Size-Exclusion Chromatography for Preparative Purification of Biomolecules

High-resolution size-exclusion chromatography (SEC) is not often used at large scales given the restrictions in sample size and flow rate. In a recent article, application data to support the use of SEC beyond small-scale operations, using Fractogel® EMD BioSEC (S) resin was presented.

The separation of 20 and 40 kDa proteins was illustrated, in which flow rate and sample size were varied, to examine the operational limits with regard to productivity. In a second application, efficient aggregate removal in monoclonal antibody purification was demonstrated. With SEC as the second step after Protein A affinity capture, the monomer with a purity of 99.5% was obtained. Purity and yield in both applications were determined by model-based peak deconvolution. For antibody purification, the productivity of SEC was compared with the productivity using cation-exchange chromatography (CEX). In addition, the pressure flow behavior of Fractogel® EMD BioSEC (S) resin was characterized on pilot scale. These data collections are key for designing an SEC operation with regard to step time, target bed height, and appropriate column hardware.

While SEC was confirmed as a low productivity operation, this method’s inherent strength to separate molecules according to their sizes should allow successful purification for whole families of molecules, as long as their size remains similar and regardless of whether they vary in charge or hydrophobic properties.

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