Isomerization and Oxidation in the Complementarity-Determining

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Isomerization and Oxidation in the Complementarity-Determining Regions of a Monoclonal Antibody: A Study of the Modification− Structure−Function Correlations by Hydrogen−Deuterium Exchange Mass Spectrometry Yuetian Yan,†,∥ Hui Wei,*,† Ya Fu,† Sutjano Jusuf,‡ Ming Zeng,† Richard Ludwig,† Stanley R. Krystek, Jr.,‡ Guodong Chen,§ Li Tao,† and Tapan K. Das† Downloaded via UNIV OF EDINBURGH on January 23, 2019 at 16:40:15 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Biologics Development, Bristol-Myers Squibb, 311 Pennington Rocky Hill Road, Pennington, New Jersey 08534, United States Molecular Discovery Technologies, and §Bioanalytical and Discovery Analytical Science, Bristol-Myers Squibb, Province Line Road and Route 206, Princeton, New Jersey 08543, United States

‡

S Supporting Information *

ABSTRACT: Chemical modifications can potentially change monoclonal antibody’s (mAb) local or global conformation and therefore impact their efficacy as therapeutic drugs. Modifications in the complementarity-determining regions (CDRs) are especially important because they can impair the binding affinity of an antibody for its target and therefore drug potency as a result. In order to understand the impact on mAb attributes induced by specific chemical modifications within the CDR, hydrogen−deuterium exchange mass spectrometry (HDX MS) was used to interrogate the conformational impact of Asp isomerization and Met oxidation in the CDRs of a model monoclonal antibody (mAb1). Our results indicate that despite their proximity to each other, Asp54 isomerization and Met56 oxidation in CDR2 in the heavy chain of mAb1 result in opposing conformational impacts on the local and nearby regions, leading directly to different alterations on antibody−antigen binding affinity. This study revealed direct evidence of local and global conformational changes caused by two of the most common degradation pathways in the CDRs of a mAb and identified correlations between chemical modification, structure, and function of the therapeutic monoclonal antibody.

M

isomerization is mainly determined by the amino acid residue in its +1 position (glycine and serine were shown to be the most destabilizing residues11−16) and its local conformational flexibility.17,18 Methionine is the most readily oxidized residue among the common amino acids, and its oxidized products are methionine sulfoxide19 or sulfone20 under harsh conditions. The most common and well-studied Met oxidation in IgG occurs at two conserved positions in the Fc, M252 and M428,21−25 while Asp isomerization has been found to occur at multiple locations within mAbs.26 Because of CDRs’ flexibility and high solvent accessibility in mAbs, they are susceptible to chemical modifications/ degradations, which can lead to decreased or even complete loss of an antibody’s antigen binding affinity and therefore its drug potency.27,28 In a previous study in which 37 mAbs were subject to stress treatments,29 all impactful degradation hot spots were found to be located in the CDR loops, of which 15

onoclonal antibodies (mAbs) have become the predominant class of protein therapeutics against cancers, infection, autoimmune, and neurodegenerative diseases over the past 20 years.1,2 To date, there are more than 40 mAbs approved by the U.S. Food and Drug administration for clinical use in various therapeutic areas,3,4 and many more are in development.5,6 Antibodies are favored for therapeutic treatments over small molecule drugs because of their high target affinity and specificity, long clearance time, as well as limited off-target toxicity.7 By varying the complementarity-determining regions (CDRs) of mAbs to different antigen target sites, mAbs provide a broad diversity of antigen specificities for treating different diseases. mAbs, however, like all other proteins, are subject to degradation processes that may impact their structure and biological activities. Two nonenzymatically driven degradations commonly monitored during antibody drug development and storage are aspartic acid (Asp) isomerization and methionine (Met) oxidation.8−10 Asp isomerization occurs via formation of a cyclic imide (succinimide) intermediate, which may hydrolyze to isoAsp or Asp, typically in a molar ratio of 3:1 at equilibrium.11 The susceptibility of an Asp residue to © 2016 American Chemical Society

Received: July 24, 2015 Accepted: January 11, 2016 Published: January 11, 2016 2041

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contained either Asp isomerization or Asn deamidation. Because Asp isomerization involves the incorporation of an extra carbon within the peptide backbone and a modification to the amino acid side group, it often leads to conformational changes and, thereby, has the potential to alter the binding affinity to drug targets.27,28,30−35 For example, isomerization of light chain (LC) CDR127,34,35/CDR231 or heavy chain (HC) CDR230,33 has been shown to reduce the binding affinity of the antibodies. Deamidation/cyclic imide formation of LC CDR136,37 or HC CDR228,32 were also reported to cause a drop in potency. However, the impact of Asp isomerization is case-dependent and hard to predict. For example, Chu et al.38 reported that succinimide formation at D30 of the LC in an IgG2 antibody has no significant impact on antigen binding. Unlike Asp isomerization, studies of Met oxidation have mainly focused on the Met residues in the Fc region. Met252 in CH2 and Met428 in CH3 are often the most easily oxidized among all methionine residues in antibodies. Oxidation at these sites has been reported to decrease mAb stability,39,40 Fc receptor binding,21 and circulation half-life.22 With the majority of oxidation studies focused on the antibody Fc region, those examining the CDRs remain limited.24,41,42 Although experiments assessing the impact of degradation on mAb attributes continue to be performed, information pertinent to the structural basis of the impact is sparse. We report here the study of the conformational impact of the Asp isomerization and Met oxidation in the CDRs of an IgG1 therapeutic antibody. Recent advances in hydrogen−deuterium exchange mass spectrometry (HDX MS) have enabled promising applications for analysis of protein higher-order structures, dynamics, interactions, and aggregation in biopharmaceutical development.43−47 Among the few studies24,25,39 assessing impact of post-translational modifications on antibody local structures, Zhang et al.24 studied the impact of various chemical modifications on local dynamics of an IgG1 mAb by HDX MS. Their experiments, however, reported no detectable local structural changes caused by the formation of isoAsp or oxidized Met in the CDRs. One possible reason is that only low abundances of the modified species were generated in their analysis (only ∼14% of Asp55 isomerization was present in their sample and Met50 oxidation was mixed with Met oxidations in the Fc region). The measured HDX differences are significantly “diluted”, since the observed HDX kinetics are weighted averages of all protein forms in solution. In this study, samples were isolated each containing enriched modified species of a mAb to avoid “diluting” the HDX differences.45 Here, we used a naturally aged IgG1 mAb developed by Bristol-Myers Squibb to generate purified fractions of isomerized (Asp) and oxidized (Met) species and applied HDX MS to evaluate the conformational impact of the two modifications in the CDRs. Computational analysis was also performed on the antibody comparing the native versus modified forms containing either Asp54 isomerization or Met56 oxidation in order to explore impact on conformation caused by chemical modifications compared to native antibody. The enriched isomerized and oxidized species were then tested for their binding affinity to the target protein. This study provides direct evidence of local and global conformational changes caused by mAb isomerization or oxidation in the CDRs, which correlates with the observed impact on antigen binding affinity of the antibody from the higher-order structure perspective.

Article

EXPERIMENTAL SECTION

Materials. The recombinant human monoclonal IgG1 antibody (mAb1) was expressed by Chinese hamster ovary (CHO) cells and purified by affinity and several ion-exchange chromatography steps at Bristol-Myers Squibb Company. The sample was stored at 4 °C for 4 years in which time a natural accumulation of isomerization on Asp54 and oxidation on Met56 occurred. H2O was purchased from Honeywell, B&J Brand (LC/MS grade) (Honeywell, Plainview, NY). Acetonitrile was purchased from J. T. Baker (LC/MS grade) (Avantor Performance Materials, Center Valley, PA). Guanidine HCl (8 M) was purchased from Thermo Scientific Pierce (Thermo Scientific Pierce, Grand Island, NY). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. HIC Enrichment. Native, isomerized, and oxidized mAb1 species were purified using hydrophobic interaction chromatography (HIC) and stored at −80 °C after enrichment. For the isomerized fraction (termed as “iso-mAb1”), greater than 97% of the sample contained isomerized antibody, with the majority of the species having Asp54 isomerization on one HC; for the oxidized fraction (termed as “ox-mAb1”), greater than 97% of the sample contained oxidized antibody, out of which ∼78% contains Met56 oxidation on one HC of the antibody; for the native fraction (termed as “native-mAb1”), 95% of the sample contained unmodified antibody (data not shown). HDX MS Protocol. HDX experiments on the native-mAb1, iso-mAb1, and ox-mAb1 samples were performed on a HDX manager system with automatic sample handling and online digestion and separation (Waters, Milford, MA). HDX control samples (non-deuterated) were prepared by diluting 5 μL of each of the protein stock solutions into 55 μL of aqueous buffer (5 mM K2HPO4, 5 mM KH2PO4 in H2O, pH 7.0). Continuous labeling with deuterium was performed by diluting 5 μL of each of the protein stock solutions into 55 μL of deuterated buffer (5 mM K2HPO4, 5 mM KH2PO4 in D2O, pD 7.0). HDX reactions were maintained at 23 °C and allowed to react for 20 s, 2 min, 15 min, and 1 and 4 h. The exchange reaction was stopped by adding 1:1 (v/v) ice-cold quench buffer (200 mM potassium phosphate, 4 M guanidine HCl, 0.5 M tris(2-carboxyethyl)phosphine), and pH was adjusted to 2.5 with NaOH). Then 50 μL of the mixture was injected into the HDX system and passed over an immobilized pepsin column (2.1 mm × 30 mm, Waters, Milford, MA) at 75 μL/min. The resulting peptides were captured on a VanGuard C18 trapping column (2.1 mm × 5 mm, Waters) and desalted with a 4 min flow at 75 μL/min of H2O containing 0.1% formic acid (FA). Peptides were then separated in a reversed-phase column (BEH C18 column, 1.0 mm × 50 mm, Waters) with an 11.5 min three-step linear gradient; its CH3CN content went from 8% to 22% in 3 min, 22−25% in 7 min, and 25−35% in 1 min. Protein digestion was carried out at 20 °C, and peptide capture and separation were carried out at 0 °C. MS detection was immediately performed after the separation on a Synapt G2-Si mass spectrometer (Waters) running in the ESI-positive mode. The instrument parameters are set as following: ESI voltage, 2.5 kV; acquisition range, m/z 50−2000; scan time, 0.3 s; cone voltage, 20 V; source temperature, 80 °C; desolvation gas nitrogen at 150 °C and 300 L/h. Data was collected at mass resolving power of 20 000 with lock spray correction using glufibrinogen peptide (Sigma-Aldrich). Each experiment was carried out in triplicate. 2042

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Figure 1. Extracted ion chromatograms of (a) native peptide 51−59 in native-mAb1; (b) iso 51−59 and native′ 51−59 in iso-mAb1; (c) ox 51−59 and native′ 51−59 in ox-mAb1 (overlaid extracted chromatograms).

HDX MS Data Analysis. To identify the peptides generated from online pepsin digestion, separate experiments without deuterium labeling were run and accurate mass precursor and product-ion spectra were acquired with MSE. The MSE data were analyzed using the ProteinLynx global server 3.0 (Waters). The created peptide list from three parallel runs were analyzed by the DynamX 2.0 software (Waters) to generate a peptide pool containing peptides with sufficient signal intensity and confidence to be reliably used for deuterium labeling analysis. The deuterium uptake level for each peptide (ΔD) at each exchange time point was calculated using the DynamX 2.0 software to generate kinetic curves for each protein state. Data obtained were used to make the HDX difference charts in which the deuterium labeling for each peptide between the native and each modified mAb states was compared. The algorithm of plotting the difference chart is described in detail by Houde et al.48 HXExpress (developed by Dr. John Engen’s group at Northeastern University) was used to calculate the deuterium uptake level of the three forms of peptide HC 51−59: native, isomerized, and oxidized. Computational Analysis. The structure of mAb1 was obtained through homology modeling using well-defined protocols of Bioluminate.49 Following model building, refinement of the model was performed using the protein preparation workflow within the Schrodinger software MAESTRO (version 10.1, Schrodinger LLC). Oxidation of methionine results in a mixture of the two diastereomers, methionine-(S)-sulfoxide and methionine-(R)-sulfoxide. The native mAb1 homology model was modified so that Met56 was modified to either methionine(S)-sulfoxide or methionine-(R)-sulfoxide. Asp54 was converted to isoAsp using the native mAb1 starting model whereby a carbon atom was inserted into the backbone of Asp54 to form isoAsp. The four models were then used for molecular dynamics (MD) simulations using DESMOND (Schrodinger LLC). Explicit solvent simulations with periodic boundary conditions were set up using an orthorhombic box with TIP3P waters and the OPLS3 force field for each protein. The system was initially relaxed with restraints on solute allowing waters to freely

equilibrate, followed by extensive simulation of the entire system without any restraints. The simulation was carried out for 10 ns with a 5 ps recording interval at 300 K using a standard protocol. Following the simulation, the two-shell water coordinates are calculated as the number of water molecules within a radius “R” (5.6 Å) from backbone N atoms in the peptide residues 52−59. In addition, the solventaccessible surface area (SASA) around the backbone N atoms for residues 52−59 was calculated at each recorded time point, and the statistical average is reported. These SASA were calculated according to the standard procedure with a water radius of 1.4 Å.50 Binding Affinity Measurement by Enzyme-Linked Immunosorbent Assay . Binding activity of the antibody variants was analyzed by an enzyme-linked immunosorbent assay (ELISA) method capable of detecting dose-dependent binding activity of antibody. Briefly, mouse monoclonal antimAb1 antigen antibody (R&D Systems, Minneapolis, MN) was first coated at 5 μg/mL in 50 μL per well on microtiter plates overnight at 2−8 °C, followed by incubation with 20 μg/mL recombinant human mAb1 antigen (R&D Systems, Minneapolis, MN) for 2 h at room temperature. After coating the plate, the native, isomerized, and oxidized samples were serially diluted 1:3 with a starting concentration of 5 μg/mL to a final concentration of 0.007 μg/mL and added to the plate followed by detection with secondary goat anti-human IgG (H + L) F(ab′)2 fragment-HRP conjugated antibodies (Jackson ImmunoResearch, West Grove, PA). All incubations were at room temperature on a microtiter plate shaker (Fisher Scientific, Pittsburgh, PA). Absorbance readings were obtained at wavelength 450 nm minus those at 650 nm within 10 min of SureBlue TMB substrate development using a SpectroMax plate reader (Molecular Devices, Sunnyvale, CA), and plotted on a four-parameter fit algorithm against the antibody concentration to yield an eight-point dose-dependent sigmoidal curve. The binding potency of the antibody variants was calculated using SoftMax Pro software (Molecular Devices, Sunnyvale, CA). 2043

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RESULTS AND DISCUSSION

Local Conformational Impact of Asp54 Isomerization or Met56 Oxidation Revealed by HDX MS. MAb1 contains one Asp-Gly motif in the flexible CDR2 (HC) at Asp54, which is susceptible to isomerization. It also contains Met56 in the same region, which is susceptible to oxidation. HDX MS was performed to assess the impact of these modifications on protein conformation. After pepsin digestion, peptide HC 51− 59, containing the residues of interest, was detected with good signal-to-noise ratio. The HDX rate of the native and isomerized forms of this peptide were compared to evaluate the local conformational impact by Asp isomerization. In order to differentiate the HDX level of isomerized and native peptides with identical molecular weight, we separated the isoAspcontaining peptide and Asp-containing peptide (from different HCs) in the iso-mAb1 sample using a shallow gradient. Figure 1a shows the elution of the unmodified peptide 51−59 from the native-mAb1 (native 51−59); Figure 1b shows partial separation of isomerized peptide 51−59 (iso 51−59) and nonmodified peptide 51−59 (native′ 51−59) from iso-mAb1. The latter eluting peak was identified as native′ 51−59 because its retention time matched with that of the native 51−59 under the same separation condition and is consistent with previous studies that showed earlier elution of the isoAsp-containing peptide relative to its Asp-containing counterpart.11 The ratio of iso 51−59 to native′ 51−59 was approximately 1:1 (Figure 1b), suggesting that only one HC is isomerized at Asp54 in isomAb1. HDX levels of the iso 51−59, native′ 51−59, and native 51−59 were obtained and were plotted against time to obtain their HDX kinetic curves, as shown in Figure 2a. The difference in the extent of back exchange for the peptides caused by their different retention times was minimum (∼0.1 Da, see Supporting Information for the calculation of back-exchange correction). This was taken into consideration by normalizing the deuterium uptake level of iso 51−59 against native′ 51−59. Figure 2a indicates a significant increase in deuterium labeling in iso 51−59 (green curve) compared to native 51−59 (blue curve) or native′ 51−59 (purple curve). This increase in exchange rate for the local region resulting from Asp54 isomerization is a result of the combined influence of the change in the intrinsic exchange rate51 of amide hydrogen atoms of Asp54 and Gly55 and the change in solvent-exposed conformation of the local region. Isomerization of Asp introduces an additional CH2 group into the peptide backbone (Scheme 1a), which not only extends the length of the backbone for Asp54 but induces additional flexibility into the CDR loop conformation. This added flexibility is correlated with the increased HDX rate of the peptide containing isoAsp54 amine and neighboring residues in the CDR2, as well as other residues proximal in three-dimensional space, as discussed below. On the basis of comparisons of native proteins versus their corresponding iso-Asp isoforms for data present in the Protein Data Bank (www.rcsb.org),52 the Asp to iso-Asp transformation induces unfolding and significant conformational changes to the iso-Asp backbone and the backbone of neighboring residues.53 Since Asp54 is part of CDR2 (HC), we expect that the conformation for the entire CDR2 would be changed substantially as a result of iso-Asp formation as is evidenced by the significant HDX exchange rate change for CDR2. In addition, because the HDX experiment was conducted under neutral pH, under which condition both Asp and isoAsp are deprotonated, the charge interaction effects

Figure 2. (a) HDX kinetics of native 51−59 from native-mAb1 (blue), native′ 51−59 in iso-mAb1 (purple), and iso 51−59 in iso-mAb1 (green). (b) HDX kinetics of native 51−59 from native-mAb1 (blue), native′ 51−59 in ox-mAb1 (purple), and ox 51−59 in ox-mAb1 (green). Back-exchange correction is applied to the uptake levels of either modified peptide.

of the Asp/isoAsp side chains on the neighboring amide hydrogen atoms are expected to be similar.51 Therefore, change of the intrinsic exchange rate upon Asp isomerization should be minimum. As a result, the observed significant increase in D uptake in iso 51−59 compared to native 51−59 is attributed to an increase in flexibility of the local backbone structure resulting from Asp isomerization. Similarly, oxidation in the same CDR2 region was monitored by comparing the HDX rate of the native and oxidized forms of peptide HC 51−59. Figure 1c illustrates the elution of the oxidized and native peptide 51−59 from ox-mAb1 (termed as “ox 51−59” and “native′ 51−59”, respectively). The ratio of the peak areas of ox 51−59 and native′ 51−59 is close to 1:1, suggesting that oxidation of Met56 is present on one of the two HCs. The HDX kinetic curves for native 51−59, ox 51−59 and native′ 51−59 were plotted (after back-exchange correction), showing that there is a slight decrease in HDX rate upon Met56 oxidation (Figure 2b). This result suggests either decreased intrinsic exchange rates of the amide hydrogen atoms of Met56 and its neighboring residues or a less flexible local region in 2044

DOI: 10.1021/acs.analchem.5b02800 Anal. Chem. 2016, 88, 2041−2050

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Analytical Chemistry Scheme 1. Chemical Reaction of (a) Asp Isomerization and (b) Met Oxidation

Figure 3. Schematic representation of the HDX propensity models. Both models depict the backbone of a three-residue peptide segment with side chain shown as X. (a) Average SASA represents the simulation average of the solvent-accessible surface area for the backbone nitrogens, which is represented as an outer blue envelope. (b) Two-shell water coordination model counts the number of water molecules (in orange) within a shell of radius R (5.6 Å) from the backbone N in fragment of interest.

Table 1. Computed HDX Propensity for HC Fragment 5159a CDR2 upon Met56 oxidation, which is opposite to the effect of Asp54 isomerization. Unlike Asp isomerization, the chemical transformation for Met oxidation is localized on the side chain (Scheme 1b). Being more polar and bulkier than Met, Met sulfoxide may cause a conformational change in CDR2 whereby new interactions stabilize the local conformation of CDR2. This change toward a more protected/less flexible structure caused by Met oxidation in the CDR of the antibody is opposite although at less significant levelcompared to those previously found in the Fc regions,39 which showed oxidation has a destabilizing effect by interrupting the backbone amide hydrogen-bonding network. This difference is possibly due to the less structured nature of CDRs compared to Fc. Local Conformational Impact of Asp54 Isomerization or Met56 Oxidation Revealed by Computational Analysis. HDX is an inherently solvent-mediated event. As such, we have explored a computational approach that was proven accurate in predicting propensity of methionine oxidation, which is also a solvent-mediated event. The approach called for performing atomistic MD simulations followed by deriving two models: (i) simulation average of the solventaccessible surface area (average SASA) of the sulfur in methionine and (ii) two-shell water coordination model which counts the number of water molecules within 6 Å from the sulfur in methionine. Both models were proven accurate in predicting the oxidation propensity when compared against experimentally measured relative rates of Met oxidation for the proteins tested.50 To evaluate the propensity of the relative HDX rates observed in our experiments, we performed atomistic MD simulations of three mAb1 species involved (native-mAb1, oxmAb1, isoAsp-mAb1) followed by deriving the average SASA and two-shell water coordination models of the backbone amide nitrogens (Figure 3) for the peptide fragment of interest, namely, HC 51−59. To be precise, amide nitrogen in 51 was excluded in the model generation since it does not contribute to the overall HDX rate of this fragment. Given that HC 51−59 is located at the flexible CDR loop region, higher numbers in the calculations of both models indicate higher solvent exposure and flexibility, and lower numbers indicate lower

average SASA (Å2) HC 51−59

median

native-mAb1 ox-mAb1-A ox-mAb1-B iso-mAb1

0.3 0.2 0.2 3.3

av (dev) 0.6 0.3 0.5 3.2

(0.7) (0.4) (0.7) (1.6)

two-shell (R = 5.6 Å) median 23 22 22 28

av (dev) 23 22 22 28

(3.2) (2.6) (2.8) (2.8)

a

Ox-mAb1-A/B refer to models that were generated by converting the methionine sulfur to the two chiral forms of methionine sulfoxide: methionine-(S)-sulfoxide or methionine-(R)-sulfoxide.

solvent exposure and flexibility. Table 1 lists the average SASA values and two-shell water coordination for HC 51−59, which shows a statistically significant increase in the average two-shell water coordination by four waters and a marked increase of SASA by average of 2.6 (Å2) when Asp54 undergoes isomerization. The results strongly indicate a significant conformational variability upon isoAsp formation which leads to a more solvent-exposed conformation within the peptide region 51−59. The results are consistent with the observed increase in HDX rates upon isoAsp formation for the same peptide. On the other hand, oxidation of Met to the chiral sulfoxide shows a minor reduction in both the SASA and twoshell water coordination for both chiral oxMet forms although changes fall within standard deviation of the mean (Table 1). This is also consistent with the HDX results where only slight decrease in HDX rates was observed upon Met oxidation. Global Conformational Impact of Asp54 Isomerization or Met56 Oxidation Revealed by HDX MS. Besides impacting the local structure, chemical modifications on antibodies sometimes lead to allosteric structural changes and impact the antibody’s overall conformation.54 In order to assess whether Asp54 isomerization or Met56 oxidation in CDR2 induces conformational changes in other regions of mAb1, the HDX levels of all mAb1 peptic peptides were evaluated comparing the two pairs of samples: native-mAb1 versus ox-mAb1 and native-mAb1 versus iso-mAb1. Overall, 210 peptic peptides were identified in the non-deuterated control experiments reproducibly, covering >99% of the mAb1 sequence. The HDX levels of these peptides were measured 2045

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Analytical Chemistry

Figure 4. HDX difference charts of mAb1 (a) HC and (b) LC. The two protein states compared are native vs isomerized samples. Deuterium uptake differences for the two states were calculated for all exchange time points: 20 s (orange), 2 min (red), 15 min (cyan), 60 min (blue), and 240 min (black). The gray vertical sticks represent the summed HDX differences of each peptide from all five exchange time points. The green dotted lines represent the criteria for reporting a significant HDX difference for all five labeling time points combined. The regions with significant HDX differences are labeled with blue (indicating less exchange in the isomerized state) or magenta (indicating more exchange in the isomerized state) colors accordingly. (c) HDX kinetic curves of the representative peptides in the segments identified in panels a and b that showed significant changes in HDX rate: peptides HC 69−80, HC 126−149, and LC 48−53 from native mAb1 (red) and iso-mAb1 (blue).

value of each peptide (along X-axis of Figures 4 and 5) at each HDX time (20 s, 2 min, 15 min, 1 h, or 4 h) was calculated by deducting the deuterium uptake value of the modified sample from that of the native sample (control) and plotted in different color traces. The summed difference for all five HDX time points for each peptide, ∑ΔD, is represented by the vertical

and the deuterium incorporation differences between the native state and either of the modified states were calculated, the values of which were used to construct the binary difference charts. Figure 4 and Figure 5 show the differential deuterium uptake levels (ΔD) between native-mAb1 versus iso-mAb1 and native-mAb1 versus ox-mAb1, respectively. The differential 2046

DOI: 10.1021/acs.analchem.5b02800 Anal. Chem. 2016, 88, 2041−2050

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Analytical Chemistry

Figure 5. HDX difference charts of mAb1 (a) HC and (b) LC. The two protein states compared are native vs oxidized samples. Deuterium uptake differences for the two states were calculated for all exchange time points: 20 s (orange), 2 min (red), 15 min (cyan), 60 min (blue), and 240 min (black). The gray vertical sticks represent the summed HDX differences of each peptide from all five exchange time points. The green dotted lines represent the criteria for reporting a significant HDX difference for all five labeling time points combined. The sequence regions with significant HDX differences are labeled with blue color accordingly, indicating less exchange in the oxidized state. (c) HDX kinetic curves of the representative peptides in the segments identified in panels a and b that showed significant changes in HDX rate: peptides HC 69−80, HC 126−149, and LC 55− 72 from native mAb1 (red) and iso-mAb1 (blue).

vertical stick was calculated and averaged, and 3 × SD (average value) was set as the minimum difference limit for significance determination (green dotted line in Figures 3 and 4). For the

sticks (Figures 4 and 5). To set the criteria for determining statistically significant HDX differences between two protein states, the standard deviation (SD) of the triplicates of each 2047

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Analytical Chemistry

Figure 6. Generic crystal structure of Fab (PDB 3f12) in IgG1 color coded with HDX differences from comparisons of (a) native vs isomerized and (b) native vs oxidized. Wheat color stands for the HC, and light gray color stands for the LC. Regions colored in magenta or blue indicate more flexibility or protection introduced, respectively, due to the modification.

(adding more flexibility), while the latter added more rigidity and/or protection to these regions. Furthermore, the extent of structural changes in CDR2 of HC (peptide HC 51−59) caused by Asp54 isomerization is much larger than that by Met56 oxidation; the former is also accompanied by more structural disruption in the subsequent region HC 69−80, which was unaffected in the oxidized state. These differences in both the location and extent of conformational changes caused by isomerization and oxidation in CDRs of mAb1 could potentially affect the antibody−antigen binding differently. Impact of Asp54/Met56 on mAb1 Antigen Binding Affinity. To study the impact of Asp54 isomerization and Met56 oxidation in CDR2 on antibody function, the binding affinity of each of the native-mAb1, iso-mAb1, and ox-mAb1 samples was determined using an ELISA assay. Table 2 shows

particular HDX system used in this study, the average SD values of ∑ΔDnative vs isomerized and ∑ΔDnative vs oxidized were both 0.3 Da (rounded to one decimal place); therefore, if all the overlapping peptides from a region have ∑ΔD values greater than 0.9 Da, this difference can be considered statistically significant in the comparison between the two samples. With the predetermined significance threshold, most regions in mAb1 from both heavy and light chains had no significant differences in HDX between the native sample and either of the two modified samples, but a few regions with overlapping peptides consistently showed significantly increased or decreased deuterium uptake levels in the modified samples, suggesting conformational changes (Figures 4 and 5). Comparing the iso-mAb1 with native-mAb1, besides the local region 51−59 (HC) containing Asp54, two other discrete regions also underwent increased deuterium labeling: HC 69− 80 and LC 48−72. Region 48−72 includes the LC CDR2. Both regions are located in the vicinity of Asp 54 in tertiary structure when mapped onto the generic crystal structure of IgG1 (colored in magenta in Figure 6a). In contrast, another region in the HC, 122−151, showed a decrease in deuterium labeling in iso-mAb1 compared to that in native-mAb1. When mapped onto the crystal structure, it is near the hinge region connecting Fab and Fc (colored in blue in Figure 6a). Interestingly, comparison of the ox-mAb1 and native-mAb1 revealed significant differences in nearly the same three regions: LC 48−72, HC 70−86, and HC 122−151 (colored in blue in Figure 6b). Region HC 122−151 showed decreased deuterium uptake in both iso-mAb1 and ox-mAb1 comparing to nativemAb. However, the other two regions, LC 48−72 and HC 70− 86, showed decrease in deuterium uptake, revealing that they became less flexible and more protected upon oxidation at Met56, opposite to the changes caused by Asp54 isomerization. It is important to note that, although both Asp54 isomerization and Met56 oxidation impact the HDX rate of the local CDR2 region (in both HC and LC), the former disrupts the local (HC) and distal (LC) structures in CDRs

Table 2. Relative Binding Affinity of Different Forms of mAb1 against the Native Form by ELISA relative binding (to native species) samples

av (%)

RSD (%)

native-mAb1 iso-mAb1 ox-mAb1

100 13 118

NA 11 12

the relative binding affinity normalized against the affinity of native-mAb1. Iso-mAb1 showed significantly reduced binding (13%) compared to the native sample, suggesting that Asp54 isomerization at CDR2 of HC significantly impaired the antibody’s binding activity. Oxidation on Met56 showed full or even slightly increased binding activity (118%) compared to the native sample. This observation agrees well with the HDX results discussed above which demonstrate relatively larger structural disruptions caused by Asp54 isomerization to CDR2 (HC) and CDR2 (LC). The isomerization of Asp54 could alter the conformation of the entire CDR2 and denature the D and E framework β strands leading to the significant loss of activity 2048

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Article

Analytical Chemistry Author Contributions

seen for this chemical modification. Methionine oxidation is most often a water-mediated event and is correlated with exposure of the specific Met side chain to solvent. While Met oxidations is generally reported to have none or negative effects on antigen interaction, our data show that oxidation of Met56 to methionine sulfoxide stabilizes the local backbone conformation and has minor enhancing effect on binding to the antigen. If Met56 is close to polar residues on the antigen, its oxidation could enhance interaction with the antigen through hydrogen-bond formation. In addition, the opposite impact of isomerization and oxidation on binding activity revealed an inverse relationship between flexibility and affinity, which in turn indicate a more enthalpy-driven binding event between the antigen and mAb1, rather than an induced fit model that is entropy-driven.

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. Reb Russell of Biologics Development, Bristol-Myers Squibb, and Dr. Jay Nair for funding of this research, Dr. Adrienne Tymiak of Bioanalytical and Discovery Analytical Science at Bristol-Myers Squibb for providing the HDX MS system used in this study, and Dr. Marcel S. Zocher and Dr. Jeffrey K. Glenn of Biologics Development, Bristol-Myers Squibb for providing the ELISA equipment used in this study. Dr. Michael Gross at the Washington University, Dr. Kelvin Bai, Dr. Jinmei Fu, and Dr. Jacob Bonders of Biologics Development at Bristol-Myers Squibb are also gratefully acknowledged for support.

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CONCLUSIONS Chemical modifications in the CDRs of monoclonal antibodies, especially those resulting from degradation, may impair antigen binding. In this study, we have applied HDX MS to examine both the local and global structural impact of two common degradation pathways, Asp isomerization and Met oxidation, at CDR2 of the HC of a therapeutic monoclonal antibody (mAb1). Asp isomerization was shown to significantly disrupt the local structure in CDR2 (HC 51−59) and a succeeding region (HC 69−80), leading to more flexibility in those regions. Moreover, the CDR2 of the LC was also impacted similarly by allosteric effects to become less structured. In contrast to isomerization, Met oxidation caused a much smaller structural perturbation to the local CDR2 (HC), the succeeding region (HC 70−86), and the allosteric effect on the LC CDR2, each of which become less flexible (more protected) upon oxidation. The impact of either modification on the conformation of the local CDR2 region was confirmed by computational analysis using both the average SASA and twoshell water coordination models. Biological activity data demonstrated isomerization impaired binding while oxidation had little to no significant impact. Furthermore, the findings in this study on the structural impact of chemical degradations on mAb1 can serve as an important reference to future studies on correlations between modifications in CDRs and antibody quality attributes, especially for IgG1 mAbs.

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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.5b02800. Calculation of back-exchange correction, zoomed-in chromatograms showing heavy chain peptide 51−59 (after 15 min of HDX) eluting at different times in two LC/MS runs (Figure S1), and raw mass spectra nondeuterated and deuterated peptides in the key segments identified in Figure 4 (Figure S2) (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 609-818-4886. Present Address ∥

Y.Y.: Department of Analytical Chemistry, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY 10591−6707. 2049

DOI: 10.1021/acs.analchem.5b02800 Anal. Chem. 2016, 88, 2041−2050

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DOI: 10.1021/acs.analchem.5b02800 Anal. Chem. 2016, 88, 2041−2050