Suspension domestication method of HEK293T cells and application of suspension domestication method in lentivirus production

Abstract

The invention provides a suspension domestication method of HEK293T cells and application of the suspension domestication method in lentivirus production, and aims to avoid the use of unknown culture medium components and animal-derived production materials in the culture process of lentivirus production cells, the suspension domestication method is simple and convenient in flow, short in domestication time and lower in cost; the adopted serum-free culture medium is clear in component and controllable in safety risk, process impurities are reduced, and quality control is relatively simple; the production process is easy to amplify, and the manpower consumption is low; an original cell bank (PCB), a main cell bank (MCB) and a working cell bank (WCB) of the suspension cells are established by the suspension cells obtained through amplification culture and domestication, the working cell bank (WCB) of the suspension system is applied to preparation and production of the lentivirus, and the yield of crude venom of the lentivirus at least can reach 1-10 * 10 < 7 > TU / mL.

Description

Technical field [0001] The present invention relates to the field of lentiviral technology, particularly to a method of suspension domestication of HEK293T cells and its application in lentiviral production. Background [0002] The harvesting of crude venom in the production process of lentivirus, usually in HEK293T as the packaging of cells, using serum-containing mixed medium, cultured in flasks or cell factories, and packaged by means of transient transfection of plasmids. This method is relatively mature in technology, simple and easy to operate, and is adopted by many laboratories. However, the use of serums of animal origin increases the risk of contamination by exogenous viral factors; The serum composition is complex, the composition is not clear, and it does not conform to the regulatory spirit of the drug production registration; Serum-introduced bovine serum albumin, which increases the difficulty of the purification process and quality control; The use of adherent cel...

AI Technical Summary

Problems solved by technology

However, the use of animal-derived serum increases the risk of contamination by exogenous virus factors; the composition of the serum is complex and the components are unclear, which does not comply with the regulatory spirit of drug production registration; the introduction of bovine serum albumin in the serum increases the purification process and quality Difficulty of control; using adherent cells to package lentivirus, it is difficult to scale up the process, improve the output, reduce the cost, and consume a lot of labor; digest adherent cells using animal-derived trypsin, which may introduce foreign virus factors and increase quality. control difficulty etc.

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