A Combination of Native LC-MS Approaches for the Comprehensive Characterization of the Antibody-Drug Conjugate Trastuzumab Deruxtecan.

BACKGROUND: Native mass spectrometry (nMS) approaches appear attractive to complement bottom-up strategies traditionally used in biopharmaceutical industries thanks to their quite straightforward and rapid workflows, especially through online hyphenation of non-denaturing liquid chromatography (LC) to nMS. The present work provides an overview of the state-of-the-art chromatographic tools available for the detailed characterization of monoclonal antibody (mAb) formats, exemplified on the antibody-drug conjugate (ADC) trastuzumab deruxtecan (T-DXd). METHODS: T-DXd was first characterized by conventional reversed phase LC (rpLC) and peptide mapping. Couplings of size exclusion chromatography (SEC), cation exchange chromatography (CEX), and hydrophobic interaction chromatography (HIC) to nMS were used to gain further insights into size, hydrophobic, and charge variants of T-DXd and its parental mAb trastuzumab, at intact and middle-up levels. RESULTS: SEC-nMS first offered a direct snapshot of the homogeneous conjugation of T-DXd, with an average drug-to-antibody ratio (DAR) of 8 in agreement with a conjugation on cysteines after reduction of all interchain disulfide bonds. Moreover, SEC-nMS afforded precise identification and quantification of aggregates and fragments. Middle-up level experiments performed after IdeS digestion confirmed that drug conjugation occurs in the Fab region of the mAb, as seen with rpLC. HIC separated two DAR8 species that could not be differentiated by nMS. Although middle-up HIC-nMS proved to be more informative for oxidized forms, the identification of minor variants was still difficult because of poor MS signal quality, showing how the coupling of HIC to nMS remains challenging. Lastly, middle-up CEX-nMS provided accurate determination and localization of post-translational modifications, with several acidic/basic variants within Fab and Fc regions of T-DXd that were also identified by peptide mapping. CONCLUSIONS: This study illustrates the strengths and drawbacks of each LC-nMS coupling. By combining SEC-, HIC-, and CEX-nMS, we were able to achieve a comprehensive characterization of T-DXd without extensive sample preparation prior to MS analysis.

Combination of IM-Based Approaches to Unravel the Coexistence of Two Conformers on a Therapeutic Multispecific mAb.

Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). Previous studies showed that these isomers might originate from a proline cis/trans isomerization in one Fab subunit of the tsAb. We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. The cis/trans equilibrium was further tackled by exploiting the IMn slicing capabilities of the cIM-MS instrument. Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challenging issues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MS methodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depth conformational characterization of these products.

High-Resolution IMS-MS to Assign Additional Disulfide Bridge Pairing in Complementarity-Determining Regions of an IgG4 Monoclonal Antibody.

Monoclonal antibodies (mAbs) have taken on an increasing importance for the treatment of various diseases, including cancers and immunological disorders. Disulfide bonds play a pivotal role in therapeutic antibody structure and activity relationships. Disulfide connectivity and cysteine-related variants are considered as critical quality attributes that must be monitored during mAb manufacturing and storage, as non-native disulfide bridges and aggregates might be responsible for loss of biological function and immunogenicity. The presence of cysteine residues in the complementarity-determining regions (CDRs) is rare in human antibodies but may be critical for the antigen-binding or deleterious for therapeutic antibody development. Consequently, in-depth characterization of their disulfide network is a prerequisite for mAb developability assessment. Mass spectrometry (MS) techniques represent powerful tools for accurate identification of disulfide connectivity. We report here on the MS-based characterization of an IgG4 comprising two additional cysteine residues in the CDR of its light chain. Classical bottom-up approaches after trypsin digestion first allowed identification of a dipeptide containing two disulfide bridges. To further investigate the conformational heterogeneity of the disulfide-bridged dipeptide, we performed ion mobility spectrometry-mass spectrometry (IMS-MS) experiments. Our results highlight benefits of high resolution IMS-MS to tackle the conformational landscape of disulfide peptides generated after trypsin digestion of a humanized IgG4 mAb under development. By comparing arrival time distributions of the mAb-collected and synthetic peptides, cyclic IMS afforded unambiguous assessment of disulfide bonds. In addition to classical peptide mapping, qualitative high-resolution IMS-MS can be of great interest to identify disulfide bonds within therapeutic mAbs.

State-of-the-Art Native Mass Spectrometry and Ion Mobility Methods to Monitor Homogeneous Site-Specific Antibody-Drug Conjugates Synthesis.

Antibody-drug conjugates (ADCs) are biotherapeutics consisting of a tumor-targeting monoclonal antibody (mAb) linked covalently to a cytotoxic drug. Early generation ADCs were predominantly obtained through non-selective conjugation methods based on lysine and cysteine residues, resulting in heterogeneous populations with varying drug-to-antibody ratios (DAR). Site-specific conjugation is one of the current challenges in ADC development, allowing for controlled conjugation and production of homogeneous ADCs. We report here the characterization of a site-specific DAR2 ADC generated with the GlyCLICK three-step process, which involves glycan-based enzymatic remodeling and click chemistry, using state-of-the-art native mass spectrometry (nMS) methods. The conjugation process was monitored with size exclusion chromatography coupled to nMS (SEC-nMS), which offered a straightforward identification and quantification of all reaction products, providing a direct snapshot of the ADC homogeneity. Benefits of SEC-nMS were further demonstrated for forced degradation studies, for which fragments generated upon thermal stress were clearly identified, with no deconjugation of the drug linker observed for the T-GlyGLICK-DM1 ADC. Lastly, innovative ion mobility-based collision-induced unfolding (CIU) approaches were used to assess the gas-phase behavior of compounds along the conjugation process, highlighting an increased resistance of the mAb against gas-phase unfolding upon drug conjugation. Altogether, these state-of-the-art nMS methods represent innovative approaches to investigate drug loading and distribution of last generation ADCs, their evolution during the bioconjugation process and their impact on gas-phase stabilities. We envision nMS and CIU methods to improve the conformational characterization of next generation-empowered mAb-derived products such as engineered nanobodies, bispecific ADCs or immunocytokines.

Glycan-Mediated Technology for Obtaining Homogeneous Site-Specific Conjugated Antibody-Drug Conjugates: Synthesis and Analytical Characterization by Using Complementary Middle-up LC/HRMS Analysis.

Conventional antibody-drug conjugate (ADC) manufacturing methods are based on the nonselective bioconjugation of cytotoxic drugs to lysine and cysteine residues. This results in highly heterogeneous mixtures of different drug-antibody ratios (DAR) that can significantly affect the safety and efficacy of the ADC product. Recently, an innovative procedure named GlyCLICK was suggested, consisting of a two-step enzymatic procedure to transform Fc-glycans present on IgG mAbs into two site-specific anchor points for the conjugation of any alkyne-containing payload of choice. Here, we evaluated the conjugation process by comparing trastuzumab and trastuzumab conjugated with DM1, following the GlyCLICK procedure. Complementary reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to high-resolution mass spectrometry (HRMS) were used to analyze the protein subunits (ca. 25-100 kDa) obtained after different levels of enzymatic digestion and chemical reduction. Our results demonstrated that the hydrophobic character of the drug molecule allows to rapidly confirm the Fc-drug conjugation at the chromatographic level. Furthermore, the hyphenation to MS detection provided accurate mass information on the ADC subunits and facilitated the DAR determination of 2.0. Therefore, this work illustrates how middle-up analysis using LC/HRMS can provide accurate and complementary information on the critical quality attributes of these novel site-specific ADC products.