FabRICATOR®

FabRICATOR® Antibody Cleavage

FabRICATOR® (IdeS)

FabRICATOR (IdeS) is a unique enzyme that specifically digests the hinge region of IgG, producing a F(ab’)2 fragment and two Fc fragments. FabRICATOR is a recombinantly modified form of IdeS from Streptocoocus pyogenes that has been developed by Genovis AB for biotech applications requiring highest standard and consistency.

The enzyme is highly specific and no other substrate besides IgG is known. Digestion of IgG with FabRICATOR rapidly generates a homogenous pool of F(ab’)2 and Fc fragments and there is no over-digestion or further degradation of the fragments typically associated with other proteolytic enzymes.

Digestion of IgG using FabRICATOR, followed by reduction produces three 25 kDa-sized fragments (Fd’, Fc and LC) that easily can be analyzed using LC/MS instrumentation. The high accuracy mass profile enables detection of antibody micro-variants such as glycosylation, C-terminal lysine clipping, oxidation and deamidation among others.

Key Characteristics

  • Precise - one cleavage site and no over-digestion. Homogenous pool of fragments
  • Works right out of the box on human IgG - no optimization needed
  • Excellent yields - close to 100%
  • Robust - works every time
  • Rapid digestion of IgG in 30 min

FabRICATOR cleaves all human, rabbit, sheep and monkey IgG subclasses at one position just below the hinge region in the CH2 domain. This generates F(ab’)2 fragments without further degradation or over-digestion commonly associated with other proteolytic enzymes like pepsin or papain.

FabRICATOR® Digestion Site

Human IgG1, IgG3, IgG4:
..CPAPELLG / GPSVF..

Human IgG2 
..CPAPPVA / GPSVF..

For more species please visit Applications

Product Formats

FabRICATOR® Enzyme

FabRICATOR enzyme supplied as lyophilized powder. The enzyme is available in formats from 8x100 units to 5x5000 units (1 unit digest 1 ug IgG).

FabRICATOR® LE

FabRICATOR enzyme lyophilized for endotoxin sensitive assays. This product formulation has extremely low levels of endotoxins per vial (<0.04 EU /vial for the 2000 Unit vials). Available in 2000 and 5000 unit vials.

FabRICATOR® Validation Kit

The FabRICATOR Validation Kit contains three lots of enzyme to make method validation easier. A certificate of analysis is included with each kit.

Immobilized FabRICATOR® - FragIT and FragIT Kit

FragIT is FabRICATOR enzyme immobilized on agarose allowing for antibody subunit fragmentation without contaminating the final preparation with FabRICATOR enzyme. FragIT Kit contains an additional product for purification of F(ab')2 and isolation of Fc fragments.

Anti-FabRICATOR™ Antibody

Anti-FabRICATOR is an antibody raised in goat directed at the FabRICATOR enzyme

Applications

FabRICATOR® in QC applications

FabRICATOR was described in a recent paper by Merck Research Laboratories for domain characterization of therapeutic antibody products. Here you can read more and download the complementary full paper.

mAb Quality in Cell Culture

Due to the extraordinary specificity and robustness of FabRICATOR and IgGZERO they are becoming a vital tool for the ever-increasing demand of analytical characterization of large complex biomolecules.

Find all applications here

Enzyme Reference Tool

FabRICATOR is used frequently for studying important quality attributes of antibody-based biotherapeutics. To help you find the reference of interest we developed an Enzyme Reference Tool that organizes all papers published using our products. With this tool you can filter by quality attribute, enzyme used, type of antibody molecule and technology.

Genovis Enzyme Reference Tool

Latest reference using this product

FAQ

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.

Yes, FabRICATOR can cleave Fc-Fusion proteins. For example Enbrel is effectivelty cleaved by FabRICATOR into Fc and TNFR. Enbrel (etanercept) is a fusion protein composed of the Fc domain of hIgG1 and the p75 tumor necrosis factor receptor (TNFR).

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.

Find all FAQ here

Patent and Disclaimer


FabRICATOR®

Genovis AB hold a license for exclusive worldwide use of biotechnical industrial applications of IdeS covered by any patents derived from International publication WO03051914, including granted US Patent No US7666582.
Other aspects of FabRICATOR technology are encompassed by pending patent applications in the name of Genovis AB.

The trademark FabRICATOR is the property of Genovis AB.


For research use only. Not intended for any animal or human therapeutic diagnostic use.