Dear Colleagues,
peptide synthesis companies offer different purities at different prices.What actually defines the purity?
Several things come into my mind:
- sequence errors (i.e. usually missing amino acids due to incomplete coupling steps)
- incomplete deprotection
- salts and incomplete removal byproducts
- products of side reactions?
Any ideas what is relevant?
Hi, Wolfgang,
I am not sure whether you are talking about solid phase peptide synthesis or solution phase peptide synthesis. I will take solid phase synthesis as an example to address your concerns:
1. Unless you peptide includes unnatural amino acids that are known to have low reactivity, usually sequence error is not a major concern;
2. Depends on the chemistry (FMOC or BOC chemistry) incomplete deprotection does happen from time to time. As a result, you end up with truncated peptide, which could be an impurity. Then again, with carefully controlled reaction condition, incomplete deprotection can be avoided. This is not a major concern;
3. I will combine the last two factors. Indeed, by products is a major contributor. Commericial peptide synthesis companies sometimes skip the final purification, which give you a 90% or so overall purity. With additional purification (typically HPLC), the purity can be improved to 95-99%. As a result, it will cost more. It is possible that your final peptide is in its salt form, for example TFA adduct after cleavage and purification.
Hope this helps.
Dear Wolfgang Schechinger.
The peptide purity is depends on satisfaction of all parameter of standard peptide.
ie., The peptide is 100%pure means exact sequence with existing concentration. but synthetically 100% is not possible when peptide reach above 6residue. due to the reducing coupling efficiency of each amino acids.
So if one amino acid attachment give reduced yield in sequence means in that un attached gap some other species will react or during next coupling process the next amino acid will attach slightly. so the purity was failure by using MASS spectra and HPLC we can able to see the purity in %.
So peptide company will do synthesis and analysis. based on analytical report. if peptide >98%pure means possible to use for therapeutic purpose. if the purity is less means they will give reduced prize.
if you request 98%purity means they will give very special care and use some special technique and achieve that increase prize.
same way the peptide not sequenced well means the TFA and Scavenger like TIS EDT etc, will be form abduct with peptide it will report in analytical report so based on that purity range they will charge.
For your question:
salts and incomplete removal byproducts
products of side reactions.
this two is relevant.
missing sequence means peptide company will able to find it and they won't provide.
incomplete removal also they can able to find.
Hope you may clear now.
Good Luck & Regards
R.Selva.....
Hello everybody,
Another problem is partial racemization - as a rule you have about 1% on each step of coupling. So, if you work with enantiopure peptides, possible impurities are diastereomers.
Almost you got clear answer from Selva Chemist.,
In addition during polypeptide synthesis less yield means possible to come more impurity and delicious peptide formation. if you request less purity means low prize because of their success based.
Hi Wolfgang,
the purity defined by the companies is the level of effort taken to purify the peptide i.e if the peptide is simply cleaved from the solid phase ( it is called crude peptide and is cheaper ). Depending on the sequence (and length) it could be relatively pure or for long sequences less than 10% pure.
there are several methods to treat the peptide depending on the final application, and these include TFA removal, desalting, HPLC purification, neutralization of acid content, and so on.
in my opinion racemization is only an issue with unnatural fluorine containing amino acids, or on Cys and His (upon application of heat or microwave).
as everyone mentioned, sequence errors are uncommon on modern machines, incomplete deprotection will also lead to deletions in the desired sequences, but this can be purified and over 95% pure peptide can be obtained by HPLC and now starts the tricky part:
since many peptides do not have W, Y or Phe, determining concentration becomes a major issue and this requires desalting followed by quantitative amino acid analysis to determine peptide concentration, which sometimes may be as low as 50-60% in a HPLC purified peptide. Again the nature of the sequence, side chains, affinity to acid/salts/ moisture would play a role here and this is not a general rule.
best regards,
Parvesh
Hi colleagues:
Perhaps, the best way to have a closer look at the by-products/impurities repertoir is to inspect the DeltaMass database, in which majority of unwanted chemical modifications are tabulated:
http://www.abrf.org/index.cfm/dm.home?AvgMass=all
Best,
Igor
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