Chromatography Forum

I have no experience with this sort of analysis(small molecule and synthetic polymer experience only) so I need beginner advice..............

I have been charged with developing an analysis to determine "purity" of antibody preparation (likely sheep) from serum. Presumably the preparation is mostly antibody protein and there may be other stuff present. So, ideally a class separation of antibody protein from other potential stuff is needed. It has been suggested that a separation on a size exclusion column would do the trick and that certainly sounds reasonable, but no further details were provided.
1. Is this the best approach?
2. Recommended column?
3. Mobile phase?

Pointing me to an easily found reference would be fine. I could do a lit search ,but I would find 1000 references and have no clue as to which was most practical. Hence, some advice based on experience would be most welcome.

Depends what else is in the preparation.

If the antibody is the only protein, then a simple colorimetric assay (with something like ninhydrin?) would be quick and easy.

If the antibody is the only protein, but there are smaller interferences (e.g., amino acids), then a quick and dirty SEC separation would do the trick quite nicely (biochemists don't call it "size exclusion", though, they call it "gel filtration" -- gotta know the lingo! ).

If there are other proteins present, things get trickier (you have to know the MW of the antibody and of the interferences), and if there is a lot of other protein material present, you may have problems picking your antibody out of the weeds by SEC.

If you know the antigen, then affinity chromatography is the way to go. Basically, you hang the antigen on the support and let it suck the antibody out of the sample, then bump off the antibody to quantitate. A biochemist friend of mine used to joke that it was great because it ". . . only requires one theoretical plate . . . but it has to be the right one!". He was only partially joking. The specificity can be good enough that you can do the analysis with a beaker and a vacuum funnel. Affinity media (and information about how to use them) are available from places like Tosoh, Sigma-Aldrich, and Bio-Rad.

Finally, hydroxyapatite has been used for years to separate immunoglobulins from other proteins. Basically gradient ion-exchange, but the material is a crystal that has both Ca++ and PO4--- - rich faces which give it a unique selectivity. Check the same vendors (e.g., BioRad).

Well the SEC was suggested because it is relatively easy: As mentioned before, I have had very good results using a Tosoh TSK-Gel Super SW 3000 SEC column for a nearly baseline separation of Mab (150 000 MW) from its Fab (100 000), keeping it in nearly natural environment of PBS as diluent and mobile phase. Besides the stuff mentioned by Tom: When one wants to get much better resolution (if you are a bit lucky) one needs gradient RP, also, and I tend to forget this as I have no experience here, ion exchange does some surprising seps. An example of the latter is given by Western Analytical Products, Inc. (and other vendors): A supposedly highly pure Mab could be resolved into at least three major peaks and several minor ones. So even Mab are not a single protein. (Used were: Column, 204CT0510, 4.6x200mm, 5µ, 1000A a PolyCAT A by PolyLC Inc.; mobile phase A: 9mM NaPhos, pH 6.2, 10% ACN, B: same as A + 0.18M NaCl, 0-100% over 70 min).

This assumes that the supplier of the antibody already used Protein L or other affinity chrom, + ion exchange etc. in purifying the stulff. Also, Pierce has a lot of products which may help.
(Bill, if you get stuck, drop a line, the blind help the lame, or so??)