School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel-Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, 1211 Geneva 4, Switzerland.
Analytical Research and Development, MRL, Merck & Co, Inc., 126 E. Lincoln Ave, Rahway, NJ 07065, United States.
J Chromatogr A. 2021 Apr 12;1642:462048. doi: 10.1016/j.chroma.2021.462048. Epub 2021 Mar 9.
The aim of this work was to expand the applicability range of UHPSFC to series of synthetic and commercialized peptides. Initially, a screening of different column chemistries available for UHPSFC analysis was performed, in combination with additives of either basic or acidic nature. The combination of an acidic additive (13 mM TFA) with a basic stationary phase (Torus DEA and 2-PIC) was found to be the best for a series of six synthetic peptides possessing either acidic, neutral or basic isoelectric points. Secondly, methanesulfonic acid (MSA) was evaluated as a potential replacement for TFA. Due to its stronger acidity, MSA gave better performance than TFA at the same concentration level. Furthermore, the use of reduced percentages of MSA, such as 8 mM, yielded similar results to those observed with 15 mM of MSA. The optimized UHPSFC method was, then, used to compare the performance of UHPSFC against RP-UHPLC for peptides with different pI and with increasing peptide chain length. UHPSFC was found to give a slightly better separation of the peptides according to their pI values, in few cases orthogonal to that observed in UHPLC. On the other hand, UHPSFC produced a much better separation of peptides with an increased amino acidic chain compared to UHPLC. Subsequently, UHPSFC-MS was systematically compared to UHPLC-MS using a set of linear and cyclic peptides commercially available. The optimized UHPSFC method was able to generate at least similar, and in some cases even better performance to UHPLC with the advantage of providing complementary information to that given by UHPLC analysis. Finally, the analytical UHPSFC method was transferred to a semipreparative scale using a proprietary cyclic peptide, demonstrating excellent purity and high yield in less than 15 min.
本工作旨在扩大 UHPSFC 在一系列合成和商业化肽中的适用性范围。首先，对不同的 UHPSFC 分析用柱化学进行了筛选，同时添加了碱性或酸性添加剂。发现将酸性添加剂（13 mM TFA）与碱性固定相（Torus DEA 和 2-PIC）组合，对于具有酸性、中性或碱性等电点的六种合成肽系列最为有效。其次，评估了甲磺酸（MSA）作为 TFA 的潜在替代品。由于其酸性更强，在相同浓度水平下，MSA 比 TFA 的性能更好。此外，使用较低百分比的 MSA（例如 8 mM），可以得到与使用 15 mM MSA 相似的结果。然后，使用优化的 UHPSFC 方法来比较不同 pI 值和肽链长度增加的肽的 UHPSFC 与 RP-UHPLC 的性能。根据其 pI 值，UHPSFC 被发现可以略微更好地分离肽，在某些情况下与 UHPLC 观察到的结果正交。另一方面，与 UHPLC 相比，UHPSFC 产生了更优异的肽分离效果，增加了氨基酸链的长度。随后，使用一组市售的线性和环状肽对 UHPSFC-MS 与 UHPLC-MS 进行了系统比较。优化的 UHPSFC 方法能够生成与 UHPLC 至少相似、在某些情况下甚至更好的性能，其优势在于提供了与 UHPLC 分析互补的信息。最后，将分析 UHPSFC 方法转移到半制备规模，使用一种专有的环状肽，在不到 15 分钟的时间内实现了优异的纯度和高收率。
