Journal of Pharmaceutical Sciences 111 (2022) 2435−2444

Contents lists available at ScienceDirect

Journal of Pharmaceutical Sciences

jo urn a l h om ep ag e : w ww.jp harms ci.o rg

Pharmaceutical Biotechnology

Intra-Micellar and Extra-Micellar Oxidation in Phosphate and

Histidine Buffers Containing Polysorbate 80
€rn-Hendrik Peters1, Yangjie Wei2, C. Russell Middaugh, Christian Scho
Bjo €neich*
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047

A R T I C L E I N F O A B S T R A C T

Article history: Polysorbate is a key excipient included in formulations of therapeutic proteins to help prevent aggregation
Received 17 March 2022 and surface adsorption. The stability of both polysorbate and therapeutic proteins can be compromised by
Revised 8 June 2022 oxidative degradation. In general, polysorbate is added to formulations at concentrations above the critical
Accepted 8 June 2022
micelle concentration (cmc). To date, however, few experiments have quantitatively addressed the extent of
Available online 16 June 2022
extra- and intra-micellar oxidation of polysorbate in pharmaceutically relevant buffers. This study utilizes
2,20 -azobis(2-methylpropionamidine)dihydrochloride (AAPH), a peroxyl radical-generating initiator, C11-
Keywords:
BODIPY(581/591), a lipid peroxidation probe, and fluorescence spectroscopy to reveal that both intra- and
Oxidation
BODIPY
extra-micellar oxidation proceed in pharmaceutically relevant phosphate and histidine buffers. It is further
Polysorbate demonstrated that the relative extent of oxidation observed in the intra- and extra-micellar compartments is
Micelle similar irrespective of the buffer system.
Radicals © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Introduction and epoxy-products of fatty acid oxidation, oxo-nonanoate, petrose-

Polysorbates are widely used in the biopharmaceutical industry for
the formulation of proteins because they help to prevent protein
aggregation and surface adsorption. They have been included in many
commercialized products and a favorable safety profile for polysor-
bates is generally expected based on historical evidence.1 While there
is no question that the inclusion of polysorbates in a biopharmaceuti-
cal formulation can provide significant benefits, the understanding of
polysorbate degradation has grown tremendously over the recent
years and it has become known that polysorbates are far from being
inert excipients. Specifically, oxidation and enzymatic hydrolysis have
been explored extensively and can be summarized in a growing list
of degradation products which includes free polyoxyethylene (POE),
POE sorbitan, POE isosorbide, various hydroperoxide-, keto-, hydroxy‑,
Abbreviations: AAPH, 2,2’-azobis(2-methylpropionamidine)dihydrochloride; BODIPY,
581/591 undecanoic acid BODIPY; cmc, critical micelle concentration; POE,
polyoxyethylene.
Declaration of interests: The authors declare that they have no known competing
financial interests or personal relationships that could have appeared to influence the
work reported in this paper.
* Corresponding author.
E-mail address: schoneic@ku.edu (C. Scho € neich).
1
Current addresses for Dr. Peters: Bristol Myers Squibb, 1 Squibb Drive, New Bruns-
wick, NJ, 08901.
2
Current addresses for Dr. Wei: Amgen Inc., 1 Amgen Center Drive, Thousand Oaks,
CA 91320.
linic acid, alcohol-, formate-, and hemiacetal-terminating groups of
POE oxidation, and free fatty acids.2-12 These degradation products
may directly or indirectly affect protein stability by physical and/ or
chemical means. For example, polysorbate degradation products can
promote the oxidation of proteins13,14 or cause the polysorbate con-
centration to drop below the critical micelle concentration (CMC)
such that the ability to efficiently inhibit protein aggregation is
lost.15,16 While a polysorbate concentration above the CMC is often
considered a requirement for efficient prevention of protein aggrega-
tion, its implications for polysorbate and/ or protein degradation are
less clear. More specifically, the presence of micelles creates intra-
micellar and extra-micellar compartments. A recent study found that
polysorbate oxidation could no longer be detected after polysorbate
micelles were disrupted by an organic solvent.17 This suggested that
polysorbate oxidation predominantly occurs in the intra-micellar
compartment. Molecular dynamics simulation shows that polysorbate
readily forms micelles without individual polysorbate molecules leav-
ing the micelle structure over simulation periods ranging from a few
nanoseconds up to 200 ns at temperatures of 24.85 °C and 26.85 °C;
it was also shown that the polar terminal groups of polysorbate face
the outside, that the POE chains are located in the middle, and that
the fatty acids make up the hydrophobic core of the micelle in an
aqueous environment.18-20 Considering that the majority of polysor-
bate molecules will form stable micelles and that the abovementioned
degradation products require oxidation of the hydrophobic

https://doi.org/10.1016/j.xphs.2022.06.011
0022-3549/© 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.
2436 B.-H. Peters et al. / Journal of Pharmaceutical Sciences 111 (2022) 2435−2444
polysorbate molecule components located in the inner micelle core,
oxygen must be present in the core. Indeed, oxygen is approximately
an order of magnitude more soluble in organic solvents.21,22 The
equilibration of singlet oxygen between intra- and extra-micellar
compartments in aqueous and organic solvent systems was demon-
strated as well.23 It is, therefore, not too surprising that polysorbate
oxidation may predominantly occur within the intra-micellar com-
partment. From a biopharmaceutical product development and stabil-
ity perspective, a more important issue to understand is the degree of
oxidation occurring in intra-micellar and extra-micellar compart-
ments since it could differentially affect individual formulation com-
ponents based on their distribution between both compartments and
their susceptibility to further react with intermediate oxidation prod-
ucts produced in either compartment.
The peroxyl radical generator 2,20 -azobis(2-methylpropionami-
dine)dihydrochloride (AAPH) was utilized in this study to induce oxi-
dative stress. The probe BODIPY 581/591 undecanoic acid (BODIPY)
has been applied in the past to monitor biologically relevant oxida-
tion reactions in model and cell membranes.24 Here we describe fluo-
rescence experiments that utilize BODIPY as a sensor molecule to
monitor the susceptibility of polysorbate-containing pharmaceutical
formulations toward oxidation and a simple approach to determine
the relative extent of intra- and extra-micellar oxidation in polysor-
bate containing buffers.
Materials and Methods
Materials
Polysorbate 80 N.F. (PS80, J.T. Baker, product #4117-04, batch
#0000190001) was purchased from Avantor (Radnor, PA). L-histidine
monohydrochloride monohydrate (product #53370), sodium phos-
phate monobasic monohydrate (product #S9638), sodium phosphate
dibasic (product #04276), 2,2’-azobis(2-methylpropionamidine)
dihydrochloride (AAPH, product #440914), and a-tocopherol (prod-
uct #T3251) were purchased from Millipore Sigma (Burlington, MA).
BODIPY 581/591 undecanoic acid (BODIPY, product #D3861) was
purchased from Thermo Fisher Scientific (Hampton, NH). Ethanol
(product #2701) was purchased from Decon Laboratories (King of
Prussia, PA). Ultra-purified water was produced fresh from a Water-
Pro PS Polishing System (Labconco, Kansas City, MO).
Sample Buffer Preparation
Buffers were 20 mM L-histidine chloride, pH 5.5 and 20 mM
sodium phosphate, pH 6.0. All were sterile filtrated with a Stericup
Millipore Express PLUS 0.22 mm PES filter system (Millipore Sigma)
and bubbled with oxygen for 1 h; the buffer pH was stable within
§0.1 pH units. Sample buffers were prepared to yield final concentra-
tions of 2 mg/ml PS80 and 0.38 mM BODIPY; final concentrations of
AAPH and a-tocopherol varied from 0 − 1 mM and 0 − 4.5 mM,
respectively.
Dynamic Light Scattering, UV vis Spectroscopy, and Fluorescence
Spectroscopy
Dynamic light scattering was performed in a flat clear bottom
384-well plate using a DynaPro Plate Reader (Wyatt Technology,
Santa Barbara, CA). A sample volume of 30 mL was added to a well.
Then, the plate was sealed to minimize evaporation and centrifuged
at 2000 rpm for 2 min to remove air bubbles. The chamber tempera-
ture was set to 40 °C and the plate was equilibrated for 10 min prior
to analysis. Then, three 10-second acquisitions were performed for
each sample every 40 min for 84 h. High polydispersity was accepted
as a true observation for the first 10 measurements of each sample
caused by the integration of a-tocopherol into micellar structures;
afterwards acquisitions with polydispersity > 0.2 were excluded
from analysis as false readings. A total of 6 samples were analyzed for
each buffer composition.
UV Vis spectroscopy was performed with an Agilent 8453 UV−vis-
ible spectrophotometer (Agilent Technologies, Santa Clara, CA). A
sample volume of 250 mL was pipetted into a 1-cm quartz cuvette
and the absorbance of each sample was obtained from 200 to 800 nm.
Fluorescence spectroscopy was performed with a PTI Fluorometer
(Photon Technology International, Birmingham, NJ), consisting of a
Xenon arc lamp light source, a water flow-through temperature con-
trolled, rotating sample holder for up to 4 cuvettes, and a photomulti-
plier detector. Sample volumes of 1 mL were pipetted into a
10 £ 10 mm quartz cuvette and placed in the sample holder. Detec-
tion of non-oxidized BODIPY was performed with excitation set to
581 nm and emission to 595 nm, and detection of oxidized BODIPY
was achieved with excitation set to 485 nm and emission to 520 nm.
The integration time was set to 1 s per data point. Emission and exci-
tation slits were set to 5 nm. The temperature setting was 40 °C for
all reactions involving AAPH and a-tocopherol, and the temperature
was set to 25 °C for measurement of the thermal stability of samples
(vide infra).
Thermal Stability
Sample buffers contained 2 mg/mL PS80 and 0.38 mM BODIPY.
Three individual samples were prepared for each storage conditions
by pipetting 0.8 mL sample buffer into 2 mL type I borosilicate glass
vials (product #68000313). The vials were stoppered (product
#19,700,317) and sealed (product #54,131,770, West Pharmaceuti-
cals, Exton, PA), placed in cardboard boxes, and incubated at 5 °C,
25 °C, and 40 °C for 1 month. Three additional vials were frozen at

20 °C as ‘T0’ samples and stored until further analysis. All buffers
and samples were protected from light and kept on ice at all times
except during incubation. Immediately following collection, samples
were frozen and stored at
20 °C until further analysis.
Results
The change of BODIPY fluorescence in 0.2% PS80 over time is
shown in Fig. 1. In this first exploratory experiment, a fluorescence
measurement was performed approximately every 77 s. Both buffer
systems, 20 mM phosphate, pH 6.0 and 20 mM histidine pH 5.5, fol-
lowed the same trend during the 40 °C thermal incubation. That is,
the fluorescence signal for non-oxidized BODIPY (ex 581 nm, em
595 nm) remained constant in the absence of AAPH and decreased
continuously in the presence of AAPH. The signal loss over time also
correlated with increasing AAPH concentration. When monitoring
the fluorescence signal of oxidized BODIPY (ex 485 nm, em 520 nm)
both buffer systems followed the same trend as well but the signal
intensity increased continuously in the presence of AAPH, i.e., its
behavior was the inverse of the non-oxidized BODIPY species, as
would be expected. No oxidized BODIPY was detected in the absence
of AAPH. The amount of oxidized BODIPY increased continuously in
the presence of AAPH in a concentration dependent manner. This first
result was critical to provide evidence that BODIPY could be used as
an oxidation sensor in a polysorbate system. However, given that the
following experiments utilized complex systems containing up to 5
components, additional verification was necessary to ensure that
none of the other components or any degradation products that
could emerge during the thermal incubation interfered with the fluo-
rescence measurements. In other words, it was necessary to provide
evidence that the system remained within certain boundary condi-
tions over the incubation duration as described hereafter and that
the fluorescence measurement was specific to the oxidized and non-
 B.-H. Peters et al. / Journal of Pharmaceutical Sciences 111 (2022) 2435−2444 2437

Figure 1. Fluorescence signal intensity change of BODIPY in 0.2% PS80 during »2 h of incubation without AAPH (black), 0.5 mM AAPH (gray), 0.75 mM AAPH (blue) and 1 mM AAPH
(red) at 40 °C. A and B show the fluorescence signal of non-oxidized and oxidized BODIPY in phosphate buffer, C and D show the fluorescence signal of non-oxidized and oxidized
BODIPY in histidine buffer, respectively. Each sample was measured every 77 s. (For a reference to color in this figure legend please see the online version).

oxidized BODIPY species. The UV−Vis spectroscopy results of individ-
ual components or relevant combinations of components prior to
incubation at 40 °C and after 4 h incubation at 40 °C are shown in
Fig 2. The buffer system had little to no effect on the absorbance of
the individual components. AAPH showed absorbance between 300
and 400 nm and PS80 showed an absorbance starting at ca. 400 nm
increasing with shorter wavelengths, consistent with observations by
Wuelfing et al..25 BODIPY alone is poorly soluble in water24 and when
attempting to dissolve BODIPY in buffer solutions not containing
PS80, even after vigorous mixing, no clear absorbance peak could be
observed in the scan range from 300 to 700 nm. a-Tocopherol was
dissolved in buffer solutions containing 0.2% PS80 since it is practi-
cally insoluble in water. The absorbance of a-tocopherol in buffer sol-
utions containing PS80 was found identical to PS80 alone at λ >
300 nm. Two absorbance peaks, the main peak at »595 nm and a
minor peak at »550 nm, were observed when BODIPY was in the
presence of PS80 alone or both PS80 and a-tocopherol. None of these
observations changed after incubation for 4 h at 40 °C.
Another consideration was to test whether PS80 micelles were
present for the entire duration of the experiment. A loss of micellar
structures during the course of the experiments would not permit
evaluation of the extent to which oxidation occurs in the intra-micel-
lar and/or the extra-micellar compartments. The DLS results describ-
ing the average micellar size of PS80 in both buffer systems and in
combination with all other components are shown in Fig. 3. At a
glance, both buffer systems followed a similar trend. That is, the
micelle size appeared to remain constant throughout the 84 h of
incubation at 40 °C in the absence of AAPH. It also appeared practi-
cally unaffected by the presence of 0.38 mM BODIPY and/or 0.9 mM
a-tocopherol. In contrast, a decrease of micelle size over time is seen
for all systems exposed to 1 mM AAPH. It is also evident from Fig. 3
that all buffer systems containing a-tocopherol showed an increased
micelle size at the beginning of the experiment before eventually
reaching the same average size as the respective buffer systems with-
out a-tocopherol. Comparing the change of micellar size over time,
oxidative degradation appeared to proceed slightly faster in phos-
phate buffer than in histidine buffer while reaching a comparable
level after 72−84 h. In phosphate buffer containing AAPH, the micelle
radius decay rate constant in the exponential phase is ca. 2.2 £ 10
6
s
1, in good agreement with the reported rate constants for unimo-
lecular AAPH decomposition at 40 °C, (1.5−2.4) x 10
6 s
1 (see Dis-
cussion). For histidine buffer containing AAPH, the micelle radius
decay rate constant for the decay of the micelle radius in the expo-
nential phase is ca. 1.3 £ 10
6 s
1, reasonably close to the reported
rate constants for unimolecular AAPH decomposition at 40 °C, (1.5
−2.4) x 10
6 s
1 (see Discussion). Table 1 summarizes the average
micellar size of 0.2% PS80 observed during the first and last 12 h of
the experiment. On average, the micellar radius was ca. 4.8 nm at T0
and remained constant in absence of AAPH but decreased to ca.
4.2 nm after 84 h of incubation at 40 °C in presence of 1 mM AAPH.
The statistically significant size difference between samples contain-
ing a-tocopherol in absence of AAPH was caused by the increased
micelle size at T0 that returned to a comparable micelle size as the
AAPH free samples after equilibration as mentioned above.
Since BODIPY alone showed a slightly higher than baseline absor-
bance in the UV−Vis experiment, it had to be considered whether it
would elicit a fluorescence signal of its own. Fig. 4 shows that a fluo-
rescence signal for BODIPY was only detected in the presence of
PS80, but not for the buffers, BODIPY, or PS80 alone. Since AAPH and
a-tocopherol showed no absorbance in the wavelength range used
for excitation, a fluorescence signal arising from either of those com-
pounds could be excluded.
Having established that the fluorescence is specific to BODIPY in
PS80 and that the average micellar size does not significantly change
during the first few hours, the determination of oxidative degrada-
tion rates became possible. Representative fluorescence signals of
2438 B.-H. Peters et al. / Journal of Pharmaceutical Sciences 111 (2022) 2435−2444

Figure 2. UV−Vis absorbance spectra of BODIPY, PS80, AAPH, and a-tocopherol individually or in admixture in phosphate and histidine buffers at T0 and after 4 h incubation at 40 °
C. BODIPY (black line), BODIPY + PS80 (gray line), BODIPY + PS80 + a-tocopherol (blue line), AAPH (black dashed line), PS80 (gray dashed line), PS80 + a-tocopherol (blue dashed
line). (For a reference to color in this figure legend please see the online version).

Figure 3. Average polysorbate micelle radius in phosphate and histidine buffers measured by DLS over a 84 h incubation at 40 °C. 0.2% PS80 alone (filled black square), 0.2%
PS80 + 0.38 mM BODIPY (filled gray circle), 0.2% PS80 + 0.38 mM BODIPY + 0.9 mM a-tocopherol (filled blue diamond); 0.2% PS80 + 1 mM AAPH (open black square), 0.2%
PS80 + 0.38 mM BODIPY + 1 mM AAPH (open gray circle), 0.2% PS80 + 0.38 mM BODIPY + 0.9 mM a-tocopherol + 1 mM AAPH (open blue diamond). The average radius was calcu-
lated from 6 independent samples. (For a reference to color in this figure legend please see the online version).

non-oxidized BODIPY in 0.2% PS80, 1 mM AAPH, 20 mM phosphate or equilibration and to remain within the boundaries described above.
histidine buffer, and increasing concentrations of a-tocopherol are In both buffer systems, a-tocopherol slows down the oxidative deg-
shown in Fig. 5. Degradation rates were calculated from the linear radation initiated by AAPH. Specifically, the degradation rate slowed
range between 1 − 2 h to account for system component down with increasing a-tocopherol concentration (0.051 mM to
 B.-H. Peters et al. / Journal of Pharmaceutical Sciences 111 (2022) 2435−2444 2439

Table 1 supported this rationale because a-tocopherol did not further

Dynamic Light Scattering (DLS): Average Micelle Radius. decrease oxidation at the highest concentrations tested. Plotting the
Buffer System Avage Micelle Radius [nm] § St Dev degradation rates reported in Table 2 against a-tocopherol concen-
trations (Fig. 6) permitted fitting of a first order decay function with
0−12 h at 40 °C 72−84 h at 40 °C
excellent correlation coefficients (R2 = 0.97 for the phosphate buffer
20 mM Phosphate pH 6.0 + system, R2 = 0.94 for the histidine buffer system). Extrapolating the
− 4.91 § 0.23 4.85 § 0.06 derived first order decay function to infinite a-tocopherol concentra-
BODIPY 4.87 § 0.10 4.87 § 0.14
tion then yielded the relative amount of oxidation occurring in the
BODIPY + a-tocopherol 5.10 § 0.33 4.86 § 0.07 *
AAPH 4.65 § 0.25 4.14 § 0.05 * extra-micellar compartment: ca. 29.6% and 36.6% of oxidative degra-
AAPH + BODIPY 4.75 § 0.09 4.19 § 0.05 * dation occurred in the extra-micellar compartments for the phos-
AAPH + BODIPY + a-tocopherol 5.03 § 0.49 4.20 § 0.05 * phate and histidine buffer systems, respectively.
20 mM Histidine pH 5.5 +
Finally, evidence was required that the use of an oxidation sensor
− 4.84 § 0.11 4.78 § 0.05
BODIPY 4.82 § 0.13 4.76 § 0.07
such as BODIPY would be feasible for stability testing under pharma-
BODIPY + a-tocopherol 5.15 § 0.47 4.78 § 0.05 * ceutically relevant conditions, e.g., during pharmaceutical product
AAPH 4.80 § 0.17 4.21 § 0.04 * development. Fluorescence results after the incubation of 0.2% PS80
AAPH + BODIPY 4.84 § 0.14 4.29 § 0.05 * in 20 mM phosphate or histidine buffer containing 0.38 mM BODIPY
AAPH + BODIPY + a-tocopherol 5.10 § 0.58 4.33 § 0.10 *
at 5 °C, 25 °C, and 40 °C for 1 month are shown in Fig. 7. Both buffer
systems showed a decreased fluorescence signal intensity for non-
72−84 h samples denoted with * were significantly different (t-test with/without
oxidized BODIPY with increasing temperature. Vice versa, the signal
Welch correction as appropriate, p < 10
9) from their corresponding 0−12 h sample.
intensity for oxidized-BODIPY increased with increasing temperature
in both buffer systems. Overall, the histidine buffer, pH 5.5, appeared
2.7 mM) until it appeared that further increases in a-tocopherol con- to be slightly more prone to oxidative degradation than the phos-
centration (3.6 mM to 4.5 mM) did not further reduce the degradation phate buffer, pH 6.0, system at 5 °C and 25 °C, while a significant dif-
rate (Table 2). The protective action of a-tocopherol is due to compet- ference was seen at 40 °C. At 40 °C, practically no non-oxidized
itive abstraction of the phenolic hydrogen by the ROS and formation BODIPY was left in histidine buffer while more than 75% of non-oxi-
of a resonance stabilized radical (Scheme 1).26 Since a-tocopherol dized BODIPY remained in the phosphate buffer system.
can be considered virtually insoluble in water based on its log P value
(see below), the compound is expected to partition fully into PS80 Discussion
micelles. Unlike a-tocopherol, BODIPY can be found in both the
intra-micellar and extra-micellar compartment. As a result, inhibition BODIPY as a Sensor Molecule for the Susceptibility of Polysorbate
of oxidative degradation by a-tocopherol is limited to the intra- Containing Formulations to Oxidation
micellar compartment while extra-micellar oxidation is expected to
continue unaffected (for further details, see the description of “Exper- BODIPY contains a conjugated diene system that is susceptible to
imental Setup” in the Discussion). The experimental results oxidative degradation.24 The degradation through hydroxyl‑, alkoxyl-
, or peroxyl-radicals yields carboxylic acid derivatives.27 As a result,
the fluorescence of the non-oxidized BODIPY is shifted from red (ex
581 em 595) to green (ex 485 em 520) in the oxidized BODIPY deriva-
tives. Scheme 2 displays a proposed mechanism for AAPH-induced
BODIPY degradation that yields the carboxylic acids, reported ear-
lier,27 and aldehydic precursors.28 The fluorescence shift is probably
an immediate result of ROS addition to the conjugated diene system
(Scheme 2, labeled “1−400 ) which shortens the overall conjugated sys-
tem of BODIPY while subsequent reactions render the shortening of
the conjugated system irreversible. Scheme 2 representatively shows
the addition of an alkoxyl radical to BODIPY because these species
have been detected during AAPH decomposition by spin trapping
and electron spin resonance (ESR) spectroscopy29,30; analogous addi-
tion reactions are possible with hydroxyl and peroxyl radicals where
the latter are readily formed during AAPH decomposition and the
degradation of PS802. Based on the reported carboxylic acid and alde-
hydic degradation products,27,28 the addition of radicals to BODIPY
appears to occur predominantly in positions 2 and 4. BODIPY further
permits the monitoring of both oxidized and non-oxidized species
simultaneously.27This feature is shown in Fig. 1 by the inverse corre-
lation of non-oxidized BODIPY fluorescence decay and oxidized BOD-
IPY fluorescence increase over time in phosphate and histidine
buffers containing 0.2% polysorbate. Generally, BODIPY was found
susceptible to various reactive oxygen species (ROS) such as
hydroxyl, alkoxyl, and peroxyl radicals as well as peroxynitrite but
not directly to superoxide, nitric oxide, transition metals, or hydro-
peroxides alone.24,31 Key applications are related to the localization
of BODIPY in lipophilic environments and include the monitoring of
Figure 4. Fluorescence signal scan of non-oxidized BODIPY in phosphate and histidine
buffers (ex 581 nm, em 585 − 610 nm). Buffers alone (black line), 0.38 mM BODIPY ROS in cell membranes of fibroblasts,24 macrophages,32 or lympho-
alone (gray line) 0.2% PS80 alone (red line), 0.2% PS80 + 0.38 mM BODIPY (blue line). cytes33 as well as cell organelles such as mitochondria.34 This feature
(For a reference to color in this figure legend please see the online version). was also used to detect the peroxidation of arachidonic acid, a
2440 B.-H. Peters et al. / Journal of Pharmaceutical Sciences 111 (2022) 2435−2444

Figure 5. Fluorescence signal of non-oxidized BODIPY (ex 581 nm, em 595 nm) in phosphate and histidine buffers containing 0.2% PS80, 0.38 mM BODIPY, 1 mM AAPH, and various
concentrations of a-tocopherol over a 2 h incubation at 40 °C. Each sample was measured every 77 s. (For a reference to color in this figure legend please see the online version).

polyunsaturated fatty acid (PUFA), in a cumene hydroperoxide /
hemin system.31 Polysorbate presents a system with similar features
when considering its ability to form micelles with a hydrophobic
core and the presence of unsaturated fatty acid esters as part of the
molecular structure. Particularly compendial grade polysorbate 80
contains significant amounts of unsaturated fatty acid esters, i.e., at
least 58% oleic acid (monounsaturated) and up to 18% and 4% of lino-
leic and linolenic acid (polyunsaturated), respectively.35 Pap et al.
also mentioned that BODIPY was sensitive to oxidation when
exposed to Fe2+/H2O2 or AAPH.31 Since AAPH is regularly used as per-
oxyl radical initiator in oxidative stress testing of polysorbates,3,36 it
appeared likely that BODIPY could serve as a sensor molecule for oxi-
dative polysorbate degradation. The AAPH-dependent oxidation of
BODIPY shown in Fig. 1 supports this hypothesis and is well aligned
with the observations made by Pap et al..31
Experimental Setup
The successful application of BODIPY as an oxidation sensor mole-
cule was in line with our expectations. However, it is quite challeng-
ing to measure oxidative degradation of polysorbate in aqueous
Table 2
Rates of BODIPY Oxidation and Linear Correlation Coefficients.
Buffer System a-tocopherol Average
Degradation Rate
[mM] § CI (95%)
[min
1] * 102
buffer solutions and attribute the extent of oxidative degradation to
the intra- and extra-micellar compartments, respectively, which
polysorbate forms at concentrations above its CMC. This challenge
required a more complex experimental setup which contained up to
five compounds in fixed and variable concentrations. Specifically, the
distribution of these compounds within the intra- and extra-micellar
compartments and their potential to interfere with the BODIPY fluo-
rescence signals was addressed. The fixed components were (I) either
20 mM phosphate or histidine buffer, (II) 0.2% w/v polysorbate, (III)
1 mM AAPH, except where specified otherwise, and (IV) 0.38 mM
BODIPY; the variable component was a-tocopherol with concentra-
tions ranging from 0.051 to 4.5 mM. The distribution of these com-
pounds within the intra- and extra-micellar compartments at
equilibrium can be approximated from their log p values. Considering
the published log p values for non-oxidized and oxidized forms of
BODIPY (2.9 and 2.8 at pH 6, respectively24) and the volume ratio of
polysorbate in aqueous buffer, it was calculated that »60% of non-
oxidized BODIPY and »55% of oxidized BODIPY are located in the
intra-micellar compartment at equilibrium, i.e., the BODIPY concen-
tration in the intra-micellar compartment was practically constant
irrespective of its oxidation state. In lieu of a published log p value for
AAPH, the value was predicted to be »0.6 using ChemDraw (Perki-
nElmer Informatics, Waltham, MA; version 16.0.1.14 (77)). Therefore,
at equilibrium approximately 0.75% of AAPH is located in the intra-
micellar compartment. a-Tocopherol has a published log p of 10.7.37
Therefore, it can be considered exclusively located in the intra-micel-
Correlation
lar compartment.
Coefficient
R2

20 mM Phosphate 0.051
0.482 § 0.008 0.989

pH 6.0 0.38 mM 0.225
0.463 § 0.010 0.984
BODIPY 1 mM 0.45
0.380 § 0.011 0.968
AAPH 40 °C 0.9
0.237 § 0.011 0.931
1.8
0.204 § 0.005 0.974
2.7
0.170 § 0.006 0.959
3.6
0.156 § 0.006 0.945
4.5
0.153 § 0.007 0.925
20 mM Histidine pH 0.051
0.414 § 0.015 0.951
5.5 0.38 mM BOD- 0.225
0.427 § 0.008 0.987
IPY 1 mM AAPH 0.45
0.374 § 0.009 0.978
40 °C 0.9
0.260 § 0.009 0.955
1.8
0.198 § 0.010 0.958
2.7
0.178 § 0.007 0.947
3.6
0.193 § 0.008 0.934
4.5
0.165 § 0.009 0.909
Each average degradation rate was calculated from 3 independent samples. Scheme 1. Reaction of a-tocopherol with ROS.
 B.-H. Peters et al. / Journal of Pharmaceutical Sciences 111 (2022) 2435−2444 2441

Figure 6. Relative degradation rates of 0.38 mM non-oxidized BODIPY in 0.2% PS80, 1 mM AAPH, and phosphate (A) or histidine (B) buffer at various concentrations of a-tocopherol
(black squares) at 40 °C, fitted with a first order decay curve (blue). All relative degradation rates are calculated by normalization to the degradation of BODIPY in the absence of
a-tocopherol (For a reference to color in this figure legend please see the online version).

Figure 7. Fluorescence signal of non-oxidized BODIPY (A, ex 581 nm, em 595 nm) and oxidized BODIPY (B, ex 485 nm, em 520 nm) in phosphate (black) and histidine (gray) buffers
containing 0.2% PS80 and 0.38 mM BODIPY at T0 and stored for 1 month at 5 °C, 25 °C, and 40 °C. Error bars represent standard deviation. (For a reference to color in this figure leg-
end please see the online version).

Each AAPH molecule decomposes into 2 carbon-centered radi-
cals during its thermal composition and each of these can react
with an oxygen molecule to form a peroxyl radical.38 The decom-
position rate is not affected significantly by pH values below < 8
and the rate constant for this first order decomposition process at
40 °C was reported as (1.5−2.4) x 10
6 s
1 in aqueous systems,38
agreeing well with the 40 °C decomposition rate constant of
2.26 £ 10
6 s
1 that may be calculated from earlier AAPH decom-
position experiments.39 While each AAPH molecule generates two
initial carbon-centered radicals, the generation of free radicals is
limited by cage recombination so that the rate constant of free rad-
ical formation at 37 °C is 1.36 £ 10
6 s
1.40 Dougherty reports
activation parameters of DH6¼ = 29.1 kcal/mol and DS6¼ = 8.5 e.u.
for the decomposition of AAPH,39 from which the rate constant of
free radical formation at 40 °C can be extrapolated to 2.13 £ 10
6
s
1, consistent with the range of rate constants, (1.5−2.4) x 10
6
s
1, provided by Werber et al.38 Hence, a 2 h incubation of 1 mM
AAPH at 40 °C forms a total of ca. 15.34 mmol/L free radicals. Con-
sidering that an air-saturated aqueous solution contains ca. 200
mmol/L oxygen at 40 °C21 and that peroxyl radicals release one
equivalent of oxygen during bimolecular reaction to generate
alkoxyl radicals,38 only ca. 3.8% of the initially dissolved oxygen
will be consumed in the 2 h fluorescence experiments (Figs. 1, 5,
and 6). Furthermore, fluorescence cuvettes were filled only with
0.8 mL solution, leaving a headspace volume of 2.7 mL. The atmo-
spheric oxygen content of 20.9% thus provides an additional 25
mmol oxygen, sufficient to fully saturate the 0.8 mL sample more
than 150 times. It is clear that oxygen availability was not limiting
at any point.
The UV−Vis spectroscopy results (Fig. 2) clearly show that neither
the buffers, polysorbate, AAPH, or BODIPY alone have a strong absor-
bance at wavelengths above 450 nm and, thus, no significant
2442 B.-H. Peters et al. / Journal of Pharmaceutical Sciences 111 (2022) 2435−2444
Scheme 2. Reaction of BODIPY with ROS affects fluorescence through shortening of the conjugated system.
absorbance of wavelengths used during the fluorescence experiment
is expected. This was the case for at least 4 h of incubation at 40 °C,
twice the duration used in the fluorescence experiments, so that the
absorbance from any oxidation product could be ruled out as well.
Interestingly, BODIPY showed only absorbance in the presence of
polysorbate. This absorbance profile closely matched the one in the
vendor’s product documentation and was not altered by the addition
of a-tocopherol. BODIPY alone showed some limited absorbance at
T0 in both buffer systems (Fig. 2A and B); it may be that BODIPY
adsorbed to the quartz cuvette due to its limited solubility in the
aqueous environment which resulted in this unexpected absorbance
signal. More importantly, this artifact did not interfere with the fluo-
rescence measurements. The fluorescence scans with excitation
wavelength set to 581 nm over an emission wavelength range from
585 nm to 610 nm clearly show that neither the buffer solutions, nor
polysorbate or BODIPY alone exhibited any fluorescence signal. Only
the combination of BODIPY with polysorbate resulted in a signal with
maximum emission at 595 nm (Fig. 4). A similar observation was
made for a dioleoyl phosphatidyl choline liposome system containing
BODIPY and 20 mM Tris−HCl buffer at pH 7.4: BODIPY was non-fluo-
rescent in buffer alone but became highly fluorescent after incorpo-
ration into the liposomes.41 Finally, a potential change of the micellar
structure during oxidative polysorbate degradation was taken into
account. Micellar size was practically constant over 3+ days at 40 °C
in either buffer system in the absence of AAPH (Fig. 3). Oxidative deg-
radation resulted in a smaller average micellar size; the size reduc-
tion progressed the fastest with AAPH alone, followed by
AAPH + BODIPY, and finally AAPH + BODIPY + a-tocopherol. Based on
the respective exponential micellar decay rate constants of ca.
2.2 £ 10
6 s
1 and 1.3 £ 10
6 s
1, degradation induced by AAPH
alone progressed faster in phosphate buffer than histidine buffer.
However, the average micellar radius after 3+ days at 40 °C was very
similar with 4.14 and 4.21 nm. Samples containing a-tocopherol also
showed an unusually large average micellar size at the beginning of
the experiment which is likely a result of an intra-micellar rearrange-
ment and equilibration phase while the sample temperature is
brought from room temperature to 40 °C at the beginning of the
experiment. Most importantly, the results confirm that the average
micelle radius remained within 4.7 − 5.2 nm irrespective of the buffer
and number of compounds used within the first 12 h while a clear
reduction is seen at later time points (Table 1). This duration easily
covers the 2 h fluorescence experiment and suggests that micelle size
changes are not a key contributor to fluorescence signal changes in
this setup.
Intra-micellar and Extra-micellar Oxidation in Polysorbate Containing
Buffers
The fundamental concept of this experiment is that (I) the fluores-
cence signal represents intra-micellar BODIPY exclusively while BOD-
IPY itself is soluble in both the intra-micellar and extra-micellar space
and that (II) intra-micellar degradation can be controlled with
a-tocopherol which is only soluble in the intra-micellar space.
Increasing the a-tocopherol concentration reduces BODIPY degrada-
tion (Fig. 5). Considering the constant degradation phase, a reduction
in degradation rate can be confirmed by visual observation alone
when increasing the a-tocopherol concentration from 0.051 mM to
1.8 mM while little to no further reduction occurs at higher concen-
trations irrespective of the buffer system. Importantly, BODIPY degra-
dation never becomes zero. This suggests that oxidation occurs in
both the intra- and extra-micellar space. Since a fluorescent BODIPY
signal is only observed for intra-micellar BODIPY, the linearity of the
change in fluorescence furthermore indicates that both the non-oxi-
dized and oxidized BODIPY species continuously equilibrate at a con-
stant rate between the intra- and extra-micellar space. This is a
critical requirement to derive the relative intra-micellar and extra-
micellar contributions to degradation displayed in Fig. 6 and consider
them meaningful. These observations and the experimental setup
also rule out the possibilities that (I) oxidation could occur only in the
intra-micellar space because in that case the degradation rate should
approach zero with increasing a-tocopherol concentrations, and that
(II) oxidation occurs only in the extra-micellar space because in that
case a-tocopherol should not have any effect and the loss of fluores-
cence signal intensity should depend only on the equilibration of
both BODIPY species between the intra- and extra-micellar space.
It was recently suggested that polysorbate oxidation predomi-
nantly occurs in the intra-micellar space.17 The authors provided a
convincing rationale for intra-micellar polysorbate oxidation that is
in agreement with the additional literature provided above. Specifi-
cally, the absence of extra-micellar polysorbate oxidation was
reported after dissolving polysorbate micelles with t-butanol.17 Our
results with a-tocopherol support intra-micellar oxidation reactions
of BODIPY in polysorbate micelles, and show that, in fact, the majority
of the AAPH-induced oxidation reactions (ca. 2/3) proceed in the inte-
rior of the polysorbate micelles. This finding is significant, as ca. 99%
of AAPH is distributed to the extra-micellar environment. At this
point we cannot specify whether the intra-micellar BODIPY oxidation
is initiated by the ca. 0.75% of AAPH distributed to the intra-micellar
environment, or radical intermediates from AAPH, polysorbate, and/
 B.-H. Peters et al. / Journal of Pharmaceutical Sciences 111 (2022) 2435−2444
or BODIPY, generated outside the micelles, which exchange with
micellar components or partition into the micelles. However, the
exclusive distribution of the chain-breaking antioxidant a-tocopherol
into the micelles, together with the maximally 70.4% inhibition of
BODIPY oxidation at a-tocopherol concentrations ≥ 4.5 mM clearly
indicate that the majority of BODIPY oxidation proceeds within the
micellar environment. The close proximity of unsaturated fatty acids
within the micelles favors chain propagation reactions, rationalizing
these results.
Applications for this Experimental Setup
Long term stability (LTS), accelerated stress testing, and selection
of formulation buffers and excipients are common tasks performed
during drug product development. To accelerate development times,
platform formulations, i.e., standardized formulations with rich his-
torical data across multiple potential drug molecules that are known
to provide sufficient stabilization under most circumstances, are
employed. However, as the pharmaceutical industry embraces novel
modalities and administration regimens, more often than not modifi-
cations to these platform approaches will become necessary to
ensure product stability. Oxidation is a key risk assessed during LTS
and accelerated stress testing, and its degree can be influenced by a
chosen formulation. From this point of view, the question is not
“does oxidation occur in intra- or extra-micellar spaces?”, but (I) “to
which extent oxidation occurs in the intra- and extra-micellar
spaces?”, (II) “how does this level of oxidation affect drug molecule
(s) and/or excipients?”, and (III) “how does variability associated
with different batches, manufacturers, and age of the surfactants
affect intra- and extra-micellar oxidation?” These questions are criti-
cal to ensure that patients are treated with a safe and high-quality
product at all times. The answer to the first question is given in Fig. 6
for the buffer systems presented here. Statistical evaluation by means
of an unpaired t-test further confirmed that the phosphate and histi-
dine buffers resulted in a similar degree of intra- and extra-micellar
oxidation. The second question, unfortunately, is product specific. In
the future, this experimental setup may be useful to identify exci-
pients that reduce intra- or extra-micellar oxidation as needed in
case a platform approach fails to provide the necessary protection
against oxidation. The micellar size decay shown in Fig. 3 may also
indicate that intra-micellar oxidation products can be released into
the extra-micellar compartment. Depending on their nature, these
oxidation products could impact protein stability. Any answer to the
third question has to await further experimental testing. The content
of mono- and polyunsaturated fatty acids in polysorbate may vary
between batches, potentially affecting the oxidation rates.
Importantly, the experimental setup is not limited to AAPH-
induced oxidation or a brief incubation period. The data in Fig. 7
show that oxidation can easily be monitored for sample buffers with-
out AAPH and a-tocopherol when stored for 1 month at various tem-
perature settings. Here, the overall oxidation increased with
temperature in both buffer systems and, in comparison, the histidine
buffer showed a higher degree of oxidation at a given temperature
than the phosphate system. Interestingly, under accelerated storage
conditions both buffer systems showed a clear difference with the
probe molecule (BODIPY) being almost fully oxidized in the histidine
buffer at 40 °C. This behavior, reduced AAPH-induced oxidative deg-
radation but accelerated oxidative degradation during accelerated
stability testing, was observed previously for polysorbate 2036. The
results here confirm this observation for PS80 as well and, most
importantly, are also identified by BODIPY as the probe molecule.
Therefore, this experimental setup may be useful to quickly assess
the oxidative burden of a formulation under LTS and accelerated sta-
bility testing conditions. Prior to any such application, it should be
assessed whether a therapeutic protein in the formulation affects
2443
BODIPY fluorescence when considering that water-soluble BODIPY
derivatives were specifically designed for their utility as hydrophobic
sensor molecules and used successfully to probe protein
surfaces.42.43 Structurally, the enhanced fluorescence of these deriva-
tives in the vicinity of hydrophobic protein surfaces depends on the
rigidity of an aryl moiety on the 8-position of the fluorophore42.43
which is absent in the BODIPY derivative used here (C11-BODIPY
(581/591)). However, potential interactions of C11-BODIPY(581/591)
with hydrophobic protein surfaces have so far not been widely inves-
tigated.
Conclusion
A fluorescence spectroscopy method was used to rapidly quantify
intra- and extra-micellar oxidation in pharmaceutically relevant
buffer solutions that contain polysorbate. This method provides unbi-
ased qualitative confirmation and quantitation of intra- and extra-
micellar oxidative stress as micellar structures are maintained
throughout the full duration of all experiments. The results clearly
demonstrate that oxidation occurs in both intra- and extra-micellar
compartments and that it occurred to a similar extent in the pre-
sented phosphate and histidine buffer systems. Specifically, in the
phosphate and histidine buffer systems exposed to AAPH, 70.4% and
63.4% of oxidation occurred in the intra-micellar space, respectively.
Finally, it was demonstrated that this fluorescence measurement
approach is a useful, simple surrogate measurement when attempt-
ing to compare the oxidative stress in buffer systems during acceler-
ated stress testing. Ultimately, this study establishes an opportunity
to gain detailed insights into oxidative stress encountered by active
pharmaceutical ingredients and excipients in pharmaceutically rele-
vant buffer systems and enables rationale drug product development
decisions to provide patients with the highest possible quality of
pharmaceuticals.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgment
Dr. Peters was the recipient of a Research Fellowship (PE 2561/1-
1) of the German Research Foundation (DFG).
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