The most significant factor contributing to the presence of host cell protein (HCP) impurities in Protein A chromatography eluates is their association with the product monoclonal antibodies (mAbs) has been reported previously, and it has been suggested that more efficacious column washes may be developed by targeting the disruption of the mAbsHCP interaction. However, characterization of this interaction is not straight forward as it is likely to involve multiple proteins and/or types of interaction. This work is an attempt to begin to understand the contribution of HCP subpopulations and/or mAb interaction propensity to the variability in HCP levels in the Protein A eluate. We performed a flowthrough (FT) recycling study with product respiking using two antibody molecules of apparently different HCP interaction propensities. In each case, the ELISA assay showed depletion of select subpopulations of HCP in Protein A eluates in subsequent column runs, while the feedstock HCP in the FTs remained unchanged from its native harvested cell culture fluid (HCCF) levels. In a separate study, the final FT from each molecule's recycling study was cross-spiked with various mAbs. In this case, Protein A eluate levels remained low for all but two molecules which were known as having high apparent HCP interaction propensity. The results of these studies suggest that mAbs may preferentially bind to select subsets of HCPs, and the degree of interaction and/or identity of the associated HCPs may vary depending on the mAb. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012