Elucidating the Interplay Between Resin Media, Feed Constituents, and Process Conditions in AAV Affinity Column...

Area of Science:

• Biotechnology
• Chromatography
• Microscopy

Background:

• Adeno-associated virus (AAV) purification relies on affinity chromatography.
• Understanding resin behavior during loading, elution, and cleaning is crucial for process optimization.
• Resin reuse impacts efficiency and purity in biopharmaceutical manufacturing.

Purpose of the Study:

• To investigate adeno-associated virus (AAV) loading and fouling in affinity resins using confocal microscopy.
• To compare the performance and reusability of PSDVB-based POROS CaptureSelect (PCS) and agarose-based AVIPure AAV9 resins.
• To elucidate the impact of resin characteristics and process conditions on column lifetime.

Main Methods:

• Confocal microscopy was used to examine AAV adsorption profiles in resin beads after various process steps.
• Commercial AAV affinity resins (PCS and AVIPure) were tested with different feed mixtures.
• High-resolution X-ray CT scans were employed to analyze resin pore structure.

Main Results:

• PCS resins allowed full AAV access initially, while AVIPure resins showed surface-limited binding.
• PCS resin fouling occurred upon reuse due to AAV and impurity carryover, leading to pore occlusion.
• AVIPure resins demonstrated effective AAV elution and removal during cleaning-in-place (CIP).

Conclusions:

• Resin pore size (PCS large pores) influences initial AAV transport.
• Fouling in PCS resins upon reuse significantly impedes solute transport in subsequent cycles.
• AVIPure resins exhibit better reusability and resistance to fouling compared to PCS resins under tested conditions.