(T1030-03-19) Calcium-Enhanced Chromatography for the Purification of Polar Ammonium and Peptide Compounds

Purpose: The purpose of this study was to evaluate calcium salts as privileged additives for the purification of highly polar ammonium and oligopeptides on normal-phase silica. The pre-clinical and Phase I batches of active pharmaceutical ingredients (APIs) often do not have the time to achieve the desired purity targets through crystallization and increased process development. Challenges in the purification of highly polar amines, ammonium salts, and oligopeptides are of particular importance as these motifs are commonly present in drug APIs. Currently, reverse-phase chromatography remains the sole chromatographic method for purification of these important compound classes. The ability to use normal-phase techniques to purify these APIs would offer significant cost savings and allow for more rapid purification at pre-clinical scale.

Methods: The separation of test compound mixtures and crude reaction mixtures in the presence of various calcium additives and solvent systems was evaluated at an analytical scale using high pressure liquid chromatography, and at a preparative scale using thin-layer chromatography (TLC) and medium pressure liquid chromatography (MPLC). Conditions were surveyed analytically and successful conditions were applied to preparative scale purifications of selected compounds of interest.

Results: To efficiently survey chromatography conditions, we began our investigation of the Ca salts by utilizing an HPLC (Thermo Fisher UltiMate 3000) for rapid data collection on an analytical scale using a normal-phase silica column (Luna 5μm Silica(2)). Our challenge test mixture of three permanently charged ammonium compounds (compounds A-C) was chosen based on the lack of purification techniques available using current normal and reverse-phase chromatography (Figure 1). The challenge mixture was eluted in the presence and absence of calcium salts. The top UV chromatogram was a control experiment with no calcium present showing a single large peak with no separation. The next three chromatograms show the individual compounds successfully eluting on a silica HPLC column with a water-methanol solvent system containing calcium (20% aqueous 0.1 M CaCl2 in MeOH). The bottom chromatogram shows that the three ammonium salts can be successfully eluted and separated from one another under the same conditions. This HPLC method was used to survey more than 10 different water-soluble salts including ions such as Na⁺, NH₄⁺, Mg²⁺, Zn²⁺, Li⁺, and K⁺, in addition to other Ca²⁺ salts. Calcium salts were found to be privileged additives which provided improved separations not seen with other cations. A variety of commonly available or easily prepared calcium salts were evaluated with varying levels of success. Calcium chloride and calcium trifluoroacetate were among the best in our analytical assay. Since the method was primarily pursued for preparative chromatography applications, we evaluated these conditions on crude reaction mixtures obtained from our previous studies. HPLC conditions were first evaluated by TLC before a Biotage MPLC instrument was used for a preparative separation. Figure 2 shows the before and after TLC plates of the crude product and the purified column fractions. Additionally, the UV chromatogram from the Biotage instrument is provided. Utilizing calcium trifluoroacetate, in a water-acetonitrile solvent system, the compound HLö7 was successfully purified on a standard silica column. HLö7 is a bis-cationic pyridinium compound which would not typically be possible to elute on a silica column with any previously reported additives such as acids or bases. We also successfully demonstrated the purification of blinded compound AS under similar chromatography conditions (Figure 3). AS was also a bis-cationic ammonium compound. We have successfully employed calcium enhanced chromatography in the synthesis of several internal syntheses of both ammonium salts and highly polar neutral poly-amine compounds. We have found this to be a general method applicable to a wide variety of highly polar compounds. Initial studies have also shown that peptides can be successfully eluted on standard silica with the help of calcium additives. The purification of peptides using this technology is still an area of active research. Successful methods for the removal of calcium from the final products have been developed and are applied on a compound-by-compound basis. Methods for calcium removal include trituration, recrystallization, ion-exchange, and the selective formation of insoluble calcium species.

Conclusion: The methods for calcium enhanced chromatography detailed above represent an advance in chromatography conditions which has not been previously reported. Calcium ions can improve the ability of normal-phase silica to separate highly polar amine and ammonium compounds which is typically not possible. Utilizing normal-phase silica for such separations can provide a significant reduction in cost and an improvement in the generality of normal-phase silica separations versus their reverse-phase counterparts. These advances have wide reaching applications for the purification of important small molecule APIs and will aid in the preclinical synthesis and manufacturing process. Additionally, preliminary results indicate that calcium enhanced chromatography is likely to be applicable to the purification of peptides for which there are currently few alternative purification methods other than reverse-phase chromatography.

Acknowledgements: The authors declare no conflicts of interest. All funding was provided by Southwest Research Institute. A patent application for this method has been submitted.


Figure 1. HPLC UV chromatogram of compounds A, B, and C both individually and in a three-component mixture with and without calcium


Figure 2. A) TLC of crude HLö7 (left), purification using Biotage MPLC (middle), and TLC of collected fractions (right). B) Biotage UV chromatogram of collected column fractions.


Figure 3. A) TLC of crude AS (left), purification using Biotage MPLC (middle), and TLC of collected fractions (right). B) Biotage UV chromatogram of collected column fractions.