Multi-component behavior of host-cell protein- and antibody-containing heteroaggregates in protein A chromatography

Abstract

Despite its high selectivity and affinity towards the monoclonal antibody (mAb) product, protein A chromatography displays persistence of impurities such as host-cell proteins (HCPs) and high molecular weight (HMW) species, necessitating further purification steps. One factor in the persistence of HCPs and HMWs is the presence of mAb-HCP heteroaggregates that can co-elute with the mAb. This work investigates the chromatographic behavior of persistent aggregate species of a number of IgG1s and IgG4s, using both industrial harvested cell culture fluids (HCCFs) and protein A eluates as feeds. For two classes of aggregates, classified as small and large by size-exclusion chromatography (SEC), the protein A chromatography behavior was determined experimentally and a multi-component, mechanistic model based on the general rate model (GRM) was developed to describe and predict chromatographic breakthrough and elution under various process conditions. The model-predicted column outlet profiles and intra-particle uptake profiles agree satisfactorily with experimental chromatographic and confocal laser scanning microscopy data, respectively. Within the model framework, the basis for separation is that the binding affinity ratio for small aggregates (SA) relative to that of the mAb increases from near 1 at pH 7 to $>4$ at pH 4.2, while for large aggregates (LA) the ratios are $\sim 25$ and $\sim 35$ respectively, resulting in lower elution pH ranges for the aggregates. These results and the model may aid in aggregate clearance and may also have broader application in multicomponent chromatographic modeling.