Willrich, Maria Alice V.

Abstract BACKGROUND Free light chain (FLC) quantification is the most analytically sensitive blood-based method commercially available to diagnose and monitor patients with plasma cell disorders (PCDs). However, instead of directly detecting monoclonal FLCs (mFLCs), FLC assays indirectly assess clonality based on quantifying κ and λ FLCs and determination of the к/λ FLC ratio. Often an abnormal FLC ratio is the only indication of a PCD, and confirmation by a direct method increases diagnostic confidence. The aim of this study was to develop an analytically sensitive method for direct detection of mFLCs. METHODS Patient sera (n = 167) previously assessed by nephelometric FLC quantification and immunofixation electrophoresis (IFE) were affinity enriched for IgG, IgA, and total and free κ and λ light chains and subjected to MALDI-TOF MS. Relative analytical sensitivity of these methods was determined using serially diluted sera containing mFLCs. RESULTS In sera with abnormal FLC ratios (n = 127), 43% of monoclonal proteins were confirmed by IFE, 57% by MALDI-TOF MS without FLC enrichment, and 87% with FLC enrichment MALDI-TOF MS. In sera with normal FLC ratios (n = 40), the FLC MALDI-TOF MS method identified 1 patient with an mFLC. Serial dilution and analysis of mFLC containing sera by IFE, nephelometry, and FLC MALDI-TOF MS demonstrated that FLC MALDI-TOF MS analysis had the highest analytical sensitivity. CONCLUSIONS FLC immunoenrichment coupled to MALDI-TOF MS enables direct detection of mFLCs and significantly increases the confirmation of abnormal serum FLC ratios over IFE and MALDI-TOF MS without FLC enrichment, thereby providing added confidence for diagnosing FLC PCDs.

Abstract Background Infliximab (IFX) is a chimeric monoclonal antibody targeting tumor necrosis factor-α, used for the management of autoimmune and inflammatory diseases. Immunogenicity to this protein drug may lead to therapeutic failure. Laboratory testing for serum IFX and antidrug antibodies (ADAs) is available for the evaluation of clinical nonresponsiveness. The purpose of this study was to compare the performance of testing methodologies used by 2 clinical reference laboratories for the quantification of IFX and detection of ADAs. Methods Deidentified serum samples submitted for clinical testing were selected (n = 120) and tested at both sites. A trypsin-based LC-MS/MS assay for IFX and a bridging electrochemiluminescent immunoassay (ECLIA) for ADAs (Mayo Clinic) and a functional cell-based reporter gene assay (RGA) to measure both bioactive drug concentrations and neutralizing ADAs (ARUP Laboratories) were compared. Results In all, 105 samples had measurable concentrations of IFX by both methods and yielded a correlation coefficient (r) = 0.917, slope = 1.028, and intercept = −0.377. One outlier measured <1.0 μg/mL by LC-MS/MS and 37 μg/mL by RGA, which was confirmed to be attributed to the presence of adalimumab. Regarding detection of ADAs, 81 of 120 samples were negative by ECLIA and RGA, whereas 30 of 120 were positive by both methods, resulting in an overall 92.5% agreement. Conclusion Although there are substantial methodological differences in the assays used for detecting IFX and ADAs, results show significant concordance between the clinically validated methodologies performed in different laboratories.

ABSTRACTTherapeutic monoclonal antibodies (MAbs) are an important class of drugs used to treat diseases ranging from autoimmune disorders to B cell lymphomas to other rare conditions thought to be untreatable in the past. Many advances have been made in the characterization of immunoglobulins as a result of pharmaceutical companies investing in technologies that allow them to better understand MAbs during the development phase. Mass spectrometry is one of the new advancements utilized extensively by pharma to analyze MAbs and is now beginning to be applied in the clinical laboratory setting. The rise in the use of therapeutic MAbs has opened up new challenges for the development of assays for monitoring this class of drugs. MAbs are larger and more complex than typical small-molecule therapeutic drugs routinely analyzed by mass spectrometry. In addition, they must be quantified in samples that contain endogenous immunoglobulins with nearly identical structures. In contrast to an enzyme-linked immunosorbent assay (ELISA) for quantifying MAbs, mass spectrometry-based assays do not rely on MAb-specific reagents such as recombinant antigens and/or anti-idiotypic antibodies, and time for development is usually shorter. Furthermore, using molecular mass as a measurement tool provides increased specificity since it is a first-order principle unique to each MAb. This enables rapid quantification of MAbs and multiplexing. This review describes how mass spectrometry can become an important tool for clinical chemists and especially immunologists, who are starting to develop assays for MAbs in the clinical laboratory and are considering mass spectrometry as a versatile platform for the task.