Supercritical Fluid Chromatography: An Essential Tool in Drug Discovery

• AbbVie Inc.

Since its early introduction in the 1960s, Supercritical Fluid Chromatography (SFC) has had a slow climb toward establishing itself as a valuable chromatographic separation tool in the pharmaceutical research industry. For decades, SFC was limited as a capillary chromatography technique; however its evolution into packed column chromatography in the 1980s, through the efforts of Terry Berger,¹ resulted in SFC emerging as a leading technique for analytical separation science, particularly in the field of chiral separations. As drug discovery continues to focus on obtaining optically pure compounds in the discovery of new therapies, SFC has enabled chiral separations on analytical and preparative scale to proceed in far greater efficiency than traditional HPLC methods. Since SFC also adds no additional carbon dioxide to the atmosphere, it also has the benefit of being known as a “green” technique.

Supercritical Fluid Chromatography of today provides a powerful toolbox to the analytical chemist in the fast-paced research environment of drug discovery. On the analytical and preparative scale, SFC enables the development of highly efficient chromatographic methods with fast equilibration, while reducing development costs associated with solvent consumption and waste removal. At Abbvie, the Analytical and Purification Sciences (APS) group has provided a chiral preparative SFC service since 2000, which has steadily grown to impact over 30 projects and over 200 samples per year.^2-4 The APS group at AbbVie has developed strategies to meet the growing need and variability of chiral separations within drug discovery, such as developing a streamlined scale-up approach and applying structure similarity software to minimize method development, providing a 3-5 day turnaround for chiral separations. Recently, analytical SFC capabilities have been expanded to include detection by mass spectrometry (MS) to detect and identify achiral impurities and expedite achiral separations request to the Analytical and Purification Sciences lab.

Impact and Discussion:

Chiral Analytical SFC Applications:

At AbbVie, SFC is the technique of choice for chiral separations supporting discovery. Analytical method development is performed within 24 hours of receiving the sample with reports emailed to the client and also linked directly to their Electronic Lab Notebook (ELN). Three analytical SFC instruments are employed to meet the screening and method development needs of the chiral separation service. Two instruments are dedicated to the Analytical and Purification Sciences lab and one system is employed as a walk-up open access system for use by discovery medicinal chemists.

Upon, submission of a racemic chiral sample to the APS lab, method development is immediately employed by screening Chiral Stationary Phases (CSPs) with a hit-rate goal of less than one business day. Twenty unique CSPs are screened, including coated and immobilized stationary phases. To effectively screen this many phases, a login wizard was developed in the lab software using macros to control column-switching valves and build a sequence table (Figure 1).

 Figure 1. Chiral SP Analytical SFC Screen Operated Using In-House ChemStation Macros

The macro streamlines the sequence-building process by allowing multiple methods and column selections to be custom matched, automatically programs an equilibration time into each run, and can be unique to each logged sample. The standard method for chiral stationary phase screening is a 5-50% methanol:CO₂ method at 150 bar over 5 minutes using 4.6 x 100 mm ID columns with a particle size of 5 microns. Since so many unique stationary phases are employed in the screen, we have found that this short screen using methanol as the sole additive to be effective for over 85% of the chiral analytical screen requests to our lab. Alternatively, methanol with 0.1% diethyl amine additive is used for polar basic analytes, where peak tailing is expected to be an issue. Combining the hit rate for methanol and methanol with 0.1% diethyl amine additive effectively allows us to achieve a hit rate of 90% for chiral analytical submissions.

In addition to the 20-column chiral stationary phase screening, additional screening for poorly selective, highly polar, or relatively insoluble samples are done on a second SFC system with single quad mass spectrometry detection, equipped with five immobilized chiral stationary phases including three polysaccharide phases and one Pirkletype chiral stationary phase. In the case of poorly soluble samples, a hit on the primary 5-50% methanol:CO₂ screen on a coated polysaccharide column can be given a short secondary screen on a separate column with a mobile phase additive of methanol:dichloromethane (8:2) to increase the analyte solubility in the mobile phase.

In addition to screening alternative modifiers and additives for chiral analytical separations, the mass spectrometer on this second system allows for investigations into achiral separations, which currently count for roughly 5% of SFC submissions to the Analytical and Purifications Sciences lab at AbbVie.

For walk-up chiral analytical SFC submissions, such as Enantiomeric purity checks, a third SFC instrument with a similar sample login macro is available to Discovery medicinal chemistry (100 users). This system has the six stationary phases we have found to have the highest percent hit rates, with dimensions 4.6 x 100 mm ID and 5 micron particle size. A walk-up user is allowed to screen up to three samples on all six columns with the 5-50% methanol:CO₂ or 5-50% methanol:CO₂ with 0.1% diethyl amine over 5 minutes at 150 bar (up to 90 minutes total run time). Clients are instructed to prepare only filtered, post workup samples, in methanol solution, at a concentration of 0.5 mg/mL to comply with coated CSP sensitivity to alternative solvents. The results, in standard report format, are emailed to each medicinal chemistry client and archived automatically to their ELN.

Chiral Preparative SFC Applications:

Preparative SFC in the APS group is performed on preparative SFC systems. In order to achieve the maximum throughput for chiral separations, analytical hits are rapidly scaled from an analytical 5-50% MeOH: CO₂ hit to an isocratic preparative run based on the retention time of the analyte on the analytical gradient method (Figure 2).

 Figure 2. Scale-up from an analytical SFC CSP hit (left) to single injection on an SFC running under isocratic control (30% MeOH:CO₂) to rapid stacked injection mode for high throughput (right).

In addition, very little time is dedicated to preparative method development. Typically, only 2-3 ‘test’ injections are executed to optimize the mobile phase and loading conditions, and often nonoptimal methods with small mixed fractions or low loading are preferred for samples less than 5g, since the efficiency of running in stacked injection mode and resubmitting mixed material typically results in higher throughput, compared to the time required to for more extensive method development. Mixed fractions can be combined, dried, and resubmitted at the end of the run to maximize product recovery.

The limitations to preparative chiral SFC are often related to the solubility of the analyte in alcohol solvent. Our group typically defines low analyte solubility as solubility of <50 mg/mL in methanol for chiral preparative submissions weighing less than 1 gram or <100 mg/ mL in methanol for submissions over 1 gram in weight. When these minimum solubility requirements are not achieved, immobilized equivalents of coated chiral stationary phases are employed and the analyte is loaded onto the column in either dichloromethane or, in extreme insolubility cases, dimethylsulfoxide (DMSO). The use of DMSO as a loading solvent is generally the least preferable to drydown conditions due to its extremely low volatility. To address analyte precipitation that occurs after injection, or poor peak shape due to limited analyte solubility and mass transfer (Figure 3), a mobile phase modifier mixture of methanol:methylene chloride (8:2) is used to increase the solubility of the analyte in the mobile phase as it is loaded onto the column and moves toward the fraction collection module. Other strategies employed for increasing analyte solubility include increasing the temperature of the column or pre-column heat exchanger from 30 degrees Celsius to 35 degrees Celsius.

Figure 3. Poor chromatographic peak shape observed as a result of low analyte solubility in mobile phase (<5 mg/mL).

Beyond poor solubility, analytes with a weak chromophore are also problematic. In these cases, applying protecting groups or separation of an earlier intermediate compound are employed to find a more suitable chiral analyte. Base-sensitive polar analytes such as free carboxylic acids often interact strongly with chiral stationary phases, leading to an observed peak widening. For these compounds, an acidic or basic additive present in the mobile phase cannot be used to overcome poor peak shape, as conversion to methyl ester in presence of acidulated or basic methanol in the mobile phase is common.

Additionally, methanol or ethanol in the mobile phase has also assisted inadvertent intramolecular conjugate addition in some analytes. To efficiently process chiral separations of analytes with two or more stereocenters, an Advanced Laser Polarimeter (ALP) is used as a secondary in-line detector on the SFC prep system. Advanced Laser Polarimeters are able to detect the net optical activity of a flowing analyte dynamically in real time on the preparative SFC. For compounds with more than one stereocenter, the use of an ALP makes it easy to determine, during the prep run, which peaks correspond to diasteriomers and which to their corresponding enantiomers (Figure 4). Additionally, an in-line ALP allows for continued identification of individual enantiomers and diastereomeric pairs when an alternative chiral stationary phase is employed, allowing the chromatographer to change CSP with ease when multiple batches with a different impurity profile are submitted for chiral separation. One limitation to the use of an in-line ALP is that strong signals on the polarimeter was observed only at high loading (<50 mg/injection) with baseline separation for all isomers in the sample.

 Figure 4. Preparative SFC UV detector signal (left) of an analyte with two non-fixed stereocenters and corresponding ALP signal (right). From the ALP signal, the enantiomeric and diasteriomeric pairs in the sample can be identified.

Data Analysis in a Chiral SFC Purification Lab:

At Abbvie, the Analytical and Purification Sciences group supports an average of 200-250 chiral separation requests annually. The samples submitted vary greatly in weight (10s of milligrams to multi-gram scale) and analyte complexity (intermediates and final products). Often, chiral separations are first performed on a small milligram scale to meet initial biology testing deadlines and then later scaled up by medicinal chemistry project teams to meet toxicology testing deadlines, which require gram quantities of purified chirally pure material. In an effort to minimize method development and consistently process chiral separation requests of identical chiral racemates across different batches, the APS lab implemented chromatography data integration software in 2014 to perform structure searches and structure similarity searches within a LIMS-based sample database. Upon medicinal chemistry client login to the APS lab, a structure similarity search is conducted and a replication or scale up of previous conditions can be applied after a fast impurity analysis on the SFC-MS system. The reduction or elimination of method development or chiral stationary phase screening often reduces the overall chiral separation throughput by 1-2 business days, allowing a substantial time savings to medicinal chemistry to meet biological testing deadlines

Conclusions

At AbbVie, the Analytical and Purification Sciences chiral separation service employs SFC technology to resolve racemic or diastereomeric mixtures to support Drug Discovery on tens of milligrams to multi-gram scale with a 3-5 business day turnaround. Relying on the chromatographic advantages of SFC instrumentation to efficiently screen and separate chiral samples on a preparative scale with high recovery and chiral purity has been a valuable asset to the service. Rapid scale up from analytical screening methods can result in large time savings rather than extending analytical method development and integrated software shortens screen time for structurally similar racemates.

References:

1. Berger, T. A. and W. H. Wilson (1995). “Separation of basic drugs by packed-column supercritical fluid chromatography. 3. Stimulants.” J Pharm Sci 84(4): 489-492.
2. Hochlowski, J., P. Searle, et al. (2011). “An Integrated Synthesis-Purification System to Accelerate the Generation of Compounds in Pharmaceutical Discovery.” J Flow Chem 2: 56-61.
3. Olson, J., J. Pan, et al. (2002). “Customization of a Commercially Available Prep Scale SFC System to Provide Enhanced Capabilities.” JALA 7(4): 69-74.
4. Searle, P. A., K. A. Glass, et al. (2004). “Comparison of preparative HPLC/MS and preparative SFC techniques for the high-throughput purification of compound libraries.” J Comb Chem 6(2): 175-180.