Chimeric antibody Pepsin digestion - (Jul/30/2009 )
Hello to all.
We want to digest a chimeric antibody (IgG1) with pepsin and then reduce with mercaptoethanol to get Fab' fragments. These fragments will be conjugated to some other molecules for drug gelivery purposes through S-S bonds. My PI suggested buying a cheap IgG1 (such as isotype control IgGs) and starting my experiments with that instead of expensive chimeric antibody.
My question is, do you think it is a good idea or not.. Can any IgG be used to optimize digestion conditions?
My other questions, if anyone can comment a little are : I've read that papain digestion could give Fab' fragments directly, and still trying to figure out what products we'll have in hand after pepsin digestion.- Definitely Fc (which I don't know how to get rid of) and Fab(2) fragment, to reduce the disulfide bonds..We don't have any affinity purification column and can not afford any kits from Pierce unfortunately. So will Pepsin digestion followed by reduction give Fab' fragments with free S- groups? Can it be that easy or am I missing major steps of reaction (which may not be written in research papers of course 
)
Probably I will be asking more questions once I start the experiments..
Best wishes to all. 
asli on Jul 30 2009, 04:45 PM said:
We want to digest a chimeric antibody (IgG1) with pepsin and then reduce with mercaptoethanol to get Fab' fragments. These fragments will be conjugated to some other molecules for drug gelivery purposes through S-S bonds. My PI suggested buying a cheap IgG1 (such as isotype control IgGs) and starting my experiments with that instead of expensive chimeric antibody.
My question is, do you think it is a good idea or not.. Can any IgG be used to optimize digestion conditions?
My other questions, if anyone can comment a little are : I've read that papain digestion could give Fab' fragments directly, and still trying to figure out what products we'll have in hand after pepsin digestion.- Definitely Fc (which I don't know how to get rid of) and Fab(2) fragment, to reduce the disulfide bonds..We don't have any affinity purification column and can not afford any kits from Pierce unfortunately. So will Pepsin digestion followed by reduction give Fab' fragments with free S- groups? Can it be that easy or am I missing major steps of reaction (which may not be written in research papers of course
)Probably I will be asking more questions once I start the experiments..
Best wishes to all.
I am sorry to be so negative, but if your lab has no experience in making antibody fragments you are in for a very, very, very........ rough time.
As a minimum you will need experience (and equipment) in affinity chromatography and PAGE analysis. You will, almost certainly, also need to be able to run anion ion-exchange chromatography (to purify the Fab´from other degradation products) . Do you have these methods up and running in the lab?
Would strongly advise you get some hands-on training in another lab that routinely make fragments.
Different monoclonal antibodies require different reaction conditions, enzyme/antibody ratios, incubation times/temps etc. These must be optimalized empirically for each monoclonal even with the same isotype. Thus, preliminary trials with a polyclonal anti-Ig reagent will probably not give you much info on how the chimeric Ab will behave. Polyclonals are also much easier to produce fragments of.
By the way, Fc is usually removed by protein A chromatography (collect flow through).
If you try this by yourself, good luck, and you have my sympathy.