FabRICATOR® cleave the IgG molecule in one specific site in the lower hinge region of the anitbody leaving a F(ab’)2 fragment and a Fc-fragment.
Human IgG1: HTCPPCPAPELLG / GPSVF
Human IgG2: VECPPCPAPP_VA / GPSVF
Human IgG3: PPCPRCPAPELLG / GPSVF
Human IgG4: PHAHHAQAPEFLG / GPSVF

FabRICATOR is very specific and small changes in the sequence (..CPAPELLGGPSVF..) may reduce or completely impair the ability to digest the IgG.

We have tested a few different modified sequences and we can see that the enzyme digest the IgG to some degree to Fab2 and Fc under somewhat modified reaction conditions. We then obtained a mixture of Fc, Fab2 and single cut mAb (one of the HC still undigested).

We used PBS buffer but with greatly reduced salt concentration (5-10 mM instead of 140 mM) which appeared to promote digestion. We also added 10X more enzyme than recommended amount and prolonged incubation time.
It is very difficult to predict the outcome for a specific modification. It might work, but we cannot guarantee it unfortunately.

There should not be any problems to use low concentrations of IgG. If you don’t get complete digestion, try to increase the incubation time. There is no risk of over digestion and the incubation time can be prolonged to over night if necessary.

An alternative would be to increase the ratio enzyme to antibody (i.e. Follow protocol for mouse IgG). We have not made any specific tests on low concentrations but we have not heard that it would be any problems.

We have not tested storing the enzymen in glycerol solutions. However, many of our customers have successfully stored the enzyme (after it was reconstituted with water) at both -70°C and -20°C for a few months.

Yes, FabRICATOR can cleave Fc-Fusion proteins as long as the digestion site below the hinge region is preserved.