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Characterization of host cell protein retention in adeno-associated virus downstream processing
Date
2025
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The advancement of adeno-associated virus (AAV) gene therapies for widespread treatment of genetic diseases is dependent on the ability to manufacture vectors at scale with favorable product quality profiles. Development and implementation of large-scale suspension culture systems for recombinant AAV production has necessitated a corresponding shift in purification techniques which must be scalable and robust to recover packaged vectors while removing process- and product-related impurities such as residual host cell proteins (HCPs). Residual HCP content is a critical quality attribute (CQA) for the clinical and commercial manufacture of biologics with reported impacts to safety, stability, and efficacy. Despite potential impacts to product quality and safety, there are limited studies which examine residual HCP retention for well-defined, scalable, and commercially relevant AAV systems. In this work, we establish methods for AAV production, capsid titer measurement, and proteomic analysis. These methods are then applied for in-depth characterization of HCP persistence in AAV chromatography processes. ☐ We first developed systems and tools to allow for assessment of residual HCP persistence in AAV bioprocessing. An expression system was established to produce four AAV serotypes (AAV2, -5, -8, and -9) through screening of multiple transfection process parameters. We developed a biolayer interferometry (BLI) capsid titer method using novel Octet® AAVX biosensors to track AAV capsids across purification processes, which offered notable advantages over enzyme-linked immunosorbent assay (ELISA) methods. For proteomics analysis, we tailored spectral library construction, data processing software, and instrument selection to overcome key challenges related to the low availability and high complexity of AAV samples. Our updated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method achieved a 78% increase in HCP identifications with an 80% reduction in sample requirement across a panel of AAV samples. ☐ These systems were applied to examine residual HCP profiles of AAV vectors across affinity chromatography processes. In-depth proteomic characterization of elution pools was performed for a base case affinity chromatography process across four AAV serotypes (AAV2, -5, -8, and -9), revealing that impurity content is dominated by a subset of highly abundant cellular proteins that are largely conserved across different serotypes. Analysis of physical and functional properties of these highly abundant residual HCPs demonstrated that their persistence coincides with distinct trends in isoelectric point, molecular weight, and binding properties. Affinity resin selection and cell culture process parameters were evaluated for their impact on intermediate vector purity. Significant divergence in HCP content was observed with the use of different commercially marketed affinity resins, driven by variable levels of single-domain antibody fragment interactions with background impurities. Viral vector lots harvested from culture media corresponded with a reduction in residual HCP content compared to lysate-harvested vectors in terms of both total cellular proteins detected and their abundances relative to capsid proteins. ☐ We then expanded our analysis of residual HCP retention in scalable AAV purification processes through the development of a three-stage downstream platform based on affinity, anion exchange (AEX), and multi-modal (MMC) chromatography. The serotype-agnostic platform achieved cumulative vector yields ranging from 44.6 – 69.2% with full capsid enrichments of 2.62 – 5.93-fold across AAV2, -5, -8, and -9. Proteomic profiling was performed across each purification stage for comprehensive tracking of individual HCPs and their abundances. Significant clearance of HCPs retained across primary purification was achieved using AEX and MMC. A subset of 21 difficult-to-remove (DTR) HCPs was identified across all samples after the three-stage purification process. Vector-mediated retention mechanisms for these DTR HCPs are supported by functional characterization, comparison to control conditions, and selective persistence across multiple modes of separation. Overall, the in-depth characterization of residual HCP retention described in this work advances the understanding of process-related impurity clearance in scalable AAV purification systems, allowing for improved downstream process design for the large-scale manufacture of safe and efficacious gene therapies.
Description
"At the request of the author or degree granting institution, this graduate work is not available to view or purchase until August 24 2026."--ProQuest abstract/details page.
Keywords
Adeno-associated virus, Chromatography, Host cell proteins, Anion exchange, Culture media