Im working on an antibody that has a strange basic peak that does not come down after CpB treatment. After treatment, the peak averages at 5%. I attached pictures below. Mainly interested in the first basic peak, second peak is small enough to not be a concern.
Peptide mapping of the total product did not help elucidate what the peak is. Now Im thinking of isolating the basic peak by repeated fractionation on CEX or HIC, so that I can run peptide map on the isolated peak. Are there any clever ways to do this efficiently?
Alternatively, are there any cheap or less time intensive methods to figure out what this thing is? (already tried out the easy tests like for methionine oxidation, free thiol analysis for incomplete disulfide bonds, and N-terminal glutamine cyclization)
Hi,
That's a fun question!
My experience with trying to identify/characterize species observed in the CEX chromatogram of a monoclonal antibody consists of two strategies: either collecting the fraction of interest from an analytical run (maybe from one, or several, injection(s) of more material than you would normally inject. I guess there is no clever way of doing that, unless you have a fraction collector..): that works if you want to study the fraction by bottom-up approaches (which seems to be your plan).
Otherwise, you could collect the fraction of interest from a semi-preparative cation exchange column; this will result in more material, allowing for a multitude of subsequent analyses.
It is always good to also collect some of the main peak and include that in your analysis, to enable a differential analysis. This can help you identifying what modification is enriched in your basic species of interest.
BTW, I think both methionine oxidation and N-terminal Glu cyclization result in an acidic shift in the chromatogram. Do you have non-glycosyated mAb in your sample?
Hope this helps!
Hi Eef,
Thanks for the response! You said one of two strategies, whats the other strategy you consider?
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