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Review
. 2025 Aug 18:16:1658366.
doi: 10.3389/fmicb.2025.1658366. eCollection 2025.

Residual host cell proteins: sources, properties, detection methods and data acquisition modes

Yifan Yao  1   2 , Xuemei Wen  1   2 , Hongjuan Pan  1   2 , Ziwei Chen  3
Affiliations
Review

Residual host cell proteins: sources, properties, detection methods and data acquisition modes

Yifan Yao et al. Front Microbiol. .

Abstract

Host cell proteins (HCPs) are process-related impurities derived from host organisms used for recombinant protein production in biopharmaceutical manufacturing. The generation of HCPs may lead to potential safety risks, such as immunogenicity, reduced drug efficacy and long-term side effects. Therefore, in the biopharmaceutical process, even trace amounts of HCPs need to be strictly regulated and controlled. The main bottlenecks associated with the detection of HCPs include a wide dynamic range of detection and instability of HCPs. Due to its high sensitivity and high resolution, mass spectrometry has attracted more and more attention in HCP detection, but it still cannot completely replace enzyme-linked immunosorbent assay (ELISA). The research in the future includes the development of more efficient sample pretreatment methods and data processing techniques to improve the sensitivity and accuracy of detection. At the same time, combined with risk assessment and process optimization, it is expected to further reduce the residual risk of HCP. This review discusses the sources, properties, pretreatment and detection of residual HCPs in therapeutic products, along with current regulatory considerations and future advancements.

Keywords: characterization; detection; host cell proteins; pretreatment; sources.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Three pretreatment methods of HCPs detection. It includes molecular weight cutoff for enrichment of HCPs, affinity depletion for isolation of HCPs and denaturing digestion for enzymatic hydrolysis of HCPs.
Figure 2
Figure 2
Four methods for detection of HCPs. ELISAs fall into either direct, indirect, competitive or sandwich capture and detection methods. 2D-PAGE is a method of separating HCPs into single components by gel. The western blot technique involves separating native or denatured proteins based on their molecular weight through gel electrophoresis. LC-MS is used to analyze the peptide products obtained by digesting HCPs.
Figure 3
Figure 3
Three methods for data acquisition modes. DDA data relies on acquisition mode. DIA mainly collects primary mass spectrum and fragment information. Full scan obtains the overall mass spectrometry spectra of all ions in the sample.
See this image and copyright information in PMC

References

    1. Beatson R., Sproviero D., Maher J., Wilkie S., Taylor-Papadimitriou J., Burchell J. M., et al. (2011). Transforming growth factor-β1 is constitutively secreted by Chinese hamster ovary cells and is functional in human cells. Biotechnol. Bioeng. 108, 2759–2764. 10.1002/bit.23217 - DOI - PubMed
    1. Bergmann U., Ahrends R., Neumann B., Scheler C., Linscheid M. W. (2012). Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging. Anal. Chem. 84, 5268–5275. 10.1021/ac203460b - DOI - PubMed
    1. Bichmann L., Gupta S., Röst H. (2024). Data-independent acquisition peptidomics. Methods Mol. Biol. 2758, 77–88. 10.1007/978-1-0716-3646-6_4 - DOI - PubMed
    1. Capito F., Skudas R., Kolmar H., Stanislawski B. (2013). Host cell protein quantification by Fourier transform mid infrared spectroscopy (FT-MIR). Biotechnol. Bioeng. 110, 252–259. 10.1002/bit.24611 - DOI - PubMed
    1. Chen G., Warrack B. M., Goodenough A. K., Wei H., Wang-Iverson D. B., Tymiak A. A., et al. (2011). Characterization of protein therapeutics by mass spectrometry: recent developments and future directions. Drug Discov. Today 16, 58–64. 10.1016/j.drudis.2010.11.003 - DOI - PubMed

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