Alain Beck
email: alain.beck@pierre-fabre.com

Guillaume Terral

François Debaene

Elsa Wagner-Rousset

Julien Marcoux

Marie-Claire Janin-Bussat

Olivier Colas

Alain Van Dorsselaer

Sarah Cianférani
email: sarah.cianferani@unistra.fr

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

Keywords: Antibody drug conjugate, bioanalysis, brentuximab vedotin, drug to antibody ratio, IdeS, ion mobility MS, native MS, mass spectrometry, trastuzumab emtansine MS, mass spectrometry, ESI, electrospray ionization, ADC, antibody-drug-conjugate, DAR, drug-to-antibody ratio, HDX, hydrogen/deuterium exchange, BV, brentuximab vedotin, T-DM1, trastuzumab emtansine, HOS, higher order structure, mAb, monoclonal antibody, HER2, human epidermal growth factor receptor 2, human immunoglobilin G, IgG, UHPLC, ultra-high performance liquid chromatography, MS/MS, tandem mass spectrometry, SEC, size exclusion chromatography, HIC, hydrophobic interaction chromatography, SMCC, succinimidyl-4- capillary electrophoresis-sodium dodecyl sulphate, IEF, isolelectrofocusing, icIEF, imaged capillary isoelectrofocusing reaction monitoring, Q-TOF, quadrupole time-of-flight, IM-MS, ion mobility mass spectrometry, ACN, acetonitrile, FA, formic acid, resonance, PK, pharmacokinetics, CD, circular dichroism, DSC, differential scanning calorimetry, UV, ultraviolet, Vis, visible, ELISA, enzyme-like immunosorbent assay

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Introduction

With advances in protein engineering technologies, monoclonal antibodies (mAbs) and their derivatives have emerged as the largest drug class in human therapeutics [START_REF] Beck | Strategies and challenges for the next generation of therapeutic antibodies[END_REF]. More than 60 antibody based pharmaceuticals such as ADCs, radio-immunoconjugates, bispecific antibodies, Fab fragments, Fc-fusion proteins and peptides have already been approved to treat cancer, autoimmune diseases, and more recently, to lower cholesterol levels [START_REF] Reiner | PCSK9 inhibitors -past, present and future[END_REF]. More than 50 others are under phase III clinical trials [START_REF] Reichert | Antibodies to watch in 2015[END_REF], with an approval rate (~20%) which compares favorably with that of other new chemical entities (~5%). The success of mAbs stems from their unique properties namely their high specificity and affinity (in the nM to pM range), their long circulating half-lives (up to 20 days) and their ability to induce immune cell effector response. For oncology therapy however, first generation mAbs are often inefficient or face resistance. To overcome these limitations, several families of armed antibodies are emerging-including radio-immunoconjugates [START_REF] Kraeber-Bodere | Radioimmunoconjugates for the treatment of cancer[END_REF], antibody-drug conjugates (ADCs, another class of immunoconjugates) [START_REF] Beck | The next generation of antibody-drug conjugates comes of age[END_REF], immunotoxins [START_REF] Madhumathi | Immunotoxin therapy for hematologic malignancies: where are we heading?[END_REF] and immunocytokines [START_REF] Danielli | Armed antibodies for cancer treatment: a promising tool in a changing era[END_REF], among which ADCs have so far proved the most successful, with two drugs already on the market.

Brentuximab vedotin (BV, marketed as Adcetris® by Seattle Genetics/Takeda) is indicated for the treatment of hematological malignancies (Hodgkin's lymphoma and systemic anaplastic large-cell lymphoma) while trastuzumab emtansine (T-DM1, marketed as Kadcyla® by Genentech/Roche) has been approved for the treatment of breast cancer patients resistant to human epidermal growth factor receptor 2 (HER2) [START_REF] Mendes | The benefit of HER2-targeted therapies on overall survival of patients with metastatic HER2-positive breast cancer -a systematic review[END_REF][START_REF] Beck | Review of Antibody-Drug Conjugates, Methods in Molecular Biology series: A book edited by Laurent Ducry[END_REF]. The ADCs in development target a wide range of cancers [START_REF] Klinguer-Hamour | World Antibody-Drug Conjugate Summit[END_REF][START_REF] Beck | Antibody-drug conjugates: present and future[END_REF]. Many of these new compounds have emerged from a better understanding of structure-function relationships, which have mainly been achieved thanks to state-of-the art mass spectrometry (MS) methods [START_REF] Sochaj | Current methods for the synthesis of homogeneous antibody-drug conjugates[END_REF][START_REF] Beck | World Antibody Drug Conjugate Summit Europe: February 21-23[END_REF], but also from lessons learned from pharmaceutical and clinical developments. An example of this process is Mylotarg® (gemtuzumab ozogamicin), which in 2000 became the first ADC approved by the FDA but was then withdrawn by the sponsor in 2010. One of the motivations for its withdrawal was its poor chemistry manufacturing and control characteristics, chiefly the presence of ~50% unconjugated mAbs competing with the ADC for antigen recognition and cell internalization [START_REF] Maeda | Charge heterogeneity of a therapeutic monoclonal antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin[END_REF][START_REF] Labrijn | Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo[END_REF][START_REF] Bross | Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia[END_REF]. This has been corrected for inotuzumab ozogamicin, another ADC from the same family, currently in clinical phase III, whose production process ensures the removal of naked antibodies.

The present review aims to draw an exhaustive picture of MS-based methodologies available for the characterization of ADCs at different levels, those of the intact conjugated protein, subunits thereof, peptides, and free drug-linker and related products (respectively the top, middle, bottom, and drug-linker levels, respectively). A particular focus will be MS approaches that allow ADCs to be analyzed in near native conditions and provide an assessment of their higher order structures.

ADC architecture

Antibody-drug conjugates (∼154 kDa) consist of a recombinant monoclonal antibody (∼148 kDa) covalently attached by a synthetic linker to a highly cytotoxic agent (0.3-1.5 kDa) [START_REF] Chari | Antibody-drug conjugates: an emerging concept in cancer therapy[END_REF].

The three structural components of an ADC are thereby (Figure 1): i) an antibody, which specifically recognizes cancer cells by binding to an overexpressed membrane antigen; ii) a payload, which is a potent warhead that inhibits the growth of the cancer cells upon its release; and iii) a cleavable or non-cleavable linker, which covalently binds the two moieties together. In ADCs therefore, the proven antigen-specific selectivity of mAbs is complemented by the potency of highly cytotoxic molecule [START_REF] Ornes | Antibody-drug conjugates[END_REF]. As a result, the systemic toxicity associated with traditional chemotherapeutic treatments is reduced and the therapeutic index improved.

Antibody carrier

From a structural standpoint, therapeutic chimeric, humanized, and human G immunoglobilins (IgGs) are tetrameric glycoproteins with molecular weights of approximately 150 kDa. They comprise two heavy chains (~50 kDa each) and two light chains (~25 kDa each). Disulfide bridges (16 for IgG1 and IgG4;18 for IgG2) and noncovalent interactions maintain their three-dimensional (3D) structure (H2L2 homoheterodimers). The heavy and light chains are linked by one disulfide bond and the heavy chains by two (for IgG1 and IgG4) or three (for IgG2) disulfide bonds located in a short hinge domain. The other 12 disulfide bridges are intramolecular and delimit six different globular domains. Antigen binding is mediated by the variable domains, mainly by three loops connecting individual β-strands in each domain named complementarity determining regions. Like natural IgGs, all recombinant antibodies contain an Asn-X-Ser/Thr-X′ (where X and X′ are any amino acid other than proline) consensus sequence for N-glycosylation in their heavy chain CH2 constant domain. On average, IgG glycans account for just 2-3 % of the total mass of the antibody.

Cytotoxic warheads

The vast majority of ADCs either approved or at the clinical trial stage utilize auristatin and maytansine derivatives-tubulin polymerization inhibitors-as payloads. Nevertheless an increasing number of clinical-stage ADCs exploit calicheamycins (DNA cleaving agents), duocarmycins (DNA alkylating agents), doxorubicins (DNA intercalating agents), pyrrolobenzodiazepines (DNA cross-linking agents), or SN38, an irinotecan metabolite (topoisomerase I inhibitor). These compounds are all highly toxic for cells and are more hydrophobic than the antibodies [START_REF] Chari | Antibody-drug conjugates: an emerging concept in cancer therapy[END_REF][START_REF] Flygare | Antibody-drug conjugates for the treatment of cancer[END_REF].

Linker properties

A plethora of linkers have been designed to connect the drug and antibody moieties. Most are stable in the bloodstream at physiological pH (7.4) and temperature but are labile once the ADC is inside the cells targeted by the antibody. They may contain conditional cleavage sites (acid-, lysosomal protease-or glutathione-sensitive) or not (depend on degradation of the mAb component in the lysosomes). In addition, their polarity (the number of charged residues) can be optimized to limit aggregation and increase or reduce the number of bystander cells killed.

Drug conjugation

Drug conjugation is most frequently achieved via reactions on side chains of two different amino acids: lysine side-chain amines or cysteine thiol groups after reduction of the interchain disulfide bonds [START_REF] Beck | Fourth World Antibody-Drug Conjugate Summit: February 29[END_REF]. The conjugated drugs already on the market-BV, a hinge cysteinelinked ADC, and T-DM1, a surface exposed lysine conjugate-are illustrative of two main ADC families currently in clinical trials (Figure 1).

Cysteine-linked ADCs are typically generated by partially reducing the interchain disulfides of the antibody hinge before alkylation with a cytotoxic agent, most frequently through maleimide chemistry [START_REF] Doronina | Erratum: Development of potent monoclonal antibody auristatin conjugates for cancer therapy[END_REF][START_REF] Senter | The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma[END_REF]. This class of ADCs forms controlled mixtures of molecular species with a variable number of drug molecules per antibody (known as the drugto-antibody ratio, DAR), a different location of the cytotoxic drug for a particular DAR, and a mixture of covalently and non-covalently associated light-and heavy-chain subdomains (Figure 2a).

Lysine conjugates are most commonly assembled via the formation of amide bonds between the epsilon amino group of endogenous lysine residues and activated esters [START_REF] Phillips | Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate[END_REF][START_REF] Blanc | SAR3419: an anti-CD19-Maytansinoid Immunoconjugate for the treatment of B-cell malignancies[END_REF].

Most IgGs contain ~90 lysine residues but only 32 cysteines, among which 8 only are involved in the interchain disulfide bridges of chimeric, humanized and human IgG1.

Therefore, although the number of drug molecules incorporated on average per antibody is similar [START_REF] Wang | Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry[END_REF], lysine conjugation yields a much more heterogeneous ADC population than cysteine conjugation does. In the reference lysine conjugate T-DM1 for instance, 40 out of the 88 lysine residues were found to be solvent-exposed. Lysine conjugation of T-DM1 proceeds through the reaction of trastuzumab with the heterobifunctional linker succinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (SMCC), producing a linker-modified intermediate. The cytotoxic agent DM1, which contains a free thiol group, is added in a second step and reacts with the maleimide to produce the thioether-linked drug conjugate. This results in an average DAR for T-DM1 of 3.5.

The instability of the chemical link between the mAb and the drug in systemic circulation is problematic (eg retro-Michael deconjugation) however. This drug loss modifies the composition of the ADC, potentially diminishing both the amount of drug delivered to the tumor site and its off-target toxicity and overall safety. Other points of concern stem from current bioconjugation methods that yield mixtures of antibodies with different DARs, and within each DAR species, different conjugation sites, each species potentially having different toxicities and ADME properties [START_REF] Roberts | Considerations for the nonclinical safety evaluation of antibody drug conjugates for oncology[END_REF]. Last but not least, the lack of high-resolution analytical techniques for their structural characterization is hampering the development of nextgeneration ADCs.

To extend the therapeutic window and to produce more homogeneous ADCs, a number of strategies have recently been developed allowing more site-specific conjugation [START_REF] Gordon | A Field Guide to Challenges and Opportunities in Antibody-Drug Conjugates for Chemists[END_REF]. This can be achieved either by adding engineered cysteine residues at specific sites without disruption of the interchain disulfide bonds [START_REF] Mcdonagh | Engineered antibody-drug conjugates with defined sites and stoichiometries of drug attachment[END_REF][START_REF] Hartley | a novel C2-aryl-substituted pyrrolobenzodiazepine dimer prodrug that cross-links DNA and exerts highly potent antitumor activity[END_REF][START_REF] Junutula | Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index[END_REF][START_REF] Sutherland | SGN-CD33A: a novel CD33targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML[END_REF] or by adding engineered peptide tags recognized by microbial transglutaminases to specifically transamidate amine-containing drug linkers attached to glutamine residues [START_REF] Dennler | Transglutaminase-Based Chemo-Enzymatic Conjugation Approach Yields Homogeneous Antibody-Drug Conjugates[END_REF][START_REF] Farias | Mass spectrometric characterization of transglutaminase based site-specific antibody-drug conjugates[END_REF]. Alternatively, unnatural amino acids can be inserted into the primary sequence of mAbs to provide a chemical handle on their conjugation [START_REF] Axup | Synthesis of site-specific antibody-drug conjugates using unnatural amino acids[END_REF][START_REF] Smith | Chemoenzymatic Fc glycosylation via engineered aldehyde tags[END_REF]. As an alternative to maleimide conjugation moreover, the ongoing development of new heterobifunctional reagents should facilitate the production of more stable ADCs [START_REF] Kolodych | CBTF: new amine-to-thiol coupling reagent for preparation of antibody conjugates with increased plasma stability[END_REF][START_REF] Koniev | MAPN: First-in-Class Reagent for Kinetically Resolved Thiol-to-Thiol Conjugation[END_REF]. To our knowledge, two to four of these next-generation ADCs have already reached the stage of phase I clinical trials [START_REF] Sutherland | SGN-CD33A: a novel CD33targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML[END_REF][START_REF] Jeffrey | A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology[END_REF][START_REF] Dorywalska | Effect of attachment site on stability of cleavable antibody drug conjugates[END_REF] and have shown an improved therapeutic index in pre-clinical toxicology studies [START_REF] Strop | Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading[END_REF].

The ADC analytical toolbox: a combination of native and denaturing methods

A large number of orthogonal analytical and bioanalytical methods are needed for the characterization of ADCs [START_REF] Beck | Review of Antibody-Drug Conjugates, Methods in Molecular Biology series: A book edited by Laurent Ducry[END_REF][START_REF] Alley | Analytical and bioanalytical technologies for characterizing antibody-drug conjugates[END_REF]. Table 1 lists the analytical, structural and functional assays reported in ADC quality control monographs as having been used either for batch release or for characterization and structural assessment [START_REF] Beck | World Antibody Drug Conjugate Summit Europe: February 21-23[END_REF][START_REF] Beck | Fourth World Antibody-Drug Conjugate Summit: February 29[END_REF][START_REF] Reichert | 9th annual European Antibody Congress[END_REF][START_REF] Hamilton | Antibody-drug conjugates for cancer therapy: The technological and regulatory challenges of developing drug-biologic hybrids[END_REF].

ADCs present an increased level of complexity compared to naked mAbs.

As discussed above, most of the current clinical-stage ADCs consist of a heterogeneous population of species with a variable number of drug molecules per antibody (Figure 2a,b).

Most frequently, an average number of 4 cytotoxic payloads are covalently attached to the mAb. For cysteine ADCs for example, conjugates with zero, two, four, six or eight cytotoxic payloads per mAb are obtained, such that the average DAR is four [START_REF] Senter | The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma[END_REF]. A discrete distribution of odd and even drug loads is observed for lysine ADCs, as a result of the greater heterogeneity of the population. However, whereas lysine ADCs are assemblies maintained by covalent interactions between heavy and light chains (no reducing step that opens the disulfide bridges), cysteine ADC populations are mixtures of covalent and non-covalent compounds due to the presence of drugs at the interchain cysteine residues, making their analysis more complex. Analytical techniques and workflows therefore need to be adapted to obtain a clear and comprehensive understanding of the relationships between conjugation and the resulting product quality and heterogeneity. inserting non-natural amino acids; engineering (3) the glycans or (4) the N-terminus of heavy and light chains, [START_REF] Beck | The next generation of antibody-drug conjugates comes of age[END_REF] adding engineered tags; [START_REF] Madhumathi | Immunotoxin therapy for hematologic malignancies: where are we heading?[END_REF] non-covalent binding using the affinity properties of Fc-binding domain; [START_REF] Danielli | Armed antibodies for cancer treatment: a promising tool in a changing era[END_REF] photoaffinity labeling on the nucleotide binding site; and [START_REF] Mendes | The benefit of HER2-targeted therapies on overall survival of patients with metastatic HER2-positive breast cancer -a systematic review[END_REF] the conjugation of a pharmacophore on the catalytic site.

Main quality attributes for the characterization of ADCs

The most important ADC characteristics required for process and formulation development, routine lot-release, and stability testing are the following [START_REF] Wakankar | Analytical methods for physicochemical characterization of antibody drug conjugates[END_REF]. The average DAR determines the amount of payload that can be delivered to the tumor cell and affects both the toxicity and safety of the ADC. The distribution of drug-loads (i.e. the fraction of antibodies containing zero, one, two, three, … n drugs) is also an important characteristic since the different forms may have different toxicological and pharmacological properties. Because the cytotoxic drugs linked to antibodies are hydrophobic, conjugates are more likely to aggregate during manufacturing and storage. Furthermore, size variants-in particular those with high molecular weights (aggregates)-can modify the pharmacokinetics (that is, accelerate clearance) and reduce drug exposure. Finally, the concentrations of free drug (from cleavable linkers) or drug-linker compounds (from non-cleavable linkers) have to be controlled, since the unconjugated drug and its related products may pose serious toxicity and safety threats.

Residual quantities of unconjugated drug or drug-related impurities may remain in the final product as a result of incomplete purification or of degradation during long-term storage. The proportion of unconjugated mAb must also be controlled.

State-of-the-art analytical methods for the characterization of ADCs

Average DAR measurements A variety of methods have been used to measure the average DAR, the most appropriate depending on the properties of the cytotoxic drug and how it is linked to the antibody. The simplest method for estimating DARs is based on ultraviolet/visible (UV/Vis) spectroscopy [START_REF] Chen | Drug-to-antibody ratio (DAR) by UV/Vis spectroscopy[END_REF], but the limitations of this approach (namely the UV photolability of calicheamicin, the lability of the linker, over-quantitation due to high concentrations of non-covalently bound drug) make confirmation using an orthogonal method necessary [START_REF] Hamblett | Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate[END_REF]. To measure the DAR of cysteine-linked drug conjugates, the most widely employed technique is hydrophobic interaction chromatography (HIC) [START_REF] Junutula | Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index[END_REF][START_REF] Hamblett | Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate[END_REF][START_REF] Le | Mathematical approach using inputs from capillary electrophoresis and hydrophobic interaction chromatography to determine the positional isomer distribution within a population of ADCs 48[END_REF][48][START_REF] Haverick | Monoclonal antibody conjugates of doxorubicin prepared with branched peptide linkers: inhibition of aggregation by methoxytriethyleneglycol chains[END_REF]. This is performed under non-denaturing conditions at neutral pH with a gradient from high to low salt concentration. A low amount of organic modifier can also be included in the (low salt) mobile phase to improve the elution of mAbs loaded with lipophilic drugs. Under these conditions, each peak in the resulting chromatogram corresponds to an intact mAb species bound to a specific number of bound drug molecules (Figure 3b). Finally, native MS has recently emerged as an alternative technique in this context, as discussed below.

Drug load distribution

As for the average DAR, chromatography is the technique most commonly used to measure the drug-load distribution, sometimes coupled to MS. Chromatographic methods separate mAbs with different payloads based on the increased hydrophobicity imparted to the antibody upon conjugation with the drug linker. When the drug is connected to the antibody through inter-chain disulfide bonds, HIC (performed under non-denaturing and non-reducing conditions) is the benchmark method used to resolve the drug-load variants [START_REF] Alley | Analytical and bioanalytical technologies for characterizing antibody-drug conjugates[END_REF]50]. Reverse phase liquid chromatography (rpHPLC) (under reducing conditions) and sodium dodecyl sulphate capillary electrophoresis (SDS-CE, under both non reducing and reduction conditions)s can serve as orthogonal techniques to confirm the drug loading profile of cysteine ADCs [START_REF] Le | Mathematical approach using inputs from capillary electrophoresis and hydrophobic interaction chromatography to determine the positional isomer distribution within a population of ADCs 48[END_REF]48]. When the conjugation is made through lysine amine residues, ion isolelectrofocusing (IEF) or imaged capillary isoelectrofocusing (icIEF) can be used to resolve the different ADC products. In this case, conjugation decreases the net positive charge of the mAb by one for each attached drug linker, if the drug-linker is itself uncharged. Finally, rpHPLC works well for ADC forms conjugated at specific sites (e.g., at inter-chain cysteines), but is less useful for more heterogeneous lysine-linked conjugates.

Size variants

These are typically determined by size exclusion chromatography (SEC), as is the case for intact mAbs. It is worth mentioning however that regular SEC using phosphate buffered mobile phase yields poor peak shapes for ADCs and an insufficient resolution between aggregates and monomeric ADC products [51]. This is probably the result of non-specific interactions between the cytotoxic drugs and the surface of the stationary phase. To overcome these difficulties, various organic modifiers have been added to the SEC mobile phase-such as 25% propylene glycol [START_REF] Hollander | Selection of reaction additives used in the preparation of monomeric antibody-calicheamicin conjugates[END_REF] or 10% DMSO [START_REF] Quiles | Synthesis and preliminary biological evaluation of high-drug-load paclitaxel-antibody conjugates for tumortargeted chemotherapy[END_REF]-to improve the shape of the resulting peaks. Alcohol-type organic modifiers may also prove valuable in improving the SEC performance of ADC products. For reduced fragments on the other hand-that is, variants with reduced charge namely light chains and heavy chains with different drug loads-generic rpHPLC has successfully been used [50].

Free drug-linker quantification:

Concentrations of free drug molecules can be determined by enzyme-like immunosorbent assay (ELISA) and CE, but rpHPLC is currently considered the gold standard technique since it often provides the best selectivity, sensitivity and precision for cytotoxic drugs, which are typically lipophilic, low molecular weight compounds [START_REF] Fleming | A reversed-phase high-performance liquid chromatography method for analysis of monoclonal antibody-maytansinoid immunoconjugates[END_REF]. This approach can be followed by UV/MS, allowing the identification and quantification of various cytotoxic species released from the conjugate in stability testing studies.

In summary, the analytical methods most appropriate to characterize a given ADC strongly depend on the chemical properties of the linker, the cytotoxic payload and the attachment anchor (i.e. lysine or inter-chain cysteines) to the antibody. One should bear in mind that the assay (cationic exchange chromatography, for instance) used for the parent mAb may not always work for the corresponding ADC.

A multi-level workflow for the comprehensive characterization of ADCs

Dozens of mAb microvariants have been identified and reported in the literature, which differ in terms of their glycoform, charge, cysteine attachment, oxidation state, size, and gene sequence [START_REF] Beck | Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry[END_REF][START_REF] Beck | Characterization of therapeutic antibodies and related products[END_REF][START_REF] Beck | Analytical characterization of biosimilar antibodies and Fc-fusion proteins[END_REF][START_REF] Beck | Cutting-edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies[END_REF]. These microvariants and payloads-mAb conjugation both increase the structural complexity of the final drug substance [START_REF] Beck | Harness the benefits of mass spectrometry for in-depth Antibody Drug Conjugates analytical characterization[END_REF], with over-deamidation typically occurring due to conjugation at basic pHs [START_REF] Beck | Harness the benefits of mass spectrometry for in-depth Antibody Drug Conjugates analytical characterization[END_REF].

Figure 3 presents a general workflow for determining the critical quality attributes of ADCs. This multi-level approach allows the quality and heterogeneity of ADCs to be assessed at the level of the primary structure, but also probes the changes to the higher order structure arising from payload conjugation. The top level of the analysis involves mass measurements of intact ADCs (> 150 kDa), typically using electrospray sources, either under native or classical denaturing conditions, which yield accuracies in the 30-100 ppm range and reveal the glycoform heterogeneity, drug-load profile and the average DAR of covalent conjugates.

If performed under classical denaturing conditions, this analysis is readily coupled to liquid chromatography (HPLC), enabling automated HPLC-MS experiments. On the other hand, native MS provides the drug or antigen binding stoichiometries. Top-down approaches can also be used to sequence intact mAbs, without proteolytic digestion [START_REF] Fornelli | Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS[END_REF][START_REF] Tsybin | Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry[END_REF][START_REF] Mao | Top-down structural analysis of an intact monoclonal antibody by electron capture dissociation-Fourier transform ion cyclotron resonance-mass spectrometry[END_REF].

The next (middle) involves the analysis of large ADC fragments . The subunits obtained by reduction or enzymatic cleavage are amenable to LC-MS analysis, which with latest-generation spectrometers provides isotopic resolution and mass accuracies under 5 ppm. Improved fragmentation capabilities (using collision induced dissociation, and/or electron transfer dissociation) make middle-level approaches less demanding than intact mAb/ADC top-down analysis [START_REF] Fornelli | Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry[END_REF].

A peptide-level (bottom level) analysis of the ADC (0.7-7 kDa fragments) is still required if information on the primary sequence and/or the conjugation site are required.

Classically, the cysteines are reduced and alkylated before the ADC is digested (with trypsin or endoproteinases such as Lys-C, Asp-N, Glu-C, or pepsin) and the resulting peptide fragments are identified through LC-MS/MS experiments and further searches in protein databases. Tandem mass spectrometry is also usually performed to sequence the peptides and to locate post-translational modifications such as glycosylation and the formation of disulfide bridges, notable at the conjugation sites.

The higher order structures (HOSs) of proteins (secondary, tertiary, and quaternary arrangements) are often responsible for their uniqueness and can govern their function, by preventing binding to antigens or Fcgamma and FcRn receptors for example. In this context, recent reports from the EMA or FDA have pointed out that knowledge of the HOSs of biopharmaceutical compounds is crucial for meaningful comparative studies between innovator products, biosimilars, and biobetters, but also, in case of ADCs, to assess the impact of conjugation on the overall structure of the mAb. As discussed below, this structural assessment can be performed by IM-MS and hydrogen/deuterium exchange coupled to MS (HDX-MS).

The fifth level of characterization presented here stems from the potential safety threats from residual drug-linker and related products in ADC batches, as mentioned above.

These compounds can be quantified using the MS strategies for selected reaction monitoring (SRM) that have been developed for similar bioanalyses.

The relevance of this multi-level ADC characterization workflow is outlined in the next sections with two example applications, for BV and T-DM1.

Hinge-cysteine linked ADCs: BV as a case study

Brentuximab vedotin is a potent ADC composed of the monoclonal antibody cAC10, which targets the CD30 antigen on Hodgkin lymphoma and systemic anaplastic large-cell lymphoma cells; a highly stable valine-citrulline linker; and a potent chemotherapeutic agent monomethyl auristatin E, which inhibits microtubule polymerization (Figure 1a). The conjugation strategy used for BV involves partially reducing the interchain disulfides of the anitbody prior to conjugation, yielding an ADC with 0-8 drug molecules loaded per antibody and an average DAR of four [START_REF] Senter | The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma[END_REF]. The presence of drug molecules at the interchain cysteine residues means that BV is a composite of covalently and non-covalently associated light and heavy chain subdomains (Figure 4a).

Top level: intact BV analysis

Classical intact mass measurements under denaturing conditions are not appropriate for the characterization of cysteine-linked ADCs

The first step in the analysis of intact proteins is often a classical ESI-MS analysis under denaturing conditions, typically in an acidified H 2 O/acetonitrile solvent, either through direct infusion or by rpHPLC-MS analysis. For BV however, these harsh solvent conditions disrupt the non-covalent interactions between the light and heavy chain, hampering the detection of the intact ADC. As a result, the most intense MS signals usually arise from drug-linked antibody fragments, from both the heavy and the light chain, while minor signals are obtained from the free unconjugated mAb (Figure 4c) [START_REF] Valliere-Douglass | Native intact mass determination of antibodies conjugated with monomethyl Auristatin E and F at interchain cysteine residues[END_REF]65]. This suggests that denaturing MS analyses are inappropriate for the characterization of intact cysteine-linked ADCs and that a one-step determination of the drug load profile and average DAR is not possible for these compounds in classical denaturing conditions.

Native and Ion Mobility MS as alternative methods for drug load profiling and average DAR measurements Native MS has been used extensively for more than 25 years to study protein/ligand complexes from a variety of systems (for reviews, see [66,[START_REF] Pacholarz | Mass spectrometry based tools to investigate protein-ligand interactions for drug discovery[END_REF]) but also to detect very large protein assemblies weighing several million Daltons [START_REF] Rajabi | Mass spectrometric methods to analyze the structural organization of macromolecular complexes[END_REF][START_REF] Bereszczak | Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids[END_REF]. It has more recently been shown to be a powerful technique for the analysis of intact mAbs [START_REF] Beck | Biosimilar, biobetter, and next generation antibody characterization by mass spectrometry[END_REF][START_REF] Beck | Analytical characterization of biosimilar antibodies and Fc-fusion proteins[END_REF][START_REF] Beck | Cutting-edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies[END_REF][START_REF] Rosati | Detailed mass analysis of structural heterogeneity in monoclonal antibodies using native mass spectrometry[END_REF][71][START_REF] Zhang | Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies[END_REF][START_REF] Thompson | Performing native mass spectrometry analysis on therapeutic antibodies[END_REF][START_REF] Debaene | Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and "Bispecific" Monoclonal Antibody Formation[END_REF][START_REF] Rosati | Exploring an orbitrap analyzer for the characterization of intact antibodies by native mass spectrometry[END_REF]. Valliere-Douglass et al. were the first to report the use of ESI-MS coupled to SEC under native conditions to record the mass of a cysteine-linked ADC and assess the relative distribution of drug-linked species [START_REF] Valliere-Douglass | Native intact mass determination of antibodies conjugated with monomethyl Auristatin E and F at interchain cysteine residues[END_REF]. The masses of the SEC-desalted ADCs were determined, using an ESI source and standard desolvation and ionization conditions, on a Q-TOF instrument for IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at inter-chain cysteine residues.

Elsewhere, Chen et al. employed nano-instead instead of conventional ESI in combination with limited enzymatic digestion to directly determine DARs [START_REF] Chen | Development of a native nanoelectrospray mass spectrometry method for determination of the drug-to-antibody ratio of antibody-drug conjugates[END_REF]. Since then, several papers have used this technique to characterize the drug load distribution and to evaluate the average DAR for several cysteine linked ADCs, with results in good agreement with those obtained by HIC or UV/vis spectroscopy [65,[START_REF] Thompson | Performing native mass spectrometry analysis on therapeutic antibodies[END_REF][START_REF] Rosati | Indepth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap[END_REF][START_REF] Marcoux | Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate[END_REF] The main advantage of using native MS to characterize cysteine-linked ADCs lies in its ability to detect non-covalent associations of light and heavy chains, as these are not revealed by classical rpHPLC-MS methods. Furthermore, native MS data obtained on most quadrupole time-of-flight or orbirtrap instruments are sufficiently resolved to confirm the identity of the ADCs, and the relative distributions of the drug-loaded species derived from these data are in good agreement with those measured by HIC. Two additional advantages of native MS are its efficiency-with measurements completed in minutes rather than an hour for HIC-and the limited sample handling that is required, which limits the risk of artifacts appearing in the data. The process could indeed be automated by using one of several commercially-available SEC-native MS workflows.

Native Top Down MS of BV

Top-down MS is an emerging approach for the amino acid sequencing of intact proteins, providing valuable information on protein isoforms (proteoforms) [START_REF] Smith | Proteoform: a single term describing protein complexity[END_REF][START_REF] Tran | Mapping intact protein isoforms in discovery mode using top-down proteomics[END_REF], with the limited sample manipulation again reducing the occurrence of artifacts in the data. Top-down approaches have so far been largely performed on Fourier transform ion cyclotron resonance and Orbitrap instruments. While top-down analysis has been used to sequence mAbs [START_REF] Fornelli | Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS[END_REF][START_REF] Tsybin | Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry[END_REF][START_REF] Mao | Top-down structural analysis of an intact monoclonal antibody by electron capture dissociation-Fourier transform ion cyclotron resonance-mass spectrometry[END_REF][START_REF] Zhang | Characterization of variable regions of monoclonal antibodies by top-down mass spectrometry[END_REF][START_REF] Bondarenko | Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer[END_REF][START_REF] Ren | Top-down Nterminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry[END_REF], their (large) size means that currently achievable resolutions only provide 30% sequence coverage at best and incomplete sequencing of the complementarity determining regions [START_REF] Fornelli | Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS[END_REF]. The top-down (middle-level) analysis of smaller mAb fragments has proved more successful however, with 70% sequence coverage having been achieved [START_REF] Fornelli | Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry[END_REF].

A recent interest in combining top-down proteoform analysis and structural characterization has lead to native top-down approaches being combined with ion mobility spectrometry either Q-TOF [START_REF] Lermyte | Electron transfer dissociation provides higher-order structural information of native and partially unfolded protein complexes[END_REF] or Orbitrap instruments [START_REF] Dyachenko | Tandem Native Mass-Spectrometry on Antibody-Drug Conjugates and Submillion Da Antibody-Antigen Protein Assemblies on an Orbitrap EMR Equipped with a High-Mass Quadrupole Mass Selector[END_REF]. Tandem native MS was successfully used by Dyachenko et al. for BV to show that drug conjugation takes place inhomogeneously at cysteine residues on both the light and heavy chains [START_REF] Dyachenko | Tandem Native Mass-Spectrometry on Antibody-Drug Conjugates and Submillion Da Antibody-Antigen Protein Assemblies on an Orbitrap EMR Equipped with a High-Mass Quadrupole Mass Selector[END_REF]. Thanks to the implementation of a high mass quadrupole on a high resolution Orbitrap instrument, precursor ions corresponding to one specific DAR were selected and then sequenced thanks to the MS/MS capabilities of the instrument. This located the conjugation site of the drugs in the antibodies and even revealed the positional isomers of the DAR 2 and DAR 6 species.

Middle level: BV subunit analysis

Even if structural insights can be obtained for intact ADCs by native MS and IM-MS, the higher mass accuracy provided by the more straightforward rpHPLC-MS analysis of their subunits remains valuable. As for unconjugated mAbs, ADC profiles can be simplified by reduction [START_REF] Beck | Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells[END_REF] (yielding the light and heavy chains at ~25 and ~50 kDa, respectively) or enzymatic treatments, such as N-deglycosylation (IgGZERO® or PNGase-F) [START_REF] Wagner-Rousset | Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion[END_REF], carboxypeptidase B digestion [START_REF] Beck | Characterization by liquid chromatography combined with mass spectrometry of monoclonal anti-IGF-1 receptor antibodies produced in CHO and NS0 cells[END_REF] or glutaminyl-peptide cyclotransferase treatment [89].

Smaller mAb fragments can also be generated by papain digestion (producing ~50 kDa Fab/Fc fragments) or IdeS digestion (Fabricator®, the immunoglobulin-degrading enzyme of Streptococcus pyogenes) followed by reduction with dithiothreitol (DTT, for Fc/2, LC and Fd fragments of ~25 kDa), as illustrated in Figure 4. This approach has the advantages of being fast (less than 2 h for the entire analysis including digestion and rpHPLC-MS analysis), informative, and inexpensive in terms of materials. Reduction experiments leading to individual light and heavy chains or IdeS treatment are to our knowledge currently mostly used for middle level analysis.

Characterization of BV subunits under denaturing and reducing conditions (middle level, 23-54 kDa fragments)

Reducing treatments are a routine way to divide the analysis of mAbs and ADCs into more manageable pieces. The drug loading profile and average DAR can then be obtained by rpHPLC with MS, as an orthogonal method to HIC [START_REF] Basa | Drug-to-antibody ratio (DAR) and drug load distribution by LC-ESI-MS[END_REF]-as for the latter, rp-HPLC relies on differences in hydrophobicity. This middle-up strategy can be implemented on any current HPLC-MS instrumentation and is therefore available in most labs. Treatment of cysteinelinked ADCs with DTT or tris(2-carboxyethyl)phosphine (TCEP) fully reduces the remaining inter-chain disulfides and yields six species: light chains with zero or one drug molecule attached (L0 and L1), and heavy chains with zero, one, two or three drug molecules attached (H0, H1, H2 and H3). These species are stable in the denaturing organic solvent and can be successfully separated on a reversed phase column. The average DAR is obtained by calculating the weighting the proportions of the total integrated intensity under the light and heavy chain peaks according to their associated drug loads (Figure 5) [START_REF] Lyon | Conjugation of Anticancer Drugs through Endogenous Monoclonal Antibody Cysteine Residues[END_REF][START_REF] Mc | Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis[END_REF]. Characterization of BV subunits after enzymatic cleavage (middle-level, 23-28 kDa fragments) Downsized mAb or ADC fragments an also be obtained by limited proteolytic cleavage under non-denaturing conditions in the hinge region of the heavy chain, yielding Fab or (Fab') 2 , and Fc fragments, whose reduction (with DTT) produces even smaller fragments of approximately 25 kDa: the light chain and the two halves of the heavy chain (Fc/2 and Fd). Formerly conducted with proteases with a limited specificity, such as papain [START_REF] Yan | Analysis of post-translational modifications in recombinant monoclonal antibody IgG1 by reversed-phase liquid chromatography/mass spectrometry[END_REF], pepsin [START_REF] Gadgil | The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations[END_REF], and endoprotease Lys-C [START_REF] Gadgil | Identification of cysteinylation of a free cysteine in the Fab region of a recombinant monoclonal IgG1 antibody using Lys-C limited proteolysis coupled with LC/MS analysis[END_REF], the enzymatic cleavage for middle level analyses is mostly conducted using IdeS, a bacterial protease that specifically cleaves IgGs under the hinge region [START_REF] Chevreux | Fast analysis of recombinant monoclonal antibodies using IdeS proteolytic digestion and electrospray mass spectrometry[END_REF]. The potency of IdeS has been demonstrated for cysteine-linked ADCs on an antibody-fluorophore conjugate [START_REF] Wagner-Rousset | Antibody-drug conjugate model fast characterization by LC-MS following IdeS proteolytic digestion[END_REF]. A rapid IdeS-and rpHPLC-MS-based procedure was also recently employed by Firth et al. for the characterization of two auristatin ADCs [START_REF] Firth | A rapid approach for characterization of thiol-conjugated antibody-drug conjugates and calculation of drug-antibody ratio by liquid chromatography mass spectrometry[END_REF].

With IdeS, a complete middle level characterization can be completed within a few hours, providing the primary sequence and the glycoprofiles of the Fab and Fc fragments. The data can also be used for biosimilar comparability studies and Fc-fusion protein studies. More recently, IdeS digestion has been shown to be preferable to DTT treatment for the characterization of BV, the LC fragments being better separated in the subsequent rpHPLC-MS analysis allowing the identification of positional isomers (Figure 6) [START_REF] Mc | Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis[END_REF]. In addition to the seven expected major peaks (from the Fc/2, L0, L1, Fd0, Fd1, Fd2 and Fd3 fragments) two minor satellite peaks with identical masses are observed close to those from Fd1 and Fd2 (Figure 6) and tentatively assigned to positional isomers. This was confirmed by peptide mapping with nanoLC-MS/MS following the digestion with endoprotease Lys-C of isolated fragments, as discussed below. Following denaturation, reduction, and alkylation of the Cys residues, mAbs or recombinant proteins are commonly digested with trypsin or endoproteinases such as Lys-C, Asp-N, or Glu-C. Samples can be prepared automatically thus facilitating multiple analyses. The extra hydrophobicity from the drug molecule means however that peptide mapping is more difficult for cysteine-linked ADCs than for mAbs, especially for species bearing two or more conjugated drugs [START_REF] Wakankar | Analytical methods for physicochemical characterization of antibody drug conjugates[END_REF]. As a result, ADCs have seldom been characterized at the peptide level.

Junutula et al. describe the site-specific conjugation of vcMMAE to an antibody through engineered cysteine substitutions on the light and heavy chains resulting in only tree isoforms with zero, one or two drug molecules and no positional isomer [START_REF] Junutula | Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index[END_REF]. The tryptic peptide mapping of these conjugates, with LC-MS detection, identified four drug-conjugated peptides via a characteristic in-source fragmentation ion (m/z = 718.5) that is observed in all the mass spectra of molecules containing vcMMAE. The peptides were all found to be fragments of either complete or partial tryptic cleavage around the engineered cysteines. More recently, Janin-Bussat et al. reported an improved BV peptide mapping protocol allowing the drugloaded peptides to be identified by LC-MS analysis [START_REF] Mc | Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis[END_REF]. Because of the hydrophobicity of the drug, all steps of the peptide mapping protocol were adjusted to maintain the hydrophobic drug-loaded peptides in solution and enable their unambiguous identification by LC-MS. In particular, solvents were added immediately before (10% acetonitrile) and after (40% isopropanol) the enzymatic digestion step. The resulting data showed that as expected, the drug molecules were linked to the inter-chain cysteines of the heavy and light chains. Furthermore, after IdeS digestion and reduction of the ADC, the positional isomers were identified for the first time by LC-MS/MS analysis, unambiguously demonstrating that when only one drug molecule binds, the link forms on Cys220 of the L15 peptide of the heavy chain.

Conversely, when two drug molecules attach to the heavy chain, they bind preferentially to Cys226 and Cys229 of the L16 peptide [START_REF] Mc | Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC-MS analysis[END_REF]. Interestingly, Birdsall et al. recently demonstrated a rapid on-line method based on MS and multidimensional chromatography, which allowed them to confirm the structure of a cysteine-conjugated ADC base on an IgG1 [START_REF] Birdsall | A rapid on-line method for mass spectrometric confirmation of a cysteine conjugated antibody-drug-conjugate structure using multidimensional chromatography. mAbs[END_REF]. The HIC peaks in the 1 st dimension were successfully assigned to specific subunits via MS following dissociation under denaturing reverse-phase conditions (the 2 nd dimension).

Elsewhere, a comparative characterization by LC-MS of the cysteine-linked conjugation profiles of IgG1 and IgG2 ADCs has been performed by Wiggins et al. [99]. The results demonstrated that IgG1 monoclonal antibodies favor conjugation to the cysteines between the light and heavy chains, whereas IgG2s link preferentially to cysteines in the hinge region.

Lysine linked ADCs: T-DM1 as a case study

Trastuzumab emtansine is the third ADC to have received market approval and is indicated for the treatment of HER2-positive breast cancer patients [START_REF] Phillips | Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate[END_REF]. Trastuzumab (Herceptin®) which mitigates the effects of HER2 overexpression, is coupled to a maytansine derivate (DM1) which inhibits the elongation of microtubules. In contrast with BV, T-DM1 can be conjugated without reduction, so that T-DM1 exists only as covalent species. Zero to eight drug molecules are attached with an average DAR of 3.5 [101].

This suggests that T-DM1 is easier to characterize than BV; however, the methods (primarily HIC) that are used to determine the average DAR for cysteine-conjugates are difficult to adapt for the analysis of lysine-conjugates. Indeed, the high degree of heterogeneity that characterizes lysine ADCs, which have at least 40 lysines available for conjugation, makes UV/vis spectroscopy and MS approaches more suitable [START_REF] Chen | Drug-to-antibody ratio (DAR) by UV/Vis spectroscopy[END_REF]. The systems amenable to the former are rather few in number as i) the drug must be detectable by UV/vis wavelengths; ii) the drug and mAb must have different maximum absorptions and iii) the absorption of light should not influence the interaction between the drug and the mAb.

Lysine-linked conjugate species with different drug loads can be resolved by icIEF, but the presence of charge-associated variants of the antibody intermediate and any cross-linking or differences in the UV responses of the differentially drug-loaded forms may prevent quantification [102,103].

Mass spectrometry based techniques are therefore pivotal for the characterization of lysine-conjugated ADCs [START_REF] Wang | Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry[END_REF][START_REF] Basa | Drug-to-antibody ratio (DAR) and drug load distribution by LC-ESI-MS[END_REF][104][105][106]. Several papers have demonstrated the potential of LC-MS and SEC-MS to respectively measure the average DAR and drug load distribution on maytansin-conjugated mAbs [START_REF] Wang | Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry[END_REF][START_REF] Basa | Drug-to-antibody ratio (DAR) and drug load distribution by LC-ESI-MS[END_REF]101]. Marcoux et al. have recently demonstrated the ability of native MS and native IM-MS to characterize lysine-conjugated ADCs, notably T-DM1 [START_REF] Marcoux | Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate[END_REF]. These authors first compared the potential of native MS versus denaturing MS for the determination of average DARs and drug load distributions. The presence of highly charged species of T-DM1 leads to charge state overlap, especially under denaturing conditions, such that only species with DARs of zero to seven (at best) can be observed (Figure 7a). High-resolution native MS is nonetheless beneficial as it reveals DAR 8 species at the intact protein level (Figure 7b). Charge reduction using imidazole also allows the detection of all the species from DAR 0 to DAR 8, and the average DAR thus obtained for T-DM1, 3.5 ± 0.1, is in agreement with the value reported by Lazar et al. [101]. As for cysteine ADCs, native IM-MS highlights the increase in heterogeneity that occurs when trastuzumab is conjugated with DM1 (Figure 7c).

Site specific ADCs

As discussed and illustrated above, the three market-approved ADCs, and most of those under clinical trial are controlled mixtures of molecules. In 2008, Junutula et al. reported the design and production of a new class of homogeneous ADCs, thiomab-drug conjugates (TDCs), prepared by conjugation to engineered IgGs containing additional exposed cysteine residues whose position were identified using phage display methods [START_REF] Junutula | Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index[END_REF]. After reducing the blocked cysteine residues and interchain disulfides, the latter were regenerated and maleimide reagents were conjugated to the reactive cysteine thiol group to generate site-specifically modified ADCs with 92.1 % of the population having two loaded molecules and only 0.2, 3.3, and 4.3 % having zero, one, or three, respectively (average DAR = 2.0), as assessed by rpHPLC-MS. Importantly, these TDCs are better tolerated at higher doses in animals, indicating that their therapeutic index is higher than that of existing ADCs. A plethora of other site specific techniques have subsequently been reported, as recently reviewed (amongst others) by Panowksi et al. [109], Agarwal and Bertozzi [110] and Dennler et al. [111], and summarized in Figure 2c. Interestingly, Junutula and colleagues published another paper, in 2012 [112],

indicating that their first-generation TDCs were not very stable in the blood stream. Thanks to MS, the degradation mechanism was shown to be a retro-Michael reaction, resulting in the transfer of the drug linker to circulating molecules bearing free thiol groups such as albumin and glutathione [113]. In addition, based again on mass shift measurements, succimide ring hydrolysis was shown to stabilize the drug linker on the antibody was observed, with a mass increase of 18 Da (H20). This ring opening was correlated with a positively charged local site. Succinimide hydrolysis has since been employed to improve the stability (and hence the safety) of several ADCs [114]; different strategies have been adopted, notably the processfavored mild method based on a basic pH treatment [115], but also self-hydrolyzing maleimides [114] and electron withdrawal N-substituents [116].

Alternatively, the specific conjugation to N-glycans of IgG (at Asn297) can be achieved by periodate oxidation of fucose followed by hydrazone condensation, metabolic incorporation of 6-thiofucose and then maleimide conjugation [117]. The process is completed using specific enzymes such as enzymatic transfer of galactose and sialic acid followed by periodate oxidation and oxime condensation, enzymatic transfer of galactose and 9-azidosialic acid followed by Cu-free click reaction, enzymatic removal of terminal galactose followed by enzymatic transfer of GalNAz and Cu-free click reaction [118]. Each step in the reaction can be monitored by LC-MS, which can then also be used to assess the structures [119], and the glyco-profiles [120].

Conjugation can also be achieved through the incorporation of non canonical aminoacids-such as p-acetylphenylalanine sometimes with an azido-lysine derivative-and then oxime condensation and Cu-free click chemistry. The cell-free incorporation of pazidomethylphenylalanine followed by Cu-free click chemistry, and the incorporation of selenocysteine followed by mild reduction and alkylation have also been described [121]. As for other site-specific conjugation techniques, the choice of attachment site may affect the stability of the resulting ADCs [START_REF] Dorywalska | Effect of attachment site on stability of cleavable antibody drug conjugates[END_REF].

Conjugation methods based on the enzymatic modification of peptide tags have also been explored extensively. One method involves glycosidase treatment-to access Q295 (and N297Q mutants)-prior to the transglutaminase-mediated conjugation of aminefunctionalized small molecules or drugs to engineered LLQGA sites [START_REF] Farias | Mass spectrometric characterization of transglutaminase based site-specific antibody-drug conjugates[END_REF]. The sortasemediated conjugation of a glycine-functionalized chelator near the C-terminus of a singlechain antibody (scFv) and the formylglycine generating enzyme-mediated conversion of cysteine to formylglycine followed by hydrazino-Pictet-Spengler ligation have also been successfully generated [122].

One of the caveats of these site-specific approaches is that many commonly used bioorthogonal reagents do not react stereospecifically and typically yield at least two diastereomeric products, resulting in several regioisomers. In addition, since these conjugation methods are applied to heterogeneous antibodies (e.g. glyco-and charge-variants), the homogeneity of the final products depends on the resolving power of the analytical and structural methods employed [START_REF] Beck | Cutting-edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies[END_REF].

ADC higher order structure and aggregates

The HOS of a protein, namely its secondary, tertiary and quaternary assemblies, is often essential for its function [123,124]. While disruption of the native fold can also impact the function of the molecule, the main immunogenic consequences of misfolded proteins stem from their propensity to aggregate [125,126]. In solution, mAbs and ADCs tend to selfassociate, forming a range species from oligomers to visible particles. Changes in the HOS of ADCs can alter their quality attributes, and thereby their safety, efficacy and pharamacokinetics. A requirement of regulatory agencies is therefore that the effects of manufacturing, storage and delivery on the HOSs of mAbs and ADCs be well characterized [127].

Determining the HOSs of proteins using analytical biophysical techniques is still challenging however. The classical methods for protein structure determination, namely X-ray diffraction (XRD) and nuclear magnetic resonance (NMR) [128], provide atomic resolution but can only tackle relatively small proteins, let alone protein assemblies, and consume large amounts of biological material. For mAbs and ADCs furthermore, the flexibility of the hinge region hinders the growth of crystals (for XRD) and complicates NMR analyses. So far, only a small number of mAb crystal structures have been published [129] and no 3D structure of an ADC has been solved. In this context, lower-resolution methods, notably those based on MS (see Figure 8 and the following sections), can provide valuable insights.

Native MS and IM-MS

Native MS has been used to reveal the presence of mAb multimers (dimers, trimers, tetramers) [130] and to study the structural dynamics of IgG4 [START_REF] Debaene | Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and "Bispecific" Monoclonal Antibody Formation[END_REF]. As non-covalent interactions are maintained, a single native MS analysis can be used to both characterize the ADC (average DAR, drug load distribution, proportion of unconjugated drug molecules) and check for the presence of multimers. Native MS has also been used to unambiguously determine antibody/antigen binding stoichiometries [131] and to monitor the dynamics of IgG4 Fab-arm exchange [START_REF] Debaene | Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and "Bispecific" Monoclonal Antibody Formation[END_REF]. Furthermore, Haberger et al. recently reported a linear correlation between results obtained by quantitative native MS and IgG-FcRn functional analysis, a larger amount of higher order complexes being detected in the absence of methionine oxidation in both cases [132].

The structural analysis of proteins can also be facilitated by . In this approach, ions are separated based on their collisional cross section (CCS) as they drift through an inert buffer gas in an electrical field, providing information on the charge and conformation of a protein complementary to the associated mass data. The measured drift times can be converted into CCSs, which correspond to the averaged rotational projection on a 2D space of the biomolecule 3D structure. IM-MS adds an additional level of conformational characterization to that provided by native MS. The analysis of the HOSs of mAbs by IM-MS remains a relatively novel endeavor [START_REF] Beck | Characterization of therapeutic antibodies and related products[END_REF][START_REF] Beck | Analytical characterization of biosimilar antibodies and Fc-fusion proteins[END_REF][START_REF] Zhang | Mass spectrometry for the biophysical characterization of therapeutic monoclonal antibodies[END_REF][START_REF] Debaene | Time Resolved Native Ion-Mobility Mass Spectrometry to Monitor Dynamics of IgG4 Fab Arm Exchange and "Bispecific" Monoclonal Antibody Formation[END_REF]131,[137][138][139][140].

To our knowledge, only three publications have so far reported the use of native IM-MS for the characterization of ADCs [65,[START_REF] Marcoux | Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate[END_REF]107]. Debaene et al. extensively analyzed the higher order structure characterization of BV by IM-MS [65], comparing the heterogeneity of BV with that of unconjugated brentuximab. Five different species were observed, having zero to eight drug molecules attached but similar gas phase conformations-viz. CCSs of 71.1 ± 0.1 nm 2 and 68.1 ± 0.1 nm 2 for the DAR 8 and DAR 0 species, respectively. The constant shift in CCS between two consecutive even drug conjugates points to a small increase in mass rather than to structural changes and indicating that the conformation of the protein is not drastically altered upon binding.

Similarly, Marcoux et al. performed a global conformational analysis of T-DM1 [START_REF] Marcoux | Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate[END_REF] and presented the first attempt 3D model of an ADC based on the structure of human IgG1 and the Fab structure of trastuzumab (PDB IDs 1HZH and 1N8Z, respectively). The CCSs derived from IM-MS were found to be slightly higher than those estimated from the masses of the species, but considerably lower than those expected from molecular modeling. These discrepancies, also observed for unconjugated mAb and attributed to a collapse of T-DM1 in the gas-phase [65,138], contrast with the results obtained for BV, for which the values calculated assuming spherical proteins match those measured by IM-MS. This suggests that the surface-exposure of lysine-linked payloads makes them more susceptible to gas-phase collapse than equivalent payloads in cysteine-linked conjugates. Each drug-loading event induced a CCS increment of ~25 Å 2 , which matches the binding contribution expected from the mass of a single DM1 molecule (~100 Å 2 ). This suggests that the conformational changes that occur in trastuzumab upon drug conjugation are very slight.

As mentioned above in the discussion of analytical methods for T-DM1, Huang et al.

[107] have also employed IM-MS to perform DAR measurements. The protein drift time was used as a second dimension to monitor the changes in DAR in lot-to-lot analyses of both a site-specifically and randomly conjugated ADC.

Although it remains an emerging technique native IM-MS offers a simple and direct means to assess the size and shape of ADCs and thereby obtain a rough conformational picture. The examples above also illustrate how IM-MS can be used to identify all the species with different drug loads present in solution and study the effects of drug conjugation on the global conformation of the mAb. In the medium term, the development of molecular dynamics algorithms able to cope with > 150 kDa proteins should facilitate the structural interpretation of IM-MS data on mAbs and ADCs.

HDX-MS

Technical developments over the past 10 years have brought HDX-MS to the forefront of methods for the structural characterization of proteins. Indeed, HDX-MS now fills a niche in their therapeutic analysis, notably for epitope mapping and comparability studies [141][142][143][144][145][146][147] In a typical HDX-MS experiment, the protein of interest is deuterated over different lengths of time, after which the reaction is quenched under strong acidic conditions at low temperature (0 °C). A classic proteolytic digestion is then performed using pepsin, which cleaves non-specifically but with a high reproducibility and under acidic conditions. The digested peptides are then analyzed by rpLC-MS to assess their level of deuteration. An additional level of IM separation can help to assign the peptides and increases the sequence coverage (Figure 8). HDX-MS can be performed on proteins or assemblies with only a dozen picomoles of material (vs. milligrams for crystallography) and no size limitations in theory (in practice HDX-MS analysis of assemblies up to 300kDa were reported [151,158] ).

Its other main strength is its ability to monitor any structural changes that occur upon labeling in solution.

A field of application of HDX-MS not mentioned above is the study of mAb HOSs.

Circular dichroism (CD), fluorescence spectroscopy and differential scanning calorimetry (DSC) are the methods most frequently used at present [159,160]. For instance, Guo et al.

reported similar CD profiles for vcMMAE and the isolated mAb (MMAE), suggesting that the secondary and tertiary structures of cysteine-linked ADCs do not differ significantly from those of the parent mAb [161]. However, a key limitation of these biophysical techniques is that the information obtained is an average both over all the protein conformers in solution and across the sequence of the protein. The better spatial resolution offered by HDX-MS (at the peptide level at least and at the amino acid level when combined with electron transfer dissociation) and its capacity to generate dynamic data for solution samples, makes HDX-MS an attractive alternative to probe the HOSs of mAbs and ADCs.

By comparing the deuterium uptake plots of the conjugated and unconjugated MMAE, Pan et al. showed that almost 90% of the primary sequence of vcMMAE has the same HDX kinetics as its parent mAb, the only differences occurring for the two peptides in the Fc domain which have faster kinetics after conjugation [162]. This suggests that vcMMAE and MMAE have very similar conformations and dynamics in solution. A detailed comparison of the HDX-MS profiles of the ADC and its corresponding TCEP-reduced mAb indicated that the partial reduction of the IgG1 interchain disulfides induced some minor, local structural differences in the conformation and dynamics of the mAb Fc region. However, alkylation of the reduced cysteine residues by the drug molecule does not further impact the local structural of the domains where the differences were observed [163]. The same group also used HDX-MS to demonstrate that site specific conjugation on engineered cysteine has only minor structural effects on other ADCs (with pyrrolobenzodiazepine or mcMMAF as the conjugated drug), the only difference being a slightly higher deuterium uptake after conjugation in the vicinity of the mutation ( 264 VDSV) because of the destabilization of hydrogen bonds between C 239 and V 264 induced by the drug-linker [164].

Since HDX-MS can provide information the conformations and dynamics of ADCs in solution-information that would be more difficult to obtain via other analytical methods-it should become a technique of choice for ADC comparisons, the optimization of conjugation sites, and the choice of the best drug-linkers in the development of therapeutic candidates.

Aggregation

As aggregation is the main cause of drastic decrease in the therapeutic efficiency of proteins, with potentially deleterious effects on patient health, a crucial characteristic of ADCs is the proportion of aggregates in the final product [124]. Both mAbs and ADCs can become unstable during conjugation [160,165,166] or under environmental stress (from changes in temperature or ionic strength, exposure to light, etc.). However, aggregation is more common for ADCs, because the solubility of the mAb is reduced upon conjugation to hydrophobic drug-linkers [START_REF] Wakankar | Analytical methods for physicochemical characterization of antibody drug conjugates[END_REF]161].

For example, Becley et al. found increased aggregation of a cysteine-linked ADC at higher temperatures and for the species with a higher DAR [166]. This trend was attributed to the drug conjugation in the hinge region, as revealed by DSC and CD data. The authors note that for the species with a higher drug loading, while conjugation does not measurably alter the secondary structure, it does reduce the thermal stability of the CH2 domain, with high molecular weight aggregates forming rapidly. Using SEC, Adem et al. showed that the aggregation propensity of an auristatin ADC increases with the ionic strength of the solution, especially again for the high drug load species [167]. The authors then used DSC enables to directly correlate the thermal unfolding of the protein with the loading of the drug.

Regarding the photosensitivity of ADCs, although those currently on the market are conjugated with non-photosensitive drugs, many of those under clinical trial contain photosensitive drugs such as doxorubicin or duocarmycin derivatives. Cockrell et al. reported that binding an eosin photosensitizer to trastuzumab promotes the formation of aggregates, with high-mass components being detected by SEC analysis and dynamic light scattering highlighting the formation of large soluble particles and aggregates [168]. Since aggregation occurred for the conjugate but not on the mAb control, this indicates that drug conjugation is specifically responsible for the observed aggregation.

The presence of charge variants can also affect the stability, solubility, pharmacokinetic and tissue distribution of an ADC. Boylan et al.

Residual drug linker and related products

As highlighted in Table 1 and already mentioned above as a potential health hazard, the amount of residual drug-linker and related products in ADC drug substance batches is a critical quality attribute. The unconjugated drug or drug-related impurities that remain in the final product are typically the result of incomplete purification down-stream of the conjugation reaction. Related forms of the unconjugated drug, such as linker-drug species or other degradation products, may also be released while the conjugate is in storage. Sensitive methods are therefore required in ADC development and production facilities to monitor and validate the cleaning procedures for cytotoxic products, both for the safety of operators and to demonstrate the absence of cross contamination in multi-cytotoxic production plants [169].

Wakanhar et al. have reviewed ELISA, HPLC-UV/Vis and CE-LIF (capillary electrophoresis laser induced fluorescence) methods that have been used to determine the concentrations of free drug molecules in various ADCs [START_REF] Wakankar | Analytical methods for physicochemical characterization of antibody drug conjugates[END_REF]. Chih et al. also emphasize the importance of monitoring drug-linker release with an example MS application [170]. This method was successfully used to explain an unexpected release of free drug during the stability testing of ADCs.

One of the major practical challenges of these kinds of analyses is removing the large ADC excess from the sample to allow the very small amounts of multiple small organic molecules to be quantified. This issue was solved by Fleming et al. for free DM1 dosages by directly injecting the ADC onto a single rpHPLC column without prior sample preparation [START_REF] Fleming | A reversed-phase high-performance liquid chromatography method for analysis of monoclonal antibody-maytansinoid immunoconjugates[END_REF]. The ADC flows through the column without interacting with the stationary phase, which combines a hydrophobic core with a hydrophilic outer layer. The hydrophilic layer shields the C18-like core from interactions with mAbs and ADCs, while the small organic molecules present in solution readily interact with the hydrophobic portion. Li et al. recently reported another elegant method based on two-dimensional LC-MS [171]. The SEC method in the 1 st dimension separated the small molecule impurities (the free drug, drug-linker, and druglinker-N-Acetylcystein adduct) from the intact ADC, and provided simultaneously information on size variants, namely monomers, dimers and aggregates. The small molecules giving rise to the peak in the 1 st dimension were isolated and further analyzed by rpHPLC in a 2 nd dimension for identification and quantitation by MS. Traditionally, pharmacokinetic studies of mAb candidates are performed using ligandbinding assays (LBAs) [172,176,177], a technique also used to study the catabolites [178] and immunogenicity of ADCs [179,180]. The limitations of this approach are well known however. The specific assay reagents required for LBA are often not available early on in a program; moreover, interference can occur from endogenous proteins, antidrug antibodies and soluble target ligands [176,181]. Complementary or alternative data from liquid chromatography coupled to MS-based methods can therefore facilitate the analysis of mAbs in biological matrixes. Selected reaction monitoring (SRM)-MS combined with stable isotope dilution is thus increasingly employed in pharmacokinetic studies of recombinant proteins, notably mAbs, in highly complex matrices (serum, plasma, tumor tissues, and other body fluids) [181][182][183][184][185][186][187]. In addition, while LC-SRM has been used for decades to quantify small molecules, tremendous improvements in the technique over the past few years have broadened its scope to include the quantitation of peptides and proteins, including ADC catabolites [188]. Optimized LC-SRM assays offer unequalled sensitivity, high analyte specificity, a high multiplexing capacity and precision, and robust quantitation of analytes down to high ng/ml-low µg/ml concentrations in unfractionated plasma. Most of the methods described above rely on enzymatic digestion of the mAb to yield at least one peptide with a unique sequence to be quantified as a surrogate for the whole mAb [189]. Multidimensional chromatography or immunoenrichment can be used to separate the mAb of interest from those of the endogenous matrix, thereby increasing the relative concentration of the analyte of interest [190,191]. The quantitative bioanalysis of ADCs in plasma has also be achieved by hybrid immuno-capture LC-MS/MS [192,193].

Bioanalysis of ADC biotransformation

ADCs are administered as intravenous infusions, and, following in vivo processing, multiple analytes are detected in systemic circulation. According to a recent paper from the American Association of Pharmaceutical Scientists ADC Working Group [194] the most commonly observed analytes are conjugated antibodies (i.e. with a DAR of one or more), total antibodies (conjugated, partially or fully deconjugated), antibody-conjugated drugs (small molecules conjugated to an antibody), and unconjugated drug molecules. Metabolites of the drug molecule, with or without the linker, may also be detected. As discussed above, most of the ADCs currently under clinical trial exist as a heterogeneous mixture of antibody species with DARs varying from zero to eight. Each of these species has its own distinct in vivo pharmacokinetic profile, efficacy, and safety [START_REF] Hamblett | Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate[END_REF] and specific quantitative analytical workflows have to be developed. Pharmacokinetic profiling highlights the impact of the DAR and other perfectible parameters on the biological properties of ADCs, notably the rate of drug loss (from deconjugation and instability) and the clearance of the species with different DARs. Ultimately through, establishing the relationships between pharamacokinetic exposures and the efficacy and toxicity of the drug is the most helpful guide for the optimization and development of ADCs. This information is also mandatory for the pharmacokinetic-pharmacodynamic modeling that helps to determine the first-in human dosage for clinical trials [195][196][197][198]. In this context, Deslandes has recently compared the pharmacokinetics of ADCs in Phase I clinical studies [199], while toxicology issues have been discussed by Saber and Leighton from the FDA [200].

Final conclusions

The development and optimization of ADCs are increasingly reliant on the analytical and bioanalytical characterization of their main quality attributes, namely the drug load distribution, proportion of naked antibody, and DAR. These needs have recently been fulfilled by a number of cutting-edge mass spectrometry methods, with workflows optimized to be used at different levels. Electrophoretic and chromatographic methods, in association or not with MS, are still the main techniques used for the characterization of mAbs and ADC. At the bottom level, high-quality ADC peptide mapping via improved (nano)HPLC-MS is now available in most analytical labs. This can be used to characterize the drug-loaded peptides and to identify positional isomers at cysteine residues. Because of the hydrophobicity of the drug molecules, organic solvents are added at all stages of the bottom-up approach, including enzymatic digestion, to ensure that the drug-loaded peptides remain in solution.

is frequently used in the second dimension as it desalts the sample before its introduction into the mass spectrometer. Recent applications of this approach reported by Li et al. [171],Stoll et al. [208] and Birdsall et al. [98], have involved SEC x rpHPLC-MS, CEX x rpHPLC-MS, and HIC x rpHPLC-MS, respectively. Recent improvements made to chromatographic methods such as SEC [209], cation exchange [210], rpHPLC [211], and HIC (Fekete S et al, submitted) will no doubt also benefit the application of 2D-LC-MS for the analysis of mAbs, ADCs and related products.

Imaging mass spectrometry has successfully been used to reveal the spatial distribution of small drugs and metabolites in biological tissues [212], and has recently been demonstrated for the study of large biological molecules [213], notably to monitor mAbs in brain tumors [214]. One assumes therefore that this technique will also be applied for the characterization of ADCs.

Focusing now on the samples under study, while on the one hand, next-generation ADCs should be more homogeneous and thereby easier to characterize, their more complex formulation will pose new analytical questions. Indeed, bi-specific antibody conjugates [215], antibody-dual-conjugates [216], treatment combinations (eg. ADCs + immune check points mAbs) as well as alternatives to IgG formats [217] have already been developed. Finally, a feasible objective for the analytical methods used to study ADCs during pharmaceutical development, is that they should be translated to the bioanalytical field, for the analysis of blood and/or tumor tissues. This would allow the quantitation of known and unknown catabolites for large and small molecules and help to optimize the next generation of ADCs.

Key issues

• High resolution native MS provides accurate mass measurements (within 30 ppm) of intact ADCs, and can also yield the average DAR and drug load distribution. Native MS is furthermore unique in its ability to simultaneous detect covalent and non-covalent species in a mixture.

• Native IM-MS reveals the drug loading profile of ADCs, the CCCs of each payload species highlighting slight conformational differences.

• As an orthogonal method, LC-MS following IdeS digestion of ADCs can be used to measure the drug load distribution on light chain and Fd fragments, as well as the average DAR for both monomeric and multimeric species. In addition, the Fc fragment can be analyzed in the same run, providing a complete glyco-profile and demonstrating the presence or absence of additional conjugation.

• Because the drug molecules are hydrophobic, all enzymatic digestion steps used to prepare the sample for peptide mapping have to be adapted to keep the drug-loaded peptides soluble. When this procedure is properly optimized however, unambiguous maps are obtained by LC-MS, while the positional isomers of the cytotoxic drug can be determined by rpHPLC after digestion with IdeS and reduction.

Financial and competing interests disclosure

This work was supported by the bioclusters of the OptimAbs network (the Lyon Biopole and Alsace Biovalley) and sponsors (DGCIS, Oseo, Feder, Région Rhône-Alpes, Région Alsace, Communauté Urbaine de Strasbourg, CNRS, and the Université de Strasbourg).

We thank GIS IBiSA and Région Alsace for financial support in purchasing respectively a Synapt G2 HDMS instrument and a Exactive Plus EMR Orbitrap spectrometer.

François Debaene is an employee of Sanofi. The other authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Table 1. Analytical, structural and functional assays reported in quality control monographs for the characterization of ADCs. They may be used for drug substance/drug product batch release or characterization as well as for comparability, stability studies and bioanalysis).

Mass spectrometry-related methods appear in bold.

General

Quantification

• Protein quantitation: UV, BCA, Protein A HPLC,

• ADCC (when applied)

Figure 1 .

 1 Figure 1. Schematic representation of the structure of the two FDA-and EMA-approved ADCs, which comprise an antibody, a linker and a payload. (a) Brentuximab vedotin (auristatin, protease cleavable linker, Cys-conjugation); (b) trastuzumab emtansine (maytansinoid, non-reducible thioether linkage, Lys-conjugation).

Figure 2 .

 2 Figure 2. Schematic representation of the different types of ADCs : (a) cysteine hinge, (b) lysine hinge and(c) site-specific ADCs, and their corresponding drug-to-antibody ratio (DAR) distributions. The strategies that have been proposed to date for site-specific conjugation include (1) the covalent labeling of engineered cysteines;[START_REF] Reiner | PCSK9 inhibitors -past, present and future[END_REF]

Figure 3 :

 3 Figure 3: Multi-level workflow affording a comprehensive characterization of ADCs.

  . More recently, Valliere-Douglas described how native SEC-MS could be used in vivo for DAR measurements and to monitoring changes over time in the drug load distribution [79]. Debaene et al. performed an extensive native MS characterization of BV in comparison with its unconjugated form brentuximab, highlighting the efficiency and accuracy (<30 ppm in routine analysis) of native MS on the latest highresolution Orbitrap instruments for drug distribution profiling and the determination of average DARs [65]. A relatively simple but efficient analytical workflow was described, consisting of desalting, optional deglycosylation, then native MS analysis of the ADC and semi-quantitative interpretation of the data obtained. The authors also highlighted the value of high-resolution native MS (Figure 4d) for one-shot ADC characterization, the limited overlap in the peak distribution obtained offering a unique description of intact glycosylated ADCs. Debaene et al. have also shown for the first time how native and IM-MS can be combined for the characterization of ADCs. The average DAR and the drug load profile were obtained through a direct extrapolation of semi-quantitative IM-MS data (Figure 4e). The native MS and IM-MS measurements were confirmed by HIC (Figure 4b) demonstrating the analytical potential of native MS strategies.

Figure 4 .

 4 Figure 4. Top level analysis of brentuximab vedotin (BV). (a) Schematic representation of cysteine ADCs with a variable number of drug molecules per antibody. S-S bonds are represented by yellow lines while drug loads appear as red stars. (b) Reference hydrophobic interaction chromatogram revealing the drug loading profile and the average drug-to-antibody ratio (DAR). (c, d) Deconvoluted electrospray ionization mass spectra of deglycosylated BV (c) under classical denaturing conditions (H 2 O:ACN:FA 50:50:1) and (d) under native conditions (AcONH 4 150 mM pH 7.4). (e) Ion mobility mass spectrometry (IM-MS) derived drug load profile for BV: relative intensities of each drug load as a function of the number of drug molecules loaded onto the mAb.

Figure 5 .

 5 Figure 5. Middle level characterization of brentuximab vedotin (BV) after reduction. (a) Schematic representation of the workflow for middle-level analysis. (b) Reverse-phase high-pressure liquid chromatogram of BV fragments obtained after reduction with dithiothreitol and alkylation with idoacetic acid. (c, d) Deconvoluted

Figure 6 .

 6 Figure 6. Middle level characterization of brentuximab vedotin (BV) after digestion with IdeS (the immunoglobulin-degrading enzyme from Streptococcus pyogenes). (a) Schematic representation of the workflow for middle level analysis. (b) Reverse-phase high-pressure liquid chromatogram of BV fragments obtained after IdeS cleavage and dithiothreitol reduction. (c-e) Deconvoluted mass spectra of (c) the light chain, (d) the Fc/2 and (e) the Fd fragments of BV. (f) Table listing the masses, proportions, and average drug-toantibody ratios of the different BV fragments, as measured from the corresponding peak areas.

Finally

  , as an example, application of new enzymes that should also prove valuable for the characterization of ADCs, Srzentic et al. used secreted aspartic protease 9 (SAP9) from C. albicans, which generates 3-4 kDa peptide fragments, and bottom-up proteomics to achieve near 100% sequence coverage on a mAb in a single LC-MS run [100].

  The drug distribution profile obtained and the average DAR (3.4 ± 0.2) are consistent with values obtained from other MS methods. Huang et al. have also illustrated the potential of IM-MS in a routine rpHPLC-MS analysis of an IgG1 lysine conjugate, showing in particular how a "cleanup" step significantly improves the signal-to-noise ratios in the mass spectra of the intact ADC, and increases the accuracy of the associated DAR measurements [107]. To assess drug loading heterogeneity and the location of the drug conjugation site, Gautier et al. have reported the combined used of native MS and bottom up proteomics for the characterization of IgG1 and IgG4 ADC-like components, using N-hydroxysuccinimide-based tandem mass tags as a mimic for ADC conjugation. Native MS allowed the detection and quantification of up to 69 conjugates while bottom up proteomics revealed some very reactive lysine conjugation sites, or "hot spots" [108].

Figure 7 .

 7 Figure 7. Top level analysis of trastuzumab emtansine (T-DM1). (a) Deconvoluted electrospray ionization mass spectra obtained under denaturing conditions (H 2 O:ACN:FA 50:50:1) by direct infusion on a Q-TOF instrument of T-DM1 after deglycosylation. (b) Deconvoluted electrospray ionization mass spectra obtained under native conditions (AcONH 4 150 mM pH 7.4) by direct infusion into a high resolution Orbitrap extended mass range instrument (nominal resolution: 17.5 k) of T-DM1 after deglycosylation and charge reduction with 40 mM imidazole. (c) Ion mobility-mass spectroscopy (IM-MS) driftscope plots obtained for trastuzumab (left) and T-DM1 (right) highlighting drug load heterogeneity. (d) Molecular model of trastuzumab conjugated with four DM1 payloads and drug load profile of deglycosylated T-DM1 obtained by IM-MS. The asterisks indicate 1220 Da linker adducts.

  , but also in the investigation of mAb/Fc receptor interactions [148]. Papers have also been published on the use of HDX-MS to study mAb aggregation [149-151], to optimize formulation stability [152], to monitor reversible mAb self-assembly [153], and to characterize the structural effects of glycosylation or chemical modifications on mAbs [154-157].

  used SEC-MS to determine the DAR and evaluate the proportion of aggregates for different charge variants of a cysteinelinked Fc fragment, the heterogeneity originating from degradation and different drug-linkers and conjugation sites[106]. For a species at a given DAR, several acidic bands are observed in the IEF data, revealing the presence of charge variants. The authors did not find any correlation between particular conjugation sites and charge variants.In the future, native MS approaches should become more widespread for the analysis of ADC and mAb aggregates, as the on-line coupling of SEC to native MS makes the the analysis of aggregates straightforward. A few groups have already exploited HDX to study mAb aggregation. Zhang et al. used HDX-MS to compare the aggregation mechanism and the resulting aggregate structures of a mAb under freeze-thaw and thermal stress, showing that bevacizumab aggregation increases with the number of F/T cycles and decreases with the protein concentration[150]. The reduced hydrogen exchange recorded for three complementarity determining regions suggests that these residues may form strong intermolecular bonds in the antibody aggregates, while the regions with enhanced HDX rates are most probably partially unfolded. An attempt was made to predict the aggregation patterns using several residue level modeling methods, but this proved unsuccessful[150].

Figure 8 .

 8 Figure 8. Mass spectrometry techniques for the analysis of higher order structure and aggregation for ADCs.

  Bioanalytical methods are rapidly being developed to quantitatively monitor the transformation of ADCs in various in vitro or in vivo biological matrices such as serum/plasma and tumor tissues [172]. Indeed, a crucial property of conjugates is their stability in biofluids as the release over time of cytotoxic drugs into the bloodstream constitutes a considerable health threat [173]. This drug loss also affects the composition of the ADC, potentially altering the amount of drug delivered to the tumor site and posing another substantial safety risk because of its off-target toxicity [174]. Tumey et al. have recently published an interesting survey of the biotransformation events that have been elucidated in recent years [175], as well as one of the resulting strategies to optimize the design of next generation ADCs [115].

  However, recent developments in native MS mean that this technique, in combination with classical MS approaches, offers the means to complete a multi-level characterization of ADCs and other protein-based therapeutic agents.At the top level, native MS and native IM-MS complement the data provided by HIC and rpHPLC-MS, the reference methods for the quality control of interchain cysteine-and lysine-linked ADCs, respectively.At the middle level, the LC-MS analysis of reduced or IdeS-digested ADCs provides structural insights into the ADC subunits. The use of IdeS ensures the complete separation of the Fc/2 and Fd fragments. This facilitates the glycoprofiling of the Fc/2 fragment and the detection of any additional conjugation.

•

  Denaturated ADC (surfactant, solvant): SDS-PAGE, SDS-CE, rpHPLC-UV/MS, MALDI-TOF • Subunits (reduced, IdeS+red.): SDS-PAGE, SDS-CE, rpHPLC-UV/MS, ETD • Peptide mapping/ disulfide bridges/ conjugation sites (Lys-C, trypsin, Asp-N, Glu-C, chemotrypsin, SAP9): rpHPLC-UV/MS, CE-MS • Higher order structures: CD, HDX-MS, DSC (Tm), FTIR, fluorescence • Immuno-identification: ELISA, immunoblots, FACS, SPR

  A4F, asymmetrical flow field-flow fractionation; ADCC, antibody-dependent cell-mediated cytotoxicity; BCA, bicinchoninic acid; CD, circular dichroism; CEX, cation exchange; DAR, drug-to-antibody ratio; DSC, differential scanning calorimetry; ELISA, enzyme-like immunosorbent assay; ETD, electron transfer dissociation; FACS, Fluorescence-activated cell sorting; FTIR, Fourier transform infrared; HDX, hydrogen/deuterium exchange; HIC, hydrophobic interaction chromatography; (i)CE, (imaged) capillary electrophoresis; (i)(c)IEF, (imaged) (capillary) isoelectrofocusing; ICP, inductively coupled plasma; IdeS, the immunoglobulin-degrading enzyme of Streptococcus pyogenes; IM, ion mobility; IR, infrared; MALDI, matrix-assisted laser desorption ionization; MALS, multiangle light scattering; MS, mass spectrometry; PAGE, polyacrylamide gel electrophoresis; rpHPLC, reverse-phase high-performance liquid chromatography; SDS, sodium dodecyl sulphate; SEC, size exclusion chromatography; SPR, surface plasmon resonance; SV-AUC, sedimentation velocity analytical ultracentrifugation; TOF, time of flight; UV, ultraviolet.

Acknowledgments

The authors would like to thank all the members of the CIPF and LSMBO that have been involved in the characterization of ADCs: A. Boeuf, M. Excoffier, L. Maurel-Chevillet, C. Klinguer-Hamour, T. Champion. M. Dillenbourg, M. Fumex, S. Tanguy, F. Delalande, H. Diemer, J.-M. Strub, C. Carapito and C. Schaeffer.

They would also like to thank Paul Guerry from Green Grow Scientific for editing of the manuscript.

For studies of aggregation and the HOSs of ADCs, native MS and IM-MS along with HDX-MS are emerging as promising techniques to complement already established methods such as SEC.

Regarding MS-based quantitation finally, it is noteworthy that SRM-MS now offers the same sensitivity as ELISA assays, but with a superior specificity.

Expert commentary & five year view

The state-of-the-art MS methods currently used to characterize mAbs, notably for bottomlevel analysis [201], are readily applicable for the study of ADCs. Capillary electrophoresis coupled to MS is now established as a valuable method, orthogonal to LC, for peptide mapping at the bottom [202][203][204] and middle [205] levels, and progress toward top level analysis is forthcoming, including under native conditions. Another emerging technique, CE-UV/MALDI-MS/MS (MALDI: matrix-assisted laser desorption ionization), has recently been applied to study mAbs [206] and should prove valuable in the future.

Top-down MS and sequencing [207], middle-down MS with electron transfer dissociation fragmentation [START_REF] Fornelli | Middle-Down Analysis of Monoclonal Antibodies with Electron Transfer Dissociation Orbitrap Fourier Transform Mass Spectrometry[END_REF], and extended bottom-up proteomics with enzymes [100] are all in constant progress. An alternative middle-down avenue toward a full sequence coverage of IdeS-digested and reduced fragments of IgG may be LC-MALDI with in-source decay [207]. A common objective of all approaches based on MS/MS is to limit both the number of sample preparation steps and the use of enzymes for peptide mapping to avoid the introduction of artifacts into the data (e.g. over-deamidation from the use of trypsin at basic pH).