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Anal Chem. 2018 Feb 6;90(3):1870-1880. doi: 10.1021/acs.analchem.7b03949. Epub 2018 Jan 8.

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics.

Author information

1
Department of Pharmaceutical Sciences, University at Buffalo, State University of New York , Buffalo, New York 14214, United States.
2
New York State Center of Excellence in Bioinformatics and Life Sciences , Buffalo, New York 14203, United States.
3
Department of Pharmacy, Huazhong University of Science and Technology , Wuhan 430030, China.
4
Division of Cardiovascular Medicine, Western New York Department of Veterans of Affairs Medical Center , Buffalo, New York 14203, United States.
5
Clinical and Translational Research Center, University at Buffalo, State University of New York , Buffalo, New York 14203, United States.
6
Thermo Scientific , San Jose, California 95134, United States.
7
Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel , Basel CH-4070, Switzerland.
8
Roche Pharmaceutical Research and Early Development, Roche Innovation Center New York , New York, New York 10016, United States.

Abstract

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

PMID:
29276835
PMCID:
PMC5960441
DOI:
10.1021/acs.analchem.7b03949
[Indexed for MEDLINE]
Free PMC Article

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