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

Abstract

Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development.

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

“…It is known that Peroxiredoxin-1 is difficult to remove and has been reported previously. [30][31][32] Cornifin-A, another commonly observed HCP, has also been identified frequently. 8,33 Nine HCP were identified in 20% or more of the drugs analyzed.…”

Characterization and implications of host‐cell protein aggregates in biopharmaceutical processing

“…However, while the global HCP titer may be below the threshold mandated by regulatory agencies, individual HCPs that persist into the drug product can cause specific challenges to product stability or patient safety (Jones et al, 2021; Krutzke et al, 2022; Rane et al, 2019; Valente et al, 2018). Among the potential mechanisms of HCP persistence, product association and co‐elution have received the most attention (Aboulaich et al, 2014; Esser‐Skala et al, 2020; Gilgunn et al, 2019; Levy et al, 2014; Valente et al, 2018). An additional mechanism that has been reported is nonspecific binding of chromatin‐derived complexes, composed of DNA, histones, HCPs, and mAbs, to protein A resins and subsequent co‐elution with the mAb product (Gagnon et al, 2014; Gagnon et al, 2015).…”

Development of a Novel HTLV-1 Protease: Human Fcγ1 Recombinant Fusion Molecule in the CHO Eukaryotic Expression System

“…Accordingly, anti-HIV protease as a therapeutic agent invalidates further research [31]. Hence, the present study intended to introduce the Fc tag of the IgG1 antibody as a cell-mediated inducer by selective targeting of the APCs [21,25,[32][33][34][35].…”

“…Amongst them, the Fc fusion protein with the receptor binding domain fragment of the virus demonstrated significant immunogenicity and clinical development for the induction and augmentation of neutralising IgG titres [39,40]. Nevertheless, from some studies of FDA-approved Fc fusion drugs [25,29,35] and suitable vaccines against infectious diseases such as Ebola [41], HIV [42], and influenza [43], there have been no efforts to construct Fc fusion vaccines against HTLV-1.…”