Despite advances in automated solid-phase peptide synthesis (SPPS), such as using elevated temperatures for complex peptides, purification remains a major bottleneck in peptide synthesis workflows. Crude peptides often contain impurities with similar retention characteristics to the target, and RP-HPLC, the standard purification method, is limited by low recoveries, long separation times, high costs, and limited loading capacity.*
A 2021 study by Prof. Knud Jensen’s research group at the University of Copenhagen investigated the use of high-performance reversed-phase flash chromatography (HPFC) as a viable method for the rapid purification of synthetic peptides. Depending on the quality of the crude peptides, HPFC was used either as the sole purification method or as part of a multi-step process to remove impurities before a final RP-HPLC purification.
The study examines various peptides, including APH3, a 44-amino acid antiparallel coiled-coil peptide. A 300 mg batch of crude APH3 (~35% initial purity) was divided into two 150 mg portions, with one purified using HPLC and the other using HPFC.
The yields proved to be similar, with HPFC yielding 30 mg of pure peptide (>97% purity) and HPLC recovering 33 mg. However, solvent consumption differed significantly. HPLC required 1777 mL of solvent A (water containing 0.1% formic acid) and 1514 mL of solvent B (acetonitrile containing 0.1% formic acid), whereas HPFC used only 1041 mL of solvent A and 477 mL of solvent B. HPLC required 1777 mL of solvent A (water containing 0.1% formic acid) and 1514 mL of solvent B (acetonitrile containing 0.1% formic acid), whereas HPFC used only 1041 mL of solvent A and 477 mL of solvent B.
HPLC required four injections, while HPFC needed only one, significantly increasing the speed and throughput of peptide purification. The total purification time for HPLC was 160 minutes, compared to just 27 minutes for HPFC.
The summary of peptide purifications with HPFC shows that Penetratin, for example, was obtained with a purity of 92% with a 66% yield, while GLP-1N was obtained with a purity of 98% with a 20% yield. This highlights the flexibility and versatility of HPFC in purifying different types of peptides.
The study concludes that HPFC is a rapid and efficient alternative to traditional RP-HPLC, enabling larger peptide loads per injection while significantly decreasing purification time, solvent consumption, and costs compared to RP-HPLC.
Beyond comparing the two methods, the study offers detailed guidelines on selecting columns, eluents, and gradients for successful HPFC, helping researchers integrate this technique into their peptide purification workflows.
For more detailed insights, read the full study (gated).
* Kasper K. Sørensen, Narendra K. Mishra,Maciej P. Paprocki, Amit Mehrotra, Knud J. Jensen. (2021). High-performance reversed-phase flash chromatography purification of peptides and chemically modified insulins. European Chemical Societies Publishing. https://doi.org/10.1002/cbic.202000826
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