To ensure product quality, methods used for release, characterisation, stability, and comparability of AAV-based gene therapy products rely on a combination of phase-appropriate readiness, in-house resources, and coordination of outsourced activities. This talk will cover challenges and specific solutions pertaining to the analysis of AAV-based gene therapy products and the alignment of analytical development strategies with regulatory guidance.

Establishing standards to enable gene therapy development is essential to maintaining safe and effective therapeutics, and to overcome the complexity and diversity of gene therapy products. In this presentation, we will provide updates on the development of documentary standards and reference materials to support the analytical development for gene therapies, process residuals, and raw materials. Case studies related to quality of plasmid DNA and AAV-based gene therapies will be discussed.

The ICH guideline for comparability was released in Nov 2004. Since then, ICH Q5E has been the only comprehensive guideline for comparability, but was written for biotech recombinant proteins. Nineteen years on, the FDA has released a draft comparability guideline specifically for cell and gene therapy products. How does this differ from ICH Q5E? Does it address the unique issues faced by these products?

Factors contributing to change in transgene expression (potency) level in rAAV are presented. For example, VP ratio is highly influential to potency and varies lot-to-lot. A reliable approach to accurately determine the ratio will be introduced. Size distribution analysis, including full and empty determination using several orthogonal methods within the limitation of each method, will be introduced.

Early analytical assays for AAV are typically established using purified high-titre reference material, yet most in-process samples have relatively low titre and are in challenging matrices such as cell lysates. This presentation will detail the challenges we have identified and improvements we have implemented to enable product characterisation throughout process development. This will include assays to determine genomic and capsid titre, potency, residual DNA concentration, and full/empty ratios.

AAV quality assessment requires monitoring several attributes from the protein capsid and genome. While several analytical technologies have been established, still there are limitations in regard to their accuracy, sensitivity, sample consumption, and user-friendliness. We have developed new chromatographic approaches for the assessment of protein capsid and genome integrity as well as empty/full ratio.

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 characterisation of the dual vectors will be presented.

Traditional capsid titre methods rely on ELISA which commonly suffer from long turnaround times, low throughput, and large volume sample requirements, which limits the application of ELISA-based methods to routine analysis, thus requiring development of alternative high-throughput (HTP) capsid titre methods to support AAV formulation screening and development studies. We have performed a comprehensive assessment on currently available orthogonal capsid titer methods using multiple serotypes at concentration range relevant in IND-enabling preclinical and first-in-human (FIH) clinical studies.

The use of adeno-associated viruses (AAVs) viral vectors as delivery systems for gene therapy is expanding rapidly. As a consequence, establishing analytical techniques able to fully characterize capsid viral proteins (VPs) and their modifications is critical to ensure product quality. Microchip-based capillary electrophoresis with mass spectrometry detection resulted in a powerful tool to decipher VPs' features and heterogeneity, even with limited sample availability, allowing extensive characterization across serotypes.

For the characterisation of AAV we need analytical methods to measure the purity of the final product. The monitoring of empty vs. full capsids is considered to be a critical quality attribute (CQA) as empty capsids result in presence of impurities, therefore it is imperative to establish robust and reliable analytical techniques. The current gold standard techniques for determining empty vs. full capsids is qPCR/ddPCR for genome titre and ELISA for capsid titre. Here we discuss and compare existing analytical tools as well as the challenges that come with AAV analysis.

• Ensuring product quality
• In-house and outsourced resources
• Regulatory implications

AviadoBio has developed a next-generation AAV gene therapy platform focused on controlling gene expression and is complemented by a suite of unique delivery solutions to deliver gene therapies directly to the brain and spinal cord. We are also building additional platforms and capabilities as well as proprietary RNA silencing and subpial delivery technologies. This talk will discuss some common CMC challenges.

Gene therapies dominate the current ATMP clinical trial landscape. In recent years we experienced a significant investment in CDMO/CRO acquiring specialised capabilities, offering a competitive market for both gene therapy manufacture and characterisation. In my talk, I will discuss the challenges in the field and address how Purespring Therapeutics strategically built a robust analytical platform while maintaining the balance between innovation and need.

This presentation will discuss what is tech transfer & why it is important. Tech transfer roadmap—key phases; fitting your process to your plant; and specific challenges/considerations for gene therapy products.

While gene therapies are offering unique and unprecedented treatment options for patients, these versatile toolboxes are coming with high complexity on a molecular level, which finally poses significant challenges to provide access to such therapies on a global scale. Gene therapies are currently where monoclonal antibodies were 25 years ago and we need a shift of our development paradigms and transformative innovation for reliable supply, more sustainable cost of manufacturing as well as enhanced molecular understanding of these next-generation therapeutics.