Rapid Highly-Efficient Digestion and Peptide Mapping of Adeno-Associated Viruses

Abstract

Adeno-associated viruses (AAVs) comprise an area of rapidly growing interest due to their ability to act as a gene delivery vehicle in novel gene therapy strategies and vaccine development. Peptide mapping is a common technique in the biopharmaceutical industry to confirm the correct sequence, product purity, post-translational modifications (PTMs), and stability. However, conventional peptide mapping is time-consuming and has proven difficult to reproduce with viral capsids because of their high structural stability and the suboptimal localization of trypsin cleavage sites in the AAV protein sequences. In this study, we present an optimized peptide mapping-based workflow that provides thorough characterization within 1 day. This workflow is also highly reproducible due to its simplicity having very few steps and is easy to perform proteolytic digestion utilizing thermally stable pepsin, which is active at 70 °C in acidic conditions. The acidic conditions of the peptic digestions drive viral capsid denaturation and improve cleavage site accessibility. We characterized the efficiency and ease of digestion through peptide mapping of the AAV2 viral capsid protein. Using nanoflow liquid chromatography coupled with tandem mass spectrometry, we achieved 100% sequence coverage of the low-abundance VP1 capsid protein with a digestion process taking only 10 min to prepare and 45 min to complete the digestion.

Figure 1.

Experimental workflow and the main outcomes of the AAV characterization using immobilized pepsin. (A) Description of experimental workflow yielding (B) 81% and 100% sequence coverage of the VP1 component of AAV2 when digesting using trypsin and pepsin, respectively. The sequence gaps that cannot be characterized using the trypsin digestion are highlighted in red, with the corresponding amino acid residue positions of these gaps indicated in the blue boxes (on the right). Deamidation and oxidation sites are highlighted in green and purple, respectively. (C) Comparative detection of process-induced deamidation and oxidation reveals that peptic proteolysis conditions practically eliminate deamidation artifacts.

Figure 2.

Improved characterization of commonly challenging protein sequence sections. Sequence gaps in the tryptic digestion experiments (162–245, 361–381, and 666–688 shown in Figure 1) contain multiple hydrophobic aromatic amino acid residues, which can be challenging to elute off the C18 column and even keep in solution. These gaps in sequence coverage are recovered with peptides released using the pepsin digestion. Representative examples of peptide sequences, fragmentation patterns, and the corresponding extracted ion chromatograms are shown in panels A-C.

Figure 3.

Peptic digestions produce a greater number of peptides with (a) a wider range of hydrophobicity scores, (b) overall smaller molecular masses, and (c) overall lower charge state distribution. (d) The experimentally determined C-terminal cleavage specificity.