A novel approach to characterize host cell proteins associated with therapeutic monoclonal antibodies

Abstract

Recombinant monoclonal antibody (mAb) products are widely produced in the pharmaceutical industry using Chinese hamster ovary (CHO) cells. Host cell proteins (HCPs) are one of many process-related impurities generated during the production of mAb products. The multi-analyte HCP enzyme linked immunosorbent assay (ELISA) is the industry standard accepted assay to measure clearance of HCPs from recombinant protein therapeutics. While similar platform processes are used for expression and purification, varying amounts of HCPs are found in final drug substances for different mAb products. Through the use of novel ELISA formats, an HCP-mAb product interaction was identified using a variety of ELISAs to investigate the underlying protein-protein interaction. This result provides evidence to explain why some mAb products have HCPs that are not cleared during purification. This ELISA technique can be used as a high-throughput screening tool to identify conditions to improve clearance of difficult HCPs during downstream processing purification. In addition, an interaction was observed to occur between anti-CHO HCP antibodies and mAb products. However, this interaction only occurs under denaturing conditions, and does not interfere with the HCP quantitation obtained by ELISA, and it was demonstrated that the cross-reactive anti-CHO HCP antibodies can be removed by affinity purification. Biotechnol. Bioeng. 2017;114: 1208-1214. © 2017 Wiley Periodicals, Inc.

Exploring sample preparation and data evaluation strategies for enhanced identification of host cell proteins in drug products of therapeutic antibodies and Fc-fusion proteins

“…HCPs, bound non-specifically either to the protein A material or to the DS, on the other hand, were eluted from the loaded affinity column by applying a wash solution that disrupts their interactions with the DS or the resin. Since it was not necessary to keep the biotherapeutic in a functional state except its binding to protein A, we applied harsh wash conditions based on previous reports [ 39 , 40 , 47 – 49 ]: 10 mM Tris, 100 mM l - arginine, 1 M NaCl, and 500 mM TMAC, pH 10.0. The obtained flow-through and wash fractions were then analyzed by bottom-up RP-HPLC-MS/MS as described for the direct workflow.…”

The agony of choice: Impact of the host animal species on the enzyme‐linked immunosorbent assay performance for host cell protein quantification

“…However, a head-tohead comparison by using an affinity-purification-based mass spectrometry (AP-MS) method remained inconclusive because different antigens were used for immunization. Other publications focused on anti-HCP antibodies from a single animal species to address HCP-related issues, whereby antibodies from goats, rabbits, or sheep were predominantly used (Baldus et al, 2017;Gunawan et al, 2018;Henry et al, 2017;Lundström et al, 2014;Rey & Wendeler, 2012;Seisenberger et al, 2021Seisenberger et al, , 2022Shukla et al, 2008;Thomson et al, 2017). Besides identifying the optimal host species, the coverage may be further enhanced by a mixture of the antibodies derived from different hosts, as certain HCPs not covered by one species could be covered by another (e.g., due to their phylogenetic distance to the origin of the HCP used).…”

Purification of monoclonal antibodies in a stirred cell with polyethyleneimine‐modified polyethersulfone ultrafiltration membrane

“…Since the licensing of the monoclonal antibody for clinical use, the industry has expanded exponentially, with mAbs accounting for almost 36% of all biopharmaceuticals under development . With this growing demand, the development of efficient and trustworthy purification processes is imperative in order to remove all kinds of impurities (high‐molecular‐weight aggregates, DNA, host cell proteins and culture media additives) with reduced yield losses. The removal of these impurities is essential, since they can affect the biological activity of the biopharmaceutical product and cause immunogenicity and other adverse side‐effects.…”