Category: Formulation and Quality
Purpose: Pharmaceutical R&D programs continue to explore molecular complexity and “three dimensionality” in efforts to achieve greater target selectivity and specificity. Related to this trend, we have seen a significant increase in requests to resolve stereoisomers from compounds with more than one variable chiral center.
Published papers in the chromatography literature on separations methods for compounds with multiple chiral centers generally report unique separations of all isomers using a single chromatographic condition. Our laboratory routinely resolves many compounds with two or three stereocenters, using preparative chromatography to isolate each of the isomers for testing. We have found that developing a single method resolving all isomers, particularly at preparative scale, is frequently challenging and time consuming.
We have used approach has been used for compounds with 2 to 5 stereocenters, but in this presentation we have chosen to illustrate its use with 3-center compounds. We present an iterative method that begins with achiral screening using rapid gradient RP-UPLC/MS/UV analysis to determine the retention times of the enantiomer pairs (the diastereomer sets) and their relative abundance.
Rapid SFC chiral screening suggests one or more chiral stationary phase and solvent choices for an SFC preparative method. Conditions are chosen not based on the determination of the best condition for resolution of all the isomers, but instead on the number of fractions that may be isolated in a single pass.
Methods: The poster will show the approach our laboratory has developed using a combination RP-HPLC (UPLC) and SFC that is significantly faster, and more directly leverages structural differences between the isomers. Chiral methods are developed and scaled to preparative scale using SFC. The UPLC is used to resolve and assess the diastereomers.
Results: RP-HPLC is capable of resolving the diastereomers but not the enantiomers. Based on this ability, the SFC fractions of the multi-chiral center crude samples can be assessed by UPLC to determine exact number of isomers in each fraction.
This poster will walk through the chiral method development and selection process. It will also illustrate the analysis of the resulting fractionation to determine the enantio-purity of the resulting fractions. This method has allowed our laboratory to purify compounds with as many as 5 chiral centers.
Conclusion: The cycle through assessment by the UPLC method (diastereomer selection) and chiral SFC (enantiomer selection) may be repeated more than once, but this cycle is significantly faster in our experience than the time consumed in developing a complete, scalable resolution of 8 isomers using a single method.