Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns. I. Experimental observations and phenomenological model

The cation exchange chromatographic behavior of three homologous bivalent bispecific antibodies (BiSAb) is characterized for two different resins, Source 15S and ProPac WCX-10, having different base matrix, particle size, pore structure, and ligand chemistry. For both resins, elution with a salt gradient results in multiple peaks for each of the three BiSAb molecules at short residence times. The peaks gradually merge into two peaks and then into one peak eluting at intermediate salt concentrations when the residence time is gradually increased. Re-injecting fractions of each the individual peaks obtained at short residence time results in nearly the same multiple peak elution pattern. This behavior, which is contrary to the behavior normally encountered in ion exchange chromatography, appears to be related to the reversible, surface -catalyzed interconversion between different conformational states of each BiSAb that interact with different strength with the chromatographic surface. This behavior is qualitatively independent of pH in the range 5-8.5, protein load in the range 0.06-5.0 mg/ml, and gradient slope, and is not associated with the formation of aggregates. Gradually increasing temperature, however, reduces the multiple peak behavior eventually resulting into a single peak at 55 °C A phenomenological model is developed that predicts the experimental behavior over a broad range of conditions using fitted rate and equilibrium constants.