Analysis of the Structure and Stability of Erythropoietin by pH and Temperature Changes using Various LC/MS

Abstract

The purpose of stability testing is to provide evidence about how the quality of a drug varies with time under the influence of a variety of environmental factors. In this study, erythropoietin (EPO) was analyzed under different pH (pH 3 and pH 9) and temperature (25 o C and 40 o C) conditions according to current Good Manufacturing Practice (cGMP) and International Conference on Harmonisation (ICH) guidelines. The molecular weight difference between intact EPO and deglycosylated EPO was determined by SDS-PAGE, and aggregated forms of EPO under thermal stress and high-pH conditions were investigated by size exclusion chromatography. High pH and high temperature induced increases in dimer and high molecular weight aggregate forms of EPO. UPLC-ESI-TOF-MS was applied to analyze the changed modification sites on EPO. Further, normal-phase high-performance liquid chromatography was performed to identify proposed glycan structures and high pH anion exchange chromatography was carried out to investigate any change in carbohydrate composition. The results demonstrated that there were no changes in modification sites or the glycan structure under severe conditions; however, the number of dimers and aggregates increased at 40 o C and pH 9, respectively.

Development of erythropoietin receptor-targeted drug delivery system against breast cancer using tamoxifen-loaded nanostructured lipid carriers

“…This is because during the experimental procedure at 37°C, the heat did not cause significant change in the structure and crystallization of NLC 15. Furthermore, aggregation of EPO such as by dimerization will only take place upon storage at 40°C for 2 weeks 40. These results also indicate that the increase in stoichiometry is likely the result of the binding of EPO to TAMNLC alone.…”

Structural Identification of Modified Amino Acids on the Interface between EPO and Its Receptor from EPO BRP, Human Recombinant Erythropoietin by LC/MS Analysis

“…Along with deamidation, another modification, oxidation, was identified at an amino acid residue, Met54. This modification was identified when rHu-EPO was under stress at pH 9 ( Chang et al, 2013 ). Met54 was placed in the hydrophobic cap of EPO.…”

Formulation and evaluation of erythropoietin-alginate microspheres at different amount of drug

“…Erythropoietin, which is a protein, has several weaknesses, such as unstable in biological fluids because of easily digested by enzymes, cannot be fully absorbed from the digestive tract due to a high molecular weight resulting to low bioavailability, and has short half-life (Wang et al, 2013). Erythropoietin is stable at pH 4-<9 and has the highest activity at pH 7-8 (Chang et al, 2013). Erythropoietin is also stable at -20°C or -70°C (Chang et al, 2013).…”

“…Erythropoietin is stable at pH 4-<9 and has the highest activity at pH 7-8 (Chang et al, 2013). Erythropoietin is also stable at -20°C or -70°C (Chang et al, 2013). At pH 9 and 40 °C, erythropoietin will undergo dimerization to form large molecules that can induce the body's immune response, which can harm patients (Chang et al, 2013).…”

“…Erythropoietin is also stable at -20°C or -70°C (Chang et al, 2013). At pH 9 and 40 °C, erythropoietin will undergo dimerization to form large molecules that can induce the body's immune response, which can harm patients (Chang et al, 2013). Some alternative is by formulatemicrospheressystem to release the drug in a sustained release manner (Chaudhary et al, 2010).…”