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

Overview

Journal J Proteome Res

Publisher American Chemical Society

Specialty Biochemistry

Date 2023 Dec 20

PMID 38123456

Authors

Josh Smith

Felipe Guapo

Lisa Strasser

Silvia Millan-Martin

Steven G Milian

Richard O Snyder

Jonathan Bones

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

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