Select host cell proteins coelute with monoclonal antibodies in protein a chromatography

Abstract

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 mAbs-HCP 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.

The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk‐based management for their control

“…Equally, proteins may recognize and bind to certain epitopes on the product molecule (e.g., chaperone proteins) giving rise to the possibility of HCPs that are associated with the product molecule being carried through the process. This is a phenomena now known to be the primary reason for HCPs making their way through protein A affinity chromatography (Nogal et al, ; Tarrant et al, ).…”

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

“…This conclusion was also supported by an elegant experiment that demonstrated large differences in protein A eluate pool HCP content depending on the order in which purified mAb and null cell harvested cell culture fluid were first applied to the column. 11 These studies left unanswered the question of whether different mAbs interact with different subpopulations of HCPs. A recent study used cross-interaction chromatography followed by two-dimensional (2D) gel electrophoresis with MALDI-TOF identification to conclude that product association constitutes an important mechanism by which specific HCPs co-purify during protein A chromatography.…”

Other Applications of Combinatorial Peptide Libraries

“…In fact, nonnegligible amounts of these host-cell proteins adsorb on the beads and co-elute with the antibody, with consequent contamination. In spite of washing treatments before the elution of antibodies, a number of impurities remain, and their amount seems to be dependent on the type of antibody produced (Nogal et al, 2012), suggesting that mAbs might represent the preferential location where subsets of HCPs (host cell proteins) bind. This is why intermediate separation processes capable of reducing the impurity presence are added, such as ion-exchange chromatography or hydrophobic chromatography (Liu et al, 2010).…”