Peptide Purification: Influenza Peptide Example
This example involves the purification of a peptide targeted for influenza vaccine development. The peptide was very hydrophobic and its solubility is limited. This translates into a high production cost using traditional means of production.
The starting point is the use of an Amberchrom™ Profile™ for initial method development. The below figure shows the analysis of the influenza peptide (Influenza peptide M1, [H]-Gly-Ile-Leu-Gly-Phe-Val-Phe-Thr-Leu-[OH]) using a standard 5 µm C18 silica column compared with the 10 µm Amberchrom™ Profile™ column.
Influenza Peptide analysis comparison
Amberchrom™ Profile™ can separate contaminants from the peak of interest and demonstrates different selectivity to the C18 column. In addition, there is less tailing of the main peak in the Amberchrom™ Profile™ chromatogram compared to the C18 silica.
The challenge was to then produce the peptide in an economical purification process. Initially the experiment reproduced literature conditions at acidic pH. High concentrations of organic solvent were needed to solubilize the peptide, and therefore only very low loading levels on the column were possible. This was true for both silica gel and the polymeric medium. Yields under acidic conditions were poor, only 43% on silica and 55% on the polymeric resin. Obviously this would not be economical process to scale up to manufacturing because of its low productivity ad high yield loss. Basic pH conditions were then investigated. This would not be possible using silica, but is enabled with a polymeric medium. At a pH 10 it was possible to increase the peptide solubility dramatically by nearly sixfold. Retention was also more favourable, meaning lower solvent consumption, and the peak shape improved considerably. Because solubility was no longer restricted, it was possible to load the column to much higher levels - giving a twelvefold increase. This improved the separation and provided a dramatic yield improvement.
When coupled with a near doubling in yield, the above means the productivity increased by a factor of 24, based on medium utilization. On a cycle time basis, this increased by yet another twofold due to shorter elution time. Finally, solvent utilisation decreased by a factor of 17. This example cannot be generalized to all situations. However, the point is that the new technology provides a chemically robust platform to enable a higher degree of process development freedom than existing technology. This can in turn enable certain types of compounds to be produced with much better productivity, economy, and environmental impact.
Amberchrom™ Profile™ XT20 - Process Optimization
Packing Material |
Column Loading mg/ml |
Linear Velocity cm/h |
Yield @ 95 % Purity |
Total Recovery % |
Acidic pH mobile phase 0.75 mg/mL conc. 4.6mm I.D. x 25cm L |
0.45 |
180 |
40 |
48 |
Acidic pH mobile phase 0.75 mg/mL conc. 4.6mm I.D. x 25cm L |
0.45 |
90 |
53 |
56 |
Basic pH mobile phase 0.75 mg/mL conc. 4.6mm I.D. x 25cm L |
0.45 |
90 |
30 |
100 |
Basic pH mobile phase 4.3 mg/mL conc. 4.6mm I.D. x 25cm L |
5.2 |
90 |
85 |
100 |
Basic pH mobile phase 4.1 mg/mL conc. 10mm I.D. x 25cm L |
4.6 |
90 |
80 |
100 |
The above table summarizes the separation optimization and scale-up of the influenza peptide purification. Peptide yield and purity were improved by decreasing linear velocity, increasing mobile phase pH, and increasing influent concentration. A minimal loss of yield at target purity was seen during scale-up (5X), however target yield and purity were still achieved.