Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins

Adeno-associated viruses (AAVs) are commonly used as
vectors for
the delivery of gene therapy targets. Characterization of AAV capsid
proteins (VPs) and their post-translational modifications (PTMs) have
become a critical attribute monitored to evaluate product quality.
Liquid chromatography–mass spectrometry (LC–MS) analysis
of intact AAV VPs provides both quick and reliable serotype identification
as well as proteoform information on each VP. Incorporating these
analytical strategies into rapid good manufacturing practice (GMP)-compliant
workflows containing robust, but simplified, data processing methods
is necessary to ensure effective product quality control (QC) during
production. Here, we present a GMP-compliant LC–MS workflow
for the rapid identification and in-depth characterization of AAVs.
Hydrophilic interaction liquid chromatography (HILIC) MS with difluoroacetic
acid as a mobile phase modifier is utilized to achieve the intact
separation and identification of AAV VPs and their potential proteoforms.
Peptide mapping is performed to confirm PTMs identified during intact
VP analysis and for in-depth PTM characterization. The intact separations
platform is then incorporated into a data processing workflow developed
using GMP-compliant software capable of rapid AAV serotype identification
and, if desired, specific serotype PTM monitoring and characterization.
Such a platform provides product QC capabilities that are easily accessible
in a regulatory setting.