Mass spectrometric evaluation of upstream and downstream process influences on host cell protein patterns in biopharmaceutical products

Abstract

For production of different monoclonal antibodies (mAbs), biopharmaceutical companies often use related upstream and downstream manufacturing processes. Such platforms are typically characterized regarding influence of upstream and downstream process (DSP) parameters on critical quality attributes (CQAs). CQAs must be monitored strictly by an adequate control strategy. One such process-related CQA is the content of host cell protein (HCP) which is typically analyzed by immunoassay methods (e.g., HCP-ELISA). The capacity of the immunoassay to detect a broad range of HCPs, relevant for the individual mAb-production process should be proven by orthogonal proteomic methods such as 2D gel electrophoresis or mass spectrometry (MS). In particular MS has become a valuable tool to identify and quantify HCP in complex mixtures. We evaluate up-and DSP parameters of four different biopharmaceutical products, two different process variants, and one mock fermentation on the HCP pattern by shotgun MS analysis and ELISA. We obtained a similar HCP pattern in different cell culture fluid harvests compared to the starting material from the downstream process. During the downstream purification process of the mAbs, the HCP level and the number of HCP species significantly decreased, accompanied by an increase in diversity of the residual HCP pattern. Based on this knowledge, we suggest a control strategy that combines multi product ELISA for in-process control and release analytics, and MS testing for orthogonal HCP characterization, to attain knowledge on the HCP level, clusters and species. This combination supports a control strategy for HCPs addressing safety and efficacy of biopharmaceutical products. K E Y W O R D S biopharmaceuticals, ELISA, host cell protein, mass spectrometry, proteomics 1 | INTRODUCTION Recombinant monoclonal antibodies (mAbs) produced in Chinese Hamster Ovarian cells are among the most important biopharmaceutical drugs. Different sub-populations of the Chinese hamster ovary (CHO) cell line are commonly used in biopharmaceutical processes for mAbs 1 . mAbs are steadily secreted into the cell culture fluid (CCF) during the fermentation process. During different steps of the DSP mAbs are purified and concentrated from CCF by separating DNA, RNA, lipids, other cell and process derived components,

Host cell protein detection gap risk mitigation: quantitative IAC-MS for ELISA antibody reagent coverage determination

“…As expected, based on previous findings, 23 the anti-HCP-Ab coverage, considering the complete bioprocess, was similar as for the CCF of the mock fermentation. This indicates that use of the Mock CCF for coverage analysis is relevant, as the majority of HCPs are covered by the anti-HCP-Ab for the complete bioprocess.…”

“… 19 , 33 , 34 As anticipated from previous study, HCP expression between both materials was observed to be highly similar. 23 Most of the HCPs exhibited a comparable abundance between Mock and Product CCF, but we found that some basic proteins or proteins with a lower MW are overrepresented, but not uniquely identified, in Product CCF. The unique HCPs identified either in Mock CCF or in Product CCF were all at low abundance, indicating a potential shift of the dynamic range of the LC-MS assay for determining HCP abundance in the drug product or in the fermentation process.…”

“…However, by introduction of the drug product into the cell line, the HCP pattern may be altered either quantitatively or by expression of different HCPs. Although it has been shown in different publications that the effect on different HCP species is “more similar than different” when comparing different transfected cell lines for production of biopharmaceuticals, 23 , 33 , 34 meaningful differences may remain between individual manufacturing processes. As such, a detailed characterization by quantitative proteomics would substantially improve the understanding of a specific bioprocess.…”

“…Recent proteomics studies have demonstrated sensitive detection for the identification of lowabundance HCPs in purified DS [17][18][19][20][21][22] and for the characterization of upstream and downstream process influences on resulting HCP patterns for different recombinant monoclonal antibody (mAb) products. 23 During the DSP, MS technology is capable of detecting individual HCPs, which is needed for thorough understanding of the process. This detailed characterization is particularly helpful for process optimization, which is achieved by monitoring persistence and depletion of individual HCPs that may pose an increased level of risk.…”

“High‐risk” host cell proteins (HCPs): A multi‐company collaborative view

“…It has been reported that some HCPs nonspecifically interact with the mAb. For example, clusterin (CLU), peroxiredoxin (PRDX), serine protease (HTRA1 isoform X2) and elongation factor 1‐alpha have been detected post protein A purification step in multiple mAb products, with PRDX persisting through polishing steps (Falkenberg et al, 2019). WIlson & Easterbrook‐Smith (1992), the following types of interaction between HCP and mAb products are possible reasons for nonspecific HCP copurification: (1) hydrophobic interactions, (2) hydrogen bonds, (3) Van der Waal's force, (4) ionic interactions, and (5) presence of immunoglobulin‐like domains (for protein A purification).…”