Integrated development of enzymatic DNA digestion and membrane chromatography processes for the purification of therapeutic adenoviruses

High titer and purity levels are key requirements for virus-based cancer gene therapy biopharmaceuticals. However, the task of removing enough host-cell DNA from therapeutic viruses in order to comply with FDA’s 10 ng/dose limit is particularly challenging. In a previous study, we demonstrated advantages using laterally-fed membrane chromatography (LFMC) adenovirus purification. Although this approach achieved >90% removal, significant amounts remained product due poor performance pre-LFMC digestion step. present attempt improve upon outcome by employing an integrated process development that examines interactions between different downstream steps (i.e., clarification, enzymatic digestion, (MC)). First, identified most efficient sequence involving clarification step followed step, as well three endonucleases (Benzonase®, Denarase®, Turbonuclease™) perform similarly respect digestion. Next, factorial design experiments (DOE) study was used evaluate how enzyme (Benzonase Denarase) concentration incubation time impact resulting virus concentrations. Since Denarase showed slightly better efficiency, it along subset DOE conditions removal via MC Sartobind Q 96-well filter plates. The lowest amount per dose conjunction feed had been digested U/mL 4 h; such, prepare lysates, which were then purified LFMC device containing 1 mL membrane. This enabled virus-recovery rate 73% residual 77 ng/dose. Ultimately, proposed resulted approximately 80-fold improvement removal.