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Development of refined helper plasmid: one step closer to the next generation rAAV vectors.
Harnessing rAAVs as viral vectors to improve delivery efficiency of therapeutic transgene to a given tissue or cell type requires improvements in yields and specificity to allow the use of lower and safer vector doses. To this end, our research is focused on developing novel technologies to ensure manufacturing of high yielding rAAV using transient transfection, as well as enhancing features of the rAAV vectors that act on the overall size of packaged material and specificity of delivery.
Here we present our state-of-the art approach to design new helper plasmids (phelper) with the aim of improving both the infectivity (TU/mL) and the quality (empty/full ratio) of the viral particle obtained from suspension cultures. We took the opportunity to exploit our proprietary DNA assembly method technology, to rationally explore the synergies of multiple genetic features modularly assembled in synthetic plasmids. Comparison of the biological activity of several versions of rationally designed pHelpers led us to identify the optimal configuration able to outperform existing helper plasmids in every tested bioproduction conditions.
Our ability to assemble tailor designed DNA plasmids and our expertise in developing scalable transfection solutions for rAAV manufacturing gives us the potential to improve both productivity and specificity of gene therapy products.
GMP AAV manufacturing process using transient transfection in a 2 000 L bioreactor (Theoretical approach)
Maxime Dumont*; Cassie-Marie Peigné*; Emmanuelle Cameau**; Guillaume Freund*; Mathieu Porte*; Malik Hellal*; Alengo Nyamay’Antu*
*Polyplus-transfection S.A: 75 rue Marguerite Perey, 67400 Illkirch (France)
** Pall Corporation: 3 Rue des Gaudines, 78102 Saint-Germain-en-Laye (France)
The approval of gene therapies delivered by Adeno-Associated Viruses (AAVs) has resulted in an increased commitment to adopt AAV as the vector of choice to address a broad range of diseases. To answer the demand in production, the industry has made investments to build dedicated facilities in parallel of working on scale-up in process development. Currently, transient transfection is used as the gold standard method because it brings key advantages with fastest process development and highest productivity. Designing a cGMP compliant AAV manufacturing process using large scale transfection is not an easy task and being advised by suppliers is a critical factor for success.
In this poster, we detail the method for achieving end-to-end AAV manufacturing process at the 2000L bioreactor scale. From raw material supply through USP/DSP manufacturing down to analytics, we review the different process stages and provide guidance for steps that need particular attention to.