High-Throughput Therapeutic Antibody Interference-Free High-Resolution Mass Spectrometry Assay for Monitoring M-Proteins in Multiple Myeloma

Abstract

Measurement of monoclonal antibodies (M-proteins) plays an important role in the diagnosis and treatment monitoring of multiple myeloma. Currently available M-protein assays have several limitations, particularly because of their lack of sensitivity and propensity to therapeutic antibody (t-mAb) interference. A previously described mass spectrometry method termed monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) is more sensitive than current clinical tests and can provide a solution for resolving t-mAb interferences. However, the original miRAMM workflow is too complex for the throughput needed to analyze a large number of samples. Here, we describe a high-throughput liquid chromatography−high-resolution mass spectrometry (HT-LC-HRMS) approach that employs a fully automated immunocapture step, significantly improved immunoglobulin recovery, simplified chromatography, and high throughput (HT) data processing. In this HT-LC-HRMS approach, raw spectra of the peaks eluting from the LC column during the predefined time period are automatically deconvoluted without the need to identify and monitor the retention time of each patient-specific M-protein. The deconvoluted peak heights of M-protein and therapeutic antibody light chain are conveniently used for quantitation. With the total LC-HRMS measurement time being only 11.0 min, this method was able to differentiate between the M-protein and elotuzumab mass signatures in 91 out of 92 (98.9%) multiple myeloma serum samples tested. The single interference case was resolved using the mass signature of a heavy chain. In addition to resolving t-mAb interference, the developed assay has a 25-fold improvement in sensitivity over immunofixation electrophoresis and can potentially provide an objective tracking of M-proteins in patients with complete response.

Validated Method Based on Immunocapture and Liquid Chromatography Coupled to High-Resolution Mass Spectrometry to Eliminate Isatuximab Interference with M-Protein Measurement in Serum

“…Although the process of quantification using XIC area is time-consuming, it was preferred to that based on deconvoluted mass intensity 18 for two reasons: first, it allows one to reach a better sensitivity as in some occasions BioPharma Finder software was not able to deconvolute low M-protein signals near the LLOQ level, and second it enables one to obtain a robust quantification in accordance with FDA requirements for bioanalytical method validation. 19 To achieve this robustness and to obtain the best compromise between specificity and sensitivity, three parameters were meticulously optimized: the number of charge states to be quantified, the number of isotopes for each charge state, and the massextraction window.…”

The potential role of mass spectrometry for the identification and monitoring of patients with plasma cell disorders: Where are we now and which questions remain unanswered?

“…The LC-MS assays provide greater sensitivity 45,46 ; however, they require more expensive equipment, greater expertise to run the samples and have lower throughput. 38,47 The clonotypic peptide approach involves a more complex and time-consuming workflow but offers very high levels of sensitivity. Using clonotypic peptides identified in the heavy chain variable region, Zajec et al reported that residual MP could be detected at <1 mg/l.…”

“…The eluates produced can also be analysed by LC–MS. The LC–MS assays provide greater sensitivity 45,46 ; however, they require more expensive equipment, greater expertise to run the samples and have lower throughput 38,47 …”

“…[52][53][54] Owing to the higher resolution of LC-MS instruments and high specificity of the clonotypic peptides, the LC-MS assays are able to provide almost universal elimination of interference from tmAbs. 47,53,55,56…”

Will Mass Spectrometry Replace Current Techniques for Both Routine Monitoring and MRD Detection in Multiple Myeloma?

“…One common approach uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to follow intact light chains and offers a high-throughput testing option [5]. Other approaches include liquid chromatography high resolution mass spectrometry (LC-HRMS) to follow the intact light chain [6,7] or clonotypic peptides [8][9][10][11]. LC-HRMS systems are more expensive and complex compared to MALDI-TOF MS but offer better analytical sensitivity.…”