Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1319, Issue -, Pages 65-71Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2013.10.028
Keywords
pH gradient; Ion-exchange chromatography (IEC); Monoclonal antibody (mAb) variants; Isoelectric point; Analytics
Funding
- Biomedical Research Council, Agency of Science, Technology and Research (A*STAR), Singapore
- Austrian Research Promotion Agency (FFG)
- A*STAR Research Attachment Programme (ARAP)
- Glyco-MEV, University of Rouen, France and BTI, A*STAR, Singapore
- Merlion initiative program called Glyco-TOOLS
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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. (C) 2013 Elsevier B.V. All rights reserved.
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