Susceptibility of antibody CDR residues to chemical modifications can

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Susceptibility of antibody CDR residues to chemical modifications can be revealed prior to antibody humanization and aid in the lead selection process Ankai Xu, Hok Seon Kim, Samarkand Estee, Sharon ViaJar, William J Galush, Avinash Gill, Isidro Hotzel, Greg A. Lazar, Paul McDonald, Nisana Andersen, and Christoph Spiess Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 17, 2018

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Molecular Pharmaceutics

Susceptibility of antibody CDR residues to chemical modifications can be revealed prior to antibody humanization and aid in the lead selection process

Ankai Xu1*, Hok Seon Kim2*, Samarkand Estee3, Sharon ViaJar2, William J. Galush3, Avinash Gill2, Isidro Hötzel2, Greg A. Lazar2, Paul McDonald1, Nisana Andersen4,5, Christoph Spiess2,5

* These authors contributed equally to this work

Department of Purification Development1, Antibody Engineering2, Early Pharmaceutical Development3, Protein Analytical Characterization4, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

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Correspondence should be addressed to [email protected] or

[email protected] (phone +1 650 467 1851, fax +1 650 467 8318)

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ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A critical part of the clinical development path for a therapeutic antibody involves evaluating the physical and chemical stability of candidate molecules throughout the manufacturing process. In particular, the risks of chemical liabilities which can impact antigen binding, such as deamidation, oxidation, and isomerization in the antibody CDR sequences, need to be controlled through formulation development or eliminated by replacing the amino acid motif displaying the chemical instability. Commonly, the antibody CDR sequence contains multiple sequence motifs (potential hotspots) for chemical instability. However, only a subset of these motifs results in actual chemical modification, and thus experimental assessment of the extent of instability is necessary to identify positions for potential sequence engineering. Ideally, this information should be available prior to antibody humanization at the stage of parental rodent antibody identification. Early knowledge of liabilities allows for ranking of clones or the mitigation of liabilities by concurrent engineering with the antibody humanization process instead of time-consuming sequential activities. However, concurrent engineering of chemical liabilities and humanization requires translatability of the chemical modifications from the rodent parental antibody to the humanized. We experimentally compared the stability of all sequence motifs by mass spectrometric peptide mapping between the rodent parental antibody and the final humanized antibody and observed a linear correlation. These results have enabled a streamlined developability assessment process for therapeutic antibodies from lead discovery to clinical development. Key words Developability assessment, deamidation, oxidation, isomerization,

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Introduction Antibodies are a successful class of biotherapeutics 1. The vast majority of antibodies that are approved or in clinical development have been identified by hybridoma technology from rodents immunized with the target protein of interest 2. The observed high immunogenicity of rodent antibodies in human clinical trials commonly necessitates humanization of the antibody 3,4. During the humanization process, the complementary-determining regions (CDRs) of the rodent antibody are grafted onto a human acceptor framework while ideally fully retaining the affinity towards the antigen 5. In vitro phage or yeast display 6 with human synthetic or natural antibody libraries or human transgenic rodents 7-9 provide alternative routes to therapeutic antibody discovery and avoid the need for humanization. However, not all targets are suitable for in vitro display technologies and due to limited access to human transgenic animals, wild-type rodents remain the predominant route to generate therapeutic antibodies in animals despite the required humanization step.

While antibody affinity is a key characteristic for a therapeutic antibody, the physicochemical stability of an antibody during the manufacturing process and upon longterm storage is an equally important attribute to ensure successful clinical development. Sustained affinity and stability in vivo is likewise important for therapeutic applications. Developability assessments during the lead selection stage commonly evaluate the physicochemical stability, viscosity, solubility, and production titer of candidate molecules under manufacturing, storage, and in vivo-relevant conditions. Ideally, the final clinical antibody does not display any liabilities and technical (CMC) development can follow a platform process. Satisfactory developability data should also support suitable physicochemical stability in simulated in vivo conditions over relevant time scales. The evaluation of the physicochemical stability of antibodies typically assesses the stability of methionine (Met), tryptophan (Trp), and certain asparagine (Asn) and aspartate (Asp) sequence motifs that are known potential sites for chemical degradation 1,10-13. While progress has been made in recent years with computational prediction tools for developability assessment 14,15, it is still required to unambiguously determine absolute stability by experimental data. This process is time and labor intensive, and thus has traditionally been done later in the discovery process, after partial or complete humanization

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of the antibody candidate. While the value of evaluating antibodies prior to humanization has been suggested 14, the impact of the humanization process and antibody isotype on the chemical liabilities in the CDRs has not been systematically investigated by quantitative analytical methods.

In this study, we compared the chemical stability of five antibodies as parental rodent mIgG1 and mIgG2a antibodies, rodent/human IgG1 chimera, and humanized hIgG1 or hIgG4 antibodies. The assessment of chemical liabilities was limited to amino acids within the antibody CDRs. Oxidation of methionine and tryptophan residues, deamidation and hydrolysis of asparagine residues, and isomerization and hydrolysis of aspartate residues were analyzed by mass spectrometric peptide mapping following accelerated oxidative and thermal stress. In addition, the impact on binding kinetics and capacity was assessed by surface plasmon resonance (SPR).

Over 30 CDR sequence motifs were analyzed as part of the study, and with one exception, susceptibility to chemical instability remained unaltered throughout the humanization process. Determination of these liabilities in the non-humanized parental antibodies (early developability screening) now justifies the experimental effort to assess chemical stability prior to humanization and provides the opportunity to rank antibodies that meet affinity requirements for subsequent humanization based on number and extent of chemical modification with high confidence. Materials and Methods Antibody expression and purification of antibodies The heavy and light chains of antibody genes were cloned by standard molecular biology techniques into separate pRK expression vectors, as described previously 16,17. The Kabat and EU numbering schemes were used to designate residue position in the antibody variable and constant domains, respectively. Antibodies were transiently expressed in Expi293T™ or CHO cells 18,19, and culture supernatants were purified using HiTrap MabSelect Sure columns (GE Healthcare Life Sciences). To enhance resin binding of antibodies with a murine-Fc, NaCl and glycine were added to culture supernatants and then pH adjusted to match the loading buffer. For antibodies with a murine-Fc, the loading buffer was composed of 1.5M Glycine, 3M NaCl

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pH 8.9. Phosphate buffered saline (PBS – 0.01M phosphate, 0.137M NaCl, pH 7.2) was used as the loading buffer for human-Fc antibody containing supernatants. Antibodies were eluted with 0.1M Citrate pH 3.0 and neutralized with 3M Tris pH 8.0 to final pH ~7.0. The eluted antibodies were dialyzed against PBS pH 7.2 prior to the next purification step. Affinity purified antibody preparations were further polished either by cation exchange (CIEX), or size exclusion chromatography (SEC) to remove any aggregates and increase homogeneity of monomeric antibody to at least 95%. For CIEX purification, samples were buffer exchanged into 0.05M NaOAc pH 4.5 and loaded onto HiTrap SP Sepharose FF columns (GE Healthcare Life Sciences) at a flowrate of 3mL/min (90cm/h). Bound antibody was eluted using a 50% linear gradient with 200mL of 0.05M NaOAc, 1M NaCl pH 4.5. The eluted antibody fractions were pooled and dialyzed against PBS pH 7.2. For SEC purification, samples were run on a HiLoad 16/600 Superdex S200 prep grade column (GE Healthcare Life Sciences) using PBS pH 7.2 load buffer at a flowrate of 1mL/min (30cm/h). Pooled fractions were filtered using a 0.2 µm filter and antibody monomer content of the final antibody preparation was assessed by analytical SEC carried out with a TSK-GEL, Super SW3000, 4.6 mm x 30 cm, 4 µm (Tosoh Bioscience) column using a Dionex Ultimate 3000 system (Thermo Fisher Scientific) at a flowrate of 0.3mL/min 20. Oxidative and accelerated stress To test the chemical stability of CDR sequence motifs, both oxidative and accelerated thermal stress was performed. Oxidatively-stressed samples were prepared by incubating 1 mM 2,2'Azobis(2-amidinopropane) dihydrochloride (AAPH) with 1.25 mg of antibody in low-ionic Histidine-Acetate, pH 5.5 for 16 hours at 40 °C. After 16 hours, the AAPH was quenched by adding to the solution a 20:1 excess of methionine (Met) to AAPH. Control samples were spiked with water instead of AAPH. Control and stress samples were buffer exchanged prior to analysis. For thermal stress, antibody samples were incubated at 1 mg/mL in low-ionic Histidine-Acetate buffer, pH 5.5 for two weeks at 40 °C or PBS, pH 7.4 for two weeks at 37 °C. Control samples were stored at -70 °C.

Analysis of antibodies by LC-MS/MS peptide mapping Tryptic Digestion

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A 250 µg sample of mAb was reduced with 20 mM DTT in 6 M guanidine hydrochloride, 360 mM Tris, and 2 mM EDTA at pH 8.6 for 1 hour. The reduced sample was cooled to room temperature and alkylated using 1 M iodoacetic acid (final concentration, 50 mM) for 15 minutes in the dark. The sample was then buffer-exchanged into digestion buffer (25 mM Tris, 2 mM CaCl2, pH 8.2). The buffer-exchanged sample was digested with trypsin for 4 hours at 37 °C using a 1:50 (w/w) enzyme to substrate ratio. The digestion was stopped by addition of 10% formic acid to a final concentration of 0.3%.

UHPLC-HRMS Analysis Tryptic digests were analyzed using an Acquity H-Class UHPLC (Waters) coupled to a Q Exactive (Thermo Fisher) mass spectrometer (MS). Separation of a 10 µg injection was performed on an Acquity UPLC Peptide CSH C18 column (Waters) with 1.7 µm, 130 Å particles running a flow rate of 0.2 mL/min at 77 °C. Mobile phase A was water, and mobile phase B was acetonitrile, each containing 0.1% formic acid. The gradient was as follows: 2 min 1% mobile phase B, 5 min 1-13% mobile phase B, 35 min 13-35% mobile phase B, 2 min 35-95% mobile phase B, 2 min 95% mobile phase B. MS data was collected in positive ion mode using a Top 8 data-dependent scan mode with resolution set to 35,000 for MS scans and 17,500 for MS2 scans. Dynamic exclusion was turned off, and the precursor scan range was set at 200-2000 m/z. External calibration of the instrument was conducted prior to sample analysis. Data was processed using instrument vendor software specific for biopharmaceutical characterization.

Relative quantitation of the chemical sequence motif was generated by integrating the extracted ion chromatograms of the monoisotopic m/z using the two most abundant charge states for the native tryptic peptide and its modified counterpart. The modified peptide peak area was divided by the sum of the modified and native peak areas and multiplied by 100 to obtain the percent modification for each chemical sequence motif. The percent change in deamidation of asparagine (N) residues, isomerization of aspartic acid (D) residues, and hydrolysis at asparagine-proline/aspartic acid-proline (NP/DP) bonds was measured following accelerated thermal stress in Histidine-Acetate and PBS buffers. The percent change in oxidation of methionine (M) and tryptophan (W) residues was measured following oxidative stress.

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Analysis of antibodies by SPR Antibodies with hIgG1 and hIgG4 constant region isotypes were captured on a Protein A capture chip (GE Healthcare). Antibodies with mIgG1 and mIgG2a constant region isotypes were immobilized using the mIgG immobilization kit (GE Healthcare). Antigen was used as analyte in multi-cycle kinetic mode (four different antigen concentrations including a zero) and the unstressed and stressed antibodies were compared in the same cycle for each same concentration series. Binding affinity (KD) was determined via the default 1:1 binding interaction fit provided in BIAevaluation software (GE Healthcare). The analyte binding capacity (Rmax) was normalized against the theoretical Rmax as described in the Biacore handbook for direct comparison of the stressed and unstressed antibodies. The normalized binding capacity (nRmax) of the stressed antibody was referenced against the unstressed antibody to determine the loss in binding capacity after the stress 21.

Results Selection of antibodies for proof of concept study For the proof of concept study, we selected five different antibodies against three antigens covering a variety of different potential chemical liabilities (total of 32 sequence motifs; Table 1). The antibodies originated from either BALB/c mice or Sprague Dawley rats and were isolated by hybridoma technology. For their recombinant production, the antibodies were expressed in Expi293TM cells. This enabled compatibility with a high throughput workflow to concurrently produce 25 individual antibodies at ≥ 15 mg after primary purification by Protein A followed by a secondary chromatography step to remove any aggregates. To study the impact of antibody variable and constant domain on susceptibility to chemical modifications in the antibody CDR, each antibody was expressed as rodentmIgG1, rodent-mIgG2a, rodent-hIgG1 chimera, humanized-hIgG1, and humanized-hIgG4. After purification, antibodies were subject to accelerated thermal stress and oxidative stress.

Peptide analysis by mass spectrometry reveals translatability The relative percent degradation of CDR motifs in 25 humanized, rodent/human chimera, and rodent parental antibody samples was determined by mass spectrometric peptide

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mapping following accelerated thermal and oxidative stress. First, we plotted the relative percent degradation of the sequence motifs in humanized-hIgG1 isotype antibodies against the relative percent degradation of the sequence motifs in humanized-hIgG4 isotype antibodies for thermal stress in Histidine-Acetate pH 5.5 (Figure 1a), thermal stress in PBS pH 7.4 (Figure 2a), and oxidative stress (Figure 3a). There are strong positive linear correlations between the relative percent degradation of sequence motifs in humanizedhIgG1 and humanized-hIgG4 antibodies, with R2=0.97 (m=1.3) for thermal stress in Histidine-Acetate pH 5.5, R2=0.89 (m=1.1) for thermal stress in PBS pH 7.4, and R2=0.99 (m=1.0) for oxidative stress.

Further, we plotted the relative percent degradation of the sequence motifs in humanizedhIgG1 antibodies against the relative percent degradation of the motif in rodent-hIgG1 chimera, rodent-mIgG1, and rodent-mIgG2a antibodies for thermal stress in HistidineAcetate pH 5.5 (Figure 1b), thermal stress in PBS pH 7.4 (Figure 2b), and oxidative stress (Figure 3b). We observed one instance of non-translatability for the hydrolytic degradation of an asparagine-proline motif in mAbB. Susceptibility to hydrolysis for this NP motif in mAbB increased after humanization (Figure 2b, blue data points). Relative percent hydrolysis in the rodent parental antibodies was < 4 % under accelerated thermal stress in both Histidine-Acetate pH 5.5 buffer and PBS pH 7.4. Under accelerated thermal stress in Histidine-Acetate pH 5.5, the relative percent hydrolysis in the humanized antibodies were 9 % (humanized-hIgG1) and 10 % (humanized-hIgG4). Under accelerated thermal stress in PBS pH 7.4, the relative percent hydrolysis in the humanized antibodies were 60 % (humanized-hIgG1) and 56 % (humanized-hIgG4). These results suggest there were possibly sequence elements located outside the mAbB CDRs modulating the stability of this NP motif. The stability modulating elements were likely altered during humanization. In such instances, determining the chemical stability of motifs in rodent parental antibodies could aid in guiding the humanization process to ensure sequence motif stability observed in the rodent parental antibodies is preserved in the humanized antibodies.

Overall, there are strong positive linear correlations between the relative percent degradation of these motifs in humanized antibodies and rodent/human chimera and rodent parental antibodies. For thermal stress in Histidine-Acetate pH 5.5 (Figure 1b), R2=0.91

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(m=0.8) for humanized-hIgG1 vs. rodent-hIgG1 chimera, R2=0.96 (m=1.0) for humanizedhIgG1 vs. rodent-mIgG1, and R2=0.95 (m=0.9) for humanized-hIgG1 vs. rodent-mIgG2a. For thermal stress in PBS pH 7.4 (Figure 2b), R2=0.31 (m=1.0) for humanized-hIgG1 vs. rodent-hIgG1 chimera, R2=0.38 (m=1.8) for humanized-hIgG1 vs. rodent-mIgG1, and R2=0.37 (m=1.5) for humanized-hIgG1 vs. rodent-mIgG2a. When data for the nontranslatable NP motif in mAbB (see above) is excluded during analysis for thermal stress in PBS, R2=0.93 (m=1.0) for humanized-hIgG1 vs. rodent-hIgG1 chimera, R2=0.93 (m=1.7) for humanized-hIgG1 vs. rodent-mIgG1, and R2=0.99 (m=1.4) for humanized-hIgG1 vs. rodent-mIgG2a, indicating strong correlations for all other sequence motifs. For oxidative stress (Figure 3b), R2=0.99 (m=1.0) for humanized-hIgG1 vs. rodent-hIgG1 chimera, R2=0.94 (m=1.0) for humanized-hIgG1 vs. rodent-mIgG1, and R2=0.98 (m=1.0) humanizedhIgG1 vs. rodent-mIgG2a. The relative percent chemical degradation of CDR sequence motifs after accelerated thermal or oxidative stress was translatable between rodent parental, rodent/human chimera, and humanized antibodies for 31 out of 32 labile and stable sequence motifs (Table 3). This strong translatability indicates chemical liabilities in rodent parental antibodies generally remain unaltered through the humanization process.

Binding affinity and capacity analysis by Biacore reveals translatability The thermal and AAPH stressed samples from the 25 antibody samples were analyzed for fold changes in binding affinity (KD) and percent loss in binding capacity (RU). Binding capacity values (RU) were normalized against the amount of stressed antibody captured on the ProteinA capture chip. A binding capacity change of 1 indicates no change. Similar results were observed for the humanized antibodies as human IgG1 and IgG4 isotypes indicating that there is minimal experimental variation or impact by the isotype (Figure 4a, c). Rodent antibodies as hIgG1 chimeras were directly plotted against the humanized IgG1 antibodies (Figure 4b, d). A significant change in KD was observed for only two antibodies (Figure 4b). For most of the stressed antibodies, relative to the controls, only a loss in binding affinity within two-fold of the humanized-IgG1 isotype was detected. This is within the experimental variation for the Biacore assay under the experimental conditions used in this study. Although there was little impact on the measured KD value after thermal and oxidative stress for most antibodies, there was a profound impact on the loss in binding capacity detected upon stress for multiple antibodies (Figure 4d). These corresponded to the

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antibodies with chemical liabilities identified by peptide mapping. Overall, there are strong linear correlations between the loss in binding capacity for thermally and oxidatively stressed samples in the humanized, rodent/human chimera and rodent parental antibodies with R2 value of ~0.8 (Figure 4d). In summary, an impact or lack of impact of the chemical modification on binding capacity of the humanized antibody or significant changes to the binding affinity can reliably be predicted at the rodent parental stage.

Applying early developability screening during the humanization of six antibodies Following the proof of concept study, the liability of sequence motifs was tracked during the humanization process of six different rodent antibodies towards three different antigens. This data set includes 58 potential CDR liabilities (Table 2). Degradation of sequence motifs of an antibody humanization variant in either IgG1 or IgG4 isotype were plotted against the relative percent degradation of the sequence motifs in the rodent/human chimera and rodent parental antibodies following thermal stress in Histidine-Acetate pH 5.5 (Figure 5a), thermal stress in PBS pH 7.4 (Figure 5b), and oxidative stress (Figure 5c). Overall, the parental antibodies accurately forecasted the stability and susceptibility of the motifs. Two instances of over prediction were observed within this data set, with a slightly higher degree of degradation observed in the rodent parental antibody compared to its humanized counterpart (Figure 5a and b). However, the results from this study underscore the value of experimentally evaluating sequence motifs already in the rodent parental antibody.

Discussion The identification of chemical modifications in CDR regions of therapeutic antibodies is a critical activity during the development process. While potential chemical liabilities can be identified based on the antibody primary sequence, they require experimental confirmation to determine the degree of actual chemical modification 14,22,23. It is highly desirable to identify these modifications as early as possible in the discovery and development process as long as they translate to the final clinical candidate. Prior to this study, the translatability of these modifications from the parental rodent antibody to the humanized clinical candidate was

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poorly understood. The results from our study clearly demonstrate that chemical instability of Met, Trp, Asn and Asp residues in the antibody CDR regions are not a consequence of manipulation during the humanization process, but already existed in the antibody selected during the immune response. The susceptibility to chemical modification may be less of a concern in an ongoing immune response because the loss of antibody binding and affinity is naturally buffered by a continuous production by plasma cells. However, therapeutic antibodies require a higher degree of stability to ensure an active antibody during the entire in vivo halflife after being subject to conditions during the manufacturing process (such as cell culture, recovery, and formulation) and product storage that can influence long–term stability. The translatability of stable and unstable sequence motifs during the antibody humanization process suggests that the sequence elements modulating chemical liability reside mostly within the antibody CDR and are not frequently affected by changes in the framework during humanization. This information enables the prioritization of activities to first focus on engineering the antibody CDR sequences rather than exploring different frameworks for humanization. Looking at the stability of the rodent parental sequence also provides the advantage of establishing a baseline. In the event that a stable residue is rendered unstable during the humanization process, the structural environment in the rodent parental antibody may provide guidance on how to possibly engineer the humanized molecule and remove the liability. Peptide map analysis combined with binding capacity and affinity determination by SPR analysis allows for ranking of variant candidate molecules beyond chemical instability. Depending on the heterogeneity and severity of the chemical modifications on residues impacting binding, it may be difficult to determine the loss in binding affinity with a heterogeneous pool of antibodies after the stress unless the modified variants are homogeneously the major species. Therefore, if a chemical modification moderately decreases the binding affinity of only a small fraction of the sample, the measured binding affinity of the sample will remain roughly unchanged due to the 1:1 binding model. The modified sample becomes negligible in the assay unless it represents the major species after the chemical stress. This behavior is illustrated in the majority of the stressed samples with limited changes in binding affinity in Figure 4 (a) and (b). However, if the chemical modifications result in complete loss in binding of antigen regardless of the fraction of the sample with such modifications, a loss in binding capacity measurements will still be reflected as a reduction of

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the absolute value of response units (RU) representing the inactive fractions. The results shown in Figure 4 (c) and (d) suggest that binding capacity (RU) is a more sensitive and reliable measure of the impact that chemical modifications can have on antigen binding compared to determining the binding affinity. A particularly good correlation between SPR and peptide map data was observed following oxidative stress. This may be due to the fact that typically only a single methionine or tryptophan in the antibody CDR was prone to oxidation, enabling a direct read out of a single residue modification. In addition, solvent accessible tryptophan and methionine residues are infrequently found in the variable domain framework region. In the future, assessment via SPR-based methods complimented by in silico prediction 10,14,15 may replace the time- and labor-intensive peptide map analysis and provide a fast and predictive oxidation risk assessment at the parental rodent antibody stage. In contrast, the correlation between absolute modification by peptide map and binding affinity or capacity loss was less tight for deamidation and isomerization with an overall higher impact by SPR for noncorrelative cases than expected by peptide map. This may be partially contributed by the higher abundance of asparagine and aspartate residues in the variable domain framework region and their potential impact on binding after deamidation and isomerization or cumulative effects by combinations of multiple events in the antibody CDRs. To exclude an impact of the antibody expression host on the extent of chemical modification, all antibodies in the study were expressed in Expi293TM cells. To investigate a potential impact of the expression host, the humanized antibodies were expressed in CHO cells and subject to peptide map prior and after accelerated stress. We did not observe any significant difference in results (data not shown), indicating that the host cell has no influence on the results. This enables an earlier developability assessment to benefit from the faster production capability of Expi293TM over CHO. In summary, the possibility to reliably assess chemical modifications in the parental rodent antibody provides antibody engineers with an early view on the developability challenges of the humanized antibody candidates. It allows additional time to mitigate the instability by sequence engineering, a process that is now off the critical path and can take place concurrently with antibody humanization. By adopting the practice of developability assessment at the rodent parental stage, antibody engineers now have additional tools available for candidate ranking which allow an informed decision on candidates based on the expected downstream liabilities for formulation work.

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Acknowledgments We thank Danielle DiCara, Farzam Farahi, Holly Yip, Kyle Hogan, Matt Chen, Michael Madonna, Mie Lansang, Sam Burns, Nicole Stephens, Scott Chamberlain and Sophia Lee and the Research Materials group at Genentech for technical insights and assistance. We also like to thank Amy Hilderbrand, Laura Simmons and Peter Day for scientific discussions. Conflict of interest All authors are current or former employees of Genentech, Inc., a member of the Roche Group, and may hold stock and options. This work was funded by Genentech, Inc..

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Table 1. Panel of antibodies used in the proof of concept study and their sequence motifs analyzed for chemical modification. The Kabat position of the sequence motif in the primary structure is indicated (H=heavy chain; L=light chain).

Variable-constant domain species and isotype

Antibody

CDR Sequence Motifs Deamidation: N(H54)G, N(L92)NG

mAbA

Isomerization: D(H96)G Hydrolysis: D(H52)P Deamidation: N(H31)Y, N(H54)G, N(H56)T

mAbB

Hydrolysis: N(L94)P Oxidation: M(L33), W(L91), W(H95), W(H100) Deamidation: N(L28)G, N(L30)T,

humanized-hIgG1 humanized-hIgG4

N(H52; H94)S

mAbC

Isomerization: D(H61)S, D(H101)Y

rodent-hIgG1

Oxidation: M(H34), M(H100),

rodent-mIgG1

W(L96)

rodent-mIgG2a Deamidation: N(H96)Y

mAbD

Isomerization: D(H61)S, D(H58)Y Oxidation: M(H34), W(H100)

Deamidation: N(H31; H96)Y

mAbE

Oxidation: M(L30), M(H34), W(L33; H100)

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Table 2. Panel of antibodies from pilot study and their sequence motifs analyzed for chemical modification. The Kabat position of the sequence motif in the primary structure is indicated (H=heavy chain; L=light chain).

Variable-constant domain species and isotype

Antibody

CDR Sequence Motifs

Deamidation: N(H31)Y

rodent-hIgG1 humanized-hIgG1

mAb1

Isomerization: D(L92; H53; H61)S, D(H101)Y Oxidation: M(L89; H34; H50)

rodent-mIgG2a rodent-hIgG1

Isomerization: D(L53; H53; H61)S,

mAb2

D(H98)A, D(L94)D Oxidation: M(H34; H57)

humanized-hIgG1

Deamidation: N(L31; H54)Y, N(H54)S

rodent-mIgG2a humanized-hIgG1

mAb3

Isomerization: D(L55; H61)S, D(H101)Y Oxidation: M(L33; H34; H100a), W(L50; L96)

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Deamidation: N(H31)A, N(L92)S,


mAb4

N(L52)N, N(H54)Y Isomerization: D(H61)S, D(H101)Y Oxidation: M(H34), W(L96)

Deamidation: N(H57)N, N(H58)Y


Isomerization: D(H53)D, D(L28; H54;

mAb5

H96)G, D(L27d)S Hydroylsis: D(L55)S, N(H60)P Oxidation: W(L89; H35)

Deamidation: N(H58; H98)Y, N(L30)S, N(L91)N


mAb6

Isomerization: D(H96)G, D(H100b)A, D(L27c)S, D(H101)Y Oxidation: M(L33; H34; H62; H100d), W(H33)

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Table 3. Fraction of labile and stable sequence motifs for which relative percent degradation is translatable between rodent parental, rodent/human chimera, and humanized antibodies.

Deamidation

Isomerization

Hydrolysis

Oxidation

Motif

NG

NNG

NT

NS

NY

DG

DS

DY

DP

NP

W

M

Fraction

3/3

1/1

2/2

2/2

4/4

1/1

2/2

2/2

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Translatable

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17


1/2

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Figure legends

Figure 1. Relative percent deamidation of asparagine (N) residues, isomerization of aspartic acid (D) residues, and hydrolysis at aspartic acid-proline (DP) or asparagine-proline (NP) bonds in the CDR following accelerated thermal stress in Histidine-Acetate measured by mass spectrometric peptide mapping. Positive linear correlations were observed between the percent motif degradation in humanized-hIgG1 antibodies and the percent motif degradation in (a) humanized-hIgG4 antibodies and (b) rodent-hIgG1, rodent-mIgG1, and rodent-mIgG2a antibodies.

Figure 2. Relative percent deamidation of asparagine (N) residues, isomerization of aspartic acid (D) residues, and hydrolysis at aspartic acid-proline (DP) or asparagine-proline (NP) bonds in the CDR following accelerated thermal stress in PBS measured by mass spectrometric peptide mapping. Positive linear correlations were observed between the percent motif degradation in humanized-hIgG1 antibodies and the percent motif degradation in (a) humanized-hIgG4 antibodies and (b) rodent-hIgG1, rodent-mIgG1, and rodent-mIgG2a antibodies.

Figure 3. Relative percent oxidation of methionine (M) and tryptophan (W) residues in the CDR following accelerated oxidative stress measured by mass spectrometric peptide mapping. Positive linear correlations were observed between the percent motif degradation in humanized-hIgG1 antibodies and the percent motif degradation in (a) humanized-hIgG4 antibodies and (b) rodent-hIgG1, rodent-mIgG1, and rodent-mIgG2a antibodies.

Figure 4. Relative binding affinity between humanized IgG1 and (a) IgG4 or (b) rodenthIgG1. Binding capacity loss between humanized IgG1 and (c) IgG4 or (d) rodent-hIgG1. Antigen binding was measured by Biacore using a 1:1 binding fit model. A positive correlation is observed between the parental rodent/human IgG1 chimeric antibodies and the humanized IgG1 antibodies.

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Figure 5. Relative percent deamidation of asparagine (N) residues, isomerization of aspartic acid (D) residues, and hydrolysis at aspartic acid-proline (DP) or asparagine-proline (NP) bonds in the CDR following accelerated thermal stress in (a) Histidine-Acetate or (b) PBS measured by mass spectrometric peptide mapping. (c) Relative percent oxidation of methionine (M) and tryptophan (W) residues in the CDR following accelerated oxidative stress measured by mass spectrometric peptide mapping. Strong correlations were observed under all stress conditions between the percent motif degradation in humanized-hIgG1 or humanized-hIgG4 antibodies and the percent motif degradation in rodent-hIgG1 or rodent-mIgG2a antibodies.

19



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Molecular Pharmaceutics (b) 15

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Antigen binding affinity loss (fold) humanized-hIgG1

Antigen binding affinity loss (fold) humanized-hIgG1

Antigen binding capacity loss (%) humanized-hIgG1

Antigen binding capacity loss (%) humanized-hIgG1

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1 2 3 10 10 4 5 6 7 5 5 8 9 10 11 12 0 0 0 5 10 15 0 5 10 15 13 Antigen binding affinity loss (fold) 14 Antigen binding affinity loss (fold) humanized-hIgG4 rodent-hIgG1 15 16 17 18(c) (d) 19 100 100 20 21 22 80 80 23 24 60 60 25 26 27 40 40 28 29 30 20 20 31 32 0 0 33 0 20 40 60 80 100 0 20 40 60 80 100 34 Antigen binding capacity loss (%) Antigen binding capacity loss (%) 35 humanized-hIgG4 rodent-hIgG1 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 ACS Paragon Plus Environment 51 52 Figure 4 53

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Motif degradation (% change) humanized-hIgG1 & humanized-hIgG4

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Rodent Trp Asn

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Chimeric Trp Asn

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Humanized Trp Asn

Labile/Stable motif


Trp Asn

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Susceptibility of antibody CDR residues to chemical modifications can

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