Structural Characterization of a Monoclonal AntibodyâMaytansinoid

Article pubs.acs.org/ac

Structural Characterization of a Monoclonal Antibody−Maytansinoid Immunoconjugate Quanzhou Luo,* Hyo Helen Chung, Christopher Borths, Matthew Janson, Jie Wen, Marisa K. Joubert, and Jette Wypych Department of Process Development, Amgen Inc., Thousand Oaks, California 91320, United States S Supporting Information *

ABSTRACT: Structural characterization was performed on an antibody−drug conjugate (ADC), composed of an IgG1 monoclonal antibody (mAb), mertansine drug (DM1), and a noncleavable linker. The DM1 molecules were conjugated through nonspecific modification of the mAb at solventexposed lysine residues. Due to the nature of the lysine conjugation process, the ADC molecules are heterogeneous, containing a range of species that differ with respect to the number of DM1 per antibody molecule. The DM1 distribution profile of the ADC was characterized by electrospray ionization mass spectrometry (ESI-MS) and capillary isoelectric focusing (cIEF), which showed that 0−8 DM1s were conjugated to an antibody molecule. By taking advantage of the high-quality MS/MS spectra and the accurate mass detection of diagnostic DM1 fragment ions generated from the higher-energy collisional dissociation (HCD) approach, we were able to identify 76 conjugation sites in the ADC, which covered approximately 83% of all the putative conjugation sites. The diagnostic DM1 fragment ions discovered in this study can be readily used for the characterization of other ADCs with maytansinoid derivatives as payload. Differential scanning calorimetric (DSC) analysis of the ADC indicated that the conjugation of DM1 destabilized the CH2 domain of the molecule, which is likely due to conjugation of DM1 on lysine residues in the CH2 domain. As a result, methionine at position 258 of the heavy chain, which is located in the CH2 domain of the antibody, is more susceptible to oxidation in thermally stressed ADC samples when compared to that of the naked antibody.

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forms may differ in their pharmacokinetic and toxicological properties.25 UV/vis spectroscopy is the simplest method to measure the DAR value of an ADC but not the drug load distribution profile.26 Depending on the conjugation process, hydrophobic interaction chromatography (HIC),25 reversed phase high performance liquid chromatography (RP-HPLC),17 imaged capillary isoelectric focusing (cIEF),27 electrospray ionization mass spectrometry (ESI-MS),18,28 and matrixassisted laser desorption ionization time-of-flight (MALDITOF) MS29 are commonly used as orthogonal methods to measure the DAR as well as to characterize the drug load distribution of ADCs. The lysine conjugation process results in ADC molecules that are heterogeneous with respect to both the distribution and loading of cytotoxic drug species on the mAb.18 Characterization of drug conjugation sites for ADCs utilizing lysine conjugation process is important in instances where modification of residues in the complementarity-determining regions (CDRs) might affect mAb binding to the target antigen. Peptide mapping with MS detection is the most powerful tool to characterize drug conjugation sites.26 For example, LC/MS analysis of an ADC composed of a humanized monoclonal IgG1 antibody (huN901) and DM1 was performed to identify

uring the last three decades, monoclonal antibodies (mAbs) have become increasingly important as cancer therapies, providing promise of targeted elimination of tumor cells without the systemic toxicity associated with conventional chemo- and radiotherapies.1−3 Despite the success, there are limitations regarding the antitumor efficacy of mAbs. One approach to enhance the cell-killing activity of antibodies is arming them with highly potent cytotoxic drugs (termed “payloads”) to create antibody−drug conjugates (ADCs).4−7 ADCs consist of three components: a mAb that is specific to a tumor antigen, a highly toxic payload, and a linker species that enables covalent attachment of the cytotoxin to the mAb. Currently, two classes of highly potent cytotoxins have been used as ADC payloads: (1) microtubule-destabilizing agents, such as auristatin derivatives monomethylauristatin E and F (MMAE8 and MMAF9) and maytansinoids DM1 and DM4;10 and (2) DNA minor-groove binders, such as calicheamicin11 and duocarmycin12 derivatives. Linkers used for ADC therapeutics can be divided into two categories: cleavable13,14 and noncleavable.15,16 Complete analytical profiling of the ADC requires other methods to evaluate specific properties, such as the average number of drug molecules attached to an antibody, drug-toantibody ratio (DAR), drug distribution, and drug conjugation sites.17−24 DAR and drug distribution profile are critical characteristics, as DAR determines the amount of payload that can be delivered to the tumor cell and various drug-loaded © XXXX American Chemical Society

Received: July 10, 2015 Accepted: December 2, 2015

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DOI: 10.1021/acs.analchem.5b03709 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. Schematic diagram of conjugation process.

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the linker-modified sites.18 Because the conjugation of DM1 at lysine residues prevents trypsin digestion, the authors first assumed that modification could occur at any lysine residue and calculated the expected mass of all the putative, uncleaved tryptic peptides with a mass increase of the conjugation. Then an extracted ion chromatogram (XIC) for each expected peptide was constructed from both naked antibody and huN901-DM1 LC/MS profiles. This approach is very timeconsuming due to the presence of a large number of potential conjugation sites. Moreover, if multiple conjugation sites are located in one peptide, this approach will not be able to distinguish which residues are linked with DM1. In this study, we performed a comprehensive characterization of a therapeutic ADC (Figure 1) composed of a small-molecule drug, DM1, and an IgG1 antibody linked by a noncleavable linker, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxylate (SMCC). Particularly, a higher-energy collisional dissociation (HCD) mass spectrometric approach was developed by using diagnostic ions generated for quick identification of conjugation sites without ambiguity. The solvent accessibility of these conjugated residues and the potential impact of DM1 modifications on ADC stability were also investigated.

EXPERIMENTAL SECTION Material. The naked IgG1 mAb was produced at Amgen Inc. (Thousand Oaks, CA) and consisted of two human heavy chains and two human λ light chains. The antibody was expressed in Chinese hamster ovary cells and was purified by well-established chromatographic procedures developed at Amgen.30 Detailed procedures for the preparation of ADC are described in Supporting Information section 1. Capillary Isoelectric Focusing Analysis. cIEF was performed on a Convergent Bioscience iCE280 analyzer by use of a fluorocarbon-coated capillary. The ADC sample was prepared by adding 0.4% methylcellulose, ampholytes, and 6.5 and 9.3 pI markers. Isoelectric focusing was carried out by applying a voltage of 3 kV to the capillary for 12 min at room temperature. The image of the whole capillary was captured with a charge-coupled device (CCD) camera at 280 nm. Intact Mass Analysis. The ADC samples were deglycosylated with peptide-N-glycosidase (PNGase) and then desalted on an Agilent 1100 HPLC system equipped with an Agilent Zorbax C8 column (2.1 × 150 mm, 300 Å pore size, 3.0 μm particle size). Solvent A was 0.1% (v/v) trifluoroacetic acid (TFA) in water, and solvent B was 0.1% (v/v) TFA in acetonitrile (ACN). A gradient from 15% to 90% B over 10 min was employed, at a flow rate of 0.2 mL/min. The eluent was diverted into a Waters Xevo G2 ESI-TOF mass spectrometer B


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and the average SAS between the two chains was reported (whenever two chains were present in the PDB structure); otherwise the SAS of only one chain was reported.

for analysis. The mass spectrum was then deconvoluted and analyzed by use of Waters Masslynx MaxEnt 1 software. Drug Load Distribution and Drug-to-Antibody Ratio. For intact mass analysis, the drug load distribution (percent) for each antibody with drug load i was calculated by comparing the peak area of antibody with drug load i to the total peak area of all species (eq 1). For cIEF analysis, the peak area (at 280 nm) of each antibody with drug load i was first divided by the molar extinction coefficient of the corresponding species at 280 nm. Then the normalized peak area was applied to eq 1 to calculate the drug load distribution (see Supporting Information section 2 for detailed calculation).

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RESULTS Drug to Antibody Ratio and Drug Load Distribution. The therapeutic ADC studied here was produced by conjugating DM1 to an IgG1 mAb via a noncleavable linker, SMCC, as shown in Figure 1. As the drug-to-antibody ratio of an ADC is a critical quality attribute that directly affects the safety and efficacy of the drug product, we first analyzed and compared the average DAR values of the ADC measured by UV/vis spectroscopy, imaged cIEF, and LC/TOF-MS. The average DAR value measured by UV/vis was 3.3, which agreed well with the values measured by cIEF (3.1) and intact mass analysis (3.2). Besides the average DAR value, the drug load distribution, which measures the homogeneity of ADC population, is also characterized by cIEF and LC/TOF-MS. Conjugation of DM1 molecules to the amino groups eliminates basic sites in the antibody and creates more acidic species. The variation in number of DM1 molecules linked to the antibody generates different charge isoforms as shown in Figure 2A. Peaks with lower pI corresponded to species with increasing levels of conjugation. Figure 2B shows the deconvoluted mass spectrum

(drug load distribution)i =

(peak area of mAb with drug loading i)normalized × 100 (total peak area of all species)normalized (1)

The average DAR was determined by using eq 2: m

DAR =

∑ i·(drug load distribution)i i=1

(2)

where i is drug load and m is maximum drug load. Liquid Chromatography/Tandem Mass Spectrometry Analysis of Enzymatic Digestion of Antibody−Drug Conjugate Samples. Enzyme digestions of reduced samples were prepared following the procedure described in a previous report.31 Online LC/MS/MS analyses were performed by use of a Waters Acuity ultra-high-performance liquid chromatography (UPLC) system (Milford, MA) directly coupled with a Thermo Scientific Orbitrap mass spectrometer (San Jose, CA) equipped with electrospray ionization source (see Supporting Information section 3 for detailed instrument setup). Enzymatic digests of the mAb and ADC samples were separated on a Waters BEH PST C4 column (2 × 150 mm, 130 Å pore size, 1.7 μm particle size) with the column temperature maintained at 50 °C. Mobile phase A was 0.1% (v/ v) TFA in water, and mobile phase B was 0.1% (v/v) TFA in ACN. A gradient (hold at 2% B for 2 min, 2−50% B for 78 min) was used to separate the digested peptides at a flow rate of 0.2 mL/min. The eluted peptides were monitored by both UV (214 and 252 nm wavelength) and mass spectrometry. Peptides were identified by use of MassAnalyzer,32 an in-house developed software. Conjugation levels at each site were quantified by determining the drop in the unmodified peptide UV peak area (or the peak area under the extracted ion chromatogram when the peptides coelute with other peaks) between the mAb and ADC samples. Tryptic map was used as the primary map for quantitation. Asp-N and Glu-C maps were used to quantify conjugation levels at lysine residues missed from the tryptic map. Molecular Modeling. Solvent accessibility calculations were performed with the molecular surface routine GETAREA in the energy minimization and Monte Carlo simulation package FANTOM (located at http://curie.utmb.edu/getarea. html),33 using a 1.4 Å solvent radius consistent with H2O, for Protein Data Bank (PDB) code 1AQK for the heavy and light chains of the Fab domain and for PDB code 1HZH for the Fc region. Framework structures were chosen from the PDB based on conservation of modified lysine residues and overall amino acid identity. The percentage of solvent-accessible surface area (SAS) of individual amino acids was calculated by the software

Figure 2. (A, B) ADC profiles determined by (A) cIEF and (B) ESITOF-MS. (C) Comparison of DM1 load distribution (%) determined by cIEF and ESI-TOF-MS. D represents DM1. C


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Figure 3. (A) XIC of DM1-attached peptide H1. (B, C) HCD MS/MS spectra of (B) H1-DM1 peptide I and (C) H1-DM1 peptide III. The portion of the spectrum marked with ×3 and a bracket is expanded 3 times for easier visualization.

of deglycosylated ADC. Species with 0−8 DM1 conjugated to the antibody were observed. The average mass difference between these species is 957 Da, which is consistent with the theoretical mass increase of 958 Da for reaction with one molecule of DM1 and one molecule of SMCC. The DM1 distribution profiles obtained by cIEF and LC/TOF-MS are similar (Figure 2C). By comparing the three methods for measuring DAR value, UV/vis spectroscopy is the simplest. However, this method cannot determine the drug load distribution. In addition, the free DM1 species in the ADC sample interfere with the measurement, which results in an overestimation of the DAR value. cIEF can be applied to measure the DAR value as well as the drug load distribution. However, cIEF cannot distinguish the antibody with linker only from the antibody with linker− DM1. LC/TOF-MS can be used to monitor multiple critical quality attributes, including average DAR value, drug load distribution, and impurities such as antibody with linker only. Nevertheless, this method requires expensive instrumentation and is the most complicated of the three methodologies. DM1 Conjugation Sites. The naked antibody used in this study contains a total of 92 potential conjugation sites,

including 90 lysine residues and two N-terminal glutamic acid residues of heavy chain. The large number of potential conjugation sites compared to the relatively small drug load results in heterogeneous product with various levels of DM1 conjugated at different sites. In this study, collision-induced dissociation (CID) fragmentation was first used to characterize the DM1-attached peptide. However, CID fragmentation of the DM1-attached peptides produced poor peptide backbone fragmentation with collision energy of 35−60% (data not shown). This challenge prompted us to employ higher-energy collisional dissociation (HCD) fragmentation technique to characterize the DM1-attached peptides. When compared with ion trap-based CID, HCD with Orbitrap detection provides several advantages including no low-mass cutoff, high-resolution ion detection, and multiple fragmentations resulting in higher quality of MS/MS spectra.34 Indeed, this HCD-based fragmentation approach allowed us to identify conjugation sites without ambiguity even in the situation where multiple modification sites are present in a single peptide. Taking one peptide H1 as an example, four peaks with the exact mass of H1-DM1 were observed, as shown in Figure 3A. MS/MS spectrum of H1-DM1 peptide I shows y6 D


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chain, were identified, covering approximately 83% of all the putative conjugation sites in the antibody, which matches 84% coverage for trastuzumab emtansine reported previously.36 It is also worth noting that the lysine residues in CH2 domain of the antibody were most prone to DM1 conjugation. The majority (97.4%) of the N-terminal glutamine of the light chain was converted to pyroglutamate, and therefore DM1 conjugation at N-terminus of light chain was not observed. The total DM1 conjugated on the antibody is 3.6, calculated by adding the conjugation levels at all sites, which agrees well with the average DAR value of 3.3 obtained by UV/vis. Maleimide exchange and succinimide ring hydrolysis in SMCC have been reported for protein conjugates.37,38 In this study, the side reaction of the conjugation process was evaluated. After hydrolysis, a mass increase of 18 Da is expected due to the addition of one water to the SMCC. Therefore, the level of the hydrolyzed succinimide was determined by comparing the peak area of the hydrolyzed DM1-attached peptide under the XIC to the peak area of the parent DM1-attached peptides. The level of hydrolyzed SMCC in the final ADC product was 2.3%, and the level increased to 31.3% after exposure to pH 8.2 and 37 °C for 4 weeks. Cysteine and tyrosine residues were found to be reactive to succinimidyl in other studies.39 However, in this case, because the cysteines in the antibody are disulfide-linked, they could not be modified. We therefore examined DM1 modification of tyrosine and found no side reactions on tyrosine due to the fact that glycine was used to quench the labile bonds formed between succinimidyl and tyrosine in our process. To understand the structural characteristics of identified conjugation sites on the ADC, the percentage of solventaccessible surface area (SAS) was calculated for each lysine residue. Since the crystal structure of the IgG1 mAb used in this study is not available, several crystallographic IgG1 model structures in the Protein Data Bank (PDB) that showed high conservation of lysine residues and amino acid identity with the IgG1 mAb were used. As expected, all of the modified lysine residues are solvent-exposed (>23% SAS, see Table 1),40 which is consistent with the results reported previously.18 Of the eight lysine residues that are not modified, four are buried (1−21% SAS) and three are exposed (>46% SAS, although two of these are arginine residues in the model crystal structures used, which may impact the SAS calculation). The remaining one solventexposed lysine residue is at the C-terminus of the heavy chain (K453) and is present at a very low level (2.9%) in the final product due to cleavage, which is the reason it is not modified. The fact that a few solvent-exposed lysine residues are not modified indicates that other factors, such as neighboring residues or hydrogen bonds or the orientation of the amino acid within the protein structure, may play a role in the reactivity of individual lysine residues.18 Impact of DM1 Conjugation on Physicochemical Stability of the Antibody−Drug Conjugate. Thermal stability and domain folding of the naked antibody and ADC was assessed by using DSC. Representative DSC thermograms of the naked antibody (blue trace) and ADC (red trace) are shown in Figure 5. The DSC profile of the naked antibody is typical of antibodies, with the thermal transitions demonstrating that the protein is properly folded into distinct domains.41 Three endothermic thermal transitions, which correspond to unfolding of the CH2, Fab, and CH3 domains, were observed for the naked antibody (blue trace in Figure 5). In contrast, only two thermal transitions corresponding to the unfolding of the

ion and a series of b ions. High-abundance y6 ions were observed due to the proline effect that proline-containing peptides ions undergo preferential fragmentation at the Ntermini of the proline residues.35 The diagnostic ions b12 and y6 conclusively identified the DM1 conjugation site of K13 (Figure 3B) for H1-DM1 peptide I. In contrast, the MS/MS spectrum of H1-DM1 peptide III showed good coverage of a series of y ions. The diagnostic ion y18 conclusively identified the DM1 conjugation site of E1 (Figure 3C) for H1-DM1 peptide III. MS/MS spectra of H1-DM1 peptides I and II were identical, suggesting the presence of two stereochemical configurations (diastereomers) of the DM1-SMCC linkage through a maleimide stereoisomers (Figure 1). The same results were obtained for H1-DM1 peptides III and IV as well. The level of DM1 conjugation at K13 is 5-fold higher than that at E1 (Figure 3A), likely due to the more nucleophilic nature of the amine group on the side chain of the lysine residue. Moreover, a diagnostic ion for DM1-attached peptides was observed with m/z 547.2200 (charge 1), which was generated due to the cleavage of the ester bond of DM1, as shown in Figure 3. The remaining parts generated after the cleavage were also detected with high abundance (m/z 2293.1858). The proposed ring structure of the diagnostic ion is also shown in Figure 3. This assignment was also confirmed by comparing the isotopic peak distribution of this observed DM1 signature ion with the theoretical isotopic peak distribution of the proposed DM1 fragment (see Supporting Information section 4). As a result, we were able to identify all DM1-attached peptides easily by extracting MS/MS spectra, using this DM1-specific signature ion as shown in Figure 4. As expected, this DM1-specific

Figure 4. (A) Base peak chromatograms of tryptic digest of ADC and (B) XIC of DM1 fragment ions (547.2200) from HCD MS/MS spectra of LC/MS/MS profile.

fragment ion was observed only in the MS/MS spectra from DM1-conjugated ADC samples but not observed from naked antibody (see Supporting Information section 5). Table 1 summarizes the DM1-attached peptides and the conjugation sites from each heavy chain and light chain by using the HCD-based method described above. A total of 76 conjugation sites, 20 from the light chain and 56 from the heavy E


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Analytical Chemistry Table 1. Conjugation Sites from Each of the Heavy and Light Chains in the Antibody−Drug Conjugate conjugation site

obsd mass (Da)

HC HC HC HC HC HC HC

E1 K13 K43 K54 K67 K78 K89

2837.3956 2837.3954 2439.1589 2474.1000 2472.0958 2656.1737 ndd


K127 K139 K153 K211 K216 K219 K220

nd 3445.6475 nd 7669.6575 8011.8517 1444.6591 1555.7275

HC K224 HC K228

1918.8012 4292.9499

K252 K254 K280 K294 K296 K323 K326 K328 K332 K340 K344

3801.7826 4618.1997 4754.1530 3115.4548 1456.6704 3183.5632 1683.6842 1691.7215 2222.0979 2223.1184 nd

HC HC HC HC HC HC HC HC HC HC HC

theor mass (Da)

SASa (%)

Lys conjugated (%)

0.3 0.3 0.1 1.8 0.2 0.2 nd

87b 76 79 42c 45 70 56

0.8 4.3 0.8 6.2 5.0 7.0 nd

nd 0.2 nd 0.2 0.7 0.8 0.8

46 23 6 65 59 31 48

nd 4.1 nd 13.4 0.5 0.7e 0.3e

0.6 0.3

49 97

0.1e 11.1

0.0 0.1 0.1 0.3 0.6 0.4 0.8 0.9 0.4 0.3 nd

74 25 81 51 51 45 35 34 70 46 12

11.8 5.2 2.6 3.9 15.9 5.6 0.3f 0.7f 0.3 5.0 nd

accuracy (ppm)

VH Domain 2837.3964 2837.3964 2439.1587 2474.0955 2472.0962 2656.1731 6322.7868 CH1 Domain 6253.7983 3445.6480 8973.2998 7669.6556 8011.8459 1444.6602 1555.7287 Hinge Domain 1918.8023 4292.9513 CH2 Domain 3801.7827 4618.1991 4754.1527 3115.4556 1456.6695 3183.5645 1683.6855 1691.7199 2222.0987 2223.1191 1602.7658

conjugation site

obsd mass (Da)

HC K346

1611.7396

K366 K376 K398 K415 K420 K445 K453

2736.2393 nd 5354.3980 nd 1773.8289 4398.9462 nd

LC K46 LC K67 LC K106

5902.8062 5534.3752 nd

K114 K133 K153 K160 K170 K175 K190 K208

3777.8874 5134.4958 3851.8390 2630.2112 2517.1388 3270.4853 3079.4069 3514.4428


LC LC LC LC LC LC LC LC

theor mass (Da)

SASa (%)

Lys conjugated (%)

0.9

73

1.5f

0.1 nd 1.0 nd 0.7 0.1 nd

53 20 47 1 31 37 99

0.5 nd 13.0 nd 9.5f 2.5 nd

0.1 0.5 nd

60 29 50

0.1 22.8 nd

0.1 0.5 0.5 0.2 0.0 0.7 0.4 0.2

64 63 28 99g 36 46 65 41

3.8 0.4 2.1 9.7 0.3 1.9 4.7 1.9

accuracy (ppm)

CH2 Domain 1611.7410 CH3 Domain 2736.2391 4643.0843 5354.3925 3385.6010 1773.8302 4398.9467 1743.8084 VL Domain 5902.8054 5534.3725 5864.6356 CL Domain 3777.8870 5134.4930 3851.8372 2630.2116 2517.1388 3270.4874 3079.4080 3514.4433

a

Solvent-accessible surface area (SAS) is shown as a percentage of total area. bThe SAS of HC E1 was calculated from the crystal structure of PDB 1HZH. cThe SAS of HC K54 was calculated from the crystal structure of PDB 1AQK. dNot detected. eConjugation levels were quantified from Asp-N map. fConjugation levels were quantified from Glu-C map. gThe SAS of LC K160 was calculated from the crystal structure of PDB 1HZH.

Figure 5. DSC thermograms of naked antibody (blue trace) and ADC (red trace).

In a separate stability study, naked antibody and ADC samples were stressed at 40 °C for 1 month. The thermally stressed mAb and ADC samples were subjected to peptide mapping to assess chemical degradation. Oxidation of heavychain (HC) methionine at position 258 (M258) in the naked antibody increased from 3.9% to 6.8% with an oxidation rate of 0.1%/day. In contrast, the level of HC M258 oxidation in the ADC increased from 4.3% to 31.6% with an oxidation rate of 0.9%/day. Therefore, the oxidation rate of M258 in ADC as compared to the naked antibody is about 10 times faster at 40 °C, which is consistent with the observation that conjugation of DM1 destabilized the CH2 domain of the antibody. Oxidation of M258, which is located in the CH2 domain of the heavy chain, may affect the efficacy by reducing neonatal Fc receptor binding, thereby impacting serum half-life.42

Fab and CH3 domains were observed for the ADC. The thermal transition of the CH2 domain is not well resolved from that of the Fab domain (red trace in Figure 5). The change of the profile and decrease in Tm for the first transition suggests that the conjugation of DM1 has greater impact on the thermal stability of the CH2 domain than on the rest of the antibody. The destabilized CH2 domain of the ADC is likely due to high levels of DM1 conjugation at lysine residues in the CH2 domain (a total of 1.03 DM1 conjugated at lysine residues located in CH2; Table 1).

CONCLUSIONS The ADC was prepared through conjugation of DM1 drugs to the solvent-exposed ε-amino groups of lysine residues in the antibody. The naked antibody contains 92 putative conjugation sites, including 90 lysine residues and two N-terminal glutamic acid residues of the heavy chain. The large number of potential conjugation sites compared to the relatively small drug load resulted in heterogeneous product with various levels of DM1 conjugated at different sites. The average DAR value of the ADC measured by UV/vis was 3.3, which agreed well with the

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values measured by cIEF and intact mass analysis. The DM1 distribution profile of the ADC determined by cIEF and intact mass analysis showed that 0−8 DM1 molecules were conjugated to one antibody molecule. With accurate mass detection of diagnostic DM1 fragment ions and high-quality HCD MS/MS spectra, a total of 76 conjugation sites were identified in the antibody. Thermal stability and domain folding of the ADC were assessed by using DSC. The DSC profile of the ADC suggests that conjugation of DM1 has greater impact on the thermal stability of the CH2 domain. As a result, M258 oxidation is about 10 times faster in thermally stressed ADC samples when compared with that of the stressed antibody samples.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03709. Additional text, equations, and figures describing (1) preparation of DM1-SMCC and ADC, (2) DAR determination by UV/vis, cIEF, and LC/TOF-MS, (3) instrument setup for LC/ESI-MS/MS analysis, (4) theoretical and observed MS spectra of signature DM1 fragment ion, and (5) base peak chromatogram of tryptic digest of naked antibody and XIC of DM1 fragment ions from HCD MS/MS (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone 805-447-6034; fax 805-376-2354; e-mail qluo@amgen. com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Izydor Apostol and Zhongqi Zhang for helpful discussion and Brent Kendrick for comments. REFERENCES

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