Insulin Formulations Characterization by Light Scattering

For studies on insulins under formulation conditions we have used the marketed U100 formulations Humalog® (Lilly, Indianapolis, USA), NovoRapid® (Novo Nordisk, Bagsveard, Denmark), as well as Apidra® and Insuman Rapid® (Sanofi-Aventis, Frankfurt, Germany). The compositions of the formulations are shown in Supplemental Table I. Lyophilized samples of the insulins (lispro, aspart, glulisine, and HI) and solvents to the corresponding formulations (placebos) were provided by Sanofi-Aventis (Frankfurt, Germany). PBS (10 mM buffer) was prepared from tablets (Calbiochem, Germany). Glycerol (85%), phenol and m-cresol were provided from Roth (Germany). ZnCl₂ was supplied from Merck (Germany). All chemicals were of analytical grade. Solvents were prepared using Milli-Q water (Merck Millipore, Germany) and were filtered and degassed after preparation using 0.45 μm Millicup filter units (Merck Millipore, Germany). In special cases, dialysis against the corresponding solvent using dialysis tubing (MW cut-off 3500, Spectra/Por©, Spectrum Laboratories, USA) was done ensuring the specified solvent conditions. Dialysis was always done, if stock solutions with high insulin concentrations were used. For measurements in Zn²⁺-free PBS, EDTA was added to the stock solutions after dissolving the drug substance, in order to remove Zn²⁺ completely. Afterwards, the samples were dialysed against PBS.
Samples for light scattering were filtered either through 0.1 μm Whatman-Anotop filters (VWR, Germany) directly into 3-mm-pathlength micro-fluorescence cells (105.251-QS, Hellma, Germany) immediately prior to use or were subjected to ultracentrifugation. Usually, about 500 μl were centrifuged at 75000 g for 30 min. An amount of 80–150 μl of the supernatant was quickly transferred into the sample cell. Peptide concentrations were determined photometrically using a specific absorption A(276 nm, 1 cm pathlength, 1 mg/ml) of 1.08.