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Materials Carbetocin (Lot no. MZ77328) was kindly donated by Ferring Pharmaceuticals A/S (Copenhagen, Denmark). Sucrose, span 20, span 80 and L-arginine were obtained from Sigma-Aldrich, USA, and tween 20, dextran sulfate, thioflavinT (ThT) were obtained from Sigma-Aldrich France, Sweden, and India respectively. Dodecanyol-D-sucrose, n-dodecyl-β-D-glucopyranoside and span 40 (from Sigma-Aldrich, USA), and n-dodecyl-β-D-maltoside (from Sigma-Aldrich, Italy) were kindly donated by Ferring Pharmaceuticals A/S. MiliQ grade water was used throughout the work. Method Preparation of Carbetocin stock solution All carbetocin samples were prepared with a target carbetocin concentration of 70 mg/ml in pure water, pH 4.6 ± 0.1. Thus depending on the requirements of each experiment a suitable stock concentration was prepared by dissolving accurately weighed amounts of pure carbetocin in miliQ water. The pH was adjusted to 4.6 ± 0.1 using 0.1 M sodium hydroxide (NaOH) solution, following which the stock solution was filtered using cellulose acetate filter of pore size 0.22 μm. Nanodrop 2000/2000c Spectophotometer (Thermo Fisher Scientific Inc.) was used to determine the exact peptide concentration by measuring UV absorbance at 280 nm using the extinction coefficient 1019.78 M-1cm-1 that was determined by the standard curve plot using the equation (Molecular weight of carbetocin ×Slope)/Pathlength Preparation of excipient stock solutions All excipient stock solutions were prepared by accurately weighing their required amounts in miliQ water. For sucrose in water, heat was applied with constant stirring to ensure that sucrose was completely dissolved. For span 20, 40 and 80, miliQ water was heated to about 80° C before adding weighed amounts to ensure complete dispersion. All excipient solutions were vortexed to achieve thorough mixing.
The target concentration of excipients are show in Table x below. For non-ionic sugar-based surfactants (Dodecanyol-D-sucrose, n-dodecyl-β-D-glucopyranoside, n-dodecyl-β-D-maltoside, span 40 and span 80) see table x. on page x. Table x: Excipient Concentration Sucrose 0.5 M 1.5 M 0.01 mM Tween 20 0.08 mM CMC is ~ 0.08 0.8 mM mMa Dextran sulfate 1 mM Span 20 0.06 mM CMC is ~ 0.06 0.6 mM mMa L-arginine 25 mM a (Kim and Hsieh, 2001) Shaking stress experiment The sample vials containing carbetocin and excipient in their target concentrations were placed horizontally on PSU-10i Grant-bio orbital shaker at 300 rpm, room temperature. The samples were stressed under this condition for 5 days. Plate reader experiment All samples were freshly prepared prior to the experiment. Both absorbance assay and ThT fluorescence were carried out using Fluostar Optima, BMG Labtech plate reader system with black 96-well polystyrene plates with optical bottom (Nalge Nunc). A total volume of 200 μL of carbetocin preparations (70 mg/ml carbetocin with excipients in their target concentrations) were filled in the wells and three replicates for each sample were measured to ensure the reproducibility of the results. The plates were covered with a clear polyolefin sealing tape (Thermo Scientific, USA) to prevent evaporation and incubated at 30 °C with double orbital shaking. Absorbance was measured first using 340 nm emission filter. Next, 20 μM ThT was added to one of the replicates and the fluorescence was measured (excitation 440 nm, emission 480 nm). The emission was detected by an optical fiber system from the bottom of the plate every 400 s. Henceforth in this report, all samples subjected to this experimental condition will be referred to as ‘stressed’ samples. Sampling of solid aggregates from the plate reader experiment The contents from each well were collected in Eppendorf tubes and centrifuged at 12000 rcf for 10 min at room temperature using Eppendorf Centrifuge 5417R (Eppendorf, Hamburg, DE) to separate the solid aggregates from the supernatant. The samples containing ThT were used for microscopic evaluation while the samples from the wells without ThT were used for FT-IR analysis.
For this purpose, the solid aggregates were isolated from the supernatant and dried for ATR-FTIR analysis while the supernatant was evaluated by transmission FT-IR. Microscopy Microscopy was performed for samples with ThT on Nikon Eclipse Ti microscope. About 10 μL of the sample containing both the solid aggregate and supernatant was taken on a microscope slide, covered using a cover slip and observed under 20x magnification lens. The images were processed using Zen 2.3 lite (Carl Zeiss Microscopy GmbH, version 126.96.36.1990). FT-IR spectroscopy All measurements were made on Bomem MB-Series spectrophotometer using the Arid-Zone light source at room temperature. For transmission FTIR, 15 μL of the liquid sample (supernatant) was placed on CaF2 cell (BioTools). The pathlength was kept constant throughout the experiment. For each spectrum, 256 scan were taken in the spectral range of 4000-1000 cm-1 at a resolution of 4 cm-1. A corresponding spectrum of the buffer as well as that of water vapor was recorded and subtracted from the peptide spectrum. For ATR-FTIR, analysis was performed on the solid aggregate using DuraScope (SensIR technologies) diamond ATR. 128 scan were taken for each spectrum at the range of 4000-500 cm-1 using 4 cm-1 resolution. A corresponding water vapor spectrum was recorded and subtracted from the peptide spectrum. In both the cases, for data analysis, second-derivative spectra were obtained using the Savitsky-Golay derivative function for a 13 point data.
The spectra were inverted by multiplying with -1 using the math function and were truncated in the amide I region from 1710 to 1590 cm-1. For the truncated region, the baseline was corrected and area-normalized to 1. GRAMS/AI software (Thermo Galactic, version 7.00) was used to obtain the spectra as well as analyze the data. To calculate the area overlap, the area-normalized spectra were exported to EXCEL. As indicated in Van De Weert et al., 2001, the area overlap of two spectra were calculated by generating a new spectrum by plotting the lower absorption value between the two spectra. The area under this curve was calculated which corresponds to the percent overlap of the two spectra. Dynamic Light scattering Dynamic light scattering (DLS) experiments were performed using Malvern zetasizer Nano series (Malvern instruments Ltd., UK). The scattered light was measured at an angle of 90° for samples taken in polystyrol cuvettes of volume 1.6 mL. The samples comprised of filtered and unfiltered span 20 (0.06 and 0.6 mM) without any peptide. For the filtered samples, filtration was carried out using cellulose acetate filter of pore size 0.22 μm. Malvern DTS v.7.03 software was used for data acquisition and analysis.