Achiral Supercritical Fluid Chromatography (SFC) for the Purification of Pharmaceuticals

With large quantities of small scale (<1000mg) compounds typically being needed to be purified and evaluated for discovery in-vitro and in-vivo testing regimes, a chromatographic purification technique needs to be easily integrated into discovery chemistry laboratories and highly reproducible.  Within Separation Sciences Departments in the Pharmaceutical Industry there are a range of methodologies focused on the analytical separation and purification to isolate pure products [1]. High performance liquid chromatography (HPLC) has been generally accepted as the main technique of choice for analysis and purification, utilising either normal phase or reverse phase conditions, with reverse phase being the generally accepted as a high throughput approach to purify small scale drug discovery products [2, 3]. Although the use of acetonitrile within reversed phase is a widely accepted modifier, it is acutely toxic to aquatic life and the effluent from laboratories has to be controlled and incinerated which increases the cost and environmental impact. At Novartis, the integration of supercritical fluid chromatography (SFC) has several advantages such as faster run times due to lower viscosity, short equilibration times, reduced solvent consumption and, in preparative applications, fast solvent removal. SFC has rapidly become a very attractive alternative to Normal Phase and Reverse Phase purification for achiral samples [4]. An example of this efficiency is shown in Figure 1 where the same sample is purified (25mg injection). The sample was processed in a third of the time by SFC with equivalent purity and recovery.

In efforts to find new and innovative ways to improve chromatographic processes, Novartis has increasingly employed Supercritical Fluid Chromatography (SFC) to decrease the overall environmental footprint and increase productivity for small to large (mg-g) scale achiral purifications. A typical example separation comparison between SFC and HPLC is shown in Table 1 where the enormous benefits in efficiency, time, solvent consumption and evaporation time are highlighted.
The combination of SFC with high performance HPLC in parallel for achiral screening has enabled us to establish an efficient SFC platform for discovery separations. Several SFC-MS directed purification systems have been set up within Novartis globally to allow for rapid and efficient purification. This successful approach has led to an increase in the use of a SFC screening platform for achiral purification submissions within Novartis. An example of this is shown in Figure 2 where routinely more submissions are purified by SFC than traditional Reverse Phase Liquid Chromatography (RP-HPLC). The improved reliability of analytical and purification SFC instrumentation and increasing availability of a range of SFC stationary phases means this methodology is becoming method of choice for more achiral separations as well as chiral small molecule purifications [5].  
The coupling of SFC-MS collection enables focused collection of products and by-products in a small fraction of methanol which can be evaporated in a fraction of the time of aqueous-acetonitrile fractions from HPLC. There are some caveats to sample preparation for purification which need to be closely adhered too, due to a high risk of precipitation and loss of product, the samples have to be fully dissolved in Methanol (MeOH) or a 1:1 mixture of Dichloromethane/Methanol (DCM/MeOH) to ensure solubility on-column.

Analytical Screening Strategy
Our standard screening approach is to maximise efficiency within a 2 minute analytical gradient using a range of SFC stationary phases. The gradient approach is as follows:
Mobile Phase
Line A: CO₂
Line B: Pure MeOH or MeOH
            (+0.1% basic modifier)
SFC Gradient
Column: 50 mm x 3mm SFC analytical
Column Temperature: 35oC
Injection volume: ml
An example where the addition of a basic modifier (0.1% n,n-diethylmethylamine) to co-solvent MeOH demonstrates the significant difference to peak shape and elution order in Figure 3.
There is no uniformly accepted ‘generic’ SFC column chemistry within the industry and therefore there is a need to select a range of chemistries in comparison to RP-HPLC where C18 is the more standard and unified approach. A demonstration of this is shown in Figure 4 for a test mix separation for 6 different column chemistries using the same generic SFC gradient.

Strategy for Purification
Based on the 2 minute gradient analytical result, we then can develop a ‘bespoke’ shallow gradient window for individual samples defining the percentage of MeOH needed to try and elute the compound of interest in the middle of the prep gradient. We have optimised the purification run times by the implementation of higher flow rates and also routine use of  3um SFC Prep columns to maximise peak efficiency. An example is shown in Figure 5 where the purification total run time is only 4 minutes using a 10-27% MeOH shallow gradient at total flow150ml mL min-1. This is now standard procedure within our laboratory and has been enabled by the recent upgrade of two waters SFC-100-MS instruments by ABsys (Oberursel, Germany).
These ABsys upgrades have enabled the laboratory to increase flow rates of up to 180 mL min-1, optimise gas-liquid separation to enhance recovery, shorten preparative methods, shorten equilibration times and implement waste collection for every run. The system is shown in Figure 6. This has enabled Achiral SFC purification processes to be optimised and has enabled an increase in the scale of separations we can employ and has been adopted by our lab as our purification system of choice.

Conclusions
By taking advantage of the advances in SFC technology, chromatographers are also able to capitalise on the improvements improving achiral separation efficiency whilst also reducing environmental impact.  Purification via SFC is ~3 to 4x faster than a traditional RP purification with purity equivalent to RP-HPLC. With the introduction of the ABsys SFC systems the reliability, speed and scale of SFC Achiral purifications has been improved. We in Novartis are now able to efficiently separate achiral novel drugs whilst significantly decreasing run times, environmental impact, maximising recovery with reduced dry down times. Analytical and Preparative Achiral SFC is standard practice and adopted by the whole Global Discovery Chemistry community. RP-HPLC will still remain a complimentary orthogonal technique for purification and first method of choice in Open Access purification.

References
1.     Kaljurand M., Koel M. “Recent Advancements on Greening Analytical Separation” Critical Reviews in Analytical Chemistry 2011, 41: 2-20,
2.     Goetzinger W., Zhang X., Bi G., Towle M., Cherrak D and Kyrano J.N “High throughput HPLC/MS purification in support of drug discovery” International Journal of Mass Spectrometry 2004, Vol         238, Issue 2, : 153-162
3.     Blom, K.F, Glass B., Sparks R. and Combs A.P. Preparative LC-MS Purification: Improved Compound –Specific Method Optimization” 2004, Journal of Comb. Chem. 6, 874-883
4.     Francotte E. SFC China 2015 (Green Chemistry Group Presentation)    SFC: A Multipurpose Approach to Support Drug Discovery
5.     Desfontaine V., Guillarme D., Francotte E., Novakova L. “Supercritical fluid chromatography in pharmaceutical analysis” Journal of Pharmaceutical
    and Biomedical Analysis,113 2015 56-71