FabRICATOR LE (Low Endotoxin) digests IgG at a single site below the hinge



FabRICATOR LE (IdeS) is an IgG-specific cysteine protease that digests antibodies or Fc-fusion proteins at one single amino acid position below the hinge region generating Fc and F(ab’)2 fragments. FabRICATOR LE contains low endotoxin levels <0.02 EU/vial and is lyophilized for digestion of 2 – 5 mg of antibodies or Fc-fusion proteins.

Detailed Information

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Reaction Conditions

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Yes, for human IgG1 molecules both enzymes can be used in combination to generate a specific hinge peptide. The incubation time is significantly longer for FabALACTICA and it is recommended to first run the FabALACTICA digest and then add FabRICATOR for the last 30 min.

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

We have tested concentrations as low 0.025 mg/ml without any measurable effect on digestion efficiency. However, low concentration samples require a longer injection time and consequently lead to a broader elution peak from the FabRICATOR-HPLC column.

We can only guarantee optimal digestion for one time use. However, depending on antibody the columns can be reused but we do not have a proper sanitization protocol. We don’t recommend that you run different antibodies on the same column as we cannot guarantee that there will be no carry-over. If desired to reuse the column store in 10-20% ethanol at +4-8°C.

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, it is possible to digest the antibody using an automation system. See more in the applications page.

Yes, it is possible to digest the antibody in the first dimension and then separate the subunit in the second dimension. See application note

To use protein A or MabSelect Sure (GE) for removal of Fc as a platform after digestion with FabRICATOR or FragIT is problematic. Therefore we have chosen CaptureSelect in the FragIT Kit for removal of Fc fragments. CaptureSelect is much more specific for Fc. Both protein A and MabSelect Sure (GE) may have affinity for Fab and the degree of affinity is antibody dependent.We have many customer reports on this subject and they usually comes back to us for the CaptureSelect columns for the above reason.

Yes, FabRICATOR works on hamster IgG using the standard protocol as for human IgG.

FabRICATOR belongs to the cysteine protease family. There should be no modification other than the cleavage of the peptide bond between the two glycines. The c-terminal will regain the carboy group – that is addition of one OH-group and the N-terminal amino group will be regained (addition of one H).

In summary, R1-CO-NH-R2 + H2O –> R1-COOH + NH2-R2

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.

According to the reference listed below, FabRICATOR does not digest IgM.

Pavel-Rammingen et al., 2002. IdeS, a novel streptococcal cysteine proteinase with uniqe specificity for immunoglobulin G.

You can indeed elute the Fc fragments from the CaptureSelect column using a low pH buffer commonly used for example during protein A/G purification of antibodies. We have successfully used 0.1 M glycine buffer pH 2.6-2.8 to release the Fc fragments. Be sure to adjust pH back to normal using a 1 M Tris-HCl buffer or similar directly after isolation of the Fc fragments from the CaptureSelect column.

The FabRICATOR-HPLC can run >400 samples of 10 ug of IgG.

If we assume that the antibody concentration in serum is 15 mg/ml you would have to add 10-15 000 units to a sample of 1 ml serum. In 50 µl serum you would have to add 500 – 750 units of FabRICATOR®.

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.

We have tested 4-6 M GuHCl which the enzyme does not withstand without significant decrease of activity. SDS have not been evaluated but 0.1% RapiGest from waters have negative influence on activity. Taken together the enzyme does not well tolerate denaturing agents although we have not tested this in more detailed studies.

Yes. FabRICATOR is compatible with EDTA. Make sure your pH is within 6-8.

Yes, columns can be reused but make sure you wash the column with buffer. It can be stored in 20% ethanol as well as buffer.

However, we do not have a proper sanitization protocol so we don’t recommend that you run different antibodies on the same column as we cannot guarantee that there will be no carry-over. From an enzymatic activity point of view the column will work for several runs but eventually enzymatic activity will decline.

If you have a technical question, please submit a support case.

The FabRICATOR MagIC magnetic beads with immobilized FabRICATOR (IdeS) enzyme.

FabRICATOR-HPLC is an HPLC column with the FabRICATOR (IdeS) protease immobilized on a HPLC compatible matrix for automated antibody subunit generation.

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.

We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR MagIC as well.

We have tested PBS and 150 mM ammonium acetate, pH7 with good results. More buffers were found to be compatible with FabRICATOR digestion in solution (more info) and should be suitable for FabRICATOR-HPLC as well. However, the ionic strength of the buffer influences retention of the antibody fragments on the FabRICATOR-HPLC column and a minimum of 100-150 mM salt is necessary for IgG1 subunits to elute as a single, narrow peak. Other IgG subclasses or fusion proteins might need some optimization of buffer composition.

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