Aggregation Analysis in AAV: Selecting Fit-for-Purpose Assays

AAV-based gene therapy samples are heterogeneous and can be composed of capsid monomer, aggregate, and fragment species. Tracking the relative abundance of these size-variant populations in the product and manufacturing process allows for greater control in delivering a consistent product to patients, and characterizing these species by size, shape, and composition provides a greater understanding of aggregation assay readouts. This study describes a framework for selecting fit-for-purpose aggregation assays based on assay context, including product release, in-process testing, characterization, and discovery/early-stage development, by carefully weighing assay parameters including specificity, precision, accuracy, software compliance, selectivity, sensitivity, turnaround time, throughput, platform compatibility, and sample requirements. This study also describes a reproducible forced-aggregation protocol for generating small, soluble capsid aggregate controls that can be used to assess assay performance across a wide variety of assays, including TEM, AUC, SEC, SEC-MALS, and FFF-MALS. Finally, this webinar summarizes results from a case study evaluating eight aggregation assays to empirically determine which assays are best suited for each assay context.

Presented by Margaret Butko, Ph.D., Senior Advisor of CMC Assay Development at Eli Lilly and Company

Margaret Butko, Ph.D., is Senior Advisor of CMC Assay Development at Eli Lilly and Company, formerly Adverum Biotechnologies, where she leads protein analytical and biophysical characterization for gene therapy programs. She has 13 years of experience in assay development spanning small molecule, therapeutic antibody, and AAV gene therapy modalities, as well as supporting programs from early discovery through post-commercialization. She has broad expertise across liquid chromatography, capillary electrophoresis, mass spectrometry, light scattering, microscopy, spectroscopy, and ligand-binding assays, and has led evaluation and implementation of new technologies for drug discovery and development applications. She currently leads the Protein Analytical Characterization and Development and Testing Analytics groups, overseeing method development, qualification, and validation for internal and outsourced assays.

Dr. Butko earned her B.A. in chemistry, summa cum laude, from the College of Wooster and her Ph.D. in biomedical sciences from the University of California, San Diego. She completed a postdoctoral fellowship in Protein Analytical Chemistry within Global Technical Development at Genentech. She is a frequent invited speaker at industry conferences, including the Gene Therapy Analytical Development Summit, and has been with Adverum, now part of Eli Lilly and Company, for 6.5 years.

Followed by Rickard Nordström Chief Operating Officer at QuTEM

Rickard Nordström is co-founder and Chief Operating Officer of QuTEM. He holds an M.Sc. in Molecular Cell Biology and conducted his Ph.D. studies at Karolinska Institutet. For over a decade, he has held a central role in building the organization and leading scientific method development of both Vironova and QuTEM. With more than 15 years of experience in adeno-associated virus (AAV) analytics, Rickard has developed proprietary methodologies for genome length determination and vector content analysis — enabling the quantification of full, empty, partially full, and overfull capsids — which are now established tools for batch release testing and regulatory submissions within the industry. He is also the originator of QuTEM’s AAV aggregation analysis method and maintains close collaborative relationships with analytical teams at leading pharmaceutical organizations. In addition to his scientific and operational contributions, Rickard has extensive experience representing organizations during Good Manufacturing Practice (GMP) audits and regulatory authority inspections.

Sponsored by QuTEM AB

QuTEM is a world-leading provider of GMP-certified TEM-services and quality control analyses of nanoparticles, based in Stockholm, Sweden. Our dedicated team of experts work in close partnership with leading pharmaceutical companies worldwide to provide analysis solutions and support. With nearly two decades of industry experience, a profound proficiency in interdisciplinary sciences, and an unwavering commitment to excellence, we proudly stand as the premier choice in providing TEM services. Using latest generation instruments together with Gridsee, our proprietary software developed in house tuned for image analysis, we developed validated methodologies to ensure the delivery of high-quality services and reliable data to our clients.

QuTEM is focused on advancing the characterization of adeno-associated viruses (AAVs) with respect to safety, efficacy, and quality attributes. In support of this mission, cryoTEM technology, combined with robust image analysis tools, has been recognized by both the U.S. Pharmacopeia (USP) as a new analytical method for full/empty capsid analysis, and by the British Pharmacopoeia — recognition that underscores its accuracy, precision, and robustness within QC and GMP-regulated environments. Building on this foundation, QuTEM is further developing new cryoTEM-based methods for AAV aggregate analysis and physical titer determination.