B-290 Development of a LCMS/MS Method for the Quantitation of Infliximab in Serum
Authors: Zhong B , Abonamah J V , Payto D , McShane A 
Published: September 2023

Abstract

Background
Quantitative bottom-up proteomics of biotherapeutics has been successfully introduced in the clinical laboratory for therapeutic drug monitoring in recent years. The general workflow includes multiple steps: sample extraction/purification, reduction, alkylation, enzymatic digestion, desalting, and LC-MS/MS analysis of signature peptides. In this study, we developed an infliximab drug assay with desired operational efficiencies needed in a clinical laboratory. Evaluations included reduction/alkylation removal, trypsin digestion length, trypsin quality, online/offline/no sample desalting, multiple/single signature peptides, and whole protein/peptide internal standardization.

Methods
For sample preparation, a general procedure was initially followed. Briefly, protein precipitation with ammonium sulfate, re-suspension with buffer, reduction, alkylation, overnight enzymatic digestion, desalting, and LC-MS/MS analysis. To simplify the workflow, the steps of reduction and/or alkylation were omitted. The trypsin digestion time was varied from 2 h, 4 h, and overnight. TPCK-modified trypsin from two different vendors (Thermo Scientific and Millipore Sigma) were also compared for digestion efficiency. SILu™MAb infliximab stable-isotope labeled monoclonal antibody (Millipore sigma) was compared with customer synthesized stable-isotope labeled signature peptides as the internal standard. Different trapping cartridges were evaluated for online sample desalting and different analytical columns were evaluated for target peptide analysis. A Transcend TurboFlow TLX UHPLC System coupled to a TSQ Altis triple-quadrupole mass spectrometer was used (Thermo Scientific) operating in multiple reaction monitoring mode (2 transitions for each peptide and internal standard). Two signature peptides YASE (Sequence: YASESMSGIPSR) and GLEW (Sequence: GLEWVAEIR) from different chains of infliximab were analyzed.

Results
For sample preparation, the omission of protein reduction/alkylation from the workflow did not significantly affect YASE quantitation. The peak area of GLEW decreased by about 30% without reduction/alkylation. The evaluation of three calibration curves corresponding to the digestion time of 2, 4, and overnight respectively showed that 4 h was optimal for all the levels of calibrators if both signature peptides were used for quantitation. TPCK-modified trypsin from two different vendors were equal in digestion efficiency. The calibration curve using SILu™MAb infliximab stable-isotope labeled monoclonal showed superior linearity (R2 &gt; 0.98) and recovery for each calibrator vs using stable-isotope labeled signature peptides as internal standard. With the optimized conditions, both signature peptides can be used for the accurate quantitation of infliximab. However, for monitoring both peptides simultaneously, the chromatography separation needed 14.5 min. On the contrary, 9 min is needed for monitoring a single peptide YASE.

Conclusion
In summary, systematic optimization of sample preparation and LC-MS/MS analysis was accomplished in this study. The entire workflow was simplified by removing the steps of reduction and alkylation and using optimal digestion time, which increases the efficiency and significantly facilitate the implementation of this assay.