Systematic and efficient method scale-up for peptide purification

Significance of the topic

Peptide-based therapeutics have gained prominence due to their high specificity, selectivity and low immunogenicity. Ensuring high purity of synthesized peptides is critical for their safe and effective application in pharmaceutical research and production. High-performance liquid chromatography (HPLC) is the method of choice for achieving analytical and preparative purification of peptides, and its linear scale-up is essential to increase throughput while maintaining method robustness and reproducibility.

Objectives and study overview

This study presents a systematic, linear scale-up strategy for purifying crude Angiotensin I from a nominal 68 % purity to > 99 % using preparative HPLC. The main goals were to adapt an analytical HPLC method to preparative scale with minimal sample loss, optimize chromatographic parameters, and validate the approach for efficient peptide fractionation.

Methodology and used instrumentation

A validated analytical HPLC method (10–100 % organic gradient over 12.5 min) was optimized through the following steps:

• Particle-size adjustment: Increasing stationary phase particle size from 5 to 10 µm to control backpressure at higher flow rates.
• Gradient focusing: Implementing a focused gradient with a high-organic wash and re-equilibration to sharpen target peak and remove late-eluting impurities.
• Volume overload study: Determining the maximum injection volume (50 µl) that preserves resolution between Angiotensin I and impurities.
• Column dimension scaling: Expanding column inner diameter from 4 mm to 20 mm and flow rate from 1.2 mL/min to 30 mL/min using linear scaling rules.

Used instrumentation

• Analytical HPLC system (KNAUER AZURA): P 8.1L pump, AS 6.1L autosampler, CT 2.1 column thermostat, DAD 2.1L detector, 150 × 4 mm Eurospher II C18 column.
• Preparative HPLC system (KNAUER AZURA): P 6.1L high-pressure pump, Assistant ASM 2.2L, fraction collector Foxy R1, 150 × 20 mm Eurospher II C18 column.
• Software: ClarityChrom® for data acquisition and Method Converter tool for scaling calculations.

Main results and discussion

The analytical method showed a 68 % crude purity of Angiotensin I. Scaling to preparative conditions with 10 µm particles and a tailored gradient reduced runtime to 9.5 min while preserving resolution. The overload study established 50 µl as the maximum injection for analytical experiments, guiding the preparative full-loop injection of 2 mL of 1 mg/mL crude. Fractionation into 2.5 mL slices yielded a pooled target fraction exceeding 99 % purity as confirmed by the original analytical method, with an output of 1.4 mg purified peptide per run.

Benefits and practical applications

• Predictable elution profiles enable rapid transition from analytical to preparative scale without extensive revalidation.
• Maintained chromatographic robustness ensures reproducible purity and yield across runs.
• Minimal sample loss during method development conserves valuable crude material.
• The approach can be applied to diverse peptide targets in pharmaceutical QC and peptide manufacturing workflows.

Future trends and potential applications

Further developments may include integration of continuous chromatography for higher throughput, automated online scale-up tools, adoption of sub-2 µm or core–shell particles for faster separations, and expansion to multi-dimensional purification schemes. Coupling preparative HPLC scale-up with process analytical technology (PAT) will enhance real-time monitoring and control of peptide production.

Conclusion

The linear scale-up methodology successfully translated an analytical HPLC purification of Angiotensin I to a preparative scale, yielding > 99 % purity with minimal sample loss. Systematic adjustments to particle size, gradient profile, injection volume and column dimensions preserved chromatographic performance, demonstrating a robust workflow for peptide purification in pharmaceutical research and production.

References

• [1] Vora D., Dandekar A. A., Banga A. K. In Peptide Therapeutics: Fundamentals of Design, Development, and Delivery; Jois S. D., Ed.; Springer International Publishing: Cham, 2022; pp. 183–201.
• [2] Bodanszky M. Principles of Peptide Synthesis; Springer: Berlin, Heidelberg, 1993.
• [3] Mant C. T. et al. In Peptide Characterization and Application Protocols; Fields G. B., Ed.; Humana Press: Totowa, NJ, 2007; pp. 3–55.
• [4] Reid I. A. Advances in Physiology Education 1998, 275, S236–S245.