Set-up and Optimization of Large-Scale Viral Stocks Preparation and Evaluation of Automation Chances in Viral Clearance

The PhD program, carried out at BQC–Viral Clearance Laboratory of Istituto di Ricerche Biomediche "A. Marxer" (R.B.M.) – Merck RBM S.p.A. (Colleretto Giacosa, TO), optimized Moloney/Amphotropic Murine Leukemia Virus (Mo/A-MuLV) and Minute Virus of Mice (MVM) purification to increase virus titer and volumes in Virus Production Lots (VPLs). To reach this goal, the conditions to use a new ultracentrifuge rotor for the purification step were defined and modifications were implemented in the propagation phase. For both MVM and Mo/A-MuLV the main objective was to increase volumes and virus titers as much as possible, always aiming at the lowest residual protein content. Regarding Mo/A-MuLV, different conditions were tested, and the parameters resulted allow to balance process efficiency and the possibility of doing more than one ultracentrifugation step in one day. This possibility will allow to achieve a more efficient way to produce bigger VPLs in terms of volumes and it will make easier to perform their qualification, needed to be used in Viral Clearance studies, both in terms of time and costs efficiency. In parallel with that, modifications to Mo/A-MuLV propagation phase were tried, obtaining a new propagation method to raise virus quantity before ultracentrifugation. Related to MVM, all ultracentrifuge settings needed too much time to perform multiple ultracentrifugation in a single day and resulted in very firm pellets. This resulted in the definition of a longer procedure to resuspend in a proper way the virus pellet, comprising a final two-steps filtration to ensure monodispersed VPL. Moreover, during the project, automation chances to enhance and to optimize laboratory workflow have been considered. A three layers system has been implemented for infectivity test results evaluation: an automated microscope to collect images from 96-well plates, containing cells infected with virus and stained using crystal violet, a collaborative robot to switch plates on the microscope and an algorithm to analyze the images, developed by Bosch in collaboration with Viral Clearance laboratory