Optimized Reversed-Phase Liquid Chromatography/Mass Spectrometry Methods for Intact Protein Analysis and Peptide Mapping of Adeno-Associated Virus Proteins

Abstract

Recombinant adeno-associated viruses (AAVs) have emerged as the leading gene delivery platform owing to their nonpathogenic nature and long-term gene expression capability. The AAV capsid, in addition to protecting the viral genome, plays an important role in viral infectivity and gene transduction, indicating the value of the constituent viral proteins (VPs) being well-characterized as part of gene therapy development. However, the limited sample availability and sequence homology shared by the VPs pose challenges to adapt existing analytical methods developed for conventional biologics. In this study, we report the development of reversed-phase liquid chromatography/mass spectrometry-based methods for characterization of AAV capsid proteins at intact protein and peptide level with reduced sample consumptions. The developed methods allowed the measurement of VP expression with fluorescence detection and intact mass/post-translational modifications (PTMs) analysis through a benchtop time-of-flight mass spectrometer. The general applicability and validity of the methods for gene therapy product development were demonstrated by applying the optimized methods to multiple common AAV serotypes. A 1-h enzymatic digestion method was also developed using 1.25 μg of AAV VPs, providing >98% protein sequence coverage and reproducible relative quantification of various PTMs of the VPs. The efficient and sensitive analyses of AAV capsid proteins enabled by the reported methods provide further understanding and offer guidance in the development and manufacturing of AAV-related therapeutics.

Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs)

“…The presented workflow demonstrates a significant improvement compared to previously reported sample preparation methods which may take up to 18 hours while also requiring larger amounts AAVs to produce satisfactory results [15]. Additionally, the number of manipulation steps required in traditional and more recent digestion protocols was also reduced [16]. By using a magnetic bead-based sample preparation for the digestion with pepsin, digestion is stopped in all samples at the exact same time, avoiding the production of different digestion patterns.…”

“…Our dataset confirms the identification of phosphorylation at S679 (S680 when the M at position 1 is considered), but the remaining modifications identified could not be confirmed using the present dataset. More recently, acetylation, deamidation, oxidation, methylation, and phosphorylation modifications were also identified in AAV5 after a 98% successful tryptic digestion [16]. General discrepancies on PTM abundance levels can be explained by differences in expression systems or overall downstream processing.…”

Development of a Two-Dimensional Liquid Chromatography-Mass Spectrometry Platform for Simultaneous Multi-Attribute Characterization of Adeno-Associated Viruses

“…Common methods for characterizing viral proteins include sodium dodecyl sulfate-polyacrylamide gel electrophoresis, capillary electrophoresis sodium dodecyl sulfate, microchip capillary electrophoresis, high-performance liquid chromatography (HPLC), and MS. HPLC coupled to MS is becoming increasingly popular for AAV protein characterization because it is high-throughput and amenable to quality control. Additionally, the separation of VP1, VP2, and VP3 allowed for higher sensitivity detection and accurate mass measurement for VP1, VP2, and their potential minor proteoforms, as ion suppression of the lower abundant VP1 and VP2 from VP3 was minimized after separation . However, separating VP1 and VP2 using HPLC-MS is particularly challenging as they are alternatively spliced and share a common C-terminal sequence.…”

“…Recent development using hydrophilic interaction chromatography (HILIC) can effectively separate not only the three VPs but also the oxidized and phosphorylated proteoforms into distinct peaks . Moreover, when difluoroacetic acid (DFA) is used as an ion-pairing reagent, reversed-phase liquid chromatography (RPLC) can separate the three VPs into three distinct peaks, compared to the multiple peaks produced by HILIC . Both chromatographic techniques enable the quantification of VP stoichiometry in the AAV capsid.…”

“…Additionally, the separation of VP1, VP2, and VP3 allowed for higher sensitivity detection and accurate mass measurement for VP1, VP2, and their potential minor proteoforms, as ion suppression of the lower abundant VP1 and VP2 from VP3 was minimized after separation. 33 However, separating VP1 and VP2 using HPLC-MS is particularly challenging as they are alternatively spliced and share a common C-terminal sequence.…”