CN116590346A - Lentiviral vector purification method - Google Patents
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Classifications
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- 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
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- 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
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- 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
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15051—Methods of production or purification of viral material
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application relates to the technical field of virus vector purification, and provides a lentiviral vector purification method which comprises the following steps: the method comprises the following steps of 1), filtering a lentivirus harvest liquid and incubating nuclease to obtain a lentivirus vector clarified harvest liquid; 2) And carrying out anion exchange chromatography and/or compound mode chromatography treatment on the lentiviral vector clarified harvest liquid to obtain a purified lentiviral vector sample. The lentiviral vector purification method provided by the application has the advantages of high lentiviral vector yield, low impurity residues such as cell host protein, host DNA, residual plasmid and the like, good safety, suitability for large-scale production and preparation, and capability of meeting the use requirements of cell therapy and gene therapy in clinic.
Description
The application is a divisional application of the original application, and the application date of the original application is as follows: 2020.09.15; the application number is: CN202010967219.4; the application is named as follows: a method for purifying lentiviral vector.
Technical Field
The application relates to the technical field of virus vector purification, in particular to a lentivirus vector purification method.
Background
In recent years, cell therapy typified by chimeric antigen receptor-modified T cells (CAR-T) and gene therapy based on gene transfer vectors have been increasingly used in clinical applications for the treatment of human diseases. Among them, one of the commonly used viral vectors is a lentiviral vector. The slow virus vector is a virus vector system modified based on HIV-1 virus, and can efficiently introduce target genes into primary cells or cell lines of animals and humans. Lentiviral vector mediated gene expression can be used to achieve continuous and stable protein production, as the gene of interest has been integrated into the genome of the host cell and replicated upon cell division. In addition, lentiviral vectors can be used to effectively infect non-dividing cells as well as those cells that are actively dividing cells, and are used in a wider range than other viral vectors.
Mature lentiviral particles were obtained by co-transfecting 293-related cells with a plurality of plasmid vectors, then packaged in cells and secreted into the extracellular culture supernatant. The laboratory generally prepares high-concentration lentiviral vector by ultracentrifugation, the method is simple but cannot be used for industrial amplification, and the prepared lentiviral vector may have high residues of endotoxin, BSA, HCP or nucleic acid and the like and cannot be directly used for human bodies. The documents "Large-Scale Production Means for the Manufacturing of Lentiviral Vectors" and "New developments in lentiviral vector design, production and purification" review that the existing chromatography method for producing lentiviral vectors mainly adopts Q membrane chromatography, CIM monolithic column or molecular sieve, and the like, and generally has the defects of high production cost, lower yield, complicated steps and the like, so that the method is difficult to meet the requirements of industrial mass production.
Therefore, there is an urgent need in the art to develop a lentiviral vector purification method that is efficient, low cost, suitable for mass production and meets GMP regulations.
Disclosure of Invention
In view of the above drawbacks of the prior art, the present application is directed to a lentiviral vector purification method, which is used for solving the problems of high production cost, low yield, complicated steps and the like in the prior art.
To achieve the above and other related objects, the present application provides a lentiviral vector purification method comprising the steps of:
1) Filtering the slow virus harvest liquid and incubating the slow virus harvest liquid with nuclease to obtain a clear slow virus vector harvest liquid;
2) Carrying out anion exchange chromatography and/or compound mode chromatography treatment on the lentiviral vector clarified harvest liquid to obtain a purified lentiviral vector sample;
optionally, the lentivirus harvest liquid is filtered by adopting a deep layer filtering membrane or a dead end filtering membrane with the pore diameter of 0.45um-1um in the step 1).
Optionally, said lentiviral harvest is incubated in step 1) with nuclease at a concentration of 5U-50U/ml and magnesium sulfate at a concentration of 0.8-2.0 mmol/L.
Optionally, the particle size of the anion exchange chromatography packing used in step 2) is greater than 60nm.
Optionally, in the step 2), one or more of the following features are further included:
the composite mode chromatography adopts a flow-through mode;
the composite mode chromatography uses Capto core 700 or Capto core 400 packing;
the loading amount of the composite mode chromatography is 2-10CV, and the preferable loading amount is 2-5CV.
As described above, the lentiviral vector purification method of the application has the following beneficial effects: the method has the advantages of simple operation, easy amplification, stable process, good repeatability, high yield of the prepared lentiviral vector, low residual impurities such as cell host protein, host DNA, residual plasmid and the like, and good safety. The lentiviral vector purification method provided by the application is suitable for large-scale production and meets the GMP regulation requirement, and the prepared lentiviral vector meets the use requirements of clinical cell therapy and gene therapy.
Drawings
FIG. 1 shows an ultraviolet absorbance graph of the purification process of the application.
Detailed Description
The application provides a lentiviral vector purification method, which comprises the following steps: the method comprises the following steps:
1) Filtering the slow virus harvest liquid and incubating the slow virus harvest liquid with nuclease to obtain a clear slow virus vector harvest liquid;
2) And carrying out anion exchange chromatography and/or compound mode chromatography treatment on the lentiviral vector clarified harvest liquid to obtain a purified lentiviral vector sample.
The application provides a lentiviral vector purification method, which comprises the following steps of 1) filtering a lentiviral harvest liquid and incubating nuclease to obtain a lentiviral vector clarified harvest liquid. The method comprises the steps of filtering the slow virus harvest liquid by using a deep filtration membrane or a dead-end filtration membrane, incubating the slow virus harvest liquid by using nuclease and magnesium sulfate for 4-20h, and reducing the content of host cell residual DNA, residual plasmid and the like in the slow virus harvest liquid to obtain a clear slow virus vector harvest liquid, wherein the nuclease incubation can be performed before filtration or after filtration.
The lentivirus harvest liquid is prepared by a method comprising the following steps: the lentivirus is packaged and produced by using different plasmid systems and corresponding culture methods, wherein three plasmids or four plasmids can be used, the culture medium can be a serum-free or serum-containing culture medium, the culture mode can be suspension or wall-attached culture, and a culture vessel can be a culture bottle, a cell factory or a bioreactor according to requirements.
The steps are the packaging and culturing process of the slow virus vector, and the slow virus vector can be cultured by referring to the documents such as Production of cGMP-Grade Lentiviral Vectors or Optimization of lentiviral vector production for scale-up in fixed-bed biological reactor.
The application provides a lentiviral vector purification method, which comprises the following steps of 2) carrying out anion exchange chromatography and/or compound mode chromatography treatment on a lentiviral vector clarified harvest liquid to obtain a purified lentiviral vector sample. The method comprises initially purifying the lentiviral vector clarified harvest using anion exchange chromatography, the anion exchange chromatography packing including but not limited to the following: unigel series (Unigel-30 DEAE, unigel-30Q, uniGel-80DEAE and Unigel-80Q) and Nano series (NanoQ-10L, nanoQ-15L and NanoQ-30L) both of which have a pore diameter of 100nm, manufactured by Sony micro-technology Co., ltd; superQ-650C, DEAE-650C, Q-600C AR, gigaCap Q-650M, gigaCap DEAE-650M, superQ-650M, DEAE-650M, superQ-650S, DEAE-650S and GigaCap Q-650S manufactured by Tosoh Corp, japan, wherein the Q-600C AR pore diameter is 75nm and the other pore diameters are 100nm; and POROS XQ manufactured by ThermoFisher corporation, pore size of 110nm, POROS HQ50, pore size of 200nm, POROS PI50, pore size of 200nm and POROS D50, pore size of 105nm. Anion exchange chromatography involves the following operations: balancing the chromatographic column by using a buffer solution with the pH value of between 7.0 and 7.7 and the concentration of between 50 and 200mmol/L NaCl and with the pH value of between 3 and 10 CV; and then loading samples, wherein the loading flow rate is 100-300cm/h, flushing the chromatographic column by using balance liquid until the ultraviolet absorption is reduced to be close to a base line after loading, eluting by using an eluent with the pH value of 7.0-7.7 and the concentration of 0.5-1mol/L NaCl, observing the ultraviolet absorption, collecting a target peak, and immediately diluting by using a diluent which is precooled to the pH value of 7.0-7.7 and does not contain NaCl at the temperature of 2-8 ℃ to ensure that the conductance of the diluted lentiviral vector sample is 5-20 ms/cm.
The method also comprises the step of purifying the lentivirus purified solution by using a composite mode chromatography, wherein the selected filler for the composite mode chromatography is Capto core 700 or Capto core 400. The composite mode chromatography adopts a flow-through mode, firstly, a buffer solution with the pH value of 7.0-7.7 and the concentration of 50-200mmol/L NaCl of 3-10CV is used for balancing the chromatographic column, then, loading is carried out, the loading flow rate is 100-400cm/h, and after loading is finished, the chromatographic column is flushed by using the balancing solution until the ultraviolet absorption is reduced to be close to a base line. The UV absorption was observed during loading and washing, and the target peaks were collected.
Further to be described is: the complex mode chromatography purification can be performed after or before anion exchange chromatography. In addition, according to the content difference of impurities in the lentiviral vector clarified harvest liquid and different quality standards of the final product, the whole purification process can be flexibly selected to carry out or not carry out the composite mode chromatography purification operation. Preferably, anion exchange chromatography and compound mode chromatography are used simultaneously, and the chromatography mode has the best effect on removing impurities, so that the content of impurities in a lentiviral vector sample can be greatly reduced.
In one embodiment, the lentivirus harvest liquid is filtered in step 1) using a deep filtration membrane or a dead-end filtration membrane pack with a pore size of 0.45um to 1um. The implementation personnel can select the aperture of dead-end filtration membrane according to the demand, and the aperture of selection can be 0.45um, 0.5um, 0.65um, 0.8um and 1um. The adoption of the deep layer filtering membrane or the dead end filtering membrane can obviously reduce host cell proteins, residual DNA and the like in the virus liquid.
In one embodiment, the step 1) uses nuclease with a concentration of 5U-50U/ml and magnesium sulfate with a concentration of 0.8-2.0mmol/L to incubate the lentivirus harvest. The practitioner can select the concentration of nuclease, the concentration of magnesium sulfate and the incubation time as desired, and the concentration of nuclease can be 5U-10U/ml, 10U-15U/ml, 15U-20U/ml, 20U-25U/ml, 25U-30U/ml, 30U-35U/ml, 35U-40U/ml, 40-45U/ml and 45U-50U/ml. The concentration of magnesium sulfate may be 0.8-0.9mmol/L, 0.9-1mmol/L, 1-1.1mmol/L, 1.1-1.2mmol/L, 1.2-1.3mmol/L, 1.3-1.4mmol/L, 1.4-1.5mmol/L, 1.5-1.6mmol/L, 1.6-1.7mmol/L, 1.7-1.8mmol/L, 1.8-1.9mmol/L, and 1.9-2.0mmol/L. The incubation conditions may be based on the recommended conditions for the nuclease product, and may be, for example, incubation at low temperature for a long period of time, for example, incubation at 2-8℃for 17-18 hours as specifically exemplified in the examples, or incubation at physiological temperature for a suitable period of time.
In one embodiment, the anion exchange chromatography microparticles used in step 2) have a pore size greater than 60nm. The practitioner can choose the anion exchange chromatography particle size, for example, 75nm, 100nm, 105nm, 110nm, and 200nm, and the particle size can be in a range, for example: 60nm-75nm, 75nm-100nm, 100nm-105nm, 105nm-110nm, 110nm-200nm, etc. Compared with the traditional small-aperture particle filler, the particle filler with large aperture has larger adsorption capacity and higher elution recovery rate for lentivirus, and is more suitable for preparing lentivirus vectors.
In an embodiment, in the step 2), one or more of the following features are further included:
the composite mode chromatography adopts a flow-through mode, and uses Capto core 700 or Capto core 400 filler. Capto Core 700 and Capto Core 400 are composite mode chromatographic packing with gel filtration chromatography and anion exchange chromatography. The particles are provided with core octylamine ligand and an inert shell layer, the inert shell layer can exclude macromolecules from entering the particles, and smaller impurities can enter the core and ligand to be combined through small holes (the exclusion molecular weight is 700KD or 400 KD) on the shell layer, so that a flow-through mode is adopted in the slow virus vector purification.
The sample loading amount of the composite mode chromatography is preferably 2-10CV, the operator can select the sample loading amount according to the requirement, the sample loading amount can be 2-3CV, 3-4CV, 4-5CV, 5-6CV, 6-7CV, 7-8CV, 8-9CV and 9-10CV, and the preferred sample loading amount is 2-5CV. When the loading amount is 2CV to 10CV, the infection titer yield of the lentiviral vector is above 85 percent; when the loading amount is increased, the removal rate of residual host protein and host DNA is gradually reduced, and the removal rate can reach more than 45%. The yield and impurity removal conditions are comprehensively considered, the loading amount in the determining process can be 2-10CV, and further, the optimal loading amount is 2-5CV.
The application provides a lentiviral vector purification method, which further comprises the following steps: and 3) concentrating the lentiviral vector sample and replacing a buffer system in the lentiviral vector sample to a preparation buffer solution to obtain the lentiviral vector.
In one embodiment, the viral vector sample is concentrated using an ultrafiltration membrane pack or hollow fiber membrane column and the buffer system in the lentiviral vector sample is replaced to formulation buffer. Lentiviral vector products are typically stored in a refrigerator at-80 ℃ for long periods of time, and suitable formulation buffers are required to maintain their infectious activity to a maximum extent, reducing the loss of infectious activity during freezing, thawing, etc.
In an embodiment, the pore sizes of the ultrafiltration membrane package and the hollow fiber membrane column are 100-1000KD, and the implementation personnel can select the pore sizes of the ultrafiltration membrane package and the hollow fiber membrane column according to the requirement, wherein the selected pore sizes can be 100KD, 300KD, 500KD, 750KD and 1000KD. The preferred pore diameters are 500KD, 750KD and 1000KD, which have larger pore diameters than those of 100KD and 300KD, and the impurity removing effect is better.
In an embodiment, said steps 1) to 3) are performed in a sterile production environment employing a fully closed operation or in a clean grade class a zone when open operation is involved; the A-level region in the application is required to meet the relevant regulations of a clean area required by the production of a ninth sterile drug in the annex of a drug production quality management Specification (revised 2010).
And/or, the method further comprises: and (3) performing sterilization filtration on the lentiviral vector.
In an embodiment, the sterilization filter with double-layer combined aperture is used, and an operator can select the double-layer combined aperture according to the requirement, for example, the combination with the aperture of 0.45um+0.22um, the combination with the aperture of 0.5um+0.22um, the combination with the aperture of 0.65um+0.22um and the combination with the aperture of 0.8um+0.22um, and the sterilization filter with the double-layer combined aperture has a loading capacity larger than that of the single-layer sterilization filter, and the yield is high.
In order to make the objects, technical solutions and advantageous technical effects of the present application more clear, the present application is described in further detail below with reference to examples. However, it should be understood that the examples of the present application are merely for the purpose of explaining the present application and are not intended to limit the present application, and the examples of the present application are not limited to the examples given in the specification. The specific experimental or operating conditions were not noted in the examples and were made under conventional conditions or under conditions recommended by the material suppliers.
Example 1
The large pore size anion exchange chromatography packing GigaCap DEAE 650M was compared with packing a for lentiviral vector purification performance. Wherein the GigaCap DEAE 650M filler is an anion exchange chromatography filler with a particle size of 100nm of TOSOH company; the filler A is an anion exchange chromatography filler of a commercial agarose gel framework of a certain brand, and the particle diameter is 28nm.
(1) Preparation of lentivirus harvest: and (3) carrying out serial passage expansion culture after resuscitating by using the 293T cells frozen in a liquid nitrogen tank, and finally carrying out passage to the Hyper flash by adopting an adherence culture mode. The four plasmids are mixed with a transfection reagent according to a certain proportion, and then cells are transfected. The supernatant was harvested 48h after transfection for a total of about 560ml.
(2) The lentivirus harvest was filtered and nuclease incubated: using a pore size of 0.6um and a membrane area of 17.5cm 2 The harvest was filtered by a bag filter. After completion of filtration, merck Benzonase was added to a final concentration of 5U/ml, and magnesium sulfate was added to a final concentration of 1.5mmol/L, and the mixture was left to stand in a refrigerator at 2-8℃overnight (18 h) for cultivation. The total amount of virus supernatant after filtration was 558ml.
(3) And (3) carrying out anion exchange chromatography on the lentiviral vector clarified harvest liquid: a chromatography column is filled with two types of fillers, namely GigaCap DEAE 650M and filler A, the diameter of the chromatography column is 0.66cm, the filling height is 8.4cm, and the filler volume is 2.874ml. The nuclease treated virus supernatant was divided into two portions, each portion was 279ml, and each portion was loaded with two prepared chromatographic columns in sequence, and the flow rate was set at 210cm/h. The chromatographic column is first equilibrated with 3CV of NaCl buffer solution with pH value of 7.2 and concentration of 100 mmol/L; and (3) loading after balancing, wherein the change of the flow-through UV280 is noticed in the loading process, and the flow-through is collected when the ultraviolet absorption begins to rise. And after loading, flushing the chromatographic column by using the balance liquid until the ultraviolet absorption reduction value is close to a base line, and ending collecting the fluid. Finally, eluting with an eluent with pH value of 7.2 and NaCl concentration of 0.8mol/L, wherein the eluting flow rate is 210cm/h, observing UV280 absorption, collecting target peaks, and immediately diluting with a diluent which is precooled to 2-8 ℃ and has pH value of 7.2 and does not contain NaCl, wherein the volume of the diluent is about 4 times of that of the diluent. In the loading process, a Beijing Boolong company lentivirus rapid titer detection card is used for detecting the flow-through liquid, and the fact that the physical particle flow-through of the lentivirus vector occurs in the packing A with the loading volume of about 200ml is found, so that the loading of 240ml of the final packing A chromatographic column is completed.
(5) And (3) detection: the collected flow-through liquid and the eluent are sent to a detection unit physical titer (p 24), the flow-through condition of the two fillers is observed, and the adsorption capacity is calculated.
(6) Test results and analysis: the specific test results are shown in the following table:
total physical titer of sample = physical titer of sample volume x sample units;
total filler adsorption = total physical titer of loading fluid-total physical titer of flow through fluid;
filler unit adsorption amount = filler total adsorption amount/filler volume;
elution yield = total physical titer of eluent/total adsorbed amount of filler x 100%.
Lentiviral vector titer detection methods are mainly divided into two types, infection titer and physical titer. Infection titer is lentiviral vector particle with infectious activity; physical titres are the content of p24 protein, which contains both infectious active lentiviral vector particles and non-infectious active lentiviral vector fragments. In lentiviral vector harvest, which contains a large amount of non-infectious active lentiviral vector fragments, it is also adsorbed on the chromatographic packing during anion exchange chromatography. Therefore, in order to compare the adsorption performance of the two types of anion exchange chromatography packing, it was examined using physical titer as an index.
As can be seen from the table, during the loading process, the p24 particles flow through both types of fillers, but the GigaCap DEAE 650M chromatographic column flows through less when the loading amount is larger, and the final result shows that the unit adsorption amount of the lentiviral vector GigaCap DEAE 650M filler is 63.3ug/ml and the unit adsorption amount of the filler A is 37.77ug/ml. The unit adsorption amount of the GigaCap DEAE 650M filler lentiviral vector is 67.59% higher than that of filler A.
In the case of lentiviral vectors adsorbed on the packing, the packing elution yield of GigaCap DEAE 650M was found to be 58.79% and that of packing A was found to be only 31.56% under the same high salt elution conditions. The elution yield of the GigaCap DEAE 650M filler lentiviral vector is 86.28% higher than that of filler A.
In summary, it can be seen that, for the purification of lentiviral vectors, the GigaCap DEAE 650M packing with large pore size has a larger adsorption capacity and higher elution recovery rate than the conventional small pore size anion exchange packing, and is more suitable for the preparation of lentiviral vectors.
Example 2
The yield and impurity removal capacity of the different loading amounts of the Capto Core 700 chromatographic packing purified lentiviral vector were examined and compared by design-related experiments.
(1) Preparation of lentivirus harvest: and (3) carrying out serial passage expansion culture after resuscitating by using the 293T cells frozen in the liquid nitrogen tank, and finally carrying out passage to a 10-layer cell culture factory by adopting an adherent culture mode. The four plasmids are mixed with a transfection reagent according to a certain proportion, and then cells are transfected. The supernatant was harvested 48h after transfection and was 959ml in total.
(2) The lentivirus harvest was filtered and nuclease incubated: the pore diameter was 0.8um and the membrane area was 17.5cm 2 Is clarified and filtered. After completion of the filtration, merck Benzonase was added to a final concentration of 20U/ml, and magnesium sulfate was added to a final concentration of 0.8mmol/L, and incubated at 2-8deg.C for about 17 hours.
(3) And (3) carrying out anion exchange chromatography on the lentiviral vector clarified harvest liquid: further purification was performed using Unigel-80DEAE packing material from Suzhou Nami, the pore size of Unigel-80DEAE being 100nm. The XK16/20 column was used and the column was loaded with a volume of about 20ml. Firstly, balancing the chromatographic column by using 5CV of NaCl buffer solution with the pH value of 7.0 and 200 mmol/L; and (3) loading samples after balancing, wherein the loading flow rate is 210cm/h, flushing the chromatographic column by using balancing liquid until the ultraviolet absorption is reduced to be close to a base line after loading, eluting by using an eluent with the pH value of 7.0 and the concentration of 0.5mol/L NaCl, observing the ultraviolet absorption at the eluting flow rate of 210cm/h, collecting a target peak, and immediately diluting by using a diluent which is precooled to 2-8 ℃ and has the pH value of 7.0 and does not contain NaCl, wherein the volume of the diluent is 2 times. After the elution was completed, 97ml of a sample of the initially pure lentiviral vector was obtained.
(4) And (3) carrying out composite mode chromatography on the initially pure lentiviral vector sample obtained in the step (3): lentiviral vector samples were further purified using Capto Core 700 chromatographic packing. The diameter of the loaded chromatographic column is 0.66cm, the height of the column is 14cm, the packing volume is 4.79ml, and the flow rate of the chromatographic process is 180cm/h. The column was first equilibrated with a pH of 7.0, 150mmol/L NaCl buffer for a total of 10CV. After the equilibration is completed, the sample of the step (3) is used for loading, and 3 experiments are carried out, wherein each experiment sequentially loads 2CV, 5CV and 10CV. After loading was completed, the column was washed with equilibration solution until uv absorbance decreased to near baseline. Ultraviolet absorption was observed during loading and washing, and flow through peaks were collected. Between each run, CIP was performed using 4CV of 0.5mol/LNaOH solution, followed by washing the column with pH7.0, 1mol/L NaCl solution until the effluent pH was neutral.
(5) And (3) detection: 3 test samples were sampled to detect lentiviral vector infection titer, residual host protein concentration and residual host DNA concentration, and the effect of different loading volumes on Capto Core 700 column chromatography performance was examined.
(6) Test results: specific test results are shown in the following table:
wherein the Unigel-80DEAE eluted sample is Capto Core 700 chromatographic loading liquid.
Infection titer yield = (fluid flow volume, fluid flow per infection titer)/(fluid loading volume, fluid flow per infection titer) x 100%.
Residual host protein removal = (loading fluid volume, residual host protein concentration in loading fluid-flow-through fluid volume, x) concentration of residual host protein in fluid)/(loading fluid volume, residual host protein in fluid) is 100% x.
Residual host DNA removal = (concentration of residual host DNA in the feed-fluid volume, concentration of residual host DNA in the flow-through fluid)/(concentration of residual host DNA in the feed-fluid volume, x) x 100%.
As can be seen from the above table, for Capto Core 700 chromatography, when the loading amount is 2CV to 10CV, the lentiviral vector infection titer yield is above 85%; when the loading amount is increased, the removal rate of residual host proteins and host DNA gradually decreases, but the removal rate can reach more than 45%. The yield and the impurity removal condition are comprehensively considered, the loading amount of the Capto Core 700 in the chromatographic process can reach 2-10CV, and the optimal loading amount is 2-5CV.
Example 3
In this example, 1L-scale lentiviral vector harvest was purified as follows:
(1) Preparation of lentivirus harvest: and (3) carrying out serial passage expansion culture after resuscitating by using the 293T cells frozen in the liquid nitrogen tank, and finally carrying out passage to a 10-layer cell culture factory by adopting an adherent culture mode. The four plasmids are mixed with a transfection reagent according to a certain proportion, and then cells are transfected. The supernatant was harvested 72h after transfection and amounted to about 965ml.
(2) Clear filtration and nuclease incubation of the lentivirus harvest: using a pore size of 1um+0.45um and a membrane area of 150cm 2 The harvest was filtered by a bag filter. After completion of the filtration, merck Benzonase was added to a final concentration of 50U/ml, and the mixture was incubated in a water bath at 37℃for 4 hours at a final concentration of 1mmol/L magnesium sulfate.
(3) And (3) carrying out anion exchange chromatography on the lentiviral vector clarified harvest liquid: purification was carried out using a POROS PI50 anion exchange chromatography packing from Thermofisher, the pore size of POROS PI50 being 200nm. The column was loaded with a volume of about 20ml using a GE company XK16/20 chromatography column. The chromatographic column is first equilibrated with 5CV of NaCl buffer having a pH of 7.5 and a concentration of 75 mmol/L; and (3) loading after the balance is finished, wherein the loading flow rate is 150cm/h, continuing to wash the chromatographic column by using balance liquid until the ultraviolet absorption reduction value is close to a base line after the loading is finished, eluting by using eluent with the pH value of 7.6 and the concentration of 1mol/L NaCl, observing the ultraviolet absorption at the elution flow rate of 150cm/h, collecting a target peak, and immediately diluting by using diluent which is precooled to 2-8 ℃ and has the pH value of 7.6 and does not contain NaCl by 9 times of volume. The lentiviral vector sample after elution was 210ml. As shown in fig. 1, peak1 is the flow through Peak during loading; peak2 is the target Peak, containing lentiviral vector sample; peak3 is an impurity Peak obtained by washing the column with 0.5mol/L sodium hydroxide solution after completion of chromatography.
(4) Concentrating a lentiviral vector sample and displacing the buffer system in the lentiviral vector sample to a formulation buffer: ultrafiltration concentration was performed using an ultrafiltration membrane pack of Merck Millipore company with a pore size of 500KD, after concentration to about 20ml, the final preparation buffer was used to change the solution, the solution was continuously changed by 5 times of volume, and after completion, the ultrafiltration membrane pack was emptied to obtain a lentiviral vector with a volume of 18 ml.
(5) Sterilizing and filtering the lentiviral vector: filtration was performed under a biosafety cabinet using Sartorius company Sartopore 2XLG sterilization filter. The aperture of the filtering membrane is 0.8um+0.2um, and the area is 4.5cm 2 . The flow rate was set at 3ml/min during filtration and the filtration pressure was monitored, the maximum pressure not exceeding 1.5bar. After filtration was completed, the filter was rinsed with about 3ml of formulation buffer. Finally, the lentiviral vector product is obtained, and 16ml of lentiviral vector product is divided into 1 ml/tube and packaged in a freezing tube for storage in a refrigerator at the temperature of minus 80 ℃.
(6) Sample feeding detection: and (3) carrying out sample feeding detection on a lentiviral vector intermediate process sample and a final product, namely detecting unit infection titer, unit physical titer (p 24), HCP, BSA, residual host DNA, residual nuclease, sterility, RCL (replication type lentivirus) and the like, calculating the total process yield and inspecting the quality of the final product.
(7) Detection result: the results showed 44.95% overall yield of infectious titer and 60.02% overall yield of physical titer for the whole purification process, with specific results shown in the following table.
Total infectious titer of sample = infectious titer in units of sample volume (ml) (IU/ml),
infection titer yield = final product total infection titer/harvest total infection titer x 100%;
total physical titer of sample = physical titer in units of sample volume (ml) (pg/ml),
physical titer yield = total physical titer of the final product/total physical titer of the harvest x 100%.
The impurity item results show that HCP <100ng/ml, BSA <100ng/ml, residual host DNA <40ng/ml, residual nuclease <0.25ng/ml, sterile detection negative, RCL detection negative all meet the quality standard requirements, and meet the use requirements of clinical cell therapy and gene therapy.
In summary, the present application effectively overcomes the disadvantages of the prior art and has high industrial utility value.
The above embodiments are merely illustrative of the principles of the present application and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all equivalent modifications and variations of the application be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. A method for purifying a lentiviral vector, comprising the steps of:
1) Filtering the slow virus harvest liquid and incubating the slow virus harvest liquid with nuclease to obtain a clear slow virus vector harvest liquid;
2) Anion exchange chromatography is adopted for the lentiviral vector clarified harvest liquid to obtain a purified lentiviral vector sample;
the anion exchange chromatography filler used in step 2) has a particle size of 75nm to 100nm and does not contain 100nm.
2. The method according to claim 1, wherein the lentivirus harvest is filtered by using a deep filtration membrane or a dead-end filtration membrane pack with a pore size of 0.45 μm to 1 μm in step 1).
3. The method of claim 1, wherein the step 1) is performed by incubating the lentivirus harvest with nuclease at a concentration of 5U-50U/ml and magnesium sulfate at a concentration of 0.8-2.0 mmol/L.
4. A lentiviral vector purification method according to claim 1, wherein step 2) comprises a compound mode chromatographic treatment and comprises one or more of the following features:
the composite mode chromatography adopts a flow-through mode;
the composite mode chromatography uses Capto core 700 or Capto core 400 packing;
the loading amount of the composite mode chromatography is 2-10CV.
5. The method of claim 4, wherein the loading of the compound-mode chromatography is 2-5CV.
6. The method of claim 1, further comprising: and 3) concentrating the lentiviral vector sample and replacing a buffer system in the lentiviral vector sample to a preparation buffer solution to obtain the lentiviral vector.
7. The method for purifying lentiviral vector of claim 6, wherein: in the step 3), the lentiviral vector sample is concentrated through an ultrafiltration membrane bag or a hollow fiber membrane column and a buffer system in the lentiviral vector sample is replaced to a preparation buffer solution.
8. The method for purifying lentiviral vector of claim 6, wherein: the aperture of the ultrafiltration membrane bag and the hollow fiber membrane column is 100-1000KD.
9. The method for purifying lentiviral vector of claim 6, wherein: said steps 1) to 3) are carried out in a sterile production environment employing a totally closed operation or in a clean grade class a zone when open operation is involved; the method further comprises the steps of: and (3) performing sterilization filtration on the lentiviral vector.
10. The method for purifying lentiviral vector according to claim 9, wherein: the sterilization filter is a double-layer sterilization filter with combined pore diameters.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003039459A2 (en) * | 2001-11-05 | 2003-05-15 | Genvec, Inc. | Viral vector production methods and compositions |
| CN102740881A (en) * | 2009-05-12 | 2012-10-17 | 特兰斯吉恩股份有限公司 | Method for orthopoxvirus production and purification |
| CN104371982A (en) * | 2014-10-21 | 2015-02-25 | 武汉维诺赛生物技术有限公司 | Method for purifying slow virus |
| US20170002332A1 (en) * | 2013-12-17 | 2017-01-05 | Genethon | Method for purifying enveloped viruses or viral vectors |
| US20180051275A1 (en) * | 2016-08-17 | 2018-02-22 | Glaxosmithkline Intellectual Property Development Limited | Method for purifying viral vectors |
| 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 |
| CN111876393A (en) * | 2020-06-30 | 2020-11-03 | 恒瑞源正（上海）生物科技有限公司 | Method for large-scale rapid production of high-purity high-activity lentiviral vector |
| CN111876392A (en) * | 2020-06-30 | 2020-11-03 | 恒瑞源正（上海）生物科技有限公司 | Method for large-scale rapid production of viral vectors |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2737730B1 (en) * | 1995-08-10 | 1997-09-05 | Pasteur Merieux Serums Vacc | PROCESS FOR PURIFYING VIRUSES BY CHROMATOGRAPHY |
| WO2007133674A2 (en) * | 2006-05-12 | 2007-11-22 | Lentigen Corporation | Lentiviral vector compositions, methods and applications |
-
2020
- 2020-09-15 CN CN202010967219.4A patent/CN114181970B/en active Active
- 2020-09-15 CN CN202310800890.3A patent/CN116590346A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003039459A2 (en) * | 2001-11-05 | 2003-05-15 | Genvec, Inc. | Viral vector production methods and compositions |
| CN102740881A (en) * | 2009-05-12 | 2012-10-17 | 特兰斯吉恩股份有限公司 | Method for orthopoxvirus production and purification |
| US20170002332A1 (en) * | 2013-12-17 | 2017-01-05 | Genethon | Method for purifying enveloped viruses or viral vectors |
| CN104371982A (en) * | 2014-10-21 | 2015-02-25 | 武汉维诺赛生物技术有限公司 | Method for purifying slow virus |
| US20180051275A1 (en) * | 2016-08-17 | 2018-02-22 | Glaxosmithkline Intellectual Property Development Limited | Method for purifying viral vectors |
| 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 |
| CN111876393A (en) * | 2020-06-30 | 2020-11-03 | 恒瑞源正（上海）生物科技有限公司 | Method for large-scale rapid production of high-purity high-activity lentiviral vector |
| CN111876392A (en) * | 2020-06-30 | 2020-11-03 | 恒瑞源正（上海）生物科技有限公司 | Method for large-scale rapid production of viral vectors |
Non-Patent Citations (4)
| Title |
|---|
| "Capto Core 400，Capto Core 700", pages 1 - 8, Retrieved from the Internet <URL:https://www.cytivalifesciences.com.cn/zh/cn/shop/chromatography/resins/multimodal/capto-core-700-multimodal-chromatography-resin-p-05642> * |
| "Capto S, Capto Q, and Capto DEAE", pages 1 - 8, Retrieved from the Internet <URL:https://www.cytivalifesciences.com.cn/zh/cn/shop/chromatography/resins/ion-exchange/capto-s-ion-exchange-chromatography-resin-p-00721> * |
| MENGRAN YU等: "Improving stability of virus-like particles by ion-exchange chromatographic supports with large pore size: advantages of gigaporous media beyond enhanced binding capacity", COMPARATIVE STUDY J CHROMATOGR A., vol. 1331, pages 72 - 73 * |
| 梁山琴: "灭活口蹄疫病毒抗原的离子交换色谱纯化", 中国优秀硕士学位论文全文数据库 农业科技辑, no. 2, pages 27 - 37 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117106713A (en) * | 2023-10-24 | 2023-11-24 | 北京恩泽康泰生物科技有限公司 | Kit, method and application for secondary purification of exosomes |
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