Interplay between resin media, feed constituents, and process conditions in aav affinity chromatographic systems
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Authors
Soni, Harshal
Issue Date
2024-12
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Chemical engineering
Alternative Title
Abstract
The emergence of gene therapies has provided a viable approach for treating previously incurable diseases and disorders. Adeno-associated virus (AAV) vector-based therapies have gained attention in clinical trials due to their efficient delivery of therapeutic genes. However, high costs of therapy, ranging from 0.8 to 3 million dollars per dose, render these treatments difficult to access for many patients.A major contributor to these exorbitant costs is the expenses incurred during the complex AAV manufacturing process. These costs arise from various sources, such as the use of expensive raw materials, low-scale production units, and poor yields during upstream processing. Additionally, the generation of high levels of impurities during the manufacturing process has led to substandard purification platforms that can lose up to 70% of the product.
Downstream processing ubiquitously relies on affinity chromatography as the AAV capture step. The expensive affinity resins used in this step are typically limited to single use in a cGMP process before needing replacement due to risks to AAV product quality in subsequent cycles. To cut down material costs, resin reuse is advantageous. However, the lack of a mechanistic understanding relating to the behavior of AAV products and feedstock constituents in existing affinity systems poses a major challenge in achieving an extended column lifetime. To fill this gap, this thesis presents the underlying factors that influence AAV affinity capture chromatography performance in relation to resin reusability.
To understand the effect of AAV capsid design, feed material constituents, and column recycling on AAV purification behavior, the elution behavior of model AAV9 vectors with varying viral protein (VP) ratio was investigated on AAV9-specific POROS CaptureSelect AAV9 and pan-serotype selective POROS CaptureSelect AAVX affinity resin columns. It was found that in the pure state, the vector types displayed consistent elution profiles with 75% product recovery. However, clarified lysate purification resulted in only 50-65% product recovery with inconsistent chromatographic profiles from column recycling. This suggested that the impurities in the feed stream significantly impacted the affinity chromatography performance and column reusability.
To discern the consequence of the in-process variabilities induced due to impurities on AAV product quality and column performance, individual fractions obtained during column recycling were examined using a combination of analytical techniques to assess purity, aggregation and process-related impurity profiles. The results showed that the critical quality attributes that were most affected by column reuse were functional product titer, aggregates, and host-related dsDNA and chromatin impurities. Moreover, the impurity content in the product eluate progressively increased from AAV9-specific affinity column for the least stable capsid types. These findings collectively demonstrated that feed impurities, affinity resin characteristics, elution pH, column clean-in-place (CIP), and vector stability impact affinity column recyclability and AAV product quality.
The next area of research investigated column fouling in affinity resins applying a series of complementary techniques, testing the hypothesis that the increased impurity co-elution in the product eluate during column recyling was likely due to residual carryover caused by incomplete column CIP, leading to fouling of columns. Confocal laser scanning microscopy (CLSM) was employed to visualize and characterize resin beads post-CIP, identifying the location and extent of fouling due to feed remnants across multiple cycles of column reuse. These studies were complemented with structural imaging and analysis of resin architecture and associated transport mechanics using nanoscale X-ray computed tomography (CT). The confocal micrographs of used resin materials confirmed fouling of POROS affinity resin materials. Fouling occurred due to blockage of resin pores by both vector-related and process-related contaminants. Further investigations revealed that although the high impurity burden in the feed contributed to reducing the lifetime of POROS AAV affinity columns, the primary factors affecting reusability were the characteristics of the resin base matrix and the ligand.
All in all, this thesis provides a comprehensive understanding of the challenges and promise of affinity-based downstream processing for AAV vector purification, offering essential insights that can help advance capture technologies and drive innovation in gene therapy vector purification for both academic and industrial applications.
Description
December2024
School of Engineering
School of Engineering
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY