Membrane chromatography of DNA: conformation-induced capacity and selectivity.
The chromatographic purification of biological macromolecules requires a novel approach to overcome some of the pore size limitations of commercially available resins. Membrane adsorbers offer the potential for better resolution as well as productivity. Sharp peaks are gained by the rapid exchange rate with the adsorbing membranes associated with the convective flow path, in contrast to the pore diffusion requirement for resin exchange. The resolution advantage is preserved even when the very short bed heights of membranes are exploited for the purpose of exceptionally high flow rates and productivity. Breakthrough experiments were used to assess the membrane dynamic loading capacities of flexible macromolecules using supercoiled (SC) DNA as a model system. In contrast to reports for smaller biomolecules such as proteins and antibodies, the dynamic capacity for DNA was found to be highly dependent on flow rates and concentrations. Increasing flow rates induced DNA elongation, which increased the surface coverage and, in turn, lowered the capacity. Increasing concentrations beyond C*, the overlap concentration, led to exclusion-volume interactions, which reduced the size of DNA and increased the membrane adsorber capacity. In the chromatographic mode, membranes with a strongly positive charge were able to resolve various isoforms of DNA, surpassing the capabilities of analogous chromatographic resins. In this study, we found that the convective-flow-induced-structural behavior of DNA is responsible for the resolution in separation.
Haber
Charbel
C
Vaccine BioProcess Engineering, Merck Research Laboratories, P.O. Box 4, West Point, Pennsylvania 19486, USA. charbel_haber@merck.com
Skupsky
Jonathan
J
Lee
Ann
A
Lander
Russ
R
D002845:Chromatography / Q000379:methods*; D004247:DNA / Q000032:analysis*; D006801:Humans; D046911:Macromolecular Substances; D008566:Membranes; D008968:Molecular Conformation
D046911:Macromolecular Substances; D004247:DNA