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FabRICATOR® IdeS Antibody Cleavage

FabRICATOR® (IdeS)

FabRICATOR (IdeS) is a cysteine protease that digests antibodies at a specific site below the hinge, generating a homogenous pool of F(ab')2 and Fc/2 fragments after 30 min incubation.

 

FabRICATOR is available in seven different product formats:

  • FabRICATOR Enzyme - lyophilized enzyme
  • FragIT - immobilized on agarose beads
  • FragIT kit -  immobilized enzyme and affinity purification resin for generation and purification of F(ab')2 and Fc/2 fragments from IgG
  • FabRICATOR LE - lyophilized enzyme with low levels of endotoxins
  • FabRICATOR Validation kit - consists of three batches of the enzyme to simplify method validation
  • FabRICATOR-HPLC - immobilized enzyme in an HPLC column for fast online digestion of monoclonal antibodies
  • FabRICATOR MagIC - immobilized enzyme on magnetic agarose beads for fast parallel digestion of multiple antibodies

Also, a method to detect the FabRICATOR Enzyme is available:

  • Anti-FabRICATOR - a goat polyclonal antibody directed towards the FabRICATOR enzyme

Product Information

FabRICATOR (IdeS) is a unique enzyme that digests IgG at a specific site below the hinge region, generating a homogenous pool of F(ab')2 and Fc/2 fragments. No other substrate besides IgG is known. The IgG is digested within 30 minutes, and there is no risk of overdigestion if the incubation time is prolonged. Since FabRICATOR digests IgG under physiological reaction conditions, the immunoreactivity is preserved. The best activity is obtained at 37°C, but digestion can also be performed at room temperature using a prolonged reaction time.

FabRICATOR digests all subclasses of human, monkey, rabbit and sheep IgG. It has limited activity on mouse IgG2a and IgG3 - for digestion of these antibody species, we recommend using FabRICATOR Z.

FabRICATOR is cloned from Streptococcus pyogenes and expressed in E. coli. The enzyme contains a His-tag and has a molecular weight of 37 725 Da. FabRICATOR is a recombinantly modified form of IdeS that has been developed by Genovis AB for biotech applications requiring the highest standards and consistency.

Key Characteristics

  • Human IgG1-4, IgG of some classes from monkey, rat, rabbit and sheep
  • 30 min reaction
  • No need for reducing agents or co-factors
  • CPAPELLG / GPSVF (below the hinge)

Applications

Product Formats

FabRICATOR® Enzyme

FabRICATOR enzyme supplied as a lyophilized powder. The enzyme is available in 8 x 100 units, 96 x 100 units, 2000 units, 5000 units and 5 x 5000 units (1 unit digests 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 batches of the enzyme to simplify method validation. A certificate of analysis is included with each kit.

FragIT - Immobilized FabRICATOR®

FragIT is FabRICATOR enzyme immobilized on agarose beads, allowing for antibody subunit generation without contaminating the final preparation with FabRICATOR enzyme.

FragIT kit

FragIT Kit contains FragIT resin for antibody digestion and an affinity resin for purification of F(ab')2 and isolation of Fc fragments.

Anti-FabRICATOR™ Antibody

Anti-FabRICATOR is a goat polyclonal antibody directed towards the FabRICATOR enzyme.

FabRICATOR®-HPLC

FabRICATOR-HPLC is an immobilized enzyme in an HPLC column for fast online digestion of monoclonal antibodies.

  • A0-FR1-250

    FabRICATOR, 5×5000 units

    A0-FR1-2503,395.00Add to cart

The FabRICATOR Movie

 

FabRICATOR workflow

Middle down work flow using FabRICATOR enzyme and subsequent reduction. Sample preparation time approximately 2 hours.

Fabricator IdeS workflow

 

Download FabRICATOR posters

Brochures and Posters

Download Genovis brochures and posters with the latest details on the SmartEnzymes

SmartStories

FabRICATOR & GingisKHAN Assist in-source decay FT-ICR Mass Spectrometry analysis

FabRICATOR IdeS Applications

Applications

Learn more about the applications of Genovis SmartEnzymes

Download FabRICATOR Poster, Appnote and External References

Download FabRICATOR poster

ASMS 2013

"Optimizing the Enzymatic Subunit Generation with IdeS for High Throughput Structure Verification of Therapeutic Antibodies by Middle-Down Mass Spectrometry"

Download FabRICATOR poster

FabRICATOR Appnote

"Analyze the Fc glycosylation profile of a therapeutic antibody by using FabRICATOR digestion in combination with LC-MS."

 

FabRICATOR IdeS references

References

Find external articles and papers using FabRICATOR

 

 

 

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 as long as the digestion site below the hinge region is preserved.

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

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Legal and Disclaimers

All rights reserved. Genovis products are covered by one or more patents, pending patent applications, trademarks and/or copyrights owned or controlled by Genovis AB.

For more information about commercial rights, please contact Genovis team at info@genovis.com.

Genovis products are intended for research use only. They are not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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