Insights into Adeno-Associated Virus Capsid Charge Heterogeneity

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Abstract
A comprehensive workflow is described to examine three contributing factors to the charge heterogeneity of Adeno-associated viruses (AAVs) from a single sample. Intact AAV9 capsids were fractionated using imaged capillary isoelectric focusing (icIEF)-based fractionation, allowing for collection of capsids with different isoelectric points (pIs). Capsid integrity of the fractions was confirmed with analytical icIEF and charge detection mass spectrometry (CD-MS). Using capillary electrophoresis (CE) immunoassays, the capsid protein ratios and capsid protein deamidation were characterized. Additionally, to analyze ssDNA content packaged in each fraction, CE-immunoassay and high-resolution CD-MS were used. This study enhances our understanding of AAVs, by examining the contributions of its attributes to capsid charge heterogeneity.

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