Hi Demetrios.

Is the Protein A column new or it has been used for long time before you used it? The efficiency of binding is affected by the integrity of the column (strong use with acid solution as Glycine for elution and then treatment with NaOH or other solution for the cleaning, dramatically affect the binding capacity).

If this is not your problem, have a look to this paper:

Protein Eng. 2003 Apr;16(4):243-5.

Single amino acid substitution in the mouse IgG1 Fc region induces drastic enhancement of the affinity to protein A.

Nagaoka M, Akaike T.

Good luck,

Barbara

Thanks Barbara,

The column was poured from fresh gel, but it is possible that the gel we are using is not good... I will read the article, which will be helpful. We are going to try with a different vendor of protein A gel, and also try a protein G gel as well. There may be interactions between this protein and the Fc tag. Have you ever tried to partially denature the protein during purification (with 1-2M Urea for example)? I am thinking about giving this a try as well.

Hi Demetrios,

As Barbara mentioned , storage / Contact with alkali (NaOH), acid and high temp. directly affect protein A ligand activity as well as resin capacity.

And also Load and equlibration condition also matters well.

1. Normally PBS is used pH 7-8 for equilibration condition

2. Filter your sample with 0.2 micron filer and also adjust the same as equilibration buffer by tris or phosphate or bycarbonate.

3. Give enough contact time to bind your protein to resin ( 6min or more).

Hi,

I never tried a mild denaturation of the protein.

If I can suggest you a Protein A column, I warmly reccomend this one:

http://www.biaseparations.com/pr/849/cim-r-protein-a-8f-ml-tube-monolithic-column

Is a different concept of column based on convective interaction media. In my experience I tried many many brands of columns, of more or less all the chromatographic techniques. Try this one, is one of the best that you can try.

Hope you will solve your problem...and happy new year :)

Barbara

You can try many things like pH washes , salt washes and other additives also. there are many publication available for this.

Urea is also used as fertilizer so if you are developing for commertial use then you need to show removal of urea from your end product. You can use this after post load wash for removing loosely associated varients or impurities.

best luck.

Barbara ,

can you please share here how CIM discs are better than traditional chromatography beads media ? Cost , time , quality and productivity ...

Dear Yamak,

disks are great, but I suggest to use the "column" format (if I'm not wrong they start from 1ml CV). The "problem" of the disks is that they are so efficient that are difficult to reproduce in big scale: to give you an example, the ion exchange CIM disks have the same (or even better) resolution of the Mono Q/S columns of GE Healthcare.

Regarding the time: the CIM columns allow you to work at so high speed and have a so high pressure tolerance that you can't imagine....usually the time limiting factor is the principle....in affinity you have to slow down to allow the maximum binding! But trust me: the runs are so efficient and fast (similar to HPLC columns).

Productivity: if you mean the binding capacity the CIM columns don't have problems if compared with classical columns, but as you know, a lot depends on the protein characteristics, folding, binding site exposure and experimental conditions....that have to be optimized as usual.

Costs: I don't know exactly the costs...we have just bought a Protein A CIM column (1 ml)....if I remember well it is a little bit more expensive than GE ones, but the CIP procedure I can perform on it makes me much more confident on re-using with other Ab.

Hope I've been clear and give you the answers you needed.

Kind regards,

Barbara

Hi Demetrios,

I have the same experience with poor binding of Fc tagged proteins to protein A columns as yours. I tried both protein A and protein G, but either has a good binding. Now I am thinking to partially denature the protein with Urea. Have you resolved this problem? If yes, what was your resolution? And for urea, do you have a protocol about when and how to add urea to the fusion protein? Thank you very much!

Hi Xia,

Yes, it turned out the construct we were using had a tag following the Fc domain. This tag folded back onto the Fc and somehow blocked binding to the protein A column.

We moved the tag to the N-terminus (away from C-terminal Fc domain) and it bound fine.

Is your Fc domain a mouse IgG1? If so, it binds weakly to protein A. I can help you with that if you need.

Good luck!

D-

Hi Demetrios,

My Fc domain is a mouse IgG2a domain. It really binds weakly to protein A and protein G. I use the new rProtein A fastflow sepharose from GE health,  I collects each fraction of every purification step. Every time there are nearly 20% protein which can bind to the beads. I tried many different methods, and I added iodoacetamide and DTT to the condition media to reduce disulfide bond. But still no help to the binding. Do you have any idea about it? Thank you!

Hi Xia,

Ugh... So, there is a binding buffer sold by BioRad that is suppose to help. I do not know exactly what the composition is, but it is composed of high salt, and high pH. I think something like 1.5 M glycine , pH=9.0, 3.0 M NaCl (exact composition is a secret I guess) . It is called MAPS II binding buffer. This is suppose to encourage binding of the mouse IgG to protein A. You may want to try this buffer and see if it increases binding. If it does, let me know - I would be interested.

On the plus side, you don't need to elute at as low pH (probably around 5.5) so that your protein will not be disrupted if it is sensitive to the low pH buffers.

The binding of our mouse IgG1 to protein A is so poor that we are abandoning and moving into ion exchange to purify.

Good luck, and let me know if that helps.

D-

Hi Demetrios,

I will try what you said. I will let you know if it helps. 

Thank you very much!