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During biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC–MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC–MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC–MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC–MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs.

A wide range of compounds that occur in the genus Hypericum are listed as effective drugs of natural origin. The main biological activities of several Hypericum representatives are due to the presence of naphthodianthrones, phloroglucinols, and other diverse groups of secondary metabolites that synergistically contribute to their therapeutic effects. The regulation of biosynthesis of hypericin as the key bioactive naphthodianthrone remains uncertain. Here, we present liquid chromatography mass spectrometry-based phenotyping of 17 Hypericum species, the results of which suggest an important role for skyrin and its derivatives in the polyketide pathway that leads to hypericin formation. Moreover, we report for the first time the presence of new metabolites in the genus Hypericum that are related to classes of anthraquinones, their derivatives, and phloroglucinols. As skyrin and other species of anthraquinones are rarely found in higher plants but frequently occur in fungal microorganisms, the obtained results suggest that further research on the synthesis pathways of hypericin and the role of anthraquinone derivatives in plant metabolism should be carried out. The fact that these compounds are commonly synthesized in endophytic fungi and perhaps there is some similarity in the metabolic pathways between these organisms should also be investigated. Electronic supplementary material The online version of this article (10.1007/s00216-018-1384-0) contains supplementary material, which is available to authorized users.