Peptide synthesis strategy

High quality reagents are essential, and as it turns out, so is a simple trick like having an acetylation cycle for each amino acid coupling step, capping incomplete sequences. It won’t matter how good you are, there will always be a certain percentage for each amino acid coupling reaction for which the reaction did not complete – the amino acid is “deleted”. If you add the next amino acid at this point then part of the material will have the previous amino acid missing. These are called deletion sequences. On the other hand, if an acetylation cycle is run in between the two reactions, then the failed sequences are effectively terminated and will no longer take part in the synthesis. Again, one might think that it is not a problem since the peptide is purified in the end, but this is not the whole story.

Peptide purification

A preparative HPLC is used in the production to purify the peptide. The principle is that the set of physiochemical properties of the full length peptide is unique in this context and that it will separate from the impurities in this process. However, when the impurities are much alike the target peptide in this respect, separation may not be possible. It is often the case with peptides that should be produced at high levels of purity that these need to be subjected to several rounds of purification, where conditions are adjusted in order to achieve the level of separation required. A more discriminating column would improve on resolution and make the separation easier, but it would also mean that the capacity dwindles to a point where it is no longer a viable option.

So, let's say that you want to make a 20-mer peptide, and you have a problem at AA #10. With no capping cycle, you get a deletion sequence, and so some of the material will be 19-mers, with the error at #10. With a capping cycle on the other hand, the peptide is cut off at #10 and you have some material that is 10-mers. It is more likely that you will have a problem sorting out the 19-mer deletion sequence compared to the 10-mer truncation, even with very fancy HPLC equipment. Here we also see the benefit of using high quality reagents, because it is simply not true that these errors always can be remedied by HPLC purification as the resolution of any preparative HPLC is limited.

Quality control: Peptide purity determination by HPLC

For quality control however, resolution is important while capacity on the other hand is not an issue. Thus the analytical HPLC should have a far more discriminating column compared to the one used in the preparative HPLC. Depending on what column is used for the HPLC analysis, you will get a different answer to what the purity of the peptide is. If you use an inferior column, the resolution is poor, and the purity shown may be far from the true value. Contaminants are more likely to co-elute with the target. You’ll see this as broad peaks, peaks with shoulders, or the use of steep gradients. Conversely, if you use a highly discriminating column, the resolution is excellent with sharp peaks using a shallow gradient. Thus you are more likely to spot any issues, and you have a chance to attempt to remedy this with additional purification rounds.