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Advantage of using scout-triggered multiple reaction monitoring mass spectrometry for robust quantification of host cell proteins in biotherapeutics matrices

ABSTRACT: The European and United States Pharmacopeias require the characterization and quantification of host cell proteins (HCPs) contaminating biological products, as these proteins pose a potential risk to clinical safety and product stability. The Enzyme-linked ImmunoSorbent Assay (ELISA) is currently the reference method for quantifying total HCPs, although it does not guarantee accurate and comprehensive detection of all HCPs. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is an alternative and orthogonal technique to ELISA, offering higher sensitivity and a broader dynamic range. In this study, we demonstrate the unmatched advantages of the Scout-triggered MRM targeted acquisition method for analyzing a 240-peptide-plex assay (720 transitions) targeting 97 HCPs in various crude or purified industrial bioproduct matrices derived from Chinese hamster ovary (CHO) cell cultures. Beyond its high multiplexing capability, the main advantage of Scout-triggered MRM, compared to conventional retention time-based methods, lies in its robustness against peptide retention time shifts across different pharmaceutical products. This method allowed us to achieve a dynamic range covering six orders of magnitude, quantifying HCPs down to 2.9 ppm.

INSTRUMENT(S):

ORGANISM(S): Cricetulus Griseus (chinese Hamster) (cricetulus Barabensis Griseus)

TISSUE(S): Cell Culture

SUBMITTER: Julie Flecheux

LAB HEAD: Emmanuelle Vulliet

PROVIDER: PXD066465 | Pride | 2026-02-23

REPOSITORIES: Pride

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			Action	DRS
	Drug_Substance_1_scheduled_MRM.mzML	Mzml
	Drug_Substance_3_Scout-triggered_MRM.mzML	Mzml
	Drug_substance_1_Scout-triggered_MRM.mzML	Mzml
	Drug_substance_2_Scout-triggered_MRM.mzML	Mzml
	Drug_substance_2_scheduled_MRM.mzML	Mzml

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Project description:Host cell proteins (HCPs) are process-related impurities that are generated by the host organism and are typically present at low levels in recombinant biopharmaceutical products, such as therapeutic antibodies. While overall HCP levels are usually monitored by enzyme-linked immunosorbent assay (ELISA), liquid chromatography coupled to mass spectrometry (LC-MS) is emerging as a powerful tool that can provide both qualitative and quantitative information about HCP levels during purification process development. However, a major challenge for LC-MS-based methods is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and therapeutic antibody in solution, which precludes the effective identification of low abundance HCPs in antibody product. This work reports a simple and powerful strategy to identify HCPs in antibody drug substance by applying molecular weight cut-off (MWCO) filtration step followed by shotgun proteomic analysis. After dissociating the interaction between HCPs and antibody with an anionic detergent, the depletion of antibody from HCPs can be easily achieved with the MWCO filtration step. Using this method, we observed that the dynamic range across proteins in the HCP samples was significantly decreased by 1,000-fold. In addition, by spiking in known amounts of HCPs to purified antibody drug substance with low levels of HCPs, we demonstrated that our method could detect HCP at a concentration as low as 1 ppm. When applying this methodology to the study of HCPs in NIST monoclonal antibody (NISTmAb), more than 150 HCPs were confidently identified, which doubles the number of identified HCPs that have been previously reported. Parallel reaction monitoring (PRM) results confirmed that the novel HCPs found using this method were present in very low abundance (0.01-8 ppm), highlighting that our method reduces the dynamic range by removing antibody interference and improving the sensitivity of HCP identification and quantification.

Project description:Targeted mass spectrometry (MS) approaches, which are powerful methods for uniquely and confidently quantifying a specific panel of proteins in complex biological samples, play a crucial role in validation and clinical translation of protein biomarkers discovered by global proteomic profiling. Common targeted MS methods, such as multiple reaction monitoring (MRM) and parallel-reaction monitoring (PRM), employ specific mass spectrometric technologies to quantify protein levels by comparing the transitions of specific endogenous (Endo) peptides with those of stable isotope labeled (SIL) peptide counterparts. These methods utilizing amino acid analyzed (AAA) SIL peptides warrants sensitive and precise measurements required for targeted MS assays. Compared to MRM, PRM provides higher experimental throughput by simultaneously acquiring all transitions of the target peptides and thereby compensate for different ion suppression among transitions of a target peptide. However, PRM still suffers different ion suppressions between Endo and SIL peptides due to poor spray stability as the Endo and SIL peptides were monitored at different liquid chromatography (LC) retention times. Here we introduce a new targeted MS method, termed as wideband PRM (WBPRM), designed for high-throughput targeted MS approach. WBPRM employs a wide isolation window for simultaneous fragmentations of both Endo and SIL peptides along with multiplexed single ion monitoring (SIM) scans for enhanced MS sensitivity of target peptides. Compared to PRM, WBPRM was demonstrated to provide increased sensitivity, precision, and accuracy of quantitative measurements of target peptides with increased throughput, allowing more target peptide measurements in a short experiment time. Its adaptability to a MS method provided by the manufacture makes it a facile method to implement for MS based assays, particularly in complex biological samples, where the needs for higher accuracy, sensitivity and efficiency are paramount.

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Advantage of using scout-triggered multiple reaction monitoring mass spectrometry for robust quantification of host cell proteins in biotherapeutics matrices

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