Development of a Mass Spectrometry-Based Method for Quantification of Ustekinumab in Serum Specimens.

Abstract

Ustekinumab (UST) is a human monoclonal antibody used to treat moderate-to-severe Crohn disease by blocking the interleukin-12/23 pathway. Although an optimized therapeutic concentration of UST is associated with clinical response and improved prognosis, the availability of clinical laboratory methods for UST monitoring is limited. Furthermore, the commercially available methods are immunoassays that are prone to interference of antidrug antibodies. This study aimed to develop a liquid chromatography-tandem mass spectrometry method for quantification of UST in human serum specimens. A tryptic peptide that is specific to the heavy chain variable region of UST was selected. Quantification of UST was performed by selective reaction monitoring on a quadrupole TQ-XS with an internal standard. After digestion with trypsin, peptides were separated by reverse-phase C18 liquid chromatography; peptides were detected by MS/MS, and analyte to internal standard peak area ratios were used for the quantification. Finally, serum samples from patients treated with UST were collected at trough levels (n = 66). The assay showed a broad dynamic range with linearity between 0.4 and 20 mg/L (R = 0.995). The lower limit of quantification was found to be 0.4 mg/L. The reproducibility was tested with 3 different UST concentrations (2, 8, and 16 mg/L). The coefficients of intra-assay and interassay variations were 2.2%-4.0% and 2.7%-5.3%, respectively. UST serum concentrations of 2-16 mg/L were stable for up to 14 days when specimens were left at room temperature (20°C). The newly developed LC/MS-based method was shown to be feasible for UST quantification. This analytical approach may lead to individualized dosing and improved patient care.