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.
Experimental
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; e-mail:[email protected].
Mr. Wang-Iverson is Associate Director, Bioanalytical
and Discovery Analytical Sciences, Pharmaceutical
Candidate Optimization (PCO),
Bristol Myers-Squibb.