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

BACKGROUND: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.

Ustekinumab serum concentrations are associated with clinical outcomes in Crohn's disease - a regional multi-center pilot study.

BACKGROUND AND AIM: The role of therapeutic drug monitoring (TDM) in ustekinumab (UST) therapy for Crohn's disease (CD) has not been established, as only few studies have analyzed the relationship between UST serum concentrations and clinical outcome. In this pilot study, we retrospectively examined the potential of UST-concentrations (cUST) 8 weeks after induction (cUSTw8) to predict clinical response at week 16. METHODS: Serum samples and clinical data from patients (n = 72) with moderate to severely active CD who received intravenous induction with UST were retrospectively analyzed. cUST were quantitated using liquid chromatography-tandem mass spectrometry (LC-MSMS). A receiver-operating characteristic (ROC) curve and area under ROC curve (AUROC) was computed to analyze the predictive potential of cUSTw8 for clinical response at week 16 and to determine the minimal therapeutic UST trough concentration. RESULTS: Forty-four patients (61 %) achieved clinical response to UST therapy at week 16. cUSTw8 was moderately effective to predict clinical response with a minimal therapeutic cUSTw8 of 2.0 mg/l (AUC 0.72, p = 0.001). CONCLUSION: Trough concentrations of UST 8 weeks after induction predict clinical response to therapy in week 16 with moderate sensitivity and specificity. TDM using LC-MSMS could prove beneficial in personalized UST therapy of patients with CD by identifying individuals with subtherapeutic concentrations who might benefit from dose escalation.