Chromatography Forum

Dear Forum,

I am having a problem with my peptide. It is a polybasic peptide (charge +6) with a myristoyl group attached to one end. There is an Aspartate-Glycine bond within this peptide, which I found out has a tendency to self cleave the peptide.

I labelled the peptide and ran it through a semi-prep HPLC column, linear gradient from 0 to 70% ACN, at 1% per minute, all solutions containing 0.05% TFA. My peptide comes off with a nice beautiful peak, labelled as the solution is yellow. At around 79 minutes, it takes I think 10 to 22 minutes to travel the column.

I lyophilized this, then ran it on the column again, same conditions, to see if the peptide had degraded during lyophilizing.

I now see two peaks of nearly equal size, that are coming off 15 minutes apart. I find that the 'yellow labelled peptide' peak is now half the size of what it is supposed to be. (One at 62-64min, and one at 79min)

I do not know how to prevent this degradation. Is it the acidic pH (4.0) thats causing this? Its the only thing I can think of and the literature is not much help.

If I need to buffer it out before I lyophilize, I would risk salts forming in the dried product.

Can I run this column without any acid at all?

Any advice would very much appreciated as I need to prevent this from happening. It is also the only thing standing in between me finish this project and graduating.

Thank you very much for your time,

Sincerely,
P

Xaelic:

The QC departments of biotech companies worry a lot about deamidation at susceptible Asn residues. Such residues tend to have a sterically unhindered residue on the C-terminal side. High pH accelerates this; it's 10x faster at pH 8 than at pH 7. The immediate product is a succinimide ring, which then hydrolyzes to give a racemic mixture of n- and iso-Asp residues. It isn't as well known that susceptible Asp residues can undergo the same reaction via dehydration (example: Asp-130 in human growth hormone). The Gly residue on the C-terminal side makes your Asp susceptible. This reaction is accelerated by low pH, precisely the conditions you're using. While the resulting succinimide residue tends to hydrolyze to yield the same racemic mixture of n- and iso-Asp residues, a high concentration of amines or NH3 ion can cause the hydrolysis of both CO-NH bonds in the succinimide ring simultaneously, leading to cleavage of the peptide backbone. People used to use hydroxylamine to chop up collagen this way. In your case, keep the pH up and keep out amines.

Dear Andy,

Thank you for your reply. I was advised to try a run using a buffered aqueous component, with 10mM Ammonium Acetate (pH 6.5), with the same gradient/conditions as above.

I was recommended ammonium acetate because it would allow me to keep the pH near neutral, and upon lyophilizing, the ammonia would evaporate and leave a peptide acetate salt, instead of the peptide trifluoroacetate salt.

From your post, would I be correct in assuming that I should not use ammonium acetate as the NH3 ion would favor hydrolysis? Is there another acetate salt I can use in order to buffer my runs between pH 6 and 7?

Thank you very much for your time,

Sincerely,
P

Try either triethylamine acetate or, better, diisopropylethylamine acetate. The salts will still be volatile. The amine components are less likely to interact with the carbonyl carbons than would ammonium ion or the ion of a less hindered amine.

Incidentally, since this is a peptide with a myristoyl tail, why not try HILIC? It works well with acylated peptides, and since the water content is lower than with reversed-phase chromatography, there should be less tendency for hydrolysis of a susceptible Asp residue. Just don't try this with uncoated silica; use a HILIC column with a thick, well-hydrated coating.