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.

Keywords: host cell protein clearance; host cell protein impurities; mAb process development; protein-protein interaction; proteomics.

Figure 1

Characterization of HCP interactions with mAbs. (A) SDS-PAGE profiles of HCPs from the null CHO cell supernatant that bound to mAb1, mAb2, mAb3, and mAb4 compared to no mAb control. (B) The levels of HCP that bound to each mAb as measured by ELISA. (C) Venn diagram comparison of the numbers of HCP species that bound in common or uniquely to each mAb. Total numbers of HCPs identified for each mAb are in parentheses. (D) Subcellular distribution of HCPs that bound to each mAb. The number of HCP species in each category is expressed as percentage of the total number of HCPs identified.

Figure 2

Effects of wash modifiers on removal of mAb1-associated HCPs. The level of HCPs remaining after each wash condition was assessed by ELISA and expressed as a percentage of the level obtained after wash with no added wash modifier.

Figure 3

Effects of wash modifiers on removal of HCPs associated with different mAbs. HCP levels were assessed by ELISA after wash with phosphate buffer (PBS), 50 mM sodium caprylate (Cap), 0.5 M tetramethylammonium chloride (TMAC), 1% (w/v) CHAPS, 1 M sodium chloride (NaCl), 0.5 M arginine-HCl (Arg), and 1 M urea. All wash modifiers were prepared in phosphate buffer at pH 7

Figure 4

Validation of the effectiveness of wash modifiers on HCP removal during purification of mAb1 on conventional Protein A chromatography. Wash modifiers were prepared in phosphate buffer at pH 7 at the indicated concentrations. The levels of HCPs in the Protein A elution pools were measured by ELISA.

Figure 5

Effects of combinations of wash modifiers on removal of mAb1-associated HCPs. All wash modifiers were prepared at the indicated concentrations in phosphate buffer at pH 7. For combined modifiers, the same concentrations were used as for the single modifiers using the same buffer system. The levels of HCPs remaining after each wash condition were measured by ELISA.

Figure 6

Effects of combinations of wash modifiers on breaking interactions of individual HCP with mAb1. The levels of individual HCPs remaining after each indicated wash were obtained from mass spectrometry analysis and are expressed as a percentage of the levels after PBS control wash as described in Method section. The x-axis shows individual HCP species.