Vedolizumab quantitation using high-resolution accurate mass-mass spectrometry middle-up protein subunit: method validation

Kendall W. Cradic; Paula M. Ladwig; Ann L. Rivard; Waddah Katrangi; Karl Florian Wintgens; Maria A.V. Willrich

doi:10.1515/cclm-2019-0862

Article

Vedolizumab quantitation using high-resolution accurate mass-mass spectrometry middle-up protein subunit: method validation

Kendall W. Cradic , Paula M. Ladwig , Ann L. Rivard , Waddah Katrangi , Karl Florian Wintgens and Maria A.V. Willrich

Published/Copyright: November 22, 2019

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 58 Issue 6

Abstract

Background

While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation.

Methods

We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity.

Results

Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory.

Conclusions

While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.

Keywords: high-resolution accurate mass (HRAM); mass spectrometry; middle-up; quantitation; vedolizumab

Corresponding author: Maria A.V. Willrich, PhD, Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA, Phone: +507-284-2511

aKendall W. Cradic and Paula M. Ladwig contributed equally to this work.

Acknowledgments

The authors would like to thank Jody L. Frinack, MT (ASCP), development technologist at the Immunology Laboratory at Mayo Clinic, for managing the sample cohort and shipping of samples for method comparisons.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2019-0862).

Received: 2019-08-14

Accepted: 2019-10-21

Published Online: 2019-11-22

Published in Print: 2020-06-25

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Supplementary material

Articles in the same Issue

https://doi.org/10.1515/cclm-2019-0862

Keywords for this article

high-resolution accurate mass (HRAM); mass spectrometry; middle-up; quantitation; vedolizumab