Lentivirus purification process

Abstract

The invention discloses a lentivirus purification process. The lentivirus purification process comprises the following steps: S1, clarifying and filtering a lentivirus-containing cell culture to obtain a lentivirus-containing cell culture supernatant; S2, concentrating the supernatant obtained in the step S1 by 8-15 times by adopting a hollow fiber ultrafiltration system; S3, washing and filtering the concentrated solution obtained in the step S2 by at least 10 volumes by using a first buffer solution; S4, carrying out nuclease Benzonase enzyme digestion treatment on the concentrated solution washed and filtered in the step S3, and controlling the treatment temperature to be 25-37 DEG C and the treatment time to be 30-60 minutes; S5, washing and filtering the concentrated solution in the step S4 by at least 10 volumes through a hollow fiber ultrafiltration system so as to remove nuclease Benzonase and nucleic acid fragments, and then adding a second buffer solution into the concentrated solution, wherein the second buffer solution contains Buffer for maintaining the stability of lentivirus particles; and S6, concentrating the lentiviral vector concentrated solution obtained in the step S5 to 5*10<7> TU/mL or above to obtain the purified lentivirus. The lentivirus purification process disclosed by the invention can obviously improve the recovery rate of the lentivirus and is suitable for large-scale production.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lentivirus purification process. Background technique [0002] Lentivirus is commonly used in Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T), which has the advantages of high transduction efficiency, integration of T cell genome and continuous expression of target proteins. Nowadays, CAR-T production is mostly based on lentiviral vector-mediated CAR gene transfer. At present, most of the processes used to produce lentivirus are adherent cell processes (such as HEK293T), and a few are suspension cell processes. Use an adherent process to clarify and filter the harvested supernatant to remove floating cells, usually using a 0.45 μm filter membrane (when the cell state is better when the supernatant is harvested) or a clarification filter membrane bag (when the cell state is poor when the supernatant is harvested) , cell debris and other impurities. For suspension cell proc...

AI Technical Summary

Problems solved by technology

However, the size of lentivirus particles is usually 80-130 nm, which is extremely fragile, not resistant to mechanical pressure and shear force, and is also relatively easy to degrade, so it is not suitable for downstream purification operations for a long time

Therefore, using more purification steps will greatly reduce its recovery rate (such as column chromatography), and long-term operation and treatment will also increase the risk of lentiviral particle degradation. Currently, the recovery rate of lentiviral particles purified based on the above method is usually only 10%. ~20%

In addition, chromatography usually requires a more expensive purification system, such as AKTA, and the purification medium used is usually more expensive

Images