Journal of Pharmaceutical Sciences 110 (2021) 719-726

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Journal of Pharmaceutical Sciences

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Drug Discovery-Development Interface

Tryptophan Oxidation of a Monoclonal Antibody Under Diverse

Oxidative Stress Conditions: Distinct Oxidative Pathways Favor
Specific Tryptophan Residues
Alex W. Jacobitz, Qian Liu, Sreekanth Suravajjala**, Neeraj J. Agrawal*

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

Article history: Tryptophan oxidation can play an important role in selecting therapeutic monoclonal antibodies for
Received 7 April 2020 commercialization. Monoclonal antibodies that harbor particularly sensitive tryptophan residues are
Revised 14 October 2020 typically discarded in favor of oxidation resistant antibodies. The susceptibility of any individual tryp-
Accepted 19 October 2020
tophan residue to oxidation is typically evaluated through forced degradation studies during the
Available online 23 October 2020
molecule development process. We compared the results of several common forced degradation “stress
tests” for each tryptophan residue in a monoclonal antibody and found that high-stress oxidation con-
Keywords:
Oxidation(s) ditions consistently provide a different ranking of oxidative sensitivity across the individual tryptophan
Liquid chromatography-mass spectrometry residues compared to long-term thermal stability or low-stress conditions. We subsequently determined
(LC-MS) that this difference in ranking is largely due to an overabundance of double oxidation (i.e. detected
Monoclonal antibody(s) as þ32 Da) of specific tryptophan residues under high stress conditions compared to single oxidation
Developability screening
Photodegradation (i.e. þ16 Da). We posit that this double oxidation is in fact mechanistically distinct from the observed
Chemical stability single oxidation and that high stress conditions favor the double oxidation mechanism (and double
oxidation sensitive tryptophan residues) while single oxidation appears to be the primary mode of
oxidation under H2O2 stress and long-term thermal stability and favors different tryptophan residues
which are more susceptible to the single oxidation mechanism.
© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Introduction
Oxidation is a chemical modification that affects many proteins
by modifying the side chains of susceptible amino acids. Cysteine,
methionine, tryptophan, histidine, tyrosine, and phenylalanine are
all susceptible to oxidative degradation under biologically relevant
conditions to various extents.1 Tryptophan oxidation is arguably the
most complex of these, as this amino acid can degrade via multiple
pathways resulting in various oxidation products including oxindo-
lealanine, (4, 5, 6, or 7)-hydroxytryptophan, dioxindolealanine,
kynurenine, N-formylkynurenine and several others.2,3
Monoclonal antibodies (mAbs) currently make up roughly half
of all FDA approved biologics and have accounted for > 70% of
biologic approvals since 2015.4 Given this growing prominence in
the marketplace, there is an increased focus on understanding the
critical quality attributes of mAbs in order to develop products with
* Corresponding author.
** Corresponding author.
E-mail addresses: ssurvajj@amgen.com (S. Suravajjala), agrawaln@amgen.com
(N.J. Agrawal).
increased safety and efficacy. For a drug candidate to be suitable for
therapeutic use it must be sufficiently resistant to all forms of
degradation such that it can be manufactured, stored, shipped, and
administered to the patient without a considerable loss in potency
or altered safety profile. Oxidation of mAbs has been shown to
reduce their stability and decrease FcRn binding5 which could
reduce the longevity of the drug both in storage and in the body.
Tryptophan oxidation in particular can be a significant impediment
to developing mAbs because tryptophan residues can often be
critical for binding to the desired target, and because those critical
tryptophan residues are often the most susceptible to oxidation.6,7
During the drug development process, mAbs are typically sub-
jected to oxidative stress tests to predict whether or not a particular
tryptophan residue will be susceptible to oxidation during
manufacturing or long-term storage.1,5,6,8,9 A wide variety of
physical and chemical stress conditions have been reported to
stress test tryptophan oxidation including visible and UV light,6
2,20 -azobis(2-amidinopropane) dihydrochloride (AAPH),5,10
H2O2,5,10,11 tert-butyl-hydroperoxide (tBHP),5,8,10 and ozone.6 In
the literature, various groups have seemingly chosen their own
favorite stress conditions, but despite the criticality of this predic-
tion, there is no clear consensus in the field to indicate which

https://doi.org/10.1016/j.xphs.2020.10.039
0022-3549/© 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
720 A.W. Jacobitz et al. / Journal of Pharmaceutical Sciences 110 (2021) 719-726

methodology is most valuable for predicting oxidation under
manufacturing conditions, and there are few examples of direct
comparisons of these different stress conditions.
Oxidation of tryptophan residues is mechanistically distinct
from other oxidation prone amino acids in that it is reportedly only
susceptible to oxidation from free radicals and excited state oxygen
species, and not via the nucleophilic mechanism that leaves some
residues vulnerable to oxidation by peroxides3,5,10 (See Fig. 1 for
primary degradation pathways). Nonetheless, several reports uti-
lize tBHP5,8,10 or H2O25,10,11 as an oxidation stress for tryptophan,
because these peroxides are thought to closely mimic the potential
for peroxide formation from decomposition of polysorbate,1,12 a
common additive in formulation buffers, and because H2O2 is
sometimes used in the cleaning of certain surfaces in the
manufacturing process. In some cases, tBHP or H2O2 stress is
coupled with the addition of soluble iron or copper as a catalyst for
free radical production which is thought to mimic the leaching of
metals off of the surface of bioreactors or other in-process contact
surfaces.1,10 Another common chemical additive, AAPH, can directly
generate alkyl-peroxides and radical oxygen species which are
thought to simulate the auto-oxidation breakdown products of
polysorbate.5,10,13 These radical oxygen species have been shown
computationally2 and via numerous experiments1,5,10 to be capable
of modifying susceptible tryptophan residues to very high levels.
The alkyl peroxides generated during the decomposition of AAPH
can catalyze the formation of radical tryptophan species14,15 which
are highly susceptible to attack by superoxide (O 2 ) which would
otherwise be a very inefficient reactant for oxidizing tryptophan.16
Photostress from UV light can also induce tryptophan oxidation via
an additional mechanism wherein the tryptophan residue directly
absorbs UV radiation to become photoionized and then resolves
this radical state through transfer of the radical to dissolved oxy-
gen,17 or to water,18 generating reactive oxygen species, and ulti-
mately yielding an oxidized tryptophan species.6,19
To better understand these oxidation stress conditions, we
performed forced oxidation of a single mAb under several different
oxidation conditions and determined the oxidation level of all
tryptophan residues by peptide mapping. We then compared the
results of each of these site-specific tryptophan oxidation profiles
and propose a mechanistic hypothesis for the observed partitioning
of these stress tests into two distinct groups.
Experimental Methods
Antibody Production
Monoclonal antibodies were produced recombinantly using an
in-house mammalian expression system and purified by standard
chromatographic methods.
Chemical Oxidation
All oxidations were performed on an antibody at 5 mg/mL in
10 mM sodium acetate pH 5.2, at room temperature, in the dark. For
AAPH oxidation experiments, AAPH (Sigma) was added to a final
concentration of 5 mM and allowed to incubate at room

Hydroxyl Radical Mediated Pathway

+ + + +

Tryptophan OH˙
~60% ~15% ~11% ~7% ~7%
oxindolealanine 4-hydroxytryptophan 7-hydroxytryptophan 5-hydroxytryptophan 6-hydroxytryptophan

4
5 3a 3 Superoxide (O2-) or Singlet Oxygen (1O2) Mediated Pathway
2
6 1
7a
7
O

O
ν , or
ROO˙

. O

N-formylkynurenine kynurenine

dioxindolealanine

Fig. 1. Main tryptophan oxidation mechanisms. Oxidative degradation of tryptophan proceeds through two main pathways: the hydroxyl radical mediated pathway (top) and the
superoxide/singlet oxygen mediated pathway (bottom). The hydroxyl radical mediated pathway produces oxindolealanine or several hydroxy-tryptophan molecules in a defined
ratio. The precise series of intermediates in the superoxide or singlet oxygen pathway (bottom) is somewhat more controversial but the stable products of the reaction, dioxin-
dolealanine, N-formylkynurenine, and kynurenine are well established, although the ratio of products is still unknown.
 A.W. Jacobitz et al. / Journal of Pharmaceutical Sciences 110 (2021) 719-726
temperature for up to 48 h. For H2O2 oxidation experiments, H2O2
(Sigma) was added to a final concentration of 0.1% (28 mM) and
allowed to incubate at room temperature for up to 24 h. Reactions
were quenched by addition of L-tryptophan and L-methionine to a
final concentration of 5 mg/mL each, then frozen.
Light Induced Oxidation
Oxidation by light was conducted by placing 200 mL of 5 mg/mL
antibody in 10 mM sodium acetate pH 5.2 into clear glass vials and
subjecting them to UV light for up to 1200 W*hrs/m2 over 5 days, to
cool white light for up to 1.2 Mlux*hrs over 5 days, or to a combi-
nation of 1.2 Mlux*hrs of cool white light and 200 W*hrs/m2 of UV
light based on the ICH Harmonized Tripartite Guideline for Pho-
tostability Testing of New Drug Substances and Products.20 All ex-
periments were conducted at room temperature, and oxidation was
terminated by moving samples to darkness.
Long-Term Thermal Stability Oxidation
The antibody was stressed at 40  C for up to 54 weeks and
further stored at 70  C until analysis.
Mass Spectrometry
Samples (500 mg) were denatured by diluting to a final con-
centration of 1 mg/mL into denaturing buffer containing 5.5 M
guanidine-HCl, 0.20 M Tris HCl, pH 8.3. Denatured samples were
reduced by adding 0.5 M dithiothreitol (DTT) to a final concentra-
tion of 9 mM and incubated at 27  C for 23 min. Post reduction, the
samples were alkylated using 0.5 M sodium iodoacetate (NaIace-
tate) and incubating in dark at room temperature for 19 min.
Reduced, alkylated samples were then buffer-exchanged into
50 mM Tris, pH 8.3 using NAP-5® columns to a final concentration
of 0.7 mg/mL. Trypsin digestion was achieved by using a ratio of
1:10 (enzyme:sample) and incubating at 37  C for 35 min. The re-
action was terminated by addition of 20% trifluoroacetic acid to a
final concentration of 0.3% (v/v). The digested samples were
analyzed by liquid chromatography tandem-mass spectrometry
(LC-MS/MS). The LC-MS/MS system consisted of a UPLC system
connected in-line to a Thermo Scientific Fusion Lumos mass spec-
trometer. A polymeric column was used to separate the peptides
with the column temperature at 50  C under typical reverse phase
conditions. The gradient started with 0% B for 10 min followed by
linear gradient of 0.25%/min over 180 min using flow rate of 0.2 mL/
min with 6 mg sample load on column. Data acquisition was per-
formed in positive mode using data-dependent acquisition (cycle
time of 2 s). MS1 resolution was set to 120,000 with a scan range of
250e2000 m/z. MS2 were acquired at 60,000 resolution using
normalized collision energy of 30. Automatic gain control was set to
4E5 and maximum injection time was set to 200 ms. Relative
quantitation was performed using Skyline v.4.2.0.19072. For each
molecule, a workbook was created containing unoxidized, i.e.
native, and oxidized versions of all tryptophan-containing tryptic
peptides. Total area of unoxidized and oxidized peptides was
determined by extracting ion chromatograms of 2 charge states per
peptide (3 isotopes per charge state). Acceptance criteria for each
peptide charge state were mass accuracy of 5 ppm and isotopic dot
product of 0.9. For some peptides, particularly at t0 time points,
isotopic dot products for one of the charge states were less than 0.9.
In these instances, only a single charge state was used for relative
quantitation. Modification percentages were calculated by dividing
the total area of the oxidized peptide by the sum of total areas from
the oxidized and unoxidized peptides.
721
Homology Modeling and Molecular Dynamics Simulations
Homology model of the Fv domain was generated using the
Antibody Modeling Cascade tool of Discovery Studio software
(Dassault Syste
mes Biovia Corp) and a 20 ns molecular dynamics
simulation was performed on this model using the AceMD soft-
ware21 as described in our previous work.22 From this molecular
dynamics trajectory, frames were extracted at every 100 ps be-
tween the 5 and 20 ns time-points of the trajectory for the solvent
accessible surface area (SASA) calculation. The average SASA of each
tryptophan side chain over the molecular dynamics trajectory was
calculated in the VMD software23 using a water molecule as a probe
(radius 1.4 Å).
Results
Tryptophan Oxidation Induced by Various Stress Conditions Reveals
that Stress Conditions Fall into Two Distinct Groups
Given the wide array of tryptophan oxidation stress conditions
employed in the literature, we were interested in understanding
how site-specific tryptophan oxidation levels might correlate to
one another. To this end, we performed stress tests on a single
antibody (mAb1) using 5 different methods garnered from the lit-
erature5e7,9,14,20 and then examined site-specific tryptophan
oxidation levels via peptide mapping (Fig. 2). We obtained com-
plete coverage of all tryptophan residues in the antibody and found
detectable levels of oxidation (% modified > 0.1%) on 4 unique
tryptophan residues in the molecule, heavy chain (HC) residues
W_H-FR2.1, W_H-CDR2, and W_H-CDR3 and light chain (LC) res-
idue W_L-CDR2, (FR: framework and CDR: complementarity
determining region) see Table 1 for complete list of tryptophan
residues and their positions in the molecule). Site-specific trypto-
phan oxidation levels for each of these oxidation-sensitive trypto-
phan residues under each condition, assayed in duplicate, are
shown in Fig. 2a. None of the constant domain tryptophan residues
undergo a detectable level of oxidation under any conditions tested.
CDR tryptophan residues on the other hand are much more sus-
ceptible to oxidation, consistent with previous accounts in the
literature.6,8 Specifically, we detected high levels of oxidation (>5%)
under several stress conditions for tryptophan residues located in
HC CDR2 and HC CDR3, and a moderate level of oxidation (>1%) for
the tryptophan in LC CDR2. We also detected low levels of trypto-
phan oxidation in one framework tryptophan in HC FR2 (between
0.1% and 0.5%). Across all of the high oxidative-stress conditions
(AAPH, white light, UV, UV þ white light), W_H-CDR2 appears to be
the most susceptible to oxidation, with oxidation levels measuring
as high as 95% under AAPH stress. It is worth reiterating that
though it has been used as a stress in other publications,5,10,11 H2O2
alone is thought to be incapable of directly oxidizing tryptophan
given the current understanding of the tryptophan oxidation
mechanism which indicates that excited state oxygen (singlet ox-
ygen, 1O2) or radical oxygen (superoxide, O 2 ) are required, since
tryptophan lacks the requisite nucleophile to attack a peroxide in
the way that cysteine or methionine can.5,10 It is plausible that H2O2
oxidation of tryptophan residues is achieved instead through trace
amounts of contaminating metals that can react with H2O2 to
stimulate the production of radical oxygen species. In our studies,
we do see a slight increase in oxidation for the H2O2 stress relative
to control, but it does not reach nearly the same level of oxidation
or share the same rank order of susceptible tryptophan residues
that the other stress conditions achieve (W_H-CDR2 > W_H-
CDR3 > W_L-CDR2 > W_H-FR2.1). H2O2 oxidation instead sees the
highest oxidation at W_H-CDR3, followed by W_L-CDR2, W_H-
CDR2, then W_H-FR2.1. Under long-term thermal stability we again
722 A.W. Jacobitz et al. / Journal of Pharmaceutical Sciences 110 (2021) 719-726

Fig. 2. Total oxidation of each tryptophan residue under various conditions. (a) Results from each assay are shown as a group of bars with each oxidation sensitive tryptophan
represented as a different color bar. Tryptophan residues that do not show any detectable oxidation under any conditions are excluded for clarity. Error bars are the standard
deviations of the measurements repeated in duplicate. (b) Example scatterplot showing the correlation between t54w long-term thermal stability and white light induced
oxidation. (c) Correlation matrix showing r2 correlation coefficients between all experimentally measured conditions and with solvent accessible surface area (SASA) represented as
an average across multiple short MD simulations.

detect the highest level of oxidation in W_H-CDR3, consistent with
H2O2 stress, and with the t0 sample. We consider this thermal stress
sample to be a good indicator of long-term storage stability, espe-
cially as a potential model of a “worst case scenario” to indicate the
maximum possible oxidation we might expect to see under long-
term storage. We choose 40C since it is well below the average
Tm of mAbs, which is typically >70C, meaning we do not expect this
condition to be significantly affected by unfolding effects. This
position is further supported for mAb1 by the fact that the oxida-
tion rate for all tryptophan residues is linear over the entire 54w
time course (Fig. S1), indicating that cooperative unfolding does not
take place even at longer time points at this temperature. We
generated a correlation matrix (Fig. 2c) by comparing the site-
specific oxidation levels across every possible pair of conditions
(see example in Fig. 2b). This matrix reveals an interesting di-
chotomy where t0 and long-term thermal stability samples corre-
late very well with each other (coefficient of determination
(r2) > 0.95) and with the H2O2 stress condition, which seems to be
an extremely low-stress condition, but correlate comparatively
poorly with all other stress conditions (r2 < 0.7). The remaining
stress conditions (all but H2O2) have r2 values above 0.7 when
Table 1
Tryptophan Residues in mAb1.
Chain Position
Heavy FR2
FR2
CDR2
CDR3
FR4
CH1
CH2
CH2
CH3
CH3
Light FR2
CDR2
CL
compared to each other, with all but one pair sharing an r2 > 0.9,
indicating that all of these high-stress conditions behave similarly.
In addition to these experimental values, we also looked at the
correlation coefficients between each of these samples and the
solvent accessible surface area (SASA) of each tryptophan side chain
represented as an average over a short MD simulation (Fig. 2c). We
see very strong correlations between long-term thermal stability
and SASA, but not with any of the high-stress conditions and SASA,
which runs contrary to some existing literature.24,25
Individual Tryptophan Oxidation Species Detected by LCMS
Correlate Well WithLong-Term Thermal Stability
Given that we observed high correlations between all possible
pairs of high-stress conditions (group 1), as well as high correla-
tions between t0, low-stress (H2O2), long-term thermal stability
conditions and SASA (group 2) but low correlations between any
two conditions across those two groups, we sought to understand
the source of this significant discrepancy. Towards this end, we
dissected the data into the 3 constituent components that were
detected via peptide mapping, referred to herein as single oxidation
(þ16 Da), double oxidation (þ32 Da) and Kynurenine (þ4 Da)
(Fig. 3a). These components could not be further characterized into
specific oxidized tryptophan species, i.e. single oxidation could
imply a single oxidation being added anywhere to the tryptophan
Residue Name
residue (2,4,5,6 or 7-hydroxytryptophan), and double oxidation
W_H-FR2.1 could be one of several products of the direct addition of molecular
W_H-FR2.2
W_H-CDR2
oxygen at a single site (e.g. dioxindolealanine, or N-for-
W_H-CDR3 mylkynurenine), or could conceivably be 2 individual oxygens
W_H-FR4 added sequentially to two distinct locations (e.g. 5,6-
W_HeCH1 dihydroxytryotophan), although we expect this latter scenario to
W_HeCH2.1
be statistically improbable. Interrogating the data in this manner
W_HeCH2.2
W_HeCH3.1 reveals a clear difference between the amount of double oxidation
W_HeCH3.2 detected at individual residues (Fig. 3a green bars) compared to the
W_L-FR2 level of single oxidation species detected at those same residues
W_L-CDR2 (Fig. 3a red bars). There is an obvious trend towards higher levels of
W_L-CL
double oxidation under all high stress conditions compared to what
 A.W. Jacobitz et al. / Journal of Pharmaceutical Sciences 110 (2021) 719-726 723

Fig. 3. LCMS analysis of individual components of tryptophan oxidation show a considerable bias to double oxidation events under high oxidative stress conditions, compared to
H2O2 and long-term thermal stress. (a) LCMS results grouped by condition, with a bar for each residue. Bars for each residue are broken down into the individual oxidized species
that were detected: single oxidation (þ16) in red, double oxidation (þ32) in green, and kynurenine (þ4) in yellow. (b-d) Example plots showing site-specific oxidation levels after
long-term thermal stability (t54w) vs. oxidation levels under white light stress for each individual oxidation component, single, double, and kynurenine, in (b, c, and d), respectively.
(e) Correlation matrix showing r2 correlation coefficients for each stress condition compared to t54w long-term thermal stability samples when split into individual oxidation
components, and with y intercept set to 0. (f) Table of line of best fit slopes from individual oxidation components under each condition when graphed against t54w long-term
thermal stability data and with y intercept set to 0.

is seen in long-term thermal stability or the H2O2 low-stress con-
dition. With this trend now visible we next explored the correla-
tions between individual oxidation components (single, double, or
kynurenine) one at a time between the different stress tests and
long-term thermal stability (examples in Fig. 3bed). The correla-
tion coefficients between long-term thermal stability samples and
each high-stress condition follow a fairly consistent pattern where
the r2 for single oxidation is generally quite high, while correlations
with double oxidation and kynurenine are generally much lower
(Fig. 3e). In addition, the slopes of these best fit lines also follow a
consistent pattern wherein the single oxidation slopes are all much
larger than double oxidation or kynurenine slopes indicating that
724 A.W. Jacobitz et al. / Journal of Pharmaceutical Sciences 110 (2021) 719-726

Fig. 4. By considering single, double, and kynurenine oxidations individually, overall equations were developed to approximate total oxidation under long-term thermal stability
based on any one of the tested oxidation methods. Each graph represents total site-specific oxidation levels determined from 54 week long-term thermal stability, compared to the
specified oxidation stress test after each oxidation component has been adjusted individually as in Equation (1). The dashed line on each plot is y ¼ x. Root mean square error
(RMSE) values are displayed on each graph.

the single oxidation species evolve to more consistent levels be-
tween stress and thermal stability samples, but the double oxida-
tion and kynurenine species tend to grow to much higher levels
under high-stress than under thermal stability. This difference
between these component slopes is extremely variable from one
type of oxidation to the next which suggests that there are differ-
ences in the oxidation mechanisms for each type of stress which
contribute to the differences in the detected levels of each
component oxidation species (vide infra).
Deconstructing Site-Specific Oxidation into Individual Components
Enables Development of Equations to More Appropriately Compare
Site Specific Tryptophan Oxidation Under Each Stress Condition to
Long-Term Thermal Stability
Once it had been established that the individual oxidation
components behaved differently under stress compared to long-
term thermal stability, we wondered whether we could use this
information to generate an equation that would individually adjust
the three oxidation components such that the total tryptophan
oxidation levels from each stress test could be shown to correlate
with oxidation under long-term thermal stability. To accomplish
this, we simply took the slope of each line of best fit from plots of
long-term thermal stability vs. each stress test, for each individual
oxidation component (i.e. the slope from a graph of the type
exemplified in Fig. 3bed) and used it as a coefficient to apply to
each component measurement from our stress test data before
summing the components to get a single value for total oxidation at
that residue (Eq. (1)). The main effect of this approach is to signif-
icantly reduce the influence of the double oxidation component in
the adjusted total oxidation result. We developed individual
equations following this methodology for each stress test. The re-
sults of applying these corrective equations to each stress test are
shown in Fig. 4 and demonstrate very close approximations to the
total oxidation levels seen under long-term thermal stability. Our
ultimate goal with this type of analysis would be to predict
oxidation levels under manufacturing conditions or long-term
storage from any individual stress-test, but given that our data
set is based on a single mAb and long-term thermal stability here is
used as a stand in for long term-storage, it would be presumptive to
assume these equations would allow us to predict long-term stor-
age for this or other mAbs. We hope to be able to validate this
analysis with additional data in the future in order to arrive at a
broadly applicable predictive solution.
Total long  term thermal stability Ox
Equation 1
¼ sOx*msOx þ dOx*mdOx þ Kyn*mKyn
sOx ¼the level of measured single oxidation (þ16 Da) from a
given stress test.
msOx ¼ the slope of a line of best fit between the single oxidation
component (þ16 Da) of the given stress and the single oxidation
 A.W. Jacobitz et al. / Journal of Pharmaceutical Sciences 110 (2021) 719-726
component (þ16 Da) of the t54w long-term thermal stability
sample.
dOx ¼ the level of measured double oxidation (þ32 Da) from a
given stress test.
mdOx ¼ the slope of a line of best fit between the double
oxidation (þ32 Da) component of the given stress and the
double oxidation (þ32 Da) component of the t54w long-term
thermal stability sample.
Kyn ¼ the level of measured kynurenine (þ4 Da) from a given
stress test.
mKyn ¼ the slope of a line of best fit between the kynurenine
(þ4 Da) component of the given stress and the kynurenine
(þ4 Da) oxidation component of the t54w long-term thermal
stability sample.
Discussion
Monoclonal antibodies are an increasingly important class of
pharmaceutical molecules and understanding their main sources of
degradation is imperative for developing stable functional mAb
based therapeutics. Tryptophan oxidation is a very common source
of pharmaceutical mAb degradation and is largely found in the CDR
regions, which are the most important regions of the mAb for
defining specificity and potency. Oxidation of tryptophan residues
at these key positions has been shown to disrupt binding in-
teractions and increase aggregation.5,6 To determine if a particular
pharmaceutical candidate is susceptible to tryptophan oxidation, a
number of stress conditions are commonly employed. By directly
comparing several of these stress conditions to each other and to
SASA for mAb1 we have uncovered a surprising trend where site-
specific oxidation levels for all high-stress conditions correlate
well with each other, but correlate poorly with low oxidative stress
conditions, (H2O2) and long-term thermal stability. We also found
that these latter conditions, H2O2 and thermal stress, correlate well
with SASA, while the high-stress conditions do not. We have
additionally shown that this clear separation into two distinct
groups appears to be based on a substantial preference for the high
stress conditions to overstimulate double oxidation (detected
as þ32 Da by MS) compared to single oxidation (þ16 Da), and that
an equation to approximate long-term thermal stability from each
individual stress test can be developed by individually considering
each separately detectable component of total tryptophan oxida-
tion (single oxidation, double oxidation, and kynurenine).
This considerable discrepancy between the two groupings of
data, high stress conditions with high double oxidation vs. long-term
thermal stability and H2O2 (low oxidative-stress) showing higher
single oxidation than double oxidation, might be based on a pref-
erence for different pathways in the mechanism. The tryptophan
oxidation mechanism is complex (Fig. 1) and has multiple distinct
pathways, with largely distinct products. The hydroxyl radical (OH_)
mediated pathway generates hydroxy-tryptophan, with the hy-
droxyl group being added to a number of possible sites with a
preference of ~3:2 for addition to the pyrrole ring over the phenyl
ring.2,26 Attack at the pyrrole ring preferentially adds to position C2,
while attack on the phenyl ring adds to C4,5,6,7 in a ratio of 4:2:2:3,
respectively.2,26 Tryptophan can also be oxidized by singlet oxygen
(1O2), or by superoxide (O 2 ) via Trp radical,
16
and this proceeds
through a distinct mechanistic path with distinct products. This
second pathway adds two oxygens to the tryptophan sidechain in a
single pass, and to our knowledge, has only ever been observed to
take place at C2 and C3 positions of the pyrrole ring, and is not ex-
pected to modify the phenyl ring.2,27,28 There are several products of
this reaction that are generally stable enough for detection, N-
formyl-kynurenine and dioxindolealanine each with a mass shift
of þ32 Da, and kynurenine, with a mass shift of þ4 Da relative to
725
tryptophan. We see considerably more of the þ32 Da double
oxidation species in our high stress conditions compared to what is
seen in long-term thermal stability or H2O2 stress, and these high
stress conditions all seem to share a consistent ranking (W_H-
CDR2 > W_H-CDR3 > W_L-CDR2 > W_H-FR2.1) (Fig. 3a). On the
other hand, long-term thermal stability and H2O2 stress conditions
strongly favor single oxidation over double oxidation and they favor
oxidation at different residues (W_H-CDR3 > W_L-CDR2 > W_H-
CDR2 > W_H-FR2.1). In addition, the levels of single oxidation
detected in the long-term thermal stability sample add up to the
second highest single oxidation level across the entire dataset
(second only to AAPH stress), but this sample has a lower level of
double oxidation than any of the high stress conditions. This in-
dicates that double oxidation is not just a result of higher oxidative
stress leading to stepwise addition of one oxygen, then another. If
that were true, we would see residues build up single oxidation
before transitioning to double oxidation at higher oxidative stress
levels. Instead, we see individual residues that are more susceptible
to only one type of oxidation or the other across all stress conditions.
W_H-CDR2 for instance doesn't see any detectable single oxidation,
despite being exceptionally sensitive to double oxidation across all
conditions tested. W_H-CDR3 on the other hand always maintains a
considerable portion of its total oxidation as single oxidation, even
under the highest oxidative stress condition (AAPH). This residue
also yields different relative levels of single and double oxidation
depending on the stress condition. Under t54w long-term thermal
stability and AAPH stress, W_H-CDR3 has nearly identical levels of
single oxidation, but it develops considerably more double-oxidation
under AAPH stress. Based on these collective observations, it seems
likely that the high-stress methods are preferentially stimulating the
superoxide/singlet oxygen pathway to yield þ32 Da products (Fig. 1.
Bottom pathway), whereas the low-stress and long-term thermal
stability conditions are seemingly favoring the hydroxyl radical
mechanism that largely yields þ16 Da products (Fig. 1. Top pathway).
The precise mechanism for hydroxyl radical generation under these
H2O2 and thermal-stability tests is not known, but hydrogen
peroxide is known to decompose to generate hydroxyl radicals in the
presence of trace metal contaminants and we believe this is the most
likely pathway to account for the observed behavior. For thermal
stress conditions, the mechanism is even less obvious, but it is
possible that over long incubations, hydrogen peroxide and hydroxyl
radicals are generated spontaneously at the air water interface as has
been demonstrated in water microdroplets previously.29 We suspect
the difference in susceptibility of an individual tryptophan to one
mechanism over the other is largely related to a combination of the
solvent accessibility of the phenyl ring, vs. the pyrrole ring (and more
specifically the C2 carbon) of the sidechain, combined with the
subtleties of the local electrostatic environment surrounding an in-
dividual tryptophan sidechain.
Oxidative stress testing of pharmaceutical candidates is generally
performed to understand the potential risk associated with a given
site on the protein, and to develop stability indicating analytical
methods. By understanding the subtle differences in each of these
different tryptophan oxidation stress conditions pharmaceutical
companies can more appropriately assess the outcomes of an indi-
vidual stress test and what that might mean for the commercializa-
tion of a candidate molecule. In this study, we observed a clear
dissociation between site-specific tryptophan oxidation levels under
high-stress and oxidation under lower oxidative stress that is likely
the result of different oxidative pathways preferentially oxidizing
different tryptophan residues. This apparent pathway preference not
only effects the type of component oxidation that is detected for the
residue, but also the absolute ranking of oxidative susceptibility
across different tryptophan residues. This latter effect is perhaps the
most pertinent to pharmaceutical development as an overemphasis
726 A.W. Jacobitz et al. / Journal of Pharmaceutical Sciences 110 (2021) 719-726
of the oxidation propensity for a tryptophan known to be involved in
binding could lead to the unnecessary rejection of an otherwise viable
candidate molecule. Finally, we provide example methodology for
converting any one of these stress tests to a predictor of long-term
thermal stability, but caution that in its present state, the method
would need be validated on each individual mAb before presuming
that the coefficients used herein are appropriate. We hope that this
publication will draw attention in the field to the subtle nuances in the
results of oxidative stress tests and provide a roadmap for other re-
searchers to advance this field of study towards the development of
improved methods for tryptophan oxidation stress testing of
pharmaceuticals.
Conflicts of Interest
All research was funded by Amgen Inc. The authors declare no
conflicts of interest.
Acknowledgements
The authors would like to acknowledge Andrew Dykstra, Oliver
Thiel, and Mike Achmatowicz for helpful discussions.
Appendix A. Supplementary Data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.xphs.2020.10.039.
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