Four Peptide Formulation Solutions in 15 Days

Background

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 forms of aggregates, associates, and other means. Using ARGEN, four peptide formulation solutions were rapidly developed 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
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.

Results
Guanidinium Chloride:

Guanidinium chloride was shown to have stabilizing effects at intermediary concentrations for the “A” group peptides. Stability maxima were observed between 2-3 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.

 

Dimethyl Sulfoxide & Acetonitrile:

Across all four peptides studied, increasing concentration of DMSO resulted in increased peptide stability. Three peptides showed stability maxima, between 0 and 20% DMSO by volume. Peptide A-2 showed maximal stability at 20%, implying if this set of assays were to be extended to a greater proportion of DMSO in solution, this peptide’s stability could further increase. Regardless of hydrophilicity, the addition of DMSO was beneficial for stability.

The stability gains achieved by the addition of DMSO were not observed in the case of the MeCN addition. MeCN caused rapid aggregation of the A group peptides, but it caused stabilization of the B group peptides. This can be accounted for by changes in solution polarity. Decreased solution polarity can decrease enthalpic penalties of solvation for hydrophobic solutes.

pH:

pH dependent stability was highly variable between samples. More extreme pHs tended to represent increased stability against aggregation, or conversion to an insoluble species. Extreme pHs could also be problematic because of acid or base catalyzed degradation pathways. In most cases, there were moderate pHs in which samples demonstrated relatively stable behavior and were not prone to hydrolysis.

Peptide Concentration:

Group A peptides showed decreasing stability with increasing concentration of peptide. Since these peptides easily dissolve into their solution, they can effortlessly explore conformations with favorable intermolecular interactions. Different from Group A peptides, Peptide B-1 shows colloidal stability. As the concentration of peptide increases, the solution is more stable. This is normally sustained by intermolecular repulsion preventing peptides from associating.

Conclusion:
  • Using ARGEN, four peptide formulation solutions were developed for long-term storage in aqueous form based on continuously collected data and a kinetic approach to analysis and data interpretation
  • Future research could utilize all 16 of ARGEN’s cells to better finely tune the formulation and probe for interactions between excipients
  • Using ARGEN’s Kinetic data, it was also possible to quickly rule out certain excipients such as guanidinium chloride for the hydrophobic B-group peptides and acetonitrile for the hydrophilic A-group peptides
  • Lastly, future research could look into the effects of more exotic additives that would be beneficial given a known primary structure

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