Implications of AAV affinity column reuse and vector stability on product quality attributes

Area of Science:

• Biotechnology
• Bioprocessing
• Viral Vector Manufacturing

Background:

• Adeno-associated virus (AAV) vectors are crucial for gene therapy.
• Ensuring high product quality during AAV manufacturing is essential.
• Affinity chromatography is a key step in AAV purification.

Purpose of the Study:

• To investigate the impact of AAV capsid design and column reuse on AAV9 vector quality.
• To evaluate the performance of POROS CaptureSelect (PCS) AAVX and AAV9 resins.
• To understand how process impurities affect AAV purification and product quality.

Main Methods:

• Utilized sf9 insect cell-derived model AAV9 vectors with varying viral protein (VP) ratios.
• Performed chromatographic experiments using PCS AAVX and PCS AAV9 affinity resins.
• Assessed vector elution profiles, aggregate content, and impurity profiles over multiple column reuse cycles.

Main Results:

• Purified AAV9 vector elution was independent of VP ratio or cycle number.
• Process impurities in clarified lysates significantly altered elution patterns and increased vector aggregation.
• The AAV9-specific column showed higher aggregation than the AAVX column over reuse cycles.
• Vector aggregates contained double-stranded DNA and histone proteins, indicating carryover due to incomplete cleaning.

Conclusions:

• Feed impurities, resin type, elution pH, and cleaning procedures critically impact AAV affinity column reusability and vector product quality.
• Incomplete clean-in-place (CIP) contributes to host cell impurity carryover.
• Optimizing purification processes is vital for consistent AAV vector production.