Author(s): Han H, Livingston E, Chen X
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Abstract A high throughput microchip capillary zone electrophoresis (CZE) method was developed for the analysis of charge heterogeneity in antibodies. The method utilizes high speed microchip electrophoresis separation and is well-suited for high throughput charge profiling of antibodies during process and formulation development. The method involves derivatization of protein molecules with Cy5 N-hydroxysuccinimide ester (NHS-ester), which does not change the protein charge profile and enables fluorescence detection on a commercial microchip instrument. The sample preparation can be performed in 96-well microtiter plates within 1 h, and each sample analysis takes only 80 s. Protein charge variants with a pI difference of 0.1 can be readily resolved in the 12.5 mm microfluidic channel. Charge profiles similar to those obtained using conventional CZE technology were found for all antibodies tested (pIs in the range of 7.5-9.2). The separation efficiency corresponds to 1.2 × 10(4) theoretical plates (1.0 μm plate height). Assay performance is assessed by demonstrating specificity, carryover, linearity, limit of detection, and precision.
This article was published in Anal Chem
and referenced in Journal of Chromatography & Separation Techniques