Potency methods remain a significant challenge for gene therapy programs and can lead to program delays. Best practices for potency method development, transfers, and validation will be discussed, including proven analytical strategies of lifecycle management for potency methods. Case studies demonstrating method comparability considerations and method optimization for validated methods will also be covered.

Potency is the Critical Quality Attribute (CQA) for Gene Therapy (GTx) manufacturing most related to the Mechanism of Action (MoA) of GTx. This presentation will discuss strategies for potency method development, phase-appropriate validation, method bridging, and life cycle management.

Potency is a critical quality attribute and potency assays are an important regulatory requirement of late-stage AAV gene therapy. A functional potency assay quantitatively measures a Mechanism-of-Action (MOA)-based biological function. In this presentation, development and optimization of a quantitative, MOA-based in vitro functional potency assay for an AAV gene therapy product will be discussed. Challenges in cell line development and strategies for their resolution will be explored as well.

The delivery of a functional gene or transgene to a target cell requires a thorough understanding of its complex mechanism of action (MOA). Among analytical methods facilitating such understanding is the cell-based in vitro bioassay and it is one the critical release and stability indicating methods.  Due to complexities around the MOA of gene therapies, more than one in vitro method is required to truly represent the MOA. To implement the in vitro methods in a GMP environment, they should be objective in their measurements and be easily executable in QC laboratories.

Establishing relevant and applicable standards that apply to gene therapy is essential to maintaining safe and effective therapeutics, but the complexity and diversity of gene therapy products present unique challenges. USP is working with stakeholders and scientific experts to address the challenges of standardizing materials and methods. This presentation will focus on the development of documentary and physical standards to support analysis of gene therapy products, process residuals, and raw materials.

This presentation will focus on the importance of demonstrating impurities clearance from the gene therapy products. In addition, some of the analytics necessary to evaluate impurities will be discussed. Topics include: importance of evaluating impurities in gene therapy products; type of impurities; analytics for measuring impurities; and demonstrating process performance for impurity clearance.

Manufacturing of AAV vectors is known to produce three types of capsids: empty, partial, and full. To specifically determine the impact of partial and empty capsid impurities on potency, an AAV preparation with a high amount of empty and partial capsids was separated into full, partial, and empty capsid populations and subjected to full analytical characterization. Capsid, genome, and potency data will be presented.

• Formulation
• Stability for Gene Therapies
• Forced Degradation
• Device Selection

The development of AAV drugs involves assessing critical quality attributes using analytical methodologies. Assessment and quantification of empty/full ratios have proven to be challenging across the industry. Analytical ultracentrifugation is a gold-standard technique that has been used for years but it has its limitations. We have explored SEC-MALS, Mass Photometry, IEX, Capsid/vg ratio, and A260/280 to mitigate the drawbacks of analytical ultracentrifugation and have expanded our AAV characterization panel.

Stressed AAV drug product was analyzed in several stability indicating assays. Results indicated that in vitro assay was much more sensitive accessing degradation than in vivo assay. Among the stress conditions, a series of studies of photodegradation of white fluorescent light (WFL) were performed. The loss of potency was associated with total WFL exposure and was independent of the intensity.

Transient transfection using HEK293 cells is one of the most common platforms for producing recombinant adeno-associated virus (rAAV). During the process development supporting multiple Phase III products, upstream parameters and raw material were fully characterized using quality-by-design approach. Critical raw materials including polyethylenimine and plasmids were identified due to potential impact on productivity and product quality. Comprehensive characterization including multiple DOEs has resulted in establishing control on these critical RMs to ensure consistent manufacture of high-quality product.

The manufacturing process for LB-001, an AAV gene editing vector designed to treat methylmalonic academia, was optimized to streamline the purification process and increase final product yield. An analytical comparability assessment was performed to evaluate the AAV gene editing material pre- and post-manufacturing process changes. Here we show that the process changes improved product quality and provide supporting analytical data.

The scope of formulation activities can range from a simple screen to confirm the performance of an optimized matrix to a full-scale development of a new buffer system from scratch. A constant across the spectrum of formulation activities is the need for analytical tools to evaluate the effectiveness of the test matrices to confer stability while avoiding deleterious effects. This presentation will highlight the analytical techniques available while exploring the effects of forced degradation and storage stability conditions.

Post-translational modifications (PTMs) of biologics can occur during the production and storage stages. Due to the chemical modification changes, the potential impact on drug safety, quality, and efficacy needs to be closely monitored. In this presentation, we will focus on PTM analysis of AAV-based gene therapy drug products and in-process samples using mass spectrometry (MS) approach, and discuss the functional implications as well.

The goal of the NGS group in Sanofi GMU (Framingham MA) is to establish NGS based assays to complement and/or replace the complicated mix of in vitro/in vivo assays, leading to accelerated development and expedited release testing. A multi-attribute NGS assay for DNA identity and residual DNA contamination was developed using a PCR free library prep method. This method improved overall genome coverage depth, the accuracy of relative quantification of residual DNA, and variant calling confidence. This presentation will discuss the development of the method, provide case studies of use, as well as provide a high level method validation strategy.