Unique Enzymatic Digestions Allow Study of Antibody Disulphides

December 12, 2017 | Products, References |

Antikropp-klyvning-över-och-under-hinge

Valegh Faid and colleagues at LFB Biotechnologies in France have developed and published an assay to study antibody disulphide bonds using middle-up LC-MS (Faid et al. 2017). In the paper, the combination of FabRICATOR® for digestion below the hinge and FabALACTICA™ for digestion above the hinge, generated three fragments from a human IgG1 antibody; the hinge peptide, the Fab and the Fc/2 fragments. These fragments were separated using RP-HPLC on a diphenyl column and analyzed using mass spectrometry. This setup enabled analysis of both inter- and intrachain disulphide bonds as well as other quality attributes.

 

The disulphide bonds of a therapeutic antibody can serve as indicators for misfolded antibodies during antibody manufacturing and has also been directly linked to mAb stability and are considered a quality parameter during antibody manufacturing. Traditionally, the methods for investigating the disulphide bonds of antibodies include labelling free cysteine residues using Ellman’s reagent or fluorescent dyes, resulting in a measure of the overall free sulfhydryls. The new assay using FabALACTICA and FabRICATOR is based on the unique digestion sites of the enzymes and utilizes the fact that both enzymes works under non-reducing conditions. The authors conclude:

 

“This method is easy to use, generic for mAbs and presents the advantage to deal with the whole complexity of a single mAb molecule by generating three well separated smaller and less heterogeneous fragments. Besides a direct application to the detection of intra- and inter-chain free sulfhydryls, this combined digestion could be promising to investigate other mAbs quality attributes“ (Faid et al. 2017).


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Read more about FabALACTICA and FabRICATOR.

 

Faid, V. et al., 2017. Middle-up analysis of monoclonal antibodies after combined IgdE and IdeS hinge proteolysis: Investigation of free sulfhydryls. Journal of Pharmaceutical and Biomedical Analysis, 149, pp.541–546.