Unique Impacts of Methionine Oxidation, Tryptophan Oxidation, and Asparagine Deamidation on Antibody Stability and Aggregation

Journal Pre-proof Unique impacts of methionine oxidation, tryptophan oxidation and asparagine deamidation on antibody stability and aggregation Magfur E. Alam, Thomas R. Slaney, Lina Wu, Tapan K. Das, Sambit Kar, Gregory V. Barnett, Anthony Leone, Peter M. Tessier PII:

S0022-3549(19)30722-1

DOI:

https://doi.org/10.1016/j.xphs.2019.10.051

Reference:

XPHS 1778

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 16 August 2019 Revised Date:

22 October 2019

Accepted Date: 28 October 2019

Please cite this article as: Alam ME, Slaney TR, Wu L, Das TK, Kar S, Barnett GV, Leone A, Tessier PM, Unique impacts of methionine oxidation, tryptophan oxidation and asparagine deamidation on antibody stability and aggregation, Journal of Pharmaceutical Sciences (2019), doi: https:// doi.org/10.1016/j.xphs.2019.10.051. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.

Unique impacts of methionine oxidation, tryptophan oxidation and asparagine deamidation on antibody stability and aggregation Magfur E. Alam1, Thomas R. Slaney2, Lina Wu3, Tapan K. Das2, Sambit Kar2, Gregory V. Barnett2, Anthony Leone2, Peter M. Tessier3,4,5,† 1

Isermann Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA 2 Biologics Development, Bristol-Myers Squibb, Pennington, NJ 08534, USA 3 Departments of Chemical Engineering, 4Pharmaceutical Sciences and 5Biomedical Engineering, Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, USA

ABSTRACT Monoclonal antibodies are attractive therapeutic agents because of their impressive biological activities and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of chemical modifications, and the impact of such modifications on antibody physical stability and aggregation remains understudied. Here we report a systematic analysis of the impact of methionine oxidation, tryptophan oxidation and asparagine deamidation on antibody conformational and colloidal stability, hydrophobicity, solubility and aggregation. Interestingly, we find little correlation between the impact of these chemical modifications on antibody conformational stability and aggregation. Methionine oxidation leads to significant reductions in antibody conformational stability while having little impact on antibody aggregation except at extreme conditions (low pH and elevated temperature). Conversely, tryptophan oxidation and asparagine deamidation have little impact on antibody conformational stability while promoting aggregation at a wide range of solution conditions, and the aggregation mechanisms appear linked to unique types of reducible and non-reducible covalent crosslinks and, in some cases, to increased levels of attractive colloidal interactions. These findings highlight that even related types of chemical modifications can lead to dissimilar antibody aggregation mechanisms, and evaluating these findings for additional antibodies will be important for improving the systematic generation of antibodies with high chemical and physical stability. Keywords: mAb, solubility, formulation, developability, protein aggregation. Running title: †

corresponding author ([email protected])

INTRODUCTION There is intense interest in developing monoclonal antibodies as therapeutics, which is evidenced by the growing number of approved antibody drugs and the large number of clinical candidates.1–3 The power of antibody therapeutics stems from their many attractive properties, including their natural ability to link antigen binding to recruitment of immune effector functions as well as their long circulation times and favorable biophysical properties.4–6 The growth in antibody therapeutics has also been aided by robust methods for generating them both in vivo (e.g., immunization) and in vitro (e.g., phage and yeast surface display) against seemingly any antigen of interest.7,8 These and other advances have led to antibodies being considered one of the standard modalities used to treat diverse human disorders. Despite the many advantages of antibody therapeutics, they are susceptible to similar chemical and physical instabilities observed for other types of biologics. For example, the Fc region of most antibodies contains conserved methionine (Met) residues (e.g., Met252 and Met428) located at the interface of the CH2 and CH3 domains, which are particularly sensitive to oxidation.9,10 Oxidation of these Met residues typically results in reduced antibody conformational stability.11–14 In some cases, this also causes decreased antibody interaction with Fc receptors such as the neonatal Fc receptor (FcRn),14–16 which can also lead to reduced antibody half-life in vivo.16 Tryptophan (Trp) is also particularly susceptible to oxidation, and oxidation of Trp residues in antibody complementarity-determining regions (CDRs) has been linked to loss of antigen binding.17–19 Moreover, Trp oxidation has been linked to reduced antibody physical stability by promoting disulfide scrambling20,21 and aggregation.17,22,23 Asparagine (Asn) deamidation is another common type of antibody chemical modification, which has been linked to reduced affinity due to deamidation of CDR residues24–26 as well as variable impacts on antibody conformational stability24,27,28 and aggregation.22,27 Nevertheless, there are a number of open questions related to how Met oxidation, Trp oxidation and Asn deamidation impact antibody aggregation that are the focus of this study. First, which type of chemical modification possesses the greatest risk for promoting antibody aggregation when compared head-to-head for a common set of antibodies? Second, how dependent are the levels of antibody aggregation induced by each type of chemical modification on the conditions (e.g., pH and temperature) used for evaluating antibody physical stability? Third, how similar or different are the aggregation mechanisms for antibodies subjected to chemical modifications of the same class (oxidation of Met versus Trp) and different classes (oxidation versus deamidation)? Here we systematically evaluate the impact of deamidation and selective oxidation of Met and Trp residues on antibody aggregation for a wide range of pHs (pH 3.8-7.4) and temperatures (4-37 °C), and report unique aggregation mechanisms for each type of chemical modification.

EXPERIMENTAL METHODS Materials 2

The mAbs used in this study (stock concentrations of 10-15 mg/mL) were an IgG1 (mAb 1) and an IgG4 (mAb 2), which were provided by Bristol-Myers Squibb (Hopewell, NJ) as formulated drug substances. Syringe filters were obtained from EMD Millipore (SLGV004SL), VWR (28145-501) and Thermo Fisher Scientific (097203). Polystyrene plates (clear 384-well, 12565506; clear 96-well, 07200656; black 96-well, 07200589) were obtained from Thermo Fisher Scientific and adhesive films (2920-0000) were obtained from USA Scientific. PCR plates (white; 04729692001) and sealing foils (04729757001) were obtained from Roche Diagnostics. Protein Thermal Shift Dye (4461146) was obtained from Applied Sciences. Zeba spin desalting columns (PI89894) and clear polystyrene inserts (13622207) were obtained from Thermo Fisher Scientific. Individually packaged cuvettes (95201005) were obtained from VWR. Potassium phosphate (205935000) was obtained from Acros Organics. Citric acid (251275), Pharmalyte (17-0456-01), urea (57-13-6) and ammonium sulfate (7783-20-2) were obtained from Sigma-Aldrich. A methyl cellulose kit (1%; 101876) was obtained from Protein Simple. Amicon 30 kDa centrifugal filters (UFC503096) were obtained from EMD Millipore. The 2x Laemmli buffer (1610737) and 4–15% mini-PROTEAN TGX gels (4561086) were obtained from Biorad. β-mercaptoethanol (200000622) was obtained from VWR. Gelcode Blue Safe Protein (1860983) and Pierce Silver Stain Kit (24612) were obtained from Thermo Fisher Scientific.

Methods Antibody sample preparation

The stock mAbs (~10 mg/mL) were provided by Bristol-Myers Squibb at pH ~6 in their respective formulation buffers. The untreated (control) mAb solutions were buffer exchanged into PBS at pH 7.4 using Zeba desalting columns (PI89894; Thermo Fisher Scientific), aliquoted (~0.2-0.5 mL) and frozen at -80 °C until evaluation. In order to achieve selective Met oxidation, H2O2 (3%, w/w) was added to the stock mAb solutions to achieve a protein:H2O2 molar ratio of 1:100. The solutions were mixed gently and incubated at 25 °C for 1 d. Afterward, a three-fold molar excess of a Met solution (200 mM) was added to quench the oxidation reaction. Next, the mAb solutions were buffer exchanged into PBS at pH 7.4 using Zeba desalting columns, aliquoted and frozen at -80 °C until evaluation. In order to achieve selective Trp oxidation, 2,2’-Azobis (2-methylpropionamidine) dihydrochloride (AAPH; 2 mg/mL) was added to the stock mAb solutions to achieve a protein:AAPH molar ratio of 1:100. Free DL-Met (10 mg/mL) was also added to the solution in order to protect the Met residues in the mAbs from oxidation. The solutions were then mixed gently and incubated at 37 °C for 3 d. At the end of the incubation period, the mAb solutions were enriched for monomer using a semi-preparative SEC column running on an ÄKTA Avant 25. The enriched monomer samples were buffer exchanged into PBS at pH 7.4 using Zeba desalting columns, aliquoted and frozen at -80 °C until evaluation. 3

In order to achieve Asn deamidation, the stock mAb solutions were titrated to pH 9.5 with Tris base. Next, the solutions were mixed gently and incubated in the absence of light at 37 °C for one week. Afterward, the mAb solutions were buffer exchanged into PBS at pH 7.4 using Zeba desalting columns, aliquoted and frozen at -80 °C until evaluation. For the accelerated stability study, the mAb samples were prepared at 1 mg/mL in citrate-phosphate buffer (15 mM citrate, 10 mM phosphate; pH 3.8-7.4) with 0.04% w/v sodium azide. The mAb samples were stored in 1.5 mL Eppendorf tubes (16155500, USA Scientific) and maintained at 37 °C in an incubator or at ~25 °C on the bench. Imaged capillary isoelectric focusing (iCIEF)

Capillary isoelectric focusing experiments were performed as described previously.27 Briefly, antibodies (theoretical pIs for mAb 1 of 8.6-8.9 and mAb 2 of 8.1-8.4) were diluted in water to 1.5-2.5 mg/mL, 4% Pharmalyte (GE Healthcare), 1% Methyl Cellulose (Protein Simple), 8 M Urea (Sigma Aldrich), and appropriate pI markers (Protein Simple). Next, the diluted samples were injected into an iCE3 (Protein Simple) system equipped with a fluorocarbon-coated capillary cartridge. Each sample was pre-focused for 1 min at 1500 V and then focused for 10 min at 3000 V. The focusing patterns were then captured by a CCD camera at 280 nm. Size-exclusion chromatography

For the concentration-dependent aggregation study, the mAb samples were analyzed via size-exclusion chromatography using a TSKgel SuperSW mAb HTP column (0.46 x 15 cm, Tosoh Bioscience) and a Waters 6000 HPLC. The column was washed with four column volumes of buffer (either PBS supplemented with 200 mM arginine at pH 7.4 or citrate-phosphate buffer at pH 3.8). Next, the samples (10 µL of mAb samples at 1 or 25 mg/mL) were injected into the column without extended storage (injected <2 h after preparation) in an order that was randomized between repeats, and the area under the curve was evaluated automatically using the HPLC software (Waters Empower Pro 2). All peaks before the main protein peak were considered to be high molecular weight (HMW) species. The %HMW species was calculated as the area under the curve of the HMW species divided by the area under the curve of the entire chromatogram. For the accelerated stability samples, aliquots (50 µL) were taken at 0, 1, 7 and 28 days, and 10 µL of each mAb solution was injected into the TSKgel SuperSW mAb HTP column equilibrated in PBS buffer supplemented with 200 mM arginine at pH 7.4. The %HMW species for each sample was calculated in the same way as described above. The total area under the curve for the entire chromatogram was also evaluated at each time point to determine the % antibody recovery. SDS-PAGE

4

The mAb samples were diluted to 2 mg/mL [1 mg/mL for the accelerated stability samples and 40 µg/mL for the deamidated (monomer and HMW) samples purified by size-exclusion chromatography] in citratephosphate buffer at pH 7.4. Next, equal volumes of the mAb solutions and 2x Laemmli buffer, prepared with and without reducing agent (β-mercaptoethanol), were mixed together. The mixtures without reducing agent were evaluated either as is (control) or after heating at 95 °C for 5 min. The mixtures with reducing agent were heated for 5 min at 95 °C. Next, the samples were centrifuged at 16000 rcf for 2 min, and 10 µL [15 µL for the accelerated stability samples and 5 µL for the purified deamidated (monomer and HMW) samples] was loaded into each well of 4–15% mini-PROTEAN TGX (4561086, Biorad) gels. Tris-glycine running buffer (25 mM Tris, 192 mM glycine and 0.1% SDS at pH 8.3) was used and the samples were run for 1 h at 120 V. Finally, the gels were stained using either Gelcode Blue Safe Protein or Pierce Silver Stain (1860983 or 24612, Thermo Fisher Scientific) and then de-stained and imaged. Differential scanning fluorimetry

The mAb samples were diluted to 0.115 mg/mL in their respective buffered solutions (15 mM citrate, 10 mM phosphate at pH 3.8 to 7.4). Protein Thermal Shift Dye (4461146, Applied Biosciences), initially provided at a concentration of 1000x, was diluted to a concentration of 40x using Milli-Q water. The mAb solutions (17.5 µL in each well) were dispensed into 96-well white PCR plates (04729692001, Roche) in triplicate. Next, dye solution (2.5 µL) was added to each well, and the solutions were mixed thoroughly. The plates were then sealed with foil (04729757001, Roche Diagnostics) and thermal melts were performed using a LightCycler 480 real-time PCR instrument (Roche Diagnostics). The fluorescence (Ex: 558 nm, Em: 610 nm) was measured as the plate was heated from 25 to 95 °C. Fifty acquisitions were collected per 1 °C, and the heating rate was ~0.7 °C /min. The apparent melting temperatures (Tm*) were determined by analyzing the first derivative of the fluorescence with respect to temperature, which involved fitting a second order polynomial to the major peak and determining the temperature at the maximum. In a few cases, the peaks could not be fit using a second order polynomial and instead were deconvoluted using a bigaussian fit (Origin 2019). Differential scanning calorimetry

The stock mAb samples were buffer exchanged into their respective buffered solutions (15 mM citrate, 10 mM phosphate at pH 3.8 and 7.4) and diluted to a concentration of 1 mg/mL. Next, the antibodies (~450 µL) were loaded into the sample cell and the corresponding buffers (~450 µL) were loaded into the reference cell of a Nano DSC calorimeter (TA Instruments). Next, the cells were heated (0.7 °C /min) from 25 to 95 °C for the samples prepared at pH 7.4 and from 15 to 75 °C for the samples prepared at pH 3.8. The apparent melting temperatures (Tm*) were determined by fitting a second order polynomial to the first peak of the thermograms (after subtracting the blank buffer baseline) and determining the temperature at the maximum. In some cases, the peaks could not be fit using a second order polynomial and instead were deconvoluted using a gaussian fit (Origin 2019). The onset temperature (Tonset) was determined by using methods described previously.29 5

Dynamic light scattering

Light scattering experiments were performed as described previously.27 Briefly, each mAb sample was buffer exchanged four times into the target buffer (15 mM citrate, 10 mM phosphate at pH 3.8 or pH 7.4). Next, the mAbs were concentrated using 30 kDa Amicon centrifuge filters (UFC503096, EMD Millipore) to ~30 mg/mL, filtered using 0.22 µm syringe filters (09-720-3, Thermo Fisher Scientific), their concentrations were measured via UV-absorbance at 280 nm using extinction coefficients of 1.53 and 1.59 mL/(mg·cm) for mAb 1 and mAb 2, respectively and were then diluted to a concentration of 25 mg/mL. The concentrated mAbs (80 µL) were transferred to disposable cuvettes (E0030106300, Fisher Scientific) and placed in a Wyatt DynaPro dynamic light scattering instrument. The size distribution was measured using a regularization algorithm (Wyatt instrument software) and was calculated as the average of 20 measurements, and each measurement was conducted for 10 s at a laser power that yielded approximately one million counts per second. *

The experiments to determine the apparent diffusion interaction parameter (kD ) were performed as described previously.27 Briefly, the mAb samples were buffer exchanged four times into the different buffers (15 mM citrate, 10 mM phosphate) at pH 3.8 and 7.4. Next, the mAbs were filtered using 0.22 µm PVDF syringe filters (SLGV004SL, EMD Millipore), and their concentrations were measured via UV-absorbance at 280 nm. The concentrated mAbs (10 mg/mL) were transferred (80 µL) to disposable cuvettes and placed in a Wyatt DynaPro dynamic light scattering instrument. The diffusion coefficients were obtained as described previously.30 Briefly, the autocorrelation function was fit using the method of cumulants (Wyatt instrument software) to determine average diffusion coefficient (DM) values, which were calculated as the average of 20 measurements and each measurement was conducted for 10 s. The mAbs were then diluted sequentially to lower concentrations (4-9 mg/mL) in their respective buffers and evaluated via light scattering. The diffusion coefficients (DM) for the mAb solutions (4–10 mg/mL) were fit to the following equation: =

1 +

∗



*

where D0 is the self-diffusion coefficient, kD is the apparent diffusion interaction parameter, and c is the antibody concentration. Hydrophobic interaction chromatography

The hydrophobic interaction chromatography experiments were performed using a ProPac HIC-10 column (063653, Thermo Fisher Scientific) and a Waters 6000 HPLC. The mAb solutions were diluted to 2 mg/mL in citrate-phosphate buffer at different pH values (pH 3.8 or 7.4) and mixed with an equal volume of 1.2 or 2 M ammonium sulfate solution (prepared at the same buffer conditions) to obtain final concentrations of 1 mg/mL mAb and 0.6 or 1 M ammonium sulfate. Next, the mAb samples (100 µL) were injected into the column equilibrated in citrate-phosphate buffer at pH 3.8 or 7.4 with 0.6 or 1 M ammonium sulfate. After the injection, an isocratic step at 0.6 or 1 M ammonium sulfate was run for 4 min followed by an ammonium sulfate 6

gradient. This consisted of a 20 min negative linear gradient (0.6 or 1 M to 0 M) at a flow rate of 1 mL/min, which was used to elute the mAbs for evaluation of their retention behavior. The area under the curve was calculated between the column dead time (void volume) and the end of the run, and the % recovery of the chemically modified antibodies was evaluated relative to the control. Ammonium sulfate precipitation

The mAb solutions were diluted to 1 mg/mL in citrate-phosphate buffer at different pH values (pH 3.87.4). Stock solutions of citrate-phosphate buffer were also prepared at the same pH values both with and without 3.5 M ammonium sulfate. Next, the mAb solutions (10 µL) were added to each well of clear 96-well plates (07200656, Fisher Scientific), and then ammonium sulfate solutions (90 µL) were added at different concentrations (0.5-2 M). The solutions were then mixed gently to prevent formation of air bubbles. The solutions were incubated at room temperature (10 min) and the absorbance (turbidity) values at 350 nm were measured. The turbidity values were normalized using the maximum and minimum turbidity values for a given set of samples (i.e., samples for the same mAb and pH). The normalized absorbance values of the mAbs (yaxis) were plotted against the ammonium sulfate concentration (x-axis), and four-parameter Boltzmann sigmoidal curves (y=min+[max-min]/[1+exp[[CS,50 -x]/Slope]]) were fit to the resulting graphs. The midpoint of transition (CS,50) was evaluated as a measure of the relative solubility of the mAbs. Intrinsic tryptophan fluorescence

The mAb solutions were diluted to 0.25 mg/mL in citrate-phosphate buffer at different pH values (pH 3.8 or 7.4). Next, the mAb solutions (100 µL) were dispensed into each well of black 96-well plates (07200589, Fisher Scientific) in triplicate. The intrinsic tryptophan fluorescence emission spectra were measured from 315 to 450 nm (in 1 nm increments) after excitation at 295 nm using a Tecan M1000 Pro plate reader. The fluorescence of the buffer solution was also measured and subtracted from the antibody fluorescence spectra.

RESULTS Oxidation and deamidation mediate unique pH- and temperature-dependent antibody aggregation behaviors Toward our goal of evaluating the effects of oxidation and deamidation on antibody stability, we performed forced degradation of two mAbs [mAb 1 (IgG1) and mAb 2 (IgG4)] using conditions that are relatively selective for modifying Met, Trp or Asn residues (hereafter referred to as Met oxidized, Trp oxidized and deamidated mAbs, respectively), while the untreated mAbs were used as controls. We performed mass spectrometry (peptide mapping) to evaluate the extent of modification of the oxidation and deamidation sites (Table 1). For the mAbs treated with H2O2, mass spectrometry analysis revealed large increases in the levels of Met oxidation relative to small increases in Trp oxidation. In particular, mAb 1 showed high levels of Met oxidation in its Fab (91%) and Fc (51%) regions, while mAb 2 displayed modest levels of Met oxidation in its 7

Fab region (12%) and high levels in its Fc region (51%). Conversely, mAbs treated with AAPH in the presence of excess methionine displayed relatively high levels of Trp oxidation (29-87% in their Fab regions) and little Met oxidation (<2%). Additionally, Trp oxidation was not observed in the Fc region for either mAb. Moreover, the samples incubated at high pH (pH 9.5) displayed relatively high levels of Asn deamidation in their Fc regions (18-20%) and minimal levels of deamidation in the Fab regions (<4%). As expected, most of the deamidation in the Fc regions occurred in the PENNY peptide (Asn384 and Asn389; 54.4% and 36.4% for mAb 1 and 2, respectively).31,32 While the three different conditions used to promote chemical degradation were relatively selective, it is notable that treatment of mAb 1 at high pH to promote deamidation also increased Met oxidation in the Fab region of mAb 1 (19% modified relative to 10% for the control), while this was not observed for mAb 2. One of the principal goals of our study was to evaluate the effects of oxidation and deamidation on the stability of antibodies due to various stresses encountered by the mAbs during manufacture, which can include changes in pH, concentration, and temperature.33,34 Therefore, we first sought to quantify the amount of soluble high molecular weight (HMW) species present without extended storage as a function of pH and concentration (Figures 1 and S1). We evaluated the aggregation behavior of the control and chemically modified mAbs via size-exclusion chromatography at pH 3.8 (Figure 1A and 1B) because low pH is frequently used for antibody purification and is often associated with antibody aggregation.35–37 At a moderately high mAb concentration (25 mg/mL), the size-exclusion chromatograms revealed the presence of HMW species for the control and chemically modified samples of both mAbs (Figure S1). The Trp oxidized samples displayed the highest levels of HMW species followed by the deamidated samples, and both the Met oxidized and control samples displayed low levels of aggregation. However, at a lower mAb concentration (1 mg/mL), only the Trp oxidized samples displayed significantly higher levels of aggregation than the control (Figure 1A and 1B). Moreover, deamidation promotes the formation of soluble aggregates at low pH in the most pronounced concentration-dependent manner, as observed previously.27 We also found that the aggregation behavior of the same mAbs at neutral pH followed a similar trend in aggregation behavior, although the levels of aggregation were higher at near neutral pH than at low pH (Figure 1C and 1D). Next, we evaluated the aggregation behavior of the mAbs using a second method (dynamic light scattering) without extended storage (<2 h) in order to determine the generality of our findings (Figures 2, S2 and S3). Light scattering revealed that the deamidated samples (25 mg/mL) displayed distinct, larger populations of particles for both mAbs at low pH (pH 3.8; Figures 2A, 2B, S2 and S3). Moreover, the Trp oxidized samples displayed a distinct population of particles for mAb 1 and modestly increased polydispersity (~21%) for mAb 2 compared to the control (~10% polydispersity; Figures 2A, 2B, S2 and S3). In contrast, the Met oxidized samples of both mAbs were similar to the controls. Increased polydispersity in dynamic light scattering may indicate the presence of dimers or trimers while distinct populations of larger species often 8

represent larger aggregates.38 At near neutral pH (pH 7.4), the deamidated samples also displayed a distinct, larger population of particles for mAb 2 (but not for mAb 1) relative to the control samples. In contrast, the same pH condition resulted in high levels of polydispersity for the Trp oxidized samples of mAb 1 (~32%) relative to mAb 2 (~13.8%) and the controls (~8% and ~9% for mAb 1 and 2, respectively; Figures 2C, 2D, S2 and S3). The Met oxidized samples at pH 7.4 showed little change relative to the control samples for both mAbs. Taken collectively, the light scattering and size-exclusion chromatography results suggest that deamidation and Trp oxidation – without prolonged incubation (<2 h) – enhance aggregation of both mAbs at a range of solution conditions. We next sought to evaluate the effects of temperature and pH on the aggregation behavior of the mAbs during extended storage. Thus, we incubated the control and modified mAbs at an elevated temperature (37 °C) and room temperature (25 °C) as a function of pH (pH 3.8-7.4) for 7-28 days (Figures 3, 4 S4-S7). We first evaluated the aggregation behavior of the mAbs at low pH (pH 3.8) during incubation for seven days via size-exclusion chromatography (Figure 3). As expected, the mAbs displayed high levels of aggregation at 37 °C and low pH during extended storage. Interestingly, Met oxidized samples displayed the largest increase in HMW species relative to the control at 37 °C after seven days (Figure 3A and 3B). The Met oxidized sample of mAb 1 also displayed reduced recovery (~75%) from the size-exclusion column relative to the control (~90%; Figure S5). This aggregation behavior was not observed when the incubation temperature was lowered to 25 °C (Figure 3C and 3D), as Met oxidized samples displayed low levels of aggregation at 25 °C that were similar to the controls. At pH 4.5, we also observed that Met oxidized samples were aggregation prone at 37 °C but not at 25 °C, although the levels of aggregation were much lower (<20% after 28 days at 25 °C; Figure S6) than at pH 3.8. At higher pH values (pH 6 and 7.4), all of the mAb samples displayed modest changes in aggregation after incubation for 28 days at 37 °C (Figures S5, 4A and 4B) and 25 °C (Figures S7, 4C and 4D). The highest levels of aggregation at pH 6 and 7.4 corresponded to the Trp oxidized samples followed by the deamidated samples, while the Met oxidized and control samples displayed the lowest levels of aggregation. At these pH values, the chemically modified antibodies were recovered from the size-exclusion column at high levels (>95%) that were similar to those observed for the controls (Figure S5). These findings demonstrate that the Met oxidized samples are particularly aggregation-prone at elevated temperature (37 °C) and low pH (pH 3.8), but are resistant to aggregation at lower temperatures (25 °C) or higher pH values (pH 6 and 7.4). Unique mechanisms mediate enhanced antibody aggregation due to oxidation and deamidation There are multiple possible mechanisms through which oxidation and deamidation can potentially enhance aggregation. One such mechanism is that these chemical modifications can lead to the formation of covalently crosslinked species between different antibody domains.20,39–41 Therefore, we used SDS-PAGE to evaluate the formation of reducible and non-reducible crosslinked species of the control, oxidized and deamidated mAbs 9

(Figure 5). We observed that the Trp oxidized samples of both mAbs displayed HMW species (>260 kDa) for non-reducing conditions and that the level of aggregation was higher for mAb 1 than for mAb 2. Intrinsic Trp fluorescence (Figure S8) and mass spectrometry (Table 1) confirmed that the Trp oxidized samples for mAb 1 were modified to a greater extent than for mAb 2, which potentially explains the higher aggregation levels for mAb 1. Moreover, the Trp oxidized samples in the presence of reducing agent did not display crosslinked species for either mAb (Figure 5). Interestingly, the deamidated samples, especially for mAb 1, displayed nonreducible crosslinked species (~80-90 kDa). Moreover, similar behavior was observed after incubating the antibodies at pH 7.4 and an elevated temperature (37 °C) for one week (Figure S9). In addition, we find that deamidated mAb 1 monomers and HMW species – which were isolated using size-exclusion chromatography – display similar levels of non-reducible species (Figure S10). These findings reveal the conditions that promote Trp oxidation lead to the formation of reducible, disulfide-linked species while the conditions that promote deamidation lead to the formation of non-reducible crosslinked species, and that mAb 1 is more prone to the formation of non-reducible crosslinks. Another potential mechanism by which oxidation or deamidation can promote aggregation is through reduction of antibody folding stability and enhancement of antibody unfolding. To test this possibility, we evaluated the apparent melting temperature (Tm*) of the mAbs as a function of pH (Figures 6 and S11). As expected, the antibody conformational stabilities decreased for all of the samples as pH was reduced. Interestingly, the Met oxidized samples displayed significant decreases in Tm* (2.5-6.5 °C) relative to the controls for a wide range of pH values (pH 3.8-7.4). For example, the apparent melting temperatures of the Met oxidized samples were significantly lower than the untreated controls at pH 3.8 (41.7 °C relative to 47.8 °C for mAb 1 and 36.2 °C relative to 42.3 °C for mAb 2). However, the differences in melting temperature between Trp oxidized, deamidated and control (untreated) samples were much smaller (<1 °C). We also evaluated the apparent melting temperatures (Tm*) and the onset temperature of unfolding (Tonset) using DSC and observed similar trends (Figures S12 and S13). These results suggest that oxidation and deamidation of the mAbs in this study result in modest decreases in antibody folding stability and, therefore, are unlikely to promote aggregation via mechanisms that involve significant antibody unfolding. The notable exception is for Met oxidized samples at low pH (pH 3.8) and elevated temperature (37 °C), a condition at which low antibody folding stability results in high levels of aggregation (Figure 3). It is also possible that chemical modification of solvent-exposed residues leads to increased antibody selfassociation and reduced colloidal stability of natively (or near natively) folded antibodies. Therefore, we evaluated the apparent diffusion interaction parameter (kD*) of the mAbs as a function of pH (Figures 7, S14 and S15). These measurements involved evaluating the diffusion coefficient of the mAbs as a function of antibody concentration using dynamic light scattering.30 Interestingly, oxidation and deamidation had unique, antibody-specific impacts on the kD* values at low pH (pH 3.8). Deamidation led to large and significant 10

decreases in kD* values for both mAbs, which corresponds to less repulsive (or more attractive) colloidal interactions. In contrast, Met oxidized samples showed either little change (mAb 1) or a significant increase (mAb 2) in kD* values. Additionally, Trp oxidation led to significantly increased (mAb 1) or decreased (mAb 2) kD* values. At a higher pH value (pH 7.4), little dependence of kD* values on chemical modifications was observed except for deamidation of mAb 2, which resulted in decreased (more negative) kD values. These findings reveal that antibody colloidal interactions are particularly sensitive to chemical modifications at low pH. Different chemical modifications uniquely impact antibody hydrophobic interactions and solubility Chemical modifications may alter several antibody properties, including their charge, charge distribution, hydrophobicity and solubility.31,42,43 Therefore, we sought to evaluate if oxidation and deamidation adversely affected these antibody properties, and thereby, contributed to aggregation. We first evaluated the presence of acidic or basic species in the control and stressed antibodies using imaged capillary isoelectric focusing (Table 2 and Figure S16). We observed that the various chemical modifications led to increased levels of acidic species compared to the control antibody samples, while the same modifications had little impact on the levels of basic species. The deamidated samples of both mAbs displayed the highest levels of acidic species, which is expected due to the formation of negatively charged moieties following deamidation.31 Additionally, oxidation typically promotes the formation of polar moieties such as methionine sulfoxide, hydroxytryptophan and Nformylkynurenine,42 and the oxidized samples of both mAbs generally displayed increased formation of acidic species. Interestingly, Met oxidation led to minor (mAb 1) and considerable (mAb 2) increases in the formation of acidic species. Given that deamidation leads to the highest levels of acidic species while Trp oxidation generally leads to the highest levels of aggregation, it appears that formation of acidic species is not a primary determinant of the aggregation behavior displayed by these antibodies. Next, we evaluated the hydrophobicity of the antibodies using analytical hydrophobic interaction chromatography (HIC) at low pH (pH 3.8) and near neutral pH (pH 7.4; Figure 8). Interestingly, the controls displayed significant differences in elution profiles (elution using a negative gradient from 0.6 M to 0 M ammonium sulfate). mAb 1 displayed higher hydrophobicity and a more complex elution profile (two main peaks) than mAb 2 (one main peak). Moreover, the hydrophobicity of mAb 1 was much more sensitive to chemical modifications than that of mAb 2. At low pH (pH 3.8), Met and Trp oxidation of mAb 1 led to significant decreases in hydrophobicity, whereas deamidation led to little change in hydrophobicity (Figure 8A). At higher pH (pH 7.4), the mAbs generally were more hydrophobic than at low pH (Figure 8C and 8D), but the relative trends in hydrophobicities were generally similar at both pH values. The chemically modified antibodies displayed relatively high (>80%) and similar recoveries from the HIC column relative to their controls (Figure S17). We also observed similar HIC results using a broader salt gradient (from 1 to 0 M 11

ammonium sulfate; Figure S18). These findings suggest that the reduced hydrophobicity of the Met oxidized samples may be responsible for their low levels of self-association and aggregation, while the aggregation behaviors observed for the Trp oxidized and deamidated samples do not appear to be mediated by increased antibody hydrophobicity. We also evaluated the relative solubilities of the chemically modified mAbs in ammonium sulfate (Figures 9 and S18). We adapted a previously reported, high-throughput method for evaluating the relative solubility of antibodies.44 This approach involves titrating antibody solutions over a wide range of ammonium sulfate concentrations to identify those that promote antibody precipitation. As expected, we found that the solubility of all the mAb samples, as judged by turbidity, decreased as a function of ammonium sulfate concentration (Figures 9 and S19). We quantified the relative solubility, in terms of the ammonium sulfate concentration that led to half maximal values of the turbidity (CS,50), at multiple pH values (Figures 9E, 9F and S19). The Met oxidized samples generally displayed little change or increased solubility, consistent with their reduced hydrophobicity relative to the control samples as judged by HIC (Figure 8). The Trp oxidized samples displayed more complex behavior, as their solubilities increased in some cases (e.g., mAb 1 at pH 7.4) and decreased in other cases (e.g., mAb 2 at pH 7.4; Figure 9E and 9F). Moreover, the deamidated samples displayed little change in solubility. Additionally, the antibody solubilities at pH 4.5 were similar to those at pH 3.8, while the solubilities at pH 6 were similar to those at pH 7.4 (Figure S19). While there is little correlation between the solubilities of the Trp oxidized and deamidated mAbs in ammonium sulfate and their propensities to aggregate (in the absence of ammonium sulfate), the findings for the Met oxidized samples suggest that their increased solubilities in ammonium sulfate may be linked to their reduced hydrophobicities and low aggregation propensities.

DISCUSSION Our experimental findings are summarized in Table 3 and deserve further consideration. One interesting aspect of our findings is that the mechanisms by which chemical modifications lead to antibody aggregation are weakly correlated with changes in antibody conformational stability. While the simplest explanation for the impact of chemical modifications on antibody aggregation is that they lead to reduced conformational stability and thereby increased aggregation, our results reveal significantly different aggregation mechanisms. The molecular basis for our findings appears explainable (at least in part) based on the sites in the mAbs that were modified. For the Met oxidized samples, the modifications were primarily observed at sites in the Fc region that are known to reduce antibody conformational stability,9,10 as we observed in this work (Figure 6). The moderate reduction in stability is not sufficient to promote aggregation except at conditions of low pH (pH 3.8; apparent Tm reduced from 47.8 to 41.7 °C for mAb 1 and from 42.3 to 36.2 °C for mAb 2) and elevated temperature (37 °C). Under these particular conditions, the conformational stability of the Met oxidized samples (apparent Tm of 36.2-41.7 °C) is too low to prevent antibody aggregation. Conversely, the methods 12

used in this work for selective Trp oxidation primarily lead to modification of a single Trp residue in the CDRs (which is different for each mAb), and this limited modification appears to promote aggregation via mechanisms that are distinct from those linked to reduced antibody conformational stability. Likewise, Asn deamidation occurs primarily in the Fc region at the well-known Asn sites in the PENNY peptide, and the lack of conformational destabilization (as observed previously27) also suggests that deamidation promotes aggregation via mechanisms distinct from those linked to reduced conformational stability. Therefore, the aggregation mechanisms for the mAbs subject to Trp oxidation and Asn deamidation – which cannot be easily explained by impacts of the chemical modifications on conformational stability – also deserve further consideration. We find that Trp oxidation leads to formation of reducible crosslinks, which are presumably disulfide bonds and may form due to disulfide scrambling. One potential mechanism for this phenomenon is that chemical oxidation of Trp can generate Trp radicals with delocalized electrons, and such delocalized electrons can in turn reduce disulfide bonds45,46 and lead to disulfide scrambling. Another potential mechanism by which Trp oxidation may lead to disulfide scrambling is through partial unfolding at or near Trp oxidation sites, which in turn may lead to the exposure of buried cysteines and thereby increase the likelihood of disulfide scrambling.47 Indeed, our mass spectrometry results are potentially consistent with this mechanism because mAb 1 displays higher levels of Trp di-oxidation – which can disrupt the Trp indole ring and cause local structural changes through formation of N-formylkynurenine – and this may lead to greater amounts of reducible aggregates for mAb 1 relative to mAb 2. It is also notable that previous studies of photooxidation of antibodies found that disulfide bond shuffling can occur due to oxidation of Trp residues located in close proximity to intramolecular disulfide bonds through electron transfer or singlet oxygen pathways, and such disulfide scrambling also promotes antibody aggregation.20,21,48 However, whether similar or unique mechanisms mediate disulfide scrambling for chemical oxidation and photo-oxidation of antibodies is unclear and requires more detailed studies in the future. It is also important to consider that the aggregation behavior of mAbs is strongly influenced by the type of stress and the specific formulation conditions. In particular, sucrose and other sugars have been shown to improve the conformational stability of proteins and antibodies through the excluded volume effect and thereby reduce the rate of aggregation.49–51 In the case of photodegraded mAbs, sucrose was also observed to reduce the rate of aggregation, although it had little effect on the rate of Met oxidation.41 Various observations have been reported for the effects of sucrose on the oxidation of other proteins, including slight protection for interleukin-7 Fc fusion protein52 and subtilisin,53 no effect for tumor necrosis factor receptor 1,54 and slight worsening for granulocyte colony-stimulating factor55 and recombinant activated factor VII.51 Moreover, sucrose has been shown to decrease the rate of Asn deamidation (or formation of acidic species) for mAbs56 and acrylodan-conjugated glucose-binding protein.50 Whether the addition of excipients such as sucrose or

13

salts would differentially modify the aggregation propensity of oxidized and deamidated mAbs requires additional studies in the future. The formation of non-reducible crosslinks during antibody storage at high pH (pH 9.5) to promote Asn deamidation also deserves further consideration. The fact that these crosslinks (which primarily form for mAb 1) are non-reducible suggests mechanisms other than disulfide scrambling, although cases of the formation of reduction-resistant disulfide bonds through disulfide scrambling have been reported.20 It is notable that previous studies have identified similar types of non-reducible crosslinks for mAbs incubated at similar conditions as those used in our study for promoting deamidation, namely elevated temperature (e.g., 37-45 °C) and high pH (pH 8-10).40,57–60 Moreover, the observed degree of crosslink formation was significantly different for different antibodies,57 as we observed in our study. One potential mechanism for the generation of nonreducible species involving antibody heavy and light chains is thioether bond formation in the hinge region. Previous studies have shown that disulfide bonds at high pH can decompose via a β-elimination reaction,61–63 which leads to intermediates that can form non-reducible thioether bonds between modified cysteines in the CH1 and CL domains.39,59 This mechanism has been reported for IgG1s,39 and the IgG1 antibody in our study (mAb 1) shows much higher levels of such non-reducible crosslinks between heavy and light chain than the IgG4 antibody in our study (mAb 2). We speculate that the different disulfide bonding patterns between CH1 and CL in IgG1 and IgG4 antibodies result in different susceptibilities to thioether bond formation, which will need to be tested using additional antibodies in the future. Another interesting aspect of our findings is the HIC retention behavior of the oxidized mAbs. Previous studies have also found that Met and Trp oxidation lead to the formation of species with reduced hydrophobicity,43 as expected based on the nature of the modifications.64,65 While we found that Met and Trp oxidation led to significantly reduced HIC retention times for mAb 1, these modifications had little impact on the HIC behavior for mAb 2. This observation suggests that local chemical or structural changes, in addition to changes in global antibody properties, caused by these chemical modifications may induce changes in the surface hydrophobicities of the mAbs in different ways and lead to distinct HIC retention behaviors.66 For Met oxidation, our mass spectrometry analysis revealed that the oxidation levels of the two mAbs were similar in the Fc regions but markedly different in the Fab regions (Table 1), which suggests that the Fab region is primarily responsible for the observed differences in hydrophobicity for the Met oxidized samples. For Trp oxidation, the oxidized samples for the two mAbs display markedly different HIC retention behavior despite the fact that both mAbs primarily undergo Trp oxidation primarily at one site in the CDRs, which is different for each mAb. One likely explanation for this behavior is that mAb 1 displays much higher levels of Trp dioxidation (N-formylkynurenine or dihydroxytryptophan) relative to mAb 2, which is expected to be more hydrophilic than Trp mono-oxidation (hydroxytryptophan) that was primarily observed for mAb 2. These speculative ideas require additional experimentation to be properly evaluated. 14

The HIC retention behavior of the deamidated mAbs also deserves further consideration. Previous studies have found that deamidation can lead to increased or decreased HIC retention times depending on the formation of succinimide67–71 or aspartic acid (and isoaspartic acid),43,70–73 respectively. The degradation condition (high pH) used in this study to generate the deamidated mAbs typically leads to the formation of aspartic acid and isoaspartic acid due to the rapid hydrolysis of the succinimide intermediate formed during deamidation of Asn at basic pH.67,70,74 The fact that we observed little impact of deamidation on the HIC retention behavior may be linked to the low levels of deamidation observed in the Fab region for both mAbs (Table 1). Furthermore, the fact that our HIC retention behavior did not correlate well with the observed aggregation propensity of the chemically modified mAbs is consistent with previous studies.75,76 The disconnect between HIC measurements and aggregation behavior suggests that hydrophobic interactions are not a primary determinant of aggregation for the mAbs in our study and that other mechanisms (e.g., covalent crosslinking and colloidal instability) play a more significant role in mediating aggregation.

CONCLUSIONS The impact of chemical modifications on antibody physical stability can be variable and difficult to predict. In this work, we found that antibody conformational stability is weakly correlated with aggregation except in extreme cases and that aggregation can occur via multiple mechanisms such as chemical crosslinking and reduced colloidal stability. Our findings underscore the importance of evaluating the physical stability of antibodies at different conditions relevant to antibody manufacture, purification and formulation to understand the effects of chemical modifications on aggregation as well as evaluating a combination of properties (e.g., crosslinking, charge, polarity, hydrophobicity and solubility) to elucidate the mechanisms of antibody aggregation. Future work should evaluate the generality of our findings by expanding these studies to include additional antibodies. We expect these studies will be important for improving the generation and production of therapeutic antibodies with high chemical and physical stability.

ACKNOWLEDGEMENTS We thank members of the Tessier lab for their assistance editing the manuscript. We would also like to thank Pritesh Patel and Shannon Flagg for providing the iCIEF data. In addition, we thank Helen Zha for allowing us to use her DSC calorimeter. This work was supported by Bristol-Myers Squibb and the Albert M. Mattocks Chair (to P.M.T.). CONFLICTS OF INTEREST None.

15

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11.

12. 13. 14. 15. 16. 17.

18.

19.

20.

21.

22.

23.

Ecker DM, Jones SD, Levine HL. The therapeutic monoclonal antibody market. mAbs. 2015;7(1):9-14. Grilo AL, Mantalaris A. The Increasingly Human and Profitable Monoclonal Antibody Market. Trends Biotechnol. 2019;37(1):9-16. Nelson AL, Dhimolea E, Reichert JM. Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov. 2010;9:767. Tiller KE, Tessier PM. Advances in Antibody Design. Annu Rev Biomed Eng. 2015;17(1):191-216. Maynard J, Georgiou G. Antibody Engineering. Annu Rev Biomed Eng. 2000;2(1):339-376. Carter PJ. Potent antibody therapeutics by design. Nat Rev Immunol. 2006;6(5):343-357. Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR. Making Antibodies by Phage Display Technology. Annu Rev Immunol. 1994;12(1):433-455. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD. Isolating and engineering human antibodies using yeast surface display. Nat Protoc. 2006;1(2):755-768. Liu D, Ren D, Huang H, et al. Structure and Stability Changes of Human IgG1 Fc as a Consequence of Methionine Oxidation. Biochemistry. 2008;47(18):5088-5100. Chumsae C, Gaza-Bulseco G, Sun J, Liu H. Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody. J Chromatogr B. 2007;850:285-294. Wang S, Ionescu R, Peekhaus N, Leung J, Ha S, Vlasak J. Separation of post-translational modifications in monoclonal antibodies by exploiting subtle conformational changes under mildly acidic conditions. J Chromatogr A. 2010;1217(42):6496-6502. Liu H, Gaza-Bulseco G, Xiang T, Chumsae C. Structural effect of deglycosylation and methionine oxidation on a recombinant monoclonal antibody. Mol Immunol. 2008;45(3):701-708. Burkitt W, Domann P, O’Connor G. Conformational changes in oxidatively stressed monoclonal antibodies studied by hydrogen exchange mass spectrometry. Protein Sci. 2010;19(4):826-835. Houde D, Peng Y, Berkowitz SA, Engen JR. Post-translational Modifications Differentially Affect IgG1 Conformation and Receptor Binding. Mol Cell Proteomics. 2010;9(8):1716-1728. Pan H, Chen K, Chu L, Kinderman F, Apostol I, Huang G. Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn. Protein Sci. 2009;18(2):424-433. Bertolotti-Ciarlet A, Wang W, Lownes R, et al. Impact of methionine oxidation on the binding of human IgG1 to FcRn and Fcγ receptors. Mol Immunol. 2009;46(8-9):1878-1882. Hensel M, Steurer R, Fichtl J, et al. Identification of Potential Sites for Tryptophan Oxidation in Recombinant Antibodies Using tert-Butylhydroperoxide and Quantitative LC-MS. PLoS One. 2011;6(3):e17708. Wei Z, Feng J, Lin H-Y, et al. Identification of a Single Tryptophan Residue as Critical for Binding Activity in a Humanized Monoclonal Antibody against Respiratory Syncytial Virus. Anal Chem. 2007;79(7):2797-2805. Hageman T, Wei H, Kuehne P, et al. Impact of Tryptophan Oxidation in ComplementarityDetermining Regions of Two Monoclonal Antibodies on Structure-Function Characterized by Hydrogen-Deuterium Exchange Mass Spectrometry and Surface Plasmon Resonance. Pharm Res. 2018;36(1):24. Wecksler AT, Yin J, Lee Tao P, Kabakoff B, Sreedhara A, Deperalta G. Photodisruption of the Structurally Conserved Cys-Cys-Trp Triads Leads to Reduction-Resistant Scrambled Intrachain Disulfides in an IgG1 Monoclonal Antibody. Mol Pharm. 2018;15(4):1598-1606. Illyés E, Staelens S, Vanhooren A, Deckmyn H, Hanssens I, Majer Z. Role of the Trp-disulfide Triads in the UV Light Induced Degradation of a Monoclonal Antibody scFv. Int J Biochem Res Rev. 2014;4:367-385. Luo Q, Joubert MK, Stevenson R, Ketchem RR, Narhi LO, Wypych J. Chemical Modifications in Therapeutic Protein Aggregates Generated under Different Stress Conditions. J Biol Chem. 2011;286(28):25134-25144. Joubert MK, Luo Q, Nashed-Samuel Y, Wypych J, Narhi LO. Classification and Characterization of Therapeutic Antibody Aggregates. J Biol Chem. 2011;286(28):25118-25133. 16

24. 25. 26. 27.

28. 29. 30. 31. 32. 33. 34. 35.

36. 37. 38.

39. 40. 41. 42. 43. 44.

45.

46. 47.

Vlasak J, Bussat MC, Wang S, et al. Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody. Anal Biochem. 2009;392(2):145-154. Yan B, Steen S, Hambly D, et al. Succinimide formation at Asn 55 in the complementarity determining region of a recombinant monoclonal antibody IgG1 heavy chain. J Pharm Sci. 2009;98(10):3509-3521. Qiu H, Wei R, Jaworski J, et al. Engineering an anti-CD52 antibody for enhanced deamidation stability. mAbs. June 2019:1-10. Alam ME, Barnett G V, Slaney TR, Starr CG, Das TK, Tessier PM. Deamidation Can Compromise Antibody Colloidal Stability and Enhance Aggregation in a pH-Dependent Manner. Mol Pharm. 2019;16(5):1939-1949. Tang L, Sundaram S, Zhang J, et al. Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry. mAbs. 2013;5(1):114-125. Menzen T, Friess W. High-Throughput Melting-Temperature Analysis of a Monoclonal Antibody by Differential Scanning Fluorimetry in the Presence of Surfactants. J Pharm Sci. 2013;102(2):415-428. Nicoud L, Lattuada M, Yates A, Morbidelli M. Impact of aggregate formation on the viscosity of protein solutions. Soft Matter. 2015;11(27):5513-5522. Pace AL, Wong RL, Zhang YT, Kao Y-H, Wang YJ. Asparagine Deamidation Dependence on Buffer Type, pH, and Temperature. J Pharm Sci. 2013;102(6):1712-1723. Yan Q, Huang M, Lewis MJ, Hu P. Structure Based Prediction of Asparagine Deamidation Propensity in Monoclonal Antibodies. mAbs. 2018;10(6):901-912. Shukla AA, Hubbard B, Tressel T, Guhan S, Low D. Downstream processing of monoclonal antibodies—Application of platform approaches. J Chromatogr B. 2007;848:28-39. Shukla AA, Thömmes J. Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends Biotechnol. 2010;28(5):253-261. Mazzer AR, Perraud X, Halley J, O’Hara J, Bracewell DG. Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold. J Chromatogr A. 2015;1415:83-90. Low D, O ’leary R, Pujar NS. Future of antibody purification. J Chromatogr B. 2007;848:48-63. Hober S, Nord K, Linhult M. Protein A chromatography for antibody purification. J Chromatogr B. 2007;848:40-47. Nobbmann U, Connah M, Fish B, et al. Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies. Biotechnol Genet Eng Rev. 2007;24(1):117128. Tous GI, Wei Z, Feng J, et al. Characterization of a Novel Modification to Monoclonal Antibodies: Thioether Cross-link of Heavy and Light Chains. Anal Chem. 2005;77(9):2675-2682. Kroon DJ, Baldwin-Ferro A, Lalan P. Identification of sites of degradation in a therapeutic monoclonal antibody by peptide mapping. Pharm Res. 1992;9(11):1386-1393. Shah DD, Zhang J, Maity H, Mallela KMG. Effect of photo-degradation on the structure, stability, aggregation, and function of an IgG1 monoclonal antibody. Int J Pharm. 2018;547(1-2):438-449. Torosantucci R, Schöneich C, Jiskoot W. Oxidation of Therapeutic Proteins and Peptides: Structural and Biological Consequences. Pharm Res. 2014;31(3):541-553. Boyd D, Kaschak T, Yan B. HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region. J Chromatogr B. 2011;879(13):955-960. Yamniuk AP, Ditto N, Patel M, et al. Application of a Kosmotrope-Based Solubility Assay to Multiple Protein Therapeutic Classes Indicates Broad Use as a High-Throughput Screen for Protein Therapeutic Aggregation Propensity. J Pharm Sci. 2013;102(8):2424-2439. Arenas A, López-Alarcón C, Kogan M, Lissi E, Davies MJ, Silva E. Chemical Modification of Lysozyme, Glucose 6-Phosphate Dehydrogenase, and Bovine Eye Lens Proteins Induced by Peroxyl Radicals: Role of Oxidizable Amino Acid Residues. Chem Res Toxicol. 2013;26(1):67-77. Hawkins CL, Davies MJ. Generation and propagation of radical reactions on proteins. Biochim Biophys Acta - Bioenerg. 2001;1504(2):196-219. Brych SR, Gokarn YR, Hultgen H, Stevenson RJ, Rajan R, Matsumura M. Characterization of antibody aggregation: Role of buried, unpaired cysteines in particle formation. J Pharm Sci. 2010;99(2):76417

48.

49. 50.

51. 52. 53.

54. 55.

56.

57. 58. 59. 60. 61. 62. 63. 64. 65.

66.

67. 68. 69.

781. Vanhooren A, Devreese B, Vanhee K, Van Beeumen J, Hanssens I. Photoexcitation of Tryptophan Groups Induces Reduction of Two Disulfide Bonds in Goat α-Lactalbumin. Biochemistry. 2002;41(36):11035-11043. Meyer JD, Nayar R, Manning MC. Impact of bulking agents on the stability of a lyophilized monoclonal antibody. Eur J Pharm Sci. 2009;38(1):29-38. Sahni N, Chaudhuri R, Hickey JM, et al. Preformulation Characterization, Stabilization, and Formulation Design for the Acrylodan-Labeled Glucose-Binding Protein SM4-AC. J Pharm Sci. 2017;106(5):1197-1210. Soenderkaer S, Carpenter JF, van de Weert M, Hansen LL, Flink J, Frokjaer S. Effects of sucrose on rFVIIa aggregation and methionine oxidation. Eur J Pharm Sci. 2004;21(5):597-606. Lim JY, Kim NA, Lim DG, Eun C, Choi D, Jeong SH. Biophysical stability of hyFc fusion protein with regards to buffers and various excipients. Int J Biol Macromol. 2016;86:622-629. DePaz RA, Barnett CC, Dale DA, Carpenter JF, Gaertner AL, Randolph TW. The Excluding Effects of Sucrose on a Protein Chemical Degradation Pathway: Methionine Oxidation in Subtilisin. Arch Biochem Biophys. 2000;384(1):123-132. Roy S, Mason BD, Schöneich CS, Carpenter JF, Boone TC, Kerwin BA. Light-induced aggregation of type I soluble tumor necrosis factor receptor. J Pharm Sci. 2009;98(9):3182-3199. Yin J, Chu J-W, Ricci MS, Brems DN, Wang DIC, Trout BL. Effects of Excipients on the Hydrogen Peroxide-Induced Oxidation of Methionine Residues in Granulocyte Colony-Stimulating Factor. Pharm Res. 2005;22(1):141-147. Alekseychyk L, Su C, Becker GW, Treuheit MJ, Razinkov VI. High-Throughput Screening and Analysis of Charge Variants of Monoclonal Antibodies in Multiple Formulations. SLAS Discov. 2017;22(8):1044-1052. Jiskoot W, Beuvery EC, de Koning AAM, Herron JN, Crommelin DJA. Analytical Approaches to the Study of Monoclonal Antibody Stability. Pharm Res. 1990;07(12):1234-1241. Zhang Q, Schenauer MR, McCarter JD, Flynn GC. IgG1 thioether bond formation in vivo. J Biol Chem. 2013;288(23):16371-16382. Cohen SL, Price C, Vlasak J. β-elimination and peptide bond hydrolysis: Two distinct mechanisms of human IgG1 hinge fragmentation upon storage. J Am Chem Soc. 2007;129(22):6976-6977. Zhang Q, Flynn GC. Cysteine Racemization on IgG Heavy and Light Chains. J Biol Chem. 2013;288(48):34325-34335. Nashef AS, Osuga DT, Lee HS, Ahmed AI, Whitaker JR, Feeney RE. Effects of alkali on proteins. Disulfides and their products. J Agric Food Chem. 1977;25(2):245-251. Florence TM. Degradation of protein disulphide bonds in dilute alkali. Biochem J. 1980;189(3):507520. Galande AK, Trent JO, Spatola AF. Understanding base-assisted desulfurization using a variety of disulfide-bridged peptides. Pept Sci. 2003;71(5):534-551. Robinson JR, Karkov HS, Woo JA, Krogh BO, Cramer SM. QSAR models for prediction of chromatographic behavior of homologous Fab variants. Biotechnol Bioeng. 2017;114(6):1231-1240. Jetha A, Thorsteinson N, Jmeian Y, Jeganathan A, Giblin P, Fransson J. Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies. mAbs. 2018;10(6):890-900. King C, Patel R, Ponniah G, et al. Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis. J Chromatogr B. 2018;1085:96-103. Ouellette D, Chumsae C, Clabbers A, Radziejewski C, Correia I. Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule. mAbs. 2013. Kwong MY, Harris RJ. Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage. Protein Sci. 1994;3(1):147-149. Valliere-Douglass J, Wallace A, Balland A. Separation of populations of antibody variants by fine tuning of hydrophobic-interaction chromatography operating conditions. J Chromatogr A. 18

70.

71.

72.

73. 74. 75. 76.

2008;1214:81-89. Cao M, Mulagapati SHR, Vemulapalli B, et al. Characterization and quantification of succinimide using peptide mapping under low-pH conditions and hydrophobic interaction chromatography. Anal Biochem. 2019;566:151-159. Valliere-Douglass J, Jones L, Shpektor D, et al. Separation and Characterization of an IgG2 Antibody Containing a Cyclic Imide in CDR1 of Light Chain by Hydrophobic Interaction Chromatography and Mass Spectrometry. Biotechnol Appl Biochem. 2002;18(2):8. Wakankar AA, Borchardt RT, Eigenbrot C, et al. Aspartate isomerization in the complementaritydetermining regions of two closely related monoclonal antibodies. Biochemistry. 2007;46(6):15341544. Harris RJ, Kabakoff B, Macchi FD, et al. Identification of multiple sources of charge heterogeneity in a recombinant antibody. J Chromatogr B. 2001;752(2):233-245. Sreedhara A, Cordoba A, Zhu Q, Kwong J, Liu J. Characterization of the Isomerization Products of Aspartate Residues at Two Different Sites in a Monoclonal Antibody. Pharm Res. 2012;29(1):187-197. Shieh IC, Patel AR. Predicting the Agitation-Induced Aggregation of Monoclonal Antibodies Using Surface Tensiometry. Mol Pharm. 2015;12(9):3184-3193. Jain T, Sun T, Durand S, et al. Biophysical properties of the clinical-stage antibody landscape. Proc Natl Acad Sci. 2017;114(5):944-949.

19

Table 1. Summary of the oxidation and deamidation levels of the antibodies evaluated in this study. Mass spectrometry analysis of the control (Con.), Methionine (Met) oxidized (H2O2 treated), Tryptophan (Trp) oxidized (AAPH + Met treated) and deamidated (high pH treated) mAbs was performed to evaluate the level of chemical modification for each sample. The data are the average (%) levels of chemical modification observed for Met, Trp and Asn residues located in the Fab or Fc region. mAb 1 modifications were detected for two Met residues (including one in the CDRs), one Trp residue (in the CDRs) and four Asn residues (including two in the CDRs) in the Fab region, as well as for three Met and three Asn residues in the Fc region. mAb 2 modifications were detected for one Met residue, one Trp residue (in the CDRs) and one Asn residue (in the CDRs) in the Fab region, and for three Met and four Asn residues in the Fc region.

Methionine residues (% modified)

mAb 1 mAb 2

Tryptophan residues (% modified)

Asparagine residues (% modified)

Con.

H2O2

AAPH + Met

High pH

Con.

H2O2

AAPH + Met

High pH

Con.

H2O2

AAPH + Met

High pH

Fab

10

91

8.7

19

<1

1.8

87

<1

<1

<1

<1

3.6

Fc Fab Fc

3.1

51

4.6

7.8

-

-

-

-

2.1

2.0

2.5

18

<1

12

1.6

<1

<1

<1

29

<1

<1

<1

<1

1.1

<1

51

3.4

2.3

-

-

-

-

2.8

2.5

2.0

20

Table 2. Summary of the levels of acidic and basic species of the antibodies evaluated in this study. Imaged capillary isoelectric focusing (iCIEF) was performed on the control, Met oxidized (H2O2 treated), Trp oxidized (AAPH + Met treated) and deamidated (high pH treated) mAbs to determine the level of charged species for each sample. The main peak represents unmodified antibody, and the acidic and basic species represent charged species. The data are for one to three replicates.

mAb 1

mAb 2

Acidic species (%)

Main species (%)

Basic species (%)

Control

41.5

52.3

6.2

H2O2 AAPH + Met High pH

43.4

50.4

6.3

61.5

29.7

8.9

91.2

6.9

1.9

Control

28.7

63.9

7.4

H2O2 AAPH + Met High pH

48.0

46.0

6.1

45.5

47.1

7.5

61.7

34.5

3.8

Table 3. Summary of the experimental analysis of the effects of chemical modifications on various antibody biophysical properties. The observed differences between the Met oxidized, Trp oxidized, or Asn deamidated mAbs relative to the control mAbs at pH 3.8 and pH 7.4 are summarized for mAbs 1 and 2. A plus sign (+) denotes an increase in the measured value of a particular biophysical property relative to the control mAb (e.g., wild-type mAb 1), while a minus sign (‒) denotes a decrease. More than one plus or minus sign indicate larger changes. Additional notations in the table include no change (NC) and not evaluated (x).

Met oxidation pH 3.8

mAb 1

pH 3.8

pH 7.4

Asn deamidation pH 3.8 **

pH 7.4

Aggregation (SEC, %HMW)

NC

NC

++

+++

Aggregation (DLS, HMW species)

NC

NC

+++

++

+++

NC

Accelerated Stability (37 °C, %HMW)

+++

+

‒

+

+++

+

Reducible HMW Species (SDS-PAGE)

×

NC

×

+++

×

NC

Nonreducible Species (SDS-PAGE)

×

NC

×

NC

×

+++

Conformational Stability (DSF, Tm )

‒‒

‒‒

NC

NC

NC

NC

Colloidal Stability (kD* )

NC

NC

++

‒

‒‒

NC

Hydrophobicity (HIC, retention time)

‒‒‒

‒‒‒

‒‒

‒‒‒

NC

NC

Solubility (ammonium sulfate, CS,50)

NC

+++

NC

++

NC

NC

*

mAb 2

pH 7.4

Trp oxidation

+

+++

**

+++

Aggregation (SEC, %HMW)

NC

NC

++

+++

Aggregation (DLS, HMW species)

NC

NC

NC

NC

+++

+++

Accelerated Stability (37 °C, %HMW)

+++

NC

NC

++

+++

+

Reducible HMW Species (SDS-PAGE)

×

NC

×

++

×

NC

Nonreducible Species (SDS-PAGE)

×

NC

×

NC

×

+

‒‒

‒‒‒

NC

‒‒

NC

NC

Colloidal Stability (kD )

+

NC

‒

NC

‒‒‒

‒

Hydrophobicity (HIC, retention time)

‒

‒

+

NC

NC

NC

+

+++

‒‒

‒‒

‒

+

*

Conformational Stability (DSF, Tm ) *

Solubility (ammonium sulfate, CS,50) **

= at high concentration only

× = not evaluated

++

NC = no change

Figure Legend Figure 1. Size-exclusion chromatography analysis of the impact of oxidation and deamidation on antibody aggregation as a function of pH. (A-D) The antibodies were prepared at 1 and 25 mg/mL in citrate-phosphate (15 mM citrate, 10 mM phosphate) buffer at (A, B) pH 3.8 and (C, D) pH 7.4, and injected into the column without extended storage (injected <2 h after preparation). The data shown are averages of three independent experiments and the error bars are standard errors. A two-tailed Student’s ttest was used to judge the statistical significance of the difference between the chemically modified and control mAbs [p-values <0.05 (*), <0.01 (**) or <0.001 (***)]. Figure 2. Dynamic light scattering analysis of the impact of oxidation and deamidation on antibody aggregation as a function of pH. (A-D) The antibodies were prepared at 25 mg/mL in citrate-phosphate buffer at (A, B) pH 3.8 and (C, D) pH 7.4, and their size distributions were measured using dynamic light scattering. The data shown are representative examples of three independent experiments. Figure 3. Size-exclusion chromatography analysis of the impact of oxidation and deamidation on antibody aggregation as a function of temperature and incubation time at pH 3.8. (A-D) The antibodies were prepared at 1 mg/mL in citrate-phosphate buffer (pH 3.8) and incubated at (A, B) 37 or (C, D) 25 °C for seven days prior to analysis by size-exclusion chromatography. The data shown are averages of three independent experiments and the error bars are standard errors. Figure 4. Size-exclusion chromatography analysis of the impact of oxidation and deamidation on antibody aggregation as a function of temperature and incubation time at pH 7.4. (A-D) The antibodies were prepared at 1 mg/mL in citrate-phosphate buffer (pH 7.4) and incubated at (A, B) 37 or (C, D) 25 °C for 28 days prior to analysis by size-exclusion chromatography. The data shown are averages of three independent experiments and the error bars are standard errors. Figure 5. SDS-PAGE analysis of the impact of oxidation and deamidation on the formation of reducible and nonreducible crosslinks. (A, B) The antibody samples for (A) mAb 1 and (B) mAb 2 at pH 7.4 in citrate-phosphate buffer were analyzed using SDS-PAGE without extended storage (<2 h after preparation). The gels shown are representative examples of three independent experiments. Figure 6. Evaluation of the impact of oxidation and deamidation on antibody conformational stability as a function of pH. (A, B) The apparent melting temperatures (Tm*) for (A) mAb 1 and (B) mAb 2 in citrate-phosphate buffer as a function of pH. An extrinsic dye (Protein Thermal Shift Dye) was used to obtain the apparent melting temperatures (first unfolding transition) using differential scanning fluorimetry. The data shown are averages of three independent experiments and the error bars are standard errors. A two-tailed Student’s t-test was used to judge the statistical significance of the difference between the chemically modified and control mAbs [p-values <0.05 (*), <0.01 (**) or <0.001 (***)]. Figure 7. Evaluation of the impact of oxidation and deamidation on antibody colloidal interactions as a function of pH. (A, B) The apparent diffusion interaction parameters (kD*) were evaluated using dynamic light scattering for (A) mAb 1 and (B) mAb 2 as a function of pH. The experiments were performed in citrate-phosphate buffer at the reported pH values. The data shown are averages of three independent experiments and the error bars are standard errors. A two-tailed Student’s t-test was used to judge the statistical significance of the difference between the chemically modified and control mAbs [pvalues <0.05 (*), <0.01 (**) or <0.001 (***)].

Figure 8. Evaluation of the impact of oxidation and deamidation on antibody hydrophobicity as a function of pH. (A-D) The antibodies were prepared at 1 mg/mL in citrate-phosphate buffer with 0.6 M ammonium sulfate at (A, B) pH 3.8 and (C, D) pH 7.4, and injected into the column without extended incubation (<2 h after preparation). The column was equilibrated at the same conditions as the antibody samples, and then a gradient of ammonium sulfate (0.6 to 0 M) was used to elute the antibodies. The chromatograms shown are representative examples of three independent experiments. Figure 9. Evaluation of the impact of oxidation and deamidation on antibody solubility in the presence of ammonium sulfate as a function of pH. (A-D) The antibodies were evaluated at 0.2 mg/mL in citrate-phosphate buffer at (A, B) pH 3.8 and (C, D) pH 7.4 as a function of ammonium sulfate concentration. The turbidity values were measured and normalized using the maximum and minimum turbidity values for a given set of samples (i.e., samples for the same mAb and pH). (E-F) The ammonium sulfate midpoints of transition (CS,50) for each mAb and pH condition. The data shown are averages of four independent experiments and the error bars are standard errors. A two-tailed Student’s t-test was used to judge the statistical significance of the difference between the chemically modified and control mAbs [p-values <0.05 (*), <0.01 (**) or 0.001 (***)].