重组艾塞那肽RP-HPLC法肽图分析

Peptide mapping analysis of recombinant Exendin-4 by RP-HPLC

目的建立并验证胰蛋白酶裂解-反向高效液相色谱法进行重组艾塞那肽肽图分析研究。方法将重组艾塞那肽样品用胰蛋白酶水解,酶解所得的肽段通过反相高效液相色谱法进行分析,采用C_8色谱柱,以0.1%三氟乙酸(TFA)的水溶液为流动相A,以0.1%TFA的乙腈溶液为流动相B,梯度洗脱70min,得到重组艾塞那肽肽图,以此进行方法的专属性、重现性、定量限、批间同一性研究。结果本实验室制备的重组艾塞那肽可以通过胰蛋白酶裂解-RP-HPLC法进行肽图分析,专属性、重现性均满足要求,定量限为0.125mg/m L,三个批次间的肽图谱也完全一致。结论表明此方法简便可靠、准确度高、重复性好,验证研究可行,可用于重组艾塞那肽的质量控制。

Objective To establish and validate a peptide mapping analysis method of trypsin hydrolysis and RPHPLC for recombinant Exendin-4. Methods Recombinant Exendin-4 was hydrolyzed by trypsin, and then analyzed by RP-HPLC with C_8 column. From a 70-minute gradient elution with mobile phase component A（0.1% TFA aqueous solution） and mobile phase component B（0.1% TFA acetonitrile solution）, the peptide map of recombinant Exendin-4 was derived. The specificity, reproducibility, quantitation limit, and batch-to-batch consistency of the method were analyzed. Results The recombinant Exendin-4 from our laboratory succeeded the peptide mapping analysis of trypsin hydrolysis and RP-HPLC, which specificity and reproducibility met the requirements, which quantitation limit was 0.125 mg/m L. And the peptide maps of three batches were also exactly the same. Conclusion The proposed method is feasible and reliable, as well as having high accuracy and good repeatability, which can be used in the quality control of the recombinant Exendin-4.