Purity determination of synthetic glucagon using a mass balance approach

Xinxue Wang,Fuhai Su,Bei Xu,Peng Xiao,Wei Sun,Hongmei Li,Dewei Song,Fangyan Zhang

doi:10.1038/s41598-020-61109-9

Abstract

Due to the widespread use of synthetic peptide drugs, their quantification and the analysis of impurities have become increasingly important in clinical and medical settings. Moreover, quantifying proteins using synthetic peptides as internal or external standards is a general approach, and the key to this approach is the knowing purities of the peptides. In this paper, synthetic glucagon was quantified using a mass balance method. The impurities in glucagon were analyzed and then accurately quantified separately. Karl Fischer (KF) titration and ion chromatography (IC) were used to determine the water and trifluoroacetic acid (TFA) contents in the samples, respectively. Furthermore, the inorganic ion content in the samples was determined by inductively coupled plasma mass spectrometry (ICP-MS). The sequence of peptide impurities was identified by a Thermo Fisher Orbitrap mass. Samples were determined to be 896.36 ± 0.68 mg/g after subtracting all impurity masses from the sample mass. The result can be traced to SI units.

Highlights

Due to the widespread use of synthetic peptide drugs, their quantification and the analysis of impurities have become increasingly important in clinical and medical settings
The principle is that the Karl Fischer reagent in the electrolysis cell of the instrument is injected into the aqueous sample when the equilibrium is reached, and the water participates in the redox reaction of iodine and sulfur dioxide
The data with a relative standard deviation (RSD) of three consecutive measurements within 3% was taken as the experimental result

Summary

Introduction

Due to the widespread use of synthetic peptide drugs, their quantification and the analysis of impurities have become increasingly important in clinical and medical settings. In addition to its ability to determine the content of only the main constituent, the mass balance method can be used to analyze and quantify impurities in peptide drugs. We chose synthetic glucagon consisting of 29 amino acids as an example and adopted the mass balance method to determine its purity. This method is a way to accurately quantify sample impurities such as organic and inorganic compounds and moisture, among others, and deduct the mass of the impurities from the total sample mass. Using the mass balance method, we accurately quantified the impurities in synthetic glucagon separately and determined the glucagon content by subtracting the masses of all impurities from the total sample mass

Methods

Results

Conclusion