title: Insulin Formulation Characterization-the Thioflavin T Assays.
abstract: The insulin molecule was discovered in 1921. Shortly thereafter, its propensity towards amyloid fibril formation, fibrillation, was observed and described in the literature as a "precipitate." In the past decades, the increased incidence of type 2 diabetes has reached global epidemic proportions. This has emphasized the demands for both insulin production and the development of modern insulin products for unmet medical needs. Bringing such new insulin drug products to the market for the benefit of patients requires that many CMC-related processes are understood, described, and controlled. One potential undesired process is insulin fibril formation. The compound thioflavin T (ThT) is known as a fluorescent probe for amyloid fibrils. As such, ThT is utilized in a versatile research assay in microtiter plate format, the ThT assay. This review will describe an experimental set-up using not only a ThT microtiter plate assay but also two orthogonal methods. The use of the ThT assay in research and characterization of insulin analogues, as well as formulations of insulin, is described by cases drawn from the scientific literature and patents. The ThT assay is compared to other physical stability tests and in conclusion the advantages and limitations of the assay are compared.
authors.affiliation: Injectable Formulation Research, Global Research, Novo Nordisk A/S, Novo Nordisk Park H6.S.09.1, DK2760, Maaloev, Denmark. mosc@novonordisk.com.
authors.forename: Morten
authors.initials: M
authors.lastname: Schlein
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D052160:Benzothiazoles; D001681:Biological Assay / Q000379:methods; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy; D015195:Drug Design; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007004:Hypoglycemic Agents / Q000008:administration & dosage / Q000009:adverse effects; D007328:Insulin / Q000008:administration & dosage* / Q000009:adverse effects; D013844:Thiazoles / Q000737:chemistry*
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D007004:Hypoglycemic Agents; D007328:Insulin; D013844:Thiazoles; C009462:thioflavin T
keywords: amyloid fibril formation; insulin; peptide and small protein drug product formulation; physical stability; thioflavin T fluorescence