title: Probing the conformational behavior of a monoclonal antibody with surfactant affinity capillary electrophoresis (SurfACE).
abstract: Multiple peaks are observed for a monoclonal antibody (mAb) when analyzed by "protein MEKC" (micellar electrokinetic capillary chromatography) using SDS-containing run buffers. We present our efforts to understand the mechanism of peak formation and the factors that affect the distribution of the mAb between these peaks. We used "intrinsic" charge ladders of the mAb to determine that peak-to-peak differences in the amount of bound surfactant are comparable to the aggregation numbers of protein-bound micelles. This suggests that the peaks represent sequential unfolding intermediates formed after collisions with micelles. Since this mechanism differs from that of small-molecule MEKC, we prefer to view this technique as a variant of affinity capillary electrophoresis and call it "SurfACE." We also find that the peak distribution is highly sensitive to pH. Lower pH favors the formation of more highly bound complexes, probably through an electrostatic effect on the kinetics. If the run buffer pH is high enough, the peak distribution appears to be set during the post-injection mixing process, as the mAb encounters surfactant during its transition from the lower-pH sample environment. Analysts who wish to interpret "protein MEKC" electropherograms should take note of these effects.
authors.affiliation: Department of Chemistry, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, NC 28223-0001, USA. btcooper@uncc.edu
authors.forename: Brian T
authors.initials: BT
authors.lastname: Cooper
authors.forename: Rohan D
authors.initials: RD
authors.lastname: Sanzgiri
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Maxey
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D016477:Artifacts; D002021:Buffers; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D002021:Buffers; D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate