Abstract Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic coagulation pathway. In patients with hemophilia A or B, inhibition of TFPI is an alternative therapeutic approach that augments the extrinsic coagulation pathway. Marstacimab is an investigational fully human monoclonal antibody that binds and neutralizes TFPI and is being evaluated as a prophylactic treatment to prevent or reduce the frequency of bleeding episodes in patients with severe hemophilia A or B, with or without inhibitors (antibodies against coagulation factors). However, the efficacy, safety, and pharmacokinetics of marstacimab may be affected by the induction of antidrug antibody (ADA) responses. Here, we describe the evolution and validation of three quasi-quantitative electrochemiluminescence-based methods to detect marstacimab ADAs, starting from their use in a first-in-human phase 1 study to their use in phase 2 and 3 clinical studies of patients with severe hemophilia. For all three methods, validation criteria evaluated the performance of the assays in screening and confirmatory cut points, precision, selectivity, drug tolerance, target interference, and stability. Additional criteria for validation were dilution linearity (Methods 1 and 2) and low positive control concentration, prozone effect, plate homogeneity, and robustness (Method 3). The three methods met validation criteria and are a potentially valuable tool in detecting the induction of marstacimab ADAs during treatment in patients with hemophilia. Graphical abstract
The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
Local inflammation elicited by Neisseria gonorrhoeae correlates closely with sensitivity to killing by normal human serum. Serum-sensitive (SS) isolates are rendered resistant in vitro by lipooligosaccharide sialylation. Differences in C3b processing on N. gonorrhoeae in vitro were found to match findings at the cervical level in vivo. Nonsialylated SS gonococci bound 5-fold more C3b than did stably serum-resistant (SR) gonococci; most was processed to iC3b, yet significant C3b persisted. Sialylated SS gonococci bound 4-fold less total C3 antigen than did SR gonococci, which was promptly converted to iC3b. C3b bound later on stably SR gonococci but again was processed swiftly to iC3b. In vivo, the iC3b/C3 ratio of SS isolates more closely resembled nonsialylated SS isolates in vitro, implying heterogeneous sialylation or desialylation in vivo. In vitro, total IgM bound was unchanged by sialylation of SS isolates, but total C4 bound decreased by 75%, suggesting that sialylation may indirectly regulate the classical complement pathway.
Background: Venous thromboembolism (VTE) is an important clinical concern in children with or without cancer. Experience with dalteparin, a low molecular weight heparin, in the treatment of VTE in adults with cancer has been extensively published, but data in children are limited. Aims: We sought to determine the twice‐daily dalteparin dose required to achieve target Anti‐Xa levels of 0.5–1.0 IU/mL, as well as its pharmacodynamics (PD), efficacy, and safety in the treatment of VTE in children <19 years old, with or without cancer. Methods: This prospective, multi‐center, Phase 2, open‐label study consisted of 3 phases: 1) Dose Adjustment Phase of up to 7 days, in which Anti‐Xa levels were measured following the first, second or third dose of dalteparin, until achievement of the target level; 2) PD Phase of up to 7 days, to obtain 2 randomized PD plasma samples for Anti‐Xa determination; and 3) Follow‐Up Phase, to complete up to 90 days of anticoagulant therapy. Patients were assessed for symptomatic new or progressive (i.e., recurrent) VTE as well as clinically relevant bleeding during treatment. Surveillance VTE imaging was performed at 90 ± 14 days. Results: The safety population consisted of 38 patients who received at least 1 dose of dalteparin (<2 years: n = 3; ≥2 to <8 years: n = 8; ≥8 to <12 years, n = 7; ≥12 to <19 years: n = 20). Twenty‐six patients (68%) had a diagnosis of cancer at baseline, of which 23 (88%) were haematological malignancies. The median [range] dalteparin dose required to achieve target Anti‐Xa levels decreased with age (<2 years: 207.5 IU/kg [201.5–213.5 IU/kg]; ≥2 to <8 years: 128.15 IU/kg [123.9–180.3 IU/kg]; ≥8 to <12 years: 125 IU/kg [124.5–152.6 IU/kg]; ≥12 to <19 years: 116.7 IU/kg [99.1–159 IU/kg]) (Figure). Therapeutic Anti‐Xa levels were achieved in 90% of patients within a mean (SD) of 2.6 days (1.54 days); the mean (SD) number of dose adjustments per patient was 0.7 (0.98). One patient (3%) developed symptomatic recurrent VTE. No patients reported clinically‐relevant bleeding. Four patients (11%) had treatment‐related serious adverse events. 62% of patients had complete resolution of their VTE at the end of the 90‐day reporting period. Summary/Conclusion: Twice‐daily dalteparin dosing achieved therapeutic levels in 90% of children with or without cancer, with a satisfactory tolerability profile. The median therapeutic doses of dalteparin were higher for the two youngest age cohort groups (<2 years and 2 to <8 years). image NCT00952380 Sources of Research Support: This study was sponsored by Pfizer Ltd.
