Steric exclusion chromatography of lentiviral vectors using hydrophilic cellulose membranes

Abstract

Enveloped viral vectors like lentiviral vectors pose purification challenges due to their low stability. A gentle purification method is considered one of the major bottlenecks for lentiviral vector bioprocessing. To overcome these challenges, a promising method is steric exclusion chromatography which has been used to purify a variety of target molecules. In this study, we successfully identified optimal process parameters for steric exclusion chromatography to purify lentiviral vectors. Lentiviral vector particle recoveries and infectious recoveries of 86% and 88%, respectively, were achieved. The process parameters optimal for steric exclusion chromatography were determined as follows: polyethylene glycol with a molecular weight of 4000 Da, a polyethylene glycol concentration of 12.5%, and a flow rate of 7 mL⋅min−1 using 5 layers of stabilized cellulose membranes as a stationary phase. High protein and dsDNA removal of approximately 80% were obtained. The remaining polyethylene glycol concentration in the eluate was determined. We defined the maximum loading capacity as 7.5 × 1012 lentiviral particles for the lab device used and provide deeper insights into loading strategies. Furthermore, we determined critical process parameters like pressure. We demonstrated in our experiments that steric exclusion chromatography is a gentle purification method with high potential for fragile enveloped viral vectors as it yields high recoveries while efficiently removing impurities.