Design of Experiment (DoE) Based Optimization of Full AAV Particle Enrichment Using Stunner® as a High-Throughput Analysis Tool

Posted: 13 November 2023

Unchained Labs invited Ascend to discuss how we use the Stunner to analyze anion exchange (AEX) chromatography as part of a design-of-experiment (DoE) study. The discussion was a great one to participate in and highlights our willingness to work with different suppliers and ensure our customers have access to the best technologies on the market.

In this webinar, Nelis Denys of Unchained Labs describes how the company’s Stunner® technology, which simultaneously leverages UV-Vis and dynamic and static light scattering (DLS and SLS) can generate viral vector capsid and genome titer, empty/full capsid ratio, %-full capsid, and aggregation data using just 2 uL of sample in less than a minute for up to 96 samples at a time.

Regina Staffler then explains how Ascend employed Stunner to analyze anion exchange (AEX) chromatography runs as part of a design-of-experiment (DoE) study implemented to develop an optimal downstream polishing step for enriching the %-full capsids generated using the company’s upstream adeno-associated viral (AAV) vector platform process.

Using two separate salt concentrations (lower than higher) it is possible to have the empty and full particles elute separately on a strong AEX resin. Baseline runs were first performed to determine the critical process parameters (CPPs) and define ranges for them. Next, a full-factorial DoE was implemented to maximize both the VG titer and %-full capsids varying the salt concentration of the empty wash buffer and the quantity of loaded capsids per column volume. Stunner was used for the analytical readouts because of its high accuracy, rapid analysis time, and lower cost compared to other methods. Six replicates were analyzed to ensure good statistical precision, and one entire plate was completed in less than 70 minutes. The entire study including buffer preparation, run performance, and evaluation of capsid and VG titer and %-full capsids was completed within 2 weeks.

It was possible to obtain a VG recovery of greater than 70% and enrichment of full capsids to greater than 52%. The process was shown to be robust with respect to varying input material and successfully upscaled 40-fold. In addition, the Stunner data showed excellent agreement with data obtained using qPCR and ELISA assays. Consequently, it has been implemented at Ascend as a universal in-process control tool for high-speed downstream, process development post-affinity capture.