Generation of Purified Fab Fragments from Mouse IgG1 using FabULOUS™

Performance Activity

Preparation of pure, homogeneous mouse IgG Fab fragments with no residual enzyme by combining FabULOUS digestion and affinity purification.

For the generation of intact and pure mouse IgG Fab fragments without residual enzyme in the final preparation, the FabULOUS enzyme is combined with affinity purification in the FabULOUS Fab Kit. With this kit, FabULOUS digestion of the mouse IgG is followed by separation of the Fab fragments from the FabULOUS enzyme and Fc fragments using the CaptureSelect™ spin column with affinity resin towards murine LC kappa chain.

Here, we demonstrate how we can use the FabULOUS Fab Kit to generate and purify homogeneous Fab fragments from mouse IgG1. The kit comprises FabULOUS lyophilized enzyme and a CaptureSelect LC-Kappa (mur) microspin column for affinity purification of the digested material. Following FabULOUS digestion, the CaptureSelect LC-Kappa column will bind the Fab fragments whilst the Fc fragments and enzyme will pass through the column.

Purification of homogeneous Fab fragments using the FabULOUS Fab Kit is highlighted with the LC chromatograms where the post-purification sample contains only Fab.

Purification of homogeneous mouse IgG1 Fab fragments using FabULOUS Fab Kit

The CaptureSelect LC-Kappa (mur) column will bind any undigested material as well as fully digested Fab. For this reason, a highly efficient enzymatic digestion step is crucial for effective performance of the FabULOUS Fab Kit. We observe robust digestion of mouse IgG1 during the first step through the lack of undigested IgG in both the pre- and post-purification sample LC chromatograms.

Fast and robust digestion of mouse IgG1 using FabULOUS

Generation of intact Fab fragments from mouse IgG1 using FabULOUS Fab Kit. Purified Fab fragments of mouse IgG1 were prepared using the FabULOUS Fab Kit. The sample was digested for 1 hr at 37 °C using FabULOUS Lyophilized before Fab purification with CaptureSelect LC-Kappa (mur). Reversed Phase LC-chromatograms are shown for the intact mouse IgG1 (top), the separated Fab and Dc frgments following FabULOUS digestion (middle) and the Fab fragments following affinity purification (bottom).

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