Generation of F(ab')2 Fragments using FabRICATOR®

Use FabRICATOR to generate specific F(ab’)2 fragments in 30 min without over-digestion or optimizing protocols.

FabRICATOR (IdeS) is a unique enzyme that specifically digests the hinge region of IgG, producing a F(ab’)2 fragment and two Fc fragments after 30 min incubation at 37°C (Figure 1). 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.

cleavage_site

Figure 1. Graphical illustration of the FabRICATOR specific site of action on antibodies.

 

Advantages of FabRICATOR® over papain and pepsin

  • Specific – one site digestion, no over-digestion
  • Generates specific F(ab’)2 fragments
  • Selective – IgG is the only known substrate
  • Protocol – 30 min incubation at 37°C
  • Ease of use – works in common buffers at neutral pH

 

FragIT™  - FabRICATOR® Immobilized

FragIT is FabRICATOR enzyme immobilized on agarose allowing for antibody subunit fragmentation without contaminating the final preparation with FabRICATOR enzyme (Figure 2). The agarose is supplied in spin columns for digestion of 0.5 mg up to 100 mg of antibody or Fc-fusion protein.

Fragit_klocka_400

Figure 2. FragIT – an easy to use spin column for rapid generation of F(ab’)2 fragments.

 

FragIT™ Kit

With the FragIT Kit it is possible to go one step further to purify the F(ab')2 from the Fc fragments (Figure 3). The kit consist of two spin column, one with FragIT resin and the other with CaptureSelect™- an agarose matrix with a 13 kDa llama antibody fragment recognizing Fc of multiple species with high affinity.

Fragit_kit_klocka_600

Figure 3. FragIT Kit – Digestion and affinity purification of antibody subunits.

 

Applications of FabRICATOR® generated F(ab’)2 fragments

  • Demonstrate the importance of Fc for a therapeutic antibody
  • Avoid binding to protein A or protein G
  • Prevent bindning to Fc receptors
  • Reduce non-specific interactions in staining, ELISA, or immunohistochemistry assays
  • Generate anti-hinge antibodies (Malia et al. 2014)
  • Sensitive antibody characterization

References

Malia, T.J. et al., 2014. Structure and specificity of an antibody targeting a proteolytically cleaved IgG hinge. Proteins: Structure, Function, and Bioinformatics, 82(8), pp.1656–1667.