Optimization of a lentiviral vector GMP manufacturing protocol to minimize unwanted residual Benzonase endonuclease and plasmid

Lentiviral vectors have recently allowed for the treatment of previously incurable acquired and genetic diseases through various gene therapy techniques, however there is a need to optimize the production process for a cleaner final product. Here we show deviations from standard operating procedures that allow for a reduction in benzonase endonuclease and lentiviral production plasmids. By adding an additional filtration step of the pre-concentration viral supernatant through a polyethersulfone .45 μm pore size filter, we significantly reduced the amount of producer cell DNA in the initial product. The reduction of DNA allows for a reduction in benzonase by allowing the endonuclease to remain available to digest the excess plasmid for which it is intended. We found that with this deviation, benzonase treatment may be reduced to as low as 1 unit/mL of supernatant down from the manufacturer recommendation of 50 units/mL while maintaining virtually complete digestion of DNA and plasmid, confirmed by 23 quantitative polymerase chain reaction. In this work, we propose that units/mL benzonase be used in cGMP compliant lentiviral production in order to account for run to run variability. Additionally, the reduction of cell debris has allowed centrifugal concentration time to be reduced from 45 to 15 minutes, freeing up centrifuges in GMP facilities typically constricted by space, allowing for higher production and more access to treat patients.