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Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield

Gene Therapy volume 6, pages 973–985 (1999)Cite this article

Abstract

Conventional methods for rAAV purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.

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Acknowledgements

This work was supported in part by grants from the National Institutes of Health to NM and BJB, PO1 HL 59412and PO1 NS 36302. NM was supported in part by the Edward R Koger, American Cancer Society Chair. We thank Terence Flotte and for helpful advice during the course of this work. We gratefully acknowledge the gift of pDG plasmid from J Kleinschmidt.

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Authors and Affiliations

  1. Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA

    S Zolotukhin, E Mason, M Potter, K Chesnut & N Muzyczka

  2. Gene Therapy Center, University of Florida, Gainesville, FL, USA

    S Zolotukhin, B J Byrne, E Mason, I Zolotukhin, M Potter, K Chesnut & N Muzyczka

  3. Department of Pediatrics, University of Florida, Gainesville, FL, USA

    B J Byrne & I Zolotukhin

  4. Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    C Summerford & R J Samulski

  5. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    R J Samulski

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  1. S Zolotukhin
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Zolotukhin, S., Byrne, B., Mason, E. et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 6, 973–985 (1999). https://doi.org/10.1038/sj.gt.3300938

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