Antibody Fragmentation and Characterization, Simplified

• Highly specific IgG cleavage
• Rapid generation of highly pure fragments
• Frozen IdeS Protease and Frozen IdeZ Protease are no longer available
   

  IgG-Digesting Enzymes for Therapeutic Antibody Characterization

  IdeS and IdeZ Proteases are immunoglobulin G (IgG)-degrading enzymes that are valuable tools for the characterization of therapeutic antibodies, Fc fusion proteins and antibody-drug conjugates.

  IdeS and IdeZ Proteases are fast and easy-to-use; complete digestion can be achieved in 30 minutes without optimization. Cleavage is highly reproducible and specific at a single site below the hinge region.

  Both IdeS and IdeZ Proteases effectively cleave human IgG1, IgG2, IgG3 and IgG4, monkey, sheep, rabbit, humanized and chimeric IgGs as well as Fc-fusion proteins. However, only IdeZ Protease cleaves mouse IgG2a and IgG3.
  
   

  Cleavage Specificity of IdeS and IdeZ Proteases

  

  IdeS and IdeZ Proteases are immunoglobulin G (IgG)-degrading proteases that cleave with high specificity.

  IdeS Protease is derived from Streptococcus pyogenes. It is an engineered, recombinant protease overexpressed in E. coli. IdeS Protease cleaves IgG at a single site below the hinge region, yielding F(ab')₂ and Fc fragments as shown here.

  IdeZ Protease is derived from Streptococcus equi subspecies zooepidemicus. It is also an engineered, recombinant protease overexpressed in E. coli. Like IdeS, IdeZ Protease specifically cleaves IgG molecules below the hinge region, yielding F(ab')₂ and Fc fragments. However, IdeZ Protease has significantly improved activity against mouse IgG2a and IgG3 subclasses compared to IdeS Protease.

  Core and Lower Hinge Sequence of Human and Mouse IgG Subclasses

  IdeS and IdeZ Protease activity against each subclass is indicated. Cut sites are indicated by bolded letters.

  Subclass	Hinge/CH2 Sequence	IdeS Activity	IdeZ Activity
  Human
  IgG1	CPPCPAPELLGGPSVF	+++	+++
  IgG2	CPPCPAPP_VAGPSVF	+++	+++
  IgG3	CPRCPAPELLGGPSVF	+++	+++
  IgG4	AHHAQAPEFLGGPSVF	+++	+++

  Subclass	Hinge/CH2 Sequence	IdeS Activity	IdeZ Activity
  Mouse
  IgG1	PCICTVPEV_SSVF	–	–
  IgG2a	CPPCAAPNLLGGPSVF	+	+++
  IgG2b	CHKCPAPNLEGGPSVF	–	–
  IgG3	GSSCPAGNILGGPSVF	+	+++

  Preparation of F(ab′)₂ Fragments using IdeS Protease and Magne™ Protein A Beads

  

  Panel A. Generation of F(ab′)₂ fragments using IdeS Protease and Magne™ Protein A