Case study 1: enrichment
A client API contained two process impurities—one identified and one unknown. Both were visible by UV detection at 260 nm,
a wavelength at which the API absorbs light less strongly. Thus, the relative absorbance chromatogram observed in the straightforward
gradient RP-HPLC method (Method A, see Figure 1) was known to overrepresent the true abundance of both impurities. The unknown impurity had a relative absorbance of 0.5%
and a true abundance estimated at less than 0.2%. Preparative isolation by scaling up Method A was rejected due to time, solvent
consumption, and the excessive fraction volumes that would be accumulated in the isolation of this low-abundance peak. It
was hoped that, by using SFC methods, the target impurity could be recovered more expediently in the milligram quantities
desired for a complete or partial structure elucidation by 2D NMR and MSn methods.
Figure 1: Reversed-phase high-performance liquid chromatography (UV 260 nm, Method A) of the API showing known and unknown
impurities. The unknown impurity (0.5% by relative absorbance) is observed at 9.7 min. [QA: please define AU]
Under preparative SFC conditions with detection at 260 nm, the impurity peak signal was difficult to identify unambiguously.
A preparative SFC method (Method B) was developed quickly and used to process several grams of API, with fractions collected
before, during, and after the main peak eluted. These fractions were concentrated by rotary evaporation and analyzed using
the RP-HPLC method for the presence of the desired peak. The desired peak was captured and enriched in a fraction collected
immediately before the main peak, indicating that the chosen SFC method adequately resolved the peak from its neighbor. To
accumulate the target peak in quantity sufficient for structural work, 50 g of the API were injected during a period of 7
h of automated stacked injection chromatography (see Figure 2) to produce a highly enriched fraction with a total mass of 240 mg (see Figure 3).
Figure 2: Preparative supercritical fluid chromatography (UV 260, Method B) chromatogram, showing the fractions collected
to enrich the target impurity. The target elutes in fraction F1.
Method C, which involves direct isolation of the target peak from the enriched fraction, was developed by screening multiple
SFC conditions in an automated method-development process. Typically, several hundred solvent and stationary phase pairings
can be screened in an overnight series of gradient runs, and a promising separation may be converted within a few minutes
to a preparative-scale isocratic process. In this case, the target peak was identified based on its relative abundance in
the HPLC chromatogram in Figure 3a, and a method resolving the target completely from its near neighbors was chosen (see Figure 3b). The 240-mg enriched isolate was dissolved in the liquid cosolvent and processed in less than 1 h to isolate the desired
compound. Evaporation of approximately 50 mL of collected solvent yielded 14 mg of the pure target peak.
Figure 3: a (left): Reversed-phase high-performance liquid chromatography (Method A) chromatogram of enriched fraction. b
(right): Analytical supercritical fluid chromatography (Method C) chromatogram of the enriched target, showing remaining active
and other impurities. The mass spectrum for target at m/z 385 is identical for the indicated peaks in each chromatogram.
The development of a controlled and well-characterized production process for a new drug is a complex task. The client producing
this API estimated that the identification of this trace impurity, efforts to synthesize it based on various hypotheses about
its source, attempts to remove it by chemical methods or through alternate synthetic approaches, and attempts to recover it
through chromatography would take 12–14 months. Isolation of the compound using SFC required less than a week, and its structure
was determined by NMR and MS.