Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion

Abstract

Here we report the design and production of an antibody-fluorophore conjugate (AFC) as a non-toxic model of an antibody-drug conjugate (ADC). This AFC is based on the conjugation of dansyl sulfonamide ethyl amine (DSEA)-linker maleimide on interchain cysteines of trastuzumab used as a reference antibody. The resulting AFC was first characterized by routine analytical methods (SEC, SDS-PAGE, CE-SDS, HIC and native MS), resulting in similar chromatograms, electropherograms and mass spectra to those reported for hinge Cys-linked ADCs. IdeS digestion of the AFC was then performed, followed by reduction and analysis by liquid chromatography coupled to mass spectrometry analysis. Dye loading and distribution on light chain and Fd fragments were calculated, as well as the average dye to antibody ratio (DAR) for both monomeric and multimeric species. In addition, by analyzing the Fc fragment in the same run, full glyco-profiling and demonstration of the absence of additional conjugation was easily achieved. As for naked antibodies and Fc-fusion proteins, IdeS proteolytic digestion may rapidly become a reference analytical method at all stages of ADC discovery, preclinical and clinical development. The method can be routinely used for comparability assays, formulation, process scale-up and transfer, and to define critical quality attributes in a quality-by-design approach.

A new tool for monoclonal antibody analysis

“…For example, there appeared to be no effect on IdeS digestion when the hinge region cysteines were replaced with serine or conjugated to small molecules. 25,30 It is thus not surprising that engineering of wild type IgG4 CPSC segment to that of IgG1 and IgG2 (CPPC) does not affect the cleavage, as shown in the current study. Domain mapping of IgG molecules by IdeS proteolysis provides a useful alternative to peptide mapping for antibody characterization.…”

“…Compared with the aforementioned proteases, IdeS provides a few advantages, including high site specificity, simple and robust digestion procedure, and high yield. [17][18][19] Application of IdeS has been demonstrated in many literature reports on IgG1 and IgG2 molecules, [20][21][22][23][24][25][26][27][28] as well as Fc fusion proteins. 29,30 Almost all the reports to date used primarily LC-MS techniques in subsequent analysis of the resulting antibody fragments.…”

“…[20][21][22][23][24][25]30,33,34,63 In most reported studies, antibody fragments were analyzed by LC-MS techniques directly to determine glycoform distributions. Whereas MS-based methods are superior in speed and sample consumption, the complexity in operating MS instruments and in data analysis often prevent them from being used in routine quality tests.…”

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

“…The proper recovery of these loaded subunits is important for the estimation of the average drug-to-antibody ratio (DAR), which is a critical quality attribute (CQA) of the antibody drug conjugates [31,[42][43][44]. Average DAR can be calculated based on the formula reported by Wagner-Rousset et al [45] and it is generally close to 4.0 [46,47]. Fig.…”

Characterization of alanine to valine sequence variants in the Fc region of nivolumab biosimilar produced in Chinese hamster ovary cells

“…Application of IdeS has been demonstrated in many literature reports on IgG1 and IgG2 molecules, as well as Fc fusion proteins. [23][24][25][26][27][28] Three predominant reduced peaks were observed in the chromatography by IdeS digestion, which represent the 3 released domains ¡Fc/2, LC and Fd, respectively. The molecular weights of the LC and Fd domains of the clone A were consistent with that of clone B.…”