A Two-Dimensional Hyphenated Ion-Exchange Chromatographic Platform for Simultaneous Assessment of AAV8 Capsids and Impurity Fingerprinting in Upstream Processes
Timotej Žvanut, Tjaša Kašček, Lara Vogrinčič, Petra Dekleva, Sandra Potušek, Anže Martinčič Celjar, Ivana Petrović Koshmak, Blaž Goričar, Mitja Martelanc, Aleš Štrancar, Andreja Gramc Livk
Journal of Chromatography B, Volume 1273, 2026
Abstract:
Monitoring full versus empty AAV capsids and impurity fingerprinting is crucial for optimizing upstream processes. Conventional methods, such as PCR-ELISA and 1D LC, often lack robustness and reliability. In this study, we achieved powerful characterization of AAV capsids and indicative impurity fingerprinting in complex upstream samples.
We developed a streamlined, high-resolution workflow using ion-exchange two-dimensional (2D) liquid chromatography (LC) on the PATfix AAV Switcher Platform that combines cation-exchange and anion-exchange. This workflow uses multi-wavelength UV–Vis, fluorescence, and light scattering to provide better control and insight into AAV8 sample heterogeneity. First, the CIM Trap SO3 cation-exchange column captures and concentrates AAV capsids. In the second dimension, the CIMac QA HR anion-exchange column provides enhanced capsid separation. Multiple detectors provide real-time, comprehensive insight into the upstream process, enabling accelerated, simplified optimization.
The system reliably distinguishes between capsids and impurities, demonstrating high recovery, negligible carryover, and reproducibility. Applicable to various AAV serotypes, this method surpasses one-dimensional (1D) LC by eliminating costly, time-consuming offline steps and filling a crucial analytical gap in AAV manufacturing.
