Exploring Development Options of a Polishing Chromatography Step for AAV7 and AAV8

Area of Science:

• Biotechnology
• Biopharmaceutical Manufacturing
• Gene Therapy

Background:

• Empty adeno-associated virus (AAV) capsids impact drug safety and quality, necessitating effective removal strategies.
• Anion-exchange chromatography (AEX) shows promise for separating empty from full AAV capsids, but challenges remain due to subtle charge differences.
• Multimodal (mix-mode) chromatography can introduce secondary interactions to improve separation of challenging AAV subpopulations.

Purpose of the Study:

• To develop a polishing chromatography step for removing empty capsids from AAV7 and AAV8 preparations.
• To investigate critical process parameters and column chemistries for optimizing AAV purification.
• To evaluate the efficacy of multimodal chromatography as an alternative to traditional AEX for difficult-to-separate AAV variants.

Main Methods:

• Utilized challenging feed materials with small gene-of-interest (GOI) and poor packaging efficiency.
• Investigated buffer matrix, salt concentration, pH, fractionation strategies, and column chemistry (strong AEX vs. mix-mode).
• Employed mass photometry (MP) and charge detection mass spectrometry (CDMS) for capsid population characterization.

Main Results:

• Optimized AEX for AAV8 achieved 80% full capsids (MP) and 90% GOI-containing capsids (CDMS).
• For AAV7, multimodal chromatography demonstrated superior resolution compared to standard AEX gradient methods.
• Multimodal chromatography proved effective for serotypes where traditional AEX struggles with separation.

Conclusions:

• Multimodal chromatography is a viable alternative for polishing AAV preparations, especially for challenging serotypes.
• Optimized chromatography strategies enhance the purity of AAV gene therapy products.
• This study provides valuable insights for developing robust downstream purification processes for AAV-based therapeutics.