Abstract
The removal of aggregates and, especially, soluble dimers and other moderate mass species, presents a significant challenge during the production of monoclonal antibody (mAb) therapeutics. These aggregate species are a key impurity attribute monitored during downstream processing, due to their lower efficacy and increased likelihood of inciting an immunogenic response. However, the chromatographic separation of these dimers and other soluble species from their monomeric form is often challenging, due in part to low selectivity and in part to slow mass transfer. Further, much of the previous work examining this topic has been largely empirical, relying on large data sets and extensive parameterization to describe the data. The overall goals of this dissertation are to provide a fundamental understanding of the multi-component chromatographic interactions of mAb monomer with its soluble dimer, and to elucidate the underlying mass transfer phenomena governing these interactions.
This work has characterized the behavior of a mAb monomer, as well as that of a process-generated mAb dimer, for a variety of experimental conditions on Nuvia HR-S, a strong cation exchange resin. The single component behaviors of the proteins were analyzed using a variety of macroscopic measurements (batch equilibrium isotherms, column experiments) and microscopic measurements (confocal laser scanning microscopy) in order to find equilibrium and kinetic parameters. Additionally, light-based techniques (dynamic light scattering, biolayer interferometry) were used to probe the size of the proteins and the kinetics of protein binding. These same techniques were later extended and adapted to investigate the two-component behaviors of mAb monomer and dimer.
A key result of the batch two-component behavior was a strong dependence on the sodium ion concentration on the observed equilibrium and kinetics of the experimental system. At low salt concentrations, the two-component behavior of the monomer and soluble dimer show very little selectivity, a departure from the expected behavior in ion-exchange theory. As the salt concentration is increased to intermediate levels where significant binding is still present, the expected selectivity behavior emerges, with the more strongly bound dimer displacing the more weakly bound monomer. These results are attributed to a kinetic resistance to displacement of the monomer by the dimer under conditions where the binding strength is high, a result corroborated by biolayer interferometry measurements of the rate of adsorption and protein-protein surface exchange.
The results of the two-component batch behavior led to the development of a separation scheme for the monomer and dimer via frontal analysis. In this process, a mixture of antibody monomer and dimer is continuously fed to a packed column under conditions where the mixture is favorably bound, resulting in two breakthrough fronts whose monomer and dimer compositions are determined by the two-component equilibrium and kinetics of the system. These experiments were performed at a variety of sodium ion concentrations, resulting in processes that work best at intermediate salt concentrations, again corroborating the observed batch results.
Finally, a numerical model was developed to describe the two-component uptake in column and batch experiments. The purpose of the model is to provide a basis to test various mechanistic models to better understand the observed two-component behavior, as well as provide a means to optimize the experimental conditions required for a successful separation of monomer and dimer species.
