Fast & High Throughput LC-MS Characterization, and Peptide Mapping of Engineered AAV Capsids Using LC-MS/MS

ABSTRACT: Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With a skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays an important role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and Western blots carry, liquid chromatography & mass spectrometry (LC-MS) allows high resolution & sensitivity with great confidence in characterizing the AAV capsid proteins to the exact masses. Our optimized LC-MS method provides quick sample preparation, fast and high-throughput 4-min run, high sensitivity (only need ~2E10 vg per run for UV detection) allowing very efficient characterization of wild-type and engineered capsids. Additionally, , as well as LC-MS/MS peptide mapping of the AAV capsid proteins, including. lLysine-targeted chemical modification of the AAV capsids, for the first time, was characterized and peptide mapped in this study, therefore elucidating the most accessible Lysine residues of the AAV tested. Our detailed protocols method areis anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency.

INSTRUMENT(S): 6545 Q-TOF LC/MS

ORGANISM(S): Adeno-associated Virus 9

SUBMITTER: Anh Lam  

LAB HEAD: Anh Kim Lam

PROVIDER: PXD035837 | Pride | 2023-03-11

REPOSITORIES: Pride