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A kind of purification method of lentivirus

A purification method, lentivirus technology, applied in the direction of viruses, biochemical equipment and methods, recovery/purification, etc., to achieve the effects of easy amplification, good repeatability and stability, and easy operation

Active Publication Date: 2020-09-08
SHEN ZHEN TSINGHUA YUANXING BIO PHARM SCI & TECHNOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the above technical problems, the present invention discloses a lentivirus purification method, which uses membrane chromatography combined with molecular sieve chromatography to purify the lentivirus harvest liquid, and finally obtains the lentivirus stock solution, which solves the problem of large-scale preparation of harvested lentivirus The purification of the culture medium, on the basis of ensuring the activity of the lentivirus, improves the recovery efficiency of the lentivirus

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  • A kind of purification method of lentivirus
  • A kind of purification method of lentivirus
  • A kind of purification method of lentivirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Optimization of virus loading by membrane chromatography purification method.

[0037] The method described in step S1 is used for lentivirus packaging and amplification, and the virus liquid is harvested. The method described in step S2 is used to pretreat the virus harvest liquid. In this example, 3ml Q performs membrane chromatography purification method for virus loading optimization, sampling and testing the flow-through liquid at different loading stages, and the specific operation of the purification process refers to step S3. Real-time fluorescent quantitative PCR was used to determine the physical titer of the lentivirus, and the number of copies of the lentivirus detected by the kit was converted into the physical titer VP / ml according to the relationship of VP titer=viral copy number / 2. See the result figure 1 .

[0038] by figure 1 It can be seen that when the sample starts to pass through the membrane column, the virus is completely adsorbed by the ...

Embodiment 2

[0042] Example 2 Comparison of the purification effect of membrane chromatography and traditional ion exchange column chromatography on lentivirus purification.

[0043] Host DNA residue is one of the key indicators for evaluating the quality of lentiviral vectors, and DNA is difficult to remove by conventional purification methods. In the past purification process, the method of adding nuclease is often used to degrade and remove the DNA, although certain effects have been achieved However, new exogenous biological reagents are introduced during the processing method, which need to be removed in the subsequent processing process. This not only increases the detection index of the lentiviral vector preparation, but also increases the quality and safety risk of the lentiviral preparation. Purification of lentivirus by membrane chromatography can remove host DNA to a certain extent. In order to evaluate the effect of membrane chromatography on the removal of host DNA and compare th...

Embodiment 3

[0051] Example 3 Scale-up of lentivirus purification process by membrane chromatography.

[0052] Refer to step S1 for lentivirus packaging and amplification, and refer to S2 and S3 for purification. The purified virus samples are sent to the quality department for testing of various indicators. The results are shown in Table 3. It can be seen from the experimental results that the quality of the recombinant lentiviral stock solution obtained by the purification process of the present invention meets the relevant regulations of the 2015 version of the "Chinese Pharmacopoeia" and the "Guiding Principles for Human Gene Therapy Research and Preparation Quality Control Technology".

[0053] Table 3 Test results of lentiviral stock after purification by a certain purification process

[0054] Test items Quality Standard Test results Physical titer vp / ml≥2×10 9

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Abstract

The invention provides a slow virus purification method. The slow virus purification method comprises the following steps: S1, culturing viruses; S2, carrying out purification pretreatment on the viruses; carrying out centrifugal separation on the slow viruses obtained through amplification, and collecting supernatant as a virus harvesting liquid; firstly carrying out primary filtration treatment on the virus harvesting liquid, carrying out secondary filtration treatment on the virus liquid obtained through filtration, collecting a virus filtrate, carrying out ultrafiltration concentration, then centrifuging, collecting the supernatant, then carrying out tertiary filtration treatment, and collecting a filtrate; and S3, carrying out membrane chromatography treatment on the collected virus purification pretreatment sample, then carrying out ultrafiltration treatment, and purifying by adopting molecular sieve chromatography, wherein matrix used by a chromatography membrane is a regenerated cellulose skeleton during the membrane chromatography. By adopting the technical scheme of the invention, virus recycling efficiency is improved on the basis of guaranteeing slow virus activity; and operation is simple, amplification is easy, the virus harvest liquid can be treated in a large scale, and the slow virus purification method provided by the invention has good repeatability and stability.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a method for purifying lentivirus. Background technique [0002] In recent years, the basic research of gene therapy has made great progress, and many research results have been applied in clinical trials of various diseases, such as liver cancer, lung cancer, melanoma, nasopharyngeal cancer, cardiovascular and cerebrovascular diseases, diabetes, and Some rare diseases have achieved remarkable research results, demonstrating its broad clinical application prospects. Viral vectors are a common and efficient gene delivery system. Due to the rapid development of viral vector gene transfer technology, the use of gene transfer technology to prevent and treat human diseases has entered the stage of clinical practical research from theoretical and laboratory research. The research, development and preparation of viral vectors requires the support of production technology, quality co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/02
CPCC12N7/00C12N2740/15051
Inventor 肖永强王巍陈彦平黄伟东
Owner SHEN ZHEN TSINGHUA YUANXING BIO PHARM SCI & TECHNOL
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