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
>>>
