The use of adeno-associated virus (AAV) in gene therapy applications involves meeting stringent standards for impurity thresholds in AAV sample formulations. Clearance of “empty” AAV capsids which lack the gene of interest at preparative purification scale presents an ever growing challenge for process scientists. Here we showcase the progress in implementing at preparative scale our novel conductivity step elution method that utilizes Mustang Q XT membrane for full AAV capsid enrichment. We previously developed this technique using AAV5, AAV8, and AAV9 with Mustang Q 0.86 and 5 mL devices. We compared absorbance measurements (260 nm vs. 280 nm) to determine the elution conditions for obtaining optimal amounts of full capsids from an affinity purified pool. The majority presence of full capsids in these elution fractions was confirmed with PCR and intact capsid ELISA. Using the stacked 0.86 and 5 mL devices, we previously demonstrated that this approach to enrichment of full AAV capsids can be used as a rapid process development tool. Herein, we extend this method to radial flow devices such Mustang Q XT50 capsule. Radial flow devices incorporate flow profile and pleated membranes that differ from flat-sheet devices present in Mustang XT 0.86 and 5 mL capsules. We present the results from our scale up trial of AAV5 capsid purification using Mustang Q XT50 capsules and compare the enrichment of full AAV5 cpasids and the clearance of host cell protein and DNA contaminants to our small scale studies.

Scale-up separation of full and empty AAV capsids using Mustang® Q XT50 capsule

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.