Journal of Pharmaceutical Sciences 109 (2020) 656-669

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Journal of Pharmaceutical Sciences

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Pharmaceutical Biotechnology

Unique Impacts of Methionine Oxidation, Tryptophan Oxidation,

and Asparagine Deamidation on Antibody Stability and
Aggregation
Magfur E. Alam 1, Thomas R. Slaney 2, Lina Wu 3, Tapan K. Das 2, Sambit Kar 2,
Gregory V. Barnett 2, Anthony Leone 2, Peter M. Tessier 1, 3, 4, 5, *
1
Isermann Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute,
Troy, New York 12180
2
Biologics Development, Bristol-Myers Squibb, Pennington, New Jersey 08534
3
Department of Chemical Engineering, Biointerfaces Institute, University of Michigan, Ann Arbor, Michigan 48109
4
Department of Pharmaceutical Sciences, Biointerfaces Institute, University of Michigan, Ann Arbor, Michigan 48109
5
Department of Biomedical Engineering, Biointerfaces Institute, University of Michigan, Ann Arbor, Michigan 48109

a r t i c l e i n f o a b s t r a c t

Article history: Monoclonal antibodies are attractive therapeutic agents because of their impressive biological activities
Received 16 August 2019 and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of
Revised 22 October 2019 chemical modifications, and the impact of such modifications on antibody physical stability and ag-
Accepted 28 October 2019
gregation remains understudied. Here, we report a systematic analysis of the impact of methionine
Available online 31 October 2019
oxidation, tryptophan oxidation, and asparagine deamidation on antibody conformational and colloidal
stability, hydrophobicity, solubility, and aggregation. Interestingly, we find little correlation between the
Keywords:
mAb impact of these chemical modifications on antibody conformational stability and aggregation. Methio-
solubility nine oxidation leads to significant reductions in antibody conformational stability while having little
formulation impact on antibody aggregation except at extreme conditions (low pH and elevated temperature).
developability
Conversely, tryptophan oxidation and asparagine deamidation have little impact on antibody confor-
protein aggregation
mational stability while promoting aggregation at a wide range of solution conditions, and the aggre-
gation mechanisms appear linked to unique types of reducible and nonreducible 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.
© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction their long circulation times and favorable biophysical properties.4-6

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 candi-
dates.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
Conflicts of interest: None.
This article contains supplementary material available from the authors by request
or via the Internet at https://doi.org/10.1016/j.xphs.2019.10.051.
* Correspondence to: Peter M. Tessier (Telephone: 734.763.1486).
E-mail address: ptessier@umich.edu (P.M. Tessier).
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 anti-
bodies 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

https://doi.org/10.1016/j.xphs.2019.10.051
0022-3549/© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669
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 disul-
fide scrambling20,21 and aggregation.17,22,23 Asparagine (Asn) dea-
midation 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 pro-
moting 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 vs. Trp) and
different classes (oxidation vs. deamidation)? Here we systemati-
cally 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
C-37
C), and report unique
aggregation mechanisms for each type of chemical modification.
Experimental Methods
Materials
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 pro-
vided 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 poly-
styrene inserts (13622207) were obtained from Thermo Fisher
Scientific. Individually packaged cuvettes (95201005) were ob-
tained 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 ob-
tained from ProteinSimple. Amicon 30 kDa centrifugal filters
(UFC503096) were obtained from EMD Millipore. The 2 Laemmli
buffer (1610737) and 4%-15% mini-PROTEAN TGX gels (4561086)
were obtained from Biorad. b-Mercaptoethanol (200000622) was
obtained from VWR. Gelcode Blue Safe Protein Stain (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
657
untreated (control) mAb solutions were buffer-exchanged into PBS
at pH 7.4 using Zeba Spin Desalting Columns (PI89894; Thermo
Fisher Scientific), aliquoted (~0.2-0.5 mL) and frozen at 80
C until
evaluation.
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
day. Afterward, a 3-fold molar excess of a Met solution (200 mM)
was added to quench the oxidation reaction. Next, the mAb solu-
tions were buffer-exchanged into PBS at pH 7.4 using Zeba Spin
Desalting Columns, aliquoted and frozen at 80
C until evaluation.
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 so-
lution to protect the Met residues in the mAbs from oxidation. The
solutions were then mixed gently and incubated at 37
C for 3 days.
At the end of the incubation period, the mAb solutions were
enriched for monomer using a semipreparative SEC column
€
running on an AKTA Avant 25. The enriched monomer samples
were buffer-exchanged into PBS at pH 7.4 using Zeba Spin Desalting
Columns, aliquoted and frozen at 80
C until evaluation.
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 1 week.
Afterward, the mAb solutions were buffer-exchanged into PBS at
pH 7.4 using Zeba Spin Desalting Columns, aliquoted and frozen
at 80
C until evaluation.
For the accelerated stability study, the mAb samples were pre-
pared 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
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 (Pro-
teinSimple), 8 M urea (Sigma Aldrich), and appropriate pI markers
(ProteinSimple). Next, the diluted samples were injected into an
iCE3 (ProteinSimple) system equipped with a fluorocarbon-coated
capillary cartridge. Each sample was prefocused 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  15 cm, Tosoh Bioscience)
and a Waters 6000 HPLC. The column was washed with 4 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 mL 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 soft-
ware (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 mL) were taken
at 0, 1, 7, and 28 days, and 10 mL of each mAb solution was injected
658 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669
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 previously. The total area under the curve for the entire
chromatogram was also evaluated at each time point to determine
the percentage antibody recovery.
SDS-PAGE
The mAb samples were diluted to 2 mg/mL (1 mg/mL for the
accelerated stability samples and 40 mg/mL for the deamidated
[monomer and HMW] samples purified by size-exclusion chro-
matography) in citrate-phosphate buffer at pH 7.4. Next, equal
volumes of the mAb solutions and 2 Laemmli buffer, prepared
with and without reducing agent (b-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 16,000 rcf for 2 min, and 10 mL (15 mL
for the accelerated stability samples and 5 mL 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
Stain or Pierce Silver Stain (1860983 or 24612, Thermo Fisher Sci-
entific) and then destained 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 Bio-
sciences), initially provided at a concentration of 1000, was
diluted to a concentration of 40 using Milli-Q water. The mAb
solutions (17.5 mL in each well) were dispensed into 96-well white
PCR plates (04729692001, Roche) in triplicate. Next, dye solution
(2.5 mL) 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
C 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 deriv-
ative 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 bi-Gaussian 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 mL) were loaded into the sample cell and the
corresponding buffers (~450 mL) were loaded into the reference cell
of a Nano DSC calorimeter (TA Instruments). Next, the cells were
heated (0.7
C/min) from 25
C to 95
C for the samples prepared at
pH 7.4 and from 15
C 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 thermo-
grams (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
Dynamic Light Scattering
Light scattering experiments were performed as described
previously.27 Briefly, each mAb sample was buffer-exchanged 4
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 and filtered using 0.22 mm syringe filters (09-720-3, Thermo
Fisher Scientific). Next, 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 then diluted to
a concentration of 25 mg/mL. The concentrated mAbs (80 mL) were
transferred to disposable cuvettes (E0030106300, Fisher Scientific)
and placed in a Wyatt DynaPro dynamic light scattering instru-
ment. The size distribution was measured using a regularization
algorithm (Wyatt instrument software) and calculated as the
average of 20 measurements. Each measurement was conducted
for 10 s at a laser power that yielded approximately 1 million
counts per second.
The experiments to determine the apparent diffusion interac-
tion parameter (kD*) were performed as described previously.27
Briefly, the mAb samples were buffer-exchanged 4 times into
buffered solutions (15 mM citrate, 10 mM phosphate) at pH 3.8 and
7.4. Next, the mAbs were filtered using 0.22 mm 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 mL) 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 con-
ducted for 10 s. The mAbs were then diluted sequentially to lower
concentrations (4-9 mg/mL) in their respective buffers and evalu-
ated via light scattering. The diffusion coefficients (DM) for the mAb
solutions (4-10 mg/mL) were fit to the following equation:


DM ¼ D0 1 þ k*D c
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 condi-
tions) to obtain final concentrations of 1 mg/mL mAb and 0.6 or 1 M
ammonium sulfate. Next, the mAb samples (100 mL) 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 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 be-
tween the column void volume and the end of the run, and the
percentage recovery of the chemically modified antibodies was
evaluated relative to the control.
 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669 659

Ammonium Sulfate Precipitation
The mAb solutions were diluted to 2 mg/mL in citrate-
phosphate buffer at different pH values (pH 3.8-7.4). Stock solu-
tions 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 mL) were added to each well of clear 96-well
plates (07200656, Fisher Scientific), and then ammonium sulfate
solutions (90 mL) were added at different concentrations (0.5-2 M).
Next, the solutions were 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 absor-
bance values of the mAbs (y-axis) were plotted against the
ammonium sulfate concentration (x-axis), and 4-parameter Boltz-
mann sigmoidal curves (y ¼ minþ[max-min]/[1 þ 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 (pH 3.8 or 7.4). Next, the mAb solutions (100
mL) 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 dea-
midation on antibody stability, we performed forced degradation of
2 mAbs [mAb 1 (IgG1) and mAb 2 (IgG4)] using conditions that are
relatively selective for modifying Met, Trp, or Asn residues (here-
after referred to as Met-oxidized, Trp-oxidized and deamidated
mAbs, respectively), whereas the untreated mAbs were used as
controls. We performed mass spectrometry (peptide mapping) to
evaluate the extent of modification of the oxidation and deamida-
tion sites (Table 1). For the mAbs treated with H2O2, mass spec-
trometry 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, whereas mAb 2 displayed modest levels of Met
oxidation in its 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%). In
addition, Trp oxidation was not observed in the Fc region for either
mAb. Moreover, the samples incubated at high pH (pH 9.5) dis-
played 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 3 different
conditions used to promote chemical degradation were relatively
selective, it is notable that treatment of mAb 1 at high pH to pro-
mote deamidation also increased Met oxidation in the Fab region of
mAb 1 (19% modified relative to 10% for the control), whereas this
was not observed for mAb 2.
One of the principal goals of our study was to evaluate the ef-
fects of oxidation and deamidation on the stability of antibodies
due to various stresses encountered by the mAbs during the
manufacturing process, which can include changes in pH, concen-
tration, and temperature.33,34 Therefore, we first sought to quantify
the amount of soluble high molecular weight (HMW) species pre-
sent without extended storage as a function of pH and concentra-
tion (Figs. 1 and S1). We evaluated the aggregation behavior of the
control and chemically modified mAbs via size-exclusion chroma-
tography at pH 3.8 (Figs. 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 (Fig. S1). The Trp-oxidized samples displayed the
highest levels of HMW species followed by the deamidated sam-
ples, 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 (Figs. 1a and 1b).
Moreover, deamidation promotes the formation of soluble aggre-
gates at low pH in the most pronounced concentration-dependent
manner, as observed previously.27 We also found that the aggre-
gation behavior of the same mAbs at neutral pH followed a similar
trend, although the levels of aggregation were higher at near-
neutral pH than at low pH (Figs. 1c and 1d).
Next, we evaluated the aggregation behavior of the mAbs using
a second method (dynamic light scattering) without extended
storage (<2 h) to determine the generality of our findings (Figs. 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; Figs. 2a, 2b, S2 and S3). Moreover, the

Table 1
Summary of the Oxidation and Deamidation Levels of the Antibodies Evaluated in This Study

Methionine Residues (% Modified) Tryptophan Residues (% Modified) Asparagine Residues (% Modified)

Con. H2O2 AAPH þ Met High pH Con. H2O2 AAPH þ Met High pH Con. H2O2 AAPH þ Met High pH

mAb 1
Fab 10 91 8.7 19 <1 1.8 87 <1 <1 <1 <1 3.6
Fc 3.1 51 4.6 7.8 e e e e 2.1 2.0 2.5 18
mAb 2
Fab <1 12 1.6 <1 <1 <1 29 <1 <1 <1 <1 1.1
Fc <1 51 3.4 2.3 e e e e 2.8 2.5 2.0 20

Mass spectrometry analysis of the control (Con.), methionine (Met)-oxidized (H2O2-treated), tryptophan (Trp)-oxidized (AAPH þ 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 2 Met residues (including 1 in the CDRs), 1 Trp residue (in the CDRs), and 4 Asn residues
(including 2 in the CDRs) in the Fab region, as well as for 3 Met and 3 Asn residues in the Fc region. mAb 2 modifications were detected for 1 Met residue, 1 Trp residue (in the
CDRs), and 1 Asn residue (in the CDRs) in the Fab region, and for 3 Met and 4 Asn residues in the Fc region.
660 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669

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 3 independent experiments and the error bars are standard errors. A 2-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 (***)].

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; Figs. 2a, 2b, S2 and
S3). By 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 represent larger ag-
gregates.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. By 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;
Figs. 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 oxidationdwithout pro-
longed incubation (<2 h)denhance 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 (Figs. 3, 4, S4-S7). We first evaluated
the aggregation behavior of the mAbs at low pH (pH 3.8) during
incubation for 7 days via size-exclusion chromatography (Fig. 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 7 days (Figs. 3a and 3b). The Met-oxidized
sample of mAb 1 also displayed reduced recovery (~75% recovery)
from the size-exclusion column relative to the control (~90% re-
covery; Fig. S4). This aggregation behavior was not observed when
the incubation temperature was lowered to 25
C (Figs. 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; Fig. 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 (Figs. 4a, 4b and S7)
and 25
C (Figs. 4c, 4d and S7). The highest levels of aggregation at
pH 6 and 7.4 corresponded to the Trp-oxidized samples followed by
the deamidated samples, whereas 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% recovery) that were
similar to those observed for the controls (Fig. 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 by which oxidation and
deamidation can potentially enhance aggregation. One such
mechanism is that these chemical modifications can lead to the
formation of covalently cross-linked species between different
 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669
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 3 independent experiments.
antibody domains.20,39-41 Therefore, we used SDS-PAGE to evaluate
the formation of reducible and nonreducible cross-linked species of
the control, oxidized, and deamidated mAbs (Fig. 5). We observed
that the Trp-oxidized samples of both mAbs displayed HMW spe-
cies (>260 kDa) for nonreducing conditions and that the level of
aggregation was higher for mAb 1 than for mAb 2. Intrinsic Trp
fluorescence (Fig. 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 ag-
gregation levels for mAb 1. Moreover, the Trp-oxidized samples in
the presence of reducing agent did not display cross-linked species
for either mAb (Fig. 5). Interestingly, the deamidated samples,
especially for mAb 1, displayed nonreducible cross-linked species
(~80-90 kDa). Moreover, similar behavior was observed after
incubating the antibodies at pH 7.4 and an elevated temperature
(37
C) for 1 week (Fig. S9). In addition, we find that deamidated
mAb 1 monomers and HMW speciesdwhich were isolated using
size-exclusion chromatographyddisplay similar levels of non-
reducible species (Fig. S10). These findings reveal the conditions
that promote Trp oxidation lead to the formation of reducible,
disulfide-linked species, whereas the conditions that promote
deamidation lead to the formation of nonreducible cross-linked
species, and that mAb 1 is more prone to the formation of non-
reducible crosslinks.
Another potential mechanism by which oxidation or deamida-
tion 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 (Fig. 6 and S11). As expected,
the antibody conformational stabilities decreased for all of the
661
samples as pH was reduced. Interestingly, the Met-oxidized sam-
ples displayed significant decreases in Tm* (2.5
C-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 apparent melting temperature
between Trp-oxidized, deamidated, and control (untreated) sam-
ples 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 (Figs. 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 tem-
perature (37
C), a condition at which low antibody folding stability
results in high levels of aggregation (Fig. 3).
It is also possible that chemical modification of solvent-exposed
residues leads to increased antibody self-association 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 (Figs. 7, S14 and
S15). These measurements involved evaluating the diffusion coef-
ficient of the mAbs as a function of antibody concentration using
dynamic light scattering.30 Interestingly, oxidation and deamida-
tion had unique, antibody-specific impacts on the kD* values at low
pH (pH 3.8). Deamidation led to large and significant decreases in
kD* values for both mAbs, which corresponds to less repulsive (or
more attractive) colloidal interactions. By contrast, Met-oxidized
662 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669

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 7 d before analysis by size-exclusion chroma-
tography. The data shown are averages of 3 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 d before analysis by size-exclusion chroma-
tography. The data shown are averages of 3 independent experiments and the error bars are standard errors.
 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669
Figure 5. SDS-PAGE analysis of the impact of oxidation and deamidation on the for-
mation 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 repre-
sentative examples of 3 independent experiments.
samples showed either little change (mAb 1) or a significant in-
crease (mAb 2) in kD* values. In addition, 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 pres-
ence of acidic or basic species in the control and stressed antibodies
using imaged capillary isoelectric focusing (Table 2 and Fig. S16).
We observed that the various chemical modifications led to
increased levels of acidic species compared to the control antibody
samples, whereas 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 after deamida-
tion.31 In addition, oxidation typically promotes the formation of
polar moieties such as methionine sulfoxide, hydroxytryptophan,
and N-formylkynurenine,42 and the oxidized samples of both mAbs
generally displayed increased formation of acidic species.
663
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 3 independent experiments and the error bars are standard
errors. A 2-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 (***)].
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 determi-
nant 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; Fig. 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 (2 main peaks) than mAb 2 (1 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
(Fig. 8a). At higher pH (pH 7.4), the mAbs generally were more
hydrophobic than at low pH (Figs. 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 (Fig. S17). We also observed similar HIC results using a
664 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669

We also evaluated the relative solubilities of the chemically

modified mAbs in ammonium sulfate (Figs. 9 and S19). 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 con-
centrations 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 (Figs. 9a, 9b, 9c, 9d 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 (Figs. 9e, 9f and S19). The Met-oxidized samples gener-
ally displayed little change or increased solubility, consistent with
their reduced hydrophobicity relative to the control samples as
judged by HIC (Fig. 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;
Figs. 9e and 9f). Moreover, the deamidated samples displayed little
change in solubility. In addition, the antibody solubilities at pH 4.5
were similar to those at pH 3.8, whereas the solubilities at pH 6
were similar to those at pH 7.4 (Figs. 9 and S19). While there is little
correlation between the solubilities of the Trp-oxidized and dea-
midated 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 hydropho-
bicities and low aggregation propensities.

Discussion

Our experimental findings are summarized in Table 3 and

deserve further consideration. One interesting aspect of our find-
ings is that the mechanisms by which chemical modifications lead
Figure 7. Evaluation of the impact of oxidation and deamidation on antibody colloidal
to antibody aggregation are weakly correlated with changes in
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 antibody conformational stability. While the simplest explanation
function of pH. The experiments were performed in citrate-phosphate buffer at the for the impact of chemical modifications on antibody aggregation is
reported pH values. The data shown are averages of 3 independent experiments and that they lead to reduced conformational stability and thereby
the error bars are standard errors. A 2-tailed Student's t-test was used to judge the
increased aggregation, our results reveal significantly different
statistical significance of the difference between the chemically modified and control
mAbs [p-values <0.05 (*), <0.01 (**), or <0.001 (***)].
aggregation mechanisms. The molecular basis for our findings ap-
pears explainable (at least in part) based on the sites in the mAbs
that were modified. For the Met-oxidized samples, the modifica-
broader salt gradient (from 1 to 0 M ammonium sulfate; Fig. S18).
tions were primarily observed at sites in the Fc region that are
These findings suggest that the reduced hydrophobicity of the Met-
known to reduce antibody conformational stability,9,10 as we
oxidized samples may be responsible for their low levels of self-
observed in this work (Fig. 6). The moderate reduction in stability is
association and aggregation, while the aggregation behaviors
not sufficient to promote aggregation except at conditions of low
observed for the Trp-oxidized and deamidated samples do not
pH (pH 3.8; apparent Tm reduced from 47.8
C to 41.7
C for mAb 1
appear to be mediated by increased antibody hydrophobicity.
and from 42.3
C to 36.2
C for mAb 2) and elevated temperature
(37
C). Under these particular conditions, the conformational sta-
Table 2 bility of the Met-oxidized samples (apparent Tm of 36.2
C-41.7
C) is
Summary of the Levels of Acidic and Basic Species of the Antibodies Evaluated in This
too low to prevent antibody aggregation. Conversely, the methods
Study
used in this work for selective Trp oxidation primarily lead to
Acidic Species (%) Main Species (%) Basic Species (%) modification of a single Trp residue in the CDRs (which is different
mAb 1 for each mAb), and this limited modification appears to promote
Control 41.5 52.3 6.2 aggregation via mechanisms that are distinct from those linked to
H2O2 43.4 50.4 6.3 reduced antibody conformational stability. Likewise, Asn deami-
AAPH þ Met 61.5 29.7 8.9
High pH 91.2 6.9 1.9
dation occurs primarily in the Fc region at the well-known Asn sites
mAb 2 in the PENNY peptide, and the lack of conformational destabiliza-
Control 28.7 63.9 7.4 tion (as observed previously27) also suggests that deamidation
H2O2 48.0 46.0 6.1 promotes aggregation via mechanisms distinct from those linked to
AAPH þ Met 45.5 47.1 7.5
reduced conformational stability.
High pH 61.7 34.5 3.8
Therefore, the aggregation mechanisms for the mAbs subject to
Imaged capillary isoelectric focusing (iCIEF) was performed on the control, Met- Trp oxidation and Asn deamidationdwhich cannot be easily
oxidized (H2O2-treated), Trp-oxidized (AAPH þ Met-treated) and deamidated
(high pH-treated) mAbs to determine the level of charged species for each sample.
explained by impacts of the chemical modifications on conforma-
The main peak represents unmodified antibody, and the acidic and basic species tional stabilitydalso deserve further consideration. We find that
represent charged species. The data are for 1 to 3 replicates. Trp oxidation leads to formation of reducible crosslinks, which are
 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669 665

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 col-
umn 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 3 independent experiments.

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 delo-
calized electrons, which 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-oxidationdwhich can
disrupt the Trp indole ring and cause local structural changes
through formation of N-formylkynureninedand this may lead to
greater amounts of reducible aggregates for mAb 1 relative to mAb
2. It is also notable that previous studies of photo-oxidation of
antibodies found that disulfide bond shuffling can occur via
oxidation of Trp residues located in close proximity to intra-
molecular disulfide bonds through electron transfer or singlet ox-
ygen 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 tu-
mor necrosis factor receptor 1,54 and slight worsening for gran-
ulocyte 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 salts, would differentially
modify the aggregation propensity of oxidized and deamidated
mAbs requires additional studies in the future.
The formation of nonreducible crosslinks during antibody
storage at high pH (pH 9.5) to promote Asn deamidation also de-
serves further consideration. The fact that these crosslinks (which
primarily form for mAb 1) are nonreducible 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 nonreducible crosslinks for mAbs incu-
bated at similar conditions as those used in our study for promoting
deamidation, namely elevated temperature (e.g., 37
C-45
C) and
high pH (pH 8-10).40,57-60 Moreover, the observed degree of
crosslink formation was significantly different for different anti-
bodies,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 b-elimination reaction,61-63 which leads to in-
termediates that can form nonreducible thioether bonds between
666 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669

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 4 independent experiments and the error bars are standard errors. A 2-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 (***)].

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 nonreducible 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 spectrom-
etry analysis revealed that the oxidation levels of the 2 mAbs were
similar in the Fc regions but markedly different in the Fab regions
(Table 1), which suggests that the Fab region is primarily respon-
sible for the observed differences in hydrophobicity for the Met-
oxidized samples. For Trp oxidation, the oxidized samples for the
2 mAbs display markedly different HIC retention behavior despite
the fact that both mAbs primarily undergo Trp oxidation 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
di-oxidation (N-formylkynurenine or dihydroxytryptophan) rela-
tive 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 experimen-
tation to be properly evaluated.
The HIC retention behavior of the deamidated mAbs also de-
serves further consideration. Previous studies have found that
 M.E. Alam et al. / Journal of Pharmaceutical Sciences 109 (2020) 656-669 667

Table 3
Summary of the Experimental Analysis of the Effects of Chemical Modifications on Various Antibody Biophysical Properties

Met Oxidation Trp Oxidation Asn Deamidation

pH 3.8 pH 7.4 pH 3.8 pH 7.4 pH 3.8 pH 7.4

mAb 1
Aggregation (SEC, %HMW) NC NC þþ þþþ þa þþþ
Aggregation (DLS, HMW species) NC NC þþþ þþ þþþ NC
Accelerated Stability (37
C, %HMW) þþþ þ ‒ þ þþþ þ
Reducible HMW Species (SDS-PAGE)  NC  þþþ  NC
Nonreducible Species (SDS-PAGE)  NC  NC  þþþ
Conformational Stability (DSF, Tm*) ‒‒ ‒‒ NC NC NC NC
Colloidal Stability (kD*) NC NC þþ ‒ ‒‒ NC
Hydrophobicity (HIC, retention time) ‒‒‒ ‒‒‒ ‒‒ ‒‒‒ NC NC
Solubility (ammonium sulfate, CS,50) NC þþþ NC þþ NC NC
mAb 2
Aggregation (SEC, %HMW) NC NC þþ þþþ þ þa þþþ
Aggregation (DLS, HMW species) NC NC NC NC þþþ þþþ
Accelerated Stability (37
C, %HMW) þþþ NC NC þþ þþþ þ
Reducible HMW Species (SDS-PAGE)  NC  þþ  NC
Nonreducible Species (SDS-PAGE)  NC  NC  þ
Conformational Stability (DSF, Tm*) ‒‒ ‒‒‒ NC ‒‒ NC NC
Colloidal Stability (kD*) þ NC ‒ NC ‒‒‒ ‒
Hydrophobicity (HIC, retention time) ‒ ‒ þ NC NC NC
Solubility (ammonium sulfate, CS,50) þ þþþ ‒‒ ‒‒ ‒ þ

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 (þ) denotes an increase in the measured value of a particular biophysical property relative to the control mAb (e.g., wild-type mAb 1), whereas a minus sign (‒)
denotes a decrease. More than one plus or minus sign indicate larger changes.
, not evaluated; NC, no change.
a
At high concentration only.

deamidation can lead to increased or decreased HIC retention times Acknowledgments

depending on the formation of succinimide67-71 or aspartic acid
(and isoaspartic acid),43,70-73 respectively. The degradation condi-
tion (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 hydropho-
bic 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 con-
ditions relevant to antibody manufacture, purification and formu-
lation 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.
The authors thank members of the Tessier lab for their assis-
tance editing the manuscript. The authors would also like to thank
Pritesh Patel and Shannon Flagg for providing the iCIEF data. In
addition, they thank Helen Zha for allowing them to use her DSC
calorimeter. This work was supported by Bristol-Myers Squibb and
the Albert M. Mattocks Chair (to P.M.T.).
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