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Improve Recovery and Reproducibility with Pre-Treatment of Analytical TSK-GEL G3000SWXL Size Exclusion Columns

T.J. Higley, Tosoh Bioscience LLC and Richard Cornell, Wyeth BioPharma


Abstract:
During a recent method optimization and investigation at Wyeth BioPharma, pre-treatment with bovine serum albumin improved reproducibility and recovery during the analytical SEC analysis of fractions pulled from a multi-step orthogonal purification of a 140 kDa apparent molecular weight protein from cell culture feedstock.


Introduction:
Ideally in size exclusion chromatography the only mechanism that provides separation of sample components is a sieving process based on the hydrodynamic radius of the sample components in combination with a porous and non-interactive stationary phase. For monodispersed samples such as proteins, spherical 5 micron silica-based materials derivatized with proprietary hydrophilic diol-type coatings have been widely used since their introduction by Kato et al. in 1987. Although exhaustively derivatized, unreacted silanol groups and the hydrophilic coating may contribute to unwanted ionic or hydrophobic interactions between the bonded phase and sample components. One technique to reduce the impact of such secondary interactions is to inject bovine serum albumin to occupy accessible reactive sites on the stationary phase.

At Wyeth BioPharma, a SEC analysis is employed at every stage of a three step cell culture purification to determine the purity and recovery of the high molecular weight species of the target protein. The cell culture is purified using affinity chromatography (protein A) and subsequent chromatographic polishing steps. The recovery and reproducibility of the peaks eluting in the region of high mole
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