mAb purification but precipitated - (Jun/23/2009 )

I producted an monoclonal antibody(Mouse IgG2b),I have terrible trouble when purifying it using protein G(GE healthcare).The antibody was always deposite,regardless the washing Ph(2.7 or more than 10).I tried many times as different washing buffer and neutralization buffer,but the result is bad luck.How can I purify the antibody?

are you saying that it binds too tightly to the protein g and you can't elute it?

are you sure it bound and didn't come off in the flow-through and wash?

have you tried protein a? the specificity is different than protein g and may not bind your mab as tightly as protein g.

Davi on Jun 23 2009, 11:23 AM said:

IgG2b binds tightly to protein G and you need a very low pH (2.7) to elute. Some monoclonals will not put up with this treatment. They bind tightly to protein A but can usually be eluted between pH 5 & 4.

I would suggest that you equilibrate a protein A column with 50 mM sodium phosphate buffer (pH 8.0). Add 1/10 of a volume of 500 mM sodium phosphate (pH 8.0) to your sample and pass through the column. Wash with 50 mM sodium phosphate, and elute first with 0.1 M sodium citrate pH 5.0, then citrate pH 4.0 then citrate 3.5. Collect each peak and adjust to pH 6.5 - 7.5 as quickly as possible.

Hope this helps

mdfenko on Jun 23 2009, 10:12 PM said:

Firstly,Thank mdfenko for fiving me the suggestion.I can elute it as the company protocol,but when adding neutralization buffer,the antibody is precipitated.In addtion,I tried Protein A,too.The result is the same.Any other suggestion?Thanks a lot.

klinmed on Jun 24 2009, 01:22 AM said:

Thank klinmed,I will try as your guidance.

Thank all of you.Any way,binding is well and it can be washed up,too.But there are always some precipitated proteins in the eluting solution,including aimed antibody.

what are you using to neutralize?

we use phosphate buffer pH 8 to neutralize from low pH and pH 6.8 to neutralize from high pH.

mdfenko on Jun 25 2009, 11:13 PM said:

Buffer we used with Protein G:
Bingding buffer:20mM sodium phosphated,pH7.0
Elution buffer:0.1 M glycine-HCl,pH 2.7.
Neutalization buffer:1M Tris-HCl,pH 9.0
Thank you very much.

Davi on Jun 26 2009, 02:25 AM said:

Hi again!
Just a thought.
In the past I was purifying a monoclonal IgG2a that precipitated at a concentration of > 1.5 mg/ml (approx).
Quite often the peak fractions from protein A columns can be >2 - 4 mg/ml.
The question is whether your antibody is precipitating because it is eluting at a concentration above it´s solubility limit. In this regard, it is important to remember that, unlike polyclonals, individual monoclonals can behave very differently during purification.

You could try loading less material on your column and collecting fraction in tubes that contain a few ml of 0.1 M tris (8.0). This will give a lower protein concentration and immediate buffering.

Alternatively, you could try removing the protein A beads from the column after the washing step and eluting in a batch mode (by incubating with elution buffer in a tube). This will lower the peak concentration during elution.

Hope this helps

klinmed on Jun 26 2009, 01:53 PM said:

Ok,thank Klinmed again.you are right,the precipitate is disappeared when decreasing the concentration.But tha concentration of aimed antibody is more lower than ascites,is the solubility changed more?