Creating an optimized and scalable production platform in suspension adapted serum-free HEK293 cells for rAAV gene delivery using a Design-of-Experime

The scope of the collaboration between the University College London and Sartorius is to establish a human embryonic kidney (HEK)293 cell line suspension platform for recombinant adeno-associated vector-based gene therapy manufacturing. Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, and as June 2022, there are two rAAV-based gene therapies approved (Luxturna, in 2017, a rAAV2-based gene therapy and Zolgesma, in 2019, a rAAV9-base gene therapy) and more than seven-hundred active clinical trials. The conventional process for the manufacture of rAAV vectors is the usage of HEK293 cell line, which is transiently transfected with the genetic material required to produce the viral vector (either for research, pre-clinical and early clinical studies purposes) (Escandell et al., 2022). The widespread use of the HEK/transient transfection platform is due to recent developments supporting the design of the molecular constructs and the introduction of the plasmid DNA (pDNA) into HEK293 cells with chemical transfection reagents. However, vector production is a complex process that requires an efficient and optimized method of introducing the necessary virus genes into the cells (Dismuke & Kotin, 2017), suitable analytics and properly defined critical quality attributes (CQAs) for processes monitoring to ensure robustness and quality of the final product (Escandell et al., 2022). Despite advances in the clinic, rAAV vector manufacturing remains a challenging strategy for therapeutic applications requiring high systemic doses due to the high cost of materials (pDNA as GMP starting material), as well as a lack of process scalability and robustness. The recent development of HEK293 cell lines optimized for transfection in suspension culture has already improved the scale of production (Chahal et al., 2014). Also, stable producer cell lines represent a potential alternative to transient manufacturing systems, being more robust and easier to scale-up (Escandell et al., 2022). Although many strategies have been developed, to introduce the virus genes into HEK293 cells and to create serum-free, suspension-adapted cell lines, no rAAV production platform has been fully optimized or standardized (Dismuke & Kotin, 2017). Efficient and scalable production of rAAV is critical to enable lower cost of goods and easier drug commercialization for gene therapy applications. Therefore, the aim of the project is to optimize a transient and stable platform for suspension adapted HEK293 for rAAV production using Design of Experiments (DoE) to study the interaction of multiple factors and its impact on CQAs affecting the product quality. The process conditions and factors that impact rAAV vector production such as temperature, agitation, pH, production cell line, cell density, culture medium, harvest time, and plasmid concentrations (Zhao et al., 2020) will be investigated using rAAV2 as a representative vector to optimize virus production. Another rAAV serotype relevant to specific target tissue will be evaluated and further optimized in a data-driven approach. Sartorius transient and stable producer cell lines will serve as basis for the project. The transient cell was successfully adapted to growth in suspension culture using a proprietary serum-free, chemically defined media by direct adaptation. The PhD thesis will comprise selection of different cell clones based on cell growth and virus titer and the evaluation of different proprietary and benchmarking media and feed supplement / strategy. Analytical methods to measure metabolites, infectious titers, AAV protein identity, vector genome quantification, AAV capsid/titer, empty to full ratio and/or other product qualities were developed in-house, and samples will be retained for analysis (when performed by Sartorius).

The CDT has a proven track record of delivering impact from its research and training activities and this will continue in the new Centre. The main types of impact relate to: (i) provision of highly skilled EngD and sPhD graduates; (ii) generation of intellectual property (IP) in support of collaborating companies or for spin-out company creation; (iii) knowledge exchange to the wider bioprocess-using industries; (iv) benefits to patients in terms of new and more cost effective medicines, and (v) benefits to the wider society via involvement in public engagement activities and impacts on policy.

With regard to training, provision of future bioindustry leaders is the primary output of the CDT and some 96% of previous EngD graduates have progressed to relevant bioindustry careers. These highly skilled individuals help catalyse private sector innovation and biomanufacturing activity. This is of enormous importance to capitalise on emerging markets, such as Advanced Therapy Medicinal Products (ATMPs), and to create new jobs and a skilled labour force to underpin economic growth. The CDT will deliver new, flexible on-line training modules on complex biological products manufacture that will be made available to the wider bioprocessing community. It will also provide researchers with opportunities for international company placements and cross-cohort training between UCL and SSPC via a new annual Summer School and Conference.

In terms of IP generation, each industry-collaborative EngD project will have direct impact on the industry sponsor in terms of new technology generation and improvements to existing processes or procedures. Where substantial IP is generated in EngD or sPhD programmes, this has the potential to lead to spin-out company creation and job creation with wider economic benefit. CDT research has already led to creation of a number of successful spin-out companies and licensing agreements. Once arising IP is protected the existing UCL and NIBRT post-experience training programmes provide opportunities for wider industrial dissemination and impact of CDT research and training materials.

CDT projects will address production of new ATMPs or improvements to the manufacture of the next generation of complex biological products that will directly benefit healthcare providers and patients. Examples arising from previous EngD projects have included engineered enzymes for greener pharmaceutical synthesis, novel bioprocess operations to reduce biopharmaceutical manufacturing costs and the translation of early stem cell therapies into clinical trials. In each case the individual researchers have been important champions of knowledge exchange to their collaborating companies.

Finally, in terms of wider public engagement and society, the CDT has achieved substantial impact via involvement of staff and researchers in activities with schools (e.g. STEMnet), presentations at science fairs (Big Bang, Cheltenham), delivery of high profile public lectures (Wellcome Trust, Royal Institution) as well as TV and radio presentations. The next generation of CDT researchers will receive new training on the principles of Responsible Innovation (RI) that will be embedded in their research and help inform their public engagement activities and impact on policy.