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FabRICATOR® Enzyme

The FabRICATOR Enzyme is supplied as a lyophilized powder with no preservatives added, and is available in 2000 and 5000 unit vials for digestion of up to 2 - 5 mg IgG.

 

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.

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Detailed 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. There is no other known substrate besides IgG. 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, and some classes of 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.

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

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.

Storage and Stability

The FabRICATOR enzyme is supplied lyophilized and reconstituted by addition of water. After reconstitution, FabRICATOR is stable for 1 month at +4-8°C. The product is shipped at ambient temperature, and should be stored at -20°C upon arrival.

Buffers

The buffers below have been tested for compatibility with FabRICATOR digestion at different pH. Note that the FabRICATOR enzyme is irreversibly inactivated below pH 5.0. Digestion above pH 8.0 requires prolonged/optimized conditions.

  • Phosphate buffer saline (PBS) 6.0 – 8.0
  • Tris buffer 7.0 – 8.0
  • MES buffer 5.5 – 6.5
  • HEPES buffer 7.0 – 8.0
  • Ammonium bicarbonate buffer 6.0 – 7.0
  • Sodium acetate buffer 6.0

Unit Definition

One unit digests ≥ 95% of 1µg human IgG when incubated in 10 mM sodium phosphate, 137 mM NaCl, 2.7mM KCl pH 7.4 at 37°C for 30min.

How to Order

For details on ordering please visit How to order. Click add to cart to buy online.

Quotations

Please do not hesitate to contact us if you need a quotation or have inquiries on price of bulk quantities.

Documents

FAQ and Support

Latest 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

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.