CN111876393A - Method for large-scale rapid production of high-purity high-activity lentiviral vector - 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 high-purity high-activity lentiviral 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 onto a Capto Core700 chromatographic column and collecting a first absorption peak; s5: and (4) carrying out ultrafiltration concentration and liquid change on the sample collected in the S4 to obtain the lentiviral vector. The method adopts a multi-mode composite layer phase separation technology mode, can obtain the high-purity and high-activity lentivirus vector by a one-step purification process, and meets the industrial use quality standard.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for rapidly producing a high-purity high-activity lentiviral vector in a large scale.
Background
The virus vector is widely applied to the three fields of cell therapy, gene therapy and virus vaccine, and has the function of introducing modified target genes into target cells to realize the expression of target proteins by the target cells so as to achieve the purpose of disease therapy. At present, gene delivery vehicles are diversified, including adenovirus, adeno-associated virus, lentivirus, retrovirus and the like, and the application of lentivirus vectors is very common, and particularly, the application in the fields of neuroscience, hematology, developmental biology, stem cell biology, transgenosis and the like is rapidly emerging.
With the increasing use of lentiviral vectors as clinical therapeutics, scalable commercial processes (especially purification) are being extensively studied and optimized to maximize the quality standards of the product. However, in large-scale production, the production process is not mature, and especially the downstream purification process is important for removing impurities such as host proteins, genomic DNA, BSA, nuclease, endotoxin and the like. The commonly used lentivirus vector purification process mainly comprises ultracentrifugation, density gradient centrifugation, PEG precipitation, ultrafiltration, tangential flow, ion exchange chromatography and other methods, and most of conventional applications, such as cell infection and common animal experiments, can be used by adopting the ultrafiltration and ultracentrifugation methods. Most of the commercial processes for preparing lentiviral vectors currently include ion exchange chromatography, affinity chromatography, and gel filtration chromatography, and the combination of the two purification processes is a relatively versatile combination. The disadvantage of molecular sieve chromatography is that the loading volume is very small, only 30% or less of the column volume can be loaded each time; the main disadvantages of ion exchange chromatography are that high salt elution is needed, and the slow virus is easy to inactivate, so that the recovery rate of the whole production process flow is low and is in the range of 10-20%.
Therefore, according to the existing industrial production capacity and quality standards, a new set of production process with the advantages of high recovery rate, high activity, high purity, process amplification, stable capacity and the like is urgently needed to meet the clinical research requirements.
Disclosure of Invention
The invention aims to provide a method for rapidly producing high-purity and high-activity lentivirus vectors in a large scale, which overcomes the defects of the prior art, adopts a mode of multi-mode composite chromatography technology, can obtain the high-purity and high-activity lentivirus vectors by one-step purification process, and meets the industrial use quality standard. The purification method provided by the invention is convenient and rapid to operate, low in cost and high in recovery efficiency, and can meet the requirements of industrial mass production.
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 onto a Capto Core700 chromatographic column and collecting a first absorption peak;
s5: and (4) carrying out ultrafiltration concentration and liquid change on the sample collected in the S4 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, and after step S5, 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, the S3 digested sample is applied to a Capto Core700 chromatography column and the first absorption peak is collected.
In some embodiments of the invention, a Capto Core700 (composite mode chromatography media) chromatography column is purchased from GE corporation.
In some embodiments of the invention, the nuclease digested sample is applied to a previously equilibrated Catpo Core700 column, the equilibration column buffer Tris-Cl is 10-100mM, NaCl is 10-300mM, and pH is 7-9, and the first elution peak, i.e., the elution peak containing the virus of interest, is collected.
Preferably, in S5, the ultrafiltration membrane has a molecular weight cut-off of 50-300 kD.
In some embodiments of the invention, the fluid exchange is followed by a sterilization step, i.e., filtration through a filter with a pore size of 0.22 μm, followed by rinsing with sterile 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.
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) at present, the commercial lentivirus vector purification process is a two-step or three-step purification process, and the purification process is mainly characterized by a one-step purification process and has the advantages of few operation steps, high treatment speed and the like compared with the commercial production process.
2) The quality of the virus vector of the production process flow and the activity titer of the final product are in the range of 1.0-9.0E +08TU/ml, and the detection of impurities such as BSA, HCP, HCD, nuclease and the like can reach the industrial use standard.
3) The purification process flow adopts multi-mode CaptoCore700 chromatography, which has the functions of hydrophobic adsorption, charged adsorption, exclusion of macromolecules of 700KDa and the like, and about 90 percent of common host protein, DNA fragments, endotoxin and nuclease can be removed by one-step purification; and the linear flow rate reaches more than 1000cm/h, and the back pressure does not exceed 3 bar. The recovery efficiency of the whole production process is over 29 percent, the relative cost is reduced by 10 to 20 percent, and the automatic production can be realized in the future direction.
Drawings
FIG. 1 is a chromatographic purification chromatogram of CaptoCore700 in an example of the present invention;
FIG. 2 is a diagram 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:
1) 300ml of collected lentivirus vector feed liquid with the activity titer of 4.50E +06TU/ml is filtered and clarified by using a 0.65 or 0.45 mu m filter PP 3;
2) 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.
3) 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 for 20 hours at the temperature of 2-8 ℃;
4) subjecting the sample obtained in step 3) to multimodal composite chromatography: and loading the sample subjected to nuclease digestion onto a Catpo Core700 chromatographic column which is pre-equilibrated, wherein the concentration of Tris-Cl in an equilibration chromatographic column buffer solution is 25m M, the concentration of NaCl is 150mM, the pH value is 7.5, and collecting a first elution peak, namely an elution peak containing the target virus, wherein the volume of the elution peak is 9.5 mL. The chromatographic column purification chromatogram of Catpo Core700 is shown in FIG. 1.
5) Concentrating the sample obtained in the step 4) by 9 times by using a membrane package or hollow fiber type ultrafiltration device, wherein the molecular weight cut-off of the ultrafiltration membrane is 50-300KD, and then replacing the sample into a virus preservation solution to obtain the volume of the lentivirus of 1.5m l;
6) the sample obtained in step 5) was filtered through a sterile filter with a pore size of 0.22 μm and rinsed with sterile PBS buffer, at which time the lentiviral vector volume was 1.7ml and the activity titer was 2.1E +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. 2 (the sampling volumes of the lentiviral vectors in the order from top to bottom in FIG. 2 are 10. mu.L, 1. mu.L and 0.1. mu.L, respectively).
The total recovery rate of the production flow of the lentivirus vector in the embodiment is 29 percent, and can meet the production requirement of GMP grade.
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:
1) 8000mL of collected slow virus vector feed liquid with the activity titer of 5.30E +06TU/mL is filtered and clarified by using a 0.65 or 0.45 mu m filter PP 3;
2) 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.
3) 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 for 20 hours at the temperature of 2-8 ℃;
4) subjecting the sample obtained in step 3) to multimodal composite chromatography: and loading the sample subjected to nuclease digestion onto a pre-equilibrated Catpo Core700 chromatographic column, wherein the concentration of Tris-Cl in an equilibration chromatographic column buffer solution is 25m M, the concentration of NaCl is 150mM, the pH value is 7.5, and collecting a first elution peak, namely an elution peak containing the target virus, wherein the volume of the elution peak is 912 mL.
5) Concentrating the sample obtained in the step 4) by 10 times by using a membrane-packed or hollow fiber type ultrafiltration device, wherein the molecular weight cut-off of the ultrafiltration membrane is 50-300KD, and then replacing the sample into a virus preservation solution to obtain 86ml of lentivirus volume;
6) the sample obtained in step 5) was filtered through a sterile filter with a pore size of 0.22 μm and rinsed with sterile PBS buffer, at which time the lentiviral vector had a volume of 88.6ml and an activity titer of 1.65E +08 TU/ml.
The total recovery rate of the lentiviral vector production flow of this example was 34.47%, which can meet the GMP-level production requirements.
The purity detection results of the virus vectors obtained in the example 1 and the example 2 are shown in the table 1, and both meet the industrial use quality standard.
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 (10)
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 onto a Capto Core700 chromatographic column and collecting a first absorption peak;
s5: and (4) carrying out ultrafiltration concentration and liquid change on the sample collected in the S4 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: reacting at 2-8 deg.c for 12-24 hr.
5. The method of claim 1, wherein in S4, the digested sample is loaded onto a pre-equilibrated Capto Core700 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 according to claim 5, 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.
7. The method of claim 1, wherein in S5, the ultrafiltration membrane has a molecular weight cut-off of 50-300 KD.
8. A lentiviral vector obtainable by the method of any one of claims 1 to 7.
9. A product comprising the lentiviral vector of claim 8.
10. Use of a lentiviral vector according to claim 8 or a product according to claim 9 in the field of gene therapy.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112899242A (en) * | 2021-02-03 | 2021-06-04 | 苏州博腾生物制药有限公司 | Lentiviral purification process |
| CN113373120A (en) * | 2021-06-18 | 2021-09-10 | 浙江康佰裕生物科技有限公司 | Purification method and application of GMP-grade retrovirus vector |
| CN113817689A (en) * | 2021-11-22 | 2021-12-21 | 北京艺妙神州医药科技有限公司 | Lentiviral purification process |
| CN113980916A (en) * | 2021-09-27 | 2022-01-28 | 湖南丰晖生物科技有限公司 | Method for purifying lentivirus |
| CN114181970A (en) * | 2020-09-15 | 2022-03-15 | 上海药明巨诺生物医药研发有限公司 | A kind of lentiviral vector purification method |
| CN116240241A (en) * | 2022-12-30 | 2023-06-09 | 上海本导基因技术有限公司 | Purification and preparation method of recombinant lentiviral vector |
| CN117025684A (en) * | 2023-09-19 | 2023-11-10 | 北京青言韦辰生物科技有限公司 | Method for purifying lentiviral vector comprising cationic chromatography |
| CN118421573A (en) * | 2024-07-02 | 2024-08-02 | 深圳源兴基因技术有限公司 | A vaccinia virus purification process for efficiently removing HCD residues |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170002332A1 (en) * | 2013-12-17 | 2017-01-05 | Genethon | Method for purifying enveloped viruses or viral vectors |
| CN107384877A (en) * | 2017-08-18 | 2017-11-24 | 深圳源兴基因技术有限公司 | A kind of purification process of slow virus |
| CN107630037A (en) * | 2017-10-19 | 2018-01-26 | 和元生物技术（上海）股份有限公司 | A kind of purifying process for obtaining high-purity gland relevant viral vector |
| CN110317832A (en) * | 2018-03-28 | 2019-10-11 | 上海赛比曼生物科技有限公司 | The method of the pure preparations of GMP grades of prepare with scale recombined lentivirus vectors |
-
2020
- 2020-06-30 CN CN202010616404.9A patent/CN111876393A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170002332A1 (en) * | 2013-12-17 | 2017-01-05 | Genethon | Method for purifying enveloped viruses or viral vectors |
| CN107384877A (en) * | 2017-08-18 | 2017-11-24 | 深圳源兴基因技术有限公司 | A kind of purification process of slow virus |
| CN107630037A (en) * | 2017-10-19 | 2018-01-26 | 和元生物技术（上海）股份有限公司 | A kind of purifying process for obtaining high-purity gland relevant viral vector |
| CN110317832A (en) * | 2018-03-28 | 2019-10-11 | 上海赛比曼生物科技有限公司 | The method of the pure preparations of GMP grades of prepare with scale recombined lentivirus vectors |
Non-Patent Citations (2)
| Title |
|---|
| 王晓娜;肖凤君;吴彬;吴祖泽;毕建进;张以芳;: "重组腺病毒Ad-GFP的纯化" * |
| 聂蓓娜;许晨光;万晓春;: "科研级嵌合抗原受体慢病毒浓缩方法对比研究" * |
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| CN113817689A (en) * | 2021-11-22 | 2021-12-21 | 北京艺妙神州医药科技有限公司 | Lentiviral purification process |
| CN116240241A (en) * | 2022-12-30 | 2023-06-09 | 上海本导基因技术有限公司 | Purification and preparation method of recombinant lentiviral vector |
| CN117025684A (en) * | 2023-09-19 | 2023-11-10 | 北京青言韦辰生物科技有限公司 | Method for purifying lentiviral vector comprising cationic chromatography |
| CN117025684B (en) * | 2023-09-19 | 2024-08-27 | 北京青言韦辰生物科技有限公司 | Method for purifying lentiviral vector comprising cationic chromatography |
| CN118421573A (en) * | 2024-07-02 | 2024-08-02 | 深圳源兴基因技术有限公司 | A vaccinia virus purification process for efficiently removing HCD residues |
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