rAAV downstream process using monoliths contains a series of purification steps that are mutually dependent, and each step should be optimized to its maximum for sufficient outcome. Lysate preparation prior to chromatography reduces impurities to increase dynamic binding capacity (DBC) on the monolith. We pretreated AAV2/9 clarified harvest from Sf9 cells in three different ways – acidification, tangential flow filtration (TFF) and TFF coupled with KryptonaseTM. Samples were then applied to capture chromatography step, which is one of crucial steps in downstream. Optimization of capture step leads to increased productivity due to high DBC, higher product purity and improves later polishing step. With CIM® SO3 0.05 mL Monolithic 96-well Plate buffers of different pH, sodium chloride concentrations and use of poloxamer 188 were screened to select optimal mobile phase for capture step. Pretreatment of sample that gave us the highest experimentally defined DBC tested using CIMmic␢ SO3 0.1 mL disc under selected conditions was TFF coupled with KryptonaseTM (1.44E14 vector capsids/mL of SO3 monolith). Results obtained with CIM® 96-well plate and CIMmic␢ screening were also verified on preparative CIMmultus␢ line, from which elution was analysed for total protein (BCA) and dsDNA (PicoGreen) content. High vector recovery and great reduction of impurities was determined for selected capture step parameters, confirming successful optimization of rAAV capture step during downstream process using monoliths.
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