With so many viral-vector-based gene therapies approved by the FDA across different types of viral vectors (AAV, lentivirus, herpes simplex virus 1) and many in late-stage development or clinical trials, the number of approved therapies is growing. There are many challenges that investor-led start-up companies navigate through, from the process of viral-vector manufacturing and CMC analytical method development, to quality issues and regulatory hurdles all in a highly competitive landscape.

Comparability of gene therapy products involves demonstrating that changes in the manufacturing process, formulation, or production site do not compromise the final product's safety, efficacy or quality. Maintaining consistent therapeutic outcomes during the clinical trial is crucial. A comprehensive approach to analytical comparability of gene therapy products considering their inherent complexity and variability is thus necessary. Key considerations include: selecting appropriate analytical methods, relevant batches, establishing acceptance criteria with limited batch availability, and designing a reasonable stability program. By addressing these challenges, the presentation will provide practical insights into ensuring robust comparability assessments for gene therapy products.

This presentation highlights how over a decade of viral vector vaccine development informs gene therapy advancements. Key insights include formulation development, surface interactions, fit-for-purpose analytical methods, modelling, and innovative approaches to create stable, robust gene therapy products.

Recently, the development of therapies based on recombinant adeno-associated viruses (rAAV) has gained huge interest in the pharmaceutical industry. To ensure a safe and effective gene therapy product, a smart control strategy for the assessment of the rAAVv genome is of utmost importance. In this presentation, different analytical techniques for rAAV genome integrity and identity assessment will be discussed, highlighting their benefits and drawbacks.

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

Methods for essential biophysical assessments of AAV vectors for in-process sample and drug product at early clinical stages will be introduced. What are the sources of heterogeneity of full particles? Which method among several biophysical techniques is suitable for full and empty ratio determination, depending on sample quality and quantity, and developmental stage?

Purespring Therapeutics utilises a hybrid platform for AAV development in the treatment of kidney diseases, integrating analytical method development at its research facilities with the efficient transfer of technology to manufacturing sites. This strategy ensures rigorous support for product release and stability evaluation. This presentation will highlight case studies of successful method transfers and their application in generating stability data, underscoring advancements in gene therapy for renal disorders.

ATMPs use cutting-edge technology about which regulatory agencies around the world are still learning what is (and is not) important with respect to product safety, identity, strength, purity, and quality. Guidance on CMC and clinical matters is limited and generally very high level. This section will explore feedback received from across multiple products in the form of meeting advice, information requests, and hold/non-hold comments.

One of the most significant challenges in adeno-associated virus (AAV) manufacturing is the effective removal of "empty" capsids that lack the gene of interest. Anion exchange chromatography has proven to be the most effective solution for scalable enrichment of full capsids. However, the removal of empty capsids is just one hurdle in this complex process. This work will highlight the critical importance of peak characterisation and its impurity profile, which includes deamidated species, empty and partially filled capsids, host cell DNA, plasmid DNA, host cell proteins, and aggregates.

Downstream processing (DSP) faces challenges due to increased impurities from intensified upstream processing. This presentation will focus on removing host-cell impurities and optimising residual DNA analysis for regulatory compliance. Data on DNA removal for secreted and intracellular AAV serotypes using high-throughput workflows will be presented. DSP strategies for intensified harvest streams, emphasising innovative impurity control and characterisation, will also be discussed.

The growing demand for plasmid DNA (pDNA) in gene therapy and vaccine production requires scalable, efficient methods. Traditional batch lysis processes struggle with long mixing times, high shear forces, and poor lysis control. Additionally, resin-based purification steps are time-consuming, requiring RNA precipitation and tangential flow filtration (TFF). This talk includes: continuous cell lysis device integrating dynamic inline mixers and a tube reactor for controlled alkaline lysis; membrane chromatography for faster, more efficient plasmid purification; a process entirely free of RNase, simplifying downstream purification.

Since 2018, twelve RNA-based therapies have been approved by the FDA, with >130 being evaluated in clinical trials, and many more in preclinical development. Designing and implementing CMC strategies for RNA-based therapies while prioritizing robust manufacturing processes and rigorous quality controls, also needs concurrent alignment with evolving regulatory guidance. This talk will provide an overview of the status and prospects for RNA-based therapies from a reg-CMC perspective.

The utility of mRNA as a vaccine or therapeutic agent is becoming increasingly more apparent as this modality is reaching the clinic in ever higher numbers. Analytical methods for this novel class of therapeutic are still in development and bring us new challenges in their characterisation. In BioPharmaceutical Development within R&D at AstraZeneca, we have been establishing a platform for monitoring mRNA drug substance, DNA starting material, and lipid-encapsulated mRNA drug product. An overview of the methods and strategies developed and applied in BPD Analytical Sciences will be presented.