Protein PI 8.6 after Ni NTA affinity it is passed through Desalting column, desalting column equilibrated with pH 8 Tris eluted with tris then IEX (DEAE), IEX equilibrated with buffer pH 8 Tris eluted with Tris.
Now IEX is run in flow through mode? but IEX is an adsorbent right doesn't my protein bind to DEAE?
Literature mentions "CEX in a bind and elute mode as the first polishing step followed by AEX in a flow-through mode of operation"
Both CEX and AEX are adsorbents how one is run in bind and elute mode and the other in flow through mode for the same protein.
"mAbs usually have a basic pI and hence bind tightly to CEX resins and tend to flow-through readily on AEX resins."
If mAbs have basic PI i.e negative charge how will they bind CEX?
There are several aspects:
- Binding of a protein to an ion exchanger - either anion or cation - depends on its own ionizable groups and pH and salt concentration of your "buffer".
- You can consider the "flow through" as a first fraction of a chromatography which separates your non-binding protein from all others which bind. For this kind of purification step you have to select between cation or anion exchanger or chose intial pH/salt conditions which result in flow through on either ion exchanger.
- Independent of its pI a protein (with either a positive or negative net charge) can bind to both anion or cation exchangers as it possesses both anionic and cationic amino acids - so that certain regions of a protein containing respective amino acids are electrostatically attracted to the ion exchanger.
Best wishes, Peter
Hi there,
There are 3 parameters to take into accout when performing IEX : pI of the protein of interest, pK of the ionic function of the matrix and working pH. mAbs have a basic pI which means they are positively charged at pHpI therefore if mAbs binds to CEX then it means the working pH is below pI.
If you work at a constant pH which is different from the protein pI then this protein should bind one of the IEX matrix (AEX or CEX) and get into the flow through of the other matrix. For instance mAb will bind CEX at neutral pH and get into the flow through of an AEX at neutral pH.
But as mentioned above the pI is a theoritical value calculated on the total content in aa of the protein considered. IEX is run in native condition which means the behavior of the protein towards IEX matrix will actually depend on the exposed ionic residues only.
If we consider the same conditions (pH), then only rarely would a protein bind both to catex and anex, simply because it is either above or below its pI and thus is either positively, or negatively charged. It cannot be both.*) And thus at the identical conditions one of them will work in bind-elute, while the other in flowthrough mode.
*) there are exceptions, when protein is in general, let's say positively charged, but it has a patch of negatively charged amino acids and thus it can bind to bond ion exchangers
There is very nicely written explanation to your question in the Roadmap to process development 1, written by reknown biochromatographer Pete Gagnon:
http://www.biaseparations.com/support/roadmap-to-process-development
Take a look, it could be a beneficial reading for you.
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