how to purify Fc fragment - Fc fragment purification after papain digestion (Jun/30/2011 )
Hi all. I am trying to purify the Fc fragment of a therapeutic human antibody after papain digestion. Protein A purification removed the Fab fragment, now I am left with a mix of the Fc fragment and whole antibodies. Does anyone have any experience with this. A while ago I had some luck when I just passed the sample through a 100 kDa MWCO membrane and was left with the Fc fragment, however this time around I want to do a large scale prep and it just doesn't work. Any ideas?
I'd recommend the ProteaPrep IgG Capture Sample Prep Kit, because it works in a way that is slightly different than a typical IgG depletion kit. It uses a reversible binding recombinant ligand (not Ab based) with an affinity for the Fc region of IgG. From the testing I have done with this kit it is capable of binding >99% of a 250 µg IgG sample. The other aspect of this product that would be beneficial for your particular application is that the binding is reversible and you should be able to recover >80% of your bound Ab. The one potential downside that I can see from what you have mentioned is the scale of the depletion. From my experiments with this product you can expect >99% depletion of IgG from about 10 µL (I estimate that might be about 250 µg of IgG, maybe less) of serum. You could probably expect it to capture IgG with roughly the same efficiency ( at least >95%) up to about 15 µL of human serum, beyond that point you start to see a fairly drastic decrease in efficiency. So my concern would be if this falls within the scale of the experiment you are performing.
Although I appreciate the input, I really need something completely different. I want to purify the Fc fragment from the whole antibody
So, your goal is to digest the Ab with papain, producing Fab and FC, then selectively capture FC and wash away the Fab?
Looking back at what I posted previously...
The IgG capture would retain anything in the sample that had an Fc region. So, I guess if the papain isn't 100% efficient at digesting your Ab then it would bind whatever amount of intact IgG still remains, as well as the Fc fragments.
Have you tried an ion exchange or RP based method of separation?
The papain digestion is never 100% efficient and if you overdigest your Abs you get a lot of small Fc fragments which was not what we wanted. So now I have a mixture of the Fc fragment and the undigested starting Abs from which I want to purify the Fc fragment. We have tried anion exchange but everything eluted more or less in one peak, we have also tried SEC but the peaks overlap. I am now thinking of preparing my own Fab-specific affinity column to deplete the whole Abs (which still containt the Fab fragment) and be left with the Fc fragment although I have never done this before and have no idea of what to expect.
I was re-reading this topic and was curious what you worked out.
BioMiha on Sat Jul 2 09:51:29 2011 said:
I am now thinking of preparing my own Fab-specific affinity column to deplete the whole Abs (which still containt the Fab fragment) and be left with the Fc fragment although I have never done this before and have no idea of what to expect.
you could use an antigen column. bind antigen to the gel and pass your mixture through. whole antibody and fab should bind with fc passing through.
We have decided to prepare an anti-Fab column. The antigen is quite expensive as it is a cytokine and it can be used to purify only this type of antibody. We'll see how the anti-Fab column works out.
I usually purify either the Fab or the entire Ab but...here is some advice and I would actually be interested if your anti-Fab column worked.
1) Be sure to work in reducing conditions as disulfide bridges keep the Fc molecules bound together. Therefore, you might have a mix of partially digested dimers (IgG) or pentamers (IgM) after papain digestion.
For that, use 25mM freshly prepared cysteine solubilised in the buffer of interest.
2) As you previously described, Amicon columns with several MWCO do exist in 50mL Falcon format for volume up to 10 mL. If your sample is bigger, you can fill the tube several times. Recovery yield is 70-80%.
3) If you really work on an semi-industrial scale, concentrate your sample in dialysis tube and perform a size exclusion column.