Abstract
The usefulness of a noncovalent, positively charged capillary coating for the efficient analysis of intact basic proteins with CE was studied. Capillaries were coated by subsequent flushing with solutions of 10% w/v Polybrene (PB), 3% w/v dextran sulfate (DS), and again 10% w/v PB. Coating characterization studies showed that stable coatings could be produced which exhibited a pH‐independent and highly reproducible EOF. The PB–DS–PB coating was evaluated with Tris phosphate BGEs of various pH using the four basic model proteins: α‐chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme. Typical migration time RSDs for the proteins were less than 0.85%, and apparent plate numbers were above 125 000 using a capillary length of 40 cm. The high separation efficiency allowed detection of several minor impurities in the model proteins. Using a BGE of medium pH, the CE system with triple‐layer coating appeared to be useful for the repeatable profiling of recombinant humanized mouse monoclonal immunoglobulin G~1~ showing a characteristic pattern of glycoforms. The CE system was also applied to the characterization of two llama antibodies, which were produced in Saccharomyces cerevisiae, revealing the presence of a side product in one of the antibodies. The high migration time stability allowed the reliable determination of antibody–antigen binding by monitoring migration time shifts. Finally, the feasibility of using the PB–DS–PB coated capillaries for CE with mass spectrometric detection was shown by the characterization of the impure llama antibody sample.