Serum Concentrations of Infliximab and IL-6 for Predicting One-Year Discontinuation of Infliximab Treatment Owing to Secondary Non-response in Patients with Rheumatoid Arthritis.

Secondary non-response to infliximab (IFX) occurs in some patients with rheumatoid arthritis (RA). Although therapeutic drug monitoring (TDM) is a useful tool to optimize IFX therapy, it is unclear whether it can help to identify the risk of secondary non-response. This study aimed to explore the utility of serum levels of IFX or other biomarkers to predict IFX discontinuation owing to secondary non-response. A single-center, retrospective study was conducted using the Kyoto University Rheumatoid Arthritis Management Alliance cohort database between 2011 and 2020. Serum IFX levels were measured using liquid chromatography-tandem mass spectrometry. An electrochemiluminescence assay was used to quantify serum levels of tumor necrosis factor-α and interleukin-6 and detect anti-drug antibodies. Eighty-four out of 310 patients were eligible for this study. The cutoff levels of biomarkers were determined by receiver operating characteristic analysis. IFX persistence was similar between groups stratified using IFX levels, tumor necrosis factor-α levels, interleukin-6 levels, and anti-drug antibodies positivity. The group with lower IFX and higher interleukin-6 levels had the worst therapy persistence (p = 0.017) and the most frequent disease worsening (90.0%, p < 0.001). Evaluating both interleukin-6 and IFX levels, not just IFX alone, enabled us to identify patients at risk of discontinuing IFX treatment. These findings support the utility of measuring IFX and interleukin-6 levels for successful maintenance therapy for RA.

A rapid and universal liquid chromatograph-mass spectrometry-based platform, refmAb-Q nSMOL, for monitoring monoclonal antibody therapeutics.

Accurate quantitation of antibodies is critical for development of monoclonal antibody therapeutics (mAbs). Therapeutic drug monitoring has been applied to measure levels of mAbs in clinics for dose adjustment for autoimmune disease. Trough levels of mAbs can be a biomarker for cancer immunotherapy. Thus, the deployment of a rapid and universal platform for mAb monitoring may benefit processes ranging from drug development to clinical practice for a wide spectrum of diseases. However, mAb monitoring often requires development and conduct of an individual ligand binding assay such as ELISA, which is impractical to scale. We streamlined quantitation of antibody therapeutics by a nano-surface and molecular-orientation limited (nSMOL) proteolysis assay using LC-MS with a universal reference antibody (refmAb-Q), for accurate multiplexed quantitation of unique signature peptides derived from mAbs. This innovative refmAb-Q nSMOL platform may provide a practical solution for quantitating an ever-increasing number of mAbs from developmental to clinical use settings.

N-terminus of Etanercept is Proteolytically Processed by Dipeptidyl Peptidase-4.

PURPOSE: Biologics are structurally heterogeneous and can undergo biotransformation in the body. Etanercept (ETN) is a fusion protein composed of a soluble tumor necrosis factor (TNF) receptor and the Fc portion of human immunoglobulin G1. The N-terminus of ETN has a putative sequence cleaved by dipeptidyl peptidase-4 (DPP-4). The purpose of this study was to investigate the biotransformation of ETN in humans and mice and evaluate its effects on functional properties. METHODS: An analytical method using liquid chromatography-mass spectrometry (LC-MS/MS) was established. The N-terminal heterogeneity of ETN was assessed in the serum of patients with rheumatoid arthritis or mice receiving ETN. The in vitro N-terminal truncation was explored using recombinant DPP-4. The binding affinity to TNF-α or TNF-ß was investigated using an in-house enzyme-linked immunosorbent assay. RESULTS: In the formulations, about 90% of ETN had an intact N-terminus, while the N-terminal truncated form was most abundant in the serum of the patients with rheumatoid arthritis and mice. Recombinant human DPP-4 cleaved two amino acids from the N-terminus of ETN in vitro. Sitagliptin, a DPP-4 inhibitor, inhibited N-terminal truncation both in vivo and in vitro. However, N-terminal truncation did not affect the binding ability to TNF-α or TNF-ß and the pharmacokinetics of ETN. ETN biosimilars exhibited similar characteristics to the reference product in vivo and in vitro. CONCLUSIONS: ETN undergoes N-terminal truncation in the body, and DPP-4 cleaves exogenous ETN via N-terminal proteolysis. The application of an MS-based assay will detect novel biotransformation of therapeutic proteins.

Potential application of measuring serum infliximab levels in rheumatoid arthritis management: A retrospective study based on KURAMA cohort data.

Infliximab (IFX) therapy has considerably improved the treatment of rheumatoid arthritis (RA). However, some patients still do not respond adequately to IFX therapy, or the efficacy of the treatment diminishes over time. Although previous studies have reported a relationship between serum IFX levels and therapeutic efficacy, the potential applications of IFX therapeutic drug monitoring (TDM) in clinical practice remain unclear. The purpose of this study was to investigate the potential applications of IFX TDM by analyzing a Japanese cohort database. Data were collected retrospectively from the Kyoto University Rheumatoid Arthritis Management Alliance cohort between January 1, 2011, and December 31, 2018. Serum IFX levels were measured using a liquid chromatography-tandem mass spectrometer. Out of the 311 RA patients that used IFX, 41 were eligible for the analysis. Serum IFX levels were significantly higher in responders than in non-responders. An optimal cut-off value was determined to be 0.32 µg/mL based on a receiver operating characteristic curve. At the IFX measurement point, a better therapeutic response was observed in the high IFX group (n = 32) than in the low IFX group (n = 9). Conversely, at the maximum effect point, when DAS28-ESR was the lowest between IFX introduction and measurement points, there were no differences in responder proportions between the low and high IFX groups. IFX primary ineffectiveness could be avoided with appropriate dose escalation without blood concentration measurement in clinical practice. In conclusion, IFX TDM could facilitate the identification of secondary non-responders and in turn, proper IFX use.

Multiplexed monitoring of therapeutic antibodies for inflammatory diseases using Fab-selective proteolysis nSMOL coupled with LC-MS.

Monoclonal antibodies have accelerated the availability of treatment options for many diseases in which the molecular mechanism has been elucidated in detail. Therefore, an assay that can universally analyze antibodies for clinical pharmacokinetics and cross-sectional studies would be indispensable. We have developed a universal antibody bioanalysis with a Fab-selective tryptic reaction, named nano-surface and molecular-orientation limited (nSMOL) proteolysis, that collects the specific antibody signature peptides in biological samples. Using the nSMOL method, we have fully validated the bioanalysis of many antibodies, Fc-fusion proteins, and their biosimilars. Inflammatory immune diseases often require long-term clinical management because of the remission and relapse observed. Accurate antibody monitoring in systemic circulation could contribute to the improvement of clinical outcomes. Because several biopharmaceuticals can be selected as practical treatment options, the assay development that quantitates many antibodies simultaneously would be applicable in many theraprutic monitoring. In this study, we have validated the LC-MS bioanalysis method for seven-mixed antibodies (Infliximab, Adalimumab, Ustekinumab, Golimumab, Eculizumab, Etanercept, and Abatacept) using the nSMOL normal reaction condition and two-mixed antibodies (Tocilizumab and Mepolizumab) using the acidified reduction acceleration condition, as reported in our previous papers. Moreover, this multiplexed assay has been verified using clinical patient samples. The nSMOL approach enables the quantitation of several immunosuppressive antibodies simultaneously in human serum, and nSMOL can potentially be applicable to the drug-drug interaction assays or therapeutic antibody monitoring of several inflammatory immune diseases to optimize administration.

Application of nSMOL coupled with LC-MS bioanalysis for monitoring the Fc-fusion biopharmaceuticals Etanercept and Abatacept in human serum.

The principle of nano-surface and molecular-orientation limited (nSMOL) proteolysis has a unique characteristic Fab-selective proteolysis for antibody bioanalysis that is independent of a variety of monoclonal antibodies by the binding antibody Fc via Protein A/G in a pore with 100 nm diameter and modified trypsin immobilization on the surface of nanoparticles with 200 nm diameter. Since minimizing peptide complexity and protease contamination while maintaining antibody sequence specificity enables a rapid and broad development of optimized methods for liquid chromatography-mass spectrometry (LC-MS) bioanalysis, the application of regulatory LC-MS for monitoring antibody biopharmaceuticals is expected. nSMOL is theoretically anticipated to be applicable for representative Fc-fusion biopharmaceuticals, because Protein A/G-binding site Fc exists on the C-terminus, and its functional domain is available to orient and interact with the reaction solution. In this report, we describe the validated LC-MS bioanalysis for monitoring Ethanercept and Abatacept using nSMOL technology. The quantitation range of Ethanercept in human serum was from 0.195 to 100 µg/mL using the signature peptide VFCTK (aa.43-47), and that of Abatacept was from 0.391 to 100 µg/mL using the signature peptide MHVAQPAVVLASSR (aa.1-14). Both proteins fulfilled the guideline criteria for low-molecular-weight drug compounds. The results indicate that the clinical and therapeutic monitoring for antibody and Fc-fusion biopharmaceuticals are adequately applicable using nSMOL proteolysis coupled with LC-MS bioanalysis.

Verification between Original and Biosimilar Therapeutic Antibody Infliximab Using nSMOL Coupled LC-MS Bioanalysis in Human Serum.

BACKGROUND: Infliximab (IFX) is a chimeric therapeutic monoclonal antibody targeting tumor necrosis factor alpha (TNFα)-mediated inflammatory immune diseases. However, despite of an initial good clinical response, decrease in response to long-term treatment is a common observation. OBJECTIVE: Recent studies suggest that IFX level in circulation has a correlation with clinical bioavailability. Therefore, the management of IFX dosage for individual manifestation by IFX monitoring may be valuable for the improvement of therapeutic response and outcomes. METHOD: In order to develop a broad IFX therapeutic monitoring in human serum, we have developed the validated IFX bioanalysis for RemicadeTM and its biosimilar product using our nano-surface and molecular-orientation limited proteolysis (nSMOL) technology coupled with liquid chromatographytandem mass spectrometry (LC-MS/MS). The nSMOL chemistry has a unique property of Fabselective proteolysis, and makes it possible a global bioanalysis for many monoclonal antibodies. RESULTS: The quantitation range of IFX in serum was from 0.293 to 300 µg/ml with good linearity. Quantitation verification at the concentrations of 0.293, 0.879, 14.1 and 240 µg/ml was within 1.56- 7.53% of precision and 98.9-111% of accuracy using H-chain signature peptide SINSATHYAESVK. Moreover, cross-verified bioanalysis of Remicade quantitation using biosimilar standard, and its opposite combination, obtained an identical and inter-comparative results. CONCLUSION: The nSMOL strategy has the potential as a practical therapeutic monitoring technology in IFX therapeutic applications.