Durapore pvdf

An excess amount of methylene blue was added to an aqueous solution containing 1 mM MES buffer and stirred in a water bath at 25 °C. An aliquot of 0.5-mL was taken and centrifuged using a centrifugal filter unit (Durapore PVDF, 0.1 μm, Merck Millipore, Darmstadt, Germany) at 2000 rpm to achieve solid-liquid separation. The liquid phase was diluted 10,000 times; then, the absorbance at 664 nm was measured using a UV/vis spectrophotometer. As a result, the absorbance was almost stabilized after about 2 h. Therefore, after stirring the liquid for more than 12 h under different temperature conditions, visible absorption was similarly measured to evaluate saturation solubility.

No formulation remained from either of the 2 phase 3 trials; formulations for the study that would be representative of the clinical material (confirmed by analytical data) were prepared at Covance Laboratories, Inc. In each formulation, lifitegrast was added to dose vehicle while stirring, and pH was adjusted with hydrochloric acid (HCl) and/or sodium hydroxide (NaOH). The formulation was stirred until a clear solution was obtained, filtered with a 0.22-μm filter (Millipore^® Millex-GV, 0.22 μm, Durapore^® PVDF; EMD Millipore, Billerica, MA) and stored at ∼5°C. Analysis of the formulations was performed by Almac Sciences (Souderton, PA), and concentrations were measured as 49.4 and 49.3 mg/mL for Formulation Nos. 1 and 2, respectively.
Formulation No. 1 (used in the OPUS-1¹⁰ trial): dose vehicle consisted of sterile water for injection, sodium chloride, sodium phosphate dibasic anhydrous, sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), and sodium thiosulfate pentahydrate, adjusted to a pH of 7.30 with HCl. After addition of lifitegrast, pH was adjusted to 6.90.
Formulation No. 2 (used in the OPUS-2¹¹ trial): dose vehicle consisted of sterile water for injection, sodium chloride, sodium phosphate dibasic anhydrous, and sodium thiosulfate pentahydrate. After addition of lifitegrast, pH of the formulation was adjusted to 7.35.

CT26, B16F10 and MC38 cells were cultured as described above for 24 h, the medium was collected and centrifuged at 500 × g for 10 min. The supernatant was transferred into a separate centrifugal filter unit (Milipore, 0.22 μm, Durapore-PVDF). The flow through was collected after centrifugation. Then 30 μl of each sample was used to measure cytokines with the MILLIPLEX^®MAP Kit (MYCTOMAG-70K-PMX). The preparation was done according to the supplier’s protocol provided with the kit. The plate was analyzed using a Luminex 200 ™ device.

The HPLC grade methanol and acetonitrile were purchased from Fisher Scientific, UK. Double-distilled, molecular grade water was obtained using a Millipore Milli-Q® (Bedford, MA, USA) water purifier. All the solvents (HPLC grade) and solutions were filtered through membrane filter (Millipore-Millex-HV® filter units, Durapore-PVDF®, polyethylene, 0.45 μm pore size) and ultrasonicated for degassing before use. Other reagents used in the present study were of AR grade.

Escherichia coli were cultured for 48 h at 37°C under static conditions. The whole cells and bacterial supernatants were collected by centrifugation at 4,310 × g for 20 min at 4°C, and filtered through a 0.45 μm Durapore PVDF (Millipore, Billerica, MA, United States). The supernatant was further subjected to ultra-centrifugation, as previously described (Wai et al., 2003a (link)), with some modifications. Soluble and insoluble fractions of the supernatant were collected as supernatant and pellets after ultra-centrifugation at 100,000 × g for 3 h at 4°C in a 45 Ti rotor (Beckman Coulter, Brea, CA, United States). Soluble fraction of the supernatant was concentrated by precipitation using 10% (w/v) trichloroacetic acid (TCA) followed by two washes with 80% acetone to remove TCA. The insoluble fraction (the pellets) of the filtrated supernatant was also collected. The whole cell samples were further subjected to subcellular fractionation by using a differential solubilization technique, as described previously (Wai et al., 2003b (link)). The protein amounts were quantified by the Bradford assay (Bradford, 1976 (link)).