Authors
Luca Ossi, Samuele Delfino, Helena Marie, Michael Schulte, Mattia Sponchioni
Published in
Biotechnology journal. Volume 21. Issue 8. Pages e70295.
Abstract
The separation of empty and full adeno-associated viruses (AAVs) is particularly critical because of the similarity between the two. Anion-exchange chromatography represents a scalable tool, leveraging their slight difference in isoelectric point. However, traditional resin chromatography can only deliver poor resolution, mainly due to the restricted accessibility of the capisds to the bead pores. To cover this gap, in this work, we designed and optimized the separation of empty and full AAVs using anion-exchange membrane chromatography. The role played by flowrate, gradient slope and wash step parameters on the chromatographic resolution was first investigated with two consecutive designs of experiments (DoE). The response surfaces indicated that high process flowrates, that is 15 membrane volumes (MVs)/min, with a gradient from 115 mM to 250 mM sodium acetate in 350 MVs maximize the chromatographic resolution. Then, preparative experiments showed that fast loading is possible, with residence time as short as 24 s, without product breakthrough. Moreover, membrane loading up to 1.2E17 viral particles (vp)/Lmembrane could be reached, providing one order of magnitude higher productivity with respect to standard column operations. With these results, we demonstrated the great potential of anion-exchange membrane chromatography in the polishing of AAVs, overcoming traditional limitations of packed-bed operations.
PMID:
42605573
Bibliographic data and abstract were imported from PubMed on 17 Aug 2026.
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