Objective:The identification of potential impurities generated during manufacturing of recombinant Arg34-GLP-1[7-37].Methods:After fermentation,the broth extraction was analyzed by HPLC,then the main product Arg34-GLP-1[7-37]and impurities were separated and purified by ion exchange chromotography and preparative HPLC respectively.The main product was treated by extreme conditions such as high temperature,acid and alkali experiments in order to figure out how the impurities generated.LC-MS,MALDI-TOF,protein sequencing and other methods were used to identify the structure of impurities.Results:The main product can be up to 97.9%after purification by Anion exchange chromatography and preparation of liquid phase.LC-MS analysis obtained the molecular weights of 6 monomer components,which were 1033.1500,1302.8308,2743.9113,3381.4459,2488.5400,and 3108.8330,and their possible structural sequences were speculated.Three main impurities were isolated and purified to 91.4%?99.2%,and 96.7%.Their MWs of[M+H]+are1033.5446,2744.2397,and 3112.4475,determined by MALDI-TOF and the amino acid sequences of Arg34-GLP-1[7-37]that impurity 1,impurity3,and impurity 9 may be E-F-I-A-W-L-V-R,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W,H-A-E-G-T-F-T-S-D-V-S-S-Y-L-E-G-Q-A-A-K-E-F-I-A-W-L-V-R.Comparison of the components before and after purification showed that the above three main impurities are generated by the fermentation process,and the content of these impurities will increase significantly by extreme conditions.The structure analysis and mechanism investigation of the three impurities analyzed in this study were all published for the first time.Conclusion:The three impurities prepared are degradation fragments of Arg34-GLP-1[7-37]under high temperature,alkaline and oxidizing conditions.In order to prevent the degradation of the main product during the purification process,the temperature should not be too high,and excessive acids and alkalis should be avoided.And some conditions that are easily oxidized.This paper shows some pitfalls to avoid for the research of GLP-1 and its analogs peptides. |