The Impact of Chiral Supercritical Fluid Chromatography in Drug Discovery: From Analytical to Multigram Scale

A significant responsibility for the analytical group within Drug Discovery Chemistry at Bristol Myers-Squibb (Princeton, NJ) is the chromatographic purification of chiral compounds, both intermediates and final products, for a wide variety of chemotypes. Initially, the main tool of choice was preparative HPLC. When numbers of requests and amounts of compound requiring chiral purification were relatively low, HPLC was quite adequate for the task. However, as chirality began to play an increasingly important role in medicinal chemistry, so too has the need for increased capacity in chiral chromatographic separations. The requests can range from tens of milligrams for a promising early lead, to tens of grams for key intermediates to be used in library syntheses, to hundreds of grams of an intermediate or final product for preclinical toxicity testing. As the demand increases, especially in the range of tens to hundreds of grams, HPLC-based separations become more and more of a bottleneck due to limitations in flow rate and the need to remove large amounts of solvent from the purified fractions.

In the mid- to late 1990s at DuPont-Merck (Wilmington, DE), researchers implemented supercritical fluid chromatography (SFC) for use in conducting large-scale chiral purifications for drug discovery. The primary advantage of SFC over HPLC is its speed, both in terms of separation time and solvent evaporation time. Since the viscosity of supercritical CO2 is approximately one-third that of typical normal-phase HPLC solvents such as hexane or heptane, SFC flow rates can be approximately three times higher that those in HPLC and still not exceed column pressure limits. Therefore, retention times are reduced and overall production rate is increased by the same factor. Since the primary component of the SFC mobile phase is CO2, the total volume of the collected fractions after separation is reduced by 70–90% relative to HPLC; thus evaporation time is reduced considerably. In many cases, preparative chiral SFC enables us to meet deadlines for in vivo animal studies when HPLC does not. Further, there is a significant cost advantage to using CO2 instead of hexane or heptane, both with respect to initial purchase and final disposal.

For these reasons, SFC has become the authors’ primary tool for chiral preparative separations, regardless of scale. HPLC is now used only in the rare cases when a separation does not work by SFC or when SFC instruments are committed to other projects. In the BMS analytical laboratory, preparative SFC combined with automated consecutive injection is now utilized routinely in chiral purification to supply several hundred grams of both final compounds and intermediates with high enantiomeric excess in support of drug discovery chemistry.


Figure 1 - Thar SFC Prep 200.

The following apparatus was employed: Thar SFC Prep 200 (flow rates up to 200 g/min at pressures up to 380 bar) (Thar Technologies, Pittsburgh, PA) (Figure 1); Chiralpak AD-H (3 × 25 cm), Chiralcel OD-H (3 × 25 cm), and Chiralcel OG (5 × 50 cm, 20 μm) columns (Chiral Technologies Inc., Exton, PA); and bulk stationary phases, Chiralpak AD (20 μm) and AS (20 μm) packed into 7.5 × 30 cm cartridges by Biotage (Charlottesville, VA). Carbon dioxide was supplied by Airgas (Piscataway, NJ).

Results and discussion

Figure 2 - Phase diagram of CO2 and principle of SFC.

Figure 3 - Preparative chiral separation of a drug discovery final product using SFC: Chiralpak AD-H (3 × 25 cm, 5 μm), CO2/methanol/acetonitrile (60/20/20), 100 mL/min at 220 nm and 36 °C, backpressure regulator set at 100 bar, 10 cc of 35 mg/mL of sample per 3.5-min cycle time.

In Figure 2, CO2 becomes supercritical fluid (neither liquid nor gas) above 31 atm and 73 °C (critical temperature and pressure). The unique characteristics of this supercritical fluid provide better mass transport and lower viscosity relative to typical normal HPLC solvents. Figure 3 shows that high flow rate combined with stacked injections results in short cycle time and high throughput (350 mg every 3.5 min or 6 g/hr). This major SFC campaign rapidly provided more than a half kilogram quantity of pure final product, enabling chemists to meet the time lines for preclinical animal studies. This experiment showed that direct mixing polar organic modifiers (methanol and acetonitrile) with CO2 resulted in excellent selectivity and solubility, which could not be achieved by HPLC.

Figure 4 - a) Preparative chiral SFC separation of a pharmaceutical intermediate using stack injections: Chiralcel OD-H (3 × 25 cm, 5 μm), CO2/ethanol (95/5), 120 mL/min at 220 nm and 36 °C, 200 mg of sample per 5-min cycle time. b) Single injection (i.e., loading study prior to stack injections): same conditions as above.

It is even more attractive to use SFC when the organic modifier is further reduced. As seen in Figure 4a and b, 5% ethanol with 95% CO2 on a Chiralcel OD-H column was utilized for chiral resolution of a pharmaceutical intermediate. This method provided the purified fractions in a much smaller volume and a very short time (5 min/injection) than would be possible by HPLC. In SFC, CO2 replaces hexane or heptane and, as a result, solvent consumption/cost is greatly reduced. Most significantly, much less time is spent evaporating solvents after the separation.

Table 1 shows the advantages of preparative chiral SFC over HPLC providing higher throughput, sample solubility, and less solvent consumption, resulting in reduced drying time and waste disposal. Clearly, SFC provides a more direct and efficient way of purifying enantiomers for drug discovery than does HPLC.

Dr. Wu is Senior Research Investigator, Ms. Leith is Senior Research Scientist, and Dr. Balasubramanian is Director, Dept. of Chemical Synthesis, Drug Discovery Chemistry, Bristol Myers-Squibb, Princeton, NJ, U.S.A. Mr. Palcic is Vice President, Instruments Div., Thar Technologies Inc., 100 Beta Dr., Pittsburgh, PA 15238, U.S.A.; tel.: 412-967-5665; fax: 412-826-3215; Mr. Wang-Iverson is Associate Director, Bioanalytical and Discovery Analytical Sciences, Pharmaceutical Candidate Optimization (PCO), Bristol Myers-Squibb.