Process Development of Recombinant Adeno-Associated Virus Production Platform Results in High Production Yield and Purity
- 1 January 2023
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 34 (1-2), 56-67
- https://doi.org/10.1089/hum.2022.153
Abstract
Optimization of recombinant adeno-associated virus (rAAV) production has important clinical implications, as manufacturing is one of the major challenges for rAAV gene therapy. In this study, we optimized upstream and downstream processing of the rAAV production platform created by an earlier Design-of-Experiment approach. Our results showed that adding peptones, yeastolate, Trypton N1 or both increased production yield by 2.8-3.4 folds. For downstream processing, a variety of wash buffers using an affinity resin, POROSTM CaptureSelectTM (PCS)-AAVX, were tested for their effects on rAAV8 purity, including NaCl, MgCl2, arginine, Triton X-100, CHAPS, Tween 20, octyl β-D-1-thioglucopyranoside (OTG), and low pH. The results showed that the OTG wash significantly improved the rAAV purity to 97% and reduced endotoxins to an undetectable level (<0.5 EU/mL), while retaining the yield at 87% of the PBS wash. The OTG wash was successfully applied to purifications of rAAV1, rAAV2 and rAAV5 using PCS-AAVX and rAAV9 using PCS-AAV9. rAAV8 purified with OTG wash showed comparable transduction efficiency in HEK293T cells to the rAAV8 purified with PBS wash. The optimized rAAV production process yielded 5.5-6.0x1014 and 7.6x1014 vector genome per liter of HEK 293T cells for purified rAAV8- and rAAV5-EF1-EGFP, respectively. The platform described here is simple with high yields and purity, which will be beneficial to both research and clinical gene therapy.This publication has 21 references indexed in Scilit:
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