Chromatography Forum

I am trying to get better resolution of a large protein using cation exchange. The molecule is a mAb scaffold attached to a peptide, and the pI ranges between 7.1 to 7.8, based on capillary electrophoresis. Right now it elutes as a very broad hump, with no discernable individual peaks. I have tried Dionex WCX and SCX, 50 mM acetate and MES buffers between pH 5-5.5, and 0-100% gradients with 1M NaCl as the salt. I have also tried adding 10% ACN, and 10mM sucrose. I get the sharpest peak at 50 deg C. Does anyone else have experience separating mAbs or have any other suggestions of what to try? I may try AEX next at about pH 10 or so.

Thanks,

Eric

If you do not need to recover the analyte, I would do RP instead of IEX. Overall better resolution...

I had very nice results for separating proteins on Dionex monolithic columns with 500 µm ID. However, the separation was performed under RP conditions.
For ion exchange, I would recommend the OligoWAX from Phenomenex and the use of "classic" phosphate buffers with pH around 6.
Success

I apologize for the untimeliness of this reply, but I have just started to participate in this forum. Our new controlled pH gradient technology gives very significantly improved results for MAb separation on both cationic and anionic stationary phases when compared with salt. We have published a detailed theoretical and experimental description of the method, Journal of Chromatography A, 1200 (2008) 166–182, and some MAb data in a paper last October in American Biotechnology Laboratory,Application of Well-Controlled pH Gradients at Variable Isocratic Salt Concentrations to IEX Chromatography. A short paper exclusively about MAb separation will appear shortly in the January, 2009 issue of American Biotechnology Laboratory. The classic "wisdom" about pH gradients is that they are confined to short ranges and, because the proteins are thought to elute from the columns very near their pI, aggregation problems are a common difficulty. The truth is much more interesting and encouraging than this picture. In fact, if you can run wide range pH gradients on both cationic and anionic stationary phases then you find that on anionic columns at typical gradient slopes, e.g. 0.1 pH units/column volume, the proteins almost always come out at least 0.5 to 1.0 pH units below their electrophoretic pI, and conversely, on cationic resins they usually emerge at least 0.5 to 1.0 pH units above their pI. As the gradient is steepened the difference between the elution pH and the “trueâ€

Below is a reversed phase application for IgG2 mAb on Intrada WP-RP:
http://www.imtakt.com/TecInfo/TI430E.pdf

Perhaps conditions similar to this is a good starting point for
reversed phase analysis.