A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Development of a Rapid Adeno-Associated Virus (AAV) Identity Testing Platform through Comprehensive Intact Mass Analysis of Full-Length AAV Capsid Proteins.

Adeno-associated viruses (AAVs) are commonly used as vectors for the delivery of gene therapy targets. Characterization of AAV capsid proteins (VPs) and their post-translational modifications (PTMs) have become a critical attribute monitored to evaluate product quality. Liquid chromatography-mass spectrometry (LC-MS) analysis of intact AAV VPs provides both quick and reliable serotype identification as well as proteoform information on each VP. Incorporating these analytical strategies into rapid good manufacturing practice (GMP)-compliant workflows containing robust, but simplified, data processing methods is necessary to ensure effective product quality control (QC) during production. Here, we present a GMP-compliant LC-MS workflow for the rapid identification and in-depth characterization of AAVs. Hydrophilic interaction liquid chromatography (HILIC) MS with difluoroacetic acid as a mobile phase modifier is utilized to achieve the intact separation and identification of AAV VPs and their potential proteoforms. Peptide mapping is performed to confirm PTMs identified during intact VP analysis and for in-depth PTM characterization. The intact separations platform is then incorporated into a data processing workflow developed using GMP-compliant software capable of rapid AAV serotype identification and, if desired, specific serotype PTM monitoring and characterization. Such a platform provides product QC capabilities that are easily accessible in a regulatory setting.

Mass spectrometry friendly pH-gradient anion exchange chromatography for the separation of full and empty adeno-associated virus (AAV) capsids.

The proportion of full and empty capsids represents a critical quality attribute of adeno-associated virus (AAV)-based therapeutics. In this study, pH-gradient anion exchange chromatography was utilized for the separation of full and empty capsid species. The developed method allowed for applicability to multiple AAV serotypes and facilitated subsequent mass spectrometric detection of intact AAVs. This is the first study demonstrating generic applicability as well as mass spectrometric compatibility, allowing for a more sophisticated analysis of AAV-based gene therapy and paving the way for future developments in the field.

SP3-based host cell protein monitoring in AAV-based gene therapy products using LC-MS/MS.

Residual host cell proteins (HCPs) represent a critical quality attribute of biotherapeutic drug products. Workflows enabling reliable HCP detection in monoclonal antibodies and recombinant proteins have been developed, which facilitated process optimization to improve product stability and safety, and allowed setting of acceptance limits for HCP content. However, the detection of HCPs in gene therapy products such as adeno-associated viral (AAV) vectors has been limited. Here, the use of SP3 sample preparation followed by liquid chromatography-mass spectrometry (LC-MS) analysis for HCP profiling in various AAV samples is reported. Suitability of the workflow is demonstrated and provided data constitutes an important reference for future work aiming towards a knowledge-driven improvement of manufacturing conditions and characterization of AAV vector products.

Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs).

Adeno-associated virus (AAV) represent a widely used delivery mechanism for gene therapy treatments currently being developed. The size and complexity of these molecules requires the development of sensitive analytical methods for detailed product characterization. Among the quality attributes that need to be monitored, characterization of the AAV capsid protein amino acid sequences and any associated post translational modifications (PTM) present, should be performed. As commonly used for recombinant protein analysis, LC-MS based peptide mapping can provide sequence coverage and PTM information to improve product understanding and the development and deployment of the associated manufacturing processes. In the current study, we report a fast and efficient method to digest AAV5 capsid proteins in only 30 min prior to peptide mapping analysis. The performance of different proteases in digesting AAV5 was compared and the benefits of using nanoflow liquid chromatography for separation prior to high resolution mass spectrometry to obtain 100% sequence coverage are highlighted. Characterization and quantitation of PTMs on AAV5 capsid proteins when using pepsin as a single protease is reported, thereby demonstrating the potential of this method to aid with complete characterization of AAV serotypes in gene therapy development laboratories.

Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells.

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.

A Native Mass Spectrometry-Based Assay for Rapid Assessment of the Empty:Full Capsid Ratio in Adeno-Associated Virus Gene Therapy Products.

Adeno-associated virus (AAV)-based gene therapy is a rapidly developing field, requiring analytical methods for detailed product characterization. One important quality attribute of AAV products that requires monitoring is the amount of residual empty capsids following downstream processing. Traditionally, empty and full particles are quantified via analytical ultracentrifugation as well as anion exchange chromatography using ultraviolet or fluorescence detection. Here, we present a native mass spectrometry-based approach to assess the ratio of empty to full AAV-capsids without the need for excessive sample preparation. We report the rapid determination of the relative amount of empty capsids in AAV5 and AAV8 samples. The results correlate well with more conventional analysis strategies, demonstrating the potential of native mass spectrometry for the characterization of viral particles.