Chromatography
Achiral Supercritical Fluid Chromatography (SFC) for the Purifi cation of Pharmaceuticals
by Thomas Wolf, Aldo Meishammer, Celine Patissier, Marcel Reck, Claudio Valente and John Reilly. Corresponding Author:
John.Reilly@
Novartis.com Novartis Institutes for BioMedical Research Inc, Fabrikstrasse 2, Basel 4056, Switzerland
With large quantities of small scale (<1000mg) compounds typically being needed to be purifi ed and evaluated for discovery in-vitro and in-vivo testing regimes, a chromatographic purifi cation 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 purifi cation to isolate pure products [1]. High performance liquid chromatography (HPLC) has been generally accepted as the main technique of choice for analysis and purifi cation, 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 modifi er, it is acutely toxic to aquatic life and the effl uent from laboratories has to be controlled and incinerated which increases the cost and environmental impact. At Novartis, the integration of supercritical fl uid 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 purifi cation for achiral samples [4]. An example of this effi ciency is shown in Figure 1 where the same sample is purifi ed (25mg injection). The sample was processed in a third of the time by SFC with equivalent purity and recovery.
In efforts to fi nd 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 purifi cations. A typical example separation comparison between SFC and HPLC is shown in Table 1 where the enormous benefi ts in effi ciency, 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 effi cient SFC platform for discovery separations. Several SFC-MS directed purifi cation systems have been set up within Novartis globally to allow for rapid and effi cient purifi cation. This successful approach has led to an increase in the use of a SFC screening platform for achiral purifi cation submissions within Novartis. An example of this is shown in Figure 2 where routinely more submissions are purifi ed by SFC than traditional Reverse Phase Liquid Chromatography (RP-HPLC). The improved reliability of analytical and purifi cation 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 purifi cations [5].
Table 1. Comparison of Prep SFC v Prep HPLC. (Courtesy of Waters Corporation) Purification by SFC
Separation Time
Organic Solvent Used Total Workup Time
Recovery 3 hours
5L of Methanol 1 hour
95%
Analytical Screening Strategy
Our standard screening approach is to maximise effi ciency 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 modifi er) SFC Gradient
Column: 50 mm x 3mm SFC analytical Column Temperature: 35o Injection volume: µl
C
Time 0
0.1 Figure 1. HPLC-RP v SFC comparison for discovery purifi cation.
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 purifi cation 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.
2.00 2.40 2.50
% Mobile Phase A 0
95 45 45 95
Figure 2. Typical 60:40 distribution for SFC v RP-HPLC for discovery achiral purifi cations.
% Mobile Phase B 5 5
55 55 5
Flow Rate (mL min-1 3.0 3.0 3.0 3.0 3.0
) 46 hours
40L of Acetonitrile 8 hours
80%
Purification by HPLC
An example where the addition of a basic modifi er (0.1% n,n-diethylmethylamine) to co- solvent MeOH demonstrates the signifi cant difference to peak shape and elution order in Figure 3.
LAB ASIA - APRIL 2022
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