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To identify the potential impurities generated during manufacturing of recombinant Arg34-GLP-1 [7-37].
After fermentation, the broth extract 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 were generated. MALDI-TOF, protein sequencing and other methods were used to identify the structures of impurities.
The purity of the main product reached 97.9% after purification by anion exchange chromatography and preparation of liquid phase. Three main impurities were isolated and purified to 91.4%, 99.2%, and 96.7%. Their MWs of [M+H]+ were 1 033.544 6, 2 744.239 7, and 3 112.447 5 as determined by MALDI-TOF. Through the destructive test and the amino acid sequences of Arg34-GLP-1 [7-37] it was speculated that the possible sequences of impurity 1, impurity 3, 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 were generated by the fermentation process, and the contents of these impurities would 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.
Since recombinant Arg34-GLP-1 [7-37] is unstable in high temperature, peracid and alkali, and oxidizing environments, care should be taken to avoid these conditions in order to prevent degradation of the main product during purification. This paper shows some pitfalls to avoid for the research of GLP-1 and its analogs peptides.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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