Estrategias de optimización y escalado para un paso de enriquecimiento de AAV reproducible en la línea CIMmultus® QA...

The advancement of adeno-associated virus (AAV) gene therapy applications faces significant challenges, particularly in the separation of empty (E), full (F), and intermediate capsids during manufacturing, as well as scaling up downstream processing. This study demonstrates the effectiveness of monolithic chromatography in maintaining high flow rates without sacrificing resolution across scales, making it ideal for large-scale applications. The CIMmultus® HR columns ensure high reproducibility at various purification scales, achieving ± 3% isoconductivity elution of empty AAV2/8 capsids.In combination with the improved method, it enriches full AAV capsids to 88% ± 9% with genome recovery of 77% ± 3% within this study. The screening of polishing columns exhibited highest enrichment and recovery of full capsids with an improved method developed on QA HR preparative column. High-throughput screening (HTS) on CIM® QA HR Monolithic Well Plate and subsequent single-column experiments confirmed these findings, facilitating predictions for implementing step elution approaches. The high reproducibility of the columns enabled the development of step elution methods in both bind-elute and flow through modes, with both approaches yielding comparable recovery and purity of full AAV capsids. Ultimately, the optimized AAV polishing method was successfully scaled up from 1 to 8000 mL monolith using step elution approach.