In Situ Neutron Scattering of Antibody Adsorption during Protein A Chromatography

Published

April 26, 2020

Author(s)

Maria Papachristodoulou, James Doutch, Hoi Sang Beatrice Leung, Andy Church, Thomas Charleston, Luke A. Clifton, Paul Butler, Christopher J. Roberts, Daniel G. Bracewell

Abstract

A deeper understanding of the nano and mesoscale structure of chromatographic adsorbents and the distribution of proteins within the media, is critical to a mechanistic understanding of separation processes using these materials. Characterisation of the media's architecture at this scale and protein adsorption within, is challenging using conventional techniques. In this study, we propose a novel resin characterisation technique that enables in-situ measurement of the structure of the adsorbed protein layer within the resin, under typical chromatographic conditions. A quartz flow-through cell was designed and fabricated for use with Small Angle Neutron Scattering (SANS), in order to measure the nano- to mesoscale structures of a silica based protein A chromatography resin during the monoclonal antibody sorption process. We were able to examine the pore-to-pore (33nm) and pore size (63nm) correlations of the resin and the in-plane adsorbed antibody molecules (4.2nm) correlation at different protein loadings and washing buffers, in real time using a contrast matching approach. When 0.03M sodium phosphate with 1M urea and 10% isopropanol buffer, pH 8, was introduced into the system as a wash buffer, it disrupted the system's order by causing partial unfolding of the adsorbed antibody, as evidenced by a loss of the in-plane protein correlation. This method offers new ways to investigate the nanoscale structure and ligand immobilisation within chromatography resins; and perhaps most importantly understand the in-situ behaviour of adsorbed proteins within the media under different mobile phase conditions within a sample environment replicating that of a chromatography column.

Citation

Journal of Chromatography A

Volume

1617

Pub Type

Journals

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Keywords

Protein A, Small Angle Neutron Scattering, SANS, Antibody, Chromatography, Adsorption, Interface

Citation

Papachristodoulou, M. , Doutch, J. , Leung, H. , Church, A. , Charleston, T. , Clifton, L. , Butler, P. , Roberts, C. and Bracewell, D. (2020), In Situ Neutron Scattering of Antibody Adsorption during Protein A Chromatography, Journal of Chromatography A, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=929204 (Accessed October 13, 2025)