An end-to-end process for large-scale adenovirus manufacturing for gene therapy

Abstract
Recent advances in personalized regenerative medicine have been significant. However, further developments are needed to support market and regulatory demands, particularly the development of manufacturing processes to support large scale batches of high quality viral vectors. Here we describe the development of a robust integrated adenoviral manufacturing platform using adherent cells. A step-by-step scalable approach was implemented starting from laboratory scale to develop a fully integrated and reproducible process. Such a well-controlled operational manufacturing process allowed the production of large amounts of high quality viral vector in compliance with GMP requirements suitable for the clinical trials. Moreover, it was also possible to apply the same manufacturing process to more than one recombinant adenoviral vector without further optimization thereby accelerating therapy development time and reducing overall development costs.

Introduction
With over 650 clinical trials reported1, and with the recent approval of autologous cell therapy products such as Kymriah* and Yescarta*, and gene therapy product Luxturna*, there is little doubt that cell and gene therapy is booming. As the technology matures scientifically, therapies with viral vectors are advancing through clinical trials towards commercialization, bringing an increasing demand for preclinical and clinical grade viral vectors2. Expansion of viral manufacturing capacity requires scalable production methods and manufacturing systems that are tailored to specific viruses. This, in turn, will facilitate the production of large quantities of virus needed to support commercial demand in full compliance with regulatory requirements3.
So, what are the limitations? The main bottleneck for viral vector manufacturing is scalability – both upstream and downstream. Adherent cell culture vessels such as flasks, cell stacks and cell factories are difficult to scale up due to the manpower required for handling, as large numbers need to be manipulated, leading to an increased contamination risk, and decreased reproducibility and yield. Thus, scale up requires switching either to a suspension system or to higher density adherent cell culture bioreactors. Downstream purification methods must also be scaled to match upstream output. This usually requires large process modifications, such as the replacement of a centrifugation step with chromatography. However, even when equipment is available in relevant sizes, maintaining product quality and yield through optimized conditions at each step often remains a challenge4. For upstream development, accurate control of parameters such as temperature, availability of nutrients, and other environmental factors is crucial throughout the process to prevent cell stress and aggregation, in particular for adenovirus production5,6.
This whitepaper describes the development of a robust, scalable and reproducible GMP-compliant virus expression and purification platform for a regenerative cell therapy company Orgenesis* to support both preclinical studies and a Phase I clinical trial with ten patients. Orgenesis is developing an adenovirus-based therapy for diabetic patients using patients’ liver biopsies to generate Autologous Insulin-Producing (AIP) cells similar to beta cells found in the pancreas. Cell reprogramming is achieved by transducing liver-derived cells with three recombinant adenoviral vectors each coding for one of the following transcription factors: PDX-1, NEUROD1 and MAFA. The manufacturing process of these three recombinant adenoviral vectors (Ad5) was transferred from cell culture flasks, to the iCELLis® Nano bioreactor, a scalable, industrial plug-and-play fixed-bed bioreactor. Furthermore, a scalable downstream purification process was developed involving depth filtration, ion exchange chromatography, tangential flow filtration and sterile filtration.

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