The Effects of SFC Preparative Scale-up on Throughput, Purity, and Recovery of an Impurity in an API Mixture

Significance of the topic

Supercritical fluid chromatography scale-up from analytical to preparative scale enables isolation of trace-level impurities in active pharmaceutical ingredients with minimal solvent consumption and high throughput. Ensuring consistent chromatographic performance across scales is crucial for process efficiency, cost reduction, and material recovery in pharmaceutical development.

Study Objectives and Overview

This study converts an analytical ACQUITY UPC2 method for separating a 0.1% 4-nitrophenol impurity from acetaminophen into a preparative scale using the Prep SFC 150 Mgm system. It evaluates scale-up calculations, system density matching, injection strategies, and compares throughput, purity, and cost between 19 mm and 30 mm preparative columns.

Methodology

• Analytical separation performed on a 4.6×100 mm Torus 2-PIC column with 80:20 CO₂/methanol at 3.5 mL/min and PDA detection at 247 nm and 306 nm.
• Scale-up to preparative 19×100 mm column using geometric formulas to convert volumetric flow rates to mass flow based on CO₂ density (0.936 g/mL).
• Density profiles matched by adjusting ABPR pressure and column temperature to maintain equivalent mobile phase density and resolution.
• Modifier-stream injection used at preparative scale to improve peak shape and avoid injection solvent effects at high sample volumes.
• Stacked injection mode applied to perform 25 sequential injections of 0.5 mL each for target compound collection.
• Pooled isolates underwent a two-cycle purification to enhance purity from 52% to 99% with approximately 90% recovery.

Instrumentation Used

• Waters ACQUITY UPC2 SFC system for analytical method development.
• Waters Prep SFC 150 Mgm system with ChromScope v2.0 for preparative separation and real-time collection.
• Waters ACQUITY UPLC H-Class system with reversed-phase CORTECS C18 column and Empower 3 software for purity and recovery analysis.

Main Results and Discussion

• Successful scale-up achieved with matched chromatographic retention times between analytical and preparative systems.
• Initial purification yielded 52% purity and 92% recovery of 4-nitrophenol; second cycle improved purity to 99% with ~90% recovery.
• Throughput on a 19 mm column was 0.37 mg/h (cycle 1) and 4.5 mg/h (cycle 2), with CO₂ and co-solvent costs of $40/mg and $3/mg respectively.
• A 30 mm column increased throughput substantially, reducing purification time for 10 mg of target from 28 hours to 11 hours, without significant change in mobile phase cost.
• Larger column diameter primarily enhances throughput and reduces process time, while solvent consumption per unit product remains stable.

Benefits and Practical Applications

Preparative SFC scale-up allows efficient isolation of low-level impurities with minimal sample and solvent use. The modifier-stream and stacking injection modes improve peak shape and throughput. Two-cycle purification strategy achieves high purity and recovery, critical for impurity profiling in pharmaceutical quality control and drug substance development.

Future Trends and Potential Applications

• Automation and real-time data monitoring to further increase throughput and reproducibility.
• Integration with continuous processing workflows for seamless scale-up.
• Expansion to a wider range of chirally pure compounds and complex mixtures.
• Development of predictive models for density and flow control across different scales and mobile phase compositions.

Conclusion

The study demonstrates a robust approach to scale up SFC from analytical to preparative scale for trace impurity isolation. Geometric scaling, density matching, and optimized injection strategies ensure high recovery and purity. Choosing appropriate column diameter significantly impacts throughput, enabling cost-effective impurity isolation workflows.

References

1. Runco J. Beginners Guide to Preparative Chromatography. Waters Corporation, 2017.
2. Runco J, Aubin AJ. Practical Strategies for Successful Scaling from UPC2 to Preparative SFC. Chromatography Today, 2018.