The document describes the process of preparative scale purification of a sample of synthetic bradykinin using a concentration overload regime.
The document details the manufacture and purification of synthetic bradykinin, a peptide used in the development of antagonists for hereditary angioedema. It outlines the method development process, starting with screening on analytical HPLC columns to determine optimal separation conditions.
The study evaluates the use of PLRP-S 100A media with varying particle sizes (5, 8, and 10 µm) and determines that isocratic elution at 21% acetonitrile is optimal. The note further describes scaling up the purification from analytical columns to a 1-inch Load&Lock column using a concentration overload technique, achieving 100% purity with 97% recovery.
| Peptide Sequence | Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg |
|---|---|
| Column Dimensions (Analytical) | 250 x 4.6 mm ID |
| Flow Rate | 1 mL/min |
| Detection Wavelength | 220 nm |
| Optimal ACN Concentration | 21% |
| Column Dimensions (Preparative) | 250 x 27 mm ID |
| Recovery | 97% |
The sequence is Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg.
The optimum concentration identified is 21% ACN.
The method achieved 100% purity with a 97% recovery.