I am trying to conjugate a 12 amino acid peptide (MW 1565Da) to PLGA-PEG-maleimide copolymer (MW: 35097Da). The conjugation reaction takes place in ACN: DMF as a solvent. Can someone please suggest a method (protocol, if you have) to purify the polymer-peptide complex from the polymer and peptide alone? Please note that for HPLC separation based on mass, the difference between that of polymer alone and the complex is not very significant.
You can try electrophoresis protocol. Regards and all the best.
Zaid
Please look at the following below links which may help you in your analysis:
https://www.beilstein-journals.org/bjoc/content/supplementary/1860-5397-11-93-S1.pdf
https://www.sigmaaldrich.com/technical-documents/articles/material-matters/protein-and-peptide-polymer-conjugates.html
Thanks
I'm guessing that you should not have a problem separating the free peptide from the conjugated peptide or the free PEG, but separating and distingushing the latter two is the main difficulty. In that case it would help to use a large excess of peptide over PEG in your reaction. That will ensure that the reaction goes to completion and you should have essentially no free PEG remaning at the end. Then use HPLC (or even size exclusion) to purify it, unreacted peptide should also be recoverable. You might need to try a couple of different types of reversed phase columns with a large pore size to see what gives you the best separation, and use a slow gradient to elute.
Precipitation might also be possible if you can find the right solvent, but you'd still need to confirm purity in the end.
As for detection, depends on how well your PEG shows up on UV. You could go by the differences in the UV absorption spectra of the polymer vs. conjugate, especially if there are aromatic side chains in your peptide. Helps if you have a PDA detector on your HPLC, but you could just look at the absorbances at two different wavelengths, one low and one high, and use the ratios to determine what is what.
Or if your PEG doesn't absorb UV, then an ELSD would be helpful. The PEG-peptide conjugate should show up on both UV and ELSD, while the unconjugated PEG would show up only on ELSD and not on UV.
Keep in mind that maleimide-thiol conjugates are not completely stable, especially in plasma. That may not necessarily be a bad thing if you want your peptide to be released from the PEG at some stage, but there are more stable alternatives out there.
Hope that helps
Zoltan
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