FabRICATOR in a Comprehensive Characterization of a Bispecific Antibody

September 9, 2021 | Applications, References |

Scientists at University of Geneva and Centre d’Immunologie Pierre-Fabre here describes a comprehensive intact and middle-level characterization of a commercial bispecific antibody by using a variety of orthogonal chromatographic methods coupled to MS.

The field of protein biopharmaceuticals is one of the most innovative and fastest growing class of therapeutics. Encouraged by the success of therapeutic monoclonal antibodies and the need for higher clinical efficacy in combination with lower adverse effects, several new antibody formats have been developed. Among new formats such as fusion proteins, antibody-drug conjugates and nanobodies, bispecific antibodies might be considered one of the rising stars of new formats in the antibody field.


One major advantage of bispecific antibodies is that they combine the antigen recognition sites of two or more antibodies in one protein construct, allowing for simultaneously binding of two or more different epitopes on the same or different antigens. During the development and production of a bispecific antibody the correct chain-association is difficult, yet critical, to monitor, as are the common chemical and enzymatic post-translational modifications (PTMs).


In this work, several LC-MS strategies in a multi-level approach were used to characterize the bispecific antibody emicizumab. Whereas analyses at intact level could be used to monitor the presence of protein size and charge variants, a major downside of intact protein analysis is the lack of site-specific information when analyzing an asymmetric bispecific antibody such as emicizumab. A middle-up approach was therefore used in order to obtain important site-specific information of the antibody.


In a middle-up level analysis, subunits of the protein of interest is generated and analyzed, enabling a more accurate peak identification. Using FabRICATOR (IdeS), the bispecific emicizumab was digested at a single site below the hinge, generating a homogenous pool of subunits suitable for in-depth characterization. The authors conclude that the analytical workflow applied here can be applied to any other bispecific antibody.


Duivelshof et al., 2021. Bispecific antibody characterization by a combination of intact and site-specific/chain-specific LC/MS techniques. Talanta. https://doi.org/10.1016/j.talanta.2021.122836









FabRICATOR – Below hinge digestion of IgG