Abstract:
OBJECTIVE To investigate the thermal stability and receptor affinity of erythropoietin(EPO) stock solutions from different companies and evaluate their biological activities through both in vivo and in vitro experiments.
METHODS The protein stability analysis system was employed to ascertain the melting temperature(Tm), particle size and polymer dispersity index(PDI) of EPO samples as the temperature increased from 25 ℃ to 95 ℃. Micro-scale thermophoresis(MST) was utilized to measure the affinity constant(KD) of EPO in relation to its receptors. Pearson correlation test was conducted to analyze the data from eight enterprises.
RESULTS The Tm of EPO samples from the eight companies ranged from 54.84 to 59.64 ℃. The mean particle sizes were 3.71−4.93 nm. PDI were 0.15−0.19, and KD were 41.37−301.40 nmol·L−1. The particle size of the samples exhibited no significant changes during the heating process. Samples from enterprise F and H had poor thermal stability and low affinity with its receptors. Samples from enterprise D displayed the best thermal stability and the best affinity with its receptors.
CONCLUSION The molecular affinity of EPO samples from various enterprises has a positive correlation with their thermal stability and biological activity molecular affinity of EPO had high correlation with biological activity both in vitro and in vivo(P<0.05). MST technology is effective for evaluating the activity of EPO, combined with the protein stability analysis system, providing valuable reference for the bioactivity evaluation and stability evaluation of protein drugs.
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