange chromatography in step (f) is 0.1-2 mol / L, preferably, 0.2-1 mol / L, preferably, 0.4-0.6 mol / L, more preferably, 0.45±0.05 mol / L.

[0032] In another preferred embodiment, in step (f), the composite chromatography and ion exchange chromatography are performed sequentially (i.e., first composite chromatography and then ion exchange chromatography).

[0033] In another preferred embodiment, in step (f), the composite chromatography is performed using Capto core 700 chromatography medium.

[0034] In another preferred embodiment, in step (f), the ion exchange chromatography is performed using DEAE chromatography medium.

[0035] In another preferred embodiment, in step (g), the final concentration is performed using a hollow fiber column.

[0036] In another preferred embodiment, in step (g), the hollow fiber column has one or more parameters selected from the group consisting of:

[0037] The material includes mPES (modified polyether sulfone);

[0038] The molecular weight cut-off is 10-500 kDa, preferably 20-200 kDa, more preferably 20-150 kDa, more preferably 20-100 kDa, and most preferably 30 kDa (kilodaltons);

[0039] The membrane area is 500-1000 cm 2 , preferably 790 cm 2 of hollow membrane module.

[0040] In another preferred embodiment, in step (g), no glycerol is added to the lentivirus storage solution.

[0041] In another preferred embodiment, in step (g), the lentivirus storage solution contains the following components: 10-100 mmol / L (preferably 20-80 mmol / L, more preferably 40-60 mmol / L) Tris-HCl, 20-500 mmol / L (preferably 50-300 mmol / L, more preferably 100-200 mmol / L) NaCl, and 0.1-4% (by volume) (preferably 0.2-2%, more preferably 0.4-2%) human blood albumin.

[0042] In another preferred embodiment, in step (g), the pH of the virus storage solution is 7-8.

[0043] In another preferred embodiment, the titer of the purified lentiviral vector is ≥ 1.0 x 10 7 TU / mL, preferably 1.0 x 10 7 TU / mL-4.0 x 10 7 TU / mL.

[0044] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and each of the technical features specifically described hereinafter (such as the examples) can be combined with each other to constitute a new or preferred technical solution. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 shows the structure of the plasmid vector, Figure 1A Figure 2 shows the structure of the packaging plasmid 1 (pMDLg / pRRE (Kan+)), Figure 1B Figure 3 shows the structure of the packaging plasmid 2 (pRSV-Rev (Kan+)), Figure 1C Figure 4 shows the structure of the envelope plasmid (pCMV-VSV-G (Kan+)), Figure 1D Figure 5 shows the structure of the shuttle plasmid (pCDH-EF1-TX103CAR (Kan+)).

[0046] Figure 2 The upstream process of the preparation of the lentiviral vector is shown.

[0047] Figure 3 The downstream process of the preparation of the lentiviral vector is shown.

[0048] Figure 4 The effect of different serum contents on the yield (titer) of lentivirus is shown.

[0049] Figure 5 The change of impurities over time under the condition of 2% FBS is shown.

[0050] Figure 6 The effect of different MOI transduction cells on the positive rate of CAR-T is shown.

[0051] Figure 7 The long-term stability of the lentivirus in the storage solution is shown. DETAILED DESCRIPTION

[0052] The present inventors have carried out extensive and in-depth research, explored the production process and screened the process parameters, and for the first time developed a purification method for lentiviral vectors. The lentiviral vectors obtained by the method of the present application have high purity, low impurities, and can obtain very high transfection efficiency. In addition, the fluctuation between batches of lentiviral vectors produced by the method of the present application is extremely small, and the overall stability in yield and recovery rate is stable, and in terms of impurity level, each batch can meet the requirements of virus injection grade. On this basis, the present application is completed.

[0053] Four-plasmid system

[0054] In the present application, the four-plasmid system includes a packaging plasmid, an envelope plasmid, and a shuttle plasmid. The packaging plasmid includes a packaging plasmid 1 and a packaging plasmid 2, the packaging plasmid 1 contains a gag gene encoding a lentivirus core and major structural proteins (such as nucleocapsid protein p7, matrix protein p17, and capsid protein p24), a pol gene encoding a virus-specific enzyme (reverse transcriptase, protease, and integrase); the packaging plasmid 2 contains a rev gene encoding a regulatory factor regulating the expression of gag and pol genes, expressing a Rev protein, forming a complex with a response element RRE to export the nucleus, ensuring efficient expression of gag and pol and high assembly of viral backbone genes; the envelope plasmid contains a gene encoding a vesicular stomatitis virus envelope G protein providing lentivirus packaging, enabling TX103 lentivirus to have a VSVG pseudo-envelope; the shuttle plasmid contains a target gene 103 CAR (scFv-H-TM-C-CD3 zeta) gene (self-inactivation), an EF1 promoter, a WPRE element, 3' and 5' end LTRs. The sequence of the target gene is shown in SEQ ID NO: 38 in the granted patent No. ZL201811125056.4.

[0055] In the four-plasmid system, the ratio of the four plasmids has a significant effect on improving the titer of lentivirus. In some embodiments of the present application, the four-plasmid system includes a packaging plasmid 1, a packaging plasmid 2, an envelope plasmid, and a shuttle plasmid, and the ratio thereof is 1-4:1-4:1:1-10, preferably 1-3:1-3:1:2-5.

[0056] Preparation of lentivirus vector

[0057] The upstream and downstream flowcharts of the preparation of the lentivirus vector are shown in Figure 2 and 3 respectively.

[0058] In the present application, the specific purification process of the lentivirus vector is as follows:

[0059] 1 Cell recovery and subculture

[0060] Adherent cells: Take one vial of frozen cells from the liquid nitrogen tank and thaw. Under sterile conditions, add 5 mL of DMEM medium containing 10% FBS to a centrifuge tube, transfer about 1 mL of cell suspension from the frozen tube to the centrifuge tube, centrifuge at room temperature for 5 min, discard the supernatant; resuspend the cells with DMEM medium containing 10% FBS, count the cells and detect the cell viability (viability should be ≥80%), and record the total number of cells. Seed the cells into a culture flask, add 37.0℃ preheated DMEM medium containing 10% FBS to a culture volume of 15 mL, and culture in a 37.0℃, 5% CO2 carbon dioxide incubator.

[0061] Under sterile operation, remove the culture medium from the above culture flask, add PBS and spread it evenly to rinse the cells. Remove PBS, then add trypsin digestion solution, spread it evenly, digest the cells at room temperature, add DMEM medium containing 10% FBS to stop the reaction and resuspend the cells, transfer to a centrifuge tube, centrifuge at room temperature, and remove the supernatant. Resuspend the cells with DMEM medium containing 10% FBS, count the cells and detect the cell viability (viability should be ≥80%), and centrifuge the cells according to (4-6)×10 4 cells / cm 2 The cells were inoculated into culture flasks at a density of 100 μg / mL and supplemented with DMEM medium containing 10% FBS preheated at 37.0°C. The cells were cultured in a 37.0°C, 5% CO2 incubator.

[0062] 2. Inoculation before transfection

[0063] Remove the culture container from the incubator. Under sterile conditions, remove the culture medium from each culture flask or cell factory, add PBS to wash the cells, and remove the PBS. Add trypsin to digest the cells at room temperature for 3 to 5 minutes. After the cells in the culture container fall off, add 10% FBS DMEM culture medium to terminate the digestion reaction and resuspend the cells. Collect them into a cell collection bottle. Take samples, count and record the cell density, viability and total cell number. The cell viability must be ≥90%. According to (4 to 6) × 10 4 cells / cm 2 The cell suspension in the roller bottle was mixed and poured into culture containers respectively, and cultured in a 37.0°C, 5% CO2 incubator for 44±4h.

[0064] 3. Transfection

[0065] The four plasmids were co-transfected into 293T cells for packaging lentivirus, 0.6 μg / cm 2 (The acceptable range is 0.6~0.8μg / cm 2 ), the ratio of the four plasmids was ZL05:ZL01:ZL02:ZL03=4:2:2:1.

[0066] Under sterile conditions, four plasmids are added to a 2.5 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (Hepes) solution, and then added to a threaded reagent bottle containing a 0.5 mol / L calcium chloride solution, mixed evenly, as transfection A liquid; the A liquid is added to the B liquid (2x HeBS: 50 mmol / L Hepes, 280 mmol / L sodium chloride, 1.5 mmol / L disodium hydrogen phosphate) in vortex, mixed evenly, and then placed at room temperature for 5 min (acceptable range is 1-10 min), as the final transfection reagent; the transfection reagent is added to fresh DMEM medium and mixed evenly; the culture medium in the culture vessel is removed, and the DMEM medium containing the transfection reagent is transferred into the culture vessel, and the cell factory is placed in a 37.0°C, 5% CO2 carbon dioxide incubator for transfection incubation for 6±1 h.

[0067] Using serum-free transfection can improve the titer and reduce the BSA content at the same time.

[0068] After the transfection incubation is completed, under sterile conditions, the culture supernatant containing the transfection reagent is removed, and 2% FBS-containing DMEM medium is added to each culture vessel. After the medium change is completed, the lentiviral vector production phase is entered. The cell factory is placed in a 37.0°C, 5% CO2 carbon dioxide incubator for 24-26 h of culture.

[0069] After the medium change, using 2% serum not only effectively reduces the BSA content, but also maintains the titer basically consistent with that of 5% or 10% serum; at the same time, 24 h of culture can effectively reduce cell death, thereby reducing the nucleic acid and host protein content and reducing the downstream purification pressure.

[0070] 4 Suspension cell recovery and subculture

[0071] According to the density of the frozen HEK293 cells, an appropriate recovery system is selected, so that the starting cell density of the recovery culture is controlled at 0.4-0.6 x 10 6 cells / mL. Taking a 100 mL culture bottle (20 mL system, the number of cells in the frozen tube is 1 x 10 7 cells) as an example, the following is shown:

[0072] The frozen HEK293 cells are quickly taken out and thawed at 37°C in a water bath (can be gently shaken in water, and the time is controlled within 1 min); the surface of the frozen tube is disinfected; 20 mL of fresh culture medium is added to the 100 mL culture bottle, and the thawed cells are all transferred into the culture bottle, and the culture bottle is gently shaken to mix the cells. Since the freshly recovered cells are fragile, the whole process should avoid violent operation and centrifugation of the cells; the resuspended cells are placed in a constant temperature incubator at 37°C, 5% CO2, and 150-175 rpm rotation speed; the cell density and viability data within 2-5 days of culture are monitored.

[0073] The medium volume used for cell passage is no more than 20% of the container volume. Count the cells, calculate the cell density and cell viability; inoculate the cells into fresh medium at a starting density of 0.3-0.4 x 10 6 cells / mL; place the culture flask in a constant temperature shaker at 37°C, 5% CO2, and 150-175 rpm; after 3-4 days of subculture, the viable cell density is generally more than 3 x 10 6 cells / mL, and the viability is greater than 90%.

[0074] After counting the cells on the day of transfection, the cell viability is greater than 95%, and the viable cell density is 3-4 x 10 6 cells / mL. Dilute the cells in the medium at a density of 2.0 x 10 6 cells / mL. According to the optimized transient transfection process, prepare the DNA and PEI mixture. For example, for 20 mL of adenovirus packaging system:

[0075] Prepare A and B solutions (A: plasmid + 1000 μL (medium or PBS); B: PEI (120 μg) + 1000 μL (medium or PBS)). After vortex mixing, let stand at room temperature for 5 min. Add B solution to A solution and mix gently by blowing. Let stand at room temperature for 10 min. Add the mixture to the culture solution and incubate. After 18-22 h of incubation, supplement the medium according to the incubation protocol. Incubate for 2-4 days, or until the viability is less than 80%, and then end the incubation (the harvest time is determined according to the type of virus).

[0076] 5 Supernatant harvest

[0077] After incubation, collect the culture solution from the cell factory under sterile conditions into a disposable storage bag.

[0078] 6 Clarification filtration

[0079] Use a capsule filter with a pore size of 0.45 μm and a polypropylene material for clarification filtration of the lentivirus harvest.

[0080] Under sterile conditions, connect the disposable storage bag to the capsule filter, and then connect the capsule filter to the filtration system. Bleed the capsule filter, and when the sample fills the entire filter, adjust the flow rate to 0.65 L / min (flux = 300 LMH). Collect the clarified sample into a disposable storage bag, control the front-end pressure to be ≤ 15 psi, and the processing capacity to be ≤ 228 L / m 2 .

[0081] 7 Hollow fiber primary concentration

[0082] The hollow membrane module is made of mPES (modified polyether sulfone) with a molecular weight cut-off of 500 kDa (kilodaltons), a hollow fiber column height of 20 cm, and a membrane area of 12500 cm 2 The lentivirus clarified solution was concentrated and exchanged.

[0083] The concentration and exchange were carried out at room temperature. The lentivirus clarified solution was placed on the balance at the sample inlet end of the hollow fiber concentration system. The flow rate was set to 5.89 L / min (acceptable range: 4.89-6.52 L / min), the shear force was controlled at 1450 -sec -1850 -sec , the transmembrane pressure was 1.5-3.0 psi, the concentration factor was 5.0 (acceptable range: 4.5-5.5), and the wash factor was 2.0 (acceptable range: 1.8-2.2). The sample was concentrated with a processing volume of ≤26 L / m 2 . After concentration, the filtrate end was closed, the sample was collected, and the hollow membrane module was washed with DMEM solution. The lentivirus primary concentrated solution was collected.

[0084] The shear force control effectively protects the lentivirus envelope and reduces the loss of lentivirus activity. The membrane pore size and wash factor can effectively remove impurities, with a removal rate of more than 90%.

[0085] 8 Nuclease enzyme digestion

[0086] Full nuclease was used for enzyme digestion to reduce the nucleic acid load in the sample. According to the volume of the lentivirus primary concentrated solution, the enzyme activity unit in the lentivirus concentrated solution was 300±3 U / mL. The solution bag was shaken and mixed, and enzyme digestion was carried out at 37.0±1.0℃ for 2.0±0.5 h.

[0087] Using high-concentration nuclease can effectively reduce the size of nucleic acid fragments.

[0088] 9 Composite chromatography

[0089] Capto core 700 chromatography medium was used to purify the lentivirus after concentration and exchange in flow-through mode to remove BSA and host protein impurities.

[0090] 10 Anion exchange chromatography

[0091] DEAE chromatography medium was used to fine purify the lentivirus vector sample after Capto core 700 purification to further remove BSA and host nucleic acid.

[0092] The purification process was carried out at room temperature, and the lentivirus vector sample purified by Capto core 700 was loaded. After the loading was completed, 3 times the column volume of the impurity washing buffer (50 mmol / L Tris, 300 mmol / L NaCl, 5% glycerol, pH 7.5) was used to wash the impurities and remove BSA and host protein impurities; and then the elution buffer (50 mmol / L Tris, 450 mmol / L NaCl, 5% glycerol, pH 7.5) was used for elution.

[0093] The use of 0.45 mol of sodium chloride for elution can further remove nucleic acids while maintaining the most active virus.

[0094] 11 Hollow fiber final concentration

[0095] The hollow membrane assembly with a material of mPES (modified polyether sulfone), a molecular weight cut-off of 30 kDa (kiloDalton), and a membrane area of 790 cm 2 was used to concentrate and replace the liquid of the anion exchange chromatography lentivirus sample.

[0096] 12 Sterile filtration and sub-packaging storage

[0097] The lentivirus solution after hollow final concentration and liquid replacement was subjected to sterile filtration using a capsule filter with a material of PES (polyether sulfone) and a pore size of 0.22 μm, and was stored in a super-low temperature refrigerator at -80±10℃ after being quickly frozen with liquid nitrogen.

[0098] In a preferred embodiment, the lentivirus vector purification method of the present application comprises the following steps:

[0099] In the plasmid transfection stage, the four plasmids were mixed in a ratio of 4:2:2:1 with DMEM medium containing 2% FBS, and the supernatant was harvested 24 hours after transfection.

[0100] In the clarification step of the purification stage, a capsule filter with a material of polypropylene and a particle size cut-off of 0.45 μm was used, the filtration speed was not higher than 343 LMH, and the unit membrane area treatment capacity should be controlled at 228 L / m 2 .

[0101] In the initial concentration and liquid replacement step of the purification stage, the parameters were in the range of: transmembrane pressure 1.5-3.0 psi, shear force 1450 -sec -1850 -sec , unit membrane area treatment capacity ≤26 L / m 2 , concentration factor 5.0, and filter washing factor 2.0.

[0102] In the nuclease enzyme cutting step of the purification stage, the nuclease concentration was set to 300 U / mL, and the enzyme hydrolysis was carried out at 37℃ for 2 h.

[0103] Purification stage composite chromatography step, Capto core 700 composite chromatography purification of Thinktoman is used, 50 times column volume (CV) of lentivirus harvest liquid sample is treated, and the linear flow rate range of chromatography is 200-300 cm / h;

[0104] Purification stage anion exchange chromatography step, Fractogel EMD DEAE (M) chromatography medium is used, and the parameter range is as follows: sample loading capacity is less than or equal to 60 CV, the NaCl concentration of washing impurities is 0.3 mol / L, the NaCl concentration of elution is 0.45 mol / L, the linear flow rate of sample loading is 100-120 cm / h, and the linear flow rate of elution is 30-50 cm / h;

[0105] Purification stage final concentration and liquid exchange step, a hollow fiber column with a cut-off size of 30 kDa is selected to perform the final concentration and liquid exchange; and the lentivirus storage solution formula is as follows: 50 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.4% human blood albumin, and the solution pH is 7.5.

[0106] The preparation process of the present application can obtain lentivirus vectors with high purity and low impurities, and can obtain very high transfection efficiency. In addition, the fluctuation between batches of lentivirus vectors produced by the method of the present application is extremely small, and the overall stability in yield and recovery rate is stable, and in terms of impurity level, each batch can meet the requirements of virus injection level.

[0107] The main advantages of the present application include:

[0108] (1) The lentivirus vectors obtained by the method of the present application have high purity, low impurities, and can obtain very high transfection efficiency. In addition, the fluctuation between batches of lentivirus vectors produced by the method of the present application is extremely small, and the overall stability in yield and recovery rate is stable, and in terms of impurity level, each batch can meet the requirements of virus injection level.

[0109] (2) The lentivirus has high purity, and for 293T cells that need to be cultured in serum, the BSA can be controlled to be less than or equal to 50 ng / mL (dose).

[0110] (3) The impurity content is low, and nucleic acid fragments can be effectively removed, so that the size of nucleic acid fragments (especially 200 bp) is controlled to be less than or equal to 10%. The lentivirus vectors purified by the present application can meet the requirements of relevant laws and regulations of China for lentivirus vectors under the premise of large-scale production.

[0111] (4) The lentivirus prepared by the present application can reach the injection level and has high purity. For impurities, especially host proteins, host nucleic acids and nucleic acid fragment size, the requirements of in vivo gene therapy are met.

[0112] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not used to limit the scope of the application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturer. Unless otherwise specified, the percentages and parts are weight percentages and weight parts.

[0113] Unless otherwise specified, the reagents and materials in the examples of the application are commercially available products.

[0114] The lentiviral vector used in the examples is a four-plasmid co-transfection 293T cell packaging lentiviral vector.

[0115] Example 1

[0116] The lentivirus harvest solution was prepared using 293T cells (purchased from Takara) cryopreserved in a liquid nitrogen tank; after resuscitation, the cells were subcultured and expanded in turn, and 4-6 x 10 4 cells / cm 2 as the cell seeding density; the cells were finally subcultured into cell factories using the adherent culture method. Four plasmids (the four plasmids were pMDLg / pRRE (Kan+), pRSV-Rev (Kan+), pCMV-VSV-G (Kan+), and pCDH-EF1-TX103CAR (Kan+), and the plasmid vector structure diagrams are shown in Figure 1A 、 Figure 1B 、 Figure 1C 、 Figure 1D ), purchased from Beijing Zhongyuan Hehe Biotechnology Co., Ltd. and System Biosciences, USA) were mixed with transfection reagents (transfection solution A: 2.5 mmol / L Hepes, 0.5 mol / L calcium chloride; transfection solution B: 50 mmol / L Hepes, 280 mmol / L sodium chloride, 1.5 mmol / L disodium hydrogen phosphate) at a certain mass ratio (the selected four-plasmid ratio was 2:2:1:4, and the total amount of plasmid used was 0.6-0.8 μg / cm 2 ) and then transfected into the cells (the optimal standing time of the transfection reagent was 5 min, and the allowed operation range was 1-10 min; the transfection incubation time was 6±1 h). According to the experiment (see Figure 4 ), the cells were cultured in DMEM medium containing 2% FBS (purchased from Corning) for 24 h, and the supernatant was harvested 24 h after transfection;

[0117] The results show that the transduction titer obtained by the medium containing 5% FBS is the highest, the transduction titer obtained by the medium containing 2% FBS is close to that of the 5% group, and the transduction titers of the 5% and 2% groups are obviously higher than that of the serum-free culture group, and are 3-4 times higher in general. With the extension of time, the lentivirus transduction titer in the culture supernatant presents a fluctuating increase, but the growth amplitude is less than 1.4 times.

[0118] According to the experiment (see Figure 5 ), it can be known that the total nucleic acid and host protein are low between 18-24h, and the host protein content in the culture supernatant increases steadily, but the total nucleic acid content increases greatly, which is not conducive to downstream purification, so 24 hours is selected as the harvesting time.

[0119] Example 2

[0120] Purification of the lentivirus sample obtained according to Example 1

[0121] Filtering of the lentivirus harvest liquid: a capsule filter (purchased from Sartorius Company) with a material of polypropylene and a particle size of 0.45 μm is used as the filter for the clarification filtering step of the lentivirus harvest liquid; the filtering speed is not higher than 343 LMH, and the treatment capacity per unit membrane area should be controlled to be 228 L / m 2 .

[0122] Concentration and medium replacement: a hollow fiber column (purchased from Repligen Company) is used to complete the concentration and medium replacement of the lentivirus sample, and the following experimental conditions are finally obtained through optimization.

[0123] Parameter range of the initial concentration of the hollow fiber

[0124]

[0125] Nuclease enzyme hydrolysis: the nuclease enzyme (purchased from Beijing Yiqiao God State Technology Co., Ltd., derived from Serratia marcescens, expressed and produced by Escherichia coli, and free of animal-derived components) hydrolysis step is placed after the initial concentration of the hollow fiber, and the nuclease concentration is 300 U / mL, the enzyme hydrolysis is carried out at 37°C for 2h,

[0126] Composite chromatography: the Capto core 700 composite chromatography purification of Thinktanks is used, which can process 50 times of column volume (CV) of the lentivirus harvest liquid sample, and linear amplification can be realized on this basis; the linear flow rate range of the chromatography is 200-300 cm / h.

[0127] Ion exchange chromatography: the Fractogel EMD DEAE (M) chromatography medium is used for the fine purification of the lentivirus, and the following experimental conditions are finally obtained through optimization, which can achieve good purification effect.

[0128] Parameter of anion exchange chromatography process

[0129]

[0130] Final concentration exchange: Hollow fiber column with 30 kDa cut-off size (purchased from Repligen) was selected for final concentration, and exchange was performed. The virus storage solution was added with 0.4% human serum albumin (HSA) and without glycerol. The specific storage solution formula was: 50 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.4% human serum albumin, and the solution pH was 7.5

[0131] According to the above implementation, the results are as follows:

[0132]

[0133]

[0134]

[0135] Comparing the results of each batch, the yield and recovery rate are generally stable. In terms of impurity level, each batch can meet the requirements of virus injection grade.

[0136] In addition, according to the long-term stability data ( Figure 7 ), it is shown that the titer does not decrease significantly within 12 months; the viability is maintained above 80%, indicating that the method of the present application has good effect on the protection of lentivirus.

[0137] Example 3

[0138] PBMC cells (collected from apheresis blood separation from different healthy subjects, obtained by cold chain transportation from blood sampling in the hospital to the production workshop and by apheresis cell separator) were transduced using different MOIs to observe the influence of transducing cells at different MOIs on the CAR-T positive rate, so as to determine the MOI value to be used in the preparation process. Considering that the difference between different individuals may affect the results, the experimental design used PBMCs from 3 different individuals (3 different healthy people with similar ages, 25 years old, 25 years old, and 26 years old) in G-REX6 well plates, and set 0.025, 0.05, 0.1, 0.25, 0.5, and 1 six MOIs; design 3 parallel experiments of PBMCs from 3 healthy subjects; 3 batches of lentivirus transduced PBMCs, 3 groups of PBMCs transduced by the same batch of lentivirus; Day 0, according to the count and CD3 + proportion, 5×10 6 CD3 + cells were inoculated in each well, the culture volume was 2.5 ml, TcellTransAct was added for activation, and the count was counted on Day 1. According to the count result, the same CD3 +Cells, different MOI for each batch of lentivirus; sample at Day5, Day7, Day9 to detect CAR-T cell transduction rate.

[0139] The results are shown in Table 1. Figure 6

[0140] The results show that for transfection of T cells, the transfection efficiency can reach more than 75% when the MOI is 0.5.

[0141] All documents referred to in this disclosure are incorporated herein by reference as if each individual document were incorporated by reference. In addition, it is to be understood that various alterations and modifications can be made to the present application upon reading and understanding the above lecture of the present disclosure, and these equivalent forms also fall within the scope of the appended claims of the present application.​

Claims

1. A method for purifying a lentiviral vector, characterized by, The method comprises the steps of: (a) providing a cell transfected by a four-plasmid system, wherein the four-plasmid system comprises a packaging plasmid 1, a packaging plasmid 2, an envelope plasmid and a shuttle plasmid, and the mass ratio of the four plasmids in the system is 1-4:1-4:1:1-10, preferably 1-3:1-3:1:2-5; (b) culturing the cell in a first culture medium containing 1-5% (by volume), preferably 2-5%, FBS, so as to obtain a culture solution containing a lentiviral vector; (c) filtering the culture solution obtained in step (b); (d) concentrating and replacing the culture solution obtained in step (c) and removing impurities; (e) enzymatically treating the culture solution obtained in step (d) with a nuclease; (f) subjecting the culture solution obtained in step (e) to complex chromatography and ion exchange chromatography, respectively; (g) performing final concentration on the culture solution obtained in step (f), and replacing the solution with a lentivirus storage solution containing 0.1-4% (by volume), preferably 0.2-2%, more preferably 0.4-2%, human blood albumin, so as to obtain a purified lentiviral vector.

2. The method of claim 1, wherein, The first culture medium comprises a DMEM culture medium.

3. The method of claim 1, wherein, The cell comprises a mammalian cell.

4. The method of claim 1, wherein, The cell comprises a human renal epithelial cell.

5. The method of claim 1, wherein, The cell comprises a human renal epithelial cell 293T or a cell derived therefrom.

6. The method of claim 1, wherein, The cell is transfected under the condition that the pH is 6.8-7.2, preferably 7.0-7.2, more preferably 7.05-7.15, and more preferably 7.12±0.

03.

7. The method of claim 1, wherein, In step (b), the culturing time is 24 h.

8. The method of claim 1, wherein, In step (e), the concentration of the nuclease is 100-2000 U / mL, preferably 100-1500 U / mL, preferably 100-800 U / mL, preferably 100-600 U / mL, preferably 200-400 U / mL, and more preferably 300±3 U / mL.

9. The method of claim 1, wherein, In step (f), when ion exchange chromatography is performed, the concentration of NaCl used for elution is 0.1-2 mol / L, preferably 0.2-1 mol / L, preferably 0.4-0.6 mol / L, and more preferably 0.45±0.05 mol / L.

10. The method of claim 1, wherein, In step (f), complex chromatography and ion exchange chromatography are performed in sequence (i.e., complex chromatography is performed first, and then ion exchange chromatography is performed).

Images