Design of Experiment (DoE) Based Optimization of Full AAV Particle Enrichment Using Stunner® as a High-Throughput Analysis Tool
Packaging heterogeneity in recombinant adeno-associated virus (rAAV) manufacturing may lead to potential product-related impurities like „empty” capsids, capsids containing partial vector genomes, and capsids containing different DNA impurities. It is technically very challenging to reduce or eliminate „empty” capsids in downstream processing, especially in a scalable manner. Here we describe a fully scalable method to efficiently enrich the percentage of full capsids by anion exchange chromatography.
An anion exchange (AEX) chromatography step was established in isocratic elution mode and optimized by design of experiment (DoE) studies. DoE data were evaluated measuring the percentage-„full” and the vg-recovery of the enriched eluate by Stunner® (Unchained Labs). Stunner was the analysis-tool of choice for this study due to its high accuracy and speed for generating the DoE response data. DoE center point run results were confirmed using additional techniques.
Based on the DoE studies it was determined that the capsid load and salt concentration during the empty particle elution phase are the most critical process parameters to achieve a high percentage of full particles and a satisfactory yield. The enrichment factor in fold-enrichment was investigated and found to depend on the proportion of „full” and „empty” capsids in the load material. For our optimized conditions we obtained an up to three-fold enrichment of full capsids compared to input material.
The AEX chromatography process is designed to be optionally plugged into downstream processing depending on required product specifications. It is readily scalable and is a tool that can be adapted for specific expression cassettes or capsids. In this case, the salt concentrations could be adapted, and an identical DSP work package applied, all within two weeks using Stunner.
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