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

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 indepth 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.

A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency

“…Since SDS-PAGE and Western blotting are only semi-quantitative methods to estimate VP levels, we set out to develop a quantitative LC workflow to accurately determine VP ratios. Using a hydrophilic interaction liquid chromatography with fluorescent detection (HILIC-FLR)-based separation approach as first described by Liu et al in 2020 [ 18 ], we developed an AAV5-specific gradient for the separation of VP proteins using difluoroacetic acid (DFA) as a mobile phase modifier, slightly adapted from the method described by Smith et al [ 19 ]. After separation ( Figure 5 ), ratios were calculated based on FLR peak areas and an assumption that the sum of VP1, 2 and 3 peaks accounted for 60 protein copies in the capsid, whereby a theoretical ratio sum of 12 (1:1:10, VP1/VP2/VP3) was used for the quantitative estimation of VP ratios [ 19 ] (full data available in Supplementary Materials Table S1 ).…”

“…Using a hydrophilic interaction liquid chromatography with fluorescent detection (HILIC-FLR)-based separation approach as first described by Liu et al in 2020 [18], we developed an AAV5-specific gradient for the separation of VP proteins using difluoroacetic acid (DFA) as a mobile phase modifier, slightly adapted from the method described by Smith et al [19]. After separation (Figure 5), ratios were calculated based on FLR peak areas and an assumption that the sum of VP1, 2 and 3 peaks accounted for 60 protein copies in the capsid, whereby a theoretical ratio sum of 12 (1:1:10, VP1/VP2/VP3) was used for the quantitative estimation of VP ratios [19] (full data available in Supplementary Materials Table S1). The ACG start codon was found to have the lowest amount of VP1 and also the lowest ratios of VP1/VP2 and VP1/VP3, which was in agreement with the profiles seen in the SDS-PAGE analysis for this construct (Table 1).…”

“…AAV capsid protein separation was performed to determine VP ratios for each construct on a Vanquish™ Horizon ultra-high pressure liquid chromatograph (UHPLC) (Thermo Fisher Scientific, Germering, Germany), slightly modified from Smith et al, 2023 [19]. In brief, separation was obtained on a Waters ACQUITY UPLC Glycoprotein BEH Amide column (300 Å, 1.7 µm, 2.1 × 150 mm) (Waters Corporation, Milford, MA, USA, p/n: 186007963) using a flow rate of 0.1 mL/min and column temperature of 45 • C. The gradient started at 85% acetonitrile (ACN) and 15% water for 3.5 min, 85% ACN to 65% ACN over the next 0.1 min, and a hold at 65% ACN for 4.4 min, followed by a 21 min gradient from 65% to 45% ACN.…”