Comparability of Five Alternative AAV9 Downstream Processes from Ultracentrifugation to Chromatography


Aleš Štrancar, Executive Managing Director, Sartorius BIA Separations
Joe Balleydier, Senior Vice President of Research and Development, Advanced Medicine Partners
Mike Hatfield, Senior Director of Research and Development, Advanced Medicine Partners

Improved analytical methods are helping improve the understanding of capsid heterogeneity in adeno-associated virus (AAV) therapeutic preparations. This understanding allows the development of manufacturing processes that produce AAV therapeutics with fewer non-functional capsids.

In this webinar, the speakers will present the results of a four-arm AAV9 comparability protocol that includes three alternative two-column downstream processes at 50-L scale and an ultracentrifugation (UC) process at 200-L scale. The UC process is the gold standard for functional capsid purification.

The expert speakers will outline simplified processes for both the capture and the empty/full (E/F) separation steps, including various combinations of cation exchange chromatography (CEX) and anion exchange chromatography (AEX) monoliths with different gradient types, as well as a process using an affinity resin and AEX monolith.

Also, the speakers will cover the analytical comparability of the drug substance lots, assessment of vector yields, host–cell proteins, plasmid and host-cell deoxyribonucleic acid (DNA) levels; capsid content by sedimentation-velocity analytical UC, charge detection mass spectrometry and mass photometry; vector protein purity by capillary electrophoresis; aggregation by size-exclusion chromatography; vector DNA purity by next-generation sequencing; potency by in vitro enhanced green fluorescent protein (eGFP) expression tests; and capsid protein charge heterogeneity and intact-capsid isoelectric point (pI) by capillary isoelectric focusing.

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