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Slow virus purification method

A purification method, lentivirus technology, applied to viruses, biochemical equipment and methods, recovery/purification, etc., to achieve good repeatability and stability, improve virus recovery efficiency, and easy operation

Active Publication Date: 2017-11-24
SHEN ZHEN TSINGHUA YUANXING BIO PHARM SCI & TECHNOL
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Optimization of virus load in membrane chromatography purification method.

[0037] Use the method described in step S1 to package and amplify the lentivirus, and harvest the virus liquid. The virus harvest liquid is pretreated by the method described in step S2. Adopt 3ml in the present embodiment Q optimizes the sample volume of the virus in the membrane chromatography purification method, and samples and detects the flow-through liquid at different sample volume stages. For the specific operation of the purification process, refer to step S3. Real-time fluorescent quantitative PCR was used to measure the physical titer of the lentivirus, and the copy number of the lentivirus detected by the kit was converted into the physical titer VP / ml according to the relationship of VP titer=virus copy number / 2. see results figure 1 .

[0038] Depend on figure 1 It can be seen that when the sample begins to pass through the membrane column, the virus is completely...

Embodiment 2

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

[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 was often used to degrade the DNA and remove it, although some results have been achieved. , but this processing method introduces new exogenous biological reagents, which need to be removed in the subsequent processing process, which not only increases the detection index of lentiviral vector preparations, but also increases the quality and safety risks of lentiviral preparations. Purification of lentivirus by membrane chromatography can remove host DNA to a certain extent. In order to evaluate the removal effect of membrane chromatography on host DNA and compare the recovery effect of membrane ch...

Embodiment 3

[0051] Example 3 Membrane chromatography lentivirus purification process scale-up.

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

[0053] Table 3 Detection results of lentiviral stock solution after purification using a definite purification process

[0054] Test items

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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, in particular 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 their 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 relevant pharmaceutical research such ...

Claims

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Application Information

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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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