Steric exclusion chromatography of lentiviral vectors using hydrophilic cellulose membranes.

Autor: Labisch JJ; Lab Essentials Applications Development, Sartorius Stedim Biotech GmbH, Göttingen, Lower Saxony, Germany; Institute of Technical Chemistry, Leibniz University Hannover, Hanover, Lower Saxony, Germany. Electronic address: jennifer.labisch@sartorius.com., Kassar M; Lab Essentials Applications Development, Sartorius Stedim Biotech GmbH, Göttingen, Lower Saxony, Germany; Karlsruhe Institute of Technology, Institute of Process Engineering and Life Sciences, Biomolecular Separation Engineering, Karlsruhe, Baden-Württemberg, Germany., Bollmann F; Marketing Separation Technologies, Sartorius Stedim Biotech GmbH, Göttingen, Lower Saxony, Germany., Valentic A; Karlsruhe Institute of Technology, Institute of Process Engineering and Life Sciences, Biomolecular Separation Engineering, Karlsruhe, Baden-Württemberg, Germany., Hubbuch J; Karlsruhe Institute of Technology, Institute of Process Engineering and Life Sciences, Biomolecular Separation Engineering, Karlsruhe, Baden-Württemberg, Germany., Pflanz K; Lab Essentials Applications Development, Sartorius Stedim Biotech GmbH, Göttingen, Lower Saxony, Germany.
Jazyk: angličtina
Zdroj: Journal of chromatography. A [J Chromatogr A] 2022 Jul 05; Vol. 1674, pp. 463148. Date of Electronic Publication: 2022 May 14.
DOI: 10.1016/j.chroma.2022.463148
Abstrakt: 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 × 10 12 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.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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