Introduction: Viral cell and gene therapies are a hot area of medical development with a rapidly maturing regulatory environment. Recently, an automated Orbitrap-based charge detection mass spectrometry platform was built to robustly measure both the precise masses of AAV therapeutics and their composition (i.e., the percentage of product containing or not containing genetic cargo). Here, we both benchmark this capability and integrate it with a companion denatured workflow, capturing precise composition at both an intact and base component level to provide a more holistic view of viral products for a variety of isotypes.

Methods: Empty and full AAV isotypes were purchased and mixed volumetrically to achieve predefined ratios. A SampleStream (Integrated Protein Technologies) fluidic device was used to desalt and inject AAV products into a Q Exactive UHMR (Thermo Fisher) enabled with Direct Mass Technology Mode. For intact mode, mixtures were buffer exchanged into the injection solution using a molecular weight cutoff membrane and injected via heated electrospray. Masses and ratios were determined using STORIBoard (Proteinaceous). For intact mode, the membrane cutoff was 100 kDa, and the injection solution was 100 mM ammonium acetate. For component mode, the membrane cutoff was 5 kDa and the injection solution was a mixture of acetonitrile, water, and formic acid.

Key Takeaways: By tracking both the intact and disassembled forms of AAVs, it is possible to more holistically track the qualities of AAV products, including different proteoforms for AAV components and RNA size for quality control purposes.

John McGee

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