(M1130-01-02) Can Microsampling Devices Coupled with Intact Mass High-Resolution Mass Spectrometry Improve M-Protein Monitoring and Simplify Minimal Residual Disease Assessment in Multiple Myeloma?

Purpose: Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of malignant plasma cells in the bone marrow (BM), which secrete a monoclonal immunoglobulin (M-protein). Measurable residual disease (MRD) is a critical prognostic marker in MM, used to assess treatment response and the risk of relapse. Traditional MRD monitoring relies on sensitive BM-based techniques such as next-generation flow cytometry (NGF) or sequencing (NGS), but these methods are invasive, limited by sampling variability, and may produce false-negative results due to spatial heterogeneity or extramedullary disease. Previous studies have demonstrated that mass spectrometry (MS)-based detection of M-protein correlates more strongly with progression-free survival than conventional MRD assessments. Based on this, we developed a patient-centric approach that allows for self-collection of blood samples using microsampling devices (20 µL Mitra®), combined with high-resolution MS for direct quantification of M-protein. This minimally invasive workflow enables more frequent and convenient MRD monitoring, potentially improving the accuracy and timeliness of disease tracking.

Methods: Blood samples collected on 20 µL Mitra® devices are processed to isolate the M-protein. The Mitra® devices are homogenized, and the extracted samples are applied to Melon Gel resin, incubated for 5 minutes, and centrifuged. The eluted proteins are then reduced under optimized conditions — including reagent type, volume, and reduction time (ranging from 5 to 30 minutes) — to maximize sensitivity, improve spectral clarity, and minimize noise during deconvolution. Reduced samples are analyzed by LC-MS on a Q Exactive mass spectrometer using a C-4 column, selected to minimize carryover and enhance the separation of light and heavy chains. Data analysis, including deconvolution of intact protein m/z spectra to determine exact masses, is performed using UniDec software for peak selection and plotting.

Results: This mass spectrometry-based workflow enables the processing of up to 96 samples in parallel within 3 hours, offering strong scalability for clinical applications. The method reliably detected and deconvoluted samples with M-protein concentrations as low as 50 µg/mL in MM patients, even in the presence of multiple clones. The assay demonstrated a linear response across the tested analytical range, meeting large molecule assay acceptance criteria for precision and accuracy (±20%, ±25% at the LLOQ) despite the absence of an internal standard. Method robustness was confirmed over a 96-hour runtime with approximately 200 injections, maintaining a %CV below 10% for processed quality controls and demonstrating strong parallelism between plasma and serum matrices. The implementation of Mitra® devices was equally successful, providing a streamlined and highly reproducible sample collection method. Samples immobilized on Mitra® tips showed nearly 100% recovery compared to matched plasma samples, with no loss of sensitivity or accuracy. Blood samples with varying hematocrit levels (35% to 55%) were tested, showing no issues with recovery or signal suppression. Additionally, the method demonstrated blood sample stability for up to 46 days when stored at room temperature. When PRM was implemented in the current method, it enabled the identification of specific IgG isoforms, facilitating more targeted affinity purification for subsequent timepoint monitoring.

Conclusion: The developed high-resolution mass spectrometry workflow enables the accurate and sensitive measurement of M-protein using just 20 µL of peripheral blood, significantly reducing the need for painful bone marrow biopsies. This method demonstrates excellent precision, accuracy, and reproducibility, successfully identifying and tracking multiple IgG isoforms across different time points with unparalleled sensitivity. The patient-centric approach using 20 µL Mitra® devices demonstrated similar recovery to plasma or serum-based purification using whole blood, while being hematocrit-independent and allowing for up to 46 days of sample stability at room temperature. This approach enables frequent blood collection at home, improving sample accessibility for monitoring minimal residual disease (MRD) and providing clinicians with better insights to tailor patient care and response strategies.

References: 1 MONTEITH, Bethany E.; SANDHU, Irwindeep; LEE, Ann S. Management of multiple myeloma: a review for general practitioners in oncology. Current Oncology, 2023, 30.5: 4382-4401.
2 PERROT, Aurore, et al. Minimal residual disease negativity using deep sequencing is a major prognostic factor in multiple myeloma. Blood, The Journal of the American Society of Hematology, 2018, 132.23: 2456-2464.
3 DERMAN, Benjamin A., STEFKA, Andrew T., JIANG, Ken, et al. Measurable residual disease assessed by mass spectrometry in peripheral blood in multiple myeloma in a phase II trial of carfilzomib, lenalidomide, dexamethasone and autologous stem cell transplantation. Blood Cancer Journal, 2021, vol. 11, no 2, p. 19.
4 MARTY, Michael T., et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Analytical chemistry, 2015, 87.8: 4370-4376.