Gene therapy offers tremendous promise to address human disease, but its complexity and diversity presents unique challenges. USP is engaging with stakeholders to identify and develop documentary and physical standards to support gene therapy. This presentation will provide an update on documentary and physical reference standards under development in areas such as plasmid DNA, AAV empty/full capsid analysis, and AAV vector genome titer.

This presentation will cover: ATMP modalities and barriers to entry; advanced therapy regulatory challenges, and CMC regulatory considerations (ex vivo and in vivo therapies).

An orthogonal method is an additional method that provides different selectivity to the primary method. The orthogonal method can be used to evaluate the primary method. The development of biotherapeutics, such as AAV vectors, requires a number of physiochemical and structural analysis in accordance with ICH Q6b guidelines. The development of analytical methods for parallel characterization of critical quality attributes (CQA) of AAV products is critical to advance the quality control of gene therapy medicines.

There are three types of in vitro cell-based assays for assessment of biological activity of viral vectors: infectivity, expression, and functional potency. Infectivity of therapeutic non-replicating vectors should be measured as their ability to deliver recombinant DNA or RNA into cells. We developed novel infectious titer methods with ddPCR readout for both lentivirus and AAV. In addition, using a relative transduction assay as a surrogate for potency will be discussed.

Transient transfection of suspension cells is the most commonly used method for AAV manufacturing. However, this method shows some limitations when upscaling the process. To address this concern, we developed a novel transfection reagent showing increased AAV titers and improved transfection protocol for large-scale bioreactors. 

This presentation will cover an overview of current strategies and gaps for determining capsid protein purity of AAV gene therapy candidates and introduce a newly developed CESDS method combined with laser-induced fluorescence (LIF) detection. This method has been validated against ICH guidelines featuring one-step sample preparation with high resolution and high sensitivity, making it an ideal next-generation method for QC analysis.

Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. This presentation will discuss some of the critical challenges and considerations.

In the gene therapy field, accurately measuring host cell DNA levels for viral vector-based programs is critical, but presents novel challenges. In addition to specificity and sensitivity, assays must be designed with sample composition and matrix interference in mind.  In this presentation, we explore the design of assays for upstream and downstream sample types, as well as the development of a quantitative assay for encapsidated DNA.

In this presentation, we discuss a size exclusion chromatography (SEC) method coupled with UV, multi-angle light scattering (MALS), and differential refractive index (dRI) detectors to measure the following three important AAV quality attributes: total number of viral capsid particles, relative capsid content, and percentage of monomer or aggregates. 

This presentation will show how multivariate analysis of transcriptomic and metabolomic data can provide a detailed understanding of cell behavior during viral vector manufacture. We will also show how this data can be used to support a PAT strategy for increased control and real-time product monitoring.

Clone selection is a critical step in AAV producer cell line manufacturing to ensure that the final clone not only has high productivity and good stability, but also the resulting AAV gene therapy products are highly potent. We used CE, LC-MS, and LCMS/MS to characterize AAV vectors produced from top clones using small-scale bioreactors and understand the potency difference among different clones.

AAV gene therapy products have complex mechanisms of action that pose unique challenges to potency assay development. Determining the true biological activity often requires multiple assays. This presentation will discuss development of potency assays for gene therapy products. We will provide a case study demonstrating a robust reporter-based assay for product lot release and stability analysis. This will include review of method qualification and comparability studies required to implement the reporter-based assay during development.