Preparation and quality control of mAb-F(ab')2

Introduction: Monoclonal antibodies (mAbs) have large molecular weights, slow metabolism, and long half-life nuclide-labeled monoclonal antibodies are difficult to clinically apply due to the large cumulative radiation dose. In this study, the specific recognition feature of IdeS enzyme was used to prepare mAb-F(ab')2 by taking Cetuximab as an example.

Methods: Cetuximab was dissolved in PBS with 0.01M pH=7.4, and an appropriate amount of IdeS enzyme was added. After 30 minutes at 37°C, a mixture of Cetuximab-F(ab')2, Cetuximab-Fc and IdeS enzymes was obtained; use excess Protein A magnetic beads to adsorb Cetuximab - Fc and 40 K Zeba desalting column purification to yield mAb-F(ab')2. In terms of quality control, product purity was assessed by 4%-15% SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining. Antibody activity identification: Conjugated with Cetuximab-F(ab')2-cy3 and Cetuximab-cy3 as fluorescent probes, added to MGC803 cell culture medium at a final concentration of 0.001 μg/mL, and observed by fluorescence microscope 12 hours later.

Results: By SDS-PAGE staining with Coomassie brilliant blue, the protein band of Cetuximab was about 150 kDa and Cetuximab-F(ab')2 was about 100 kDa. Through fluorescence microscope observation, the binding amount of MGC803 cells to Cetuximab-F(ab')2-cy3 and Cetuximab-cy3 fluorescent probes is similar (p>0.05), which proves that the binding force of Cetuximab-F(ab')2 is similar to that of Cetuximab-F(ab')2. Total resistance is similar.

Conclusions: IdeS enzymatic cleavage can obtain mAb-F(ab')2 with similar activity and smaller molecular weight.