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

Abstract

In this work, the implications of AAV9 capsid design and column reuse on AAV9 vector product quality were assessed with POROS CaptureSelect (PCS) AAVX and AAV9 resins using sf9 insect cell-derived model AAV9 vectors with varying viral protein (VP) ratios. Chromatographic experiments with purified drug substance AAV9 model feeds indicated consistent vector elution profiles, independent of adeno-associated virus (AAV) VP ratio, or cycle number. In contrast, the presence of process impurities in the clarified lysate feeds resulted in clear changes in the elution patterns. This included increased aggregate content in the vector eluates over multiple cycles as well as clear differences in the performance of these affinity resin systems. The AAV9-serotype specific PCS AAV9 column, with lower vector elution pH, resulted in higher aggregate content over multiple cycles as compared to the serotype-independent PCS AAVX column. Further, the results with vectors of varying VP ratio indicated that while one vector type eluate displayed higher aggregation in both affinity columns over column reuse, the eluate with the other vector type did not exhibit changes in the aggregation profile. Interestingly, vector aggregates in the affinity eluates also contained double-stranded DNA impurities and histone proteins, with similar trends to the aggregate levels. This behavior upon column reuse indicates that these host cell impurities are likely carried over to subsequent runs due to incomplete clean-in-place (CIP). These results indicate that feed impurities, affinity resin characteristics, elution pH, column CIP, and vector stability can impact the reusability of AAV affinity columns and product quality.