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Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillaries.
- Source :
-
Journal of separation science [J Sep Sci] 2009 Jul; Vol. 32 (14), pp. 2408-15. - Publication Year :
- 2009
-
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.
- Subjects :
- Animals
Dextran Sulfate chemistry
Electrophoresis, Capillary instrumentation
Hexadimethrine Bromide chemistry
Hydrogen-Ion Concentration
Immunoglobulin G isolation & purification
Immunoglobulin Heavy Chains isolation & purification
Mice
Saccharomyces cerevisiae chemistry
Surface Properties
Antibodies, Monoclonal isolation & purification
Antigen-Antibody Complex isolation & purification
Chymotrypsinogen isolation & purification
Cytochromes c isolation & purification
Electrophoresis, Capillary methods
Muramidase isolation & purification
Ribonuclease, Pancreatic isolation & purification
Subjects
Details
- Language :
- English
- ISSN :
- 1615-9314
- Volume :
- 32
- Issue :
- 14
- Database :
- MEDLINE
- Journal :
- Journal of separation science
- Publication Type :
- Academic Journal
- Accession number :
- 19557816
- Full Text :
- https://doi.org/10.1002/jssc.200900164