Improved performance of protein separation by continuous annular chromatography in the size-exclusion mode

In size-exclusion chromatography (SEC), proteins and peptides are separated according to their molecular size in solution. SEC is especially useful as an effective fractionation step to separate a vast amount of impurities fr om the components of interest and/or as final step for the separation of pu rified proteins from their aggregates, in a so-called polishing step. Howev er, the throughput in SEC is low compared to other chromatographic processe s as good resolution can be achieved only with a limited feed volume (i.e., maximal approximately 5% of the column volume can be loaded). This limitat ion opposed widespread application of conventional SEC in industry despite its excellent separation potential. Therefore a continuous separation proce ss (namely preparative continuous annular chromatography) was developed and compared to a conventional SEC system both using Superdex 200 prep grade a s sorbent. An immunoglobulin G sample with a high content of aggregates was chosen as a model protein solution. The influence of the feed how-rate, el uent flow-race and rotation rate on the separation efficiency was investiga ted. The height equivalent to a theoretical plate was lower for preparative continuous annular chromatography which could be explained by reduced extr a column band broadening. The packing quality was proved to be identical fo r both systems. The productivity of conventional batch SEC was lower compar ed to continuous SEC, consequently buffer consumption was higher in batch m ode. (C) 2001 Elsevier Science B.V; All rights reserved.