Approaches to Interchain Cysteine-Linked ADC Characterization by

Review pubs.acs.org/molecularpharmaceutics

Approaches to Interchain Cysteine-Linked ADC Characterization by Mass Spectrometry John F. Valliere-Douglass,* Shawna M. Hengel, and Lucy Y. Pan Seattle Genetics, Inc., 21823 30th Drive SE, Bothell, Washington 98021, United States ABSTRACT: Therapeutic antibody−drug conjugates (ADCs) harness the cell-killing potential of cytotoxic agents and the tumor targeting specificity of monoclonal antibodies to selectively kill tumor cells. Recent years have witnessed the development of several promising modalities that follow the same basic principles of ADC based therapies but which employ unique cytotoxic agents and conjugation strategies in order to realize therapeutic benefit. The complexity and heterogeneity of ADCs present a challenge to some of the conventional analytical methods that industry has relied upon for biologics characterization. This current review will highlight some of the more recent methodological approaches in mass spectrometry that have bridged the gap that is created when conventional analytical techniques provide an incomplete picture of ADC product quality. Specifically, we will discuss mass spectrometric approaches that preserve and/or capture information about the native structure of ADCs and provide unique insights into the higher order structure (HOS) of these therapeutic molecules.

KEYWORDS: hydrogen−deuterium exchange, mass spectrometry, HDX-MS, antibody−drug conjugate, ADC, higher order structure, drug distribution, pharmacokinetics, auristatin

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INTRODUCTION The therapeutic efficacy of ADCs is derived from cytotoxic small molecule agents that are attached to tumor-recognizing monoclonal antibodies (mAbs) via chemical linkers. ADCs have a relatively broad therapeutic index because the mAb component of the therapeutic delivers cytotoxic agents to tumors with great specificity, thus reducing systemic toxicities to noncancerous cells.1−3 There are currently two approved ADC therapies for the treatment of cancer. ADCETRIS (brentuximab vedotin) is approved for the treatment of relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma, and KADCYLA (ado-trastuzumab emtansine) is approved for treatment of HER2 positive metastatic breast cancer. These ADCs illustrate some of the different chemical approaches that are taken to conjugate cytotoxic drugs to mAbs. ADCs can be generated by alkylating endogenous interchain cysteine residues on the mAbs with cytotoxic agents via maleimide chemistry following partial reduction of disulfides (Figure 1).4,5 This approach, while targeted exclusively to © XXXX American Chemical Society

interchain cysteine residues, typically yields a heterogeneous ADC with a variable number of drugs per antibody.6 The epsilon amino group of endogenous lysine residues in the primary sequence of mAbs may be utilized as a conjugation site for drug incorporation7,8 Conjugation at lysine residues adds considerable complexity to the resulting ADC relative to interchain cysteine-linked ADCs despite the similar overall level of drugs incorporated per antibody.9 This is largely due to the greater number of lysine residues that are available for conjugation. Alternatively, interchain cysteine residues can be changed to serine to reduce the complexity of interchain cysteine conjugate ADCs. This approach gives rise to molecules with a uniform drug load and distribution.10 Recently, siteSpecial Issue: Antibody-Drug Conjugates Received: September 12, 2014 Revised: November 26, 2014 Accepted: December 4, 2014

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dx.doi.org/10.1021/mp500614p | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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Figure 1. Interchain cysteine-linked ADCs are manufactured by partially reducing mAb interchain disulfide bonds and adding maleimidyl drug linkers which alkylate the reduced interchain cysteine residues (top panel). The chemical structures of vcMMAE and mcMMAF drug linkers are shown in the bottom panel. Reprinted with permission from ref 57. Copyright 2014 American Chemical Society.

characterize therapeutic molecules and monitor relevant product quality attributes. The potency of an ADC is due in part to the extent of druglinker incorporation on the mAb. Methods that can structurally characterize the drug load and distribution have been developed and proven to be critically important for understanding ADC product quality. Wakankar et al. have summarized several considerations for the development of analytical methods that measure quality attributes which are unique to ADCs such as drug load and drug distribution.20 ADC drug load can be readily determined by UV spectroscopy when the absorbance maximum of the drug linker is sufficiently different from that of the mAb.21−23 Capillary electrophoresis (CE) and chromatographic methods utilizing hydrophobic interactions chromatography (HIC) and reversed-phase chromatography have also been used to quantitate drug load and to characterize the distribution of heterogeneous ADCs.6,9,16,24 Siegel et al. have used matrix-assisted laser desorption ionization (MALDI) MS to determine the drug load and distribution of ADCs by assessing the mass shift that was evident upon conjugation, however, the drug distribution on the intact ADC could only be estimated on the basis of the peak shape observed in the low resolution MS data.25,26 More recently, intact electrospray-ionization (ESI) mass spectrometry of protein therapeutics, particularly mAbs and ADCs, has become an integral component of molecular characterization. The deconvoluted intact mass spectrum of a protein provides a top-down view of the relative purity of the molecule as well as information about the distribution of covalent chemical and posttranslational modifications (PTM), including the number of conjugated drugs.27−33 Intact molecule ESI-MS data from large proteins is typically more highly resolved and accurate than the corresponding MALDI-MS data.34,35 In the context of ADCs, estimation of overall drug load and the distribution of

specific conjugation ADC modalities have emerged whereby unpaired cysteine residues are engineered into the constant domain sequence of mAbs to provide discrete conjugation sites for drug incorporation. Examples include SGN-CD33A and SGN-CD70A, ADCs which have unpaired cysteine residues that are conjugated with pyrrolobenzodiazepine (PBD) dimer11,12 containing drug linkers in the CH2 domain below the IgG1 hinge region.13,14 Similarly, unpaired cysteine residues have been engineered into the constant domain of Fab heavy chain for facile conjugation of these residues with valinecitrulline monomethyl auristatin E (vcMMAE).15,16 Other site specific conjugation approaches involve the use of microbial transglutaminases to specifically transamidate amine containing drug linkers to mAb glutamine residues17 or engineering unnatural amino acids into mAb primary sequence to provide a chemical handle for conjugation.18,19 By design, ADCs are a composite of recombinant protein and synthetic small-molecule intermediates and thus contain an underlying heterogeneity that is a product of the heterogeneity of the intermediates. Additionally, as mentioned above, the ADC conjugation process wherein the drug linkers are chemically linked to the mAb backbone can also be a source of heterogeneity in the final product.6 It is important to have a clear understanding of the relationships between the conjugation/manufacturing process and the resulting product quality and heterogeneity of the ADC. In order to define these relationships, analytical methods that can be applied to ADCs and the parent mAb to elucidate the relationships between the manufacturing process and product quality are necessary. Regulatory agencies are increasingly emphasizing the importance of defining relevant product quality attributes that have an impact on the clinical performance of the therapeutic. The Quality by Design approach highlights the importance of analytical assays which can be used to comprehensively B



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Figure 2. Characterization of vcMMAE-ADC by HIC and SEC-MS. (A) HIC separation of the vcMMAE-ADC showing the distribution of individually loaded forms. (B) Deconvoluted mass spectra for the deglycosylated vcMMAE-ADC. The 5 different mass values correspond to 5 ADC subpopulations with 0, 2, 4, 6, or 8 drugs per mAb, respectively. Reprinted with permission from ref 57. Copyright 2014 American Chemical Society.

shown that proteins dissolved in D2O buffer undergo backbone amide hydrogen exchange with deuterium and that the rate of deuterium incorporation depends on solvent exposure and hydrogen bonding networks that involve the backbone amides.50−52 While HDX-MS cannot be used to define absolute structure in the manner of X-ray crystallography, it is well suited for assessing local, domain-specific HOS comparability.48 It has been used in this context on mAbs that have been subjected to a variety of accelerated stress conditions to understand how those conditions impact mAb HOS.53−55 The impact of PTMs and discrete chemical modifications on the HOS structure of mAbs has also been investigated by HDXMS,56 and, more recently, HDX-MS has been applied to the HOS characterization of interchain linked cysteine ADCs to assess whether conjugation processes and/or the presence of drug linker itself changes the HOS of the parent mAb.57 Presently, mass spectrometry is widely used during the manufacturing and process development life cycle for the molecular and structural characterization of protein therapeutics. This review will highlight some of these recent applications of mass spectrometry to the analysis and characterization of interchain cysteine linked ADCs. Particular emphasis will be placed on analytical MS techniques which maintain the intact native ADC structure during analysis and MS techniques which can be used to comparatively probe HOS such as HDX-MS.

the individual drug-loaded forms can be directly determined from ESI-MS data.30,33,36−38 Modern, commercially available time-of-flight (TOF) and Orbitrap mass detectors are routinely capable of measuring the mass of large proteins and peptides at approximately 15 ppm (ppm) levels of accuracy.34,39 This is often sufficient to unambiguously confirm the molecular formula for drug linkers and primary sequences of ADCs and to identify the drug conjugation sites on the mAb.9,30,33 Recently, the application of native mass spectrometric techniques to interchain cysteine linked ADCs has made it possible to obtain intact mass spectra on bioconjugates that are composites of covalent and noncovalent assemblies.36−38 This work has been extended to the analysis of this class of ADCs in vivo to monitor chemical changes to ADCs that arise over time, in biological matrices.40,41 The functional properties of protein based therapeutics depend on the higher order structure (HOS) of the protein(s). Perturbations to the HOS of protein based therapeutics may produce undesired changes to the safety and efficacy of the therapeutic.42,43 X-ray crystallography and nuclear magnetic resonance spectroscopy have traditionally been used to study protein structure and dynamics, however, these applications are not practical and, in the case of NMR, can be limited by molecule size.44−46 Recently, mass spectrometry has made inroads into the study of protein HOS where it is used as a detector for monitoring the kinetics of hydrogen−deuterium exchange (HDX) at protein backbone amides.47−49 It has been C



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NATIVE MASS SPECTROMETRY OF ADCS Interchain cysteine linked ADCs are manufactured by treating endogenous IgG interchain disulfide bonds with sufficient reducing agent to generate a conjugation intermediate in which a subpopulation of interchain disulfides are reduced. The intermediate reduced mAb is subsequently alkylated at the cysteine free thiols with cytotoxic agents such as vcMMAE and mcMMAF (Figure 1). The resulting ADCs typically have an average molar ratio of drugs to antibody (MRD) equal to 4 but exist as a heterogeneous distribution of species ranging from 0 to 8 drugs incorporated per antibody. Interchain cysteine ADCs are more challenging to analyze by intact MS than lysine-linked or site-specific ADCs because they are composed of molecule subpopulations which lack disulfide bonds linking heavy chain (HC) and light chain (LC) subdomains. As a consequence, the ADC dissociates into the constituent drug-linked HC and LC components when analyzed with conventional LC−MS methods that denature the molecule during chromatographic separation. The number of cytotoxic agents incorporated onto the ADC is a very important attribute because ADC activity depends on the amount of drug that is delivered to target cells.22,58 Intact molecule MS techniques can be leveraged to estimate interchain cysteine linked ADC drug load, but careful sample preparation prior to MS analysis is critical to ensure that denaturation of the ADC does not occur. Prior to analysis, removal of nonvolatile buffers and excipients that may be present in the ADC formulation is necessary because these formulation components are incompatible with MS. Many suitable sample preparation methodologies such as centrifugal concentration, dialysis, and off-line size-exclusion chromatography (SEC) have been developed to facilitate investigations of noncovalent protein complexes by MS.59−63 A subset of the sample preparation methodologies listed above has been used to develop native MS techniques which preserve the HOS of ADCs. These approaches enable the direct analysis of intact interchain cysteine linked ADCs to provide an estimate of the drug load and distribution.36−38 Our approach, which utilizes an SEC column connected directly to a TOF-MS, differs from previous applications of SEC-MS to mAbs and immunoconjugates due to the use of ammonium acetate at neutral pH as the SEC mobile phase.30,64 Applied in this manner, the SEC column serves mainly as a means to exchange the ADC from the formulation buffer which contains nonvolatile (MS incompatible) buffers and excipients into a volatile, MS compatible buffer system. When coupled to MS, the native SEC mode of operation preserves the monomeric native structure of the ADC and minimizes dissociation of noncovalently associated HCs and LCs that occurs during LC−MS. Application of MS in this manner is an important addition to the repertoire of techniques that are leveraged to characterize ADCs because the abundances of the individual drug-loaded species obtained from intact MS can be used to estimate the antibody MRD. In previous work, we have observed that overall drug load and distribution obtained from the intact MS of the ADC is comparable to results from off-line chromatographic techniques such as HIC which have historically been used to calculate MRD.37,65 A qualitative comparison of the drug distribution of an IgG1 vcMMAE conjugate by HIC and SECMS shows that similar levels of ADC species MR0−8 are observed in both methods (Figure 2). There are some advantages to utilizing HIC over MS in a manufacturing and

development environment where simplicity, cost, and ruggedness are all influencing factors. Nevertheless, there are also applications that are well suited to an MS based approach. For example MS based approaches for measuring drug load and distribution may offer specific advantages over established offline methods when greater sensitivity or lower sample consumption is required.

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APPLICATION OF NATIVE MASS SPECTROMETRY TO THE ANALYSIS OF ADCS IN VIVO Bioanalysis of pharmaceutical agents is the measurement of the concentration of the agent in vivo, over time. Typically, bioanalysis is carried out using ligand binding (ELISA) or multiple reaction monitoring (MRM) MS assays. These assays are performed routinely for pharmacokinetic modeling, dose response and toxicity profiling. For ADCs, both the protein and the nonconjugated small molecule are typically quantified.66 In a traditional in vivo pK study, ELISA and antibody peptide backbone LC−MS/MS methods are used to quantitate ADCs through specific detection of the antibody backbone. However, these methods do not provide a measure of the actual amount of cytotoxin available for delivery and release in targeted cells. It is also important to note that cysteine linked ADCs contain a mixture of differentially loaded species and, it has been previously demonstrated, that for some technologies, the higher loaded species are cleared at a faster rate in vivo than lower loaded species.22,67 The biological clearance bias due to drug load is further complicated by deconjugation and release of the maleimidyl containing drug linker.68 The net observed effect is a decrease in the average drug load of circulating ADCs due to the combined effects of clearance and deconjugation. Even in the case of ADCs dosed as a single drug load species, deconjugation has been observed leading to a changing drug load and distribution over time.33 In practice, the contribution of deconjugation to total clearance in vivo can be estimated using in vitro plasma stability studies; however, it is preferable to assess deconjugation directly, in the context of the relevant in vivo study. While drug load and distribution can be readily measured during product testing and characterization (Figure 2), an in vivo assessment of drug load and distribution presents considerable additional challenges. Mass spectrometry methods that are used to determine the drug load and distribution call for concentrated, pure starting materials.36,37 However, the circulating concentration of an administered ADC is typically low relative to endogenous IgG and this necessitates the application of affinity purification workflows to purify the ADC from the biological matrix. Coupling biotinylated antigen or anti-idiotypic antibodies to streptavidin magnetic beads has been successfully used to purify various types of ADCs from plasma in an unbiased manner.40,41,69 Xu et al. demonstrated that engineered cysteine-linked ADCs purified using affinity reagents could be characterized by LC−MS analysis to specifically track changes in drug distribution.41 Affinity purification of interchain cysteine linked ADCs is challenging, however, because native conditions must be maintained during elution of the ADC from the affinity resins; otherwise the ADC will dissociate into its constituent drug-linked HCs and LCs. Once ADCs have been purified from the biological matrix, the absolute quantity of sample available for analysis is usually relatively small. It is then important to microscale existing analytical methods to minimize sample consumption. In our recent work, we describe a microscale SEC-MS method which D



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reduced sample consumption 100 fold compared to the analytical scale method.37,40 We subsequently applied microscale SEC-MS to the analysis of intact ADC captured from in vivo studies in Sprague−Dawley rats and gained important additional insight into the mechanisms of drug loss that cannot be ascertained from typical denaturing LC−MS analyses.40 The results from our studies indicated that deconjugation and posttranslational modifications of the dosed ADC were occurring in vivo. We found that deconjugation was giving rise to odd-loaded ADC species over time, particularly ADC with 1 and 3 drugs per mAb (Figure 3). Interestingly, the mass

Figure 4. Summary of mass shifts and asymmetric tailing observed in cysteine-linked ADCs in vivo from (A) control to (B) 6 h postdose. Reprinted with permission from ref 40. Copyright 2014 American Chemical Society.

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HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY (HDX-MS) OF ADCS As therapeutic antibodies and ADCs progress toward commercialization, a greater burden is placed on understanding how product quality attributes affect the clinical performance of the therapeutic. The conformation of a protein therapeutic is an important component of product quality and changes to, or disruption of native-folded conformation can impact the function of the molecule. Additionally, misfolded proteins may be more likely to form aggregates and particulates which in turn may prove immunogenic.42,70,71 For purposes of simplicity, protein conformation is often used interchangeably with protein higher order structure to refer to the native folded state of the protein. However, it is worth noting that in solution, proteins are highly dynamic and protein functionality can be linked to conformational fluctuations occurring in millisecond time scales.72,73 In spite of the importance of HOS to protein function, approaches for characterizing biotherapeutic protein HOS continue to rely heavily on spectroscopic techniques such as circular dichroism (CD), fluorescence spectroscopy and differential scanning calorimetry (DSC).54,74,75 While some domain specific information about protein heat capacity (Cp) can be obtained with DSC, the data obtained from most traditional biophysical spectroscopic techniques represents an average of the protein conformers in solution and also an average across the sequence of the protein. It is especially important to consider new approaches to HOS characterization for ADCs. The novelty and complexity of these compounds highlights the importance of performing investigative characterization studies to gain an understanding of HOS changes associated with various modalities. Recent years have witnessed the emergence of hydrogen/ deuterium exchange mass spectrometry as a technique uniquely suited to investigations of protein HOS. The technique involves monitoring the kinetic exchange rates of protein backbone amide hydrogen atoms for deuterium when incubated in D2O under native conditions (Figure 5). The labeled protein is

Figure 3. Deconvoluted mass spectra of cysteine-linked ADCs from an in vivo study. (A) Control ADC, (B) ADC from 30 min postdose, (C) ADC from 6 h postdose. Dashed lines indicate theoretical mass of ADCs with 0−8 drugs per antibody. Reprinted with permission from ref 40. Copyright 2014 American Chemical Society.

of the odd-loaded species observed in vivo was over 100 Da higher than the (expected) theoretical mass, which was consistent with previous studies where deconjugation was investigated using engineered cysteine-linked ADCs.33,41 Using reversed phase (rp) LC−MS we found that the mass shift observed on the odd-loaded ADC in vivo was likely due to cysteinylation of the cysteine free thiol on the ADC which resulted from drug loss. Over time in vivo, we also observed asymmetric tailing of the native intact MS of the individual drug loaded species which suggested other covalent modifications were occurring on the ADC that increased the underlying heterogeneity of the molecule (Figure 4). Results from rp-LC− MS analysis indicated that the broadening observed in the microscale SEC-MS of the ADC was due to hydrolysis of the thio-succinimidyl ring which links vcMMAE to the mAb. These hydrolysis events have previously been shown to enhance the stability of the drug-mAb linkage.33 Overall, these results indicate that native intact MS can contribute important insights into the relative rates of clearance and drug loss in vivo. These results also highlight the ways in which MS can be leveraged to monitor changes in product quality attributes and heterogeneity over time, in a physiologically relevant context. E



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Figure 5. Schematic representation of protein-amide hydrogen−deuterium exchange. Scissors represent proteolytic cleavage of the protein aminoacid backbone following incubation in D2O.

some conformational differences that are evident in mAbs and interchain cysteine linked ADCs under extreme temperature stress conditions.79,80 Our results, initially communicated in Pan et al., indicated that there was a very high degree of conformational similarity between the vcMMAE-ADC and the corresponding parent mAb.57 Indeed, the exchange kinetics of the mAb and ADC in phosphate buffer at physiological pH was indistinguishable over ∼90% of the primary sequence. However, 2 peptides within the Fc domain were found to have increased exchange kinetics in the ADC relative to the parent mAb which indicated that these specific-local domains were more structurally dynamic and/or solvent accessible in the ADC (Figure 7). As noted earlier, interchain cysteine linked ADCs are comprised of molecule subpopulations which lack interchain disulfide bonds due to drug conjugation. This characteristic of the ADC prompted an inquiry into the question of whether the kinetic differences observed in mAbs and ADCs was due to the absence of interchain disulfides, the presence of small molecule drugs or some combination thereof. To address this question, the parent mAb was treated with tris(2-carboxyethyl)phosphine (TCEP) to generate a molecule with an average of 2 reduced interchain disulfides. The mAb, ADC and partially reduced mAb were then analyzed by HDXMS in parallel. Similar to the previous ADC results, the partially reduced mAb also showed increased exchange kinetics for the same 2 peptic peptides located in the Fc domain (Figure 7). The peptides with increased exchange kinetics in the partially reduced mAb and the ADC were located in the CH2 domain just below the IgG1 hinge (244FLFPPKPKDTLM) and in the CH2-CH3 domain interface (337KTISKAKGQPREPQV). These initial findings indicated that the partial reduction of IgG1 interchain disulfides induced some minor, local structural differences in the conformation and dynamics of the mAb Fc region, and that the subsequent alkylation of the reduced cysteine residues with vcMMAE does not f urther impact the local structural domains where the differences were observed. A similar HDX study was also carried out on an IgG1 ADC conjugated with mcMMAF drug linkers. The results from the mcMMAF-ADC were consistent with the vcMMAE-ADC indicating that conjugation of reduced interchain cysteine residues with mcMMAF does not produce additional changes to local Fc domains above that which is observed as a consequence of partial reduction.57 These studies indicated that conjugation of interchain cysteine residues with vcMMAE or mcMMAF had only minor-local impact to CH2 domain and no impact to any other mAb domains, including CDRs. Additional insight into the conformational impact of interchain disulfide reduction was gained by contextualizing

subsequently digested by a protease (typically pepsin) under acidic conditions and the mass shifts of the corresponding peptic peptides are quantitated by mass spectrometry.76 Unlike traditional biophysical approaches, HDX-MS provides detailed information about the extent of deuteration and, by extension, the local domain-specific structural features of the protein. HDX-MS is particularly well suited for assessing domain specific comparability between proteins and protein therapeutics.48,77 There are several examples in the literature describing the application of HDX-MS to understanding conformational changes brought on by aggregation of proteins and mAbs.55,78 The impact of PTMs and chemical modifications has also been studied and it has been found that Fc domain glycosylation structure and Fc domain oxidation can have impacts on the exchange kinetics of discrete, structurally distinct regions of the Fc.53,56 Against this backdrop of work done on therapeutic mAbs, we sought to apply HDX-MS to the assessment of the conformational comparability of ADCs and the corresponding parent mAbs. The basis for probing this question was the observation that CD comparison of mAbs and vcMMAE-ADC showed no differences, indicating that there was a very high degree of conformational similarity between the mAbs and ADCs.79 A typical example of a mAb and corresponding vcMMAE-ADC far UV CD spectrum show a negative band with a minima at 217 nm which was consistent with a high degree of β-sheet content (Figure 6). However, it has been noted that there are

Figure 6. Far-UV CD spectra comparison of mAb (black) and the corresponding vcMMAE-ADC (blue). F



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Figure 7. Comparison of the HDX kinetics for two peptides FLFPPKPKDTLM (CH2 domain, left) and KTISKAKGQPREPQV (CH2−CH3 interface, right) in different samples. Black, mAb; blue, vcMMAE-ADC; red, partially reduced mAb. Reprinted with permission from ref 57. Copyright 2014 American Chemical Society.

interact with drug-linker molecules conjugated to the IgG1 hinge and Fab regions. The physiological relevance of the 2 local Fc domain differences observed in mAbs and ADCs is not known at this time. Based on the HDX-MS results, it is apparent that there are no observable conformation differences over ∼90% of the primary sequence, including the variable domains, which contain the CDR regions. From a practical standpoint, these findings indicate that ADC manufacturing processes that involve partial reduction of mAb interchain cysteine residues followed by conjugation with drug linkers do not significantly impact the conformational integrity of the mAb.

the HDX-MS results with the crystal structure of a model IgG1 (PDB: 1HZH).46 As noted above, the increased exchange kinetics was observed in peptides which are separated in the linear sequence by 82 amino acids. Nevertheless, it is quite clear from the model IgG1 shown in Figure 8 that these regions are

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SUMMARY Mass spectrometry is an increasingly important core component of therapeutic protein characterization. The specific applications and approaches we have described were developed to overcome limitations imposed by traditional analytical assays or to supplement the information provided by these assays. Mass spectrometry has previously been leveraged to explore protein therapeutic clearance and chemical modification rates of mAbs in vivo,81,82 and we have sought to extend these types of studies to the analysis of relevant ADC quality attributes such as drug loading and distribution. Development and application of native MS methods which preserve the intact structure of the ADC have provided mechanistic insights into how ADC drug loading and distribution changes over time, in a physiological context. These findings are especially important for constructing pharmacokinetic models that reflect the actual chemical processes occurring on the ADC. In contrast to the above approach where the native structure of the ADC is necessarily preserved during MS to facilitate the determination of drug loading and distribution, HDX-MS is used to directly assess the native structure in a comparative fashion. As we have noted, HDX-MS is gaining acceptance as a useful approach for the comparative assessment of higher order structure in mAbs. It follows that there are opportunities to extend HDX-MS to ADCs to address questions that revolve around HOS. In order to address these questions, it is first necessary to understand how the conjugation process and subsequent incorporation of drug linkers onto the ADC impact the HOS of the parent mAb. Different modalities such as interchain cysteine linked and engineered cysteine linked ADCs may demonstrate characteristic HDX-MS fingerprints when compared to the parent mAb. This concept is supported by our findings that interchain cysteine linked ADCs display a slight

Figure 8. Crystal structure of the Fc domain of a humanized IgG1 (PBD: 1HZH). Residues of the C H 2 domain peptide FLFPPKPKDTLM and the CH2−CH3 interface peptide KTISKAKGQPREPQV that exhibit increased HDX in ADCs and partially reduced mAbs relative to the intact mAbs are highlighted in red and blue, respectively. Glycans are depicted as yellow sticks, and the four intrachain disulfides are depicted as spheres. Reprinted with permission from ref 57. Copyright 2014 American Chemical Society.

proximally adjacent. The CH2 domain peptide FLFPPKPKDTLM, which is colored red, starts as a β-strand and transitions to a short α-helix in the lower part of the CH2 domain. This region is adjacent to the CH2−CH3 domain interface peptide KTISKAKGQPREPQV which is represented in blue in Figure 8. Both regions are spatially separated from the IgG1 hinge region and thus would not be expected to G



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(11) Hartley, J. A.; Hamaguchi, A.; Coffils, M.; Martin, C. R.; Suggitt, M.; Chen, Z.; Gregson, S. J.; Masterson, L. A.; Tiberghien, A. C.; Hartley, J. M.; et al. SG2285, a novel C2-aryl-substituted pyrrolobenzodiazepine dimer prodrug that cross-links DNA and exerts highly potent antitumor activity. Cancer Res. 2010, 70, 6849−58. (12) Hartley, J. A.; Spanswick, V. J.; Brooks, N.; Clingen, P. H.; McHugh, P. J.; Hochhauser, D.; Pedley, R. B.; Kelland, L. R.; Alley, M. C.; Schultz, R.; et al. SJG-136 (NSC 694501), a novel rationally designed DNA minor groove interstrand cross-linking agent with potent and broad spectrum antitumor activity: part 1: cellular pharmacology, in vitro and initial in vivo antitumor activity. Cancer Res. 2004, 64, 6693−9. (13) Jeffrey, S. C.; Burke, P. J.; Lyon, R. P.; Meyer, D. W.; Sussman, D.; Anderson, M.; Hunter, J. H.; Leiske, C. I.; Miyamoto, J. B.; Nicholas, N. D.; et al. A potent anti-CD70 antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine drug with site-specific conjugation technology. Bioconjugate Chem. 2013, 24, 1256−63. (14) Kung Sutherland, M. S.; Walter, R. B.; Jeffrey, S. C.; Burke, P. J.; Yu, C.; Kostner, H.; Stone, I.; Ryan, M. C.; Sussman, D.; Lyon, R. P.; et al. SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drugresistant AML. Blood 2013, 122, 1455−63. (15) Dornan, D.; Bennett, F.; Chen, Y.; Dennis, M.; Eaton, D.; Elkins, K.; French, D.; Go, M. A.; Jack, A.; Junutula, J. R.; et al. Therapeutic potential of an anti-CD79b antibody-drug conjugate, antiCD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma. Blood 2009, 114, 2721−9. (16) Junutula, J. R.; Raab, H.; Clark, S.; Bhakta, S.; Leipold, D. D.; Weir, S.; Chen, Y.; Simpson, M.; Tsai, S. P.; Dennis, M. S.; et al. Sitespecific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 2008, 26, 925−32. (17) Strop, P. Versatility of Microbial Transglutaminase. Bioconjugate Chem. 2014, 25 (5), 855−62. (18) Axup, J. Y.; Bajjuri, K. M.; Ritland, M.; Hutchins, B. M.; Kim, C. H.; Kazane, S. A.; Halder, R.; Forsyth, J. S.; Santidrian, A. F.; Stafin, K.; et al. Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 16101−6. (19) Smith, E. L.; Giddens, J. P.; Iavarone, A. T.; Godula, K.; Wang, L. X.; Bertozzi, C. R. Chemoenzymatic Fc glycosylation via engineered aldehyde tags. Bioconjugate Chem. 2014, 25, 788−95. (20) Wakankar, A.; Chen, Y.; Gokarn, Y.; Jacobson, F. S. Analytical methods for physicochemical characterization of antibody drug conjugates. MAbs 2011, 3, 161−72. (21) Chari, R. V.; Martell, B. A.; Gross, J. L.; Cook, S. B.; Shah, S. A.; Blattler, W. A.; McKenzie, S. J.; Goldmacher, V. S. Immunoconjugates containing novel maytansinoids: promising anticancer drugs. Cancer Res. 1992, 52, 127−31. (22) Hamblett, K. J.; Senter, P. D.; Chace, D. F.; Sun, M. M.; Lenox, J.; Cerveny, C. G.; Kissler, K. M.; Bernhardt, S. X.; Kopcha, A. K.; Zabinski, R. F.; et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate. Clin. Cancer Res. 2004, 10, 7063−70. (23) Laguzza, B. C.; Nichols, C. L.; Briggs, S. L.; Cullinan, G. J.; Johnson, D. A.; Starling, J. J.; Baker, A. L.; Bumol, T. F.; Corvalan, J. R. New antitumor monoclonal antibody-vinca conjugates LY203725 and related compounds: design, preparation, and representative in vivo activity. J. Med. Chem. 1989, 32, 548−55. (24) Le, L. N.; Moore, J. M.; Ouyang, J.; Chen, X.; Nguyen, M. D.; Galush, W. J. Profiling antibody drug conjugate positional isomers: a system-of-equations approach. Anal. Chem. 2012, 84, 7479−86. (25) Siegel, M. M.; Hollander, I. J.; Hamann, P. R.; James, J. P.; Hinman, L.; Smith, B. J.; Farnsworth, A. P.; Phipps, A.; King, D. J.; Karas, M.; et al. Matrix-assisted UV-laser desorption/ionization mass spectrometric analysis of monoclonal antibodies for the determination of carbohydrate, conjugated chelator, and conjugated drug content. Anal. Chem. 1991, 63, 2470−81. (26) Siegel, M. M.; Tabei, K.; Kunz, A.; Hollander, I. J.; Hamann, R. R.; Bell, D. H.; Berkenkamp, S.; Hillenkamp, F. Calicheamicin

increase in HDX kinetics in local domains located in CH2 and the CH2−CH3 transition regardless of drug-linker type. The establishment of ADC modality specific conformational “fingerprint” libraries derived from HDX-MS data could serve as a valuable resource for demonstrating process consistency and also be informative for investigational purposes. The clinically validated success of ADCETRIS and KADCYLA and the increasing number of ADCs in early phase clinical trials indicate that ADCs are a highly effective means for delivering cytotoxic agents to cancer cells with great specificity. The relative complexity of ADCs underscores the importance of developing analytical approaches which can be used to assess relevant quality attributes such as drug load and higher order structure. Our results highlight the utility of MS for characterization of relevant quality attributes of ADCs.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (425) 527-2412. Notes

The authors declare no competing financial interest.

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REFERENCES

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