Enhancing the sensitivity of peptide quantification for the targeted host cell proteins analysis


Using SCIEX Triple Quad™ 7500 LC-MS/MS System – QTRAP® Ready, powered by SCIEX OS Software

Ian Moore1 and Lei Xiong2
1SCIEX, Canada: 2SCIEX, USA

Abstract


Host cell proteins (HCPs) are process-related impurities monitored in downstream biotherapeutic manufacturing and QC, requiring highly sensitive and robust assays. Here, the simultaneous quantification of 48 proteins in an HCP-like sample was demonstrated using the SCIEX Triple Quad™ 7500 LC-MS/MS System, with an 8 min LC-MS analysis, and LLOQs ranging from 0.02 to 4.54 ppm.

RUO-MKT-02-11820-A1

Introduction


Host cell proteins (HCPs) are a major class of process-related impurities that accompany a recombinant biotherapeutic product during production. As their levels impact the potential toxicity and efficiency of the therapeutics, there are significant requirements for the quantitative measurement of HCP’s across the entire development paradigm, from discovery to quality control.

In downstream manufacturing and QC stages, the HCP targets for quantification are typically predefined, and their levels in the final drug substance or late purification steps can be at the trace level. This results in the increased need for targeted HCP analysis methods with high sensitivity, reduced analysis time, robustness and multiplexing capability (quantify significant numbers of analytes in one injection). Triple quadrupole and QTRAP LC-MS/MS Systems are ideal instrument platforms for such work due to their high quantitative performance.

Herein, a targeted HCP analysis workflow utilizing the SCIEX Triple Quad 7500 LC-MS/MS System – QTRAP Ready has been developed. Multiple hardware improvements on the ion source and the front end of the mass analyzer significantly boost the instrument sensitivity.1

The Scheduled MRMTM Algorithm2 was used to enable the simultaneous quantification of 48 proteins (4 transitions per protein) in an 8 min LC-MS analysis, with LLOQs ranging from 0.02 to 4.54 ppm.

Figure 1. Representative calibration curves for selected peptides from 48 host cell proteins.

Key features of targeted HCP quantification workflow
 

  • Enhanced sensitivity over previous generation systems, with a 4-fold S/N improvement compared to the SCIEX Triple Quad 6500+ LC-MS/MS System1 

  • Hardware advances in ion generation (OptiFlow® Pro Ion Source with E Lens™ Technology) and ion sampling (D Jet™ Ion Guide)

  • SCIEX OS Software for data acquisition and processing— single easy to use platform for acquisition, processing and management

  • Scheduled MRM Algorithm enabled high multiplexing to measure over 200 MRM transitions in an 8 min LC-MS/MS run by maximizing duty cycle

  • The assay shows a high level of reproducibility, accuracy, and linearity, proving the robustness and performance of the developed method