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.Graphical abstract
Highlights
Therapeutic monoclonal antibodies and Fc-fusion proteins are conventionally produced in mammalian expression systems such as Chinese hamster ovary (CHO) or human embryonic kidney cells
We describe an optimized data evaluation protocol that enhances process-independent host cell proteins (HCPs) identification based on established analytical techniques, i.e., drug substance (DS) depletion via protein A affinity chromatography followed by reversed phase-highperformance liquid chromatography (HPLC)-MS/MS
We performed HCP identification in these commercial drug product (DP) via a direct workflow involving tryptic digestion of the respective DP, peptide analysis by RP-HPLCMS/MS and protein identification against a CHO cell database supplemented with sequences of the respective DS, protein A, trypsin, and standard proteins (Fig. 1)
Summary
Therapeutic monoclonal antibodies (mAbs) and Fc-fusion proteins are conventionally produced in mammalian expression systems such as Chinese hamster ovary (CHO) or human embryonic kidney cells. Despite rigorous clean-up procedures during downstream processing, minor amounts of these host cell proteins (HCPs) may be co-purified with the therapeutic protein and remain in the final drug product (DP) [1,2,3,4,5]. Since these contaminating proteins may affect DP quality [6,7,8,9,10,11,12] or provoke immune responses when the drug is administered [13, 14], HCPs are generally considered in the context of critical quality attributes (CQAs) and product quality attributes (PQAs) [15]. Challenges in HCP characterization arise from low amounts of the contaminating proteins at large excess of DS, requiring a wide dynamic range to be covered by analytical methods
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