Experts Webinar Series: Adeno Associated Viruses Characterization

Protein Characterization of AAV Vectors using Mass Spectrometry

Adeno associated viruses (AAV) are gene-delivery vectors carrying their genetic payload inside a protein capsid comprised of three viral proteins (VP1/2/3). The viral protein coat interacts with structural elements on various target-cell types where the gene of interest is transported. Aspects of VP primary structure including post-translational modifications (PTMs) may impact AAV activity and gene-delivery efficacy. To capture important aspects of AAV protein primary structure, approaches to peptide mapping and intact protein subunit mass analysis are discussed in relation to low-protein content samples. Complete sequence and PTM characterization remain a requirement, and key structural elements need to be established with high-quality MS2 spectra. MS-based analyses of host cell proteins (HCPs) from AAV expression systems is also discussed as LC-MS HCP assays may be critical where compressed development timelines and the lack of product-specific ELISAs are gating factors.

Presenter: Andrew Hanneman, Ph.D. (Charles River Laboratories Biological Testing Solutions, Shrewsbury, Massachusetts)

Andrew Hanneman, Ph.D. is Scientific Advisor at Charles River Laboratories Biologics Testing Solutions in Shrewsbury, Massachusetts. Dr. Hanneman has been an Analytical chemist specializing in biological molecules for over 30 years, with more than 15 years’ experience in biotherapeutic protein characterization using mass spectrometry. Dr. Hanneman has led groups specializing in glycoprotein analysis and conducted projects covering diverse range of molecules at various biopharma companies in the Greater Boston area.

An insight into CE Method Development to assist implementing a cIEF separation of Adeno Associated Virus (serotype 8): a promising bioanalytical technique to determine empty, partial and full capsids.

Capillary isoelectric focusing (cIEF) is a well-established bioanalytical technique capable of separating closely related species based on their isoelectric points. Adeno-associated viruses (AAV) have rapidly become suitable0 cIEF candidates for empty vs full determination, due to pI differences: full AAV capsids generally have lower isoelectric points due to their negatively charged DNA load, compared to empty or partially filled capsids. Our preliminary results show the separation of three well-resolved peaks from proprietary formulations of both empty and full AVV-GFP serotype 8 samples. While further method optimization for each serotype and complimentary characterization techniques are required to confirm empty, partial and full identities, data to be presented confirms cIEF is a promising tool exhibiting great resolving power to separate this species. Separation conditions, method development tips & tricks on how to overcome well-known cIEF technique-related challenges will be discussed to help implementing a cIEF method for this purpose.

Presenter: Lorena Lopez (Application Scientist, Agilent Technologies, Inc.)

Lorena has a degree in Chemical Engineering and an MBA focused on Life Sciences and is currently based out of the Agilent facilities in Lexington, Massachusetts. She has been with Agilent for 7 years tightly involved within Liquid Chromatography, Capillary Electrophoresis and Dissolution instrumentation. She acquired her strong background expertise in the bio and pharmaceutical industry working for companies such as Merck and Eli Lilly where she started her early career as a QC laboratory scientist, followed by leader roles in analytical Method Development of brand-new small molecule formulations. She also spent several years as a member of a Life Sciences Research Institute, which gave her a broad perspective on the analytical LC and biopharma fields where she has been focusing for the last couple of years within her current role as an LC Application Scientist.

LC/MS of Intact Adeno-Associated Virus Capsid Proteins for Rapid Confirmation of Product Identity

Adeno-associated viruses (AAVs) are a promising new class of biotherapeutic. The FDA requires AAV capsid proteins to be unambiguously identified before release, especially in facilities where multiple serotypes or engineered variants are produced. This can be challenging with affinity-based measurements due to the small differences that exist between serotypes or variants. In this webinar we describe a method for rapidly analyzing AAV capsid proteins with Agilent’s 6545XT AdvanceBio LC/Q-TOF high-resolution mass spectrometer. In addition, we demonstrate its versatility by successfully analyzing a number of different serotypes. This method has easy sample preparation requirements and is robust to high salt conditions as well as the common nonionic detergent additive Pluronic F-68.

Presenter: Brian Liau, Ph.D. (Solution Development Scientist, Singapore, Agilent Technologies, Inc.)

Dr. Brian Liau is a graduate of Johns Hopkins University with a background in Biomedical Engineering. He is currently a Scientist at Agilent’s Global Solution Development Center in Singapore. There, he develops new analytical techniques for emerging treatment modalities, including adeno-associated viral vectors and mRNA vaccines. Prior to joining Agilent, he was a research fellow at the Bioprocessing Technology Institute (A*STAR) where he created new spectroscopic and LC-MS based technologies for glycan analysis.