Abstract：
A macrocyclic peptide A was successfully purified in large quantities (∼30 g) in >95 % purity by an integrated two-step orthogonal purification process combining supercritical fluid chromatography (SFC) with medium-pressure reverse-phase liquid chromatography (MP-RPLC). MP-RPLC was used to fractionate the crude peptide A, remove unwanted trifluoroacetic acid (TFA) originating from the peptide A cleavage off the resin, and convert the peptide A into ammonium acetate salt form, prior to the final purification by SFC. A co-solvent of methanol/acetonitrile containing ammonium acetate and water in CO2 was developed on a Waters BEH 2-Ethylpyridine column. The developed SFC method was readily scaled up onto a 5 cm diameter column to process multi-gram quantities of the MP-RPLC fraction to reach > 95 % purity with a throughput/productivity of 0.96 g/h. The incorporation of SFC with MP-RPLC has been demonstrated to have a broader application in other large-scale polypeptide purifications.
摘要：
通过将超临界流体色谱（SFC）与中压反相液相色谱（MP-RPLC）相结合的综合两步正交纯化工艺，成功地以>95％的纯度大量（〜30g）纯化了大环肽A。使用MP-RPLC分级分离粗肽A，去除不需要的三氟乙酸（TFA）源自肽A裂解树脂，并将肽A转化为乙酸铵盐形式，在SFC最终纯化之前。在WatersBEH2-乙基吡啶色谱柱上开发了在CO2中含有乙酸铵和水的甲醇/乙腈共溶剂。将开发的SFC方法容易地放大到5cm直径的柱上,以处理多克量的MP-RPLC级分,以达到>95%的纯度,产量/生产率为0.96g/h。已证明SFC与MP-RPLC的掺入在其他大规模多肽纯化中具有更广泛的应用。
