Rapid methodology for basal system selection of therapeutic proteins during the early stage biopharmaceutical development

Purpose: This study aimed to introduce a quick method for developing formulations during the early stages of biopharmaceutical drug development, starting from the isoelectric point (pI). Methods: Human insulin and etanercept, an Fc fusion protein, were used as model proteins to observe the effects of pH, buffer, and buffer concentration on their physicochemical properties. For biophysical analysis, dynamic light scattering (DLS), micro differential scanning calorimetry (μDSC), and circular dichroism (CD) were utilized to measure zeta average size with zeta potential, transition melting temperature, and secondary structure, respectively. Furthermore, an accelerated storage test was performed at 4 °C, 25 °C, and 40 °C using size exclusion chromatography (SEC) to evaluate storage stability. Results: Zeta potential measurements showed that the practical pI of human insulin in solution was 5.8 to 6.0, and the pI of etanercept was 5.0 to 5.1. Acetate and phosphate were selected as buffering agents, and the pH range was pH 3.4 to 7.4 with different buffer concentrations, 10 mM and 50 mM. Protein stability was increased when the pH was away from the pI with a relatively high absolute zeta potential, resulting in increased conformational stability and storage stability. Response surface methodology (RSM) was also used to identify a suitable pH and buffer. Conclusion: The determined pH and buffer were able to suppress protein aggregation and to sustain the monomer fraction, thereby increasing protein stability. Therefore, the approach in selecting basal system could be very useful during the early stage biopharmaceutical development.