Optimization and validation of a quantitative capillary electrophoresis sodium dodecyl sulfate method for quality control and stability monitoring of monoclonal antibodies.

In previous work, a capillary electrophoresis sodium dodecyl sulfate (CE-SDS) method using precolumn labeling and laser-induced fluorescence (LIF) detection was developed at Genentech Inc. as part of the control system for the quality control release of a recombinant monoclonal antibody (rMAb) (Hunt, G.; Nashabeh, W. Anal. Chem. 1999, 71, 2390-2397.). In the current work, a generic and quantitative CE-SDS assay with LIF detection of rMAbs with improved accuracy and precision is described. The implementation of an alkylating step with iodoacetamide and optimization of the incubation temperature and time, in the presence of SDS, greatly decrease any thermally induced fragmentation of nonreduced labeled rMAb samples. In addition, a quantitative study of the effects of sample buffer pH on rMAb fragmentation is also presented. Furthermore, the performance of alternative CE-SDS polymer solutions and instrumentation for quantitative analysis of rMAbs is shown in this article. The validation of this method, under the guidelines of the International Committee on Harmonization (ICH), demonstrates that the assay quantitatively determines the consistency of rMAb manufacture as it relates to size heterogeneity and product purity.