Antibody adsorption in protein‐A affinity chromatography – in situ measurement of nanoscale structure by small‐angle X‐ray scattering

Abstract: Protein‐A chromatography is the most widely used chromatography step in downstream processing of antibodies. A deeper understanding of the influence of the surface topology on a molecular/nanoscale level on adsorption is essential for further improvement. It is not clear if the binding is homogenous throughout the entire bead network. We followed the protein absorption process and observed the formation of a protein layer on fibers of chromatography resin in a time‐resolved manner in nanoscale. To characterize the changes in the antibody‐protein‐A ligand complex, small angle X‐ray scattering was employed using a miniaturized X‐ray‐transparent chromatography column packed with a MabSelect SuRe resin. Antibody‐free MabSelect SuRe resin fiber had an average radius of 12 nm and the protein layer thickness resulting from antibody adsorption was 5.5 and 10.4 nm for fiber and junctions, respectively under applied native conditions. We hypothesize that an average of 1.2 antibodies were adsorbed per protein‐A ligand tetramer bound to the outermost units. In contrast to previous studies, it was therefore possible for the first time to directly correlate the nanostructure changes inside the column, which is otherwise a black box, with the adsorption and elution process.

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch

References and Relationships

• Bibliographic citation: Antibody adsorption in protein‐A affinity chromatography – in situ measurement of nanoscale structure by small‐angle X‐ray scattering ; volume:41 ; number:22 ; year:2018 ; pages:4122-4132 ; extent:11 ; Journal of separation science ; 41, Heft 22 (2018), 4122-4132 (gesamt 11)

Contributors, Places and Time

• Creator: Plewka, Jacek ; Silva, Gonçalo L. ; Tscheließnig, Rupert ; Rennhofer, Harald ; Dias‐Cabral, Cristina ; Jungbauer, Alois ; Lichtenegger, Helga

Further information

• DOI: 10.1002/jssc.201800776
• URN: urn:nbn:de:101:1-2022090618461380311240
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 15.08.2025, 7:32 AM CEST