Plasmid DNA primary recovery from E. coli lysates by depth bed microfiltration

Abstract

Recently, several studies have been published on the application of plasmid DNA (pDNA) in gene therapy and vaccine production. The bioprocess to obtain pDNA involves the steps of fermentation, primary recovery, secondary recovery and final purification. The pDNA primary recovery, which is the key step to the rest of the process, includes biomass separation, alkaline lysis and clarification of the lysate. In this work, the clarification by depth bed microfiltration of lysates of E. coli DH5α containing the plasmid pVAX1-NH36 was investigated. The studies were conducted using filter capsules with nominal 8.0 µm pore size using fluxes of 0.0027 and 0.004 cm(3)/(cm(2)-s). The results were compared with the conventional clarification by centrifugation. A fiber coating model was used to describe the behavior of the microfiltration system. A 99 % of solids elimination of the lysates was achieved with depth bed filtration method. The removed solids occupied 23 and 43 % (for 4 and 6 cm(3)/min, respectively) of the void volume of the depth bed microfiltration capsule since an early breakthrough curve is characteristic of these processes. The depth bed microfiltration process for removal of solids from the cell lysate showed competitive results compared to clarification by centrifugation.

Separation Properties of Plasmid DNA Using a Two-Stage Particle Adsorption-Microfiltration Process

“…Membrane processes have immense potential for large-scale plasmid purification. Several studies [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ] have demonstrated that membrane-based processes are effective for the purification of plasmid DNA. Microfiltration membranes are mainly used to remove contaminants such as chromosomal DNA, proteins, and aggregates of bacteria because capturing nanosized plasmid DNA is difficult [ 11 , 14 ].…”