Integrated metrology approaches for the characterization of viral vectors: From mass spectrometry to molecular assays

Viral vectors are key components used in biotherapeutic applications including gene therapy and as vaccine delivery agents. The measurement of their critical quality attributes: identity, purity, potency, titre, safety, and stability and associated sources of analytical bias and error are essential, making the role of sound measurement science in viral vector characterization of utmost importance. This study focuses on developing and investigating the performance of methods used for the characterization of recombinant adeno-associated virus (rAAV) using a combination of mass spectrometry, PCR-based molecular assays. Mass spectrometry was employed to identify a unique peptide specific to each AAV9 viral capsid protein and a peptide present in every capsid protein, enabling absolute quantification of the viral capsid proteins using Stable Isotope Dilution (SID) and measurement of the ratio VP1:VP2:VP3 in AAV9. We developed improved digital PCR (dPCR) approaches to assess genome integrity and provide linkage information between different genome targets. Furthermore, we identified potential sources of bias and error in our developed dPCR approach, and we demonstrate the critical role of genome target selection in dPCR measurements of AAV titre, emphasizing its impact on measurement variability. Additionally, we show that multiplex dPCR can be used effectively for genome integrity assessment of AAV vectors, surpassing the limitations of current methods.