Generating Antibody Mimetics with GingisKHAN

Antibodies formulated as solid-state depots can potentially be used for local treatments and minimize the need for large systemic doses. Bevacizumab may for instance be administered locally to control post-operative scarring following glaucoma filtration surgery. A solid-state form would however be required in order to obtain a proper slow release of the antibody.


In order to study the possibility of such a solid-state formulation, scientists at the School of Health in London developed antibody mimetics and compared the stability after lyophilization to that of bevacizumab. Consisting of two Fabs linked together by a flexible polyethylene glycol (PEG) scaffold, the mimetics functionality is similar to that of IgG, but without the Fc-mediated effector functions. In addition, the mimetic format enhances binding affinity while reducing the propensity for aggregation.


To prepare the antibody mimetics, Fab fragments from bevacizumab were obtained by digestion above the hinge using either papain or GingisKHAN®. While both enzymes were able to produce Fabs without impacting the binding site, LC-MS analysis showed that the unspecific papain digestion resulted in three different isomers, and a free light chain due to disulfide reduction. In contrast, the GingisKHAN enzyme generated a homogenous pool of Fab fragments as was indicated by one single mass peak.


Antibody mimetics produced with the different Fab fragments were then subjected to freeze-drying for a head-to-head comparison with lyophilized bevacizumab. The results showed that only the mimetic produced with GingisKHAN generated Fabs was unsusceptible to aggregation and remained intact without heavy or light chain dissociation. In short, the scientists concluded that the antibody mimetic was more stable than IgG when subjected to lyophilization, and that pure homogenous Fabs could play an important part for the stability of the final product.


Khalili. 2020. Using different proteolytic enzymes to digest antibody and its impact on stability of antibody mimetics. Journal of Immunological Methods. doi: 10.1016/j.jim.2020.112933





Generation of human IgG1 Fab fragments.


GingisKHAN Fab kit
Generation and purification of human IgG1 Fabs.