Papachristodoulou, M;
Doutch, J;
Leung, HSB;
Church, A;
Charleston, T;
Clifton, LA;
Butler, PD;
... Bracewell, DG; + view all
(2020)
In situ neutron scattering of antibody adsorption during protein A chromatography.
Journal of Chromatography A
, 1617
, Article 460842. 10.1016/j.chroma.2019.460842.
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Abstract
A deeper understanding of the nanoscale 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 nanoscale 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 (˜133 nm) and pore size (˜63 nm) correlations of the resin and the in-plane adsorbed antibody molecules (˜ 4.2 nm) correlation at different protein loadings and washing buffers, in real time using a contrast matching approach. When 0.03 M sodium phosphate with 1 M 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.
| Type: | Article |
|---|---|
| Title: | In situ neutron scattering of antibody adsorption during protein A chromatography |
| Location: | Netherlands |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.chroma.2019.460842 |
| Publisher version: | https://doi.org/10.1016/j.chroma.2019.460842 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher’s terms and conditions. |
| Keywords: | Protein A, Small Angle Neutron Scattering, Antibody, Chromatography, Adsorption, Interface |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10089972 |
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