Anticorps thérapeutiques et dérivés : une palette de structures pour une pléthore d’indications

Les anticorps monoclonaux (Acm) et dérivés constituent la classe de principes actifs qui connaît le plus fort taux de développement actuel dans les domaines biotechnologique et pharmaceutique. En un peu plus de vingt ans, plus de 30 immunoglobulines (IgG) et dérivés ont obtenu une autorisation de mise sur le marché (AMM) dans des indications thérapeutiques très variées (oncologie, inflammation et auto-immunité, transplantation, angioplastie, hématologie, ophtalmologie, infections virales, allergie). La palette de structures comporte des anticorps murins, chimériques, humanisés et humains d’isotypes différents (IgG1, 2 et 4), ainsi que des dérivés d’IgG (immunoconjugués, radio-immunoconjugués, fragments Fab, protéines et peptides de fusion Fc, bispécifiques souris/rat). De plus, du point de vue structural, leur glycosylation dépend du système de production et peut influencer les fonctions effectrices et l’immunogénicité. À partir des connaissances actuelles des relations structure-fonction, quels formats d’anticorps est-il préférable de choisir ? Avec quels types de glycosylation ? Pour quelles indications 

Native Mass Spectrometry, Ion Mobility, and Collision-Induced Unfolding for Conformational Characterization of IgG4 Monoclonal Antibodies

International audienceAlthough the majority of FDA and EMA approved therapeutic monoclonal antibodies (mAbs) are IgG1, the number of IgG4-based formats reaching the market is increasing. IgG4 differs from other mAb isotypes by its specificity to form half mAbs that recombine into bispecific (bsAbs) molecules, through a process termed fab-arm exchange (FAE). We report here the complementarity of native mass spectrometry (MS), ion mobility (IM), and collision-induced unfolding (CIU) experiments for the structural characterization of members of the IgG4 subfamily (wild-type (wt), hinge-stabilized (hs, S228P mutation), and the resulting bsAb IgG4s). Native MS allows confirming/invalidating the occurrence of FAE as a function of these different types of IgG4. While IM-MS was unable to distinguish iso-cross-section IgG4 species, CIU experiments provide unique specific structural signatures of each individual IgG4 based on their specific unfolding pathways. Common CIU features of IgG4 formats include the observation of three conformational states and two transitions. In addition, CIU experiments demonstrated that S228P mutation stabilizes gas phase conformations of hsIgG4, in agreement with increased stability related to more rigid hinge regions. CIU patterns also appear to be more informative than IM-MS for bsAb structural characterization, unfolding signature of the bsAb being intermediate to the ones of the former parent wt-IgG4s, highlighting that bsAb CIU profiles keep the memory of their origins. Altogether, our results demonstrate that CIU patterns can serve as mAb specific structural signatures and are mature to be included in MS-based analytical workflows for conformational/structural characterization of mAb formats in early development phases and for multiple attribute monitoring

Protocols for the analytical characterization of therapeutic monoclonal antibodies. I - Non-denaturing chromatographic techniques

Size-, charge- and hydrophobicity-related variants of a biopharmaceutical product have to be deeply characterized for batch consistency and for the assessment of immunogenicity and safety effects. Size exclusion chromatography (SEC) and ion exchange chromatography (IEX) are considered as the gold standard for the analysis of high molecular weight species (HMWS) and charge-related variants, respectively. Hydrophobic interaction chromatography (HIC) has drawn renewed attention to monitor the small drug payload distribution in the cysteine-linked antibody-drug conjugates (ADC). These three chromatographic techniques, namely SEC, HIC and IEX, are historical, non-denaturing and robust approaches widely used for the characterization of biopharmaceutical proteins. Despite the broad spectrum of monoclonal antibodies (mAbs) structures, isoelectric points (pIs) and hydrophobicities, generic protocols can be applied to separate their size-, charge- and hydrophobicity-related variants, using the last generation of chromatographic columns and appropriate mobile phase conditions. Straightforward protocols are described in this manuscript with representative chromatograms of ten distinct Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved therapeutic mAb products to illustrate the performance of the SEC, IEX and HIC methods

Drug Loading and Distribution of ADCs After Reduction or IdeS Digestion and Reduction

International audienceHigh-resolution native mass spectrometry (MS) provides accurate mass measurements (within 30 ppm) of intact ADCs and can also yield drug load distribution (DLD) and average drug to antibody ratio (DAR) in parallel with hydrophobic interaction chromatography (HIC). Native MS is furthermore unique in its ability to simultaneously detect covalent and noncovalent species in a mixture and for HIC peak identity assessment offline or online.As an orthogonal method described in this chapter, LC-MS following ADC reduction or IdeS (Fabricator) digestion and reduction can also be used to measure the DLD of light chain and Fd fragments for hinge native cysteine residues such as brentuximab vedotin. Both methods allow also the measurement of average DAR for both monomeric and multimeric species. In addition, the Fc fragments can be analyzed in the same run, providing a complete glycoprofile and the demonstration or absence of additional conjugation of this subdomain involved in FcRn and Fc-gammaR binding

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

Characterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precisio

Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate

International audienc

Development of a fast workflow to screen the charge variants of therapeutic antibodies

Chemical or enzymatic modifications of therapeutic monoclonal antibodies (mAbs) having high risktowards safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic mAbsprocess development, a variety of analytical techniques have to be used for the thorough characteriza-tion and quantitative monitoring of CQAs. This paper describes the development of a rapid analyticalplatform to assess and rank charge variants of mAbs. The workflow is first based on a cation exchangechromatography (CEX) comparative analysis of intact IgGs versus F(ab)’2 and Fc sub-domains generatedby IdeS digestion. This analytical procedure was validated with FDA and EMA approved mAbs. Then,functional assays and peptide mapping can be performed in a second instance. This approach can be usedduring the early stage of drug research and development to screen lead molecules and select optimizedcandidates (best clone, best formulation) which could be “easily” developed (OptimAbs)

Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates

International audienceAntibody drug conjugates (ADCs) are highly cytotoxic drugs covalently attached via conditionally stable linkers to monoclonal antibodies (mAbs) and are among the most promising next-generation empowered biologics for cancer treatment. ADCs are more complex than naked mAbs, as the heterogeneity of the conjugates adds to the inherent microvariability of the biomolecules. The development and optimization of ADCs rely on improving their analytical and bioanalytical characterization by assessing several critical quality attributes, namely the distribution and position of the drug, the amount of naked antibody, the average drug to antibody ratio, and the residual druglinker and related product proportions. Here brentuximab vedotin (Adcetris®) and trastuzumab emtansine (Kadcyla®), the first and gold-standard hinge-cysteine and lysine drug conjugates, respectively, were chosen to develop new mass spectrometry (MS) methods and to improve multiplelevel structural assessment protocols