Antibody adsorption in protein‐A affinity chromatography – in situ measurement of nanoscale structure by small‐angle X‐ray scattering. Issue 22 (16th October 2018)

• Record Type: Journal Article
• Authors: Plewka, Jacek; Silva, Gonçalo L.; Tscheließnig, Rupert; Rennhofer, Harald; Dias‐Cabral, Cristina; Jungbauer, Alois; Lichtenegger, Helga C.
• Is Part Of: Journal of separation science. Volume 41:Issue 22(2018)
• Page Start: 4122
• Page End: 4132
• Publication Date: 2018-10-16
• Subjects: agarose -- immunoglobulin -- protein layer thickness -- protein‐A chromatography -- small angle X‐ray scattering; Separation (Technology) -- Periodicals; Chromatographic analysis -- Periodicals; 543.089
• DOI: 10.1002/jssc.201800776
• Languages: English
• ISSNs: 1615-9306

Abstract

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.