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