Capillary electrophoresis sodium dodecyl sulfate nongel sieving analysis of a therapeutic recombinant monoclonal antibody: a biotechnology perspective.

With the increasing interest in the therapeutic use of recombinant monoclonal antibodies (rMAbs), a generic analytical approach for the analysis of size-based rMAb variants is desired. Such a method using capillary electrophoresis (CE) with laser-induced fluorescence detection is described. The assay was developed as a replacement for silver-stained SDS-PAGE and was validated according to the guidelines of the International Committee on Harmonization for use in routine lot release testing of a rMAb pharmaceutical. In this assay, the rMAb solution is first derivatized with a neutral fluorophore, e.g., 5-carboxytetramethylrhodamine succinimidyl ester. The labeled sample is then incubated with SDS, and the SDS-protein complexes are then separated by CE using a hydrophilic polymer as a sieving matrix. The precolumn labeling conditions described in this study allowed the detection of rMAb at a low-nanomolar concentration (9 ng/mL), with no apparent loss in resolution or changes to the distribution of rMAb analyte species, when compared to an unlabeled sample. In addition, the traditional practice of heating proteins at elevated temperatures in the presence of SDS to facilitate SDS-protein binding resulted in the generation of significant levels of rMAb fragmentation, and alternative conditions to minimize this artifact are discussed. Illustrations of the uses of this assay in monitoring consistency of bulk manufacture of a protein pharmaceutical, and in providing a size-based separation of product-related variants, as well as nonproduct impurities are shown. In brief, the assay described in this paper demonstrated comparable resolution and sensitivity to silver-stained SDS-PAGE but offered the advantages of enhanced precision and robustness, speed, ease of use, and on-line detection.