High and low ionic force
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
> Thanks a lot.
> monoclon at infomed.sld.cu
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