10 August / September 2019
Walk-up SFC-MS for Fast Purification of reaction Mixtures within Discovery Chemistry
by Craig White, Analytical Technologies, Eli Lilly and Company Limited, Lilly Research Centre, Erl Wood Manor, Sunninghill Road, Windlesham, Surrey, GU20 6PH, UK
Purification is often regarded as the bottleneck to delivering pure compounds for biological assays within Discovery Research. Medicinal chemists depend on simple processes and robust instrumentation for rapid purification of intermediates and final products, whilst expending as little time and effort as possible. Reverse phase LC-MS platforms are typically used, often with an open-access interface which for the chemist, simplifies sample submission, method selection and batching of samples. The widely accepted limitation of reverse phase LC purification, however, is the time required to remove the post purification aqueous solution and provide a dry isolated product. Supercritical Fluid Chromatography (SFC) is a technique that delivers fractions in a small volume of organic solvent, significantly reducing the post purification processing time. SFC is an established technique within Lilly for queue-based chiral and achiral purification submissions. Until recently, SFC instrumentation has not had a robust hardware and software interface, and as such, has been generally inaccessible to the medicinal chemist in a walk-up environment. In this short application note, we describe our experience with the deployment of SFC-MS for walk-up purification, considering both the challenge to the analytical chemist, and the benefit it affords to the medicinal chemist.
Within our Discovery Chemistry synthesis laboratories, normal phase low pressure flash purification tends to be used as the first pass purification method by the medicinal chemist, for front line achiral purification of intermediates and final products. In an attempt to reduce purification touch time for the chemist, and to free up time for synthetic design, we have explored and tested different purification service concepts. One example is a fee for service flash purification laboratory where a third party provides instrumentation and staff to deliver a two-hour turnaround for flash purifications, using a method provided by the chemist. Although this workflow delivers speed, we occasionally find the quality of collected material to be compromised due to the selection of inappropriate solvent conditions, resulting in no elution of target compound or co-elution of target compound with impurities. Medicinal chemists have limited time for method development; the selection of generic solvent conditions increases the probability of unsuccessful purification, which then impacts productivity through purification re-work. Instead, we have implemented a screening strategy using open access analytical instrumentation, to quickly identify the right technique and the right method for each reaction mixture.
The benefits and application of SFC are well documented and recently published [1-2]. Farrell’s group was one of the first adopters of SFC for fast track purification [3]. The speed, separation orthogonality and solvent handling benefits of SFC offer a win-win scenario for both the analyst and medicinal chemist. To complement reverse phase and normal phase HPLC, we have implemented a four-column SFC-MS screen to quickly scout for mass directed SFC purification conditions. Both analytical SFC- MS and preparative SFC-MS instruments are located in a shared equipment zone for easy access, and configured with walk-up software for simple sample submission. The walk-up (open access) interface enables a chemist to submit samples into a queue, without interacting with the instrument operating system, using methods set up by an administrator. Generating analytical data in open access, across multiple techniques and methods, offers a diverse and fast screening approach, which significantly improves the probability of a successful first pass purification within an acceptable timeframe. Generating the analytical screening data ‘up-front’ also provides the flexibility to the chemist of running their own open-access purification, or to hand the sample over to experts where method selection, purification, evaporation and characterisation is delivered as a service.
Experimental CO2
Carbon dioxide was supplied by BOC Gases (Worsley, Manchester, UK). CO2
gas (99.9%
purity) was delivered from a bulk tank and pressurised to 1500 psi using a booster system supplied from Va-Tran Systems, Inc. (Chula Vista, CA, USA).
Instrumentation The analytical (UPC2
) and preparative
(Prep100) SFC systems were both configured with a QDa MS, supplied by Waters. The analytical configuration included a column oven and switching valve to accommodate eight columns in total, supporting four chiral columns for enantiomeric excess (EE) determination, in addition to the four achiral columns used for purification method screening. The preparative configuration includes column switching between four columns (achiral only).
SFC Stationary Phase
A four-column analytical screen was established using a selection of commercially available stationary phases including (i) GreenSep Naphthyl, (ii) Torus 1-Aminoanthracene (1-AA), (iii) Propyl-
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