06/24/2024
By Yong Suk Lee

The Zuckerberg College of Health Sciences, Department of Biomedical & Nutritional Sciences, invites you to attend a doctoral dissertation defense by Yong Suk Lee on "Separation of full, empty, and partial adeno-associated virus capsids via fluorescence-based anion-exchange chromatography with continuous recycling and accumulation."

Candidate Name: Yong Suk Lee
Degree: Doctoral
Defense Date: Wednesday, July 3, 2024
Time: 9 to 11 a.m.
Location: TBD
Thesis/Dissertation Title: Separation of full, empty, and partial adeno-associated virus capsids via fluorescence-based anion-exchange chromatography with continuous recycling and accumulation.

Advisor: Seongkyu Yoon, Professor, Chemical Engineering, University of Massachusetts Lowell

Committee

  • Jonghan Kim, Associate Professor, Biomedical & Nutritional Sciences, University of Massachusetts Lowell
  • Shannon L. Kelleher, Professor, Biomedical & Nutritional Sciences, University of Massachusetts Lowell
  • David McNally, Assistant Professor, Medicine, UMass Chan Medical School
  • Raghu Shivappa, Vice President, Global Biologics and Gene Therapy Process Development, Takeda

Brief Abstract:

The field of recombinant adeno-associated virus (rAAV) gene therapy has attracted increasing attention over decades. Within the ongoing challenges of rAAV manufacturing, the co-production of impurities, such as empty and partial capsids containing no or truncated transgenes, poses a significant challenge. Due to their potential impact on drug efficacy and clinical safety, it is imperative to conduct comprehensive monitoring and characterization of these impurities prior to the release of the final gene therapy product. Nevertheless, existing analytical techniques encounter notable limitations, encompassing low throughput, long turnaround times, high sample consumption, and/or complicated data analysis. Chromatography-based analytical methods are recognized for their current Good Manufacturing Practice (cGMP) alignment, high repeatability, reproducibility, low limit of detection, and rapid turnaround times. Despite these advantages, current anion exchange high pressure liquid chromatography (AEX-HPLC) methods struggle with baseline separation of partial capsids from full and empty capsids, resulting in inaccurate full-to-empty capsid ratio, as partial capsids are obscured within peaks corresponding to empty and full capsids. In this study, we present a unique analytical AEX method designed to characterize not only empty and full capsids but also partial capsids. This method utilizes continuous N-Rich chromatography with recycling between two identical AEX columns for the accumulation and isolation of partial capsids. The development process is comprehensively discussed, covering the preparation of reference materials representing full (rAAV-LacZ), partial (rAAV-GFP), and empty (rAAV-empty) capsids, N-rich method development, fraction analysis, determination of fluorescence response factors between capsid variants, and validation through comparison with other comparative techniques.