NOVEMBER 2022LIFE SCIENCES REVIEW 19As this space grows and newapproaches are being developed, theneed for a standardized "plug and play"system that meets the unique and custom needs of each developer helps standardize these processes in order to make it easier to manufacture these substancesflexibility of this approach also offers possibility to replace the GFP gene with any therapeutic genes through a simple cut and sew cloning strategy. The Tailored Development ProcessThe upstream (US) process we developed leverages the scale-down iCELLis®Nano system (Packed bed Bioreactor) where key process parameters were optimized in terms of productivity and a contaminants profile. The development activities were designed to define the producer cell line, identify the best transfection settings, the harvest and/or lysis strategy and the timing of each single step. Different surface areas (packed bed sizes) were tested to obtain the desired batch size.Downstream (DS) parameters were set at small-scales to be perfectly representative of the full-scale process and designed to remove contaminants (Host Cell Proteins, Host Cell DNA/plasmid DNA and BSA) and preserve vector infectivity.For LVV, the DS process consists of an AEX chromatography step that captures and concentrates the vector, removing HCPs, DNA and BSA. The eluted vector is then concentrated and diafiltered with tangential flow filtration using hollow fibers, and the sterile filtered and filled in vials. The total process recovery is approximately 30%.For AAV, the DS process couples a capture step with the affinity chromatography and concentration and formulation steps, based on tangential low filtration. Polishing can be applied where needed to increase the full/empty particles ratio, according to serotypes peculiarity. Several resins and membranes were tested to find the best performing ones for both affinity and AEX chromatography. The whole yield of DS for AAV processes ranges from 30% to 50%, depending on the presence or absence of the polishing step with a percentage of full particles higher than 80%.For the LVV platform, we performed 50+ US experiments in iCELLis® Nano and 9 US+DS runs in the final setting, obtaining great results that highlight the little variability from batch-to-batch.Data obtained on the full-scale iCELLis®500 system confirmed good scalability and equivalent performance of the process (Figure 1).LVV potency assays, in terms of VCN, on target CD34+ and T-cells are shown in the Figure 2. For AAV we set the process with one of the most challenging serotypes AAV6, obtained by a transient transfection of HEK293 cell line.Figure 1: LVV Infectious viral titer and residuals data obtained in iCELLis®Nano and iCELLis®500 (bulk vector and final filled vector)Anna StornaiuoloMargherita Neri
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