Analysis of monoclonal antibody chimeric BR96-doxorubicin immunoconjugate by sodium dodecyl sulfate-capillary gel electrophoresis with ultraviolet and laser-induced fluorescence detection.
Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CAGE), the capillary version of SDS-polyacrylamide-based slab gel electrophoresis, has been utilized for the separation and analysis of monoclonal antibody chimeric BR96 and the corresponding immunoconjugate prepared between BR96 and the anticancer drug doxorubicin (BR96-DOX). SDS-CAGE was performed in a coated capillary column filled with a polymer solution-based gel network matrix. Two detection formats, a uv absorbance detector and an argon-ion laser-based fluorescence detector, were incorporated into this system, providing complementary information for the determination of conjugated species. Both monoclonal antibody and immunoconjugates were studied in their native, denatured, and denatured and reduced states, respectively. Six peaks were identified following separation of the denatured BR96-DOX. These peaks were confirmed to correspond to all the possible conjugated species as expected. Analysis of the resulting "fingerprint" maps indicated that the light, heavy, and light-heavy chain conjugates are the predominant species. SDS-CAGE offers an alternative approach to the conventional slab gel electrophoresis and other chromatographic techniques, providing rapid, efficient, sensitive, and accurate information for the analysis of antibody and bioconjugates.
D000911:Antibodies, Monoclonal; D002197:Capillary Action; D004317:Doxorubicin / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D000922:Immunotoxins / Q000032:analysis*; D007202:Indicators and Reagents; D007834:Lasers; D008970:Molecular Weight; D011489:Protein Denaturation; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D000911:Antibodies, Monoclonal; D000922:Immunotoxins; D007202:Indicators and Reagents; D012967:Sodium Dodecyl Sulfate; D004317:Doxorubicin