mAb purification but precipitated - (Jun/23/2009 )
Davi on Jun 27 2009, 02:49 PM said:
klinmed on Jun 26 2009, 01:53 PM said:
Davi on Jun 26 2009, 02:25 AM said:
mdfenko on Jun 25 2009, 11:13 PM said:
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.
we use phosphate buffer pH 8 to neutralize from low pH and pH 6.8 to neutralize from high pH.
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.
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
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?
Hi Davi,
The most likely explanation is that the binding to protein G (and/or the exposure to extremes of pH) slightly alters the conformation of the antibody. This could expose hydrophobic regions that can induce aggregation when the mab is at high concentrations.
This conformational change may be temporary. Thus after a period at neutral pH you may be able to concentrate your protein if you need to (for example to label it).
Hope this helps
-klinmed-
Thanks for your suggestion.
Anyway,I need to list 3 points.
1.The precipitation will be formed as long as neutralizing.
2.When the antibody concentration is low,we can not know that there are no precipitation or there are a little precipitation that we can not see?
3.The pricipiation is formed,too,when anding sodium acetate buffer(pH 4.8) to ascites while purification using caprylic acid.
-Davi-
Davi on Jun 30 2009, 11:07 AM said:
Thanks for your suggestion.
Anyway,I need to list 3 points.
1.The precipitation will be formed as long as neutralizing.
2.When the antibody concentration is low,we can not know that there are no precipitation or there are a little precipitation that we can not see?
3.The pricipiation is formed,too,when anding sodium acetate buffer(pH 4.8) to ascites while purification using caprylic acid.
Anyway,I need to list 3 points.
1.The precipitation will be formed as long as neutralizing.
2.When the antibody concentration is low,we can not know that there are no precipitation or there are a little precipitation that we can not see?
3.The pricipiation is formed,too,when anding sodium acetate buffer(pH 4.8) to ascites while purification using caprylic acid.
Well yes...
All antibody solutions contain some aggregates, especially those exposed to extremes of pH (eg. protein G purified!). You are indeed right that a clear solution does not mean your material is free of aggregates.
After protein A purification we always fractionate our monoclonals on a 2.6 x 60 cm Superdex 200 gel filtration column using an Åkta Explorer purification system.
We do this just to remove aggregates.
Usually, the gel filtration results suggest that most of our preps (after protein A) contain >95 % monomer.
-klinmed-
Hi,We resolved this problem using caprylic acid method,thank everyone.
-Davi-