A sensitive high-resolution mass spectrometry method for quantifying intact M-protein light chains in patients with multiple myeloma.

To determine the disease status and the response to treatment for patients with multiple myeloma, measuring serum M-protein levels is a widely used alternative to invasive punctures to count malignant plasma cells in the bone marrow. However, the quantification of this monoclonal antibody, which varies from patient to patient, poses significant analytical challenges. This paper describes a sensitive and specific mass spectrometry assay that addresses two objectives: to overcome the potential interference of biotherapeutics in the measurement of M-proteins, and to determine the depth of response to treatment by assessing minimal residual disease. After immunocapture of immunoglobulins and free light chains in serum, heavy and light chains were dissociated by chemical reduction and separated by liquid chromatography. M-proteins were analyzed by high-resolution mass spectrometry using a method combining a full MS scan for isotyping and identification and a targeted single ion monitoring scan for quantification. This method was able to discriminate M-protein from the therapeutic antibody in all patient samples analyzed and allowed quantification of M-protein with a LLOQ of 2.0 to 3.5 µg/ml in 5 out of 6 patients. This methodology appears to be promising for assessing minimal residual disease with sufficient sensitivity, specificity, and throughput.

Absorption, Metabolism, and Excretion of [14C]-Tolebrutinib After Oral Administration in Humans, Contribution of the Metabolites to Pharmacological Activity.

BACKGROUND AND OBJECTIVE: Tolebrutinib is a covalent inhibitor of Bruton's tyrosine kinase, an enzyme expressed in B lymphocytes and myeloid cells including microglia, which are thought to be major drivers of inflammation in multiple sclerosis. This excretion balance and metabolism study evaluated the metabolite profile of tolebrutinib in healthy male volunteers. METHODS: Six healthy volunteers received a 60-mg oral dose of [14C]-tolebrutinib, and metabolite profiling of 14C-labeled metabolites was performed using a combination of liquid chromatography, mass spectrometry, and radioactivity assay methods. RESULTS: Tolebrutinib was rapidly and completely absorbed from the gastrointestinal tract, followed by rapid and extensive metabolism. Excretion via feces was the major elimination pathway of the administered radioactivity (78%). Tolebrutinib was highly metabolized, with 19 metabolites identified in human plasma. Phase 1 biotransformations were primarily responsible for the circulating metabolites in plasma. Seven metabolites that achieved exposure in plasma similar to or higher than the parent compound were characterized biochemically for inhibition of Bruton's tyrosine kinase activity. Metabolite M8 exceeded the exposure threshold of 10% (18%) of the total radioactivity but had little if any pharmacological activity. Metabolite M2 (4% of circulating radioactivity) retained the ability to irreversibly and potently inhibit Bruton's tyrosine kinase in vitro, similar to the parent compound. Tolebrutinib and metabolite M2 had short (3.5-h) half-lives but durable pharmacodynamic effects as expected for an irreversible antagonist. CONCLUSIONS: Tolebrutinib was extensively metabolized to multiple metabolites. The hydroxylated metabolite M2 demonstrated similar inhibitory potency toward Bruton's tyrosine kinase as the parent compound. Both tolebrutinib and metabolite M2 likely contributed to pharmacological activity in vivo.

Immunocapture LC-MS(/MS) assays for biotherapeutic and biomarker proteins: the European Bioanalysis Forum continuing discussions on scientific and regulatory challenges.

The use of LC-MS(/MS) assays to quantify (biotherapeutic or biomarker) proteins is commonplace and well accepted across industry. There is a good understanding on the added value over conventional analytical technologies (i.e., ligand-binding assays). In fact, the impact of combining small- and large-molecule technologies for large-molecule analysis has played a significant part in bringing the bioanalytical communities closer together and building a mutual respect and understanding between scientists. This paper from the European Bioanalysis Forum presents a history of the journey and future perspectives for hybrid assays, with focus on the unanswered scientific questions, including regulatory discussions to be had. Hybrid assays are essentially a combination of ligand-binding assays and MS, and the ICH M10 guideline does not address this approach directly. Decision-based acceptance criteria are still being discussed, and the industry should continue to do so.

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

In multiple myeloma (MM) disease, malignant plasma cells produce excessive quantities of a monoclonal immunoglobulin (Ig), known as M-protein. M-protein levels are measured in the serum of patients with MM using electrophoresis techniques to determine the response to treatment. However, therapeutic monoclonal antibodies, such as isatuximab, may confound signals using electrophoresis assays. We developed a robust assay based on immunocapture and liquid chromatography coupled to high-resolution mass spectrometry (IC-HPLC-HRMS) in order to eliminate this interference. Following immunocapture of Ig and free light chains (LC) in serum, heavy chains (HC) and LC were dissociated using dithiothreitol, sorted by liquid chromatography and analyzed using HRMS (Q-Orbitrap). This method allowed the M-proteins to be characterized and the signals from isatuximab and M-proteins to be discriminated. As M-protein is specific to each patient, no standards were available for absolute quantification. We therefore used alemtuzumab (an IgG kappa mAb) as a surrogate analyte for the semiquantification of M-protein in serum. This assay was successfully validated in terms of selectivity/specificity, accuracy/precision, robustness, dilution linearity, and matrix variability from 10.0 to 200 µg/mL in human serum. This method was used for clinical assessment of samples and eliminated potential interference due to isatuximab when monitoring patients with MM.

Inter-Laboratory Robustness of Next-Generation Bile Acid Study in Mice and Humans: International Ring Trial Involving 12 Laboratories.

BACKGROUND: The increasing relevance of individual bile acids quantification in biological samples requires analytical standardization to guarantee robustness and reliability of laboratory results. We have organized the first international ring trial, carried out in 12 laboratories, to evaluate the newly developed LC-MS/MS-based test kit for bile acid analysis. METHODS: Each laboratory received a Biocrates® Bile Acids Kit including system suitability test (SST) protocol. The kit is designed to analyze 16 individual human and 19 mouse bile acids. A set of 9 human and mouse plasma samples was measured in replicates. Laboratories were first required to pass the acceptance criteria for the SST. Within the subset of laboratories passing SST criteria, we evaluated how many laboratories met the target criteria of 80% of reported values with a relative accuracy within the 70%-130% range and analytical precisions (%CV) below 30%. RESULTS: A total of 12 of 16 participating laboratories passed the SST as the prerequisite to enter the ring trial. All 12 laboratories were then able to successfully run the kit and ring trial samples. Of the overall reported values, 94% were within 70%-130% relative accuracy range. Mean precision was 8.3% CV. The condition of CV <30% was fulfilled by 99% of the reported values. CONCLUSIONS: The first publically available interlaboratory ring trial for standardized bile acids quantification in human and mouse plasma samples showed very good analytical performance, within acceptance criteria typically applied in the preclinical environment. The kit is therefore suitable for standardized quantitative bile acid analysis and the establishment of reference values.