IgM anti-GM2 antibodies in patients with multifocal motor neuropathy target Schwann cells and are associated with early onset.

BACKGROUND: Multifocal motor neuropathy (MMN) is a rare, chronic immune-mediated polyneuropathy characterized by asymmetric distal limb weakness. An important feature of MMN is the presence of IgM antibodies against gangliosides, in particular GM1 and less often GM2. Antibodies against GM1 bind to motor neurons (MNs) and cause damage through complement activation. The involvement of Schwann cells (SCs), expressing GM1 and GM2, in the pathogenesis of MMN is unknown. METHODS: Combining the data of our 2007 and 2015 combined cross-sectional and follow-up studies in Dutch patients with MMN, we evaluated the presence of IgM antibodies against GM1 and GM2 in serum from 124 patients with MMN and investigated their binding to SCs and complement-activating properties. We also assessed the relation of IgM binding and complement deposition with clinical characteristics. RESULTS: Thirteen out of 124 patients (10%) had a positive ELISA titer for IgM anti-GM2. Age at onset of symptoms was significantly lower in MMN patients with anti-GM2 IgM. IgM binding to SCs correlated with IgM anti-GM2 titers. We found no correlation between IgM anti-GM2 titers and MN binding or with IgM anti-GM1 titers. IgM binding to SCs decreased upon pre-incubation of serum with soluble GM2, but not with soluble GM1. IgM anti-GM2 binding to SCs correlated with complement activation, as reflected by increased C3 fixation on SCs and C5a formation in the supernatant. CONCLUSION: Circulating IgM anti-GM2 antibodies define a subgroup of patients with MMN that has an earlier onset of disease. These antibodies probably target SCs specifically and activate complement, similarly as IgM anti-GM1 on MNs. Our data indicate that complement activation by IgM antibodies bound to SCs and MNs underlies MMN pathology.

Human IAPP is a contributor to painful diabetic peripheral neuropathy.

Peripheral neuropathy is a frequent complication of type 2 diabetes mellitus (T2DM). We investigated whether human islet amyloid polypeptide (hIAPP), which forms pathogenic aggregates that damage pancreatic islet ß cells in T2DM, is involved in T2DM-associated peripheral neuropathy. In vitro, hIAPP incubation with sensory neurons reduced neurite outgrowth and increased levels of mitochondrial reactive oxygen species. hIAPP-transgenic mice, which have elevated plasma hIAPP levels without hyperglycemia, developed peripheral neuropathy as evidenced by pain-associated behavior and reduced intraepidermal nerve fiber (IENF) density. Similarly, hIAPP Ob/Ob mice, which have hyperglycemia in combination with elevated plasma hIAPP levels, had signs of neuropathy, although more aggravated. In wild-type mice, intraplantar and intravenous hIAPP injections induced long-lasting allodynia and decreased IENF density. Non-aggregating murine IAPP, mutated hIAPP (pramlintide), or hIAPP with pharmacologically inhibited aggregation did not induce these effects. T2DM patients had reduced IENF density and more hIAPP oligomers in the skin compared with non-T2DM controls. Thus, we provide evidence that hIAPP aggregation is neurotoxic and mediates peripheral neuropathy in mice. The increased abundance of hIAPP aggregates in the skin of T2DM patients supports the notion that hIAPP is a potential contributor to T2DM neuropathy in humans.

Anti-PEG antibodies compromise the integrity of PEGylated lipid-based nanoparticles via complement.

PEGylation of lipid-based nanoparticles and other nanocarriers is widely used to increase their stability and plasma half-life. However, either pre-existing or de novo formed anti-PEG antibodies can induce hypersensitivity reactions and accelerated blood clearance through binding to the nanoparticle surfaces, leading to activation of the complement system. In this study, we investigated the consequences and mechanisms of complement activation by anti-PEG antibodies interacting with different types of PEGylated lipid-based nanoparticles. By using both liposomes loaded with different (model) drugs and LNPs loaded with mRNA, we demonstrate that complement activation triggered by anti-PEG antibodies can compromise the bilayer/surface integrity, leading to premature drug release or exposure of their mRNA contents to serum proteins. Anti-PEG antibodies also can induce deposition of complement fragments onto the surface of PEGylated lipid-based nanoparticles and induce the release of fluid phase complement activation products. The role of the different complement pathways activated by lipid-based nanoparticles was studied using deficient sera and/or inhibitory antibodies. We identified a major role for the classical complement pathway in the early activation events leading to the activation of C3. Our data also confirm the essential role of amplification of C3 activation by alternative pathway components in the lysis of liposomes. Finally, the levels of pre-existing anti-PEG IgM antibodies in plasma of healthy donors correlated with the degree of complement activation (fixation and lysis) induced upon exposure to PEGylated liposomes and mRNA-LNPs. Taken together, anti-PEG antibodies trigger complement activation by PEGylated lipid-based nanoparticles, which can potentially compromise their integrity, leading to premature drug release or cargo exposure to serum proteins.

Anti-C2 Antibody ARGX-117 Inhibits Complement in a Disease Model for Multifocal Motor Neuropathy.

BACKGROUND AND OBJECTIVES: To determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN. METHODS: iPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed. RESULTS: iPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs. DISCUSSION: Binding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

A generic sample preparation method for the multiplex analysis of seven therapeutic monoclonal antibodies in human plasma or serum with liquid chromatography-tandem mass spectrometry.

Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total mAb concentrations in human plasma for clinical studies and therapeutic drug monitoring. We developed an easy, rapid, and robust sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated for infliximab (IFX), rituximab (RTX), cetuximab (CTX), dupilumab (DPL), dinutuximab (DNX), vedolizumab (VDZ), and emicizumab (EMZ). Saturated ammonium sulfate (AS) was used to precipitate immunoglobulins in human plasma. After centrifugation, supernatant containing albumin was decanted, and the precipitated immunoglobulin fraction was re-dissolved in buffer containing 6M guanidine. This fraction was then completely denatured, reduced, alkylated, and trypsin digested. Finally, signature peptides from the seven mAbs were simultaneously quantified on LC-MS/MS together with their internal standards stable isotopically labeled peptide counterparts. The linear dynamic ranges (1 - 512 mg/L) of IFX, CTX, RTX, and EMZ showed excellent (R2 > 0.999) linearity and those of DPL, DNX, and VDZ showed good (R2 > 0.995) linearity. The method was validated in accordance with the EMA guidelines. EDTA plasma, sodium citrate plasma, heparin plasma, and serum yielded similar results. Prepared samples were stable at room temperature (20°C) and at 5°C for 3 days, and showed no decline in concentration for all tested mAbs. This described method, which has the advantage of an easy, rapid, and robust pre-analytical sample preparation, can be used as a template to quantify other mAbs in human plasma or serum.

Cytokine receptor clustering in sensory neurons with an engineered cytokine fusion protein triggers unique pain resolution pathways.

New therapeutic approaches to resolve persistent pain are highly needed. We tested the hypothesis that manipulation of cytokine receptors on sensory neurons by clustering regulatory cytokine receptor pairs with a fusion protein of interleukin (IL)-4 and IL-10 (IL4-10 FP) would redirect signaling pathways to optimally boost pain-resolution pathways. We demonstrate that a population of mouse sensory neurons express both receptors for the regulatory cytokines IL-4 and IL-10. This population increases during persistent inflammatory pain. Triggering these receptors with IL4-10 FP has unheralded biological effects, because it resolves inflammatory pain in both male and female mice. Knockdown of both IL4 and IL10 receptors in sensory neurons in vivo ablated the IL4-10 FP-mediated inhibition of inflammatory pain. Knockdown of either one of the receptors prevented the analgesic gain-of-function of IL4-10 FP. In vitro, IL4-10 FP inhibited inflammatory mediator-induced neuronal sensitization more effectively than the combination of cytokines, confirming its superior activity. The IL4-10 FP, contrary to the combination of IL-4 and IL-10, promoted clustering of IL-4 and IL-10 receptors in sensory neurons, leading to unique signaling, that is exemplified by activation of shifts in the cellular kinome and transcriptome. Interrogation of the potentially involved signal pathways led us to identify JAK1 as a key downstream signaling element that mediates the superior analgesic effects of IL4-10 FP. Thus, IL4-10 FP constitutes an immune-biologic that clusters regulatory cytokine receptors in sensory neurons to transduce unique signaling pathways required for full resolution of persistent inflammatory pain.

ARGX-117, a therapeutic complement inhibiting antibody targeting C2.

BACKGROUND: Activation of the classical and lectin pathway of complement may contribute to tissue damage and organ dysfunction of antibody-mediated diseases and ischemia-reperfusion conditions. Complement factors are being considered as targets for therapeutic intervention. OBJECTIVE: We sought to characterize ARGX-117, a humanized inhibitory monoclonal antibody against complement C2. METHODS: The mode-of-action and binding characteristics of ARGX-117 were investigated in detail. Furthermore, its efficacy was analyzed in in vitro complement cytotoxicity assays. Finally, a pharmacokinetic/pharmacodynamic study was conducted in cynomolgus monkeys. RESULTS: Through binding to the Sushi-2 domain of C2, ARGX-117 prevents the formation of the C3 proconvertase and inhibits classical and lectin pathway activation upstream of C3 activation. As ARGX-117 does not inhibit the alternative pathway, it is expected not to affect the antimicrobial activity of this complement pathway. ARGX-117 prevents complement-mediated cytotoxicity in in vitro models for autoimmune hemolytic anemia and antibody-mediated rejection of organ transplants. ARGX-117 exhibits pH- and calcium-dependent target binding and is Fc-engineered to increase affinity at acidic pH to the neonatal Fc receptor, and to reduce effector functions. In cynomolgus monkeys, ARGX-117 dose-dependently reduces free C2 levels and classical pathway activity. A 2-dose regimen of 80 and 20 mg/kg separated by a week, resulted in profound reduction of classical pathway activity lasting for at least 7 weeks. CONCLUSIONS: ARGX-117 is a promising new complement inhibitor that is uniquely positioned to target both the classical and lectin pathways while leaving the alternative pathway intact.

PBPK model reporting template for chemical risk assessment applications.

Physiologically-based pharmacokinetic (PBPK) modeling analysis does not stand on its own for regulatory purposes but is a robust tool to support drug/chemical safety assessment. While the development of PBPK models have grown steadily since their emergence, only a handful of models have been accepted to support regulatory purposes due to obstacles such as the lack of a standardized template for reporting PBPK analysis. Here, we expand the existing guidances designed for pharmaceutical applications by recommending additional elements that are relevant to environmental chemicals. This harmonized reporting template can be adopted and customized by public health agencies receiving PBPK model submission, and it can also serve as general guidance for submitting PBPK-related studies for publication in journals or other modeling sharing purposes. The current effort represents one of several ongoing collaborations among the PBPK modeling and risk assessment communities to promote, when appropriate, incorporating PBPK modeling to characterize the influence of pharmacokinetics on safety decisions made by regulatory agencies.

Quantification of T Cell Binding Polyclonal Rabbit Anti-thymocyte Globulin in Human Plasma with Liquid Chromatography Tandem-Mass Spectrometry.

The addition of rabbit anti-human thymocyte globulin (ATG) to the conditioning regimen prior to allogeneic hematopoietic cell transplantation has significantly reduced the risk of graft-versus-host disease (GvHD) and graft failure. However, ATG has a small therapeutic window. Overexposure of ATG post-HCT hampers T cell immune reconstitution and has been associated with increased relapse rates and viral reactivations, whereas underexposure has been associated with an increased incidence of GvHD, both of which lead to increased mortality. Therapeutic drug monitoring of T cell binding ATG plasma levels provides a means to optimize dosing for patients at high risk for graft failure to ensure timely T cell immune reconstitution and subsequently increase survival chances. This manuscript describes the first liquid chromatography tandem-mass spectrometry (LC-MS/MS) method to quantify the pharmacologically active fraction of polyclonal ATG in plasma. This was achieved through immunoaffinity purification of active ATG from plasma with Jurkat T cells. After the binding and washing, samples were eluted, denatured, and trypsin-digested. Signature peptides originating from the IgG constant chain were measured with LC-MS/MS. Critical method parameters were optimized, and the method was successfully validated following European Medicines Agency (EMA) guidelines. The method covered the therapeutic range of ATG and was validated at a lower limit of quantification (LLOQ) of 1 AU/mL with an overall CV and bias of 11.8% and - 2.5%, respectively. In conclusion, we developed a LC-MS/MS-based method to quantify active polyclonal rabbit ATG in human plasma. We suggest that this novel assay can be used to monitor and optimize dosing of ATG in clinical practice.

Evidence for bradykinin release in chronic spontaneous urticaria.

BACKGROUND: Chronic spontaneous urticaria (CSU) is characterized by recurrent itchy weals and/or angioedema and is believed to be driven by mast cell activation. It was shown that excessive mast cell activation during anaphylaxis initiates contact activation, resulting in bradykinin release. Evidence for bradykinin release was never demonstrated in CSU. OBJECTIVE: To study biomarkers of bradykinin release in CSU. METHODS: Plasma samples of CSU patients were collected during routine visits at the outpatient clinic. Cleaved high molecular weight kininogen (cHK) was used as a biomarker for bradykinin release. cHK, factor XIIa-C1-inhibitor (FXIIa-C1-INH), kallikrein-C1-INH, plasmin-antiplasmin (PAP) complexes and soluble urokinase-type plasminogen activator receptor (suPAR) levels were determined by ELISA. Clinical data and data on tryptase levels were collected from medical records. cHK levels were compared to previously determined levels in hereditary angioedema (HAE). RESULTS: One hundred seventeen samples from 88 CSU patients and 28 samples from healthy controls were analysed. Median cHK level in CSU was 9.1% (range: 1.4%-21.5%), significantly increased compared to healthy controls (median 6.0% range: 0%-19.9%; P = .0005) and comparable to HAE (n = 46, median 10.3%, range 0%-44.3%, P > .9999). cHK levels normalized in patients during disease remission (median 6.5% range 1.5%-20.8%) but were not dependent on the presence of angioedema, acute angioedema attacks or response to antihistamines. Surprisingly, cHK levels were inversely correlated to serum tryptase (r = -0.65 P = .0137). C1-INH complexes and suPAR levels were not elevated in patients compared to healthy controls. PAP-complex levels in patients were elevated compared to healthy controls but there was no correlation between PAP-complex and cHK levels. CONCLUSIONS: cHK levels are elevated in symptomatic CSU patients compared to healthy controls, indicating increased bradykinin production. Increased cHK levels are not limited to patients with angioedema. CLINICAL RELEVANCE: If elevated bradykinin generation has clinical implications in the pathology of CSU is open to debate.

Complementary and alternative medicine and musculoskeletal pain in the first year of adjuvant aromatase inhibitor treatment in early breast cancer patients.

BACKGROUND: Patients with breast cancer (BC) show strong interest in complementary and alternative medicine (CAM), particularly for adverse effects of adjuvant endocrine treatment - e.g., with letrozole. Letrozole often induces myalgia/limb pain and arthralgia, with potential noncompliance and treatment termination. This analysis investigated whether CAM before aromatase inhibitor (AI) therapy is associated with pain development and the intensity of AI-induced musculoskeletal syndrome (AIMSS) during the first year of treatment. PATIENTS AND METHODS: The multicenter phase IV PreFace study evaluated letrozole therapy in postmenopausal, hormone receptor-positive patients with early BC. Patients were asked about CAM use before, 6 months after, and 12 months after treatment started. They recorded pain every month for 1 year in a diary including questions about pain and numeric pain rating scales. Data were analyzed for patients who provided pain information for all time points. RESULTS: Of 1396 patients included, 901 (64.5%) had used CAM before AI treatment. Throughout the observation period, patients with CAM before AI treatment had higher pain values, for both myalgia/limb pain and arthralgia, than non-users. Pain increased significantly in both groups over time, with the largest increase during the first 6 months. No significant difference of pain increase was noted regarding CAM use. CONCLUSIONS: CAM use does not prevent or improve the development of AIMSS. Pain intensity was generally greater in the CAM group. Therefore, because of the risk of non-compliance and treatment discontinuation due to the development of higher pain levels, special attention must be paid to patient education and aftercare in these patients.

Internal Threshold of Toxicological Concern (iTTC): Where We Are Today and What Is Possible in the Near Future.

The Threshold of Toxicological Concern (TTC) is a risk assessment tool for evaluating low-level exposure to chemicals with limited toxicological data. A next step in the ongoing development of TTC is to extend this concept further so that it can be applied to internal exposures. This refinement of TTC based on plasma concentrations, referred to as internal TTC (iTTC), attempts to convert the chemical-specific external NOAELs (in mg/kg/day) in the TTC database to an estimated internal exposure. A multi-stakeholder collaboration formed, with the aim of establishing an iTTC suitable for human safety risk assessment. Here, we discuss the advances and future directions for the iTTC project, including: (1) results from the systematic literature search for metabolism and pharmacokinetic data for the 1,251 chemicals in the iTTC database; (2) selection of ~350 chemicals that will be included in the final iTTC; (3) an overview of the in vitro caco-2 and in vitro hepatic metabolism studies currently being generated for the iTTC chemicals; (4) demonstrate how PBPK modeling is being utilized to convert a chemical-specific external NOAEL to an internal exposure; (5) perspective on the next steps in the iTTC project.

A mutation in the kringle domain of human factor XII that causes autoinflammation, disturbs zymogen quiescence, and accelerates activation.

Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin. Pathological mutations in the F12 gene, which encodes FXII, provoke acute tissue swelling in hereditary angioedema (HAE). Interestingly, a recently identified F12 mutation, causing a W268R substitution, is not associated with HAE. Instead, FXII-W268R carriers experience cold-inducible urticarial rash, arthralgia, fever, and fatigue. Here, we aimed to investigate the molecular characteristics of the FXII-W268R variant. We expressed wild type FXII (FXII-WT), FXII-W268R, and FXII-T309R (which causes HAE), as well as other FXII variants in HEK293 freestyle cells. Using chromogenic substrate assays, immunoblotting, and ELISA, we analyzed expression media, cell lysates, and purified proteins for FXII activation. Recombinant FXII-W268R forms increased amounts of intracellular cleavage products that are also present in expression medium and display enzymatic activity. The active site-incapacitated variant FXII-W268R/S544A reveals that intracellular fragmentation is largely dependent on autoactivation. Purified FXII-W268R is highly sensitive to activation by plasma kallikrein and plasmin, compared with FXII-WT or FXII-T309R. Furthermore, binding studies indicated that the FXII-W268R variant leads to the exposure of a plasminogen-binding site that is cryptic in FXII-WT. In plasma, recombinant FXII-W268R spontaneously triggers high-molecular-weight kininogen cleavage. Our findings suggest that the W268R substitution influences FXII protein conformation and exposure of the activation loop, which is concealed in FXII-WT. This results in intracellular autoactivation and constitutive low-grade secretion of activated FXII. These findings help to explain the chronically increased contact activation in carriers of the FXII-W268R variant.

Increased intra-articular granzyme M may trigger local IFN-λ1/IL-29 response in rheumatoid arthritis.

OBJECTIVES: Granzymes are serine proteases involved in eliminating tumour cells and virally infected cells. In addition, extracellular granzyme levels are elevated in inflammatory conditions, including several types of infection and autoimmune diseases, such as rheumatoid arthritis (RA). While GrA and GrB have been associated with RA, a role for the other three granzymes (GrH, GrK, and GrM) in this disease remains unclear. Here, we aimed to investigate the presence and role of GrM and GrK in serum and synovial fluid of patients with RA, psoriatic arthritis, and osteoarthritis. METHODS: Granzyme levels were determined in serum, synovial fluid, peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) of RA patients and relevant control groups. In addition, the link between GrM and inflammatory cytokines in synovial fluid was investigated. RESULTS: Serum GrM and GrK levels were not affected in RA. GrM, but not GrK, levels were elevated in synovial fluid of RA patients. GrM was mainly expressed by cytotoxic lymphocytes in SFMCs with a similar expression pattern as compared with PBMCs. Intra-articular GrM expression correlated with IL-25, IL-29, XCL1, and TNFα levels. Intriguingly, purified GrM triggered the release of IL-29 (IFN-λ1) from human fibroblasts in vitro. CONCLUSIONS: These data indicate that GrM levels are increased in RA synovial fluid and that GrM can stimulate proinflammatory IL-29 release from fibroblasts, suggesting a role of GrM in the pathogenesis of RA.

Sialic Acid-Engineered IL4-10 Fusion Protein is Bioactive and Rapidly Cleared from the Circulation.

PURPOSE: Modulating sialylation of therapeutic glycoproteins may be used to influence their clearance and systemic exposure. We studied the effect of low and high sialylated IL4-10 fusion protein (IL4-10 FP) on in vitro and in vivo bioactivity and evaluated the effect of differential sialylation on pharmacokinetic parameters. METHODS: CHO cell lines producing low (IL4-10 FP lowSA) and high sialylated (IL4-10 FP highSA) fusion protein were generated. Bioactivity of the proteins was evaluated in an LPS-stimulated whole blood assay. Pharmacokinetics were studied in rats, analyzing plasma levels of IL4-10 FP upon intravenous injection. In vivo activity was assessed in an inflammatory pain mice model upon intrathecal injection. RESULTS: IL4-10 FP lowSA and IL4-10 FP highSA had similar potency in vitro. The pharmacokinetics study showed a 4-fold higher initial systemic clearance of IL4-10 FP lowSA, whereas the calculated half-life of both IL4-10 FP lowSA and IL4-10 FP highSA was 20.7 min. Finally, both IL4-10 FP glycoforms inhibited persistent inflammatory pain in mice to the same extent. CONCLUSIONS: Differential sialylation of IL4-10 fusion protein does not affect the in vitro and in vivo activity, but clearly results in a difference in systemic exposure. The rapid systemic clearance of low sialylated IL4-10 FP could be a favorable characteristic to minimize systemic exposure after administration in a local compartment.

Simultaneous Quantification of Free Adalimumab and Infliximab in Human Plasma Using a Target-Based Sample Purification and Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Therapeutic drug monitoring of tumor necrosis factor alpha (TNF-α) inhibitors such as adalimumab (ADM) and infliximab (IFX) is considered of added value for patients with systemic inflammatory diseases. In contrast to enzyme-linked immunosorbent assay methods, liquid chromatography-tandem mass spectrometry methods allow for simultaneous quantification of multiple target antibodies in 1 run and thus providing a higher sample throughput. We describe a fast sample work-up strategy for the absolute and simultaneous quantification of ADM and IFX therapeutic monoclonal antibodies in human plasma samples using a target-specific sample purification in combination with liquid chromatography-tandem mass spectrometry. METHODS: The sample purification was based on the selective capture of ADM and IFX in human plasma or serum using biotinylated TNF-α (b-TNF-α), which was coated on a streptavidin 96-well plate. After elution, analytes were heat denatured and trypsin digested to obtain signature peptides for quantification. Stable isotopically labeled ADM and IFX were introduced as internal standard before sample purification. RESULTS: The method was successfully validated following current European medicines agency guidelines. The linear dynamic rage for both analytes were 1-32 mcg/mL with an excellent mean coefficient of determination, R = 0.9994 for ADM and 0.9996 for IFX. Within-run and between-run imprecision and accuracy were within acceptance criteria. Cross-validation against enzyme-linked immunosorbent assay method showed a high between-method correlation R = 0.962 for ADM and R = 0.982 for IFX. CONCLUSIONS: This method provides an easy, efficient, and cost-effective workflow for therapeutic drug monitoring patients treated with ADM or IFX.

Six-step workflow for the quantification of therapeutic monoclonal antibodies in biological matrices with liquid chromatography mass spectrometry - A tutorial.

The promising pipeline of therapeutic monoclonal antibodies (mAbs) demands robust bioanalytical methods with swift development times for pharmacokinetic studies. Over the past decades ligand binding assays were the methods of choice for absolute quantification. However, the production of the required anti-idiotypic antibodies and ligands limits high-throughput method development for sensitive, accurate, and reproducible quantification of therapeutic mAbs. In recent years, high-resolution liquid chromatography tandem mass-spectrometry (LC-MS) systems have enabled absolute quantification of therapeutic mAbs with short method development times. These systems have additional benefits, such as a large linear dynamic range, a high specificity and the option of multiplexing. Here, we briefly discuss the current strategies for the quantification of therapeutic mAbs in biological matrices using LC-MS analysis based on top-down and middle-down quantitative proteomics. Then, we present the widely used bottom-up method in a six-step workflow, which can be used as guidance for quantitative LC-MS/MS method development of mAbs. Finally, strengths and weaknesses of the bottom-up method, which currently provides the most benefits, are discussed in detail.

High occurrence of antihistamine resistance in patients with recurrent idiopathic angioedema.

Antihistamines are the most prescribed therapy in recurrent idiopathic angioedema, yet little is known about their efficacy. Herein, we report on clinical improvement with antihistamine therapy in 120 patients evaluating angioedema attack frequency. A high incidence (36%) of antihistamine refractory cases was observed. Forty percent of patients on antihistamine prophylaxis suffered from 1 or more angioedema attacks per month. Our findings stress the need for additional treatment options for recurrent idiopathic angioedema.