Factorial approach for the development of stability indicating HPLC assay of recombinant human insulin: Application to its stability study

摘要

This paper describes the development and validation of a most economical and sensitive isocratic stability indicating HPLC method for the assay of recombinant human insulin (RHI) from yeast origin (Hansenula polymorpha). The method employs a Thermo Biobasic, C-18 (250×4.6 mm, 300 A°, 5 μm) column with a mobile phase of acetonitrile potassium dihydrogen phosphate (pH 2.5; 50 mM) (35:65, v/v) and UV detection at 242 nm. Factorial design was used to facilitate method development. Buffer pH and concentration of acetonitrile were considered as the independent variable to study capacity factor of RHI. Sixteen experiments were undertaken, and a quadratic model was derived for the capacity factor of RHI. The method produced well defined, sharp peaks having lower tailing factor (1.114). A linear response with a correlation coefficient (r2) of 0.999 was observed over the range of 0.025 2.0 IU mL-1. Developed method was employed to analyze RHI samples from forced degradation and stability study. Degradation of RHI under different ICH prescribed stress conditions was carried out. Four batches of RHI formulation were exposed to different conditions of temperature and humidity for forty-five days. Degradation of RHI during stability study followed first-order kinetics. Data obtained from degradation kinetics were employed to find out the rate constant; time left for 50% potency, and time left for 90% potency. Rate constants obtained from different conditions were employed to plot the Arrhenius plot.