Structural Characterization of Cross-Linked Species in Trastuzumab

Article pubs.acs.org/bc

Structural Characterization of Cross-Linked Species in Trastuzumab Emtansine (Kadcyla) Yan Chen,* Michael T. Kim, Laura Zheng, Galahad Deperalta, and Fred Jacobson Department of Protein Analytical Chemistry, Genentech, Inc., 1 DNA way, South San Francisco, California 94080-4990, United States ABSTRACT: The antibody−drug conjugate, trastuzumab emtansine (Kadcyla), is produced by attachment of the antitubulin drug, DM1, to lysine amines via a heterobifunctional linker, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Following the reaction of the Nhydroxysuccinimide activated linker with antibody lysines to produce a linker-modified intermediate (Tmab-MCC), DM1 is added to yield the desired product. In addition to the expected distribution of drug-linked forms (from 0 to 8), mass spectrometry also demonstrates the presence of a second distribution shifted by about +222 Da. This series is consistent with the presence of a population containing a bound linker without DM1 (“unconjugated linker”). Extended characterization of trastuzumab emtansine was performed using capillary isoelectic focusing, CE-SDS, peptide mapping, and LC/MS following 18O labeling of peptide digests to identify this family of product variants. These studies demonstrate that the presence of these +222 Da species is due to an unexpected reaction of the maleimide moiety in the MCC linker with antibody lysine residues to produce cross-linked species that cannot conjugate to DM1.

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INTRODUCTION Chemical cross-linking reagents are widely used to study the structure and interaction of proteins or protein complexes, for immobilization of proteins to solid supports, and in preparing bioconjugates for biological imaging or for human therapeutics.1 Linkers can incorporate a variety of chemistries to preferentially target specific residues on the proteins or on the small molecules being attached. In addition, linkers can also incorporate molecular functionality to facilitate product solubility or site-specific/mechanism-based delivery of drugs. The development of improved cross-linking reagents, including cleavable and noncleavable linkers, has contributed significantly to recent successes in the development of antibody−drug conjugates (ADCs).2,3 The 2011 approval of Adcetris,4 an ADC targeting CD30+ Hodgkin’s lymphoma, and the 2013 approval of Kadcyla,5 an ADC targeting HER2+ breast cancer, have confirmed the clinical benefit that these therapeutic products can deliver. Linker selection for a given ADC is typically based on the desired reactivity of specific activated groups with amino acids or drug functionalities6 (such as NHS-esters toward lysine or drug amines and maleimides toward cysteine or drug thiols). However, the possible occurrence of side reactions must also be characterized. Trastuzumab emtansine (Kadcyla) is an ADC5 that contains the humanized anti-HER2 IgG1, trastuzumab (Herceptin), linked to the microtubule inhibitory drug maytansinoid DM1 via a thioether bond introduced by the succinimidyl 4-(N-malemidomethyl) cyclohexane-1-carboxylate (SMCC) linker. SMCC, a commonly used heterobifunctional cross-linker, contains a primary amine-reactive N-hydroxysucci© 2016 American Chemical Society

nimide (NHS) ester on one end and a sulfhydryl-reactive maleimide group on the other end.1 NHS esters are highly reactive toward primary amines, including the free N-terminus and the ε-amino group of lysine side chains of a protein. Maleimide, on the other hand, is most frequently used for coupling to sulfhydryl groups, such as the thiol in DM1. Figure 17,8 shows the two-step manufacturing process for trastuzumab emtansine. In the first step (the modification reaction), the NHS ester of SMCC reacts with antibody amines to form the linker-modified intermediate, Tmab-MCC. In the ensuing conjugation reaction, the maleimide moiety in the intermediate undergoes a Michael addition with the sulfhydryl (-SH) group in DM1 to form a stable, thioether-bound drug. The final product is heterogeneous with respect to both the number and location of the lysine residues that are conjugated. The average drug to antibody ratio (DAR), as determined using UV spectroscopy, is 3.5.8 Mass spectrometry (MS) has been shown to be a powerful tool for characterizing the distribution of different drug-loaded forms in trastuzumab emtansine.8−12 As shown by the LC-MS analysis of the deglycosylated sample (Figure 2), the main series of peaks corresponds to trastuzumab emtansine forms with zero to eight conjugated drugs per antibody in an approximate Poisson distribution.8 The heterogeneity observed in trastuzumab emtansine is not surprising since there are many surface-accessible lysine residues on the antibody that can react with SMCC. Received: June 16, 2016 Revised: July 19, 2016 Published: July 26, 2016 2037

DOI: 10.1021/acs.bioconjchem.6b00316 Bioconjugate Chem. 2016, 27, 2037−2047

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Bioconjugate Chemistry

predicted mass of MCC bound to the antibody, but without a conjugated DM1 (“unconjugated linker”). Various analytical techniques including MS, capillary isoelectric focusing (iCIEF), and capillary electrophoresis in the presence of SDS (CE-SDS) were applied to characterize the species that contain the unconjugated linkers. We have demonstrated that these species correspond to ADCs with linkers that are bound at both ends to antibody lysines, generating cross-links between antibody chains. Our studies show that these cross-linked species are generated during the modification reaction as a result of the maleimide moieties in the Tmab-MCC intermediate reacting with nearby lysine residues on the antibody.

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RESULTS AND DISCUSSION The deconvoluted mass spectrum in Figure 2 shows that, in addition to the primary peak series corresponding to the expected drug-linker with a mass of ∼957 Da, a second series of less abundant peaks are observed, separated by an offset of approximately +222 Da. The molecular weight shift of these species correlates well to the predicted mass of MCC bound to the antibody, but without a conjugated DM1 (219 Da). Chen et al.12 reported the detection of linker-modified species that contain active/unreacted maleimide moieties in the peptide map analysis of trastuzumab emtansine. However, no such species were positively identified in our own peptide map analysis of a typical sample. Our finding is further supported by the MS analysis of the reduced protein. No linker-modified light chain or heavy chain without a conjugated DM1 was detected (data not shown). Given the efficiency of thiol/ maleimide reactions and the molar excess of DM1 used in the conjugation reaction, it is unlikely to have active/unreacted linker present after the completion of the conjugation reaction. Hydrolysis of the maleimide ring in the MCC linker, which would produce an unreactive linker with a molecular weight of 237 Da, is not expected to be responsible for these peaks given the difference in the observed shift and the mass accuracy of the instrument. Similarly, reactions of the maleimide with other reagents introduced in the modification and conjugation process would give a higher mass shift relative to the observed one, and are therefore also ruled out. Although the intact MS analysis of the deglycosylated protein does not provide much insight into the structure of these species, it can be used to estimate the level of these unconjugated linker (UL) species relative to the total linker content from the deconvoluted mass spectrum (Figure 2). Under target process conditions, each drug-containing peak has only a single higher mass (unconjugated linker) peak. However, in some multivariate process characterization and process hold time studies, species containing multiple unconjugated linkers can also be found in the MS profiles (Figure 3). A weighted area for each peak is calculated based on the number of linkers it contains (both with and without conjugated drug).

Figure 1. Schematic of trastuzumab emtansine conjugation: modification of lysines on trastuzumab with SMCC linker and subsequent reaction with the sulfhydryl of the DM1 drug. Figure adapted from Wakankar et al.7

Figure 2. Deconvoluted mass spectrum of a typical deglycosylated trastuzumab emtansine sample from LC-MS. Starred peaks correspond to unconjugated linker species that are approximately 222 Da heavier than the associated drug-containing peaks.

Wt Area total linker = (Number of Linkers) × (Peak Area)

For each peak that contains unconjugated linker, a weighted area of unconjugated linker is calculated.

Figure 2 also reveals the presence of a second, less abundant series of peaks (labeled with * in the figure) separated from the primary peak series by a mass of approximately +222 Da (Figure 2 inset). This second series corresponds to the

Wt Area unconjugated linker = (Number of Unconjugated Linkers) × (Peak Area) 2038


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Figure 3. Deconvoluted mass spectrum of a deglycosylated trastuzumab emtansine sample derived from a process hold time study with ∼14% unconjugated linker. Starred peaks correspond to unconjugated linker species that are approximately 219−222 Da heavier than the associated drug containing peaks. There is clear evidence of species containing two unconjugated linkers.

Figure 4. Reduced CE-SDS analysis of trastuzumab (unconjugated antibody) and trastuzumab emtansine. Note: Peak assignments (HH, HL, HHL) are based on apparent molecular weight relative to standards. L = light chain; H = heavy chain; HL = heavy chain-light chain; HH = heavy chain-heavy chain; HHL = heavy chain-heavy chain-light chain; NGHC = nonglycosylated heavy chain.

Therefore, for the entire mixture of species, the unconjugated linker fraction (ULfxn) relative to the total linker content is calculated by ULfxn = ΣWt Area unconjugated linker /ΣWt Area total linker

This is consistent with what is observed in the intact MS profiles (Figures 2 and 3). The same mechanism is also hypothesized to contribute to formation of high-molecularweight species in Tmab-MCC intermediate after 14 days at 40 °C.7 To further strengthen the hypothesis that both CE-SDS cross-links and MS “unconjugated linker” are derived from the same side reaction, samples from multivariate process characterization studies were analyzed by both methods. Both target and nontarget process conditions were evaluated in these studies. As shown in Figure 5, these samples span a range of MS-quantified unconjugated linker from 6% to 30% and the

Approximately 7% of the total linker species bound to the antibody was quantified as being unconjugated linkers in the sample shown in Figure 2, while the example shown in Figure 3 gave ∼14% unconjugated linkers. As part of the effort to fully characterize the structure of trastuzumab emtansine, CE-SDS analysis was conducted to evaluate the size heterogeneity and purity of the product. Under reducing conditions, the CE-SDS profile (Figure 4) of trastuzumab emtansine showed two main peaks corresponding to the heavy chain (H, molecular weight ∼50 kDa) and light chain (L, molecular weight ∼25 kDa), as expected. However, several other peaks showing molecular weights of ∼75, 100, and 125 kDa were also observed in the reduced trastuzumab emtansine, but not in the corresponding unconjugated antibody (trastuzumab). Based on their apparent molecular weights, these peaks are identified as interchain cross-linked species such as HL, HH, and HHL. The presence of these species at significant levels in the reduced CE-SDS analysis suggests that the formation of interchain cross-links involves mechanisms other than disulfide bonds. The sum of these species by CESDS (about 7%) is consistent with the level of the unconjugated linkers observed in the MS analysis. Based on these observations, it was hypothesized that the unconjugated linker species observed in the MS analysis correspond to interchain cross-links resulting from SMCC reacting with sites on two adjacent protein chains during the modification reaction. Following initial reaction of the NHS activated ester with protein amines, some of the maleimides may react with a proximal amino acid. In the subsequent conjugation step, only those maleimides that have not reacted with a protein side chain would be available to react with DM1. The resulting product would have a mixture of MCC-linked DM1 moieties (with incremental masses of 957 Da) and unconjugated linkers (with incremental masses of 219 Da).

Figure 5. Correlation between % unconjugated linker (UL) by MS versus cross-linked species by reduced CE-SDS. Note: Samples derived from small scale multivariate process study and hold time studies performed using a range of process conditions. 2039


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Bioconjugate Chemistry correlation (R2 = 0.978) between the data obtained from the two methods is strong. This supports the hypothesis that the MS species correspond to linkers that are bound at both ends to the antibody, generating the cross-links between antibody chains that are detected by reduced CE-SDS. Additional evidence on the relationship between unconjugated linker determined by MS and cross-linked species measured by reduced CE-SDS was obtained from MS analysis of a reduced sample derived from a study where the TmabMCC intermediate was held for an extended time at elevated temperature and pH. In this sample, with about 30% unconjugated linker, it was possible to detect masses that correlate with those expected for HL (heavy chain-light chain) with both one and two unconjugated linkers. Figure 6 shows three different mass range windows from the same deconvoluted mass spectrum allowing the identification of HL cross-linked species containing 0 drugs (Panel A), 1 drug (Panel B), and 2 drugs (Panel C). Even in this highly crosslinked sample, evidence of other postulated species (such as HH and HHL) cannot be directly observed by MS due to their low abundance, relatively poor ionization, and additional complexity (from extent of drug loading). Theoretically SMCC cross-links may occur either between separate antibodies (intermolecular cross-links) or between amino acids of the same mAb monomer (intramolecular crosslinks). Intermolecular cross-links are expected to result in covalent aggregates, detectable by nonreduced CE-SDS. Trastuzumab emtansine contains a slightly elevated level of high-molecular-weight species (∼1.6% mAb dimer, data not shown) compared to the unconjugated antibody (≥0.2% dimer), suggesting that a portion of the total cross-links (7% in a typical sample) may be intermolecular. However, the majority of the cross-linking is believed to be intramolecular (between separate chains of the mAb). Within the same molecule, unconjugated linker may also theoretically involve links between two sites on the same antibody chain (intrachain links) or between two different chains. Although not detectable by the reduced CE-SDS method, evidence of intrachain links was not observed in the LC-MS analysis of the reduced samples. The strong correlation between intact LC-MS and reduced CE-SDS values, and the slope near 1.0 in Figure 5, suggest that the majority of cross-links in trastuzumab emtansine are between different antibody chains within the same molecule. These intramolecular, interchain cross-linked species in trastuzumab emtansine are generated during the modification reaction. Following initial reaction of the NHS activated ester, some of the maleimides react with a proximal amino acid on an adjacent antibody chain. Among all nucleophilic amino acid side-chains, sulfhydryl groups of cysteine residues are the strongest nucleophiles, especially in their ionized form. They are followed by primary amino groups such as the α-amino group of the N-terminus and the ε-amino groups of lysine residues. Secondary amines of histidine and tryptophan and the guanidine group of arginine exhibit less nucleophilicity compared to primary amines and are not expected to react with maleimide.1 In addition, maleimides do not react with weak nucleophiles such as tyrosine, aspartic acid, and glutamic acid.13 Based on the literature,1 maleimide addition to sulfhydryls is the expected reaction at a pH of 4−8. To investigate whether there are free sulfhydryl/thiol groups present in the starting unconjugated antibody that might be available to react with

Figure 6. Reduced mass spectra of a highly cross-linked sample (∼30% by reduced CE-SDS). (A) HL cross-linked species without any MCCDM1 drug attached to the protein. (B) HL cross-linked species containing 1 drug. (C) HL cross-linked species containing 2 drugs. Note: HL = heavy chain-light chain; UL = unconjugated linker; D = drug.

protein-bound MCC, trastuzumab was analyzed using Ellman’s reagent, 5,5′ dithiobis(2-nitrobenzoic acid) (DTNB).14 Samples were tested both in the absence and in the presence of a denaturing agent (8 M urea) to ensure that any inaccessible protein thiols would be detected. The free thiol content was 2040


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Bioconjugate Chemistry found to be ≤0.13 (moles sulfhydryl per mole of protein) and therefore could not account for the 7% cross-linking (equivalent to ∼0.25 mol sulfhydryl per mole of protein) observed. When the pH is above 8.0, however, maleimide is known to react with primary amines.1 The ε-amino group in a lysine amino acid has a pKa of 9.3−9.5.1 At pH 7, it is expected that ≥99% is protonated and therefore not a strong nucleophile. The reaction of maleimide with lysine is expected to proceed at a very slow rate, about 1000 times slower than its reaction with sulfhydryl group.13 Interestingly, Brewer and Riehm15 reported that more than 50% of the lysine residues in lysozyme reacted with 0.1 M N-ethylmaleimide (NEM) at pH 7. They suggested that some amines in proteins may undergo rapid reactions with maleimide due to favorable steric and electrostatic interactions. It is known that the pH of the solution and the side chain’s microenvironment influence the charge on each side chain in a folded protein. Isom et al.16 reported that large shifts in pKa values were found for lysine residues buried inside a protein. In their study, 19 out of the 25 lysine residues showed pKa values significantly lower than that of free lysine, some as low as 5.3. There findings indicate that the ε-amino group in a lysine residue may still act as a nucleophile even in a neutral or slightly acidic environment. In the case of manufacturing trastuzumab emtansine where pH is never above 8.0, it is still possible that the ε-amino groups in some lysine residues could react with maleimide in the MCC linker and generate cross-linked species. Analysis of heavily cross-linked samples using a charge-based method, imaged Capillary Isoelectric Focusing (iCIEF), supports the hypothesis that the lysine ε-amino groups are involved in the interchain cross-linking. iCIEF17 is an electrophoretic technique that separates species primarily on the basis of a molecule’s intrinsic net charge and has been successfully applied for the examination of charge-based isoforms of ADCs.18 Attachment of each uncharged linkerdrug (MCC-DM1) to a lysine through the NHS ester reduces the net positive charge on the protein by one. Therefore, the iCIEF profile of trastuzumab emtansine shows a distribution of charged forms (Figure 7, Panel A) that mimics the MS based profile (Figure 2). The most basic peak with an apparent pI at 8.9 corresponds to the main peak of the unmodified antibody, trastuzumab (Figure 7, Panel A). Species with higher drug loads are more acidic and exhibit lower pI values. Similarly, if a lysine ε-amino group is modified by reaction with maleimide, the protein will become more acidic. Cross-linking involving cysteine sulfhydryl groups, on the other hand, will not alter the protein charge. To assess the impact of cross-linking on the charge distribution, four trastuzumab emtansine samples with similar average DAR values (3.2−3.4) but significantly different percentages of cross-linking (7−25% as determined by CESDS), were analyzed by iCIEF where the charge variants were separated in an ampholyte mixture. As shown in Figure 7B, the iCIEF profile shifts in the acidic direction as the result of increasing cross-linking, suggesting a loss of positive charges such as lysine ε-amines. Chemical cross-linking in combination with peptide map analysis using mass spectrometry is often applied to structural analysis of protein−protein interactions and protein threedimensional structures.19 In a bottom-up approach, the protein reaction mixture is proteolytically digested after the crosslinking reaction, and mass spectrometric identification of the cross-linked products is performed in order to map structural adjacencies.20 However, since the number of unmodified

Figure 7. (A) iCIEF analysis of trastuzumab and trastuzumab emtansine samples. (B) Effect of cross-linking on iCIEF profiles. Samples were derived from small scale multivariate process characterization and hold time studies.

peptides or fragments greatly exceeds the number of crosslinked ones, the identification of the cross-linked peptides can be quite cumbersome.19 Enzyme-facilitated 18O labeling is a simple technique21 for identifying cross-linked peptides in the complex digestion product. 18O exchange takes place at the C-terminal carboxyl group (COO−) of proteolytic peptides. Non-cross-linked peptides or intrapeptide cross-link species contain only one C-terminus with one COO− group. Therefore, a mass shift of 4 Da is expected due to the incorporation of two 18O atoms in the C-terminus. Interpeptide cross-linked species, which contain two C-termini, will exhibit a shift of 8 Da compared to their nonlabeled counterparts. The retention time of the 18O labeled peptide, however, remains the same and is often used as a criterion for identifying the cross-linked peptides. 18 O labeling during proteolytic digestion often introduces a variable degree of 18O incorporation.22 Decoupling the digestion and 18O labeling can significantly improve labeling efficiency.23 In this study, postdigestion 18O labeling of a trastuzumab emtansine sample was performed and results were compared to that of the 16O control sample. However, using this approach one also needs to be aware of the additional incorporation of 18O atoms during nonenzymatic deamidation of Asn and Gln.24,25 Therefore, postdigestion 18O labeling of an unconjugated antibody sample was included in the study as an 2041


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Bioconjugate Chemistry additional control to eliminate potential false positives from side-chain exchange or incorporation. The masses corresponding to cross-linked tryptic peptides were readily differentiated from unmodified tryptic peptides in mass spectra by their characteristic 8 Da shift (as opposed to 4 Da shift) between the 18O labeled and 16O labeled samples. An example is shown in Figure 8. Using scripts developed in-house,

Figure 9. Crystal structure of IgG1 (PDB ID: 1HZH) showing four proposed cross-links (dashed yellow) involving lysine 225 on heavy chain. The numbers on each cross-link signify the distance (in angstroms, Å) between the Cα of the connected lysine residues. Heavy chain is depicted in green and light chain in blue. Image generated using PyMOL computer software.

Figure 8. MS spectrum of a cross-linked peptide (monoisotopic m/z 1189.07) showing a shift of 8 Da (m/z shift of 2 Da at a charge state (z) of 4) after 18O labeling.

a theoretical library of in silico cross-linked tryptic peptides was constructed. While the iCIEF data seemed to suggest that lysines were predominantly cross-linked (via MCC) to other lysines, the search was expanded to also consider lysines crosslinked to cysteines. Experimental masses with the characteristic 8 Da shifts were then matched to this theoretical library of cross-linked peptides and a 5 ppm mass accuracy was enforced. The search space of all possible lysine-to-lysine and lysine-tocysteine combinations is quite large. In order to effectively manage the false positive rate, an additional distance constraint was applied to determine whether the proposed cross-link was physically relevant. Based on the respective amino acid residue and SMCC linker lengths, it was determined that the α-carbons of the proposed cross-linked amino acid residues should be no more than 20 Å apart for lysine-to-lysine cross-links or 17 Å apart for lysine-to-cysteine cross-links. The full-length IgG (PDB ID: 1HZH)26 and trastuzumab Fc (PDB ID: 3D6G)27 crystal structures were used to extract α-carbon x,y,z coordinates. Candidates that did not satisfy the distance constraint were eliminated.28 Figure 9 is an example of the crystal structure showing four proposed cross-links involving heavy chain lysine 225. Table 1 shows the list of cross-linked peptides identified as described above. All candidates contain lysine-to-lysine linkage. No cysteine-to-lysine cross-links were found. To further confirm the identity of the cross-linked peptides, MS/MS spectra of each peptide under collision-induced dissociation (CID)29 with electrospray ionization (ESI)30 were examined. MS/MS analysis of protonated peptides often provides unambiguous sequence information and has been widely used for protein identification,31 determination of posttranslational modifications (PTM),32 and de novo sequencing.33 It was successfully applied in studying structures of homodimeric glutathione S-transferase and monomeric bovine serum albumin34 where MS/MS analysis enabled unequivocal identification of the cross-linking site(s) and determination of

the amino acid sequence of each peptide chain in the crosslinked peptide(s). As mentioned above, SMCC is a heterobifunctional linker with an NHS ester on one end and maleimide on the other. To differentiate the two ends, we refer to the two peptides in a cross-linked peptide as α- and β-chains, where α-chain is the peptide that is linked by the NHS ester and the β chain is attached through the maleimide. Linking the primary amines in the antibody to the NHS ester in the linker generates a new amide bond that is equally susceptible to cleavage in CID as the amide bonds35 in the peptide backbone. Consequently, MS fragments from the α chain are often observed as if α chain was not modified. On the other hand, reaction between maleimide and amine does not produce a labile amide bond. Therefore, fragment ions from the β chain may contain the MCC linker as a nonfragmenting modification. It was possible to verify the presence of the intact α and/or MCC-modified β chains in some of the ESI MS/MS data. The difference in the fragment patterns in the α and β chains allows possible identification of the orientation of the linker in the cross-linked peptides. Examples of MS/MS spectra of two cross-linked peptides and the proposed sequences are shown in Figures 10 and 11. The most intense charge state obtained for each cross-linked peptide was selected for MS/MS experiments. The spectra were labeled according to the notation proposed by Schilling et al.36 Assignment of the α and β chains is proposed based on the fragmentation pattern as described above. Longer peptide chains often carry additional protons in ESI; therefore, the relative sizes of α and β chains can affect the fragmentation pattern.37 When the β chain is much smaller than the α chain (Figure 10), the majority of peptide backbone dissociations take place on α-chain as if it was not modified, whereas the MCC-modified β chain behaves as a regular, nonfragmenting modification. In this case, the identification of the α chain by its 2042


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Table 1. Cross-Linked Peptides Identified in a Trastuzumab Emtansine Sample Containing 14% Cross-Linked Speciesa monoisotopic m/z (experimental) z

ppm

1189.06654+

−0.1

1273.63934+

0.3

1306.90044+

5.1

1425.19104+

−0.1

1512.24924+

−1.3

1724.07564+

−1.5

1821.14764+

−0.5

sequence (α peptide chain)

distance between Cα (Å)

sequence (β peptide chain)

TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK (Heavy Chain Lys 277) GPSVFPLAPSSKSTSGGTAALGCLVK (Heavy Chain Lys 136) THTCPPCPAPELLGGPSVFLFPPKPK (Heavy Chain Lys 249) SCDKTHTCPPCPAPELLGGPSVFLFPPKPK (Heavy Chain Lys 225) SCDKTHTCPPCPAPELLGGPSVFLFPPKPK (Heavy Chain Lys 225) SCDKTHTCPPCPAPELLGGPSVFLFPPKPK (Heavy Chain Lys 225) SCDKTHTCPPCPAPELLGGPSVFLFPPKPK (Heavy Chain Lys 225)

sequence identified based on MS/ MS

CKVSNK(Heavy Chain Lys 325)

8.4

α

VYACEVTHQGLSSPVTKSFNR (Light Chain Lys 207) FNWYVDGVEVHNAKTKPR (Heavy Chain Lys 291)

7.3

α, β

17.8

α

HKVYACEVTHQGLSSPVTK (Light Chain Lys 190)

17.5

α, β

GPSVFPLAPSSKSTSGGTAALGCLVK (Heavy Chain Lys 136) SCDKTHTCPPCPAPELLGGPSVFLFPPKPK (Heavy Chain Lys 225) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR (Light Chain Lys 126)

17.9

α, β

9.4 17.2

α(β)b α, β

All peptides shown in the table satisfied the space constraint. bα and β have identical sequences, corresponding to the two heavy chains in an antibody molecule.

a

Figure 10. CID MS/MS spectrum of a cross-linked peptide detected in a trastuzumab emtansine sample with ∼14% cross-linked species. The precursor ion at m/z 1189.824+ was selected in the CID experiment. Based on the fragmentation pattern, cross-linking between heavy chain Lys 277 (α chain) and heavy chain Lys 325 (β chain) is proposed. The spectra were labeled according to the notation proposed by Schilling et al.36 The boxed fragments correspond to intact α chain and the MCC-modified β chain.

fragmentation pattern plus the mass of the β chain was used for the identification of the cross-linked peptide. In another example (Figure 11), when α and β chains have similar, large sizes, fragmentation was observed in both α and β chains in the product ion spectra. Process studies have shown that the cross-linking reaction rate (and overall extent of cross-linking) depends on conditions used in the modification step. For trastuzumab emtansine manufactured under target conditions, the distribution and levels of the cross-linked species are found to be consistent

between batches and production scales (data not shown). To understand the impact of cross-links on biological properties of the conjugate, trastuzumab emtansine containing 25−30% cross-links, derived from nontarget process conditions, was compared with the reference standard produced using the target process (∼9% cross-links). The binding to recombinant HER2 ECD as determined by ELISA, and the cell killing activity toward a HER2 overexpressing breast cancer cell line (BT-474) were similar (Table 2). In addition, the impact of cross-linking on FcRn binding was assessed to evaluate 2043


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Figure 11. CID MS/MS spectrum of a cross-linked peptide detected in a trastuzumab emtansine sample with ∼14% cross-linked species. The most abundant isotopic peak at m/z 1425.954+ was selected as the precursor ion in the CID experiment. Based on the fragmentation pattern, cross-linking between heavy chain Lys 225 (α chain) and light chain Lys 190 (β chain) is proposed. The spectra were labeled according to the notation proposed by Schilling et al.36 The boxed fragments correspond to intact α chain and the MCC-modified β chain.

Table 2. Potency of Trastuzumab Emtansine Samples with Different Levels of Cross-Linked Species

Table 3. FcRn Binding Activities of Trastuzumab Emtansine with a High Level of Cross-Linked Species

activitya (% of reference standard) sampleb

cross-linked speciesc (%)

HER2 binding ELISA

BT-474 cell killing

1 2 3 4 5 6

7 8 9 15 21 26

96 109 95 109 94 93

107 100 93 114 114 104

sample

cross-linked speciesa (%)

activityb (% of reference standard)

Controlc 1d 2d

9 21 26

88 73 87

a

Cross-linked species analyzed by reduced CE-SDS. bA trastuzumab emtansine reference standard containing 9% cross-linked species was used for these studies. cControl was an independent dilution of the reference standard. dSample derived from process characterization studies with similar average DAR values (3.3−3.4).

a

A trastuzumab emtansine reference standard containing 9% crosslinked species was used for these studies. bSamples derived from smallscale multivariate process characterization studies with similar average DAR values (3.2−3.4). cCross-linked species analyzed by reduced CESDS.

modulate the pKa of an individual protein-bound amino acid side chain and thus increase or decrease its nucleophilicity relative to its free version.38 Although the primary reaction of maleimides with proteins is through cysteine sulfhydryls, reactivity toward lysine has been reported at basic pH. Despite the use of a low pH in the trastuzumab emtansine manufacturing process, our findings demonstrated that protein-bound maleimides can also react with lysine residues leading to chemical cross-linking between mAb peptide chains. In the case of trastuzumab emtansine, the initial reaction of trastuzumab and SMCC produces the MCC-linked antibody intermediate. Subsequent reaction of some of the introduced maleimides with proximal lysines (potentially ones exhibiting decreased pKa values) leads to covalent cross-linking. Crosslinking reactions are typically second-order. Therefore, significant rate acceleration of maleimide reaction may be due to the high local concentration relative to the reaction of two free species. An interesting observation is that five out of the

potential impact on ADC pharmacokinetics. A comparison of reference standard with two highly cross-linked samples (containing 21−26%) did not indicate a significant difference in binding (Table 3). This suggests there should be minimal impact to clearance of total antibody due to intramolecular, interchain cross-linking.

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CONCLUSIONS It is well-known that protein side chains can exhibit a wide range of chemical reactivities. Factors that contribute to this variability include location-specific pKa differences between residues, steric constraints, and relative solvent accessibility. Hydrogen bonding and other noncovalent interactions can 2044


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NAP-5 sizing columns (GE Healthcare), sample buffer (citrate/ phosphate, pH 6.6) was added, and the samples were reduced with 48 mM DTT (dithiothreitol) and 1% SDS at 70 °C for 20 min. The samples were analyzed using a Beckman PA 800 Plus CE system. After introducing the labeled samples into a 50-μmdiameter uncoated fused silica capillary (31 cm total length, 21 cm effective length) by electrokinetic injection (40 s at 10 kV), the electrophoretic separation was performed at a constant voltage of 15 kV for 35 min. Separation was accomplished using SDS MW gel buffer (Beckman Coulter) as the sieving matrix, with the capillary maintained at a temperature of 40 °C. The migration of labeled components was monitored by laserinduced fluorescence (LIF); the excitation was at 488 nm using an argon ion laser, and the emission was monitored at 560 nm. The resulting electropherograms were integrated to obtain the corrected peak areas (AU*min) using the 32 Karat software v 8.0 (Beckman Coulter). Free Thiol Analysis under Nondenaturing Conditions. Free thiol content of the unconjugated trastuzumab was quantified using Ellman’s reagent, 5,5′ dithiobis(2-nitrobenzoic acid) (DTNB, Sigma, 98+%). Samples containing 3 mg of trastuzumab were exchanged into assay buffer (0.1 M sodium phosphate, 1 mM ethylenediaminetetraacetic acid (EDTA); pH 7.3) using 10 kDa MWCO Microcon spin filters (Millipore) and diluted to 150 μL. A solution containing 4 mg/mL of Ellman’s reagent DTNB was prepared in the same buffer. Protein concentrations were determined for all buffer exchanged samples using absorbance at 280 nm. Sample (125 μL), buffer (125 μL), and DTNB (14.5 μL) were mixed and incubated for 30 min at room temperature. Absorbance at 412 nm was then measured in a 1 cm path length cuvette against a buffer control. A standard curve was generated with cysteine at 1 μM, 5 μM, 10 μM, 50 μM, and 75 μM using the same procedure. The cysteine standards were measured immediately after adding the DTNB reagent. Imaged Capillary Isoelectric Focusing (iCIEF). Imaged capillary isoelectric focusing (iCIEF) provides a means of assessing the charge heterogeneity of a protein. Analysis of trastuzumab emtansine or trastuzumab by iCIEF was carried out using an ICE 280 instrument (ProteinSimple). Samples were diluted to a final concentration of 0.5 mg/mL in a solution containing 1.6 M urea, 4% carrier ampholytes (85% Pharmalytes pH 8−10.5, 15% Pharmalytes pH 3−10, GE Healthcare), 0.28% methylcellulose, and isoelectric point (pI) markers. The mixture was introduced into the capillary under pressure and then focused at 1500 V for 1 min followed by 10 min at 3000 V, at which time a charge-coupled device camera captured an image of the entire capillary illuminated by a 280 nm light source. Software provided by the instrument vendor converts the image into an electropherogram showing absorbance as a function of position in the capillary or of pI based on the position of calibration standards. 18 O Labeled Peptide Map. Samples containing 1 mg (50 μL) of trastuzumab emtansine or trastuzumab were diluted to 1 mg/mL into a pH 5.0 buffer containing 6 M guanidine and 0.3 M sodium acetate. DTT (Thermo Scientific) was added to a final concentration of 10 mM, and the samples were then incubated at 37 °C for 1 h. Iodoacetic acid (Sigma) at 3.5 M, freshly prepared in 1.0 M sodium hydroxide, was then added to a concentration of 42 mM. After a 20 min incubation at ambient temperature in the dark, the alkylation was quenched by adding DTT to a final concentration of 40 mM.

seven cross-linked peptides identified (Table 1) involve the lysine residues (HC 225 or HC 249) in the hinge region. Previous studies have demonstrated that the hinge region is highly flexible,39 therefore potentially making these lysine residues even more susceptible to cross-linking. Although the presence of nonreducible, interchain cross-links resulting from nonspecific reaction of maleimides with lysine side chains was unexpected, biological characterization studies demonstrated that trastuzumab emtansine samples containing up to 26% cross-links showed no loss in HER2 ECD binding, cell killing, and FcRn binding activities relative to the control. In addition, the distribution and levels of the cross-linked species are tightly controlled under the well-defined manufacturing conditions used to produce trastuzumab emtansine (Kadcyla).

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EXPERIMENTAL PROCEDURES LC-ESI-MS. Mass spectrometric analysis was used to characterize the drug distribution and determine levels of unconjugated linker species (MCC moieties that do not contain DM1) present on trastuzumab emtansine. MS analysis of intact and deglycosylated trastuzumab emtansine was performed using an AB-Sciex QSTAR Pulsar i mass spectrometer. The capillary and declustering potentials of the mass spectrometer were optimized for maximum sensitivity. The optimized instrument conditions included an electrospray voltage of 5200 V, a first declustering potential (DP1) of 60 V, a second declustering potential (DP2) of 15 V, a focusing potential of 200 V, and a capillary temperature of 350 °C. Protein was diluted to 1 mg/mL in 100 mM Tris-HCl buffer pH 7.5 and deglycosylated by incubation overnight at 37 °C in the presence of PNGase F (New England Biolabs) at a ratio of 5000 units per mg antibody. Samples (20 μg) were introduced into the mass spectrometer using an RP-HPLC column (Agilent POROSHELL 300SB, C8, 1.0 × 75 mm, 5 μm, 300 Å pore) run at 0.2 mL/min and equilibrated at 75 °C with 82% solvent A (0.1% formic acid in water). After 10 min at initial conditions, samples were eluted using a 16 min linear gradient from 18% to 95% solvent B (0.1% formic acid in acetonitrile). For analysis after reduction, deglycosylated samples prepared as described above were treated with 20 mg/mL TCEP (tris(2carboxyethyl)phosphine) at 37 °C for 60 min. Approximately 15 μg of a reduced, deglycosylated sample was loaded onto a PLRP-S RP-HPLC column (Agilent, 2.1 × 150 mm, 8 μm, 1000 Å pore) equilibrated at 78 °C with 85% solvent A (0.1% formic acid and 0.025% TFA (trifluoroacetic acid) in water). Samples were eluted using a 37 min linear gradient from 15% solvent B (0.1% formic acid and 0.025% TFA in acetonitrile) to 47% solvent B at a flow rate of 0.2 mL/min. Mass spectra were derived by deconvolution of the multiply charged ions using BioAnalyst QS 1.1 software (Sciex). Integration of the resulting peak areas was performed using the same software package to generate the percent peak area associated with each drug-loaded species and to derive a value for the unconjugated-linker content. Reduced CE-SDS. Capillary electrophoresis was performed in the presence of SDS (sodium dodecyl sulfate) in a capillary filled with a sieving matrix to assess the molecular size distribution of proteins under denaturing conditions. Prior to analysis, samples were diluted to 1 mg/mL in 0.1 M sodium bicarbonate, pH 8.3, and labeled with 0.17 mg/mL 5-TAMRA (5-carboxytetramethylrhodamine), an amine reactive fluorescent dye. After removing the excess dye by passage through 2045


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emtansine concentrations and a parallel line program was used to estimate the activity of the trastuzumab emtansine samples relative to the reference standard with an assigned activity of 100%. Alphascreen-Based FcRn Binding Assay. The binding activities of the trastuzumab emtansine samples for the human neonatal receptor (FcRn) were assessed using a proximitybased luminescent assay format (AlphaScreen). In this assay, streptavidin donor beads, biotinylated FcRn, and varying concentrations of trastuzumab emtansine samples were added to a 96 well plate. This was followed by the addition of acceptor beads conjugated to trastuzumab. In the absence of competing trastuzumab emtansine samples, donor bead-bound FcRn and acceptor bead-conjugated trastuzumab interact to produce a signal. Increasing concentrations of trastuzumab emtansine samples compete with the trastuzumab/acceptor bead to FcRn/ donor bead interaction and reduce the luminescent signal. Changes in luminescence are proportional to the amount of competing sample(s). The results, expressed in relative luminescence units, were plotted against trastuzumab emtansine concentrations, and a 4-parameter curve fitting program was used to estimate the activity of the sample(s) relative to the reference standard with an assigned activity of 100%.

Following incubation for 5 min at room temperature, the reduced and S-carboxymethylated samples were exchanged by gel filtration using PD-10 columns (GE Healthcare) into a pH 6.7 buffer containing 25 mM ammonium acetate. Twenty microliters of CaCl2 at 106 mM was also added. Trypsin at 1 mg/mL (sequencing grade, Roche) was then added to the samples at a 1:40 (trypsin:protein, w:w) ratio. Digestion was allowed to proceed for 4 h at 37 °C. Each peptide digest was divided into 2 fractions for labeling in 16O water (control) and 18 O water, respectively. These fractions were first lyophilized before the labeling reaction. For 18O labeling, the peptides were dissolved in 18O water containing 2.5 μg trypsin in 50 mM ammonium acetate and 2 mM CaCl2 (pH 6). After labeling at 37 °C overnight, residual trypsin activity was quenched by boiling for 10 min followed by the addition of 3% TFA (v/v). Digested samples (100 μL, approximately 50 μg) were analyzed by RP-HPLC on an Agilent Zorbax 300SB C8 column (2.1 × 150 mm, 3.5 μm particle) equilibrated with 100% solvent A (0.1% TFA in water) using an Agilent 1200 HPLC system. The column was eluted at a flow rate of 0.25 mL/min and maintained at 45 °C. Peptides were resolved with a linear gradient from 0% to 45% solvent B (0.09% TFA in acetonitrile) over 225 min. The chromatogram was developed with a multistep gradient as follows: 0−45% solvent B in 225 min, 45−95% in 5 min, equilibration at 95% solvent B for 5 min, and back to 0% solvent B in 0.1 min with equilibration at 0% for 14.9 min. Absorbance was monitored at both 214 and 252 nm and column flow was directed into the source of a Thermo LTQ Orbitrap XL hybrid Ion Trap-Orbitrap mass spectrometer. Data were analyzed using Xcalibur 3.0 (Thermo Scientific). HER2 Binding ELISA. Briefly, a 96-well microtiter plate was coated with anti-maytansine (anti-DM1) antibody (100 μL/ well) and incubated for 18−72 h at 4 °C. The plate was then blocked and washed, and then increasing concentrations of trastuzumab emtansine samples (100 μL/well) were added. Following an incubation step at 25 °C, the plate was washed and diluted biotinylated HER2 extracellular domain (ECD) at 100 μL/well is added. Following an incubation period, the plate was washed, and bound HER2 ECD was detected using streptavidin-horseradish peroxidase (HRP) and a tetramethylbenzidine (TMB) substrate solution, which produces a colored product. The results, expressed in OD (optical density), were plotted against the trastuzumab emtansine concentrations, and a parallel line program was used to estimate the activity of the trastuzumab emtansine samples relative to a reference standard with an assigned activity of 100%. BT-474 Cell Killing. This assay measures the ability of trastuzumab emtansine to induce killing of the breast cancer cell line BT-474. Briefly, BT-474 cells were seeded at a density of 1 × 105 cells/well (100 μL) into a 96-well microtiter plate and incubated in a humidified incubator set at 37 °C/5% CO2. Following incubation, increasing concentrations of trastuzumab emtansine standard, control, and sample(s) were added and the plates incubated. Twenty-five microliter aliquots of alamarBlue were added to each well, and the plates were incubated. AlamarBlue is blue and nonfluorescent in its oxidized state, but is reduced by the cell’s intracellular environment to a pink form that is highly fluorescent. The changes in color and fluorescence are proportional to the number of viable cells. Fluorescence was determined at 530 nm excitation and 590 nm emission on a fluorescence plate reader. The results, expressed in relative fluorescent units (RFU), were plotted against the trastuzumab

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (650) 225-1718. Fax: (650) 742-4966. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Yaning Wang and Max Tejada for performing the potency assays and Pat Rancatore for her insightful comments and review.

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REFERENCES

(1) Hermanson, G. T. (2013) Bioconjugate techniques, Academic press. (2) Chari, R. V. J. (2008) Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc. Chem. Res. 41, 98−107. (3) Wu, A. M., and Senter, P. D. (2005) Arming antibodies: prospects and challenges for immunoconjugates. Nat. Biotechnol. 23, 1137−1146. (4) Younes, A., Bartlett, N. L., Leonard, J. P., Kennedy, D. A., Lynch, C. M., and Sievers, E. L. (2010) Brentuximab vedotin (SGN-35) for relapsed CD30-positive lymphomas. N. Engl. J. Med. 363, 1812−1821. (5) Lewis Phillips, G. D. L., Li, G., Dugger, D. L., Crocker, L. M., Parsons, K. L., Mai, E., Blattler, W. A., Lambert, J. M., Chari, R. V. J., Lutz, R. J., et al. (2008) Targeting HER2-positive breast cancer with Trastuzumab-DM1, an antibody cytotoxic drug conjugate. Cancer Res. 68, 9280−9290. (6) Ducry, L., and Stump, B. (2010) Antibody−drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjugate Chem. 21 (1), 5−13. (7) Wakankar, A. A., Feeney, M. B., Rivera, J., Chen, Y., Kim, M., Sharma, V. K., and Wang, Y. J. (2010) Physicochemical stability of the antibody-drug conjugate Trastuzumab-DM1: Changes due to modification and conjugation processes. Bioconjugate Chem. 21, 1588−1595. (8) Kim, M. T., Chen, Y., Marhoul, J., and Jacobson, F. (2014) Statistical modeling of the drug load distribution on trastuzumab emtansine (Kadcyla), a lysine-linked antibody drug conjugate. Bioconjugate Chem. 25 (7), 1223−1232. 2046


Article


IGG against HIV-1: a template for vaccine design. Science 293 (5532), 1155−1159. (27) Moiani, D., Salvalaglio, M., Cavallotti, C., Bujacz, A., Redzynia, I., Bujacz, G., Dinon, F., Pengo, P., and Fassina, G. (2009) Structural characterization of a Protein A mimetic peptide dendrimer bound to human IgG. J. Phys. Chem. B 113 (50), 16268−16275. (28) Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) The Protein Data Bank. Nucleic Acids Res. 28, 235−242. (29) Wells, J. M., and McLuckey, S. A. (2005) Collision-induced dissociation (CID) of peptides and proteins. Methods Enzymol. 402, 148−185. (30) Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, C. M. (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246 (4926), 64−71. (31) MacCoss, M. J., McDonald, W. H., Saraf, A., Sadygov, R., Clark, J. M., Tasto, J. J., Gould, K. L., Wolters, D., Washburn, M., Weiss, A., et al. (2002) Shotgun identification of protein modifications from protein complexes and lens tissue. Proc. Natl. Acad. Sci. U. S. A. 99 (12), 7900−7905. (32) Biemann, K. (1988) Contributions of mass spectrometry to peptide and protein structure. Biol. Mass Spectrom. 16 (1−12), 99− 111. (33) Seidler, J., Zinn, N., Boehm, M. E., and Lehmann, W. D. (2010) De novo sequencing of peptides by MS/MS. Proteomics 10 (4), 634− 649. (34) Soderblom, E. J., and Goshe, M. B. (2006) Collision-induced dissociative chemical cross-linking reagents and methodology: applications to protein structural characterization using tandem mass spectrometry analysis. Anal. Chem. 78 (23), 8059−8068. (35) Loo, J. A., Edmonds, C. G., and Smith, R. D. (1990) Primary sequence information from intact proteins by electrospray ionization tandem mass spectrometry. Science 248 (4952), 201−204. (36) Schilling, B., Row, R. H., Gibson, B. W., Guo, X., and Young, M. M. (2003) MS2Assign, automated assignment and nomenclature of tandem mass spectra of chemically crosslinked peptides. J. Am. Soc. Mass Spectrom. 14 (8), 834−850. (37) Santos, L. F., Iglesias, A. H., and Gozzo, F. C. (2011) Fragmentation features of intermolecular cross-linked peptides using N-hydroxy-succinimide esters by MALDI-and ESI-MS/MS for use in structural proteomics. J. Mass Spectrom. 46 (8), 742−750. (38) Guo, X., Bandyopadhyay, P., Schilling, B., Young, M. M., Fujii, N., Aynechi, T., Guy, R. K., Kuntz, I. D., and Gibson, B. W. (2008) Partial acetylation of lysine residues improves intraprotein crosslinking. Anal. Chem. 80 (4), 951−960. (39) Jefferis, R., and Lund, J. (2002) Interaction sites on human IgGFc for FcγR: current models. Immunol. Lett. 82 (1), 57−65.

(9) Marcoux, J., Champion, T., Colas, O., Wagner-Rousset, E., Corvaïa, N., Van Dorsselaer, A., Cianférani, S., and Beck, A. (2015) Native mass spectrometry and ion mobility characterization of trastuzumab emtansine, a lysine-linked antibody drug conjugate. Protein Sci. 24 (8), 1210−1223. (10) Beck, A., Terral, G., Debaene, F., Wagner-Rousset, E., Marcoux, J., Janin-Bussat, M. C., Colas, O., Dorsselaer, A. V., and Cianférani, S. (2016) Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates. Expert Rev. Proteomics 13 (2), 157−183. (11) Sandra, K., Vanhoenacker, G., Vandenheede, I., Steenbeke, M., Joseph, M., and Sandra, P. (2016) Multiple heart-cutting and comprehensive two-dimensional liquid chromatography hyphenated to mass spectrometry for the characterization of the antibody-drug conjugate ado-trastuzumab emtansine. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci., DOI: 10.1016/j.jchromb.2016.04.040. (12) Chen, L., Wang, L., Shion, H., Yu, C., Yu, Y. Q., Zhu, L., Li, M., Chen, W., and Gao, K. (2016) In-depth structural characterization of Kadcyla®(ado-trastuzumab emtansine) and its biosimilar candidate. mAbs, 00. (13) Mattson, G., Conklin, E., Desai, S., Nielander, G., Savage, M. D., and Morgensen, S. (1993) A practical approach to crosslinking. Mol. Biol. Rep. 17 (3), 167−183. (14) Riener, C. K., Kada, G., and Gruber, H. J. (2002) Quick measurement of protein sulfhydryls with Ellman’s reagent and with 4, 4′-dithiodipyridine. Anal. Bioanal. Chem. 373 (4−5), 266−276. (15) Brewer, C. F., and Riehm, J. P. (1967) Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins. Anal. Biochem. 18 (2), 248−255. (16) Isom, D. G., Castañeda, C. A., Cannon, B. R., and GarciaMorena, E. B. (2011) Large shifts in pKa values of lysine residues buried inside a protein. Proc. Natl. Acad. Sci. U. S. A. 108 (13), 5260− 5265. (17) Wu, J., Li, S. C., and Watson, A. (1998) Optimizing separation conditions for proteins and peptides using imaged capillary isoelectric focusing. Journal of Chromatography A 817 (1), 163−171. (18) Michels, D. A., Salas-Solano, O., and Felten, C. (2011) Imaged capillary isoelectric focusing for charge-variant analysis of biopharmaceuticals. BioProcess Int. 9 (10), 48−54. (19) Sinz, A. (2006) Chemical cross-linking and mass spectrometry to map three dimensional protein structures and protein-protein interactions. Mass Spectrom. Rev. 25 (4), 663−682. (20) Trakselis, M. A., Alley, S. C., and Ishmael, F. T. (2005) Identification and mapping of protein-protein interactions by a combination of cross-linking, cleavage, and proteomics. Bioconjugate Chem. 16 (4), 741−750. (21) Back, J. W., Notenboom, V., de Koning, L. J., Muijsers, A. O., Sixma, T. K., de Koster, C. G., and de Jong, L. (2002) Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling. Anal. Chem. 74 (17), 4417−4422. (22) Valkenborg, D., and Burzykowski, T. (2011) A Markov-chain model for the analysis of high-resolution enzymatically 18O-labeled mass spectra. Stat. Appl. Genet. Mol. Biol. 10 (1), 1−37. (23) Yao, X., Afonso, C., and Fenselau, C. (2003) Dissection of proteolytic 18O labeling: endoprotease-catalyzed 16O-to-18O exchange of truncated peptide substrates. J. Proteome Res. 2 (2), 147− 152. (24) Liu, H., Wang, F., Xu, W., May, K., and Richardson, D. (2013) Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis. Anal. Biochem. 432 (1), 16−22. (25) Du, Y., Wang, F., May, K., Xu, W., and Liu, H. (2012) Determination of deamidation artifacts introduced by sample preparation using 18O-labeling and tandem mass spectrometry analysis. Anal. Chem. 84 (15), 6355−6360. (26) Saphire, E. O., Parren, P. W., Pantophlet, R., Zwick, M. B., Morris, G. M., Rudd, P. M., Dwek, R. A., Stanfield, R. L., Burton, D. R., and Wilson, I. A. (2001) Crystal structure of a neutralizing human 2047


Structural Characterization of Cross-Linked Species in Trastuzumab

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