2.8. Stability Studies by Size Exclusion Chromatography (SEC)

The stability of the samples was determined by measuring the level of protein aggregates using SEC. Samples were sealed in a desiccator at 40°C over a saturated solution of magnesium chloride, which generated an environment of 33% relative humidity (RH). At each time point (15, 30, 60, and 90 days), three vials were removed and diluted to protein concentrations of 1 mg/mL. Solutions were centrifuged at 12,000 rpm and 4°C for 10 min to remove insoluble aggregates, and the supernatant was then removed and placed in HPLC vials for analysis. Samples were analyzed on a high performance liquid chromatography system (HPLC, 1200 series, Agilent Technologies, Santa Clara, CA) using isocratic flow of 50 mM sodium phosphate, 100 mM sodium chloride solution (pH 6.8) over 15 min at a flow rate of 1 mL/min. The column used was a TSKgel® G3000SWXL HPLC Column from Sigma Aldrich (St. Louis, MO). Instability was determined as a percentage of loss of the area under the curve for the monomeric peak of a sample before storage under accelerated conditions, with the exception of BSA, where initial aggregate peaks were also included in determining monomer content.