High and low ionic force

Dave Ring Dave_Ring at cc.chiron.com
Wed Sep 21 17:49:39 EST 1994


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

>  
>  
>    Does anybody know any practical experience in the purification of 
>  
>    monoclonal antibody by protein-A chromatography using  buffer 
>  
>    systems of high and low ionic force?
>  
>    
>  
>    a-Which one is better?
>  
>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.    
>  
>    b-Can I obtain monoclonal antibodies with diferent caracteristics 
>  
>    like isoelectric point and estability if I use the same monoclonal 
>  
>    with diferent system of purification like the already mentioned ?
>  
>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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