Production of Fab Fragments

Fab fragments from human IgG1 can be generated in 1 hour using GingisKHAN (KGP) enzyme - Read more here

For other IgG subclasses and species there is another procedure using a combination of our FabRICATOR enzyme with 2-MEA (2-mercaptoethylamine or Cysteamine). This procedure is simple and usually gives good yields. It can be completed in two hours - Read more here.

Intact Fab with GingisKHAN® - Human IgG1

GingisKHAN (KGP) is an enzyme that digests human IgG1 yielding intact Fab and Fc fragments. GingisKHAN digests human IgG1 at one single site in the upper hinge. A second cleavage site on the Fc may appear if the N-glycans are removed. GingisKHAN is a cysteine protease and requires reducing environment to be active. Intact Fab and Fc fragments are obtained with GingisKHAN digestion of human IgG1 since mild reducing conditions (i.e. 2 mM cysteine) is sufficient for enzyme activity.

gingis-digestions-1000px

Figure 1: GingisKHAN digestion site in human IgG1. 

Procedure in short:

  1. Prepare the human IgG1 in 0.1M Tris pH 8 at a concentration of 0.5-5 mg/ml
  2. Add solubilized GingisKHAN and GingisKHAN Reducing Agent (supplied with the enzyme) to the reaction mixture.
  3. Incubate at 37°C for 1 hour.

The sample is now ready for further use or analysis.

Digestion of human IgG1 by GingisKHAN is negatively affected at denaturing conditions i.e. in the presence of chaotropic agents and/or detergents. If analysis of digestion efficiency is done with SDS-PAGE take care to run samples immediately after SDS sample preparation. Preferentially, run the SDS-Page under reducing conditions or stop the reaction with 10 mM iodoacetamide before SDS sample preparation.

Go from Antibody F(ab')2 to Fab'

A beautiful way to circumvent the micro-heterogeneity and boost the low yield that is often the result of fragmentation using other proteolytic enzymes like pepsin or papain is to combine our FabRICATOR enzyme with 2-MEA (2-mercaptoethylamine or Cysteamine). This procedure is simple and usually gives good yields. It can be completed in two hours.

For the researchers who wish to do bioconjugation of the generated Fab' fragments to other biomolecules or surfaces there’s good news: The generated Fab fragments contains the hinge thiols available for conjugation through standard maleimide chemistry. You don’t even need to remove the Fc fragments before conjugation, just perform a simple desalting of the fragments and then proceed directly with your conjugation reaction. The protocol below is adapted from Hermanson, Bioconjugate Chemistry 2nd Ed.

1. Generate F(ab')2 fragments using FabRICATOR or FragIT

fab2_fragmentation

Use FabRICATOR or FragIT to cut the IgG just below the hinge region. This procedure takes approximately 30 minutes. You will end up with a homogenous pool of F(ab')2 fragments and Fc fragments. Reaction should be performed in a buffer conatining EDTA, for example, 50 mM Phosphate, 150 mM NaCl, 5 mM EDTA, pH 7.2.

Next, add 2-MEA (2-mercaptoethylamine) to a final concentration of 50 mM and incubate at 37°C for 90 minutes. There is no need to remove the FabRICATOR enzyme if present. (Make a stock solution of 2-MEA) in the same buffer as above).

2. Mild reduction of hinge thiols

Fab2_to_Fab_spot

Now your solution conatins Fab fragments, Fc fragments, 2-MEA and possibly FabRICATOR enzyme. To remove the 2-MEA a desalting column can be used for rapid and convenient processing. We have tested desalting using columns from GE Healthcare (NAP 5, NAP 10, NAP 25) and similar G25 Sephadex™ columns.

3. Final step.

fragments_done

Now your solution conatins Fab fragments, Fc fragments, 2-MEA and possibly FabRICATOR enzyme. To remove the 2-MEA a desalting column can be used for rapid and convenient processing. We have tested desalting using columns from GE Healthcare (NAP 5, NAP 10, NAP 25) and similar G25 Sephadex™ columns.

Fab2_toFab_web

SDS-PAGE of Lane 1: MW marker Lane 2: Trastazumab Lane 3: After enzymatic fragmentation using FabRICATOR enzyme Lane 4: Fab' fragments after mild reduction of hinge thiols.

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