The removal of soluble dimers from monoclonal antibodies (mAbs) is a significant downstream processing challenge. Despite the large difference in molecular mass, monomer-dimer resolution is often difficult, in part because of low selectivity and in part because of slow mass transfer. Single and multicomponent protein adsorption in cation exchange resins have been studied extensively using both macroscopic and microscopic methods for a variety of model systems. However, little has been published regarding the behavior of actual mAb monomer-dimer mixtures with respect to their competitive binding properties at high protein loadings. In this work, we have examined the single and two-component binding behavior on the cation exchange resin Nuvia HR-S (BioRad Laboratories) of a purified mAb monomer and of its dimer, which was isolated from a process feedstock using size-exclusion chromatography. The CEX resin used in this work contains relatively large pores allowing both monomer and dimer to diffuse freely within the particles. Single and two-component adsorption isotherms were obtained at different salt concentrations from batch measurements while single and two-component adsorption kinetics were obtained by confocal laser scanning microscopy (CLSM) using fluorescently labeled forms of each protein. While preferential binding of the more highly charged dimer occurs at intermediate salt concentrations, the single component isotherms are nearly indistinguishable and little selectivity is observed at low salt concentrations where binding is strongest. The adsorption kinetics are dominated by pore diffusion, suggesting that the kinetics of competitive interactions of the two species with the resin’s charged ligands is relatively fast. A model is developed to describe the competitive binding process, providing a tool to optimize column design and explain how process parameters such as feed composition and salt concentration affect resolution.

Competitive binding of antibody monomer-dimer mixtures on CEX resins: Equilibrium and kinetics

249th National Meeting (2015)

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