ABSTRACT
Deamidation is one of the most common degradation impurities in protein and peptide drugs. The deamidation of glutamine and asparagine in the protein sequence can lead to changes in the chemical and biological properties of the protein. However, the rutine trypsin-based pretreatment process can significantly increase the artificial deamidation impurities during the digestion process, resulting in high determination level. In this study, after optimizing the conditions of Glu-C enzymatic hydrolysis, we obtained the best enzymatic conditions under acidic condition and the artificial deamidation impurities significantly reduced in digestion process, identified the deamidation site (N48). Through the methodological investigation and comparison of the measurement results of different methods, the specificity, reproducibility and accuracy of the method are verified. The method established in this research has laid a solid foundation for the accurate determination of deamidation impurities in cobratide and its similar protein peptide biochemical drugs.
ABSTRACT
The paper aims to establish the method to determine the monosaccharide composition and monosaccharide ratio in propylene glycol alginate sodium sulphate (PSS). Samples were hydrolyzed with trifluoroacetic acid, neutralized with sodium hydroxide solution after the reaction conditions for sample pretreatment were optimized via orthogonal analysis. A high performance anion exchange chromatograghy (HPAEC) coupled with pulsed amperometric detector (PAD) was performed on a CarboPac®PA20, using 200 mmol·L-1 sodium hydroxide solution and 1 mol·L-1 sodium acetate solution as mobile phase. The established HPAEC-PAD method was validated by testing the linear relationship, precision and accuracy, and showed exclusive, sensitive, rapid and wide use. The monosaccharide composition of PSS from different manufacture can be accurately determined with great significance for the structural identification of PSS.