In this presentation, we explore the multifaceted comparability challenges facing late-stage gene therapy programs, including regulatory standards, analytical methodologies, and product consistency.

The complexity of gene therapy makes production and characterization challenging. These challenges are amplified by the lack of applicable reference standards. USP is developing physical and documentary standards to support gene therapy, with a focus on AAV. Several AAV-related standards are currently in development, with the intent of supporting AAV manufacturers from production through release testing. These standards will focus on three areas—raw materials, impurities, and viral vector characterization. 

This presentation will discuss emerging analytics for AAV characterization, including: early vs. late-state characterization; examples of emerging analytics, all supported by a case study.

AAV-based gene therapies present a considerable analytical challenge due to their molecular size and complexity. Strategies for the characterization of various quality attributes of AAV using liquid phase separations and mass spectrometry will be presented. Examples include the characterization of intact viral proteins using LC-MS and CE-MS, determination of the capsid full state using LC-MS, and charge-detection mass spectrometry for mass-based analysis of capsid fill state and heterogeneity.

We evaluated the platform for AAV quantification and characterization, comparing it to established analytical methods. The platform offers empirical, data-driven measurements with minimal sample requirements. Upon testing hundreds of rAAV vectors comprising diverse serotypes and transgenes, the data showed strong correlations with established analytical methods and exhibited high reproducibility. Its capability also extends to in-process samples from various purification processes, meeting the demand for rapid, high-throughput analysis.

Residual host-cell DNA is a safety concern in gene therapy products, necessitating analytical tools for evaluating residual DNA mass and size. This presentation will report on development of a linker-ligation/PCR-based method for characterizing residual DNA size distribution, as well as on evaluation of PCR-based methods for quantifying residual host-cell DNA. Using these tools, a performance comparison of commercial nucleases during lentiviral downstream processing will be presented. 

Sensorion is a biotech company dedicated to the development of therapies for genetic forms of hearing loss. Two novel gene therapy programs include deafness due to otoferlin deficiency as well as GJB2 mutation. Since the otoferlin gene is large and exceeds the AAV packaging capacity, two AAV vectors have been developed. The product manufacturing and a deep characterization of the dual vectors will be presented.

Development of a functional potency is one of the major analytical challenges facing the sponsors of the cell and gene therapy products. An MOA-based potency bioassay is expected to be a part of a release panel prior to the pivotal clinical studies. Strategies to streamline development of the potency assays for the rAAV therapeutics will be presented with the emphasis on building a toolbox and leveraging platform knowledge.

Modalis Therapeutics employs novel AAV capsid variants with enhanced tissue targeting and transduction to deliver our epigenetic gene modulation technology, known as Guide Nucleotide Directed Modulation (GNDM). Among the many challenges faced in process development is ensuring consistent product quality throughout process iterations. Here, we will delve into key factors affecting in-process product stability, different testing methodologies, and their implications on final product quality.

Advancements in manufacturing strategies and platform processes for producing adeno-associated virus (AAV) have significantly contributed to the successful clinical outcomes of this therapeutic approach. There is an increasing demand for analytical techniques that are rapid, high-throughput, and reliable to facilitate the quick iteration of process development. This presentation will explore the creation of a suite of straightforward yet robust analytical methods designed to expedite the characterization process, thereby shortening developmental timelines. Additionally, case studies will illustrate how these technologies have been applied in downstream process development to swiftly adapt to changing bioreactor conditions, ultimately reducing resource expenditure and project timelines.

AAV pre-development testing has been minimal causing developability challenges that could be avoided. An understanding of where degradation transitions occur is vital before development begins. This talk introduces high-throughput, microplate-based methods to aid in the measurement of degradation transitions based on intrinsic and extrinsic fluorescence in addition to anisothermal dynamic light scattering, and shows how they have been used to solve developability challenges and increase our product understanding.

Studies of the exposure of the AAV product to extreme conditions help in understanding the intrinsic stability of the molecule and the degradation pathways. Temperature changes, pH variations, and oxidation conditions were tested to evaluate exposure to harsh conditions. The impacts on critical quality attributes (CQAs) such as quantity, purity, and activity are summarized. The primary impacts observed were genome loss, aggregation, PTM changes, and loss of activity.

The mAAVRx manufacturing process utilizes a 2-plasmid transient transfection along with several design optimizations to greatly increase bioreactor productivity while reducing packaged impurity levels. Transfected cell culture samples were collected over time to identify the potential effects that the 2-plasmid mAAVRx system had on AAV production. In-depth analytical characterization demonstrated several noteworthy differences in product quality and vector production kinetics between the two systems.

The inverted terminal repeats (ITRs) are essential elements of the recombinant rAAV genome and are involved in several key steps in the AAV life cycle. The repetitive nature of these segments can create some challenges during the development of rAAV, and in-depth characterization is of great importance. In this study, we employed next-generation sequencing to investigate the effects of ITR deletions at the plasmid level on AAV vector yield and genomic quality.