title: Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range.
abstract: Recombinant antibodies with high isoelectric point are frequent since most of them are constructed from the same framework. Classically, cation exchange chromatography is used as a standard method for the determination of antibody charge heterogeneity. In contrast, in this study highly linear pH gradients were achieved by keeping the buffering capacity over the length of the gradient constant. The buffering compounds were selected to be unretained on the column and their respective concentration was adjusted in the start and end buffer of the pH gradient to achieve constant buffering capacity. This helps conserve linearity and stability of the gradient. The method allows quantification of charge variant distribution and the determination of chromatographic isoelectric point. To demonstrate the effectiveness of this novel method, a ProPac WCX-10 column was used to separate isoforms of trastuzumab biosimilar antibodies. Effects of pH gradient linearity and of varying the analytical amount of sample on the separation are shown.
authors.affiliation: Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria; Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, No. 06-01 Centros, 138668 Singapore, Singapore.
authors.forename: Nico
authors.initials: N
authors.lastname: Lingg
authors.forename: Eddy
authors.initials: E
authors.lastname: Tan
authors.forename: Beate
authors.initials: B
authors.lastname: Hintersteiner
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point
chemical_list: D000911:Antibodies, Monoclonal
keywords: Analytics; Ion-exchange chromatography (IEC); Isoelectric point; Monoclonal antibody (mAb) variants; pH gradient