Separation of antibody monomer-dimer mixtures by frontal analysis.

The removal of aggregates, particularly soluble dimers, from monoclonal antibodies (mAbs) remains a persistent challenge in downstream processing. In this work, we have examined the separation of an antibody monomer from its dimer on the cation exchange resin Nuvia HR-S (Bio-Rad Laboratories) using frontal analysis. In this process, a mixture of monomer and dimer is continuously fed to the column under conditions where the mixture is favorably bound, resulting in two breakthrough fronts whose monomer and dimer compositions are determined by the multi-component equilibrium and kinetics of the system. Experimentally, the selectivity for dimer was found to vary substantially with ionic strength, being lowest when conditions favor the strongest binding, and increasing to a maximum at intermediate ionic strengths where rapid exchange with the bound monomer can occur. A mechanistic model is developed to describe the competitive binding frontal analysis process, assuming pore diffusion and a significant kinetic resistance to binding as a function of ionic strength. The model was solved numerically and was able to describe both the frontal analysis processes and batch adsorption experimental data, accounting for process parameters such as feed composition and salt concentration. The resulting model can be used to optimize column operating conditions for yield and purity.