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J Chromatogr A. 2017 May 19;1498:80-89. doi: 10.1016/j.chroma.2016.11.056. Epub 2016 Nov 27.

Evaluation of size exclusion chromatography columns packed with sub-3μm particles for the analysis of biopharmaceutical proteins.

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School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.
Center of Immunology Pierre Fabre, 5 Avenue Napoléon III, BP 60497, 74160, Saint-Julien-en-Genevois, France.
Research Institute for Chromatography (RIC), President Kennedypark 26, 8500, Kortrijk, Belgium.
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address:


The aim of this study was to evaluate the practical possibilities and limitations of several recently introduced size exclusion chromatographic (SEC) columns of 150×4.6mm, sub-3μm (Agilent AdvanceBioSEC 2.7μm, Tosoh TSKgel UP-SW3000 2.0μm, Phenomenex Yarra SEC X-150 1.8μm and Waters Acquity BEH200 1.7μm) for the separation of biopharmaceutical proteins. For this purpose, some model proteins were tested, as well as several commercial therapeutic monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs). Calibration curves were drawn to highlight the applicability of these new SEC columns for the separation of mAbs, ADCs and their aggregates, despite some differences in their nominal pore diameter (vary from 150 to 300Å). The kinetic performance (van Deemter curves and kinetic pots) was evaluated. Columns packed with 1.7-2.0μm particles improved the plate count by a factor of 1.5-2 compared to 2.7μm particles, which is in agreement with theoretical expectations. Finally, possible secondary hydrophobic and/or electrostatic interactions between the SEC stationary phases and biopharmaceutical proteins were systematically studied. Significant differences in nonspecific interactions were observed, with hydrophobic interactions generally exerting more influence than electrostatic interactions. The use of a novel bond chemistry with the AdvanceBioSEC column was found highly effective to limit non-specific interactions and pave the way to further improvements for column provider. At the end, the average resolutions achieved on the four sub-3μm SEC columns between monomer and dimer structures were comparable for ten approved mAbs products.


Aggregates; Antibody-drug-conjugates; Kinetic efficiency; M SEC columns; Monoclonal antibodies; Size exclusion chromatography

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