Intact AAVs act as a vehicle to protect and deliver oligonucleotide therapeutics for gene therapies. The full capsid needs to be carefully characterized to ensure product efficacy, and impurities such as host cell DNA and proteins need to be accurately characterized and controlled to ensure product quality and safety.

Separation and analysis of AAVs

Thermo Scientific ProPac 3R SAX 3 μm Columns provide excellent separation of full and empty AAV capsids and other impurities using both a linear salt gradient and a linear salt gradient with an isocratic hold as demonstrated in the application note Salt gradient separation and analysis of adeno-associated virus samples using a 3 μm monodisperse strong anion exchange chromatography column. Their unique column design provides the high resolution, robust performance, and lot-to-lot reproducibility needed for AAV analysis.

Host cell protein and peptide mapping of AAVs and their post translational modifications

Besides the full AAV capsid containing the desired gene material, the final product can contain many different types of process and product related impurities. Among the quality attributes that need monitoring, characterization of the AAV capsid proteins’ amino acid sequences and any associated post translational modifications (PTM) present should be performed. This full characterization should also include process-related impurities, such as residual host cell proteins (HCPs) derived from the production cells, to mitigate immunogenicity and ensure the safety, quality, and efficacy of AAV products. LC-MS based peptide mapping commonly used for recombinant protein analysis can provide sequence coverage, PTM information, and identification of HCPs to improve product development and deployment of associated manufacturing processes.


As much as 100% sequence coverage can be achieved by using Thermo Scientific SMART Digest Pepsin Kits for the digestion of AAV capsids, as described in the application note Adeno-associated virus capsid protein characterization and host cell protein profiling using micro-flow UHPLC-Orbitrap MS. Furthermore, Thermo Scientific Hypersil GOLD C18 Columns are excellent for peptide mapping analysis as the columns contain secondary interaction-preventing ultrapure silica with a unique pore size that is beneficial for the peak shape of the peptide.

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