Category: Formulation and Quality
Purpose: To investigate the role of sugars and polyglycols in the stabilization of helical peptides
Methods: Peptide Synthesis: Model peptide NH3-(AAQAA)3Y-Ac (AQY) peptides were synthesized using Fmoc synthesis with a rink-amide resin and capped with an acetal group. Peptides were characterized using MS and HPLC and purified.
CD Analysis: CD-UV analysis was done on a Jaso-810 CD spectrometer with an attached Peltier, and 0.1 cm quartz cuvette. CD spectra were recorded in triplicate with a scan rate of 100min/min, 1mm slit width with excipient/buffer solutions as blanks. HT values did not exceed 600 V for recorded data. Full scan spectra were acquired from 200-260 nm and 2-25°C. Thermal denaturation were carried by ramping the temperature from 1°C to 55°C at a rate of 0.5°C/min. Samples were prepared at 130 μM peptide concentration with varied concentrations of carbohydrate (0 to 1.5M) and PEG (0 to 0.187M) excipients in water or PBS buffer.
Data Analysis: Percentage of helicity was calculated according to the following equation:
where θexp is the recorded molar ellipticity (MRE) in deg cm2dmol at 222 nm, θu is the theoretical max MRE of an uncoiled peptide, θh is the theoretical max MRE of 100% helix peptide, were θu is calculated from (T in °C) and (x is a constant used to correct nonhydrogen bonded carbonyls with a value of 1 in this study). The data of similar peptide is assumed to follow a two-state model and be monomeric over a wide concentration range(1-4). ΔH, Tm were estimated from monitoring MRE θ222 change over temperature. ΔH was estimated from plotting In Keq over 1/T, where Keq = [F]/[U] (folded/unfolded) determined by calculating fH at each temperature point.
Results: The purities of peptides were greater than 90%. Effects of excipients on AQY peptide helicity with varying concentrations at 5°C is given in table 1. It was observed at this temperature that AQY in water had a helicity of approximately 9%. The helicity of AQY was around 15% regardless the concentration of PEG from 25 mM to 187 mM, while mannitol, sucrose and trehalose showed concentration dependence at the lower concentrations, plateauing between 250mM and 750mM, with mannitol and trehalose showing increased helicity at 1 M. Overall, AQY in trehalose solution had the lowest helicity percentages of the carbohydrate excipients at different concentrations followed by sucrose and mannitol. Temperature affected helicity as shown in table 2, with percentage of helicity decreasing by 64% from 3 to 25°C for sucrose. Effect of excipient concentration on AQY full scans is illustrated in figure 1 for PEG, mannitol, sucrose and trehalose.
Thermal denaturation yielded AQY Tm values of 11.29 °C and 16.62°C for peptide in water and 1M sucrose solution, with ΔH values of -259 J/mol and -452 J/mol, respectively.
Thermal denaturation yielded AQY Tm values of 11.29 °C and 16.62°C for peptide in buffer and 1M sucrose solution, with ΔH values of -259 J/mol and -452 J/mol, respectively.
Conclusion: All four excipients enhanced the helix folding of the 16-residue peptide. Moreover, the excipients used in this study stabilized the helix formation in the thermal denaturation of the AQY peptide in sucrose solution.