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

In this work, confocal microscopy is employed to study the loading and fouling behavior in AAV affinity resins as well as the implications of resin reuse with several commercial chromatographic materials and feed mixtures. Resin samples are obtained from both batch and column experiments, and confocal microscopy is carried out to examine the adsorption profiles in the beads after loading, wash, elution, and CIP steps. A comparison of PSDVB-based POROS CaptureSelect (PCS) AAV resins with agarose-based AVIPure AAV9 resins revealed distinct differences in both AAV transport and resin fouling. While AAVs were able to fully access the entire PCS AAV resin under loading onto virgin resin materials, they were restricted to the surface region of the AVIPure AAV9 resin. High resolution X-ray CT scans indicate that the large pores of the PCS media are likely responsible for this enhanced transport. However, AAV transport into full PCS AAV resin volume was found to only occur during the first AAV loading experiment and was not observed when clarified lysate was employed or upon resin reuse. Further, confocal microscopy indicated that fouling of the PCS AAV affinity resins occurred due to deposition and carryover of AAV and residual impurities upon column re-use, likely due to incomplete elution and ineffective column CIP. Interestingly, this residual binding in the region near the resin surface resulted in limiting solute transport in subsequent cycles, likely due to pore occlusion. In contrast, AAV bound to the AVIPure resin was readily eluted and removed during CIP. This study elucidates the impact of resin characteristics, feed constituents, and process conditions on the lifetime and fouling of AAV affinity columns.