Sunteți pe pagina 1din 8

RESEARCH ARTICLE – Pharmaceutical Biotechnology

Selective Oxidation of Methionine and Tryptophan Residues in a


Therapeutic IgG1 Molecule
EMILIEN FOLZER,1,2 KATHARINA DIEPOLD,3 KATRIN BOMANS,3 CHRISTOF FINKLER,4 ROLAND SCHMIDT,1 PATRICK BULAU,3
JÖRG HUWYLER,2 HANNS-CHRISTIAN MAHLER,1 ATANAS V. KOULOV4
1
Pharmaceutical Development and Supplies, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel,
Switzerland
2
Division of Pharmaceutical Technology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
3
Analytical Development and Quality Control, Pharma Technical Development Biologics Europe, Roche Diagnostics GmbH, Penzberg,
Germany
4
Analytical Development and Quality Control, Pharma Technical Development Biologics Europe, F. Hoffmann-La Roche Ltd., Basel,
Switzerland

Received 9 January 2015; revised 5 April 2015; accepted 23 April 2015


Published online 25 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24509

ABSTRACT: Oxidation of methionine and tryptophan are common degradation pathways for monoclonal antibodies and present major
analytical challenges in biotechnology. Generally, protein oxidation is detectable in stability and/or stressed samples (e.g., exposed to
hydrogen peroxide, UV light, or metal ions). The induced chemical modifications may impact the biological activity of antibodies and may
have biological consequences. However, these effects and the contribution of individual protein modifications are difficult to delineate
as different amino acids are often oxidized simultaneously and accompanied by other degradants such as aggregates, especially in
forced degradation studies. Here, we report a new method to obtain selective oxidation of methionine or tryptophan by using oxidation
reagents combined with large excess of free tryptophan or methionine, correspondingly. More specifically, using hydrogen peroxide or
tert-butyl hydroperoxide in combination with addition of free tryptophan allowed for selective oxidation of methionine. Conversely, the
use of 2,2-azobis(2-amidinopropane) dihydrochloride in combination with free methionine resulted in selective tryptophan oxidation,
whereas methionine oxidation was not significantly altered. This novel stress model system may prove to be valuable tool in future
mechanistic studies of oxidative degradation of protein therapeutics.  C 2015 Wiley Periodicals, Inc. and the American Pharmacists

Association J Pharm Sci 104:2824–2831, 2015


Keywords: therapeutic proteins; protein oxidation; methionine; tryptophan; mass spectrometry; protein A binding

INTRODUCTION idized methionine or tryptophan in the complementary deter-


mining regions may induce loss of potency and antigen-binding
Oxidation is a common degradation pathway that may occur
capabilities.14,15
during the life cycle of proteins and peptides.1–5 The resulting
During the last 30 years, cysteine, histidine, methionine,
chemical modifications may affect in vitro stability and in vivo
phenylalanine, tryptophan, and tyrosine residues were identi-
biological functions.5–17 The position of oxidized amino acids
fied as major oxidation sites in proteins because of the high
in the protein sequence is crucial for their impact on protein
reactivity of sulfur atoms and aromatic rings toward various
structure, function, and biological response, especially in the
reactive oxygen species.5,18 Oxidation might be induced during
case of monoclonal antibodies (mAbs) where binding proper-
production, processing, or storage (e.g. because of the possi-
ties are essential to reach target antigen. It has been previ-
ble reactive impurities present or formed in excipients such
ously reported that oxidation of methionine from the Fc region
as polyethylene glycol or polysorbate).19,20 The generation of
can reduce binding to the neonatal Fc receptor and biologi-
peroxides through autooxidation of these polymeric compounds
cal half-life of the concerned mAb.8,10,11 Such a change in the
might favor oxidation of these amino acid residues. The pres-
pharmacokinetic profile is highly dependent on the position of
ence of transition metals may also lead to oxidation, for ex-
the oxidation. A recent study reported that M252 oxidation
ample, via the Fenton reaction.21,22 One option to minimize or
was the dominant contributor to PK impairment observed in
prevent oxidation of the active protein is the addition of antioxi-
mice transgenic for the human FcRn receptor. In this study,
dants, chelating agent, or radical scavengers to the formulation
an increased plasma clearance was only observed if both IgG
buffer, if acceptable from a toxicological and regulatory per-
HCs were oxidized at M252.17 Moreover, the formation of ox-
spective and thus, if considered acceptable and safe to clinical
use.20,22,23
In order to assess the potential susceptibility of protein
Correspondence to: Atanas Koulov (Telephone: +41-616877402; Fax: +41- products to oxidation, different oxidation stress conditions
616870280; E-mail: atanas.koulov@roche.com) have been studied, such as hydrogen peroxide (H2 O2 ), tert-
This article contains supplementary material available from the authors upon butyl hydroperoxide (t-BHP), 2,2-azobis(2-amidinopropane)
request or via the Internet at http://wileylibrary.com.
dihydrochloride (AAPH), transition metal ions, or UV
Journal of Pharmaceutical Sciences, Vol. 104, 2824–2831 (2015)

C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association exposure.15,22–25 Such relatively harsh treatments often

2824 Folzer et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2824–2831, 2015


RESEARCH ARTICLE – Pharmaceutical Biotechnology 2825

MATERIALS AND METHODS


An IgG1 therapeutic mAb was chosen as a model protein for
the present study. The mAb (IgG1) solution was provided at
25 mg/mL in 51 mM sodium phosphate, 6 % trehalose, and 0.04
% polysorbate 20 (pH 6.2) by F. Hoffmann-LaRoche Ltd. (Basel,
Switzerland).
Hydrogen peroxide, t-BHP, AAPH, ethylenediaminete-
traacetic acid (EDTA), DTT (dithiothreitol), iodoacetic acid,
and cysteine were purchased from Sigma–Aldrich (St. Louis,
Missouri), and Dulbecco’s phosphate-buffered saline 10× from
GIBCO (Invitrogen, San Diego, California). Guanidine hy-
drochloride (Gdn-HCl) from AppliChem (Darmstadt, Germany)
was used during the study.
Trifluoroacetic acid (TFA), formic acid (FA), acetic acid
(AcOH), acetonitrile, hydrochloric acid (HCl), potassium
Figure 1. Schematic representation of experimental strategy to gen-
chloride (KCl), and sodium chloride (NaCl) were pur-
erate selectively oxidized antibody species. Combination of hydrogen chased from Merck (Whitehouse Station, New Jersey).
peroxide (H2 O2 ) or tert-butyl hydroperoxide (t-BHP) with addition of Tris(hydroxymethyl)-aminomethan (Tris) was obtained from
free tryptophan under well-defined experimental conditions leads to se- Ajinomoto (Raleigh, North Carolina).
lective oxidation of methionine residues. Combination of 2,2-azobis(2- Papain from Carica papaya (10 mg/mL) was delivered by
amidinopropane) dihydrochloride (AAPH) with addition of free methio- Roche Diagnostic (Indianapolis, Indiana). Trypsin Proteomics
nine allows to selective tryptophan oxidation under well-defined exper- grade was provided by Roche Diagnostics GmbH (Penzberg,
imental conditions. Germany).
L-tryptophan was purchased from Sigma–Aldrich and was
dissolved directly in mAb solution to reach a final tryptophan
concentration of 4 mg/mL in solution (19.6 mM). H2 O2 or t-
result in a myriad of modifications on several amino-acid
BHP were used as oxidants and added into the mAb solution
residues, such as histidine, phenylalanine, and tyrosine, for
with or without prior addition of free tryptophan to obtain final
example.5,22,26 However, methionine and tryptophan are the
concentrations of oxidants between 0% and 5%.
residues that are oxidized most commonly and to the highest
L-methionine was purchased from Sigma–Aldrich and was
extent when exposed to such conditions.23,24,27–29
dissolved directly in mAb solution to reach a final methionine
In recent investigations, antibody samples containing both
concentration of 40 mg/mL in solution (0.26 M). As oxidant,
methionine and tryptophan oxidation products were used to
AAPH was then added into the mAb solution in the presence
draw conclusions regarding impact on target binding and re-
or absence of free methionine to final oxidant concentrations
duced binding affinity.14–16 Still, one major impediment to the
between 0% and 5%.
interpretation of such studies is the fact that frequently both
The final mAb concentration for each prepared sample was
methionine and tryptophan residues are oxidized concurrently,
18.5 mg/mL. Samples were incubated under protection from
making the definition of the individual contribution of these
direct light at 5°C, 25°C, or 40°C and aliquots were collected at
residues to the overall effects difficult to delineate. Thus, the
1, 6, 24, or 120 h. A control sample, without oxidant, was also
preparation of proteins in which methionine and tryptophan
incubated and analyzed for each time point and temperature.
have been selectively oxidized would be very helpful, in or-
The oxidation was stopped by buffer exchange using disposable
der to improve the understanding of the relationship between
PD-10 desalting columns (GE Healthcare, Buckinghamshire,
structure and functional consequences.
UK).
In a recent report, it has been demonstrated that the addi-
tion of free L-methionine and L-tryptophan to forced oxidation
reactions of parathyroid hormone can effectively limit the ox-
idation of methionine and tryptophan residues, respectively.22 INSTRUMENTATION AND SAMPLE ANALYSIS
However, so far, this observation has not been further tested A Waters Alliance 2695 HPLC system equipped with a 2487 UV
and reported when mAbs are used. In the present study, we detector and Empower2 software (version 7) (Waters, Milford,
demonstrate the utility of this competition approach using a Massachusetts) was used throughout the study. The determi-
new method for selective oxidation of methionine or tryptophan nation of protein concentration before injection was performed
residues in a model mAb from the IgG1 subclass (the experi- on a SoloVPE spectrometer from C-Technologies (Bridgewater,
mental strategy is depicted in Fig. 1). In order to achieve this New Jersey).
aim, a large excess of free amino acids was added to the re-
action conditions, in order to protect the corresponding amino
Size-Exclusion HPLC
acid in the mAb when applying different oxidants (t-BHP, H2 O2 ,
AAPH). Different reaction conditions such as incubation tem- Samples were analyzed by UV absorbance detection at 280 nm.
perature, reaction time, and oxidant concentration were evalu- A TSK G3000 SWXL column (5 :m, 250A, 7.8 × 300 mm2 )
ated. Oxidation products in the model IgG1 were characterized (Tosoh Bioscience, Tokyo, Japan) was used for separation. The
using size-exclusion chromatography (SEC), protein A-based mobile phase (200 mM phosphate, 250 mM KCl, pH 7.0) was
affinity chromatography, reversed-phase high-pressure liquid pumped at a flow rate of 0.5 mL/min. Sample size for analysis
chromatographic (RP-HPLC), and mass spectrometry. was 25 :g. The stationary phase was kept at 25 ± 2°C.

DOI 10.1002/jps.24509 Folzer et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2824–2831, 2015


2826 RESEARCH ARTICLE – Pharmaceutical Biotechnology

Protein A Chromatography Table 1. Experimental Parameters for Artificial Oxidation of Model


IgG1
Analytical protein A chromatography was performed, as de-
scribed previously on a POROS R
A (4.6 × 50 mm2 ) column Parameter Value/Range
(Applied Biosystems, Foster City, California).12 The stationary
Oxidant H2 O2 , t-BHP, AAPH
phase was kept at 23 ± 2°C. Oxidant concentration 0.0%, 0.05%, 0.1%, 0.2%,
0.5%, 1.0%, 2.0%, 5.0%
Reversed-Phase HPLC
Antioxidant None, L-methionine (0.26 M),
Protein samples were diluted to a final concentration of L-tryptophan (19.6 mM)
1 mg/mL in Tris buffer (0.1 M Tris, 4 mM EDTA, and 1 mM cys- Incubation temperature 5°C, 25°C, 40°C
teine, pH 7.4). Fifty microliter of 0.1 mg/mL papain was added Incubation time 1, 6, 24, 120 h
Model mAb concentration 18.5 mg/mL
to 500 :L of the mAb1 solution before incubation at 37°C for
2 h. Following incubation, 11 :L of 1 M DTT was added to each The oxidant, oxidant concentration, antioxidant, incubation temperature, and
tube and samples were incubated for another 30 min at 37°C. time were varied to find optimal condition for selective oxidation of methionine
Analysis of oxidized and intact mAb was carried out using a or tryptophan, respectively. The mAb concentration was kept constant for the
complete set of samples.
BioBasic Phenyl column (5 :m, 300A, 2.1 × 250 mm2 ) from
Thermo Fischer Scientific (Dreieich, Germany). Mobile phase
A was 0.09% TFA in water. Mobile phase B was 0.08% TFA
in acetonitrile. Flow rate was 0.45 mL/min. The chromatogra- The fragmentation was induced by low-energy CID using he-
phy was performed using a linear gradient from 35% to 40% lium as collision gas (“top5” mode). Data acquisition was con-
solvent B in 16 min. Mobile phase B (40%) was maintained trolled by Xcalibur software (Thermo Fisher Scientific) using a
during 4 min and finally 35% solvent B was pumped during manual acquisition mode. Oxidized amino acids were identified
10 min. Sample size for analysis was 8 :g. The fragment elu- from the MS/MS data using the software Proteomics Discoverer
tion was monitored by UV absorption at 215 nm. As the method (Thermo Fisher Scientific). The collision energy was adjusted
described above cannot be used to identify the tryptophan ox- according to stability and mass of the parent ion.
idation site in the protein sequence, it was utilized only as a Peptides of interest were identified manually by searching
rapid screening tool for tryptophan oxidation present in the for their theoretical m/z values within the experimental mass
fragment antigen-binding part of the heavy chain (HC-Fab). spectrum. For their quantification, specific ion current chro-
The tryptophan oxidation fraction was calculated by dividing matograms of peptides of interest were generated on the ba-
the area of the prepeak by the total peak area of the HC-Fab sis of their monoisotopic mass and detected charge states us-
peaks. ing GRAMS AI software (Thermo Fisher Scientific). Relative
amounts of nonoxidized and oxidized peptides were calculated
Proteolytic Digest of mAb
by manual integration of the corresponding peaks.
For detection and quantification of oxidized amino acids, the Besides the summarized large number of methionine and
mAb was digested with trypsin. First, the protein was dena- tryptophan oxidation sites (Table 1; Tables S1 and S2), no sig-
tured in 0.4 M Tris–HCl, 8 M Gdn-HCl, at pH 8.5 by diluting nificant levels of additional amino acid oxidations events (e.g.,
350 :g of oxidized mAb in a total volume of 300 :L. For re- at histidine residues) were detected by careful inspection of the
duction, 10 :L of 100 mg/mL DTT were added and incubated experimental mass spectra.
at 50°C for 1 h. After alkylation of free cysteines by adding
10 :L of 330 mg/mL iodoacetic acid and incubation at room
temperature in the dark for 30 min, the buffer was exchanged
RESULTS AND DISCUSSION
to digestion buffer (0.1 M Tris–HCl, pH 7.0) by using a NAP5
gel filtration column. Subsequently, NAP5-eluate (500 :L) was In forced oxidation studies of therapeutic proteins, widely vary-
mixed with 10 :L of a solution of 0.25 mg/mL trypsin in 10 mM ing extents of methionine oxidation (from 0% to 100%) were
HCl and incubated at 37°C for 18 h. The digestion reaction was reported. In these reports, the most commonly used oxidative
stopped by adding 50 :L of a 10% TFA solution. reagents were H2 O2 and t-BHP with reaction conditions span-
The tryptic peptide mixture was separated by reversed- ning a broad range, including different temperatures (room
phase UPLC (ACQUITY; Waters, Manchester, UK) on a C18 temperature or 40°C), different concentrations of the oxida-
column (BEH C18 1.7 :m, 2.1 × 150 mm2 ; Waters) and the elu- tive agent (0.0005%–0.2% for H2 O2 , 0.05%–5% for t-BHP), and
ate was analyzed online on a Q-TOF SYNAPT G2 instrument different reaction times (6 h to 7 days).12–14,22 One drawback of
(Waters). The mobile phases of RP-HPLC consisted of 0.1% FA such oxidation studies is the possible co-oxidation of tryptophan
in water (solvent A) and 0.1% FA in acetonitrile (solvent B). The and methionine. This effect was reported for the oxidation of
chromatography was performed using a linear gradient from a recombinant antibody performed with 0.05% t-BHP at room
1% to 35% solvent B in 45 min and finally from 35% to 80% temperature for 7 days where 65% methionine oxidation and
solvent B in 3 min using a flow rate of 300 :L/min. Three mi- 25% tryptophan oxidation were detected in the same sample.14
crogram digested protein was applied. UPLC-system and mass One other example of co-oxidation of tryptophan and methion-
spectrometer were connected by PEEK capillary tubes. Data ine was shown during the reaction of parathyroid hormone with
acquisition was controlled by MassLynxTM software (Waters). AAPH. Indeed, incubation of 0.1 mg/mL parathyroid hormone
Tandem mass-spectrometry (MS/MS) experiments were per- with 0.1 M AAPH at 40°C for 24 h resulted in 84% of tryptophan
formed on a LTQ Orbitrap Velos electrospray mass spectrome- oxidation and 58% of methionine oxidation.22 After reacting hu-
ter (Thermo Fisher Scientific) using the chromatographic sys- man growth hormone with AAPH, Steinmann et al.30 reported
tem described above in order to identify the oxidized peptides. a multitude of oxidized residues, including 20% tryptophan

Folzer et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2824–2831, 2015 DOI 10.1002/jps.24509


RESEARCH ARTICLE – Pharmaceutical Biotechnology 2827

Figure 2. Analysis of oxidized and nonoxidized monoclonal antibody


(mAb) species by analytical protein A chromatography. (a) The chro-
matogram overlay shows the elution profile of control material (black),
mAb oxidized in the presence of 1% hydrogen peroxide for 24 h at 5°C
with (blue) or without addition of free tryptophan (red). The red and
blue curves are perfectly overlapping. (b) The chromatogram overlay
shows the elution profile of control material (black), mAb oxidized in
the presence of 2% t-BHP for 120 h at 25°C with (blue) or without
addition of free methionine (red). The red and blue curves are per-
fectly overlapping. (c) The chromatogram overlay shows the elution
profile of control material (black), mAb oxidized in the presence of 5%
2,2-azobis(2-amidinopropane) dihydrochloride with (blue) or without
addition of free methionine (red) for 120 h at 40°C.

Figure 3. Overlay of RP-HPLC chromatograms used to quantify tryp-


oxidation and significant methionine oxidation (100% M14; 80%
tophan oxidation present in the heavy chain Fab part (HC-Fab) of stud-
M125; 70% M170).
ied IgG1 for control material versus oxidized samples. The analyses
Indeed, the use of established conditions for forced oxi- were performed after papain digest followed by dithiothreitol reduc-
dation available in literature did not allow us to selectively tion. (a) Complete RP-HPLC chromatogram for control material (black).
oxidize neither methionine nor tryptophan amino acids in (b) Zoom in of HC-Fab region; control material (black), 1% hydrogen per-
a mAb model protein as depicted in Figures 2–4, and Sup- oxide oxidation for 24 h at 5°C with (blue), or without addition of free
plementary Tables 1 and 2. Instead, extensive co-oxidation tryptophan (red). (c) Zoom in of HC-Fab region; control material (black),
of both methionine and tryptophan was present under these 2% t-BHP oxidation 120 h at 25°C with (blue), or without addition of
conditions. free tryptophan (red). (d) Zoom in of HC-Fab region; control material
To identify optimal conditions leading to selective oxidation (black), 5% 2,2-azobis(2-amidinopropane) dihydrochloride oxidation for
120 h at 40°C with (blue), or without addition of free methionine (red).
of methionine or tryptophan in a mAb, a large set of parame-

DOI 10.1002/jps.24509 Folzer et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2824–2831, 2015


2828 RESEARCH ARTICLE – Pharmaceutical Biotechnology

Table 2. Liquid chromatography - mass spectrometry (LC-MS) Results for the Oxidation with H2 O2 or t-BHP with or Without Addition of Free
Tryptophan

Oxidation with/Without Tryptophan Protection

Modified 1% H2 O2 1% H2 O2 2% t-BHP 2% t-BHP 25°C


Amino Acids Control 5°C 24 h 5°C 24 h with Tryptophan 25°C 120 h 120 h with Tryptophan

LC M4ox 0.3 4.4 0.9 4.1 3.8


HC M34ox 0.7 7.4 2.5 6.9 7.4
HC M83ox 0.6 4.1 0.7 4.2 4.0
HC M258ox 4.4 99.6 99.6 99.7 99.7
HC M364ox 0.4 90.8 92.9 91.0 91.6
HC M434ox 1.2 96.1 96.1 96.1 96.7
LC W35ox+4 0.2 0.6 0.5 0.6 0.5
LC W35ox+16 0.0 0.0 0.0 0.0 0.0
LC W96ox+4 0.1 0.3 0.2 0.4 0.2
LC W96ox+16 0.1 0.2 0.1 0.2 0.2
HC W50ox+4 0.1 0.2 0.2 0.2 0.2
HC W50ox+16 0.2 0.3 0.2 0.3 0.3
HC W50ox+32 0.0 0.0 0.0 0.0 0.0
HC W108ox+4 0.0 0.0 0.0 1.5 0.3
HC W108ox+16 0.2 0.3 0.2 2.6 0.6
HC W108ox+32 0.1 0.1 0.1 0.1 0.1
HC W283ox+4 0.1 0.1 0.1 0.2 0.1
HC W283ox+16 0.1 0.2 0.2 0.2 0.2
HC W319ox+4 0.1 0.2 0.1 0.2 0.2
HC W319ox+16 0.4 0.8 0.6 0.8 0.7
HC W319ox+32 0.0 0.0 0.0 0.0 0.0

The mAb was denatured and the disulfide bridges were reduced using DTT. Alkylation of free cysteines was performed using iodoacetic acid. After a buffer
exchange, the mAb was finally digested with trypsin. LC stands for light chain, HC stands for heavy chain. Methionine (M) and tryptophan (W) oxidation levels were
quantified.

ters were evaluated based on published studies and previous 2% t-BHP for 120 h at 25°C allowed for the nearly complete
in-house experiments (data not shown).12,14,22 A model IgG1 oxidation of the methionine residues from the Fc region.
was incubated with different oxidants while the reaction times The extent of methionine oxidation was also determined by
and incubation temperatures were varied as described in Ta- selected ion current chromatogram analysis of the oxidized pep-
ble 2. To curtail oxidation of the amino acids methionine or tides (Table 1). We found more than 91% oxidation for M364,
tryptophan, the corresponding free amino (L-methionine or L- 96% oxidation for M434, and 99% oxidation for M258 according
tryptophan) acids were added to the reaction mixtures. After to LC–MS results.
completion of all reactions, samples were screened for total ox- In order to assess the total tryptophan oxidation of the HC-
idation levels using protein A analytical chromatography and Fab, the oxidized samples were first digested with papain and
RP-HPLC. Selected samples were further analyzed using LC– then the disulfide bridges were reduced with DTT. The gen-
MS and LC–MS/MS in order to identify and semiquantify the erated LC, HC-Fc, and HC-Fab were then separated by RP-
methionine and tryptophan oxidation sites in the amino-acid HPLC (for details see Materials and Methods) as presented in
sequence. Figure 3a, and the tryptophan oxidation of the HC-Fab was
In the model IgG1 used in this study, several methionine quantified. The oxidized species because of tryptophan oxida-
residues were identified as susceptible to oxidation (M4, M34, tion elute before the HC-Fab peaks. The peak with a retention
M83, M258, M364, and M434). Basal levels of oxidation of time of 19.5 min represents in that case mainly oxidized tryp-
these susceptible residues were identified in the starting ma- tophan W108 of the HC-Fab (Figs. 3b and 3c; signal labeled as
terial (Table 1). From the oxidized tryptophan identified by “Trp-ox”).
LC–MS/MS (W35, W50, W96, W108, W283, and W319), only As previously reported, the oxidation of mAb with H2 O2 or
surface-exposed W50 and W108 showed significant changes be- t-BHP induced relatively low tryptophan oxidation levels (see
tween unstressed and stressed samples. Figs. 3b and 3c).22 The well-resolved signal with a retention
time of 19.5 min (corresponding to tryptophan oxidation prod-
ucts) increased not only with increasing H2 O2 or t-BHP con-
Selective Methionine Oxidation centrations, but also when increasing the reaction time and
Oxidation levels of Fc methionine were first quantified by the temperature of incubation (data not shown). In order to
protein A chromatography. The nonoxidized antibody species exclude the possibility of metal ion contamination resulting in
eluted at 23 min (Fig. 2). The oxidized species formed upon in- Fenton-like reactions, protein oxidation reactions were carried
cubation with oxidant eluted earlier (before 19.8 min) because out in the presence and absence of EDTA. As no differences
of the lower affinity of oxidized Fc methionine to protein A.7,12 in the oxidation levels of Trp were detected (data not shown),
As depicted in Figures 2a and 2b, the incubation of the mAb the mechanism of Trp oxidation in these reactions remains un-
with 1% H2 O2 for 24 h at 5°C as well as the incubation with clear. To avoid this unwanted tryptophan oxidation, reaction

Folzer et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2824–2831, 2015 DOI 10.1002/jps.24509


RESEARCH ARTICLE – Pharmaceutical Biotechnology 2829

Figure 5. Size-exclusion HPLC chromatograms of control material


versus oxidized samples. Monomer peak elutes at 17 min. The ag-
gregated species elute below 15.5 min. (a) The chromatogram overlay
shows the elution profile of control material (black), 1% hydrogen per-
oxide oxidized sample for 24 h at 5°C with tryptophan addition (blue)
and 2% tert-butyl hydroperoxide oxidized sample for 120 h at 25°C with
tryptophan addition (red). The blue and red curves overlap the black
curve. (b) The elution profile of control material (black) is represented
Figure 4. Comparison of the relative abundance of methionine and with the aggregation profile of 5% 2,2-azobis(2-amidinopropane) dihy-
tryptophan residues susceptible to oxidation quantified by LC–MS drochloride oxidation for 120 h at 40°C with (blue) or without addition
peptide map. The oxidation was performed with 5% 2,2-azobis(2- of free methionine (red).
amidinopropane) dihydrochloride (AAPH) at 40°C for 120 h with or
without addition of free methionine. Relative abundance is calculated
as relative percentage using specific ion current chromatogram anal-
should be investigated in detail before use to avoid unexpected
ysis of tryptic peptides for M258, M364, and M434 (a), W50 (b), and
effects.31
W108 (c). The tryptophan modifications kynurenine (+4), hydroxytryp-
tophan (+16), and N-formylkynurenine (+32) are represented in b Size-Exclusion HPLC was used to assess the sample in-
and c. tegrity after oxidation. Both H2 O2 (1%, 5°C, 24 h with free tryp-
tophan) or t-BHP (2%, 25°C, 120 h with free tryptophan) oxida-
tion induced only minimal changes in aggregate and fragment
formation. In comparison to the reference material, the incuba-
conditions were set as H2 O2 oxidation at 5°C for 24 h and t-
tion with 1% H2 O2 induced 0.8% soluble aggregates, whereas
BHP oxidation at 25°C for 120 h. Moreover, the tryptophan ox-
the oxidation with 2% t-BHP induced 1.4% soluble aggregates
idation could be inhibited using addition of free tryptophan at
(Fig. 5a). Thus, the application of the developed model stress
concentrations of 19.6 mM during the incubations as depicted
system does not significantly impact molecular integrity and
in Figures 3b and 3c (for detailed LC–MS, results see Table 2).
the biological consequences of selective methionine oxidation
LC–MS analyses confirmed the low abundance of tryptophan
could be further analyzed by functional testing.
oxidation (Table 1), and as a consequence the protective effect
of free tryptophan against tryptophan oxidation. According to
Selective Tryptophan Oxidation
LC–MS results, the total oxidation of W50 and W108 was lower
than 1% when adding free tryptophan before oxidation with t- 2,2-Azobis(2-amidinopropane) dihydrochloride is a reagent that
BHP (2%, 25°C, 120 h). In the case of H2 O2 oxidation (1%, 5°C, has been used for forced tryptophan oxidation studies.22 In
24 h), where tryptophan oxidation products were present at our hands, the simultaneous oxidation of several tryptophan
very low level (<1%), the addition of free tryptophan could not residues upon stress (mass additions of +4, +16, +32, +48,
protect against the formation of low levels of residual trypto- +64) resulted in a complex mixture of reaction products that
phan oxidation products (see Table 1). were difficult to separate well by RP-HPLC (Fig. 3d). The pre-
In addition to free tryptophan, which has been shown peaks were broad and poorly resolved. Nevertheless, it was
to protect tryptophan residues in proteins from oxidation, possible to detect tryptophan-oxidized species in the oxidized
other antioxidants could potentially be used to avoid tryp- sample as early-eluting peaks (retention times from 16 to
tophan oxidation.20,21 However, the addition of antioxidant 19.6 min in Fig. 3d) and to identify the different tryptophan

DOI 10.1002/jps.24509 Folzer et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2824–2831, 2015


2830 RESEARCH ARTICLE – Pharmaceutical Biotechnology

oxidation sites by subsequent LC–MS analyses. Upon incuba- still present in the sample containing free methionine during
tion at 40°C for 120 h with AAPH (0%–5%), a considerable incubation with 5% AAPH for 120 h at 40°C. The content of
tryptophan oxidation (between 12% and 95%) was observed, as soluble fragments remained below 0.3% for the control and the
seen in Supplementary Tables 2 and 3. stressed samples in all studies. The correlation between Met
As reported in previous studies, the reaction of proteins oxidation and protein aggregation observed in this study was
with alkyperoxyl radicals (generated by AAPH that reacts not unexpected, as such correlation has been reported previ-
with oxygen) also induced methionine oxidation.22,32 As seen in ously by others and studied in some detail.11,33 These studies
Figures 2c and 4a, oxidation with 5% AAPH lead to almost have demonstrated that Met oxidation in mAbs may result in
complete oxidation of Fc methionine measured by protein A changes of the secondary and tertiary structure and in turn
chromatography and LC–MS peptide mapping. significantly affect the physical stability of the protein result-
The addition of a large excess of free methionine into solu- ing in increased aggregation propensity. Thus, the decrease of
tion before oxidation at 40°C for 120 h allowed protection of Fc aggregation upon the addition of free Met can be attributed to
methionine as seen in the protein A chromatogram (Fig. 2c). As the oxidation protective effect of Met.
depicted in Supplementary Figures 1 and 2, the low levels or The experimental approach of using combinations of oxida-
the absence of peptide signal in the TIC plots and extracted ion tive reagents and antioxidants presented here provided an ef-
chromatograms for M258ox (RT = 12.65 min), M364ox (RT = ficient strategy for selective oxidation of Met and Trp residues
1.78 min), and M434ox (RT = 18.45 min) when adding free me- in therapeutic IgG molecules. Although residual low levels of
thionine into the mAb solution before oxidation with 5% AAPH undesirable oxidation were detected in some cases (which pre-
(at 40°C for 120 h) is a clear confirmation of the protective effect sumably would need to be accounted for in follow-up biological
of free methionine against methionine oxidation. studies), we believe that this approach will find broad applica-
The results from LC–MS analyses of AAPH-oxidized sam- tion in studying the biological effects of protein oxidation.
ples are presented in Tables 1 and 2 of the Supplementary
Material. These results clearly demonstrate that methionine
residues otherwise susceptible to oxidation by AAPH can be se- CONCLUSIONS
lectively protected by addition of free methionine, even in the
Free methionine and/or tryptophan are used as pharmaceuti-
harshest reaction conditions used in this study [longest incu-
cal antioxidants because of their protective action against ox-
bation time (120 h), highest oxidant concentration (5%), and
idation of amino acids in protein therapeutics. In the present
highest temperature of incubation (40°C)]. This specific sample
report, we explore the potential of these antioxidants to selec-
showed 10.3% oxidation for M258, whereas the same sample
tively generate methionine- or tryptophan-oxidized residues. A
without methionine addition showed oxidation of 99.7%. Simi-
combination of oxidative (H2 O2 , t-BHP, and AAPH) and protec-
larly, 6.3% versus 98.8% M434ox was verified by LC–MS. The
tive (free tryptophan or methionine) components will serve as a
oxidation of M364 was lower than 1% in the protected sample
strategy to address questions related to the impact of oxidation-
(99% oxidation in the sample without addition of free methio-
induced changes on the biological properties of protein thera-
nine). It has to be mentioned that 4.5% M258ox, 0.4% M364ox,
peutics (e.g., bioactivity, PK/PD, immunogenicity, etc.). Such
and 1.1% M434ox were found in the nonstressed reference ma-
mechanistic studies will be instrumental to better understand
terial (Fig. 4a).
and control oxidative degradation of antibodies.
Figures 4a–4c show a comparison of the methionine (M258,
M364, and M434) and tryptophan (W50 and W108) distribution
between AAPH-stressed samples with and without addition of
ACKNOWLEDGMENT
free methionine. Over and above the decrease of methionine
oxidation, the presence of free methionine in the mAb solution We thank Y. John Wang, Ulla Grauschopf, and Kishore Ravuri
for AAPH incubation slightly changed the oxidation profile of for insightful discussions and comments on the manuscript.
tryptophan residues as seen in Figures 4b and 4c. As already
observed in the RP-HPLC data, the total amount of trypto-
phan oxidation (as quantified by LC–MS) slightly decreased REFERENCES
but remained considerable when adding free methionine to the
1. Mohammadian-Mosaabadi J, Naderi-Manesh H, Maghsoudi N,
solution (Figs. 4 and 5). On the one hand, a higher percentage
Khalilzadeh R, Shojaosadati SA, Ebrahimi M. 2005. Effect of oxida-
of hydroxytryptophan (+16) for W50 and W108 was measured tive stress on the production of recombinant human interferon-gamma
in the samples containing free methionine; on the other hand, in Escherichia coli. Biotechnol Appl Biochem 41(Pt 1):37–42.
a lower percentage of N-formylkynurenine (+32) was found for 2. Krishnamurthy R, Madurawe RD, Bush KD, Lumpkin JA. 1995.
W50 and W108 when adding free methionine (Supplementary Conditions promoting metal-catalyzed oxidations during immobilized
Figs. 3 and 4). Presumably, this effect is related to shift in the cu-iminodiacetic acid metal affinity-chromatography. Biotechnol Progr
equilibrium between reaction intermediates caused by the ad- 11(6):643–650.
dition of the antioxidant. Further elucidation of this mechanism 3. Kerwin BA. 2008. Polysorbates 20 and 80 used in the formulation of
may be of interest as a topic of future studies. protein biotherapeutics: Structure and degradation pathways. J Pharm
Sci 97(8):2924–2935.
Significant formation of soluble aggregates was detected by
4. Takenawa T, Yokota A, Oda M, Takahashi H, Iwakura M. 2009.
size SEC correlating well with the increase in AAPH concentra-
Protein oxidation during long storage: Identification of the oxidation
tion. The addition of free methionine, however, had a protective sites in dihydrofolate reductase from Escherichia coli through LC-MS
effect against aggregation as seen in Figure 5b. This effect cor- and fragment studies. J Biochem 145(4):517–523.
relates well with overall reduction on oxidation. The monomer 5. Torosantucci R, Schoneich C, Jiskoot W. 2014. Oxidation of thera-
content represented 30% of the total area in the AAPH sample peutic proteins and peptides: Structural and biological consequences.
without methionine addition, whereas 65% of monomers were Pharm Res 31(3):541–553.

Folzer et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2824–2831, 2015 DOI 10.1002/jps.24509


RESEARCH ARTICLE – Pharmaceutical Biotechnology 2831

6. Khor HK, Jacoby ME, Squier TC, Chu GC, Chelius D. 2010. Iden- 18. Li S, Schoneich C, Borchardt RT. 1995. Chemical instability of pro-
tification of methionine sulfoxide diastereomers in immunoglobulin tein pharmaceuticals: Mechanisms of oxidation and strategies for sta-
gamma antibodies using methionine sulfoxide reductase enzymes. bilization. Biotechnol Bioeng 48(5):490–500.
MAbs 2(3):299–308. 19. Wu Y, Levons J, Narang AS, Raghavan K, Rao VM. 2011. Reac-
7. Gaza-Bulseco G, Faldu S, Hurkmans K, Chumsae C, Liu H. 2008. tive impurities in excipients: Profiling, identification and mitigation of
Effect of methionine oxidation of a recombinant monoclonal antibody drug–excipient incompatibility. AAPS PharmSciTech 12(4):1248–1263.
on the binding affinity to protein A and protein G. J Chromatogr B 20. Wang W. 1999. Instability, stabilization, and formulation
Analyt Technol Biomed Life Sci 870(1):55–62. of liquid protein pharmaceuticals. Int J Pharm 185(2):129–
8. Pan H, Chen K, Chu L, Kinderman F, Apostol I, Huang G. 188.
2009. Methionine oxidation in human IgG2 Fc decreases bind- 21. Mozziconacci O, Ji JA, Wang YJ, Schoneich C. 2013. Metal-
ing affinities to protein A and FcRn. Protein Sci 18(2):424– catalyzed oxidation of protein methionine residues in human parathy-
433. roid hormone (1-34): Formation of homocysteine and a novel
9. Bertolotti-Ciarlet A, Wang W, Lownes R, Pristatsky P, Fang Y, methionine-dependent hydrolysis reaction. Mol Pharm 10(2):739–755.
McKelvey T, Li Y, Drummond J, Prueksaritanont T, Vlasak J. 22. Ji JA, Zhang B, Cheng W, Wang YJ. 2009. Methionine, tryptophan,
2009. Impact of methionine oxidation on the binding of human IgG1 and histidine oxidation in a model protein, PTH: Mechanisms and sta-
to Fc Rn and Fc gamma receptors. Mol Immunol 46(8–9):1878– bilization. J Pharm Sci 98(12):4485–4500.
1882. 23. Lam XM, Yang JY, Cleland JL. 1997. Antioxidants for prevention
10. Wang W, Vlasak J, Li Y, Pristatsky P, Fang Y, Pittman T, Roman J, of methionine oxidation in recombinant monoclonal antibody HER2. J
Wang Y, Prueksaritanont T, Ionescu R. 2011. Impact of methionine ox- Pharm Sci 86(11):1250–1255.
idation in human IgG1 Fc on serum half-life of monoclonal antibodies. 24. Keck RG. 1996. The use of t-butyl hydroperoxide as a probe for
Mol Immunol 48(6-7):860–866. methionine oxidation in proteins. Anal Biochem 236(1):56–62.
11. Liu D, Ren D, Huang H, Dankberg J, Rosenfeld R, Cocco MJ, Li L, 25. Wasylaschuk WR, Harmon PA, Wagner G, Harman AB, Templeton
Brems DN, Remmele RL Jr. 2008. Structure and stability changes of AC, Xu H, Reed RA. 2007. Evaluation of hydroperoxides in common
human IgG1 Fc as a consequence of methionine oxidation. Biochemistry pharmaceutical excipients. J Pharm Sci 96(1):106–116.
47(18):5088–5100. 26. Luo Q, Joubert MK, Stevenson R, Ketchem RR, Narhi LO, Wypych
12. Loew C, Knoblich C, Fichtl J, Alt N, Diepold K, Bulau P, J. 2011. Chemical modifications in therapeutic protein aggregates gen-
Goldbach P, Adler M, Mahler HC, Grauschopf U. 2012. Analytical pro- erated under different stress conditions. J Biol Chem 286(28):25134–
tein a chromatography as a quantitative tool for the screening of me- 25144.
thionine oxidation in monoclonal antibodies. J Pharm Sci 101(11):4248– 27. Qi P, Volkin DB, Zhao H, Nedved ML, Hughes R, Bass R, Yi SC,
4257. Panek ME, Wang D, Dalmonte P, Bond MD. 2009. Characterization of
13. Schlothauer T, Rueger P, Stracke JO, Hertenberger H, Fin- the photodegradation of a human IgG1 monoclonal antibody formulated
gas F, Kling L, Emrich T, Drabner G, Seeber S, Auer J, Koch S, as a high-concentration liquid dosage form. J Pharm Sci 98(9):3117–
Papadimitriou A. 2013. Analytical FcRn affinity chromatography for 3130.
functional characterization of monoclonal antibodies. MAbs 5(4):576– 28. Liu H, Gaza-Bulseco G, Zhou L. 2009. Mass spectrometry analysis
586. of photo-induced methionine oxidation of a recombinant human mono-
14. Hensel M, Steurer R, Fichtl J, Elger C, Wedekind F, Petzold A, clonal antibody. J Am Soc Mass Spectrom 20(3):525–528.
Schlothauer T, Molhoj M, Reusch D, Bulau P. 2011. Identification 29. Rao PE, Kroon DJ. 1993. Orthoclone OKT3. Chemical mechanisms
of potential sites for tryptophan oxidation in recombinant antibod- and functional effects of degradation of a therapeutic monoclonal anti-
ies using tert-butylhydroperoxide and quantitative LC–MS. PloS One body. Pharm Biotechnol 5:135–158.
6(3):e17708. 30. Steinmann D, Ji JA, Wang YJ, Schoneich C. 2012. Oxidation of
15. Wei ZP, Feng JH, Lin HY, Mullapudi S, Bishop E, Tous GI, Casas- human growth hormone by oxygen-centered radicals: Formation of
Finet J, Hakki F, Strouse R, Schenerman MA. 2007. Identification of a Leu-101 hydroperoxide and Tyr-103 oxidation products. Mol Pharm
single tryptophan residue as critical for binding activity in a humanized 9(4):803–814.
monoclonal antibody against respiratory syncytial virus. Anal Chem 31. Grewal P, Mallaney M, Lau K, Sreedhara A. 2014. Screening
79(7):2797–2805. methods to identify indole derivatives that protect against reactive
16. Haberger M, Bomans K, Diepold K, Hook M, Gassner J, Schlothauer oxygen species induced tryptophan oxidation in proteins. Mol Pharm
T, Zwick A, Spick C, Kepert JF, Hienz B, Wiedmann M, Beck H, Metzger 11(4):1259–1272.
P, Molhoj M, Knoblich C, Grauschopf U, Reusch D, Bulau P. 2014. As- 32. Werber J, Wang YJ, Milligan M, Li X, Ji JA. 2011. Anal-
sessment of chemical modifications of sites in the CDRs of recombinant ysis of 2,2 -azobis (2-amidinopropane) dihydrochloride degradation
antibodies: Susceptibility vs. functionality of critical quality attributes. and hydrolysis in aqueous solutions. J Pharm Sci 100(8):3307–
MAbs 6(2):327–339. 3315.
17. Stracke J, Emrich T, Rueger P, Schlothauer T, Kling L, Knaupp A, 33. Zhang A, Hu P, MacGregor P, Xue Y, Fan H, Suchecki P, Olszewski
Hertenberger H, Wolfert A, Spick C, Lau W, Drabner G, Reiff U, Koll L, Liu A. 2014. Understanding the conformational impact of chemical
H, Papadimitriou A. 2014. A novel approach to investigate the effect modifications on monoclonal antibodies with diverse sequence variation
of methionine oxidation on pharmacokinetic properties of therapeutic using hydrogen/deuterium exchange mass spectrometry and structural
antibodies. MAbs 6(5):1229–1242. modeling. Anal Chem 86(7):3468–3475.

DOI 10.1002/jps.24509 Folzer et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:2824–2831, 2015

S-ar putea să vă placă și