科研之友
微信
新浪微博
Facebook
分享链接
摘要
Glycosylated proteins formed during expression are present as impurities in yeast based recombinant production of Human Insulin (HI). These closely related glycosylated impurities pose challenge in purification of HI by RP-HPLC. In this work, separation of glycosylated Human Insulin (gHI) and HI in presence of other HI related impurities under preparative loading conditions was studied at pH between 2.8 to 4.0. Sodium salts of acetate, citrate, formate, perchlorate, and succinate were used as ion-pairing agents in mobile phase. The study was performed by varying the concentration of ion-pairing agent, pH of mobile phase, and HI loading on the column. The effect of ion-pairing agent, in conjunction with pH, on resolution of gHI and HI was evaluated based on recovery, purity of HI, and percent reduction of gHI. Ion-pair formation of sodium perchlorate in the presence of acetonitrile as the organic modifier resulted in effective separation of gHI and HI yielding HI purity of 98.5% under preparative loading conditions (5 to 15 g/L). In order to comply with the purity and impurity specifications as per pharmacopeia, development of additional purification methods would be required, based on analysis results of the product using pharmacopeia methods. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.
- 出版日期2011
