A proof of concept study on the use of online two-dimensional SEC-UV-RPLC-MS method for the multi-attribute characterization of gene therapy products

Abstract

Background: Recombinant adeno-associated virus (rAAV) vectors have emerged as a leading platform for gene therapy, supported by several FDA-approved products and numerous clinical trials. Due to their biological complexity and heterogeneity, rigorous analytical methods are essential to ensure the quality, safety, and efficacy of rAAV-based products. Critical quality attributes (CQAs) such as aggregation, full/empty capsid ratios, viral protein composition, and post-translational modifications (PTMs) must be monitored comprehensively. While several chromatographic techniques address individual CQAs, there is a growing industrial need for techniques that enable multi-attribute analysis in a single run.

Results: In this study, we developed a multidimensional size-exclusion chromatography-ultraviolet detection-reverse-phase liquid chromatography-mass spectrometry (SEC-UV-RPLC-MS) method for the comprehensive characterization of rAAV vectors. The method enables simultaneous assessment of high molecular weight species (aggregates), capsid identity, and PTMs. In the first dimension, SEC separates intact viral particles and quantifies aggregates, followed by a targeted heart-cut of the monomeric fraction. This fraction is then subjected to RPLC for protein separation under denaturing conditions, with high-resolution MS used to identify viral proteins and their modifications. Initially optimized for rAAV8, the method was successfully extended to other rAAV serotypes, demonstrating its adaptability across serotypes. A forced degradation study under thermal stress conditions further highlighted the method's ability to monitor structural integrity and stability of the viral vectors.

Significance: This study presents a novel 2D-LC-MS approach for streamlined, multi-attribute analysis of viral vector products. By combining multiple analyses into a single run, this 2D-LC-MS method can be considered as a powerful tool for comprehensive analysis of gene therapy vectors in the future. Potential applications include real-time in-process control, comparability studies, stability testing, support during process development for aggregate or PTM profiling, and drug substance or product characterization.

Keywords: AAV; Adeno-associated virus; Gene therapy; Heart-cutting; Multi-attribute method; Two-dimensional liquid chromatography.