Obtention of F(ab)2 fraction - (Apr/10/2008 )
May anybody please describe a protocol of obtenting, by pepsin digestion, the F(ab)2 fraction of a mAb preparation, including the subsequent purification method [since this F(ab)2 fraction will be administered to animals]? The amounts of mAb will be within the range of mg, and we have a FPLC and are experienced in using it for purification purposes.
Thanks in advance
First of all, you should do a screening procedure, to obtain optimal (or at least satisfying) digestion condition. Try digestion at different pH values, using a suitable buffer (I used a simple acetate buffer). Start with pH 5 and go lower, down to whatever your antibody can stand). Take care that you never put pepsin in a neutral buffer, as it will lose its activity. Also test different times of digestion (up to 24h) and possible different antibody/pepsin ratios (most protocols state 1:100, I used 1:20 because my antibody was quite resistant to digestion). Reaction is usually run at 37°C.
Analyze all fractions with SDS-PAGE and coomassie or silver staining. Western blotting and detecting with anti-Fab antibody is also a good idea. This should tell you which conditions give you best F(ab)2 yield. Be prepared that you might also see some undesired fragments of varying sizes, as pepsin is really not very specific. You should also check that digestion didn't damage your antibody too much by testing its activity in whatever assay you normally use.
Once you establish all this, you can upscale the digestion using the determined pH, time and enzyme concentration. There are no limits here, just use whatever amount is sensible to you.
Purification with FPLC shouldn't be a problem, you have several options: a. affinity purification using protein A (binds undigested antibody and possible Fc region), F(ab)2 flows through; b. SEC: just collect the fraction containing the protein of the correct size; c. IEX: F(ab)2 probably has different pI from all other fragments created by the digestion, so you can use this to separate them.
From personal experience, I would prefer b. or c.
Hope this helps,