Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillaries.
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: alpha-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.
Haselberg
Rob
R
Department of Biomedical Analysis, Utrecht University, Utrecht, The Netherlands. r.haselberg@uu.nl
de Jong
Gerhardus J
GJ
Somsen
Govert W
GW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000936:Antigen-Antibody Complex / Q000302:isolation & purification*; D002919:Chymotrypsinogen / Q000302:isolation & purification*; D045304:Cytochromes c / Q000302:isolation & purification*; D016264:Dextran Sulfate / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D006583:Hexadimethrine Bromide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification; D007143:Immunoglobulin Heavy Chains / Q000302:isolation & purification; D051379:Mice; D009113:Muramidase / Q000302:isolation & purification*; D012259:Ribonuclease, Pancreatic / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D006583:Hexadimethrine Bromide; D045304:Cytochromes c; D002919:Chymotrypsinogen; D016264:Dextran Sulfate; D012259:Ribonuclease, Pancreatic; D009113:Muramidase