FabRICATOR® Xtra – Below Hinge Digestion of Mutated IgG

FabRICATOR Xtra LALA is an enzyme that digests hinge-mutated antibodies, including those containing the LALA mutation.

The enzyme digests below the hinge, generating F(ab’)2 and Fc fragments, which can be reduced into Fd’, LC and scFc fragments. FabRICATOR Xtra LALA digests a number of hinge-mutated antibody formats at two potential sites below the hinge (between residues 235 and 236 or between residues 236 and 237; Table 1).

Table 1. Recommended Reaction Conditions and Digestion Sites for Different Hinge-mutated IgG Formats

Antibody	Enzyme Amount	Reaction Time	Digestion Site(s)
hIgG1-LALA (L234A, L235A)	1 unit / μg IgG	1 h	CPAPEAAG / GPSVF (primary), CPAPEAA / GGPSVF (secondary)
hIgG4-FALA (F234A, L235A)	1 unit / μg IgG	1 h	CPAPEAA / GGPSVF (primary), CPAPEAAG / GPSVF (secondary)
hIgG1-LAGA (L235A, G237A)	2 units / μg IgG	2 h	CPAPELA / GAPSVF (primary), CPAPELAG / APSVF (secondary)
hIgG1-FES (L234F, L235E, P331S)	3 units / μg IgG	O/N	CPAPEFEG / GPSVF

The enzyme is active at pH 7.0-9.0 and at a salt concentration up to 200 mM. No additional cofactors are required for digestion, however, adding a reducing agent (e.g. up to 20 mM DTT) will slightly increase enzymatic activity. The incubation time required for digestion using FabRICATOR Xtra LALA varies depending on the specific hinge mutations present in the antibody (Table 1).

FabRICATOR Xtra LALA is derived from Streptococcus castoreus and expressed in E. coli. The enzyme contains a His-tag and has a molecular weight of 41 kDa.