Performance metrics for evaluating system suitability in liquid chromatography—Mass spectrometry peptide mass mapping of protein therapeutics and monoclonal antibodies

Abstract

The use of liquid chromatography – mass spectrometry (LC-MS) for the characterization of proteins can provide a plethora of information related to their structure, including amino acid sequence determination and analysis of posttranslational modifications. The variety of LC-MS based applications has led to the use of LC-MS characterization of therapeutic proteins and monoclonal antibodies as an integral part of the regulatory approval process. However, the improper use of an LC-MS system, related to intrinsic instrument limitations, improper tuning parameters, or poorly optimized methods may result in the production of low quality data. Improper system performance may arise from subtle changes in operating conditions that limit the ability to detect low abundance species. To address this issue, we systematically evaluated LC-MS/MS operating parameters to identify a set of metrics that can be used in a workflow to determine if a system is suitable for its intended purpose. Development of this workflow utilized a bovine serum albumin (BSA) digest standard spiked with synthetic peptides present at 0.1% to 100% of the BSA digest peptide concentration to simulate the detection of low abundance species using a traditional bottom-up workflow and data-dependent MS² acquisition. BSA sequence coverage, a commonly used indicator for instrument performance did not effectively identify settings that led to limited dynamic range or poorer absolute mass accuracy on 2 separate LC-MS systems. Additional metrics focusing on the detection limit and sensitivity for peptide identification were determined to be necessary to establish system suitability for protein therapeutic characterization by LC-MS.

Keywords:

• system suitability
• protein therapeutics
• liquid chromatography
• mass spectrometry
• quality control

Disclosure of Potential Conflicts of Interest

The ideas, findings, and conclusions in this publication have not been formally disseminated by the US. Food and Drug Administration (FDA) and should not be construed to represent any agency determination or policy.

Acknowledgments

The authors acknowledge manuscript preparation assistance from Dr. Michaella Levy and Anneliese Faustino.

Funding

This study was supported in part by an appointment to the Research Participation Program at the FDA Center for Drug Evaluation and Research, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and FDA (MZ).This work was funded by the FDA Center for Drug Evaluation and Research Critical Path program (MTB).

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