Chemical Production of Cytotoxic Bispecific Antibodies Using the Ugi Multicomponent Reaction.

Bispecific antibodies (bsAbs) have recently emerged as a promising platform for the treatment of several conditions, most importantly cancer. Based on the combination of two different antigen-binding motifs in a single macromolecule; bsAbs can either display the combined characteristics of their parent antibodies, or new therapeutic features, inaccessible by the sole combination of two distinct antibodies. While bsAbs are traditionally produced by molecular biology techniques, the chemical development of bsAbs holds great promises and strategies have just begun to surface. In this context, we took advantage of a chemical strategy based on the use of the Ugi reaction for the site-selective conjugation of whole antibodies and coupled the resulting conjugates in a bioorthogonal manner with Fab fragments, derived from various antibodies. We thus managed to produce five different bsAbs with 2:1 valency, with yields ranging from 20% to 48%, and showed that the affinity of the parent antibody was preserved in all bsAbs. We further demonstrated the interest of our strategy by producing two other bsAbs behaving as cytotoxic T cell engagers with IC50 values in the picomolar range in vitro.

Denaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions.

Chemical cross-linking reactions (XL) are an important strategy for studying protein-protein interactions (PPIs), including low abundant sub-complexes, in structural biology. However, choosing XL reagents and conditions is laborious and mostly limited to analysis of protein assemblies that can be resolved using SDS-PAGE. To overcome these limitations, we develop here a denaturing mass photometry (dMP) method for fast, reliable and user-friendly optimization and monitoring of chemical XL reactions. The dMP is a robust 2-step protocol that ensures 95% of irreversible denaturation within only 5 min. We show that dMP provides accurate mass identification across a broad mass range (30 kDa-5 MDa) along with direct label-free relative quantification of all coexisting XL species (sub-complexes and aggregates). We compare dMP with SDS-PAGE and observe that, unlike the benchmark, dMP is time-efficient (3 min/triplicate), requires significantly less material (20-100×) and affords single molecule sensitivity. To illustrate its utility for routine structural biology applications, we show that dMP affords screening of 20 XL conditions in 1 h, accurately identifying and quantifying all coexisting species. Taken together, we anticipate that dMP will have an impact on ability to structurally characterize more PPIs and macromolecular assemblies, expected final complexes but also sub-complexes that form en route.

SEC-MS in denaturing conditions (dSEC-MS) for in-depth analysis of rebridged monoclonal antibody-based formats.

Disulfide rebridging methods are emerging recently as new ways to specifically modify antibody-based entities and produce future conjugates. Briefly, the solvent-accessible disulfide bonds of antibodies or antigen-binding fragments (Fab) thereof are reduced under controlled conditions and further covalently attached with a rebridging agent allowing the incorporation of one payload per disulfide bond. There are many examples of successful rebridging cases providing homogeneous conjugates due to the use of symmetrical reagents, such as dibromomaleimides. However, partial rebridging due to the use of unsymmetrical ones, containing functional groups with different reactivity, usually leads to the development of heterogeneous species that cannot be identified by a simple sodium dodecyl sulfate-polyacrylamide gel eletrophoresis (SDS-PAGE) due to its lack of sensitivity, resolution and low mass accuracy. Mass spectrometry coupled to liquid chromatography (LC-MS) approaches have already been demonstrated as highly promising alternatives for the characterization of newly developed antibody-drug-conjugate (ADC) and monoclonal antibody (mAb)-based formats. We report here the in-depth characterization of covalently rebridged antibodies and Fab fragments in-development, using size-exclusion chromatography hyphenated to mass spectrometry in denaturing conditions (denaturing SEC-MS, dSEC-MS). DSEC-MS was used to monitor closely the rebridging reaction of a conjugated trastuzumab, in addition to conjugated Fab fragments, which allowed an unambiguous identification of the covalently rebridged products along with the unbound species. This all-in-one approach allowed a straightforward analysis of the studied samples with precise mass measurement; critical quality attributes (CQAs) assessment along with rebridging efficiency determination.

Site-Selective Protein Conjugation by a Multicomponent Ugi Reaction.

The chemical bioconjugation of proteins has seen tremendous applications in the past decades, with the booming of antibody-drug conjugates and their use in oncology. While genetic engineering has permitted to produce bespoke proteins featuring key (un-)natural amino acid residues poised for site-selective modifications, the conjugation of native proteins is riddled with selectivity issues. Chemoselective strategies are plentiful and enable the precise modification of virtually any residue with a reactive side-chain; site-selective methods are less common and usually most effective on small and medium-sized proteins. In this context, we studied the application of the Ugi multicomponent reaction for the site-selective conjugation of amine and carboxylate groups on proteins, and antibodies in particular. Through an in-depth mechanistic methodology work supported by peptide mapping studies, we managed to develop a set of conditions allowing the highly selective modification of antibodies bearing N-terminal glutamate and aspartate residues. We demonstrated that this strategy did not alter their affinity toward their target antigen and produced an antibody-drug conjugate with subnanomolar potency. Excitingly, we showed that the high site selectivity of our strategy was maintained on other protein formats, especially on anticalins, for which directed mutagenesis helped to highlight the key importance of a single lysine residue.

Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows.

Antibody-drug conjugates (ADCs) are highly complex proteins mainly due to the structural microvariability of the mAb, along with the additional heterogeneity afforded by the bioconjugation process. Top-down (TD) and middle-down (MD) strategies allow the straightforward fragmentation of proteins to elucidate the conjugated amino acid residues. Nevertheless, these spectra are very crowded with multiple overlapping and unassigned ion fragments. Here we report on the use of dedicated software (ClipsMS) and application of proton transfer charge reduction (PTCR), to respectively expand the fragment ion search space to internal fragments and improve the separation of overlapping fragment ions for a more comprehensive characterization of a recently approved ADC, trastuzumab deruxtecan (T-DXd). Subunit fragmentation allowed between 70 and 90% of sequence coverage to be obtained. Upon addition of internal fragment assignment, the three subunits were fully sequenced, although internal fragments did not contribute significantly to the localization of the payloads. Finally, the use of PTCR after subunit fragmentation provided a moderate sequence coverage increase between 2 and 13%. The reaction efficiently decluttered the fragmentation spectra allowing increasing the number of fragment ions characteristic of the conjugation site by 1.5- to 2.5-fold. Altogether, these results show the interest in the implementation of internal fragment ion searches and more particularly the use of PTCR reactions to increase the number of signature ions to elucidate the conjugation sites and enhance the overall sequence coverage of ADCs, making this approach particularly appealing for its implementation in R&D laboratories.

Rapid Access to Potent Bispecific T Cell Engagers Using Biogenic Tyrosine Click Chemistry.

Bispecific antibodies as T cell engagers designed to display binding capabilities to both tumor-associated antigens and antigens on T cells are considered promising agents in the fight against cancer. Even though chemical strategies to develop such constructs have emerged, a method that readily converts a therapeutically applied antibody into a bispecific construct by a fully non-genetic process is not yet available. Herein, we report the application of a biogenic, tyrosine-based click reaction utilizing chemoenzymatic modifications of native IgG1 antibodies to generate a synthetic bispecific antibody construct that exhibits tumor-killing capability at picomolar concentrations. Control experiments revealed that a covalent linkage of the different components is required for the observed biological activities. In view of the highly potent nature of the constructs and the modular approach that relies on convenient synthetic methods utilizing therapeutically approved biomolecules, our method expedites the production of potent bispecific antibody constructs with tunable cell killing efficacy with significant impact on therapeutic properties.

Cysteine-Cysteine Cross-Conjugation of both Peptides and Proteins with a Bifunctional Hypervalent Iodine-Electrophilic Reagent.

Peptide and protein bioconjugation sees ever-growing applications in the pharmaceutical sector. Novel strategies and reagents that can address the chemo- and regioselectivity issues inherent to these biomolecules, while delivering stable and functionalizable conjugates, are therefore needed. Herein, we introduce the crosslinking ethynylbenziodazolone (EBZ) reagent JW-AM-005 for the conjugation of peptides and proteins through the selective linkage of cysteine residues. This easily accessed compound gives access to peptide dimers or stapled peptides under mild and tuneable conditions. Applied to the antibody fragment of antigen binding (Fab) species, JW-AM-005 delivered rebridged proteins in a one-pot three-reaction process with high regioselectivity, outperforming the standard reagents commonly used for this transformation.

Recent advances in structural mass spectrometry methods in the context of biosimilarity assessment: from sequence heterogeneities to higher order structures.

Biotherapeutics and their biosimilar versions have been flourishing in the biopharmaceutical market for several years. Structural and functional characterization is needed to achieve analytical biosimilarity through the assessment of critical quality attributes as required by regulatory authorities. The role of analytical strategies, particularly mass spectrometry-based methods, is pivotal to gathering valuable information for the in-depth characterization of biotherapeutics and biosimilarity assessment. Structural mass spectrometry methods (native MS, HDX-MS, top-down MS, etc.) provide information ranging from primary sequence assessment to higher order structure evaluation. This review focuses on recent developments and applications in structural mass spectrometry for biotherapeutic and biosimilar characterization.

Advanced mass spectrometry workflows for accurate quantification of trace-level host cell proteins in drug products: Benefits of FAIMS separation and gas-phase fractionation DIA.

Therapeutic monoclonal antibodies (mAb) production relies on multiple purification steps before release as a drug product (DP). A few host cell proteins (HCPs) may co-purify with the mAb. Their monitoring is crucial due to the considerable risk they represent for mAb stability, integrity, and efficacy and their potential immunogenicity. Enzyme-linked immunosorbent assays (ELISA) commonly used for global HCP monitoring present limitations in terms of identification and quantification of individual HCPs. Therefore, liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged as a promising alternative. Challenging DP samples show an extreme dynamic range requiring high performing methods to detect and reliably quantify trace-level HCPs. Here, we investigated the benefits of adding high-field asymmetric ion mobility spectrometry (FAIMS) separation and gas phase fractionation (GPF) prior to data independent acquisition (DIA). FAIMS LC-MS/MS analysis allowed the identification of 221 HCPs among which 158 were reliably quantified for a global amount of 880 ng/mg of NIST mAb Reference Material. Our methods have also been successfully applied to two FDA/EMA approved DPs and allowed digging deeper into the HCP landscape with the identification and quantification of a few tens of HCPs with sensitivity down to the sub-ng/mg of mAb level.

Online Collision-Induced Unfolding of Therapeutic Monoclonal Antibody Glyco-Variants through Direct Hyphenation of Cation Exchange Chromatography with Native Ion Mobility-Mass Spectrometry.

Post-translational modifications (PTMs) not only substantially increase structural heterogeneity of proteins but can also alter the conformation or even biological functions. Monitoring of these PTMs is particularly important for therapeutic products, including monoclonal antibodies (mAbs), since their efficacy and safety may depend on the PTM profile. Innovative analytical strategies should be developed to map these PTMs as well as explore possible induced conformational changes. Cation-exchange chromatography (CEX) coupled with native mass spectrometry has already emerged as a valuable asset for the characterization of mAb charge variants. Nevertheless, questions regarding protein conformation cannot be explored using this approach. Thus, we have combined CEX separation with collision-induced unfolding (CIU) experiments to monitor the unfolding pattern of separated mAbs and thereby pick up subtle conformational differences without impairing the CEX resolution. Using this novel strategy, only four CEX-CIU runs had to be recorded for a complete CIU fingerprint either at the intact mAb level or after enzymatic digestion at the mAb subunit level. As a proof of concept, CEX-CIU was first used for an isobaric mAb mixture to highlight the possibility to acquire individual CIU fingerprints of CEX-separated species without compromising CEX separation performances. CEX-CIU was next successfully applied to conformational characterization of mAb glyco-variants, in order to derive glycoform-specific information on the gas-phase unfolding, and CIU patterns of Fc fragments, revealing increased resistance of sialylated glycoforms against gas-phase unfolding. Altogether, we demonstrated the possibilities and benefits of combining CEX with CIU for in-depth characterization of mAb glycoforms, paving the way for linking conformational changes and resistance to gas-phase unfolding charge variants.

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.

A Novel Family of Acid-Cleavable Linker Based on Cyclic Acetal Motifs for the Production of Antibody-Drug Conjugates with High Potency and Selectivity.

Cleavable linkers have become the subject of intense study in the field of chemical biology, particularly because of their applications in the construction of antibody-drug conjugates (ADC), where they facilitate lysosomal cleavage and liberation of drugs from their carrier protein. Due to lysosomes' acidic nature, acid-labile motifs have attracted much attention, leading to the development of hydrazone and carbonate linkers among several other entities. Continuing our efforts in designing new moieties, we present here a family of cyclic acetals that exhibit excellent plasma stability and acid lability, notably in lysosomes. Incorporated in ADC, they led to potent constructs with picomolar potency in vitro and similar in vivo efficacy as the commercially available ADC Kadcyla in mouse xenograft models.

Large-Scale Conformational Changes of FhaC Provide Insights Into the Two-Partner Secretion Mechanism.

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane ß barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes on a slow time scale, with parts of the POTRA2 domain approaching the lipid bilayer and the protein's surface loops. Specifically, our data indicate that an amphipathic POTRA2 ß hairpin can insert into the ß barrel. We propose that these motions enlarge the channel and initiate substrate secretion. Our data propose a solution to the conundrum how TpsB transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase.

In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.

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.

Resistance to glycation in the zebra finch: Mass spectrometry-based analysis and its perspectives for evolutionary studies of aging.

In humans, hyperglycemia is associated with protein glycation, which may contribute to aging. Strikingly, birds usually outlive mammals of the same body mass, while exhibiting high plasma glucose levels. However, how birds succeed in escaping pro-aging effects of glycation remains unknown. Using a specific mass spectrometry-based approach in captive zebra finches of known age, we recorded high glycaemia values but no glycated hemoglobin form was found. Still, we showed that zebra finch hemoglobin can be glycated in vitro, albeit only to a limited extent compared to its human homologue. This may be due to peculiar structural features, as supported by the unusual presence of three different tetramer populations with balanced proportions and a still bound cofactor that could be inositol pentaphosphate. High levels of the glycated forms of zebra finch plasma serotransferrin, carbonic anhydrase 2, and albumin were measured. Glucose, age or body mass correlations with either plasma glycated proteins or hemoglobin isoforms suggest that those variables may be future molecular tools of choice to monitor glycation and its link with individual fitness. Our molecular advance may help determine how evolution succeeded in associating flying ability, high blood glucose and long lifespan in birds.

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.

The Importance of Charge in Perturbing the Aromatic Glue Stabilizing the Protein-Protein Interface of Homodimeric tRNA-Guanine Transglycosylase.

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since it is only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each mutated variant with respect to the dimer and thermal stability or enzyme activity by applying native mass spectrometry, a thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate a strong influence of pH on the homodimer stability. Apparently, protonation of a histidine within the aromatic cluster supports the collapse of an essential structural motif within the dimer interface at slightly acidic pH.

Toward Automation of Collision-Induced Unfolding Experiments through Online Size Exclusion Chromatography Coupled to Native Mass Spectrometry.

Ion mobility (IM)-based collision-induced unfolding (CIU) has gained increasing attention to probe gas-phase unfolding of proteins and their noncovalent complexes, notably for biotherapeutics. CIU detects subtle conformational changes of proteins and emerges as an attractive alternative to circumvent poor IM resolution. However, CIU still lacks in automation for buffer exchange and data acquisition, precluding its wide adoption. We present here an automated workflow for CIU experiments, from sample preparation to data interpretation using online size exclusion chromatography coupled to native IM mass spectrometry (SEC-CIU). Online automated SEC-CIU experiments offer several benefits over nanoESI-CIU, among which are (i) improved and fast desalting compared to manual buffer exchange used for classical CIU experiments; (ii) drastic reduction of the overall data collection time process; and (iii) maintaining the number of unfolding transitions. We then evaluate the potential of SEC-CIU to distinguish monoclonal antibody (mAb) subclasses, illustrating the efficiency of our method for rapid mAb subclass identification at both intact and middle levels. Finally, we demonstrate that CIU data acquisition time can be further reduced either by setting up a scheduled CIU method relying on diagnostic trap collision voltages or by implementing mAb-multiplexed SEC-CIU analyses to maximize information content in a single experiment. Altogether, our results confirm the suitability of SEC-CIU to automate CIU experiments, particularly for the fast characterization of next-generation mAb-based products.