Lab Equipment - November 2018

With custom-designed oligopeptides becoming an increasingly popular option in pharmaceuticals, it’s important to prevent spoilage of any kind. Formulation of Four Peptide Solutions in

15 Days

» by Fluence Analytics

Custom-designed oligopeptides are an increasingly accessible option in peptide-based technologies for pharmaceuticals. Following their synthesis, peptides are often subjected to HPLC purification and synthetic modification, which can require the use of non-aqueous media or salts requiring later removal. During the production of pharmaceutical peptides, spoilage can occur in the form of aggregates, associates and other means. Using ARGEN (Fluence Analytics), this app note demonstrates the rapid formulation of four peptide solutions that are based on excipient effects correlated to increasing molecular weight. The first peptide was formulated in five days for optimal stability. The remaining three peptides were formulated in parallel over 10 days.

Methodology

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 to 4. 3 M, DMSO from 0 to 20 percent by volume, and MeCN from 25 to 75 percent by volume. All solutions were held at 30 C for up to 48 hours under constant monitoring by ARGEN.

Once the aforementioned 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 aggre-
gation after 48 hours of monitoring correlates to the optimal stor-
age condition (variables included varied pH and varied concentra-
tions of additives). Solubility testing determined that all peptides
struggled to dissolve at concentrations greater than 10 mg/mL.

Results

Guanidinium chloride was shown to have stabilizing effects at intermediary concentrations for the “A” group peptides. Stability maxima were observed between 2 to 3 M of salt (Figure 1). The light scattering signature of aggregation is steady and relatively linear. This denotes a steady increase of normalized molecular weight from its monomeric, unaggregated state, to that of a population of dimerized aggregates. Kinetic analysis showed that peptides A-1 and A- 2 increased in stability by 250 and 350 times, respectively, with the addition of the optimal concentration of guanidinium chloride.

Both “B” group peptides, B-1 and B- 2, precipitated almost immediately following the addition of saline solution. With the understanding that the B group peptides are hydrophobic, this is not unexpected. Though guanidinium is a chaotrope, its nature as a salt increases the solution’s polarity, destabilizing the sparingly dissolved hydrophobic peptides. Further investigation would greatly reduce the concentration of GnCl for these peptides to analyze stability in a micromolar regime.