Development of LC-MS methods for AAV capsid protein quantification and host cell protein profiling.
Theodoros Kontogiannis1,2, Christopher McElroy2, Milena Quaglia3
1School of Natural Sciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Molecular Therapy. Methods & Clinical Development
|September 8, 2025
Summary
Accurate quantification of recombinant adeno-associated virus (rAAV) capsid proteins is essential for gene therapy. A new mass spectrometry method precisely measures rAAV9 capsid stoichiometry and content, offering improved characterization over traditional methods.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Molecular Biology
Background:
- Accurate quantification and characterization of recombinant adeno-associated virus (rAAV) capsid proteins are critical for product quality, safety, and process development.
- rAAV capsids are composed of VP1, VP2, and VP3 proteins, with variations in stoichiometry potentially affecting vector infectivity.
Purpose of the Study:
- To develop and validate a mass spectrometry (MS)-based method for precise quantification and stoichiometry determination of rAAV9 capsid proteins (VP1, VP2, VP3).
- To assess the utility of MS for measuring total capsid content and characterizing host cell proteins in rAAV products.
- To establish MS as a potential reference method for standardized rAAV measurements.
Main Methods:
- Development of a mass spectrometry (MS)-based assay for quantifying VP1, VP2, and VP3 proteins in rAAV9 capsids.
- Application of the MS method to determine capsid stoichiometry and total capsid content.
- Identification and characterization of host cell proteins present in rAAV samples.
Main Results:
- The developed MS method accurately quantifies VP1, VP2, and VP3 proteins and determines capsid stoichiometry in rAAV9.
- The methodology provides precise measurements of total capsid content, offering more detailed information than ELISA.
- Consistent detection of relevant host cell proteins was achieved, highlighting their importance in gene therapy products.
Conclusions:
- Mass spectrometry is a powerful tool for precise characterization of rAAV, including capsid protein quantification and stoichiometry.
- The developed MS approach offers a standardized method for measuring rAAV across different drug products, surpassing traditional methods.
- Monitoring host cell proteins using MS is crucial for ensuring the safety and quality of gene therapy products.
Keywords:
AAVcapsidcritical quality attributesdeamidationhost cell proteinsmass spectrometryprotein quantificationserotypestoichiometryviral vectorsMore Related Videos
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