AAV Characterization

SMART Digest Pepsin Mag Beads (60109-110-MB)

• Adeno-associated virus (AAV) is a small, non-pathogenic virus used as a gene therapy vector due to its ability to deliver genetic material into cells

• Characterization of AAV capsid and corresponding post translational modifications (PTM), should be performed to confirm the correct production sequence, product purity, and storage stability

• Conventional peptide mapping of viral capsids is time-consuming and often irreproducible due to their high structural stability and lack of optimal trypsin cleavage sites

• Presented here is an optimized peptide mapping-based workflow using SMART Digest Pepsin that provides thorough characterization within 1 day

“We anticipate that our workflow will greatly benefit fast paced pharmaceutical environments that aim to quickly and completely characterize AAV based gene therapy products through peptide mapping analysis.” Journal of Pharmaceutical and Biomedical Analysis 207 (2021) 114427

 Example protocol

• A volume of 25μL of water was added to the 25 μL AAV sample and then diluted 1:4 (v/v) with the Pepsin SMART Digest buffer (pH 2) provided with the kit.

• A volume of 5 μL of 0.5 M TCEP was added to the digestion mixture.

• To initiate digestion, 15 μL of the Magnetic SMART digest pepsin resin (Part no. 60110-101-MB) was then added to the sample (corresponding to 14 μg of heat-stable immobilized pepsin), and the reaction vessels were placed on an Eppendorf thermomixer equilibrated at 70 °C.

• In all samples, the peptic digestion was allowed to proceed at 70°C for 45 min at 1400 rpm.

• After the digestion, the digestion vessel was placed in a side magnetized plate, and the supernatant was transferred to a fresh tube.

• To ensure no residual resin was present in the sample, the side magnetic attraction of enzymatic beads and transfer of supernatant was repeated.

• All samples were diluted with 0.1% formic acid (FA) in water to a final protein concentration of 1 μg in 200 μL, and 5 μL of this digest mixture was loaded on the column for each chromatographic analysis.

 Select publications

• Toole, E. N., et al. (2021). Rapid Highly-Efficient digestion and peptide mapping of Adeno-Associated viruses. Analytical Chemistry, 93(30), 10403–10410.

• Smith, J., et al. (2023). Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins. Journal of Proteome Research, 23(1), 161–174.

• Guapo, F., et al. (2021). Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs). Journal of Pharmaceutical and Biomedical Analysis, 207, 114427.

• Takino, R., et al. (2024). Physicochemical and biological impacts of light stress on adeno-associated virus serotype 6. Molecular Therapy — Methods & Clinical Development, 32(4), 101362.

• Smith, J., et al. (2023). Rapid characterization of adeno-associated virus (AAV) capsid proteins using microchip ZipChip CE-MS. Analytical and Bioanalytical Chemistry.