I am trying to achieve baseline separation for antibody charge heterogeniety using cation exchange. The PI for this antibody is about 6.5. I am looking for few column options using weak cation exchange. Any suggestions on what columns are out there would be appreciated.

I would recommend you to separatate it by chromatofocusing in Mono-P column (rather than a cation exchanger)using a polybuffer PBE 74 or PBE 96. It is quite resolutive and it can resolve pI differences of 0.02 pH units.
Check the apbiotech website at http://www.apbiotech.com

Dionex (ProPac WCX-10), PolyLC (Poly Cat A)and Tosoh all have products suitable for this application. The Dionex product was specifically developed targeting this application and is used by a number of antibody manufacturers as a QC tool for the purpose you have in mind.

I did try the ProPac WCX column. I see about 5-7 charge variants. However, I don't get baseline separation varying Buffer strengths, pH, salt conc, and types of buffers. I also learned that the ProPac column is micro porous. I guess the interaction between the stationary phase and protein is on the surface. Would it be better using columns with large pore size so that antibody can get inside?

What is chromatofocusing? I have never heard of it. What PBE74 or 96 consists of?

Chromatofocusing is a chromatographic method for separating proteins according to their isoelectric point. The stationary phase is a weak anion exchanger (polybuffer exchanger: PBE 94) that is tritated with a polybuffer (polybuffer 96 or 74, in my first comment I made a mistake writing PBE 96 and 74 instead of PB 96 and 74 ) made by a mixture of several synthetic charged compounds with different pK. The result of passing this polybuffer through the column is a pH gradient that allows a very resolutive separation according to the isoelectric point. For example: first, one equilibrates the column at pH 9; after injection and reequilibration, the polybuffer 96 is passed through the column until pH 6 is reached. Proteins with pI from 9 to 6 are eluted. You don't need to use a gradient pump. Inside the column a pH gradient is formed due to the tritation of the ion exchanger with the polybuffer. Diluting the PB you can improve the resolution.
You can find more information at apbiotech website http://www.apbiotech.com in the chromatography area.
Additionally apbiotech sells a small and practical handbook on chromatofocusing.

I was just looking at this technique from Amersham. Sounds like it is used more as preparative method for applications like purification. I am interested in developing an analytical method using only minimal amount of sample (in ug loading range). What is the typical protein load in using this technique?

Yes, it is used mainly for purification. The typical protein load is 1-10 mg protein/ml gel.
Amersham sells a small column of 1ml, I think it
can be used in the range of ug depending on the protein and detection method. The commonly used detection method is UV absortion, and the main limitation of polybuffer is its absorption at low wavelenght, for this reason the UV detection is at 280nm, with lower sensitivity. I can not predict the lowest amount of protein you can detect, you should try it, if it is technically possible, or search for more information. Chromatofocusing is resolutive and easy to use, surely somebody has used it as analytical tool.

I use the common UV dection at 280nm. I definitely will look into this technique. I already ordered the handbook. Thanks a lot for all the info you have provided.

Anon

The ProPac WCX column is actually nonporous. The terminology "microporous" is a polymer chemistry term referring to polymers without discrete separate pores. However, the material in the case of this column is better described as nonporous with pore size too small to be measured by any available technique. Given the confusion this term obviously engendered, this terminology will be removed from the next release of the column manual.

Have you tried varying the gradient slope? This approach should be the most effective for improving resolution.

Chris is correct shallow the gradient way down. Gradients as low as 1-3mM/min can yield some outstanding results.

I am aware of several researchers who have achieved baseline resolution on intact MAb's (up to 9 or so components) using the ProPac WCX-10. In those cases pH optimization was essential. Try also to run at couple of pH's close to the pI of the molecule (0.2-1.0pH unit away). Care with setting of the pH and temperature are required in this parameter envelope, however the results can be very gratifying.

Yes, I tried to shallow out the salt gradient about 7 -15 mM/min and I did see the peaks are pulled out more, but not to the extent where baseline separation had occured. Of cause, band broadening was observed when shallowing out the salt gradient. I wonder what kind of sensitivity one would get and amount of protein needs to be loaded if gradient goes as low as 1-3 mM/min.

I also tried to add ACN to mobile phases without any success.

I am also wondering if any of the work by the reseachers mentioned by you are publised. If so, would you be able to give me some references? Thanks.

Anon,
Please feel free to contact me directly about your application I would be delighted to try and help you achieve a similar separation to those achieved elsewhere.

Regretably I am not aware of any publications those researchers made describing the work they have undertaken with the ProPac columns. There was a previous publication by Santora et al. Anal. Biochem. 275, 98-108, (1999). Although, I suspect that sample was simpler than the case you are working with.