Purification process for obtaining high-purity adeno-associated virus vector

Abstract

The invention provides a process for obtaining a high-purity adeno-associated virus vector. The process comprises the following steps: A) collecting an adeno-associated virus vector for culturing a supernatant, filtering to remove cell debris, and using a clarified sample for chromatographic purification; B) purifying by a cation column; C) purifying by an adsorption gel filtration column; D) purifying by an anion exchange column; and E) concentrating by an ultrafiltration membrane. In the process provided by the invention, purification is carried out from cell culture of the supernatant, so that a harvesting process of the adeno-associated virus vector is greatly simplified; the process does not involve difficult-to-amplify unit operation such as cracking, centrifugation, precipitation and ultracentrifugation, thereby having great significance for large-scale production and promotion of clinical application of adeno-associated viruses.

Description

technical field [0001] The invention relates to the field of biotechnology and gene therapy, in particular to a purification process for large-scale acquisition of high-purity type 2 adeno-associated virus vectors. Background technique [0002] Gene therapy is a method of safely and stably introducing foreign genes into patients. Entering the cell, the gene fragment expresses the desired product to achieve a therapeutic effect. The key to gene therapy is to choose the appropriate gene carrier. Recombinant adeno-associated virus (rAAV) is currently recognized as a safe and reliable gene therapy vector, which can mediate the long-term stable expression of foreign genes in vivo, and has low immunogenicity, so it has broad application prospects. [0003] However, one of the obstacles limiting the use of rAAV is the development of a scalable purification process. The traditional laboratory-scale rAAV purification method is ultracentrifugation. The biggest drawback of this ...

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Problems solved by technology

There are currently some literature and patent reports using chromatography to purify rAAV, but these reported technologies mainly have the following two problems: 1) Chromatography is a supplement to ultracentrifugation, and the entire process still contains ultracentrifugation steps, which is difficult to scale up; 2 ) The rAAV harvesting solution is a cell lysate, which means that the rAAV harvesting process is relatively complicated, and a series of unit operations including lysis, centrifugation, and filtration are required

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