Protein Fingerprinting of a Viral Vector, AAV5

by | Apr 16, 2024

Explore the analysis of capsid proteins using intact mass analysis and peptide mapping to determine critical quality attributes for gene therapy.

This article from issue 16 of the Analytix Reporter discusses the analysis of capsid proteins using intact mass analysis and peptide mapping to determine critical quality attributes for gene therapy, utilizing various chromatography columns.

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The culture and use of adeno-associated virus (AAV) as a gene delivery device has seen much interest in recent years as a strategy for delivering targeted gene therapies towards muscle, nerve, liver, and eye disorders. This includes different AAV serotypes that target specific tissues as well as genetically engineered hybrid types with altered tissue specificity.

In-depth characterization of the viral capsid proteins, as well as the genomic content, is essential to verify critical quality attributes of these particles. Both the amino acid sequence as well as post-translational modifications (PTMs) that are found on the viral capsid proteins can have an impact on the tissue tropism, efficacy, and immunogenicity of AAV.1 PTMs that have been identified on AAV include phosphorylation, SUMOylation, ubiquitination, acetylation, methylation, and glycosylation.2

The USP released draft guidelines3 for the analytical characterization of viral vectors in 2022 to provide method starting points for determination of critical quality attributes. A variety of tests are described to characterize the identity, purity, concentration, and potency of these viral vectors, among other traits. For determination of capsid identity, starting methods are provided that use Western blotting, reversed-phase HPLC with UV detection, and HPLC with MS detection. The latter approach describes the use of both intact mass analysis of the capsid proteins, as well as amino acid sequence analysis using peptide mapping. Here we describe our work to develop methods for protein fingerprinting of AAV serotype 5 using both intact mass analysis and peptide mapping. Several post- translational modifications of the viral proteins VP1, VP2 and VP3 were identified.


A system suitability mix, MSRT1, was prepared according to the instructions on the data sheet but with a final acetonitrile concentration of 1.6% (v/v). The injection volume was 10 µL. This solution is a mix of 14 isotopically labelled peptides injected prior to injection of samples to verify instrument performance.

See the full article for AAV production, intact mass analysis, and peptide mapping specifications.


With respect to protein fingerprinting, the draft USP guidelines provided a starting point for MS characterization of viral vectors by both intact capsid fingerprinting and peptide mapping. The draft guideline also describes an additional method, using UV detection and a 2-hour chromatographic run, for determination of capsid stoichiometry. While we did not replicate this method, we did use UV detection in conjunction with mass spectrometry fingerprinting of the intact viral capsids over a shorter 30-minute run. Integration of the UV detected peaks was then used to evaluate capsid stoichiometry. The combined LC- UV-MS analysis provided a convenient, one-method assessment of fingerprint and stoichiometry in a shorter run time.

The article also describes the intact mass analysis of the viral proteins VP1, VP2, and VP3, and includes a peptide mapping comparing three different columns.

Read the full article for comprehensive results and discussion.


Several column comparisons were shown to demonstrate uses of the BIOshell™ line of columns for characterizing viral vectors, in this case AAV serotype 5. Conditions outlined in the USP draft guideline were used for both intact mass fingerprinting of viral capsids and for peptide mapping experiments, but we suggest that further improvements in chromatography might be made with additional gradient optimization.

The BIOshell™ columns, particularly the A400 Protein C4 column, have proven to be effective in separating the three capsid proteins of AAV5 for intact mass analysis and stoichiometry evaluation. In addition, the BIOshell™ A400 Protein C4 column provides partial separation of the VP3- clip from VP3. The competitor column also shows partial separation of VP3-clip from VP3 but with coelution of VP1.

The BIOshell™ A160 Peptide C18 columns, in both the 2.7 and 2.0 µm particle sizes, proved to be useful in retaining short, polar peptides to provide slightly improved sequence coverage over the competitor column. Retention of the N-terminus of VP2 was only provided by the BIOshell™ columns.

The USP draft conditions for the mobile phase and gradient conditions were used in both approaches for characterizing capsids, but we suggest these conditions might benefit from further optimization with other AAV serotypes or specific PTMs.

*The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

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  1. Yan Z, Zak R, Luxton GWG, Ritchie TC, Bantel-Schaal U, Engelhardt JF. 2002. Ubiquitination of both Adeno-Associated Virus Type 2 and 5 Capsid Proteins Affects the Transduction Efficiency of Recombinant Vectors. J Virol. 76(5):2043-2053.
  2. Mary B, Maurya S, Arumugam S, Kumar V, Jayandharan GR. 2019. Post‐translational modifications in capsid proteins of recombinant adeno‐associated virus (AAV) 1‐rh10 serotypes. The FEBS Journal. 286(24):4964-4981.
  3. USP. Analytical Procedures for Viral Vectored Vaccine Quality 2022.

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