A two-dimensional hyphenated ion-exchange chromatographic platform for simultaneous assessment of AAV8 capsids and impurity fingerprinting in upstream processes

Reliable monitoring of full (F) versus empty (E) adeno-associated virus (AAV) capsids, along with impurity fingerprinting, is essential for optimising upstream processes (USPs). Conventional analytical workflows for USP monitoring, including PCR-ELISA and one-dimensional (1D) liquid chromatography (LC), often lack robustness, selectivity, reliability, and throughput, especially when analysing complex and impurity-rich samples. To overcome these challenges, we introduce a two-dimensional (2D) LC workflow that combines cation-exchange (CEX) capture in the first dimension with subsequent anion-exchange (AEX) separation in the second. The system incorporates multi-wavelength UV-Vis, fluorescence, and light scattering (LS), offering greater control over analysis and deeper insights into AAV8 sample heterogeneity while simplifying production analysis. The platform facilitates direct analysis of complex AAV8 USP samples-without the need for offline purification-achieving high sensitivity, high throughput, and robust separation. The two-dimensional system consistently resolved E and F capsids, distinguished partially filled intermediates, and enabled concomitant impurity monitoring. Validation studies demonstrated high recovery rates, negligible carryover, exceptional linearity, and reproducibility across matrices relevant to USP standards. Furthermore, the 2D method optimised for AAV8 was demonstrated to be applicable to other AAV serotypes. Compared to conventional one-dimensional liquid chromatography, orthogonal techniques confirm the superior selectivity and practicality of this method by obviating the need for costly, time-consuming, multi-step offline procedures. The integrated multi-detector two-dimensional workflow addresses a critical analytical gap in AAV manufacturing and provides a compelling tool for process monitoring, method standardisation, and quality assurance within biopharmaceutical analytics.