Infliximab Therapeutic monitoring by tryptic peptide LC-MS/MS method improvements lead to improved accuracy with decreased imprecision and turnaround time.

Introduction: Therapeutic drug monitoring of infliximab has become the standard of care for inflammatory bowel disease in the setting of loss of response to therapy, and occasionally in proactive therapy personalization. Measurement of infliximab by tryptic peptide HPLC-MS/MS has been available since 2015, mostly in reference laboratories. Objectives: Here, we present method improvements to our original published method leading to a more efficient, robust, and high throughput tryptic peptide HPLC-MS/MS assay for infliximab quantitation. Methods: Deidentified residual serum samples submitted for clinical testing were used for method comparison and infliximab was spiked into normal human serum for performance studies. Improvements included the addition of a stable isotope labeled full length infliximab internal standard (IS) replacing a surrogate IS, and immunoenrichment using Melon Gel for immunoglobulins replacing the saturated ammonium sulfate precipitation. Digestion and chromatography were optimized, and automation was added. The method improvements were validated to include precision, accuracy, reportable range, linearity, and analytical sensitivity. Results: The digestion time was reduced from overnight to 1 h. The assay analytical measuring range (AMR) remained the same throughout improvements, 1-100 µg/mL, with linearity of 0.98x + 0.50, R2 = 1.00. Intra- and inter-assay imprecision were less than 5 % CV at four different concentrations. Accuracy was assessed with 106 patients within the AMR; Passing-Bablok Regression yielded a slope of 1.00 and a y-intercept of 0.25. Turnaround time was reduced by 1 day, and imprecision of three levels of quality control trended down after new method implementation. Conclusions: Method improvements including automation have allowed for assay completion in half a day, improving robustness and turnaround time.

Alpha-1-Antitrypsin (A1AT) Proteotyping by LC-MS/MS.

The diagnosis of alpha-1-antitrypsin (A1AT) deficiency is established by quantitation of protein concentration in serum (immunoassay) followed by determination of specific allelic variants by phenotyping (isoelectric focusing (IEF) gel electrophoresis) and/or allele-specific genotyping. Various phenotyping and genotyping methodologies are available, and each has their own advantages and disadvantages. As an alternative, mass spectrometry is emerging as a powerful tool in the identification and quantitation of proteins and peptides. The method described here, referred to as proteotyping, is a proteomic method using trypsin digestion and tandem mass spectrometry that detects the most common deficiency alleles, S and Z, associated with A1AT deficiency.This qualitative mass spectrometry method is based on the principle that the S and Z mutations lead to amino acid changes which result in a change in the mass of the A1AT protein. When the A1AT protein is proteolytically digested, multiple peptides are generated, two of which include the sites of the S and Z mutations, respectively. Peptides generated from wild-type A1AT (M alleles) differ in sequence and mass from peptides generated from the S and Z alleles at these two specific locations. The mass difference allows for differentiation of S and Z peptides, representing the deficiency alleles, from non-S and non-Z peptides, representing the wild-type alleles (M). Interpretation of the peptide patterns in conjunction with A1AT quantitation by immunoassay allows for an accurate assessment for the presence of deficiency alleles in the majority of patients.