13 replies to this topic

[Davi]

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?

Edited by Davi, 23 June 2009 - 01:24 AM.

[mdfenko]

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.

[klinmed]

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

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?

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

[Davi]

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

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.

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.

[Davi]

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

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

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?

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

Thank klinmed,I will try as your guidance.

[Davi]

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.

Edited by Davi, 24 June 2009 - 12:29 AM.

[mdfenko]

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.

[Davi]

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.

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.

[klinmed]

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.

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

[Davi]

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.

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?

[klinmed]

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.

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

[Davi]

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.

[klinmed]

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.

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.

[Davi]

Hi,We resolved this problem using caprylic acid method,thank everyone.