MSACL 2024 Abstract

Antibody Light-Chain Quantitation with LC-MS: Advantages of Specificity to Improve Accuracy and Precision

Kayd Meldrum (1), Anthony Maus (2), Paula Ladwig (2), Andrew Swansiger (1), Jacob Koscho (1), Michael Knierman (3), Crystal Cody (3), John Sausen (3), Maria Willrich (2), James Prell (1)
(1) Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR (2) Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN (3) Agilent Technologies, Santa Clara, CA

James Prell, PhD (Presenter) University of Oregon

Presenter Bio: Associate Professor James S. Prell has been on the faculty of the Department of Chemistry and Biochemistry at the University of Oregon since 2014, where his laboratory develops ion mobility-mass spectrometry experiments, computational methods, and theory to explore both fundamentals and applications of bioanalytical IM-MS. With a background in ion spectroscopy and nanocalorimetry and ultrafast solid-state nanoscience from his PhD and postdoctoral work at UC Berkeley, he has investigated mechanisms of electrospray ionization, patterning of nanodroplet water networks by embedded ions, and the relationship between condensed-phase and gas-phase structures of proteins and their complexes. His work collaborates with academic, clinical, and industry researchers around the world to advance novel mass spectrometry signal processing methods, tissue imaging, pharmaceutical drug quantitation, and understanding of biomolecular ion structure-thermochemistry relationships.

Relevant Financial Disclosures (within past 24 months, reported on Feb 23, 2024) Grant/Research Support National Institute of General Medical Sciences, National Science Foundation, Agilent Technologies Discussing Products? YES, presenter will present educational content related to products/services of one or more ineligible company(ies) listed above. Conflict mitigation IN PROCESS.

INTRODUCTION: Highly accurate and precise quantitation of antibodies in the clinical setting, especially at levels below a few micrograms per milliliter, can be very challenging due to interferents. High-resolution mass spectrometry (MS) and state-of-the-art spectral deconvolution software offer major advantages to address this challenge.

OBJECTIVES: Vedolizumab light chain from human serum samples was quantitated using liquid chromatography-MS (LC-MS) and two different data analysis methods. Major objectives were to introduce Gábor Transform deconvolution software, which has never before been demonstrated for protein quantitation, and to compare light chain concentrations determined using this method to those determined using commercial software.

METHODS: Nivolumab standard is added to human serum samples containing vedolizumab before Melon Gel purification and cleavage to produce light chains. LC-MS data for quantitation were acquired for ~400 samples in parallel at Mayo Clinic and the University of Oregon, where they were analyzed using an extracted ion chromatogram-based method or Gábor-Transform-based software (“iFAMS Quant+”), respectively. These samples ranged in concentration from below 1 microgram/milliliter to ~200 micrograms/milliliter (~3 orders of magnitude). Concentrations determined using the two methods were plotted against one another to determine method bias and Pearson R2 coefficients, and Bland-Altman analysis was used to identify concentration-dependent differences in the results from the two methods.

RESULTS: Due to the high mass accuracy of the MS instrumentation used, signals for vedoluzimab and nivolumab light chain were easily distinguished from those of endogenous IgG light chains and other interferents on both instruments. Quantitation was highly consistent between the two instruments and software methods, with R2 coefficients for each sample set comparison between Mayo and U Oregon results exceeding 0.9. iFAMS Quant+ was able to establish a Lower Limit of Quantitation (LLOQ) of less than 2 microgram/milliliter for these samples. As expected, Bland-Altman analysis revealed that the largest discrepancies between the two methods typically occur at the lowest concentrations studied and are consistent with the LLOQ.

CONCLUSION: Highly chemically specific antibody drug quantitation can be achieved using LC-MS and state-of-the-art quantitation software. Excellent agreement between results for vedolizumab light chain in human serum at two different institutions with different instrumentation and data analysis software, especially at low-microgram per milliliter concentrations, highlights the advantages of this technique to address the challenge of antibody quantitation. These results also constitute the first demonstration of Gabor Transform deconvolution tools for protein quantitation.