Tell us about your project
To extend the utility of both IgG and Fc-fusion proteins and overcome their stability issues which is a need to develop longer acting medicines, we have developed IgG and Fc-fusion mimetics called Fab-PEG-Fab (FpF) and receptor-PEG-receptor (RpR). Antibody fragments (Fabs) and receptor-binding fragment (VEGFR1-VEGFR2) are obtained by proteolytical digestion of IgG and aflibercept respectively. FpF and RpRs are prepared using disulfide bridging reagents, PEG-di(mono-sulfone) reagent. Each terminus in the PEG reagent undergoes site-specific conjugation with the two cysteine thiols from an accessible disulfide in a Fab or VEGFR1-VEGFR2 by a sequence of addition-elimination reactions resulting in conjugation by the insertion of a 3-carbon methylene bridge to re-anneal the original disulfide into a more stable re-bridged disulfide.
How did SmartEnzymes enhance your project?
To prepare the RpR, we needed to generate receptor-binding fragment (VEGFR1-VEGFR2). We first used immobilised pepsin but the yield and efficiency of obtained fragment was very poor. Using of IdeS enzyme (FabRICATOR®, Genovis), we were able to obtain pure fragment with a yield of above 90%.
In case of GingisKHAN, we used it to generate Fab fragment from IgG to prepare Fab-PEG-Fab mimetics. While we initially used immobilized papain to obtain Fab, we later found out that the stability profile of FpF prepared from Papain-Fab was very different from FpF prepared from GingisKHAN-Fab. The Fab generated by GingisKHAN was pure and homogenous and resulted in increased stability of FpF.
