Integrated metrology approaches for the characterisation of recombinant adeno-associated viral vectors: from molecular assays to mass spectrometry

Characterisation of recombinant adeno-associated viral (rAAV) vector products for therapeutic use is essential to ensure the safety and efficacy of gene therapies. However, several current analytical techniques used to assess the critical quality attributes (CQAs) have limitations, such as low precision, limited resolution, and poor comparability between methods. To overcome these limitations and support the assessment of CQAs, a comprehensive measurement framework based on metrology is needed. This includes the development of advanced reference methods to enable comparability and traceability of measurements, support the optimisation of existing assays, and guide the development of reference materials. This thesis describes the development of higher-order analytical methods for measuring CQAs of rAAVs. Initially, rAAV preparations were characterised using standard and widely used techniques for quantification of genome and capsid titres, as well as assessment of the purity of rAAV samples and percentage of full particles. These current practice techniques, consisting of quantitative polymerase chain reaction (qPCR) for genome titre analysis, enzyme-linked immunosorbent assay (ELISA) for capsid titre analysis, and sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) for purity analysis, formed the basis for comparison with more advance analytical methods developed in this study for comprehensive characterisation of both the rAAV capsid and genome. For genome characterisation, single-target digital polymerase chain reaction (dPCR) assays were initially developed for quantifying the genome titre and were compared to qPCR. However, while single-target assays provide information on genome titre, they do not offer insights into genome integrity. To address this limitation, multiplex dPCR assays were developed for a more comprehensive characterisation of rAAV preparations, enabling evaluation of genome integrity alongside quantification of genome titre across different regions of the rAAV genome. Genome integrity data obtained through multiplex dPCR in rAAV preparations were then compared to genome integrity data generated using nanopore sequencing. For impurity characterisation, residual DNA originating from the host cells and production plasmids was identified using nanopore sequencing, while mass spectrometry (MS) was employed to detect host cell proteins (HCPs). A total of 100 HCPs were identified, 24 of which have previously been reported in rAAV preparations in the literature. For capsid characterisation, an MS-based approach, traceable to the International System of Units (SI), was developed for absolute quantification of individual capsid proteins of rAAV9 (VP1, VP2, and VP3), determination of total capsid content, and measurement of capsid stoichiometry. The findings indicate that the measured capsid stoichiometry of the analysed rAAV9 sample deviates from the widely assumed 1:1:10 ratio of VP1:VP2:VP3. Additionally, MS analysis detected deamidations in both rAAV8 and rAAV9 capsids. Together, the findings of this study will contribute to improved measurement of rAAV CQAs, which can, in turn, inform process development, enhance understanding of how these attributes influence the overall quality of rAAV-based gene therapy products, and ultimately support improved product quality and batch-to-batch consistency.