Methodology
Monitoring Peptide Aggregation with ARGEN:
Four peptides of unknown primary structure were examined against a panel of solution additives. Excipients included dimethyl sulfoxide (DMSO), acetonitrile (MeCN), and guanidinium chloride (GnCl). Other conditions investigated were pH and peptide concentration.
Each peptide was dissolved to 2 mg/mL in a 1:1 H2O:MeCN cosolvent solution which was adjusted to pH 7 before peptide dissolution, as prescribed by the peptide’s provider. This solution was used to prepare 1 mg/mL solutions of the peptide with an excipient. These conditions included guanidine hydrochloride from 0-4.3 M, DMSO from 0-20% by volume, and MeCN from 25-75% by volume. All solutions were held at 30 °C for up to 48 hours under constant monitoring by ARGEN.
Once the afore described assays were analyzed, a stock solution with each additive at its optimal concentration was prepared for each peptide. Each peptide was dissolved in its respective buffer at 1 mg/mL, and pH was adjusted with dilute NaOH and HCl solutions to identify the optimal pH between 2 and 10. This was determined by characterizing the degree of aggregation for each peptide at each pH level over a 48-hour period of aggregation monitoring at 30 °C. The formulated sample with the least aggregation after 48 hours of monitoring correlates to the optimal storage condition (variables included varied pH and varied concentrations of additives). Solubility testing determined that all peptides struggled to dissolve at concentrations greater than 10 mg/mL.
