High and low ionic force

[Dave Ring]

In article <9409140009.AA12443 at infomed>, monoclon%infomed at GN.APC.ORG (Ctro
Inmunologia Molecular) wrote:

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>    Does anybody know any practical experience in the purification of 
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>    monoclonal antibody by protein-A chromatography using  buffer 
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>    systems of high and low ionic force?
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>    
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>    a-Which one is better?
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>From past experience with a lot of mouse monoclonal antibodies, you may
have to experiment with conditions for each.  Don't put too much faith in
dogma about which subclasses will bind or not bind.  I found that most
murine G2a, G2b and G3 bound, less than half G1 bound, and (against dogma)
quite a few IgM bound.  Some of the commercial protein A systems that come
with proprietary buffers will probably force more monoclonals to bind, but
our experience has been that protein G works more often.  We use GammaBind
from Pierce, and Zymed has a new agent called KappaLock that is supposed
to bind all mouse antibodies with kappa light chains.    
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>    b-Can I obtain monoclonal antibodies with diferent caracteristics 
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>    like isoelectric point and estability if I use the same monoclonal 
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>    with diferent system of purification like the already mentioned ?
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>I would not use protein A or related affinity systems to separate MABs on
isoelectric point.  Mono S or S sepharose with a pH gradient would
probably be the best bet.  This tends to split MABs into a mess of peaks
which represent alternate glycoforms with different numbers of sialic acid
residues.  I'm not sure how one would separate MABs according to
stability.
  
> Thanks a lot.
>  
> Lobvi
> monoclon at infomed.sld.cu
> CIM
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