期刊
JOURNAL OF CHROMATOGRAPHY A
卷 1093, 期 1-2, 页码 126-138出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2005.07.077
关键词
ion exchange chromatography; protein separation; chromatography models; human antibodies
Rational methods for predicting the chromatographic behavior of human monoclonal antibodies (hMabs) in protein A affinity chromatography and cation exchange chromatography from the amino acid sequences information were proposed. We investigated the relation between the structures of 28 hMabs and their chromatographic behavior in protein A affinity chromatography and cation exchange chromatography using linear gradient elution experiments. In protein A affinity chromatography, the elution pH of the hMabs was correlated with not only the structure of the Fc region (subclass), but also that of the variable region. The elution pH of hMabs that have LYLQMNSL sequences in between the CDR2 and CDR3 regions of the heavy chain became lower among the same subclass of hMabs. In cation exchange chromatography, the peak salt concentrations I-R of hMabs that have the same sequences of variable regions (or that have a structural difference in their Fc region, which puts them into a subclass) were similar. The IR values of hMabs were well correlated with the equilibrium association constant K, and also with the surface positive charge distribution of the variable region of the heavy chain (corrected surface net positive charge (cN) of the VH region). Based on these findings, we developed rational methods for predicting the retention behavior, which were also tested with eight additional hMabs. By considering the information on the number of binding sites associated with protein adsorption as determined experimentally, and the surface positive charge distribution from the three-dimensional structure of Mab A, we hypothesized that hMabs is separated by cation exchange chromatography as the surface positive charge distribution of the VH region is recognized. (c) 2005 Elsevier B.V. All rights reserved.
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