One of the biggest challenges for AAV gene therapy products is establishing an appropriate analytical strategy to support product manufacture, release, stability, comparability and characterization at different stages of development. This presentation will highlight some of the existing analytical challenges and provide guidance on development of an appropriate strategy to help overcome these.

Gene therapy delivery of a drug product requires precise determination of vector titre by a suitably qualified analytical method. Despite the importance of titre assays for product release, optimisation of R&D methods is also crucial to allow for better understanding of the changes in rAAV titre and yield from upstream to downstream unit operations. qPCR is widely considered the gold standard for this purpose. In addition, we adopted orthogonal analytical tools including ddPCR and HPLC as robust and rapid titration alternatives to qPCR.

Currently, static and shake flasks for suspension processes are widely used in the process development of advanced therapies. However, these cell expansion approaches are labor-intensive and require a high level of highly skilled operator manipulation and offer only a low level of cell culture monitoring and control. Here we demonstrate the utility of the ambr250 modular and the newly designed unbaffled single impeller vessel as a process evaluation tool for the expansion of CAR-T cells.

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 (i.e. a matrix approach), and the strategy for implementing these assays may evolve through the product lifecycle. This presentation will discuss phase-appropriate development and qualification of different in vitro potency assays.

Ultragenyx has established a fast-to-clinic CMC development strategy leveraging two distinct production platforms, implementation of high-throughput centers of excellence, and a state-of-the-art pilot plant to streamline and standardize the development and technology transfer of preclinical and clinical candidates. Process development has resulted in significant volumetric productivity increase and improvement in yield across chromatography and filtration steps. Scalability of new products to 250L has been demonstrated within 3 months.

A major bottleneck of gene therapy is the large-scale manufacturing of viral vectors. The most common platform for producing AAV is the transient transfection of adherent HEK293 cells, however, suspension cultures are easy to scale up. Here, we used Sartorius ambr15 system to adapt two HEK293 cell lines into 7 media and to screen transfection conditions in suspension using DoE with the MODDE® software to maximize AAV productivity.

Droplet digital PCR is a powerful technique, which allows vector genome titer quantification without the necessity of a reference. In this talk, the benefits, like an enhanced precision compared to RT qPCR, the power of absolute quantification but also potential drawbacks of this innovative technique will be in the focus. Moreover, different strategies of designing the transgene target sequences and the individual influence on the obtained results will be discussed.

Viral vector processing at scale requires a high level of control which can be achieved via the introduction of PAT. This presentation will present a case study of successful PAT implementation within the viral vector space to drive both process intensification and increase process robustness.

Reliable biophysical characterization is a central challenge in development of gene therapies. Robust analytical tools based on light scattering, essential in the development and commercialization of monoclonal antibodies and virus-based vaccines, are also suitable for gene vectors, whether viral or non-viral. This talk will highlight the applications of multi-angle and dynamic light scattering for the determination of critical quality attributes of gene vectors including among others analysis of AAVs, lentiviral vectors and synthetic vectors 

Lentiviral vector manufacturing is transitioning from transient to stable production systems. The establishment of stable producer cell lines has been a challenge due to the cytotoxicity of the vector. This work presents strategies enabling the generation of constitutive lentiviral cell lines showing sustained production over 1 week which supports effective upstream processes. Detailed characterization of producer cell clones was performed revealing critical parameters.

In this presentation I will describe our approach to generate packaging cells for lentiviral vectors. The approach entails the generation of pools that are cloned by limiting dilution into nanowells and isolation with a robotic cell picker. I will also provide examples of process development using producer clones derived from our packaging cells to increase the yield of vectors in serum-free suspension culture.

The purification of adeno-associated virus has become an increasingly important topic in the field of biomanufacturing as the prevalence of AAV gene therapies increases. One of the major hurdles facing AAV purification is the separation of empty capsids from full capsids. This presentation will address Precision BioSciences’ advances in downstream chromatography for both capture and empty full separation steps.