A New Approach for Increasing Speed, Loading Capacity, Resolution, and Scalability of Preparative Size-Exclusion Chromatography of Proteins

Abstract

Low speed, low capacity, and poor scalability make size-exclusion chromatography (SEC) unattractive for use in the preparative separation of proteins. We discuss a novel z2 cuboid SEC device that addresses these challenges. A z2 cuboid SEC device (~24 mL volume) was systematically compared with a conventional SEC column having the same volume and packed with the same resin. The primary objective of this study was to use the same volume of SEC medium in a much more efficient way by using the novel device. At any given flow rate, the pressure drop across the z2 cuboid SEC device was lower by a factor of 6 to 8 due to its shorter bed height and greater cross-sectional area. Under overloaded conditions, the peaks obtained during protein separation with the conventional column were poorly resolved and showed significant fronting, while those obtained with the z2 cuboid SEC device were much better resolved and showed no fronting. At any given flow rate, better resolution was obtained with the z2 cuboid SEC device, while for obtaining a comparable resolution, the flow rate that could be used with the z2 cuboid SEC device was higher by a factor of 2 to 3. Hence, productivity in SEC could easily be increased by 200 to 300% using the z2 cuboid SEC device. The scalability of the z2 cuboid SEC device was also demonstrated based on a device with a 200 mL bed volume.