sodium-dodecyl-sulfate and 5-carboxytetramethylrhodamine-succinimidyl-ester

LEDs present an alternative to lasers in LIF detection with CE, resulting in LED-induced fluorescence (LEDIF). LEDs are much less expensive, consume less energy and are more stable. In addition, LED light sources allow a greater range of wavelengths to better match the maximum wavelength for the fluorescence of the dye. Antibodies were largely studied in SDS capillary gel electrophoresis (SDS-CGE) and LIF detection with different dyes to label the proteins. In this work, our goal is to show that LEDs can advantageously replace lasers. We used 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRA.SE), 3-(2-furoyl)-quinoline-2 carboxaldehyde (FQ), and naphthalene-2,3-dialdehyde (NDA) to label IgG and we compared the LIF sensitivity with that obtained from LEDIF. We measured that the LOD values of LEDIF are identical to that obtained with the wavelength equivalent laser, and for 5-TAMRA.SE analysis, LOD values are about six times better than when the classical 488 nm laser was used.

Topics: Amines; Amino Acids; Electrophoresis, Capillary; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Furans; Humans; Immunoglobulin G; Lasers; Limit of Detection; Naphthalenes; Quinolines; Rhodamines; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence

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.

Topics: Antibodies, Monoclonal; Electrophoresis, Capillary; Quality Control; Recombinant Proteins; Rhodamines; Sodium Dodecyl Sulfate; Spectrometry, Mass, Electrospray Ionization