Mechanistic modeling of empty‐full separation in recombinant adeno‐associated virus production using anion‐exchange membrane chromatography

Abstract

Recombinant adeno‐associated viral vectors (rAAVs) have become an industry‐standard technology in the field of gene therapy, but there are still challenges to be addressed in their biomanufacturing. One of the biggest challenges is the removal of capsid species other than that which contains the gene of interest. In this work, we develop a mechanistic model for the removal of empty capsids—those that contain no genetic material—and enrichment of full rAAV using anion‐exchange membrane chromatography. The mechanistic model was calibrated using linear gradient experiments, resulting in good agreement with the experimental data. The model was then applied to optimize the purification process through maximization of yield studying the impact of mobile phase salt concentration and pH, isocratic wash and elution length, flow rate, percent full (purity) requirement, loading density (challenge), and the use of single‐step or two‐step elution modes. A solution from the optimization with purity of 90% and recovery yield of 84% was selected and successfully validated, as the model could predict the recovery yield with remarkable fidelity and was able to find process conditions that led to significant enrichment. This is, to the best of our knowledge, the first case study of the application of de novo mechanistic modeling for the enrichment of full capsids in rAAV manufacturing, and it serves as demonstration of the potential of mechanistic modeling in rAAV process development.

Removal of empty capsids from high‐dose adeno‐associated virus 9 gene therapies

“…A similar NaCl-driven rAAV9 elution order was previously reported (Lock & Alvira, 2016b). Conversely, other serotypes elute "empty, then full," in-line with expected higher negativity of full capsids (Dickerson et al, 2021;Gomis-Fons et al, 2023;Joshi et al, 2021;Lavoie et al, 2023;Nass et al, 2018;Qu et al, 2007). The unintuitive rAAV9 elution order reported here and by Lock and Alvira might be driven by capsid structure (DiMattia et al, 2012).…”

“…Most literature reports of scalable, reproducible EC reduction have leveraged anion-exchange chromatography (AEX) (Dickerson et al, 2021;Khanal et al, 2023;Lavoie et al, 2023;Okada et al, 2009;Qu et al, 2007;Tomono et al, 2018), which has been employed for many rAAV serotypes (Joshi et al, 2021;Nass et al, 2018). AEX resolves rAAV capsid species based on charge via differential interaction with a cationic ligand immobilized on a resin (Urabe et al, 2006), membrane (Gomis-Fons et al, 2023;Hejmowski et al, 2022), or monolith (Dickerson et al, 2021).…”