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Method for purifying slow virus

A purification method, lentivirus technology, applied in the biological field, to achieve the effect of low host DNA residue, high purity and titer, and good safety

Inactive Publication Date: 2015-02-25
武汉维诺赛生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is a major challenge to scale up the purification method for clinical use, so that its purity and titer can meet the standards of clinical gene transfer therapy, while ensuring its safety and efficacy.

Method used

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  • Method for purifying slow virus
  • Method for purifying slow virus
  • Method for purifying slow virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the preparation of virus liquid

[0040] (1) Large-scale culture of packaging cells.

[0041] First, the frozen 293T cells were taken out from the liquid nitrogen tank, and immediately placed in a 37°C water bath for recovery. After it is completely melted, add 5mL DMEM complete medium (DMEM+10%FBS+P / S) and mix well, centrifuge at 1000rpm for 5min, discard the supernatant, mix well with 5mL DMEM complete medium for the precipitate, and transfer to T- 25cm 2 In a cell culture flask, cultivate overnight at 37°C in a 5% CO2 incubator; then, when the above cells grow to 80% confluence, discard the medium, add 5mL PBS, shake slightly, discard the PBS, and add 1mL 0.25 %EDTA-trypsin, put it at room temperature for 2-3min, when the cells are completely detached from the bottle wall, add 10mL DMEM complete medium, mix well, centrifuge at 1000rpm for 5min, discard the supernatant, and culture the cell pellet completely with 5mL DMEM Mix well, transfer to T-75cm ...

Embodiment 2

[0048] Embodiment 2: the purification of virus liquid:

[0049] (5) Nuclease treatment of virus liquid.

[0050] After the virus liquid in Example 1 was concentrated and filtered in step (4), 250 U / mL Benzonase was added and reacted in a water bath at 37°C for 6 hours.

[0051] (6) The virus liquid is purified by anion exchange chromatography and concentrated by centrifugation.

[0052] Specifically, the steps are as follows: ① first wash the 500mL HiTrap Q-XL strong negative spin column with 1L ddH2O, and set the flow rate to 50mL / min. ②Equilibrate a 500mL HiTrap Q-XL strong negative spin column with 1L loading buffer Buffer A (50mM Tris-HCl, pH 8.0), and set the flow rate to 50mL / min. ③ Load the virus supernatant after 0.45um filtration, and set the flow rate to 50mL / min. ④ Rinse 500mL HiTrap Q-XL strong negative spin column with 1.5L loading buffer Buffer A (50mM Tris-HCl, pH8.0) until the baseline is stable to remove non-specifically bound proteins, and set the flow rat...

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Abstract

The invention relates to a method for purifying a slow virus. The method comprises the following steps: (S10) providing a cell culture containing the slow virus; (S20) carrying out centrifugal concentration on the cell culture, and filtering to obtain virus suspension; (S30) purifying the virus suspension by virtue of anion exchange chromatography; and (S40) purifying the virus suspension by virtue of gel filtration chromatography. A process for purifying the slow virus is easy to amplify, can be used for processing large amounts of virus liquid produced by cell factories or bioreactors on a large scale, can meet production requirements of slow virus liquid, is stable and is good in repeatability; furthermore, the purified slow virus liquid prepared by virtue of the method is not polluted by exogenous factors, is low in residues of cell host protein and host DNA, good in safety, and high in purity and titer and can completely meet requirements of clinic gene therapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying lentivirus. Background technique [0002] With the development of biotechnology, the method of gene transfer in virology has been more and more used in gene therapy. It can introduce exogenous genes into somatic cells including mice, primates and humans, and integrate them into the cell genome to achieve long-term stable expression. There are very good prospects for applications in many fields. [0003] Retroviral vectors and adenoviral vectors are the most mature gene transfer vectors in virology. Common retroviral vectors are modified from mouse leukemia virus (MLV). Although they can integrate the target gene into the target cell genome and achieve stable expression, they can only transduce dividing cells, so they can only be used in isolated cells for gene therapy. In vivo scheme; adenovirus vector can transduce both dividing cells and quiescent cells, a...

Claims

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Application Information

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IPC IPC(8): C12N7/02C12R1/93
Inventor 黄俊
Owner 武汉维诺赛生物技术有限公司
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