Session: Parallel session 8 - Quantification

Advantage of using Scout-triggered MRM for reliable detection and quantification of Host Cell Proteins in biotherapeutics matrices

Julie FLECHEUX^1,2, Chloé BARDET², Laura HERMENT², Tanguy FORTIN², Jérôme LEMOINE¹

¹Universite Claude Bernard Lyon1, ISA, UMR5280, CNRS, Villeurbanne, France
²ANAQUANT, Lyon, France

The pharmacopoeias require the characterization and quantification of host cell proteins (HCPs) contaminating biological products as these proteins have a potential risk for clinical safety and stability of the product itself. ELISA (Enzyme-linked ImmunoSorbent Assay) is currently the standard method for quantifying total HCPs, though it does not, however, guarantee the accuracy and completeness of HCPs determination. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has proven an alternative and orthogonal technique to ELISA, with higher sensitivity and dynamic range, especially when deployed in the targeted Multiple Reaction Monitoring (MRM) mode.

However, the analysis of the 97 selected HCPs (240 peptides (heavy and light), 720 MRM transitions) by scheduling methods turned out to be problematic in the different matrices, leading to a systematic revision of the method per sample. High Retention Time (RT) shifts have been found between matrices: up to 8min between all samples.

The recently release Scout-triggered MRM mode allows the targeted detection and absolute quantification of the 97 HCPs in different steps of purification of drug substances. The Scout-triggered mode can detect 100% of heavy peptides in a single run, compared with 60% for the Scheduled MRM.

Drug substances samples from Chinese Hamster Ovary (CHO) cell cultures were collected by various biomanufacturing industries at different purification steps. 500µg of total protein content were denaturated, reduced, alkylated and digested with trypsin. LC-MS/MS analysis was performed on an Exion LC system coupled to a QTRAP® 6500+ SCIEX.

The acquisition mode Scout-triggered MRM was applied to increase the multiplexing capacity through the implementation of a 97-protein multiplex. This study shows the application to the HCPs analysis and the robustness of the Scout-triggered MRM method compared to the time window scheduling methods, limiting the loss of information due to potential RT shifts.