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

Abstract

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 bioavailabil-ity. 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 molecu-lar-orientation limited proteolysis (nSMOL) technology coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The nSMOL chemistry has a unique property of Fab-selective prote-olysis, 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. Quan-titation 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 combina-tion, obtained an identical and inter-comparative results.Conclusion: The nSMOL strategy has the potential as a practical therapeutic monitoring technology in IFX therapeutic applications.

High‐throughput untargeted screening of biotherapeutic macromolecules in equine plasma by UHPLC‐HRMS/MS: Application to monoclonal antibodies and Fc‐fusion proteins for doping control

“…The highest human IgG concentration reached 1.2 μg/mL, 36 h after administration. This concentration remains constant until Day 10 (about 1 μg/mL) and rapidly decreased between Days 10 and 15 as for this time point, and in the forthcoming ones (i.e 16,18,20,21,24,26,28,. and 30 days), human IgG peptides were no longer detected (Figure…”

“…Thus, the detection and quantification of mAbs or Fc-proteins surrogate peptides after enzymatic digestion (bottom-up strategy) is still the preferred approach in such research areas. [21][22][23][24] For human doping controls, several electrophoretic techniques (IEF/SDS-and SAR-PAGE) and Western blotting or LC-HRMS/MS approaches have been described. These methods target inhibitors of the activin receptor signaling pathways, including therapeutic antibodies (such as domagrozumab 25 and bimagrumab 26 ), ActRII-Fc fusion proteins (such as sotatercept and luspatercept 27 ), and follistatin-Fc fusion proteins.…”

“…Although feasible, the top‐down proteomics‐based detection of mAbs 20 presents a major shortcoming in terms of sensitivity despite constant improvements in MS instruments and related techniques. Thus, the detection and quantification of mAbs or Fc‐proteins surrogate peptides after enzymatic digestion (bottom‐up strategy) is still the preferred approach in such research areas 21–24 …”

Best Practice for Therapeutic Drug Monitoring of Infliximab: Position Statement from the International Association of Therapeutic Drug Monitoring and Clinical Toxicology

“…145,146 Advances in proteomics research have demonstrated that liquid chromatography-tandem mass spectrometry methods are also capable of analyzing peptides and proteins in biological matrices with high selectivity and specificity. 143,147,148 The lower limit of quantification varies between assays but is generally well below the therapeutic threshold, making it suitable for TDM applications, with reported LLOQs ranging from 0.001 to 0.06 mg/L.…”

Potential Application of Measuring Serum Infliximab Levels in Rheumatoid Arthritis Management: A Retrospective Study based on KURAMA Cohort Data

“…Blood samples for measuring trough serum IFX levels were collected immediately before a new infusion. Serum IFX levels were measured using an LCMS-8060 quadrupole mass spectrometer (SHIMADZU, Kyoto, Japan), as previously reported, with some modifications [24][25][26]. Briefly, to obtain the peptides from the fragment antigen-binding (Fab) region of immunoglobulin G, serum samples were pretreated using the nSMOL TM Antibody BA Kit (SHIMADZU, Kyoto, Japan) according to the provided protocol.…”