Reductive Desulfuration as an Important Tool in Detection of Small

Article Cite This: Anal. Chem. XXXX, XXX, XXX−XXX

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Reductive Desulfuration as an Important Tool in Detection of Small Molecule Modifications to Payload of Antibody Drug Conjugates Jianyao Wang,* Wei Zhang, Rhys Salter, and Heng-Keang Lim Department of Drug Metabolism and Pharmacokinetics, Janssen Research & Development, Welsh & McKean Roads, Spring House, Pennsylvania 19477, United States

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S Supporting Information *

ABSTRACT: A high resolution accurate mass LC−MS method was developed to facilitate the characterization of a subset of antibody drug conjugate (ADC) biotherapeutics, where the payload is linked to the antibody by a thioether bond. Desulfuration of the thioether linker was optimized for release of the payload to take advantage of the high resolution and high mass accuracy of the Orbitrap to characterize metabolism of the payload. Two model ADCs, trastuzumab emtansine (T-DM1) and SigmaMAb dansyl-cadavarine-SMCC (SigmaMAb ADC mimic) were selected for optimization of the desulfuration reaction as a function of reaction time, pH, organic solvent, and chaotropic reagents (urea, guanidine HCl) by monitoring the yield of released desulfurated DM1 from T-DM1 and desulfurated dansyl-cadaverine-SMCC from SigmaMAb ADC mimic, respectively. The optimized desulfuration technique was successfully applied to enable characterization of the ADC following its incubation in hepatocytes, liver microsomes, and buffers, as illustrated by the identification of a hydrolyzed thiosuccinimide ring of SigmaMAb ADC mimic following incubation in buffer for 48 h. The results from this study demonstrate that the chemical cleavage of thioether bond by desulfuration is simple, efficient, and specific. This technique is useful in characterization of metabolism on the payload of ADC to provide guidance for improvement of its biopharmaceutical profile. This is the first report on characterization of modification to payload of ADC following desulfuration.

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INTRODUCTION Antibody−drug conjugates (ADCs) are fast becoming one of the major biotherapeutics for the treatment of certain cancers.1 This is illustrated by two marketed ADCs, brentuximab vedotin and trastuzumab emtansine (T-DM1), which are effective against hematological and solid malignancies, respectively. In addition, since 2016, there have been over 55 ADCs advanced into clinical trials.2 ADCs are three-part complexes consisting of a cytotoxic drug, also called payload, linked to an antibody through a degradable chemical linker. This targeted drug delivery system selectively targets cancer cells while reducing systemic exposure of the cytotoxic drug and, together with the relative stability of the linker, minimizes general cytotoxicity.3 ADCs are internalized into cells by receptor-mediated endocytosis and then fused with lysosomes for degradation by proteases.4 The cytotoxic payload can then be released into the cancer cells while the antibodies are degraded to amino acids by lysosomal proteases for eventual recycling back to the amino acid pool. The homobifunctional © XXXX American Chemical Society

maleimide cross-linker is commonly used for the Michael addition of a thiolate to the double bond of the maleimide to form a thiosuccinimide bond that connects the antibody to the ADC payload. Interestingly, both marketed ADCs such as brentuximab vedotin and T-DM1 are examples that utilize a thiosuccinimide bond linking the antibodies to the cytotoxic agents. T-DM1 is an ADC for the treatment of breast cancer overexpressing human epidermal growth factor receptor 2 (HER2).5,6 In vitro studies suggest that the cytotoxic agent emtansine (DM1) and its derivatives are 25- to 4000-fold more potent than the currently used chemotherapeutic agents.7,8 In two phase III studies involving patients with HER2-positive metastatic breast cancer, T-DM1 significantly improved overall and progression free survival9,10 together with a favorable toxicity profile relative to control treatment regimens. Received: November 6, 2018 Accepted: January 4, 2019 Published: January 4, 2019 A

DOI: 10.1021/acs.analchem.8b05134 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

payload attaching to the Fd′ fragment is within the mass resolution of Q/TOF or Orbitrap for mass resolution into monoisotopic peak for assignment of the chemical formula for definitive structural elucidation. The maleimide linker is one of the many linkers employed to couple a payload to an antibody34 as indicated by the commercialization of maleimide-link biotherapeutics like trastuzumab emantasin, brentuximab vedotin etc. Desulfuration is a well-established chemical reaction which had been successfully utilized to convert glutathione conjugates of xenobiotics for characterization by gas chromatography−mass spectrometry (GC−MS) analysis.35 This reaction has been recently applied to convert the cysteine residue of a protein to its dehydroalanine derivative prior to tryptic digestion for sequencing by mass spectrometry.36,37 Raney nickel has been used for desulfuration of drug−glutathione conjugate for analytical characterization by GC−MS in trace amounts.38 Another reagent that is routinely used for desulfuration is nickel or cobalt boride, which was used in desulfuration of thioethers containing amino acids or biotin methyl ester.39 The desulfuration reaction was further extended to thioketals and thiophenes40 and more comprehensive description of its applications was recently reviewed.41 Furthermore, this desulfuration technique has found increasing use in characterization of peptide structures.42,43 This report describes the development of a high resolution accurate mass LC−ESI/MS method to better characterize a subset of ADC biotherapeutic where the payload is linked to antibody by a thiosuccinimide bond. Desulfuration of the thiosuccinimide linker was optimized to efficiently release the payload and leverage the high resolution and high mass accuracy of Orbitrap for characterization of molecules within the mass range (m/z 2000) of the instrument. This desulfuration procedure provided evidence for hydrolysis of the succinimidyl ring on the payload of SigmaMAb dansylcadavarine-SMCC (SigmaMAb ADC mimic). Furthermore, the desulfuration procedure elicited adequate sensitivity for investigation of in vitro metabolic stability of T-DM1.

The metabolism of each payload on an ADC is often characterized by both top-down11−13 and bottom-up14,15 mass spectrometric analysis. Hence, due to its high sensitivity, a mass spectrometry-based method is essential for the characterization of ADC because it only requires nanogram amounts of material for structural elucidation of the biotransformation pathways under investigation.16−24 However, it is challenging to distinguish a minor change in molecular weight from oxidative metabolism of a payload (plus 16 Da) by mass spectrometric analysis of intact ADC (MW ∼ 150 kDa) without any chromatographic separation. This is because the mass spectrometer (quadrupole/time-of-flight [Q/TOF] or Orbitrap) typically used for characterization of intact ADC does not have an adequate mass resolution to separate the m/z of oxidative metabolite from unchanged ADC. It is even more challenging to identify a 1 Da change from the metabolic conversion of, for example, glutamine to glutamic acid or asparagine to aspartic acid in an ADC. This analytical complexity is further compounded by the addition of PEG linkers to ADC’s due to the polydispersity of PEG that superimposes the protein charge pattern of the ADC. Charge stripping by postcolumn addition of organic base has been successfully utilized to analyze PEGylated biotherapeutics.25,26 Unfortunately, the postcolumn addition of organic base has the undesirable effect of reducing the mass spectrometry signal and ultimately the sensitivity of detection.27,28 Quantitative analysis of PEGylated proteins in animal tissues has been reported using liquid chromatography−mass spectrometry (LC−MS) coupled with in-source collision-induced dissociation (CID).27 Thus, the development of a biotherapeutic relies heavily on the highly sensitive immunoassay to quantify the low concentration of biotherapeutic from the low dose given due to its high potency. However, such an immunoassay method suffers from lower selectivity and an inability to differentiate itself from its metabolic products. This is reflected in the limited report on characterization of the metabolic products until the discovery of the utility of high resolution and accurate mass electrospray ionization/mass spectrometry (ESI/MS) in structural elucidation of low concentration of metabolic biotherapeutic products.28,29 Strategic implementation of appropriate mechanistic drug metabolism and pharmacokinetic (DMPK) experiments followed by identification of an absorption, distribution, metabolism and excretion (ADME) profiles from routine screening of compounds in discovery has been successful in designing out the ADME liability after several iterations. Such a strategy had worked for small organic drug molecule and recently reported to be equally useful in the optimization of the stability of a drug molecule of an ADC.30,31 This improvement in the in vitro metabolic stability of the drug molecule of an ADC was successfully translated into improvement in its PK and PD properties. It is without doubt that the characterization of metabolism of the payload of ADC is important in rational drug design of ADC. However, the size of the payload relative to the size of the half-life extension protein can present a challenge for characterization of metabolic changes to payload from direct analysis of ADC by LC−MS if the monoisotopic m/z cannot be resolved by the mass resolution of the instrument. The current approach to reducing the size of the ADC is by selective cleavage of immunoglobulin G (IgG) at a single site containing G-G below the hinge region by protease IdeS and followed by disulfide bond reduction to give 3 approximately 25 kDa fragments (Fd′, Fc, and LC).32,33 The

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EXPERIMENTAL SECTION Materials and Reagents. All chemicals and materials used in this experiment are described on page S-3 of the Supporting Information. Preparation of Stock Solutions. Stock solutions of TDM1, DM1, and SigmaMAb ADC mimic, at 1 mg/mL, were prepared in 50/50 methanol (MeOH)/water and stored at 4 °C until use. NiCl2·6H2O at 100 mg/mL was prepared in water and stored at 4 °C until use. Sodium borohydride (NaBH4) at 40 mg/mL was freshly prepared in water before use. Optimization of Desulfuration of ADC. The desulfuration reaction was optimized for maximum yield of released desulfurated DM1 and desulfurated dansyl-cadaverine-SMCC, respectively, as a function of reaction time, pH, organic solvents, and chaotropic reagents (urea and guanidine HCl). The optimized procedure was then applied to desulfuration of immunoaffinity-captured ADC from plasma or serum. The experimental details on optimization of the desulfuration reaction are described on pages S-3−S-5 in the Supporting Information. Optimized Conditions for Desulfuration Reaction. The optimized reaction used for subsequent desulfuration is as described. ADC solution (1 mg/mL) is diluted 10-fold by B


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Figure 1. High resolution accurate full scan (MS) and product ion (MS2 and MS3) mass spectra of desulfurated T-DM1 (A), scheme of fragmentation pathway (B), and proposed structure of desulfuration of T-DM1 (C).

adding 10 μL of ADC stock solution with 90 μL of 1 M urea in pH 2.5 buffer prior to heating at 90 °C for 30 min. The tube is then chilled in an ice-bath for 5 min before adding 100 μL of methanol and hand-vortexing to mix thoroughly. The desulfuration of the above ADC solution should be accomplished using nickel boride (Ni2B) generated in situ by adding 40 and 50 μL of aqueous solution of 100 mg/mL nickel(II) chloride hexahydrate (NiCl2·6H2O) and 40 mg/mL NaBH4, respectively. The tube is capped and vortexed at room temperature for 3 h. The desulfuration reaction should be

carried out in excess Ni2B, as indicated by the presence of black particles in green solution. The remaining solid particles in the reaction mixture are removed by filtration using 0.45 μm nylon filter at 2000g at room temperature for 1 min at the end of reaction. The filtrate is dried under a gentle stream of N2 and reconstituted with 200 μL of 20% acetonitrile (ACN) in H2O prior to LC−MS analysis. The desulfuration procedure can be directly applied to ADC released from immunoaffinitycaptured resins by acidic buffer, pH 2.5 (0.2 M glycine/HCl). It is recommended to increase the amounts of Ni2B for C


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Figure 2. High resolution accurate full scan (MS) and product ion (MS2) mass spectra (A), scheme of fragmentation pathway (B), and proposed structure of desulfuration of SigmaMAb ADC mimic (C).

desulfuration of immunoaffinity-captured ADC from plasma or serum samples if observed fewer black particles of Ni2B in the green solution. If needed, more NaBH4 can be added to generate more Ni2B during the 3 h reaction time. Immunoaffinity Capture ADC from Plasma or Serum. In this experiment, ADC spiked in serum or plasma was captured using antibody selective for Fc domain (CaptureSelect human IgG-FcXL) or CH1 domain (CaptureSelect IgG-CH1) of all human IgG subclasses. Briefly, aliquots of 200 μL slurry of CaptureSelect affinity resins were transferred to 0.45 μm centrifuge filter insert (0.5 mL) (Pall Biotech, Port Washington, NJ) and centrifuged at 2000g to remove storage solvent prior to washing with 400 μL of 10 mM PBS buffer, pH 7.4 by centrifugation at 1000g for 1 min. This step was repeated for a total of three times. Then, an aliquot of 200 μL of ADC spiked plasma or serum at 0.1 mg/mL was loaded into the inset containing washed affinity resins and mixed by rotating for 15 min for affinity capturing. The remaining plasma or serum was separated from the captured ADC on affinity resins by centrifugation at 5000g for 2 min. The remaining plasma or serum residues on the affinity resins were removed by washing with 3 × 400 μL 10 mM PBS buffer (pH 7.4) as described above. Finally, the captured ADC on the affinity resins was released by adding 400 μL of 0.2 M glycine/ HCl buffer (pH 2.5) into the inset and mixing by rotating for 10 min prior to centrifugation as described above. The recovered ADC from plasma or serum by immunoaffinity capture was used directly for desulfuration prior to LC−MS

analysis as described above. Similarly, the effect of biological matrix on the efficiency of desulfuration reaction was evaluated by spiking the same concentration of ADC to 0.1 M phosphate buffer (pH 7.4) to the inset for immunoaffinity capture in the same procedure as described above for serum or plasma prior to processing for LC−MS analysis. In vitro Stability of ADC. The experimental details on application of desulfuration to investigation of the in vitro stability of ADC in 0.1 M phosphate buffer (pH 5.5−7.4), serum, liver microsomes, and hepatocytes are described on pages S-5 and S-6 in the Supporting Information LC−MS Analyses. All LC−MS analyses were performed using an Accela UHPLC (Thermo Scientific, San Jose, CA, United States) interfaced with an LTQ/Orbitrap XL (Thermo Scientific, Bremen, Germany). Chromatographic separations of desulfuration products of ADCs were carried out using a Phenomenex Jupiter C4 column (250 × 2.0 mm ID, 300 Å, 5 μm; Torrance, CA) thermostated at 35 °C. The column was eluted with a linear gradient from 5 to 95% ACN containing 0.1% formic acid from 5 to 20 min at 200 μL/min and held at 95% ACN for 5 min before returning to equilibrate with an initial gradient of 5% ACN% for 5 min. The entire eluent was sprayed into the mass spectrometer at 4.5 kV with sheath, auxiliary, and sweep gas set at 60, 15, and 5 arbitrary units, respectively. Desolvation of the solvent droplets was aided by heated capillary temperature at 300 °C. The LTQ/Orbitrap XL was operated in positive electrospray ionization mode; the resolution was set at 30 000 with full scan mass range from m/z D


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449.2573 (C23H37N4O3S+, 449.2581, +1.8 ppm) and 336.1738 (C17H26N3O2S+, 336.1740, −0.6 ppm), which further undergo neutral loss of NH3 to form product ions at m/z 432.2300 (C 23 H34 N3 O 3 S+ , 432.2315, −3.4 ppm) and 319.1471(C17H23N2O2S+, 319.1475, −1.3 ppm), respectively. The cleavage of sulfonamide bond combined with neutral loss of H 2 O resulted in the product ion at m/z 419.3017 (C23H39N4O3+, 419.3017, 0 ppm). As such, the structure of the desulfurated product is proposed as shown in Figure 2C. Optimization of the Desulfuration Reaction. There was no significant change in the yield of desulfurated product from T-DM1 or the SigmaMAb ADC mimic as a function of reaction time as shown in Figure S7. The desulfuration of SigmaMAb ADC mimic and T-DM1 resulted in maximum yield at pH 2.5 (Figure S8). For the effect of organic solvents, the data showed that 25% of acetonitrile to water or 0.1 M phosphate buffer (pH 7.4) gave the maximum yield of desulfurated products from both ADCs compared to 100% water or water containing 25 or 50% methanol, see Figure S9. Also, the efficiency of the desulfuration reaction was not affected by 4 M urea or guanidine-HCl for both ADCs studied as shown in Figure S10, suggesting that there is no need to reduce the denaturant concentration by dilution prior to desulfuration. That the desulfuration reaction was quantitative is demonstrated in Table S1, which appeared to be linear with about 3−4 orders of magnitude proportional to the amount of starting material, ranging from 5 to 5000 ng/mL and 5 to 50 000 ng/mL for SigmaMAb ADC mimic and T-DM1, respectively. The peak area of desulfurated product from SigmaMAb ADC mimic is about 1−2 orders of magnitude greater than that from T-DM1 over the concentration range investigated despite the similarity in molecular weight of ADC, their small drug molecule payload, and DAR. However, there is no explanation for the observation. Immunoaffinity capture did not affect the desulfuration reaction based on quantitative recovery of T-DM1 from plasma as indicated by detection of level of desulfurated DM1 comparable to that from 0.1 M phosphate buffer (pH 7.4), as shown in Figure S11. Applications of Desulfuration. The linker stability is the key determinant of the efficacy and toxicity of an ADC.44,45 The thiosuccinimide linker is widely used to connect payloads to antibodies (Ab) and is also recognized as one of the soft spots for thiosuccinimide linked ADCs because it undergoes a retro Michael addition reaction, leading to a decrease in the DAR. As a result, adverse effects were suspected from the premature release of the payload due to its intrinsic cytotoxicity. Moreover, the exposed resulting electrophilic maleimide on the antibody could potentially form a hapten with circulating nucleophiles produced under cell stress. This retro Michael addition reaction is thought to be responsible for the observed long half-life of about 7 days for ADC’s coupled via maleimide-caproyl monomethyl auristatin F in rat due to transfer of the payload to the cys-34 of serum albumin.45 Another reaction recently reported for the thiosuccinimide linker itself is hydrolysis of the thiosuccinimide ring to form two isomeric ring-opened thiosuccinamic acids.43−46 This ringopened thiosuccinamic acid has unexpectedly improved stability in vitro and in vivo, but there is limited structure− activity relationship on its formation and stability.46 Therefore, the desulfuration reaction should also be applicable to release the payload from thiosuccinamic acid in addition to closed analogues to increase versatility of desulfuration reaction in characterization of thiosuccinimide-linked ADC.

300−2000, and the resolution was at 7500 in tandem mass spectrometry mode. Ions were detected with electron multiplier 1 and 2 set at 1260 and 1250 V, respectively. Data acquisition and processing were carried out using Xcalibur 2.0 (Thermo Scientific, San Jose, CA). The full scan raw data were deconvoluted using ProMass software (Novatia LLC, PA).

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RESULTS AND DISCUSSION The chemical structures of trastuzumab emtansine (T-DM1) and SigmaMAb dansyl-cadavarine-SMCC (SigmaMAb ADC mimic) are shown in Figure S1. The desulfuration reaction was initially applied to emtansine (DM1), and the structural elucidation of the desulfurated DM1 was performed by using the high resolution accurate full scan and product ion mass spectra of DM1 as shown in Figure S2, and Figure S3. The interpretation of the full scan and product ion mass spectra of both DM1 and its desulfurated product are described in pages S-9 and S-11, respectively. The identification of desulfurated product provided evidence for desulfuration of DM1 (Figure S4). Desulfuration of T-DM1. The desulfuration of T-DM1 was conducted and analyzed under the same conditions used for desulfuration of DM1. A major desulfurated product of TDM1 with a similar retention time of desulfurated DM1 at 19.2 min (Figure S5) possessed an identical chemical formula of C35H48O10N3ClH+ (0.8 ppm) at m/z 706.3107 as that of the desulfurated product of DM1 by high resolution accurate mass measurement, as shown in Figure 1A. This was further corroborated by good agreement of product ion mass spectra of the desulfurated product from T-DM1 (Figure 1B) with those from DM1 (Figure S2) as well as its desulfurated product (Figure S3). The protonated molecule of desulfurated T-DM1 at m/z 706.3107 (C35H48O10N3ClH+, 706.3101, +0.8 ppm) lost an H2O molecule to form the abundant dehydrated product ion at m/z 688.3003 (C35H46O9N3ClH+, 688.2995, +1.2 ppm), which further lost a CO2 molecule to generate the product ion at m/z 644.3076 (C34H47ClN3O7+, 644.3097, −3.2 ppm). In addition, other fragmentation pathways of this desulfurated product were the formation of product ions at m/ z 529.2080 which corresponded to cleavage of C−O bond linking the side chain and the ring system with a chemical formula of C28H34ClN2O6+ (m/z 529.2100, −3.8 ppm), then loss CO2 to form m/z 485.2180 (C27H34ClN2O4+, 485.2202, 4.5 ppm). In the MS3, fragment ions of m/z 453.1926 correspond to neutral loss of CH3OH (C26H30ClN2O3+, 453.1939, −2.9 ppm), and its consecutive neutral losses of H2O and CO to form m/z 435.1823 (C26H28ClN2O2+, 435.1834, −2.5 ppm) and 425.1979 (C25 H30ClN 2O2+ , 425.1990, −2.6 ppm), respectively. As such, the desulfuration product from T-DM1 could be proposed as shown in Figure 1. As such, the desulfurated product from T-DM1 could be proposed as shown in Figure 1C. Desulfuration of SigmaMAb ADC Mimic. High resolution accurate mass analysis of the desulfurated products of SigmaMAb ADC mimic by LC−UV/MS revealed that the product at ∼16.1 min (Figure S6) resulted in the full scan mass spectrum and product ion mass spectrum shown in Figure 2A. A protonated molecule is observed at m/z 670.3619 that was 2.1 ppm less than the calculated value of m/z 670.3633 based on the chemical formula (C35H51N5O6SH+) of the postulated desulfurated product of SigmaMAb ADC mimic. The product ion mass spectrum (Figure 2B) was characterized in the cleavage of two amide bonds resulted in product ions of m/z E


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Figure 3. RIC of desulfurated product of SigmaMAb ADC mimic and its hydrolyzed product at m/z 670.3633 and 688.3738, respectively, from LC−MS analysis of incubation of 0.1 mg/mL SigmaMAb ADC mimic in 0.1 M phosphate buffer at pH 7.4 (A) and 5.5 (B) up to 96 h at 37 °C and proposed structure of desulfurated product with m/z 688 (C).

Stability of SigmaMAb ADC Mimic in 0.1 M Phosphate Buffer. LC−MS analysis of the desulfurated products from the incubation of the SigmaMAb ADC mimic in 0.1 M phosphate buffer, pH 7.4 for 48 h revealed a degradation product at ∼17.4 min; the only product detected at 96 h (Figures 3A and 3B). However, this degradation product began

to appear only following incubation in 0.1 M phosphate pH 5.5 buffer at 96 h (Figure 3B), suggesting that the rate of hydrolysis of the thiosuccinimide ring of ADC was in fact faster at neutral pH of 7.4 than at acidic pH of 5.5. The assignment of protonated molecule and its product ions by mass spectrometric analysis support that the degradant (m/z F


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unambiguous identification based on a correct assignment of its chemical formula. This approach can readily identify small molecular weight changes to a drug payload compared to the analysis of >30 kDa intact ADCs with same small change in mass,51 which is beyond the resolution of TOF mass analyzers. This desulfuration procedure is suitable for the detection and characterization of an ADC containing thiosuccinimide linker spiked at low microgram/mL in buffer and also in biological matrixes because the desulfuration reaction is compatible with antibody-based enrichment procedures. As such, the desulfuration reaction is complementary to selective protease-based middle-down proteomic LC−MS methods for the characterization of ADCs.

688.3739) was produced by hydrolysis of thiosuccinimide to form a ring-opened species in Figure S14. The details of the interpretation are described in Figure S14. The proposed degradation of SigmaMAb ADC mimic by hydrolysis is illustrated in Figure 3C. There are two possible isomers from hydrolysis of the thiosuccinimide ring of ADC, and both ended with the same desulfurated product. It consisted of about 30% ADC hydrolyzed when incubated in 0.1 M phosphate buffer pH 7.4 for 48 h based on peak area and completely hydrolyzed at 96-h incubation. Interestingly, there were no other degradation products observed in the 96-h incubation mixture, suggesting that the ring-opened analogue is relatively more stable than ring-closed analogue. This clearly illustrates that the desulfuration reaction is suitable for detecting hydrolyzed thiosuccinimide ring of ADC. Stability of T-DM1 in Serum. Information on the metabolism of ADC is important for understanding of its pharmacokinetic and pharmacology.47,48 LC−MS analysis of the desulfurated product from rat and human serum stability studies are as shown in Figure S12. The desulfurated DM1 product was the only observed payload-related component in all samples analyzed. The accompanying time-dependent decrease in desulfurated DM1 led to an estimation of the disappearance half-life of 16 and 17 h of T-DM1 in rat and human serum, respectively. The disappearance of T-DM1 may be due to cleavage of the thiosuccinimide ring linkage to ADC via retro-Michael addition reaction accompanied by transfer of the exposed maleimide linker and free sulfhydryl of DM1 to albumin.49 Stability of T-DM1 in Microsomes and Hepatocytes. Hepatic metabolism is an important clearance pathway for many small organic drug molecules. The limitation of high resolution accurate mass spectrometer to resolve the monoisotopic m/z of intact ADC for the assignment of its chemical formula led to the investigation, in rats, of the metabolism and disposition of the payload (DM1) instead of the entire ADC.50 The payload (DM1) was reported to undergo metabolism but may not be totally reflective of metabolism of DM1 on ADC. The desulfuration procedure may offer an alternative approach to interrogate metabolism of DM1 of T-DM1. Therefore, the unchanged desulfurated DM1 from T-DM1 was the only payload-related component detected in both extracts of hepatocytes (Figure S13A) and liver microsomal (Figure S13B) incubations from all species. This suggests that the thiosuccinimide linked payload of TDM1 was stable and did not undergo any appreciable metabolism due to inaccessibility of T-DM1 to the catalytic sites of enzymes and consistent with a previous report.51 However, the absence of metabolism cannot be fully ruled out due to poor uptake into hepatocytes.39

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b05134.

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Materials, reagents, and experimental conditions, fourteen figures and a table showing the structures of model ADCs, mass spectra, and interpretation of fragmentation pathway of desulfurated product of DM1 and hydration led ring opened of succinimide of SigmaMAb ADC mimic, optimization of experimental conditions such as pH, organic solvent, surfactant, and dynamic range of desulfuration, compatibility with immunoaffinity capture techniques; and results of in vitro stability of ADC in 0.1 M phosphate buffer, serum, liver microsomes and hepatocytes (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel.: 215-540-4055; E-mail: [email protected]. ORCID

Jianyao Wang: 0000-0003-2559-8849 Author Contributions

H.-K.L. provided technical advice and support in composition of the manuscript. W.Z. and R.S. contributed equally. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS LC−MS liquid chromatography−mass spectrometry Fd 220 amino acids from the Nterminus of the heavy chain of antibody Fc fragment crystallizable region is the tail region of an antibody LC light chain of antibody GC−MS gas chromatography−mass spectrometry ADC antibody−drug conjugate RIC reconstructed ion chromatogram BPC base peak chromatogram T-DM1 trastuzumab emtansine SigmaMAb ADC mimic SigmaMAb dansyl-cadavarineSMCC DAR drug-antibody ratio

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CONCLUSION A desulfuration reaction using nickel boride was successfully developed and optimized for the quantitative release of drug payloads coupled via a thiosuccinimide linker to an antibody. The desulfuration reaction was selective for reductive cleavage of the C−S bond accompanied by the resultant formation of alanine from the cysteinyl residue. The desulfuration reaction will take place if S is a sulfhydryl (−SH), sulfalkyl (−SR), sulfinyl (−SO), or sulfonyl (−SO2) form. The molecular weight of the desulfurated drug payload is within the mass resolution of most mass spectrometers (Orbitrap and Q/TOF) for baseline separation of its monoisotopic peak to facilitate its G


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Analytical Chemistry ESI-MS CID

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(28) Su, D.; Ng, C.; Khosraviani, M.; Yu, S. F.; Cosino, E.; Kaur, S.; Xu, K. Anal. Chem. 2016, 88, 11340−11346. (29) Rosati, S.; Rose, R. J.; Thompson, N. J.; van Duijn, E.; Damoc, E.; Denisov, E.; Makarov, A.; Heck, A. J. Angew. Chem., Int. Ed. 2012, 51, 12992−12996. (30) Tumey, L. N.; Leverett, C.; Vetelino, B.; Li, F.; Rago, B.; Han, X.; Loganzo, F.; Musto, S.; Bai, G.; Sukuru, S.; Graziani, E.; Puthenveetil, S.; Casavant, J.; Ratnayake, A.; Marquette, K.; Hudson, S.; Doppalapudi, V.; Stock, J.; Tchistiakova, L.; Bessire, A.; Clark, T.; Lucas, J.; Hosselet, C.; O’Donnell, C.; Subramanyam, C. ACS Med. Chem. Lett. 2016, 7, 977−982. (31) Bessire, A.; Ballard, T. E.; Charati, M.; Cohen, J.; Green, M.; Lam, M.; Loganzo, F.; Nolting, B.; Pierce, B.; Puthenveetil, S.; Roberts, L.; Schildknegt, K.; Subramanyam, C. Bioconjugate Chem. 2016, 27 (7), 1645−1654. (32) An, Y.; Zhang, Y.; Mueller, H.; Shameem, M.; Chen, X. MAbs. 2014, 6 (4), 879−893. (33) Beck, A.; Wagner-Rousset, E.; Ayoub, D.; Dorsselaer, A. V.; Sanglier-Cianférani, S. Anal. Chem. 2013, 85 (2), 715−736. (34) Jain, N.; Smith, S. W.; Ghone, S.; Tomczuk, B. Pharm. Res. 2015, 32 (11), 3526−3540. (35) Balazy, M.; Murphy, R. C. Anal. Chem. 1986, 58, 1098−1101. (36) Wang, Z.; Rejtar, T.; Zhou, Z.; Karger, B. L. Rapid Commun. Mass Spectrom. 2010, 24, 267−275. (37) Bernardes, G. J. L.; Chalker, J. M.; Errey, J. C.; Davis, B. G. J. Am. Chem. Soc. 2008, 130, 5052−5053. (38) Zaikin, V. G.; Halket, J. M. Eur. J. Mass Spectrom. 2005, 11, 127−60. (39) Davis, J. A.; Rock, D. A.; Wienkers, L. C.; Pearson, J. T. Drug Metab. Dispos. 2012, 40, 1927−1934. (40) Schlesinger, H. I.; Brown, H. C.; Finholt, A. E.; Gilbreath, J. R.; Hoekstra, H. R.; Hyde, E. K. J. Am. Chem. Soc. 1953, 75, 215−219. (41) Ganem, B.; Osby, J. O. Chem. Rev. 1986, 86, 763−780. (42) Back, T. G.; Baron, D. L.; Yang, K. J. Org. Chem. 1993, 58, 2407−2413. (43) Rentner, J.; Kljajic, M.; Offner, L.; Breinbauer, R. Tetrahedron 2014, 70, 8983−9027. (44) Ackermann, B.; Berna, M. Protein Analysis using Mass Spectrometry: Accelerating Protein Biotherapeutics from Lab to Patient; John Wiley & Son, Inc.: United States, 2017. (45) Fontaine, S.; Reid, R.; Robinson, L.; Ashley, G.; Santi, D. Bioconjugate Chem. 2015, 26, 145−152. (46) Baldwin, A. D.; Kiick, K. L. Polym. Chem. 2013, 4, 133−143. (47) Han, T.; Zhao, B. Drug Metab. Dispos. 2014, 42, 1914−1920. (48) Kraynov, E.; Kamath, A.; Walles, M.; Tarcsa, E.; Deslandes, A.; Iyer, R.; Datta-Mannan, A.; Sriraman, P.; Bairlein, M.; Yang, J.; Barfield, M.; Xiao, G.; Escandon, E.; Wang, W.; Rock, D.; Chemuturi, N.; Moore, D. Drug Metab. Dispos. 2016, 44, 617−623. (49) Bender, B.; Leipold, D.; Xu, K.; Shen, B.; Tibbitts, J.; Friberg, L. AAPS J. 2014, 16, 994−1008. (50) Wei, C.; Zhang, G.; Clark, T.; Barletta, F.; Tumey, N.; Rago, B.; Hansel, S.; Han, X. Anal. Chem. 2016, 88, 4979−4986. (51) He, J. T.; Su, D.; Ng, C.; Liu, L.; Yu, S.-F.; Pillow, T. H.; Del Rosario, G.; Darwish, M.; Lee, B.-C.; Ohri, R.; Zhou, H. X.; Wang, J. W.; Kaur, K.; Xu, K. Y. Anal. Chem. 2017, 89, 5476−5483.

electrospray ionization mass spectrometry collision-induced dissociation

REFERENCES

(1) Peters, C.; Brown, S. Biosci. Rep. 2015, 35, e00225. (2) Chari, R. ACS Med. Chem. Lett. 2016, 7, 974−976. (3) Chari, R. V. Acc. Chem. Res. 2008, 41, 98−107. (4) Ezan, E. Adv. Drug Delivery Rev. 2013, 65, 1065−1073. (5) Lewis Phillips, G. D.; Li, G.; Dugger, D. L.; Crocker, L. M.; Parsons, K. L.; Mai, E.; Blättler, W. A.; Lambert, J. M.; Chari, R. V.; Lutz, R. J.; Wong, W. L.; Jacobson, F. S.; Koeppen, H.; Schwall, R. H.; Kenkare-Mitra, S. R.; Spencer, S. D.; Sliwkowski, M. X. Cancer Res. 2008, 68 (22), 9280−90. (6) Burris, H. A., 3rd; Tibbitts, J.; Holden, S. N.; Sliwkowski, M. X.; Lewis Phillips, G. D. Clin. Breast Cancer 2011, 11 (5), 275−82. (7) Junttila, T. T.; Li, G.; Parsons, K.; Phillips, G. L.; Sliwkowski, M. X. Breast Cancer Res. Treat. 2011, 128, 347−356. (8) Kovtun, Y. V.; Audette, C. A.; Mayo, M. F.; Jones, G. E.; Doherty, H.; Maloney, E. K.; Erickson, H. K.; Sun, X.; Wilhelm, S.; Ab, O.; Lai, K. C.; Widdison, W. C.; Kellogg, B.; Johnson, H.; Pinkas, J.; Lutz, R. J.; Singh, R.; Goldmacher, V. S.; Chari, R. V. Cancer Res. 2010, 70, 2528−2537. (9) Verma, S.; Miles, D.; Gianni, L.; Krop, I. ; Welslau, M.; Baselga, J.; Pegram, M.; Oh, D. Y.; Diéras, V.; Guardino, E.; Fang, L.; Lu, M. W.; Olsen, S.; Blackwell, K. N. Engl. J. Med. 2012, 367, 1783−1791. (10) Krop, I. E.; Kim, S.-B.; González-Martín, A.; LoRusso, P. M.; Ferrero, J. M.; Smitt, M.; Yu, R.; Leung, A. C.; Wildiers, H. Lancet Oncol. 2014, 15, 689−699. (11) Reid, G.; McLuckey, S. J. Mass Spectrom. 2002, 37, 663−75. (12) Bondarenko, P.; Second, T.; Zabrouskov, V.; Makarov, A.; Zhang, Z. J. Am. Soc. Mass Spectrom. 2009, 20, 1415−1424. (13) Mazur, M.; Seipert, R.; Mahon, D.; Zhou, Q.; Liu, T. AAPS J. 2012, 14, 530−41. (14) Kelleher, N.; Lin, H.; Valaskovic, G.; Aaserud, D.; Fridriksson, E.; McLafferty, F. J. Am. Chem. Soc. 1999, 121, 806−812. (15) Zhang, Y.; Fonslow, B.; Shan, B.; Baek, M.; Yates, J. Chem. Rev. 2013, 113, 2343−2394. (16) Firth, D.; Bell, L.; Squires, M.; Estdale, S.; McKee, C. Anal. Biochem. 2015, 485, 34−42. (17) Birdsall, R. E.; Shion, H.; Kotch, F. W.; Xu, A.; Porter, T. J.; Chen, W. mAbs 2015, 7, 1036−1044. (18) Wagner-Rousset, E.; Janin-Bussat, M. C.; Colas, O.; Excoffier, M.; Ayoub, D.; Haeuw, J. F.; Rilatt, I.; Perez, M.; Corvaia, N.; Beck, A. mAbs 2014, 6, 173−184. (19) Hengel, S. M.; Sanderson, R.; Valliere-Douglass, J.; Nicholas, N.; Leiske, C.; Alley, S. C. Anal. Chem. 2014, 86, 3420−3425. (20) Debaene, F.; Boeuf, A.; Wagner-Rousset, E.; Colas, O.; Ayoub, D.; Corvaia, N.; Van Dorsselaer, A.; Beck, A.; Cianferani, S. Anal. Chem. 2014, 86, 10674−10683. (21) Xu, K.; Liu, L.; Dere, R.; Mai, E.; Erickson, R.; Hendricks, A.; Lin, K.; Junutula, J. R.; Kaur, S. Bioanalysis 2013, 5, 1057−1071. (22) Kaur, S.; Saad, O.; Xu, K. Analysis of antibody drug conjugates by beads-based affinity capture and mass spectrometry, US Patent US8541178, 2013. (23) Xu, K.; Liu, L.; Saad, O. M.; Baudys, J.; Williams, L.; Leipold, D.; Shen, B.; Raab, H.; Junutula, J. R.; Kim, A.; Kaur, S. Anal. Biochem. 2011, 412, 56−66. (24) Huang, R. Y.; Chen, G. Drug Discovery Today 2016, 21, 850− 855. (25) Huang, L.; Gough, P.; Defelippis, M. Anal. Chem. 2009, 81 (2), 567−77. (26) Cheng, T.; Chuang, K.; Chen, B.; Roffler, S. Bioconjugate Chem. 2012, 23, 881−899. (27) Gong, J.; Gu, X.; Achanzar, W.; Chadwick, K.; Gan, J.; Brock, B.; Kishnani, N.; Humphreys, W. G.; Iyer, R. A. Anal. Chem. 2014, 86, 7642−7649. H


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