VectorMOD: Method for Bottom-Up Proteomic Characterization of rAAV Capsid Post-Translational Modifications and Vector Impurities

Abstract

Progress in recombinant AAV gene therapy product and process development has advanced our understanding of the basic biology of this critical delivery vector. The discovery of rAAV capsid post-translational modifications (PTMs) has spurred interest in the field for detailed rAAV-specific methods for vector lot characterization by mass spectrometry given the unique challenges presented by this viral macromolecular complex. Recent concerns regarding immunogenic responses to systemically administered rAAV at high doses has highlighted the need for investigators to catalog and track potentially immunogenic vector lot components including capsid PTMs and PTMs on host cell protein impurities. Here we present a simple step-by-step guide for academic rAAV laboratories and Chemistry, Manufacturing and Control (CMC) groups in industry to perform an in-house or outsourced bottom-up mass spectrometry workflow to characterize capsid PTMs and process impurities.

Keywords: AAV (adeno-associated virus); PTM (post-translational modification); adverse drug reaction; bottom-up approach; glycosylation; immunogenicity; mass spectrometry - LC-MS/MS; proteomics.

Figure 1

Bottom-up rAAV proteomic workflow. Schematic of key steps in the vector preparation for LC-MS/MS and subsequent data analysis steps.

Figure 2

Example Digestion Efficiency Validation. Trypsin cleaves peptides on the C-terminal side of lysine and arginine residues (unless followed by a proline). All rAAV capsid serotypes have regular Lys and Arg amino acids throughout VP1 so you should see regular cleavage that produces peptides ~10-45 amino acids in length (shown in Preview as green bars with PTMs noted in red) throughout the entire length of VP1. Good digestion efficiency and MS/MS runs should exhibit >90% sequence coverage of rAAV VP1. This screenshot highlights typical results for the first 100 amino acids of a prototypical rAAV8 run.

Figure 3

Representative HexNAc fingerprints. (A) GlcNAc-containing glycopeptides will have a higher abundance of the ion at 138 m/z than the ion at 144 m/z. This fingerprint will be present in peptides with N-glycans, O-GlcNAc, and/or GlcNAc-containing core 2 O-glycans. (B) GalNAc-containing glycopeptides will have nearly equal abundance of 138 m/z and 144 m/z ions. This fingerprint will be present in mucin-type glycopeptides that do not contain GlcNAc.