Antibody Subunit Fragmentation

In many applications antibody fragments are desired over the entire antibody. Fc fragments may cause unwanted interactions in cell and tissue based applications and assays, both in vitro and in vivo, and thus F(ab’)2 or Fab fragments are preferred. Antibody subunit fragmentation significantly improves the information retrieved in antibody characterization with LC-MS. Fabricator is an excellent enzymatic tool in generating antibody subunit fragments. The enzyme cleaves IgG at one specific site yielding perfect F(ab’)2 and Fc fragments.

Introduction

Fragmentation of antibodies is typically associated with heterogeneity of produced fragments, time-consuming reaction condition optimization, low yield and potential loss of immunoreactivity due to cleavage at low pH. Traditionally papain, pepsin or ficin are used to generate antibody fragments and it is a laborious process (Andrew, et al). Cleavage of IgG with pepsin requires extensive optimization and it is done at low acidic pH, thus compromising immunoreactivity. Papain needs an activator and both F(ab’)2 and Fab can be obtained depending on the reaction conditions resulting in a heterogenous pool of fragments (Table 1). These shortcomings can be circumvented using the novel enzyme, FabRICATOR® (IdeS). The cleavage procedure is very fast and simple whereas no optimization is needed. It is performed at neutral pH, preserving the immunoreactivity and generating precise F(ab’)2 and Fc fragments. No further degradation or over-digestion commonly associated with other proteolytic enzymes like pepsin or papain is obtained.

   FabRICATOR Pepsin Papain Ficin
 pH  7 (6-8)  1 (1-5)  6.5 (4-9.5)  6.5 (4-9.5)
 Yield  95-100%  20-50%  50-70%  50-70%
 Cofactor  No  No  Cystein
sulfides
sulfite
cyanide
EDTA
 Cystein
sulfides
sulfite
cyanide
EDTA
 Inhibitors  Iodoacetate (20 mM)
Iodoacetamide (1 mM)
 pH >6
Epoxides
 Heavy metals
carbonyls
NEM
 Heavy metals
carbonyls
NEM
 Advantages
  •  Fast reaction
  • No optimization
  • Robust
  • Extremely specific
  • No over-digestion
  • High yield
  • "Specific"
  • Easy to control reaction
  • No thiols required
  • Neutral pH digestion
  • Broad species range
  • Produces Fab and F(ab')2
  • Neutral pH digestion
  • Broad species range
  • Produces Fab and F(ab')2
 Disadvantages
  • Limited species range
  •  Acidic pH
  • Requires optimization
  • pH affects immunoreactivity
  • Low yield
  • Requires thiol conc optimization
  • Mixture of Fab and F(ab')2
  • Yield
  • Requires thiol conc optimization
  • Mixture of Fab and F(ab')2
  • Yield

Table 1. Overview of FabRICATOR, Papain, Pepsin and Ficin characteristics.

 

F(ab')2 and Fc with FabRICATOR®

FabRICATOR is a unique proteolytic enzyme which cleaves IgG at one single defined site in the lower hinge region of the antibody generating F(ab’)2 and Fc fragments. IdeS, Immunoglobulin G degrading enzyme of S. pyogenes, was originally isolated from the human pathogen Streptococcus pyogenes, its biological role being to circumvent the immune defense mainly by preventing opsnization (von Pawel-Rammingen, et al). The enzyme is very specific for IgG and to date no other substrates has been reported. FabRICATOR, recombinant IdeS, is a modified cysteine protease which rapidly cleaves IgG. A homogenous pool of fragments is obtained through the site specific cleavage after Gly236 in the hinge region (Vincents, et al).

cleavage_site

Fig. 1 Antibody subunit digestion using FabRICATOR showing the cleavage site in the lower hinge region.

FabRICATOR cleaves IgG under physiological reaction conditions thus preserving the immunoreactivity. In all commonly used buffers, in pH ranging from 6.0 to 8.0, FabRICATOR cleaves human IgG efficiently (Table 2).

 Buffer Lower pH   Upper pH
 Phosphate buffer saline (PBS)  6.0  8.0
 Tris buffer  7.0  8.0
 Ammonium bicarbonate buffer  6.0  7.0
 MES buffer  5.5  6.5
 HEPES buffer  7.0  8.0
 Sodium Acetate buffer  6.0  

 

Table 2. Buffers tested for compatibility with FabRICATOR enzyme. Note! FabRICATOR enzyme is irreversibly inactivated below pH 5.0. Digestion above pH 8.0 requires prolonged/optimized.

 

 

All human subclasses as well as humanized and chimeric IgG are cleaved by FabRICATOR. Other species fragmented by FabRICATOR are rabbit, sheep and monkey. Mouse IgG3 is efficiently cleaved whereas mouse IgG2arequires increased FabRICATOR concentration and prolonged incubation time.

 Species cleaved by FabRICATOR  Species NOT cleaved by FabRICATOR 
 Human IgG (1, 2, 3 & 4)  Mouse IgG1
 Humanized  Mouse IgG2b
 Chimeric  Bovine
 Monkey  Porcine
 Rabbit  Goat
 Sheep  
 Mouse IgG2a*  
 Mouse IgG3*  
 Rat IgG2b  

 

Table 3. Species cleaved by FabRICATOR. * Increased incubation time required - Recommend to use FabRICATOR Z (IdeZ) instead.

Advantages of using FabRICATOR for F(ab')2 or Fab' production

  • Rapid - IgG is cleaved within 30 minutes
  • No optimization of reaction conditions is needed - works right out of the box
  • Homogenous pool of fragments – site specific cleavage
  • Physiological reaction conditions preserves the immunoreactivity of the generated fragments

 

F(ab')2 and Fc with FragIT Kit

In applications where pure F(ab’)2 or Fc fragments are desired FragIT kit is a most convenient and economical solution. FabRICATOR immobilized on agarose beads, FragIT, cleaves IgG molecules generating F(ab’)2 and Fc fragments. Fc fragments are separated on CaptureSelect™, an affinity column containing agarose beads with an immobilized Fc specific lama antibody, leaving pure F(ab’)2 in the flow through. Fc fragments, if desired, can be isolated in one step by a simple lowering of pH. A spin column format provides for quick and easy experimental handling.

SDS_FragIT_Kit

Fig. 2 SDS-Page F(ab')2 of digeststed Avastin.

  • Speed - the time for completion is very fast, usually less than one hour even for first-time users.
  • Yield - usually more than 95% of starting material is recovered.
  • Purity - the included affinity separation column binds selectively to the Fc fragment leaving very pure F(ab')2 fragments in the flow-through.
  • Immuno reactivity - gentle handling of the fragments throughout the procedure minimizes reduction of immunoreactivity.
  • Cost - highly cost-effective method due to high yield and the excellent quality of the fragments generated. The short handling time increases the value of this fragmentation solution for the end user.

 

Fab' Fragments with a Single Reduction Step

Fab fragments are easily obtained from F(ab’)2 produced by cleavage with FabRICATOR. A single reduction step with 2-MEA (2-mercaptoethylamine) or Cysteamine yields Fab. This procedure is simple and quick, it is completed within two hours with excellent yield.

If bioconjugation is desired, the generated Fab fragments contain the hinge thiols available for conjugation through standard maleimide chemistry. There is no need to remove Fc fragments before conjugation, a simple desalting step of the fragments is performed before proceeding directly with the conjugation reaction.

Fab2_toFab_web

Fig. 3 Herceptin® (Trastazumab) converted to F(ab')2 and subsequently to Fab' using selective hinge thiol reduction using 2-MEA. Lane 1: Herceptin® (Trastazumab) undigested. Lane 2: FabRICATOR treated. Lane 3: Selective reduction of hinge thiols using 2-MEA (2-mercaptoethylamine).