Exposure Concentrations of Infants Breastfed by Women Receiving Biologic Therapies for Inflammatory Bowel Diseases and Effects of Breastfeeding on Infections and Development.

BACKGROUND & AIMS: Exposure to biologic and immunosuppressant agents during breastfeeding is controversial, and there are limited data on safety. We investigated whether biologics are detectable in breast milk from women receiving treatment for inflammatory bowel diseases (IBDs) and whether breastfeeding while receiving treatment is associated with infections or developmental delays. METHODS: We performed a multicenter prospective study of women with IBD and their infants, collecting breast milk samples (n = 72) from patients receiving biologic therapy from October 2013 to November 2015. Drug concentrations were measured in all breast milk samples at several time points within 48 hours of collection and within 168 hours for some samples. Child development was assessed using the Ages and Stages Questionnaire 3, completed by 824 women with IBD (treated or untreated) during pregnancy (620 breastfed, and 204 did not). Data on children's health and development were obtained from mothers and pediatricians, along with information on mothers' medication exposure, IBD history, activity, pregnancy, and postpartum complications. We used chi-squared method or Fisher exact test to determine associations between categorical values and compared differences in continuous outcomes between groups using analysis of variance models. The primary outcome was drug concentration of biologic agents in breast milk (from 72 women) at 1, 12, 24, and 48 hours after dosing and also at 72, 96, 120, and 168 hours for available samples. Secondary outcomes were a range of infant infections and Ages and Stages Questionnaire 3-defined developmental delays among all breastfed infants. RESULTS: We detected infliximab in breast milk samples from 19 of 29 treated women (maximum, 0.74 µg/mL), adalimumab in 2 of 21 treated women (maximum, 0.71 µg/mL), certolizumab in 3 of 13 treated women (maximum, 0.29 µg/mL), natalizumab in 1 of 2 treated women (maximum, 0.46 µg/mL), and ustekinumab in 4 of 6 treated women (maximum, 1.57 µg/mL); we did not detect golimumab in breast milk from the 1 woman receiving this drug. Rates of infection and developmental milestones at 12 months were similar in breastfed vs non-breastfed infants: any infection, 39% vs 39% in control individuals (P > .99) and milestone score, 87 vs 86 in control individuals (P = .9992). Rates of infection and developmental milestones did not differ among infants whose mothers received treatment with biologics, immunomodulators, or combination therapy compared with unexposed infants (whose mothers received treatment with mesalamines or steroids or no medication). CONCLUSIONS: In a study of women receiving treatment for IBD and their infants, we detected low concentrations of infliximab, adalimumab, certolizumab, natalizumab, and ustekinumab in breast milk samples. We found breastfed infants of mothers on biologics, immunomodulators, or combination therapies to have similar risks of infection and rates of milestone achievement compared with non-breastfed infants or infants unexposed to these drugs. Maternal use of biologic therapy appears compatible with breastfeeding. Clinicaltrials.gov no.: NCT00904878.

Optimization of Receptor Occupancy Assays in Mass Cytometry: Standardization Across Channels with QSC Beads.

Receptor occupancy, the ratio between amount of drug bound and amount of total receptor on single cells, is a biomarker for treatment response to therapeutic monoclonal antibodies. Receptor occupancy is traditionally measured by flow cytometry. However, spectral overlap in flow cytometry limits the number of markers that can be measured simultaneously. This restricts receptor occupancy assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. We therefore developed a receptor occupancy assay suitable for mass cytometry. Measuring more markers than currently available in flow cytometry allows simultaneous receptor occupancy assessment and high-parameter immune phenotyping in whole blood, which should yield new insights into disease activity and therapeutic effects. However, varying sensitivity across the mass cytometer detection range may lead to misinterpretation of the receptor occupancy when drug and receptor are detected in different channels. In this report, we describe a method for optimization of mass cytometry receptor occupancy measurements by using antibody-binding quantum simply cellular (QSC) beads for standardization across channels with different sensitivities. We evaluated the method in a mass cytometry-based receptor occupancy assay for natalizumab, a therapeutic antibody used in multiple sclerosis treatment that binds to α4-integrin, which is expressed on leukocyte cell surfaces. Peripheral blood leukocytes from a treated patient were stained with a panel containing metal-conjugated antibodies for detection of natalizumab and α4-integrin. QSC beads with known antibody binding capacity were stained with the same metal-conjugated antibodies and were used to standardize the signal intensity in the leukocyte sample before calculating receptor occupancy. We found that QSC bead standardization across channels corrected for sensitivity differences for detection of drug and receptor and generated more accurate results than observed without standardization. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Analysis of Monoclonal Antibody Glycopeptides by Capillary Electrophoresis-Mass Spectrometry Coupling (CE-MS).

Glycosylation is a crucial posttranslational modification (PTM) that might affect the safety and efficacy of monoclonal antibodies (mAbs). Capillary electrophoresis-mass spectrometry (CE-MS) enables the characterization of the primary structure of mAbs. A bottom-up proteomic workflow is designed to provide detailed information about the glycosylation. In this chapter, we describe the validated experimental protocol applied for the characterization and relative quantification of mAbs N-glycosylation at the glycopeptide level.