Fab and Fc Antibody Fragments

Intact Fab and Fc Antibody Fragments using FabALACTICA or GingisKHAN

For applications involving intact Fab or Fc antibody fragments, digestion at the antibody hinge is necessary. Traditional enzymes such as papain and Lys-C can be used to generate Fab fragments, but the unspecific enzymatic activity needs careful optimization to minimize the risk of several digestion sites. The SmartEnzymes IgG proteases GingisKHAN and FabALACTICA have one digestion site to generate homogenous Fab and Fc antibody fragments from human IgG1 (Table 1). The generated fragments can be used for crystallization and NMR studies for higher order structure (HOS), binding studies and much more. Here, we present different strategies for obtaining intact Fab and Fc antibody fragments are discussed.

Intact Fab and Fc Generation with FabALACTICA or GingisKHAN

Table 1. Comparison of enzyme strategies for obtaining Fab fragments from human IgG1 antibodies.

Intact IgG1 Fab with using FabALACTICA®

Most therapuetic antibodies carry a human IgG1 backbone and the development of bi- or multispecific formats are rapidly increasing. The analysis of such antibodies requires specific enzymes to characterize properties such as monovalent binding, disulfide scrambling and paired Fc glycosylation. Traditional protocols include enzymatic digestion using papain and Lys-C, which require extensive optimizations to minimize over-digestion and obtain sufficient yields. The cysteine protease FabALACTICA (IgdE) digests human IgG1 at one specific site above the hinge to generate intact Fab and Fc fragments. The enzyme is also active on most human IgG1 where mutations below the hinge region have been introduced. To demonstrate the performance of FabALACTICA, we digested different human IgG1 antibodies and analyzed the resulting fragments using RP-HPLC (Fig. Z). The defined peaks observed after digestion with FabALACTICA shows the homogenous fragment generation and robust enzymatic performance on different human IgG1 antibodies. The FabALACTICA enzyme can also be used to generate intact Fc fragments.

 

Paired Glycan Analysis of mAbs using FabALACTICA

Intact Fab with using FabALACTICA

Figure 1. LC separation of Fab and Fc fragments of therapeutic antibodies after FabALACTICA digestion.

Fab Generation with using Immobilized FabALACTICA®

In preparative and analytical applications for antibody fragments, it is often advantageous to have a pure sample without any residual enzyme. A scalable and robust preparation of Fab and Fc fragments from monoclonal antibodies can be challenging and require extensive optimization. Using the Immobilized FabALACTICA Fab kit, preparation of milligram quantities (0.5 -100 mg) of pure fragments can easily be obtained with high yields.To demonstrate the performance of Immobilized FabALACTICA, a monoclonal antibody was digested, and the Fab and Fc fragments purified before SDS-PAGE analysis (Fig. 2). The antibody was incubated on the digestion column overnight at RT followed by affinity purification using the CaptureSelect spin column. The flow-through contained pure Fab fragments suitable for structural analyses and pure Fc fragments in the flow-through easily were captured with affinity purification for human CH1.

 

Figure 2. SDS-PAGE analysis of antibody digestion using Immobilized FabALACTICA. Lane 1) kDa marker, Lane 2) intact antibody, Lane 3) Fab and Fc fragments after digestion using Immobilized FabALACTICA, Lane 4) Flow through of Fc fragments, Lane 5) Eluted and pure Fab fragments. Figure text missing.

Intact IgG1 Fab with GingisKHAN®

Intact Fab with GingisKHAN® first image

Figure 3. LC separation of Fab and Fc fragments from a selection of therapeutic antibodies and a Fc-fusion protein after GingisKHAN digestion.

 

The generation of Fab and Fc fragments can be challenging for Fc-fusion proteins or antibodies carrying a mutated hinge. The GingisKHAN enzyme digests human IgG1 at one accessible lysine site above the hinge under reducing conditions mild enough to preserve the intra and inter-chain disulphide bridges, resulting in intact Fab and Fc fragments. For fusion proteins, digestion is often obtained above the hinge, but the structure and amino acid sequence of the fusion partner may result in other exposed digestions sites. A second cleavage site in the Fc may also appear with GingisKHAN if the conserved Fc N-glycans are removed.

 

Intact Fab with GingisKHAN® second image

 

 

A selection of monoclonal human IgG1 antibodies and a Fc-fusion protein were incubated with GingisKHAN for 1 hour at 37°C and analyzed by HPLC (Fig. 3). The resulting fragments show the efficient digestion of GingisKHAN for the generation of intact Fab fragments or separation of the Fc from the fused TNFR domain on etanercept. Since the specific digestion of GingisKHAN is dependent on the native fold of the IgG the specificity is negatively affected at denaturing conditions. For analysis of digestion efficiency with SDS-PAGE, the reaction was stopped in the analytical sample with iodoacetamide before added SDS-loading buffer.

 

Pure Fab Fragments with GingisKHAN® Fab kit

To obtain pure and intact Fab fragments, digestion of human IgG1 and subsequent purification of the antibody fragments is carried out using the GingisKHAN Fab kit. The intact fragment generation was demonstrated on trastuzumab by incubation with GingisKHAN followed by affinity purification on the CaptureSelect human CH1 spin column (Fig. 4). The enzyme and Fc portion of the sample is found in the flow-through of the column and the Fab is easily eluted by lowering of the pH. Using this kit, Fab fragments form up to 2 mg of human IgG1 can be prepared

 

Figure 4. SDS-PAGE analysis under non-reducing conditions of the human IgG1 mAb trastuzumab digested by GingisKHAN.

  1. Molecular weight marker
  2. Intact trastuzumab
  3. Fab and Fc generated by GingisKHAN
  4. Flow-through of Fc
  5. Eluted Fab fragments

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