Host cell protein profiling of commercial therapeutic protein drugs as a benchmark for monoclonal antibody-based therapeutic protein development

Abstract

Therapeutic proteins including monoclonal antibodies (mAbs) are usually produced in engineered host cell lines that also produce thousands of endogenous proteins at varying levels. A critical aspect of the development of biotherapeutics manufacturing processes is the removal of these host cell proteins (HCP) to appropriate levels in order to minimize risk to patient safety and drug efficacy. During the development process and associated analytical characterization, mass spectrometry (MS) has become an increasingly popular tool for HCP analysis due to its ability to provide both relative abundance and identity of individual HCP and because the method does not rely on polyclonal antibodies, which are used in enzyme-linked immunosorbent assays. In this study, HCP from 29 commercially marketed mAb and mAbbased therapeutics were profiled using liquid chromatography (LC)-MS/MS with the identification and relative quantification of 79 individual HCP in total. Excluding an outlier drug, the relative levels of individual HCP determined in the approved therapeutics were generally low, with an average of 20 ppm (µmol HCP/mol drug) measured by LC-MS/MS, and only a few (<7 in average) HCP were identified in each drug analyzed. From this analysis, we also gained knowledge about which HCP are frequently identified in mAb-based products and their typical levels relative to the drugs for the identified individual HCP. In addition, we examined HCP composition from antibodies produced in house and found our current development process brings HCP to levels that are consistent with marketed drugs. Finally, we described a specific case to demonstrate how the HCP information from commercially marketed drugs could inform future HCP analyses.

Characterization of a SARS-CoV-2 spike protein reference material

“…Both the UV–Vis ID-MS techniques are sensitive to the presence of host-cell proteins (HCPs), in this case CHO cell proteins co-purified with the spike protein. We were able to detect low-level HCPs in SMT1-1, similar to work on other CHO-based biologics [ 28 ]; however, we did not perform detailed quantitation. Therefore, akin to the NISTmAb reference material, we did not include a purity correction for HCPs.…”

Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines

“…Liquid chromatography coupled with tandem mass spectrometry (LC-MS/ MS) and two-dimensional electrophoresis (2DE) have enabled proteomic profiling with quantitative output and have been applied to determine the relative or absolute abundance of individual HCPs (Valente et al, 2018). These methods have been used to track HCP clearance through multiple stages of downstream processing (Chiverton et al, 2016;Falkenberg et al, 2019;Zhang et al, 2014), as well as identify and quantify residual HCPs present at ppm levels in final drug substances (Falkenberg et al, 2019;Kreimer et al, 2017;Molden et al, 2021). Substantial effort has been dedicated to cataloging difficult-toremove CHO HCPs that persist through chromatography steps in standard downstream purification platforms because of co-elution or product-association mechanisms.…”

Toward optimal clearance: A universal affinity‐based mass spectrometry approach for comprehensive ELISA reagent coverage evaluation and HCP hitchhiker analysis

“…In contrast, a tryptic digestion using native conditions reduces the dynamic range of the sample resulting in improved sensitivity, as demonstrated in several recent publications. [20][21][22] All crucial steps of the optimized AP-MS method were carefully evaluated in terms of functionality and efficiency. The effect of a threshold application on false negative and false positive coverage results was analyzed in detail.…”

“…Also, applying denaturing conditions for digesting the bound HCPs directly from the antibody affects the sensitivity by LC–MS/MS analysis due to a large excess of antibody peptides in the digested sample. In contrast, a tryptic digestion using native conditions reduces the dynamic range of the sample resulting in improved sensitivity, as demonstrated in several recent publications 20–22 …”