Gene therapy has emerged as a cutting-edge modality with transformative potential for treating a broad range of genetic disorders. Despite significant advancements, the purification and separation of the product of interest from process- and product-related impurities remains a major challenge in the development, particularly for adeno-associated virus (AAV) vectors.
The main bottlenecks were development cost and timelines, according to Kevin Vera, Scientist II, process development, pharma services group, Thermo Fisher Scientific. Conventional approaches require large feed volumes. By implementing high-throughput purification technologies, we reduced feed requirements by five to 10 times, enabling far more efficient use of material,” he said. “In parallel, the throughput for downstream chromatography optimization studies increased by nearly 10 times, giving us the ability to evaluate many more conditions and design spaces in a fraction of the time.”
Leveraging QbD principles, the high-throughput downstream development platform for viral vectors increased the throughput of downstream purification operations to over 100 purifications per week, supported by at-line analytical tools to generate rapid readouts of product quality. These include:
• UV absorbance ratios (260/280 nm) for nucleic acid to protein content
• Size exclusion chromatography with multi-angle static light scattering (SEC-MALS) for size distribution and aggregation, and titer measurement
• Mass photometry for percent full determination
“These technologies deliver near real-time readouts of key quality attributes—capsid titer, genome content, and percent full capsids—enabling rapid process adjustments and faster, data-driven decisions,” noted Vera, adding that automation and robotics played a key role in achieving over 100 purifications per week.
“Automation and robotics were essential in scaling throughput. We implemented robotic liquid handling for sample preparation and automated column handling systems, all coordinated under a centralized scheduling framework,” Vera told Gen. “This eliminated manual bottlenecks, minimized operator variability, and enabled runs to proceed in parallel with minimal intervention. As a result, we were able to scale consistently to over 100 purifications per week—a level of throughput that would be impossible with manual workflows—while maintaining reproducibility and data integrity.”
The results highlight advancements in high-throughput downstream purification for AAVs as well as formulation development for process intermediates, pointed out Vera.
“This platform enables rapid process characterization and optimization, significantly reducing development timelines while supporting robust process understanding and consistent product quality,” he said.
“I would like to thank Pouria Motevalian, Director, Process Development, Viral Vector Services, Thermo Fisher Scientific, for his expert collaboration on this project.”