Advances in analytical approaches have the potential to gather novel information about gene therapy vectors and characterize the same better. Here, I will be talking about the application of biolayer interferometry (BLI) to characterize adeno-associated virus (AAV) to antibody binding. Additionally, I will be talking about flow virometry which is emerging due to more sensitive instruments for the analysis of larger vectors like lentiviruses.

In gene therapy a better understanding of the AAV content, in terms of transgene integrity and unwanted DNA contaminations, is fundamental to achieve the best possible therapy outcome and reduce the risk of side effects. Nanopore long read sequencing and multiplex digital PCR are two state-of-the-art technologies, which enable a detailed analysis of the DNA cargo and can help to improve the production process towards better gene therapy products.

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 Usher Syndrome type 1. Since the Otoferlin gene is large and exceeds the AAV packaging capacity, two AAV vectors have been developed. The product characterization of the dual vectors will be presented.

Because there are limited options to apply viral reduction or elimination steps in the manufacture of viral vectors, viral control becomes more important. As a consequence, viral testing likely needs to be more comprehensive. Multiple orthogonal methods increase the chances of detection and examples of viral contamination events will be presented.

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. We will also show that multidimensional approaches offer additional possibilities for automation and reduced sample consumption.

Developing high-yielding, scalable, and commercially viable processes for AAV manufacture, with associated analytical methods for assessing the CQAs, presents challenges. Here, we present work being performed at CPI to address some of the upstream challenges, including development of an intensified N-1 seed train step to reduce inoculum requirements. Further, we discuss some of the approaches we have been working on for full and empty capsid determination, including mass photometry.

• Linking analytics to quality
  
• Capsid capture and analysis
• Emerging analytical methods

Dramatic increase in mergers and acquisitions within the global CDMO market has raised concerns about delays in bringing the disruptive innovative gene therapy target development and delays in clinical trials. This has encouraged CDMOs to provide integrated services to advance drug development but has become a financial burden on the clinical stage companies, e.g., higher cost for services, licensing fees, and ad delays in timelines for IND/BLA submissions.

Alignment of upstream and downstream processes and analytical methods from preclinical to GMP manufacturing reduces time to market and de-risks drug development. The use of standardized assays for the characterization of preclinical vectors provides critical insights on potency, identity, and purity, and strengths the value of PoC and IND-enabling studies performed with those vectors.

Adeno-associated virus (AAV) vectors are one of the most versatile gene therapy platforms. However, AAV vectors have a small packaging capacity impairing delivery of therapeutic genes over 3.5 kb in size. This limits their use in the treatment of innumerous genetic diseases. Herein dual AAV co-delivery strategies are discussed, namely the use of protein trans-splicing systems, to deliver larger genes.