A novel approach to monitor clearance of host cell proteins associated with monoclonal antibodies

Abstract

Co-purification of a subset of host cell proteins (HCPs) with monoclonal antibodies (mAbs) during the capture of mAbs on Protein A affinity chromatography is primarily caused by interactions of HCPs with the mAbs. To date, there is limited information about the identity of those HCPs due to the difficulty in detecting low abundance HCPs in the presence of a large amount of the mAb. Here, an approach is presented that allows identification of HCPs that specifically associate with the mAb, while avoiding interference from the mAb itself. This approach involves immobilization of purified mAb onto chromatography resin via cross-linking, followed by incubation with HCPs obtained from supernatant of non-mAb producer cells that are representative of the expression systems used in mAb manufacturing. The HCPs that bind to the mAb are recovered and identified using mass spectrometry. This approach has not only allowed a comprehensive comparison of HCP subpopulations that associate with different mAbs, but also enabled monitoring of the effects of a variety of wash modifiers on the dissociation of individual HCP–mAb interactions. The dissociation of HCPs that associated with the mAb was monitored by enzyme-linked immunosorbent assay and mass spectrometry. This approach can be utilized as a screening tool to assist the development of effective and targeted wash steps in Protein A chromatography that ensures not only reduction of HCP levels copurified with the mAb but also removal of specific HCPs that may have a potential impact on mAb structural stability and patient safety. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1114–1124, 2014

Contributions of depth filter components to protein adsorption in bioprocessing

“…Which proteins are likely to adsorb can be assessed to a first approximation from electrostatic considerations; for instance, 46% of the identified host cell proteins identified in mAb production from Chinese hamster ovary cells have isoelectric points below 7.3 (Aboulaich et al, 2014), so they would be expected to bind at pH values typical for cell culture. These complement the current body of work on depth filtration in bioprocessing and extend our understanding of a depth filter as a composite unit.…”

Purification process of recombinant monoclonal antibodies with mixed mode chromatography

“…Thanks to the third step based solely on ionic interactions, their amount fell to acceptable levels. It has been previously shown that some HCPs can directly interact with mAbs [38,39]. Such is the case for glutathione S-transferase P1, complement C1r-A subcomponent, peroxiredoxin-1, lipoprotein lipase and procollagen C-endopeptidase enhancer 1.…”

Knockout of a difficult‐to‐remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations

“…Specifically, one of the difficult-to-remove HCPs noted above, lipoprotein lipase (LPL), which was also identified in other studies of HCP persistence (Aboulaich et al, 2014; Doneanu et al, 2012), hydrolyzes ester bonds within triglycerides to form alcohol and fatty acid molecules (Nilsson-Ehle et al, 1980). Given the structural similarities between polysorbates and triglycerides, it is hypothesized that LPL may enzymatically degrade polysorbates and consequently negatively impact mAb stability.…”