Purification of plasmid DNA therapeutics: New opportunities for membrane processes.

摘要

Recent progress in the field of DNA therapeutics has created a need for new separation technologies suitable for the large-scale production of highly purified plasmid DNA. This includes the removal of endotoxins, host cell proteins, RNA, and DNA fragments, as well as the separation of the desired supercoiled isoform from the open-circular and linear isoforms generated by a nick/break in one or both strands of the DNA double helix. The objectives of this dissertation were to examine the potential of using membrane ultrafiltration (UF) for purification of the supercoiled plasmid and to compare the behavior of the different plasmid isoforms in UF with that in size exclusion chromatography (SEC) and agarose gel electrophoresis.;The effective size of the different plasmid isoforms was examined by evaluating the radius of gyration (RG) of different sized plasmids using static light scattering. For any given plasmid size, the RG of the linear isoform was greater than that of the open-circular which was in turn greater than that of the supercoiled. A new analytical expression was developed for the radius of gyration of the supercoiled isoform, providing accurate estimates of the RG values over a wide range of plasmid size.;Experimental data for the partitioning of plasmid DNA isoforms in SEC were obtained over a wide range of conditions. The partition coefficient (K p) for each isoform decreased with increasing plasmid size, with the behavior for the different isoforms consistent with the measured RG values and with theoretical calculations for the partitioning of different polymer structures in slit-shaped pores. However, the very low resolution between the different plasmid DNA isoforms made separations using SEC extremely challenging.;The main focus of this work was to develop a fundamental understanding of the factors affecting transmission of the different plasmid DNA isoforms through UF membranes, including the role of filtrate flux, plasmid size, pore size, and solution ionic strength. At all conditions, the extent of plasmid transmission was a very strong function of the filtrate flux, with the sieving coefficient increasing from near zero at low flux to nearly 100% transmission at filtrate flux values well above the critical filtrate flux for plasmid transmission. This behavior was explained in terms of the deformation/elongation of the plasmid in the converging flow field above the UF membrane pores. A modified form of the classical elongational flow model for polymer transmission was developed and shown to be in good agreement with experimental data over a wide range of conditions.;For any given plasmid size, the critical filtrate flux was lowest for the linear isoform followed by the supercoiled and then the open-circular isoform. These surprising differences in critical flux provided a unique opportunity for the separation of the plasmid DNA isoforms based on a completely novel strategy that relies on differences in molecular flexibility. The results from sieving and diafiltration experiments with binary and ternary mixtures of the plasmid isoforms clearly demonstrate the feasibility of this approach, providing an initial framework for the design of novel membrane processes suitable for the large-scale production of DNA therapeutics.