(416e) Preparative-Scale Dynamic Field Gradient Focusing: Proof of Concept

Filtration and chromatography, the principal methods used for preparative scale purification of biopharmaceuticals, have difficulty separating highly similar molecules, like a recombinant protein with inconsistent post-translational modifications. Separating such molecules is often easier when using an electrophoretic approach, like dynamic field gradient focusing (DFGF), which exploits small differences in net charge and size to separate molecules by their electrophoretic mobilities. Purification by DFGF is accomplished in free-solution within a vertical, annular separation chamber filled with a low-conductivity buffer. Unlike isoelectric focusing, no pH gradient is used in the separation.

Dynamic field gradient focusing uses two opposing forces to focus a molecule. One force, due to an electric field, is proportional to the molecule's electrophoretic mobility and decreases in intensity as the molecule moves down the chamber. Pumping buffer into the bottom of the separation chamber pushes the molecule up the chamber in opposition to the force of the electric field. The molecule will then focus at the height in the chamber where the opposing forces are equivalent. Thus, DFGF separates molecules by electrophoretic mobility and concentrates them in one step.

A preparative-scale DFGF instrument designed for 100 mg protein loads has now successfully separated equine hemoglobin (Hb) and FITC labeled bovine serum albumin (BSA) in 2.5 hours. Samples taken at the end of the separation were run on pH 3-10 isoelectric focusing gels and show no overlap between Hb and BSA bands. The Hb-BSA separation, although simple, demonstrates that the equipment and concepts involved are sound and functional. A non-linear model is being developed to help assess the capabilities of the preparative-scale dynamic field gradient focusing apparatus. The resolution between Hb and BSA bands suggests that more difficult separations are possible.