CN111876392A - Method for large-scale rapid production of viral vectors - Google Patents
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Abstract
The invention belongs to the field of biology, and particularly relates to a method for rapidly producing a virus vector in a large scale. The method comprises the following steps: s1: filtering and clarifying a cell culture solution containing the lentiviral vector by using a filter; s2: carrying out ultrafiltration concentration and liquid replacement on the clarified liquid obtained in the step S1 to obtain a concentrated lentivirus vector; s3: adding nuclease into the lentivirus concentrated solution obtained in the step S2 for digestion; s4: loading the digested sample of S3 on a Sepharose6FF chromatographic column, and collecting a first absorption peak; s5: loading the sample collected in S4 to an anion exchange chromatography column, and eluting and collecting by using eluent; s6: and (4) carrying out ultrafiltration concentration and liquid change on the sample collected in the S5 to obtain the lentiviral vector. The invention adopts a mode of combining molecular sieve and ion exchange membrane chromatography, and obtains the lentiviral vector which has high purity and high activity and meets the pharmaceutical standard of biological products by a two-step purification process.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for rapidly producing a virus vector in a large scale.
Background
The gene therapy is to introduce normal genes into pathological cells or somatic cells to express target proteins, so as to treat related diseases caused by gene defects/abnormalities, thereby realizing source therapy, and particularly aiming at partial indications with poor curative effects in partial traditional treatment modes. The current indications for successful gene therapy are mainly focused on monogenic-deficient genetic diseases. Viral vectors are also increasingly being used in clinical studies, and common types of vectors include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and the like. Lentiviral vectors are capable of transforming a variety of cell types without the need to divide the host cell. Therefore, researchers have used it as a gene delivery vector in clinical applications. Despite the routine use of these vectors in many research laboratories, large-scale production using Current Good Manufacturing Practice (CGMP) methods faces a series of challenges, and regulatory approval must be obtained in view of the increased number of clinical trials using lentiviral vectors.
Because the lentivirus vector always keeps higher activity titer in the production process and has more quality influence factors, such as HCP, DNA, BSA, nuclease, endotoxin and the like, the production process of the lentivirus vector is more difficult. The prior industrial production process of the lentiviral vector comprises an ultracentrifugation method and a chromatographic purification method: 1) ultracentrifugation: although the recovery efficiency is high, the virus titer is high; but the disadvantages are also very obvious, the processing volume is limited, the production process is difficult to be amplified, and other toxic substances such as cesium chloride, organic solvents (ethanol or isopropanol) and the like are also introduced. 2) The chromatographic purification method comprises the following steps: the adopted purification processes are various, two-step or three-step chromatography is the most common, most production process flows have long operation time and high cost, and the production process is insufficient due to low product recovery rate (10-20%).
From the consideration of market acceptance and high cost, the development of the production process with lower production cost, high recovery efficiency and qualified product quality is necessary for future market application, is also industrialized production and meets the requirement of GPM-level production and preparation.
Disclosure of Invention
The invention aims to provide a method for rapidly producing a lentivirus vector on a large scale, and overcomes the defects of the prior art. The method adopts a mode of combining molecular sieve and ion exchange membrane chromatography, and obtains the high-purity and high-activity lentivirus vector by a two-step purification process, and the high-purity and high-activity lentivirus vector meets the medicinal standard of biological products. The purification method provided by the invention is convenient and rapid to operate, low in cost, high in recovery efficiency, suitable for large-scale production and capable of meeting the requirements of GMP-level production and preparation.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the present invention, there is disclosed a method for producing a viral vector, comprising:
s1: filtering and clarifying a cell culture solution containing the lentiviral vector by using a filter;
s2: carrying out ultrafiltration concentration and liquid replacement on the clarified liquid obtained in the step S1 to obtain a concentrated lentivirus vector;
s3: adding nuclease into the lentivirus concentrated solution obtained in the step S2 for digestion;
s4: loading the digested sample of S3 on a Sepharose6FF chromatographic column, and collecting a first absorption peak;
s5: loading the sample collected in S4 to an anion exchange chromatography column, and eluting and collecting by using eluent;
s6: and (4) carrying out ultrafiltration concentration and liquid change on the sample collected in the S5 to obtain the lentiviral vector.
It should be understood that the present invention is not limited to the above steps, and may also include other additional steps, for example, before step S1, between steps S1 and S2, between steps S2 and S3, between steps S3 and S4, between steps S4 and S5, between steps S5 and S6, and after step S6, without departing from the scope of the present invention.
Preferably, in S1, the clarification is performed by filtration using a 0.65 or 0.45 μm glass fiber filter.
It should be understood that the pore size of the filter membrane is not limited to the above range, and one skilled in the art can select any suitable filter membrane to implement the present invention as required and all fall within the protection scope of the present invention.
Preferably, in S2, the ultrafiltration membrane has a molecular weight cut-off of 100-500 kD.
Preferably, in S3, the obtained lentivirus concentrate is digested with a digestion reaction solution containing a totipotent nuclease; more preferably, the digestion reaction solution includes: 10-100mM Tris-Cl, 1-10mM MgCl2 and 5-200U/ml totipotent nuclease; more preferably, the digestion reaction conditions are: the reaction is carried out at 37 ℃ for 30-120 minutes.
It should be understood that the components or digestion conditions of the digestion reaction solution are not limited to the above ranges, and those skilled in the art can select any suitable components or conditions as required to complete the present invention and all fall within the protection scope of the present invention.
Preferably, in S4, the digested sample is applied to a Sepharose6FF column at a set linear flow rate.
In some embodiments, the linear flow rate is in the range of 60-100 cm/h.
More preferably, in S4, the digested sample is applied to a pre-equilibrated Sepharose6FF column; the concentration of Tris-Cl in the buffer solution of the equilibrium chromatographic column is 10-100mM, the concentration of NaCl is 10-300mM, and the pH value is 7-9.
In some embodiments of the invention, Sepharose6FF columns are purchased from GE.
Preferably, in S5, the sample obtained in S4 is directly loaded onto a pre-equilibrated anion exchange chromatography column, eluted with an eluent, and diluted 3-5 times with a culture medium after collecting the first absorption peak; more preferably, the medium is DMEM medium.
Preferably, in S5, the ion exchange chromatography column is an anion exchange chromatography column.
More preferably, the anion exchange chromatography column is selected from the following classes:
anion exchange packing: SOURCE 15Q, SOURCE 30Q, Capto Q ImpRes, Capto Q, Capto DEAE, Q Sepharose HP, Q Sepharose FF, DEAE Sepharose FF, Q Sepharose 4 FF, MacroCap Q, Q Sepharose XL, Q Sepharose,
EMD TMAE(strong AEX)、
EMD TMAEHicap、
EMD DEAE(weak AEX)、
Q、
CPX、
HCX、POROS XQ、POROS XQ50、POROS PI50、POROS D50；
Anion exchange membrane chromatography: natrifloTMHD-Q、Sartobind Q、Mustang Q。
It should be understood that the above-mentioned ion exchange chromatography column is not limited to anion exchange chromatography column, and further, is not limited to the kind of anion exchange chromatography column listed above, and any suitable ion exchange chromatography column can be selected by those skilled in the art as required to complete the present invention, and all such columns are within the scope of the present invention.
Preferably, the concentration of Tris-Cl in the buffer solution of the equilibrium chromatographic column is 10-100mM, the concentration of NaCl is 10-300mM, and the pH value is 7-9; preferably, the eluent has a Tris-Cl concentration of 10-100mM, a NaCl concentration of 0.3-1.0M and a pH value of 7-9.
Preferably, in S6, the ultrafiltration membrane has a molecular weight cut-off of 50-300 kD.
In some embodiments of the invention, the fluid change further comprises a sterilization step, i.e., filtering the mixture with a filter having a pore size of 0.22. mu.M, and rinsing the filtered mixture with sterile PBS buffer to obtain the lentiviral vector.
In a second aspect, the invention discloses a lentiviral vector obtained by the above method.
In a third aspect, the invention discloses a product comprising the lentiviral vector described above. Preferably, the product may be a kit.
In a fourth aspect the invention discloses the use of a lentiviral vector according to above or a product according to above in the field of gene therapy. Preferably, the lentiviral vector described above or the product described above is used in a monogenic defective genetic disorder.
On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.
Compared with the prior art, the invention has the following remarkable advantages and effects:
1) the purification process adopts a purification process combining a molecular sieve and membrane chromatography, and the molecular sieve used in the first step has higher recovery rate and relatively simple operation; the second step uses membrane chromatography, which is characterized by high loading capacity and high flow rate, the flow rate is 10 times/min of the membrane volume, the purification time is 30-50 times faster than the traditional filler chromatography, and the cost is 20-40% of the column chromatography.
2) The quality and the activity titer of the viral vector of the production process flow are 0.9-5.0E +08TU/ml, and the detection of the impurities such as BSA, HCP, HCD, nuclease and the like in the obtained lentiviral vector reaches the industrial use standard.
3) Compared with the production process flow on the market, the overall recovery efficiency is 30 percent, and the relative cost is reduced by 20 to 30 percent.
Drawings
FIG. 1 is a Sepharose6FF purification chromatogram of an example of the invention;
FIG. 2 is a Mustang Q purification chromatogram of an example of the present invention;
FIG. 3 is a graph showing the results of flow-based assay of the activity titer of the final product obtained in the examples of the present invention.
Detailed Description
The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.
The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.
Example 1
The embodiment discloses a method for rapidly producing high-purity and high-activity lentivirus vectors in a large scale, which comprises the following steps:
300ml of collected slow virus vector feed liquid with the activity titer of 6.30E +06TU/ml is filtered and clarified by using a 0.65 or 0.45 mu m glass fiber filter;
concentrating the lentivirus clarified liquid obtained in the step 1) by using a membrane package or a hollow fiber type ultrafiltration device, wherein the cut-off molecular weight of the ultrafiltration membrane is 100-fold and 500KD, and the concentration is 10 times.
Carrying out totipotent nuclease digestion on the lentivirus concentrated solution obtained in the step 2), and adding a reaction solution: 25mM Tris-Cl (pH8.0), 2mM MgCl2Benzonase all-round nuclease 100U/ml; reacting for 60 minutes at 37 ℃;
subjecting the sample obtained in 3) to molecular sieve 6FF chromatography: loading a sample digested by nuclease to a pre-equilibrated Sepharose6FF chromatographic column, wherein the concentration of Tris-Cl in a buffer solution of the equilibrated chromatographic column is 25mM, the concentration of NaCl is 150mM, the pH value is 7.5, collecting a first elution peak, namely an elution peak containing the target virus, and the purification chromatogram of Sepharose6FF is shown in figure 1, wherein the volume of the elution peak is 17 mL;
directly loading the sample obtained in the step 4) to a prestabilized Mustang Q membrane chromatographic column, eluting by using eluent, collecting the first absorption peak, eluting to collect the volume of 6ml, and immediately diluting by 5 times by using DMEM. The concentration of Tris-Cl in the buffer solution of the equilibrium chromatographic column is 25mM, the concentration of NaCl is 150mM, and the pH value is 7.5; the eluent has a Tris-Cl concentration of 25mM, a NaCl concentration of 1.0M and a pH value of 7.5. The Mustang Q purification chromatogram is shown in FIG. 2.
Concentrating the sample obtained in step 5) by 6 times by using a membrane-packed or hollow fiber type ultrafiltration device, wherein the cut-off molecular weight of the ultrafiltration membrane is 50-300KD, and then replacing the sample into a virus preservation solution to obtain the lentivirus with the volume of 5.6 ml;
the sample obtained in step 6) was filtered through a sterile filter with a pore size of 0.22 μm and rinsed with sterile PBS buffer, at which time the plasmid DNA volume was 5.2ml and the activity titer was 1.03E +08 TU/ml. The results of performing the activity titer flow assay on 10. mu.L, 1. mu.L and 0.1. mu.L of each lentiviral vector are shown in FIG. 3 (the sampling volumes of the lentiviral vectors in the order from top to bottom in FIG. 3 are 10. mu.L, 1. mu.L and 0.1. mu.L, respectively). The total recovery rate of the lentiviral vector production flow of this example was 28.3%, which can meet the GMP-level production requirements.
Example 2
The embodiment discloses a method for rapidly producing high-purity and high-activity lentivirus vectors in a large scale, which comprises the following steps:
6000ml of collected lentivirus vector material with the activity titer of 5.230E +06TU/ml is filtered and clarified by using a 0.65 or 0.45 mu m glass fiber filter;
concentrating the lentivirus clarified liquid obtained in the step 1) by using a membrane package or a hollow fiber type ultrafiltration device, wherein the cut-off molecular weight of the ultrafiltration membrane is 100-fold and 500KD, and the concentration is 10 times.
Carrying out totipotent nuclease digestion on the lentivirus concentrated solution obtained in the step 2), and adding a reaction solution: 25mM Tris-Cl (pH8.0), 2mM MgCl2Benzonase Universal nuclease: 100U/ml; reacting at 37 ℃ for 60 minutes;
subjecting the sample obtained in 3) to molecular sieve 6FF chromatography: loading a sample digested by nuclease to a pre-balanced Sepharose6FF chromatographic column, wherein the concentration of Tris-Cl in a buffer solution of the balanced chromatographic column is 25mM, the concentration of NaCl is 150mM, and the pH value is 7.5, collecting a first elution peak, namely an elution peak containing target virus, and obtaining the volume of the elution peak of 750 mL;
directly loading the sample obtained in the step 4) to a prestabilized Mustang Q membrane chromatographic column, eluting by using eluent, collecting the first absorption peak, eluting to collect the volume of 260ml, and immediately diluting by 4 times by using DMEM. The concentration of Tris-Cl in the buffer solution of the equilibrium chromatographic column is 25mM, the concentration of NaCl is 150mM, and the pH value is 7.5; the eluent has a Tris-Cl concentration of 25mM, a NaCl concentration of 1.0M and a pH value of 7.5.
Concentrating the sample obtained in step 5) by 15 times by using a membrane package or hollow fiber type ultrafiltration device, wherein the cut-off molecular weight of the ultrafiltration membrane is 50-300KD, and then replacing the sample into a virus preservation solution to obtain the lentivirus with the volume of 70 ml;
the sample obtained in step 6) was filtered through a sterile filter with a pore size of 0.22um and rinsed with sterile PBS buffer, at which time the plasmid DNA volume was 65ml and the activity titer was 1.53E +08 TU/ml.
The total recovery rate of the production flow of the lentivirus vector in the embodiment is 31.6%, and can meet the production requirement of GMP level.
The purity of the virus vectors obtained in example 1 and example 2 was checked, the results are shown in table 1, and it can be seen from table 1 that the obtained virus vectors all meet the standards of biological products.
TABLE 1 detection results of virus purified samples
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (11)
1. A method of producing a viral vector, comprising:
s1: filtering and clarifying a cell culture solution containing the lentiviral vector by using a filter;
s2: carrying out ultrafiltration concentration and liquid replacement on the clarified liquid obtained in the step S1 to obtain a concentrated lentivirus vector;
s3: adding nuclease into the lentivirus concentrated solution obtained in the step S2 for digestion;
s4: loading the digested sample of S3 on a Sepharose6FF chromatographic column, and collecting a first absorption peak;
s5: loading the sample collected in S4 to an anion exchange chromatography column, and eluting and collecting by using eluent;
s6: and (4) carrying out ultrafiltration concentration and liquid change on the sample collected in the S5 to obtain the lentiviral vector.
2. The method according to claim 1, wherein in S1, the clarification is performed by filtration using a 0.65 or 0.45 μm glass fiber filter.
3. The method as claimed in claim 1, wherein the ultrafiltration membrane has a molecular weight cut-off of 100-500KD in S2.
4. The method according to claim 1, wherein in S3, the resulting lentivirus concentrate is digested with a digestion reaction solution containing a totipotent nuclease; preferably, the digestion reaction solution comprises: 10-100mM Tris-Cl, 1-10mM MgCl2And 5-200U/ml of a totipotent nuclease; preferably, the digestion reaction conditions are: the reaction is carried out at 37 ℃ for 30-120 minutes.
5. The method of claim 1, wherein in S4, the digested sample is applied to a pre-equilibrated Sepharose6FF chromatography column; the concentration of Tris-Cl in the buffer solution of the equilibrium chromatographic column is 10-100mM, the concentration of NaCl is 10-300mM, and the pH value is 7-9.
6. The method of claim 1, wherein in S5, the sample obtained in S4 is directly applied to a prestabilized Mustang Q membrane chromatography column, eluted with an eluent, collected at the first absorption peak, diluted 3-5 times with a medium; preferably, the medium is DMEM medium.
7. The method of claim 6, wherein the equilibrium chromatography column buffer used has a Tris-Cl concentration of 10 to 100mM, a NaCl concentration of 10 to 300mM, and a pH of 7 to 9; preferably, the eluent has a Tris-Cl concentration of 10-100mM, a NaCl concentration of 0.3-1.0M and a pH value of 7-9.
8. The method of claim 1, wherein in S6, the ultrafiltration membrane has a molecular weight cut-off of 50-300 KD.
9. A lentiviral vector obtainable by the method of any one of claims 1 to 8.
10. A product comprising the lentiviral vector of claim 9.
11. Use of a lentiviral vector according to claim 9 or a product according to claim 10 in the field of gene therapy.
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| CN114317464A (en) * | 2021-12-27 | 2022-04-12 | 武汉汇研生物科技股份有限公司 | Separation and purification method of adeno-associated virus rAAV9 |
| CN115141813A (en) * | 2022-07-29 | 2022-10-04 | 深圳源兴基因技术有限公司 | Adenovirus purification method for efficiently removing residual proteins of host cells |
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| CN114317464A (en) * | 2021-12-27 | 2022-04-12 | 武汉汇研生物科技股份有限公司 | Separation and purification method of adeno-associated virus rAAV9 |
| CN115141813A (en) * | 2022-07-29 | 2022-10-04 | 深圳源兴基因技术有限公司 | Adenovirus purification method for efficiently removing residual proteins of host cells |
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