How did you come up with the idea to combine FabRICATOR (IdeS) and FabALACTICA (IgdE)?
The middle-up analysis is fast and very informative regarding the protein sequence integrity and post-translational modifications. However, investigating the oxidative state of disulfide bridges is tricky and often involved a time-consuming peptide mapping in non-reducing conditions. The combination of FabRICATOR and FabALACTICA in non-reducing conditions presents the advantage to generate specifically three fragments i.e. hinge, Fc/2 and Fab that are both easily separated by RP-HPLC and analysed by MS.
How does the new enzymatic assay compare to previous methods to study antibody disulphide bonds?
Peptide mapping in non-reducing condition is time consuming even if dedicated software to improve the treatment of data has largely improved. The combined FabRICATOR and FabALACTICA middle-up approach increases the throughput for the investigation of free thiols and disulphide scrambling.
Would the assay be used in a QC setting relying solely on liquid chromatography separation?
The analytical workflow is robust and requires mere handlings of the antibody samples. Once the identification of each peak of the chromatogram is confirmed by MS, quantitation based on the UV detection is a current practice. Such analytical configuration involving an HPLC and a UV detection is actually common in most of QC labs and thus easily and robustly implementable.
Why are antibody disulphide bonds important?
Disulphide bonds are highly important because of their critical role in the stabilization of protein conformations. Breaking and/or scrambling of disulphide bond occur during manufacturing and storage of biotherapeutics which is a concern in terms of safety and efficacy. The monitoring of these product-derived impurities is mandatory during development operations in order to minimize these forms.
