pH-dependent isoform transitions of a monoclonal antibody monitored by micellar electrokinetic capillary chromatography

Anal Chem. 1997 Feb 1;69(3):338-43. doi: 10.1021/ac9606517.

Abstract

Within the pH range 2-12, the monoclonal chimeric antibody BR96 can be separated into one to five isoforms by micellar electrokinetic capillary chromatography (MECC). The distribution of the immunoglobulin between these isoforms is pH dependent and apparently reversible. Some of the changes in the electrophoretic profile are represented by alterations in the immunoglobulin secondary structure. MECC and CD data demonstrate that, in other cases, differences in electrophoretic mobilities of the intact and acid-stressed antibody molecules were not due to differences in the ionization of the protein functional groups or changes in secondary structure, but rather resulted from differences in the exposure of the molecule's structural elements to the solvent. The results indicate that the interaction of the isoforms with sodium dodecyl sulfate micelles plays a crucial role in MECC isoform separations. The formation of analyte-micelle complexes was postulated to make electrophoretic mobilities, especially of large protein molecules, susceptible to subtle conformational changes that are not detectable by other methods.

MeSH terms

  • Antibodies, Monoclonal / analysis*
  • Calorimetry, Differential Scanning
  • Circular Dichroism
  • Electrophoresis, Capillary
  • Hydrogen-Ion Concentration
  • Isomerism

Substances

  • Antibodies, Monoclonal