The final steps of peptide production, i.e., purification and isolation, are critical regardless of the synthetic approach used
A number of methods are potentially available for the purification of peptide, however, the following have been considered:
- countercurrent distribution
- partition chromatography
- gel permeation chromatography
- low-pressure, hydrophobic interaction chromatography
- ion exchange chromatography
- reverse-phase, high performance liquid chromatography
Since peptides are considered hard to crystallize, crystallization is rarely used as final purification step, except in the small molecules. Historically countercurrent distribution and partition chromatography have been used but recently for peptide purification, they have largely been replaced by more powerful techniques, such as those based on reverse-phase HPLC.
In comparison to most other drugs, pharmaceutical peptides are usually isolated and purified by chromatographic procedures due to the complications in the products and the strict purity regulations from regulatory agencies.
The most common chromatographic techniques in use are:
- low pressure hydrophobic
- gel permeation
- ion exchange
- reverse-phase HPLC chromatography
Varied ranges of chromatographic media are available and the choice of the combination of purification steps and individual media used is determined by the nature of the peptide and impurities. Since chromatographic techniques, in general, are time consuming, the purification of the final product should be performed in as fewest of steps possible. A single purification step is ideal case, since every chromatographic step potentially decreases the yield, but this is often difficult to achieve in practice.
In a usual purification scheme, the product from the production step is first subjected to ion exchange, gel permeation, or hydrophobic interaction chromatography—which is aimed to remove by-products from the final deprotection step, most of those by products are uncharged low molecular weight. If further purification is required, a final “polishing” step is performed using techniques, such as reverse-phase HPLC. Finally, the organic modifier from the reverse-phase HPLC step is evaporated and the final product is isolated by Lyophilization