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.

A general method for the analysis of asparaginyl-linked (N-linked) carbohydrate moieties of an IgG1 monoclonal antibody is described here. The antibody, rituximab, is a mouse/human chimeric antibody to human CD20 antigen. The glycans present on rituximab are neutral complex biantennary oligosaccharides with zero, one, and two terminal galactose residues (G0, G1, and G2, respectively). To monitor the variation of the glycosylation during manufacture, the glycans were first enzymatically released from the antibody via digestion with peptide-N-glycosidase F, then derivatized with a charged fluorophore, 8-aminopyrene-1,3,6-trisulfonic acid and further separated by capillary electrophoresis with laser-induced fluorescence detection. All observed glycans were fully resolved, including the positional isomers of G1. The exact nature of the isomers in terms of the location of the terminal galactose was further characterized via multiple enzymatic digestion steps including mannosidase with activity toward specific Man(alpha 1,3) linkage. The optimization and several key parameters, i.e., enzymatic digestion and derivatization, in the assay development will be discussed. Moreover, to ensure that the assay can be used in routine lot release testing, the assay was validated and found to be accurate and precise. The analytical approach described is suitable for characterization as well as routine testing of the N-linked glycan content in any IgG1 monoclonal antibody and glycoproteins in general.

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A competitive immunoassay for cortisol based on capillary electrophoresis (CE) and laser-induced fluorescence is described. The work involved the production of assay reagents and the development of separation conditions allowing for routine analysis of serum samples. Fluorescein-labeled cortisol was synthesized and purified. Fab fragments were produced from mouse monoclonal anticortisol antibody and purified using a POROS cation exchange chromatography column. After incubation of these reagents with serum, free and bound labeled antigen were separated by CE with high reproducibility. No prior sample cleanup of the serum samples was necessary. Serum calibration curves were established and used for the quantitation of cortisol in serum. The results demonstrate feasibility for a cortisol assay based on CE operating directly on serum samples.

The purpose of this article is to review and evaluate the uses and potentials of capillary electrophoresis (CE) in immunoassay analysis (IA). This review will be divided into four sections. First, a brief summary of the fundamentals, applications and requirements of immunoassays in both research and clinical diagnostics will be given. This section will also cover the rationale behind the current research to combine CE and IA. Also, the specific needs to CE in this field will be addressed. Second, the modes of use of CE in IA will be discussed and typical applications for each will be given. Third, a separate section will be devoted to the investigation of performing immunoassays on micro fabricated devices, an interesting alternative to the conventional capillary-based approach. Finally, a critical assessment of the current status and merits of this technology will be presented.