The development of biosimilar products is expected to grow rapidly over the next five years as a large number of approved biologics reach patent expiry. The pathway to regulatory approval requires that similarity of the biosimilar to the reference product be demonstrated through physiochemical and structural characterization, as well as within in vivo studies that compare the safety and efficacy profiles of the products. To support nonclinical and clinical studies pharmacokinetic (PK) assays are required to measure the biosimilar and reference products with comparable precision and accuracy. The most optimal approach is to develop a single PK assay, using a single analytical standard, for quantitative measurement of the biosimilar and reference products in serum matrix. Use of a single PK assay for quantification of multiple products requires a scientifically sound testing strategy to evaluate bioanalytical comparability of the test products within the method, and provide a solid data package to support the conclusions. To meet these objectives, a comprehensive approach with scientific rigor was applied to the development and characterization of PK assays that are used in support of biosimilar programs. Herein we describe the bioanalytical strategy and testing paradigm that has been used across several programs to determine bioanalytical comparability of the biosimilar and reference products. Data from one program is presented, with statistical results demonstrating the biosimilar and reference products were bioanalytically equivalent within the method. The cumulative work has established a framework for future biosimilar PK assay development.

Background and Purpose AMG 139 is a human anti‐IL‐23 antibody currently in a phase II trial for treating Crohn's disease. To support its clinical development in humans, in vitro assays and in vivo studies were conducted in cynomolgus monkeys to determine the pharmacology, preclinical characteristics and safety of this monoclonal antibody. Experimental Approach The in vitro pharmacology, pharmacokinetics (PK), pharmacodynamics and toxicology of AMG 139, after single or weekly i.v. or s.c. administration for up to 26 weeks, were evaluated in cynomolgus monkeys. Key Results AMG 139 bound with high affinity to both human and cynomolgus monkey IL‐23 and specifically neutralized the biological activity of IL‐23 without binding or blocking IL‐12. After a single dose, linear PK with s.c. bioavailability of 81% and mean half‐life of 8.4–13 days were observed. After weekly s.c. dosing for 3 or 6 months, AMG 139 exposure increased approximately dose‐proportionally from 30 to 300 mg·kg−1 and mean accumulation between the first and last dose ranged from 2‐ to 3.5‐fold. Peripheral blood immunophenotyping, T‐cell‐dependent antigen responses and bone formation markers were not different between AMG 139 and vehicle treatment. No adverse clinical signs, effects on body weight, vital signs, ophthalmic parameters, clinical pathology, ECG, organ weights or histopathology were observed in the monkeys with the highest dose of AMG 139 tested (300 mg·kg−1 s.c. or i.v.). Conclusions and Implications The in vitro pharmacology, PK, immunogenicity and safety characteristics of AMG 139 in cynomolgus monkeys support its continued clinical development for the treatment of various inflammatory diseases.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that plays an important role in the regulation of serum low-density lipoprotein (LDL) cholesterol by downregulation of LDL receptor, and as such is considered a novel target in cholesterol lowering therapy. In support of the drug development program for Evolocumab, a fully human IgG2 antibody that targets PCSK9, a quantitative ELISA to measure free PCSK9 in human serum was developed. PCSK9 serves as a biomarker of pharmacological response during treatment, and measuring levels of the free ligand post-dosing was of interest as an aid to establishing the pharmacokinetic and pharmacodynamic properties of the therapeutic. Given the complexities associated with the measurement of free ligand in the presence of high concentrations of circulating drug, it was important to challenge the method with experiments designed to assess ex vivo conditions that have the potential to affect the binding equilibrium of drug and ligand within test samples during routine sampling handling and assay conditions. Herein, we report results of experiments that were conducted to characterize the assay in alignment with regulatory guidance and industry standards, and to establish evidence that the method is measuring the free ligand in circulation at the time serum was collected. A robust supporting data package was generated that demonstrates the method specifically and reproducibly measures the free ligand, and is suitable for its intended use.

Hu714MuXHu is a recombinant chimeric murine‐human monoclonal antibody directed against interleukin‐15 (IL‐15), a proinflammatory cytokine associated with memory CD8+ and natural killer (NK) T‐cell activation and implicated in the pathogenesis of inflammatory diseases. A pharmacokinetic‐pharmacodynamic (PK/PD) model was developed to describe the NK cell count reduction in cynomolgus monkeys after treatment with Hu714MuXHu. Cynomolgus monkeys were dosed with Hu714MuXHu in three studies: as a single dose at 0.1 or 1 mg·kg−1 i.v.; weekly for 5 weeks at 0, 30, 60, or 150 mg·kg−1 i.v. or 150 mg·kg−1 s.c.; weekly for 13 weeks at 0, 5, 30, or 150 mg·kg−1 s.c. Serum Hu714MuXHu concentration‐time data were analyzed using noncompartmental analysis and the PK/NK cell count relationship was assessed via simultaneous PK/PD modeling. Hu714MuXHu PK was approximately dose‐proportional between 0.1–150 mg·kg−1 for i.v. and 5–150 mg·kg−1 for s.c. administration with an elimination half‐life of 12.7–18 days. Hu714MuXHu administration resulted in rapid and marked reductions in NK cell counts after the first dose which recovered fully after the serum Hu714MuXHu concentrations approached 0.1 μg·mL−1 (assay limit of quantification). PK/PD modeled Hu714MuXHu effects on NK cells had an EC 50 of 0.09 μg·mL−1. In summary, weekly i.v. or s.c. doses with Hu714MuXHu for up to 3 months in cynomolgus monkeys demonstrated linear PK and significant NK cell count reduction, which was described using PK/PD modeling. This approach may be used to guide investigative product dose selections for inflammatory diseases where NK cell count alterations are quantifiable.