Chromatographic and adsorptive behavior of a bivalent bispecific antibody and associated fragments.

The elution and adsorptive behavior of a bivalent bispecific antibody (BiSAb), comprising an IgG1 framework with a scFv domain genetically fused to each heavy chain C-terminus via flexible linkers, and of two associated fragments were studied on two cation exchange chromatography media - ProPac WCX-10, which is pellicular and suitable for analytical use, and Nuvia HR-S, which is macroporous and suitable for preparative and process scale uses. Both fragments were identified by MS as missing one of the two scFv domains and its flexible linker, but one of them also contains an additional C-terminal lysine. The separation of these fragments on both resins occurs as a result of differences in non-specific ligand-protein interactions that are modulated by the salt concentration. For the ProPac WCX-10 column, complex, multipeak elution behaviors are observed, since, as a result of the linker flexibility, both the intact molecule and the fragments appear to exist in multiple binding configurations with each scFv domains either collapsed onto the IgG framework or extended away from it. With a residence time of 2.5 min and at 21 °C, two peak elution is observed for the fragments which contain a single linked scFv and three peak elution for the intact molecule which contains two linked scFvs. This behavior is affected by residence time, temperature, and hold time. Increasing the residence time to 25 min or increasing temperature to 40°C results in elution of a single, merged peak for each of the protein species. For Nuvia HR-S, the broader peaks, obtained as a result of mass transfer limitations, tend to obscure the multipeak elution behavior. Nevertheless, even for this resin, the effects of configurational flexibility are still manifested at the single-particle scale and affect the evolution of the patterns of protein binding within individual resin particles as evident from confocal microscopy observations.