Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillaries.

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: 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.

• Authors: Rob Haselberg, Gerhardus J de Jong, Govert W Somsen
• Journal: Journal of separation science (J Sep Sci) Vol. 32 Issue 14 Pg. 2408-15 (Jul 2009) ISSN: 1615-9314 [Electronic] Germany
• PMID: 19557816 (Publication Type: Journal Article)

Chemical References

• Antibodies, Monoclonal
• Antigen-Antibody Complex
• Immunoglobulin G
• Immunoglobulin Heavy Chains
• Hexadimethrine Bromide
• Cytochromes c
• Chymotrypsinogen
• Dextran Sulfate
• Ribonuclease, Pancreatic
• Muramidase

Topics

• Animals
• Antibodies, Monoclonal (isolation & purification)
• Antigen-Antibody Complex (isolation & purification)
• Chymotrypsinogen (isolation & purification)
• Cytochromes c (isolation & purification)
• Dextran Sulfate (chemistry)
• Electrophoresis, Capillary (instrumentation, methods)
• Hexadimethrine Bromide (chemistry)
• Hydrogen-Ion Concentration
• Immunoglobulin G (isolation & purification)
• Immunoglobulin Heavy Chains (isolation & purification)
• Mice
• Muramidase (isolation & purification)
• Ribonuclease, Pancreatic (isolation & purification)
• Saccharomyces cerevisiae (chemistry)
• Surface Properties