(M1430-12-77) A Rational and Data-Driven Approach to Formulation of Therapeutic Peptides

Purpose: A rational development strategy is crucial to obtain chemically and physically stable formulations of therapeutic peptides. The field has gained increasing interest due to the unique advantages of peptides as therapeutic agents, however, the development of peptide-based drugs faces challenges related to their inherent instability. This study aimed to design a pre-formulation strategy to support the choice of formulation for a model peptide with 40 amino acids and an albumin binding tag. Albumin binding improves the peptide’s circulating half-life but presents challenges in the formulation process, since the hydrophobic tag can induce multimerization and potential aggregation. To meet the specific requirements of the target formulation of this peptide, it was essential to optimize for long-term stability under extreme and unpredictable conditions, including high and/or fluctuating temperatures and physical stress during transportation. This optimization was critical considering the challenging storage conditions in certain regions where the formulation is intended for administration. Consequently, a freeze-dried (FD) formulation offering enhanced stability, resistance to physical stress, and temperature variations, was selected and optimized aiming to demonstrate stability exceeding 3 months (3M) at 40 °C.

Methods: Initially the solubility, buffering capacity, and acid-base characteristics of the peptide in water and saline was investigated by pH titrations. With this knowledge, a simple matrix design was employed to investigate the influence of pH, salt, and surfactant on the formulation. Three pH values were selected, and formulations were prepared with and without addition of salt and surfactant. Physical and chemical stability tests were conducted, including rotational stress, thermal stress, freeze-thaw cycles, and oxidation stress. Concurrently, the fibrillation tendency of the peptide was assessed using a thioflavin T assay. These studies laid the foundation for choosing four formulations for the FD formulation development where the effects of different bulking agents and excipients were examined. A systematic screening and long-term stability test finalized the pre-formulation study.

Results: The peptide showed good solubility in both pure water and saline, maintaining an intrinsic pH of approximately 4 without buffer components (Figure 1, left). The peptide solution remained clear until reaching a pH of 10, where precipitation was observed. For the initial matrix study a systematic assessment of the effects of pH (3, 6, 9), salt (150 mM NaCl), and surfactant (Tween 80) revealed a clear destabilizing effect of NaCl and high pH (Figure 1, right). Tween 80 appeared stabilizing, in particular for the samples with higher pH, but did not significantly increase the stability in the presence of NaCl. In the fibrillation study, the peptide also demonstrated the highest physical stability at lower pH (pH 3 > pH 6 > pH 9), and pure water exhibiting better stability than 150 mM NaCl, cementing the need for a non-ionic tonicity agent. The chemical stability was assessed by forced degradation using oxidative and thermal stress. The oxidation tests revealed that the peptide was prone to oxidation under accelerated conditions (addition of H₂O₂), suggesting a potential need for inclusion of antioxidants in the formulation. A pH of 4 was chosen for the FD formulation study to align better with physiological conditions and enhance compliance during intravenous administration. In an initial FD test mannitol emerged as the preferred bulking agent for FD formulations, providing the best cake appearance and physical stability after reconstitution based on freeze-thaw cycles, rotation studies, and thermal stress. A long-term stability study was prepared with four formulations all based on mannitol as the bulking agent and with or without sucrose as a potential lyoprotectant. In this study the potential benefits of addition of an antioxidant (methionine) and/or a surfactant (Tween 80) were also evaluated (Figure 2). The FD samples were incubated at 5 and 40 °C for 3M. Samples incubated at 5 °C showed no significant degradation after 3M (Figure 3) by RP-HPLC with UV detection. For the samples incubated at 40 °C a differentiation was observed, in particular for the sample lacking methionine (formulation 4), which performed the worst. A formulation containing methionine but lacking Tween 80 was the second worst performing formulation (formulation 3). The two best formulations contained both methionine and Tween 80 but the presence of sucrose in the formulation did not stabilize the peptide; i.e., formulation 1 performed better than formulation 2.

Conclusion: The conducted pre-formulation studies supported the selection of formulations for the stability study. Initial FD experiments showed that mannitol provided good cake appearance and combined with low pH was protective against physical and thermal stress. The results also indicated the importance of incorporating methionine as an antioxidant to mitigate oxidation. Furthermore, Tween 80 was stabilizing against physical stress. The FD formulation that performed best contained Tween 80, methionine and mannitol. In conclusion, the pre-formulation studies provided essential data optimization and stability assessment, facilitating the development of a robust and stable freeze-dried formulation for the peptide compound with at least 3M stability at 40 °C.

Figure 1. Left: titration curves in water (grey) and saline (black). The x-axis gives the volume added of either 0.1 M NaOH or 0.1 M HCl. The added volume of NaOH is shown with negative values in order to construct a continuous curve. The pH at 0 µL added acid/base represents the intrinsic pH. Right: data from the 12-formulation matrix study. The samples were incubated at 40 °C and subjected to end-over-end rotation for 1 h/day. The green bars indicate the number of days where the formulation appeared clear and colorless without visible precipitation and the red bars indicate the time when precipitation was observed.

Figure 2. Freeze-dried formulation setup

Figure 3. HPLC chromatograms of the FD formulations after 3M incubation at 5°C (insert) and 40 °C (main chromatogram). The bar chart gives the purity of the main peptide peak at t0 and after 2W, 1M and 3M. The purity of the peptide peak is given as the area of the main peptide peak relative to all peaks detected. The sample compositions are given in Figure 2.