International immunopharmacology · 2015
Quantitation of infliximab using clonotypic peptides and selective reaction monitoring by LC-MS/MS.
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Willrich Maria Alice V, Murray David L, Barnidge David R, Ladwig Paula M, Snyder Melissa R
Аннотация
OBJECTIVES: Although therapeutic concentrations of infliximab (chimeric IgG1 kappa) are associated with improved clinical prognosis, clinical laboratory methods for monitoring are limited. Therefore, we aimed to develop a LC-MS/MS method to measure infliximab in serum.
METHODS: Infliximab was measured using isotope-labeled peptides and horse IgG as a surrogate internal standard. After trypsin digestion, peptides were separated by reverse-phase C8 LC and detected by MS/MS on an ABSciex API 5000; analyte-to-internal standard peak area ratios were used for quantitation. Sera from patients receiving infliximab were collected at different time points in treatment and compared with a commercially available ECLIA method.
RESULTS: The linear dynamic range of the assay was 1-100 μg/mL (R(2)>0.998); both intra- and inter-assay imprecisions were <20%. Patients undergoing infliximab therapy had trough concentrations of 8.5 ± 8.8 μg/mL (mean ± SD), which substantially increased 48-72 h after infusion (77 ± 40 μg/mL), then fell after 28-32 days (15 ± 11 μg/mL). A comparison of LC-MS/MS and ECLIA methods demonstrated a slope of 0.967 (95% CI: 0.894-1.034, r=0.970).
CONCLUSIONS: We have demonstrated the ability to quantify infliximab in patients using clonotypic peptides. This approach has the potential to be quickly adaptable to other monoclonal antibodies and to expand the availability of testing for this class of therapeutics in routine clinical practice.
METHODS: Infliximab was measured using isotope-labeled peptides and horse IgG as a surrogate internal standard. After trypsin digestion, peptides were separated by reverse-phase C8 LC and detected by MS/MS on an ABSciex API 5000; analyte-to-internal standard peak area ratios were used for quantitation. Sera from patients receiving infliximab were collected at different time points in treatment and compared with a commercially available ECLIA method.
RESULTS: The linear dynamic range of the assay was 1-100 μg/mL (R(2)>0.998); both intra- and inter-assay imprecisions were <20%. Patients undergoing infliximab therapy had trough concentrations of 8.5 ± 8.8 μg/mL (mean ± SD), which substantially increased 48-72 h after infusion (77 ± 40 μg/mL), then fell after 28-32 days (15 ± 11 μg/mL). A comparison of LC-MS/MS and ECLIA methods demonstrated a slope of 0.967 (95% CI: 0.894-1.034, r=0.970).
CONCLUSIONS: We have demonstrated the ability to quantify infliximab in patients using clonotypic peptides. This approach has the potential to be quickly adaptable to other monoclonal antibodies and to expand the availability of testing for this class of therapeutics in routine clinical practice.