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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Chemical Modifications of Therapeutic Proteins Induced by
Residual Ethylene Oxide
LOUISE CHEN, CHRISTOPHER SLOEY, ZHANG ZHONGQI, PAVEL V. BONDARENKO, HYOJIN KIM, DA REN, SEKHAR KANAPURAM
Process and Product Development, Amgen Inc., Thousand Oaks, California
Received 10 September 2014; revised 13 October 2014; accepted 17 October 2014
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24257
ABSTRACT: Ethylene oxide (EtO) is widely used in sterilization of drug product primary containers and medical devices. The impact of
residual EtO on protein therapeutics is of significant interest in the biopharmaceutical industry. The potential for EtO to modify individual
amino acids in proteins has been previously reported. However, specific identification of EtO adducts in proteins and the effect of
residual EtO on the stability of therapeutic proteins has not been reported to date. This paper describes studies of residual EtO with two
therapeutic proteins, a PEGylated form of the recombinant human granulocyte colony-stimulating factor (Peg-GCSF) and recombinant
human erythropoietin (EPO) formulated with human serum albumin (HSA). Peg-GCSF was filled in an EtO sterilized delivery device and
incubated at accelerated stress conditions. Glu-C peptide mapping and LC–MS analyses revealed residual EtO reacted with Peg-GCSF
and resulted in EtO modifications at two methionine residues (Met-127 and Met-138). In addition, tryptic peptide mapping and LC–MS
analyses revealed residual EtO in plastic vials reacted with HSA in EPO formulation at Met-328 and Cys-34. This paper details the work
conducted to understand the effects of residual EtO on the chemical stability of protein therapeutics. C2014 Wiley Periodicals, Inc. and
the American Pharmacists Association J Pharm Sci
Keywords: drug delivery systems; injectables; protein delivery; chromatography; HPLC; mass spectrometry; proteins; peptides; formula-
tion; analytical biochemistry
INTRODUCTION
Common sterilization methods include moist heat (i.e., steam),
dry heat, ionizing radiation (gamma or e-beam), hydrogen per-
oxide, and ethylene oxide (EtO). Each method has its advan-
tages and limitations. Ionizing radiation, hydrogen peroxide,
and EtO often leave residuals that are damaging to proteins
and DNA.1–4 EtO sterilization is particularly useful for steriliz-
ing delicate medical devices and heat and/or moisture sensitive
equipments or materials.5,6 However, the difficulties caused by
the potential hazards of EtO to patients, staff, and the envi-
ronment as well as risk associated with handling a flammable
gas make EtO sterilization a challenging task.5Because dif-
ferent materials adsorb and desorb EtO differently,7the allow-
able levels of residual EtO remaining in sterilized apparatus
and materials need to be specified for individual products per
regulations. Therefore, the sterilization process, including EtO
concentration, sterilization time, temperature, and humidity
as well as the aeration process conditions and time must be
optimized for effectively eliminating residual EtO.8
Although EtO has been utilized for decades, protein degra-
dation by EtO has not been widely reported in the literature. An
Abbreviations used: ACN, acetonitrile; CEX, cation-exchange chromatog-
raphy; CID, collision-induced dissociation; DTT, dithiothreitol; EDTA,
ethylenediaminetetraacetic acid; EPO, recombinant human erythropoietin;
EtO, ethylene oxide; GC/FID, gas chromatography equipped with flame ion-
ization detector; GC–MS, gas chromatography coupled with MS; Gdn-HCl,
guanidine-HCl; HSA, human serum albumin; IAA, iodoacetic acid; LC–MS,
liquid chromatography coupled with MS; MS/MS, fragmentation mass spectra;
Peg-GCSF, pegylated recombinant human granulocyte colony-stimulating factor;
PFS, prefilled syringe; PP1, post peak 1; PP2, post peak 2; PP3, post peak 3;
RP-HPLC, reversed-phase HPLC; RT, retention time; TFA, trifluoroacetic
acid.
Correspondence to: Louise Chen (Telephone: +805-447-6488; Fax: +805-376-
8505; E-mail: louise.chen@amgen.com)
Journal of Pharmaceutical Sciences
C
2014 Wiley Periodicals, Inc. and the American Pharmacists Association
initial model system found it forms adducts with methionine,
cysteine, and histidine amino acid residues. The model experi-
ments conducted by reaction of EtO with commercial proteins
also reported a decrease in biological activity. The reduction in
bioactivity appeared to be correlated with the electrophilic hy-
droxyethylation of an atom with one or more lone pairs of elec-
trons, particularly nitrogen and sulfur.1Gas chromatography
coupled with MS (GC–MS) and liquid chromatography coupled
with MS (LC–MS) were applied in measuring small-molecule
DNA and protein adducts released during EtO treatments,
such as N-(2-hydroxyethyl) valine, N1-(2-hydroxyethyl) histi-
dine, and S-(2-hydroxyethyl) cysteine.4Thelackofinformation
about tandem MS (MS/MS) analysis of EtO-modified peptides
in proteins made site identification in protein sequence and
quantitation of the modification analytically challenging. The
challenges were because of the poor gas-phase fragmentation
of peptides containing methionine residues modified by EtO. To
date, identification of this modification is not widely described
in the literature.
In this report, detection and identification of the protein
degradation caused by residual EtO (low levels of EtO remain-
ing in the different medical containers) using two therapeutic
proteins, a PEGylated form of recombinant human granulo-
cyte colony-stimulating factor (Peg-GCSF), and a recombinant
human erythropoietin (EPO) formulated with human serum
albumin (HSA), were studied. EtO sterilized delivery devices
were filled with Peg-GCSF from a prefilled syringe (PFS) for-
mulated at 10 mg/mL in 10 mM sodium acetate, 5% sorbitol,
pH 4.0, 0.004% polysorbate 20 and incubated at an accelerated
condition of 42 ±1◦C for 54 h. Samples were analyzed by sev-
eral analytical assays to evaluate the impact of residual EtO
on Peg-GCSF. Assay results showing the impact of EtO, in-
cluding cation-exchange chromatography/HPLC (CEX–HPLC)
and reversed-phase HPLC (RP-HPLC) are highlighted in this
Chen et al., JOURNAL OF PHARMACEUTICAL SCIENCES 1
2RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Figure 1. Identification of all minor species present in the retention time window of 40–56 min in Glu-C peptide map of Peg-GCSF. Blue trace,
filled device control; red trace, stressed filled device; green trace, PFS control; pink trace, stressed PFS.
report. Endoproteinase Glu-C digested peptide maps analyzed
by LC/MS were used for identification and characterization of
any EtO modifications.
In a separate experiment, EPO was formulated at
0.0168 mg/mL in 20 mM sodium citrate buffer pH 6.9 with
100 mM sodium chloride and 2.5 mg/mL HSA. A 1.0 mL sam-
ple was filled in plastic vials which had been custom sterilized
using an EtO process. Vials were incubated at 37 ±1◦Cforup
to 2 months after which they were analyzed by several assays
to evaluate the impact of residual EtO on EPO with HSA.
EXPERIMENTAL PROCEDURES
Materials and Equipment
Chemicals and Materials
Sodium acetate, sodium chloride, glycerol, trifluoroacetic
acid (TFA), tris(hydroxymethyl)aminomethane (Tris), Tris
hydrochloride (Tris–HCl), urea, hydroxylamine, dithiothre-
itol (DTT), iodoacetic acid (IAA), ethylenediaminetetraacetic
acid (EDTA), and guanidine-HCl (Gdn-HCl) were ACS grade
(Sigma–Aldrich, St. Louis, Missouri). All organic solvents were
of analytical or HPLC grade. Trypsin and Endoproteinase Glu-
C was Roche sequencing grade (Roche Diagnostics Corporation,
Indianapolis, Indinana). Vydac C-4 columns (250 ×2.1 mm2,
5-:m particle size) were purchased from Grace Co. (Columbia,
Maryland). PLRS-S columns (4.6 ×150 mm2)andVarianPo-
laris C18 Ether columns (2.0 ×250 mm2,3-:mparticlesize)
were purchased from Agilent Technologies (Santa Clara, Cal-
ifornia). Cation exchange columns (TSK gel SP-NPR, 4.6 ×
35 mm2, 2.5-:m nonporous particle) were from TosoHaas Bio-
science Company (Phenomenex, part # CHO-8039; King of
Prussia, Pennsylvania). Delivery devices were manufactured
from Insulet Company (USA).
Instruments
A Thermo Scientific (Waltham, Massachusetts) Velos Ion Trap
(LTQ) mass spectrometer was used for Glu-C peptide map-
ping analysis and CEX post peak characterization and an Ag-
ilent 1100 series HPLC was used for the RP-HPLC assay. A
Dionex (Sunnyvale, California) Ultimate 3000 HPLC was used
for the CEX–HPLC assay. Agilent headspace gas chromatogra-
phy equipped with flame ionization detector (FID) was used for
EtO analysis.
Protein Formulations
Peg-GCSF (pegfilgrastim) was formulated at 10 mg/mL in
10 mM sodium acetate, 5% sorbitol, pH 4.0, and 0.004% polysor-
bate 20. Epoetin alfa (recombinant human EPO) was formu-
lated at 0.0168 mg/mL in 20 mM sodium citrate buffer (pH 6.9)
containing 100 mM sodium chloride and 2.5 mg/mL HSA.
Methods
EtO-Degraded Peg-GCSF Sample Preparation
Peg-GCSF was loaded into an EtO sterilized delivery device
from a glass PFS. The filled devices along with the initial PFS
were subjected to storage at an accelerated condition of 42 ±
1◦C for 54 h. The initial PFS was known to contain no residual
EtO and served as a control at the stressed condition. Post
incubation, the Peg-GCSF in the stressed containers and PFS
control samples were each collected for testing. The PFS and
filled containers at time zero (unstressed) were also included as
controls. All samples were digested with endoproteinase Glu-C
Chen et al., JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24257
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 3
Figure 2. The fragmentation mass spectrum (MS/MS) of the 4+ion with m/z1019.2 (43.6 min) and the 3+ion with m/z1357.9 (46.2 min)
suggested a 92 Da loss in the side chain during collision-induced dissociation (CID).
Figure 3. MS/MS for the 4072 Da mass peak at 43.6 min (4+ion of m/z1019.2) shown in the square root ion intensity scale was used to assign
the peaks and identify the site of the modification in G7 native peptide (L125-E163).
followed by analysis using a RP-HPLC coupled to the Velos Ion
Trap mass spectrometer.
Glu-C Peptide Mapping
Peg-GCSF test samples were digested with endoproteinase Glu-
C in digestion buffer [0.5 M Tris, 0.5 M Tris–HCl, 0.4 M methy-
lamine hydrochloride (CH3NH2·HCl), 0.2 M DTT, and 6 M urea]
and incubated at 25◦C for 18 h. A Vydac C-4 (250 ×2.1 mm2,
5-:m particle) reversed phase column was used and maintained
at 40◦C with a flow rate of 0.2 mL/min. Mobile phase A was
0.1% TFA in water. Mobile phase B was 0.09% TFA in 90%
acetonitrile (ACN). Peptides were separated by ramping the
mobile phase B from 2% to 60% over 75 min and followed by
column cleanup and system equilibration. The total run time is
DOI 10.1002/jps.24257 Chen et al., JOURNAL OF PHARMACEUTICAL SCIENCES
4RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Scheme 1. EtO and methionine adduct formation in the G7 peptide
(L125-E163) of Peg-GCSF and the process for gas-phase side-chain loss.
120 min. Eluted peptides were detected by UV at 214 nm, fol-
lowed by mass spectrometric analysis.
CEX–HPLC Analysis
A SP-NPR (4.6 ×35 mm2, 2.5-:m nonporous particles) CEX
column was used and maintained at 25◦Cwithaflowrateof
1.0 mL/min. Mobile phase A was 5 mM sodium acetate and 5%
glycerol, pH 5.6. Mobile phase B was 5 mM sodium acetate,
100 mM NaCl, and 5% glycerol, pH 5.6. The separation of the
charged variants was carried out by ramping the mobile phase
B from 0% to 33% over 60 min.
Tryptic Peptide Mapping of EPO/HSA
Each EPO sample containing 0.5 mg HSA was diluted in a
solution containing 7.5 M Gdn-HCl, 0.25 M Tris, and 2 mM
EDTA. Then, 30 :L of 0.5M DTT was added and sample was
incubated at room temperature for 30 min. Next, 7 :Lof
0.5 M IAA was added and samples were incubated for 15 min in
the dark. Then, 4 :L of 0.5 M DTT was added to stop the alky-
lation reaction. Samples were desalted using NAP-5 columns
(GE Healthcare, Piscataway, New Jersey) prior to adding of re-
combinant trypsin solution (Roche). Incubation was conducted
at 37◦C for 30 min. Mobile phase A was 0.1% TFA in water.
Mobile phase B was 0.085% TFA in 90% ACN. The HPLC col-
umn used was a Varian Polaris Ether, C18, 2.0 mm ID ×250
mm length, 3-:m particle size, with a 300 ˚
A pore size. The col-
umn temperature was maintained at 50◦C, and the flow rate
was 0.2 mL/min. Peptides were separated by ramping mobile
phase B from 0% to 50% over 195 min. Detection of peptides
was performed using a UV detector set at 214 nm, followed by
mass spectrometric analysis using the LTQ Velos instrument.
The mass spectrometric conditions were at positive mode, full
scan followed by a zoom scan and MS/MS with collision-induced
dissociation (CID) scans, with dynamic exclusion.
RESULTS
EtO Analysis by Gas Chromatography
A delivery device with EtO sterilized in a cGMP like process
was used as the residual EtO source. The residual EtO level
in the experimental devices were quantified at approximately
40 :g based on the analysis by headspace gas chromatography
equipped with FID (GC/FID).9Similarly, the residual EtO level
in the sterilized plastic vials were determined approximately
30 :g per vial.
Detection and Identification of EtO Modification in Peg-GCSF
The LC/UV traces in the stressed filled device and controls from
the Glu-C peptide mapping analysis were overlaid for compari-
son (Fig. 1). No differences were observed for the major peptides
(G1–G8) in the Glu-C peptide map. However, in the selected re-
tention time (RT) window of 40–56 min (zoom scale), two minor
species at 43.6 and 46.2 min showed a significant increase in
the filled device samples. The peak at 43.6 min [ions: m/z1358.0
(3+), m/z1019.2 (4+), and m/z1354.4 (3+)] was identified as a
coelution of methionine-oxidized peptide L125-E163 (contain-
ing methionine oxidation at both sites of Met-127 and Met-138,
mass 4060 Da)10,11 and an unknown species (mass 4072 Da).
The peak at 46.2 min [ions: m/z1357.9 (3+), m/z1019.0 (4+),
and 1128.5 (2+)] was identified as a coelution of Met-122Ox
Figure 4. CEX profiles of Peg-GCSF for stressed filled device compared with PFS and device controls. Black trace, PFS control; blue trace, filled
device control; pink trace, stressed PFS; brown trace, stressed filled device.
Chen et al., JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24257
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 5
Figure 5. Characterization of the CEX–HPLC fractions by Glu-C peptide map. (a) No modified species were found in the main peak fraction.
(b) The PP1 fraction was enriched with Met-127 EtO adducts. (c) The PP3 fraction was enriched with Met-138 EtO adducts. (d) The PP2 fraction
was a mixture of various oxidized methionines. Peak (#1) represents bothOx (OxMet-127 and OxMet-138) for the G7 peptide (L125-E163) with a
monoisotopic mass of 4057 Da. Peak (#2) represents OxMet-138 for the G7 peptide with a monoisotopic mass 4041 Da. Peak (#3) represents OxMet-
122 for the peptide T106-E124 with a monoisotopic mass of 2254 Da. Peak (#4) represents OxMet-127 for the G7 peptide with a monoisotopic
mass of 4041 Da. Peak (#5) represents OxMet-122 for the G6 peptide (L100-E124) with a monoisotopic mass of 2850 Da. Several minor species
were observed in the overlaid traces of (b) and (c) and the sources may be because of the impurities from the neighboring peaks in the collected
fractions of PP1 and PP3.
(methionine sulfoxide formed at methionine-122) for peptide
T106-E124 (mass 2255 Da) and an unknown species (mass
4072 Da).
To identify the unknown 4072 Da species, a Thermo Scien-
tific LTQ Velos ion trap mass spectrometer was operated in full
scan (MS) mode followed by a fragmentation scan (MS/MS). The
MS/MS spectra of the 4+ion with m/z1019.2 (43.6 min) and
3+ion with m/z1357.9 (46.2 min) were both found to contain
an unusual peptide fragmentation pattern. MS/MS of each ion
resulted in a single major fragment indicating a loss of 92 Da
from the 4072 Da species (Fig. 2) inferring that an EtO-modified
side chain of an amino acid residue may be susceptible to losing
the modified side chain with a 92 Da loss (CH3SCH2CH2OH)
during CID. A similar fragmentation pattern was reported for
multiply charged ions containing oxidized methionine12,13 in-
dicating that the modified side chain of methionine is suscep-
tible to fragmentation and is easily loss of 64 Da (CH3SOH).
It was noticed that the expected m/zare 1018.9 Da (4+)and
1358.2 Da (3+) (theoretical average mass 4071.6 Da), which
differs to the measured m/zby 0.3 amu approximately. This
difference is common for low-resolution spectrum of multiply
charged ions in Velos ion trap Mass Spectrometer.
The MS/MS confirmed that the 4072 Da species at 43.6 and
46.2 min correspond to peptide L125-E163 with EtO-modified
methionine side chains at Met-138 and Met-127, respectively.
Figure 3 showed the MS/MS and fragment assignments of the
4+ion at 43.6 min for Met-138. To magnify the low-abundance
fragments, the MS/MS spectra were displayed in square-root
scale. Data for Met-127 are not shown. An illustration of the
EtO and methionine reaction as well as gas-phase side-chain
loss is shown in Scheme 1.
Characterization of the Post Peaks Found by CEX–HPLC
The Peg-GCSF that had been stressed at an elevated tempera-
ture in the device displayed a different CEX profile when com-
pared with the controls. Three post peaks were found in the
stressed samples by CEX–HPLC. The impurities of post peaks
in the stress samples were quantified approximately 4.4%–5.8%
(Fig. 4). Where post peak #1 (PP1) increased relative to controls
and post peaks #2 and #3 (PP2 and PP3) were new peaks. These
two new peaks were neither found in historical stability data
nor in previous forced degradation studies for Peg-GCSF. It was
suspected that these post peaks were related to the residual
EtO in the stressed device samples.
DOI 10.1002/jps.24257 Chen et al., JOURNAL OF PHARMACEUTICAL SCIENCES
6RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Figure 6. Tryptic peptide map of EPO showed two EtO modifications in EtO sterilized plastic vial (green) when compared with the unsterilized
plastic (red) and glass vial (blue) controls.
Figure 7. MS/MS for m/z834 at RT 131 min indicates a 44 Da mass increase of the parent ion resulting from ETO reaction with Met-328 of
HSA peptide D324-R336 followed by loss of the modified methionine side chain (−92 Da) from fragment ions.
To characterize the three CEX post peaks, fractions were col-
lected from the CEX elution using an analytical CEX column,
SP-NPR (4.6 ×35 mm2, 2.5-:m nonporous particles). The frac-
tions included the main peak and the three post peaks (PP1,
PP2, and PP3). Each collected fraction was concentrated and
buffer-exchanged with product formulation buffer containing
no polysorbate. The main peak fraction contained the expected
intact molecule without any protein modifications and was used
as a study control for the entire collection and characterization
process. The post peak fractions (PP1, PP2, and PP3) were rel-
atively low in concentration, and therefore were each added
to the PFS sample (which contained no residual EtO) to make
up the final sample concentration needed for Glu-C peptide
mapping analysis. These solutions were then verified by CEX–
HPLC to confirm the identity and purity prior to performing the
characterization. The CEX–HPLC traces confirm the integrity
of the fraction collecting process performed on the starting Peg-
GCSF material from the stressed devices (data not shown).
The PFS-fraction solutions were then each digested by Glu-
C endoproteinase followed by reversed-phase LC–MS analyses.
The LC–UV traces from individual digested solutions were each
overlaid with the digested solution from PFS only to identify the
major changes in the Glu-C peptide profile. No changes were
identified in the chromatogram of the main peak fraction Glu-C
Chen et al., JOURNAL OF PHARMACEUTICAL SCIENCES DOI 10.1002/jps.24257
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 7
Figure 8. HPLC–UV chromatogram overlay. The 14 Da difference between two peaks at 162 and 163 min is from +44 EtO modification of
Cys-34 in the absence of the +58 modification from alkylation.
digest, which confirmed that the collection and characterization
process had no impact (Fig. 5a). One major change was seen in
the PP1 fraction Glu-C digest (RT: 45.4 min) which was identi-
fied as being enriched with Met-127 EtO adducts based on its
RT (Fig. 5b), mass and MS/MS data (data not shown). Simi-
larly, the major change seen in the PP3 fraction Glu-C digest
(RT: 42.9 min) was identified as enriched Met-138 EtO adducts
(Fig. 5c). Note that several minor changes were also observed
in PP1 and PP3 fraction Glu-C digests, which may be because
of the impurities from adjacent peaks in the collected fractions.
For the PP2 fraction Glu-C digest, multiple peaks were found
with higher intensity in the overlaid UV traces. These peaks
were identified as several oxidized methionine residues (Met-
138, Met-127, and Met-122) (Fig. 5d).
The CEX fractions were also tested by an intact protein
RP-HPLC method to compare the hydrophobicity of the EtO
adducts. The RP-HPLC traces indicated that all post peak frac-
tion chromatograms are eluted at the prepeaks region on the
RP-HPLC. The fractions PP1 (Met-127 EtO adducts) and PP3
(Met-138 EtO adducts) were eluted earlier than fraction PP2
(multiple oxidation species) and the main peak (no modifica-
tion). This indicates that the EtO adducts are less hydrophobic
than the oxidation species and main peak, presumably because
of the introduction of the charged side chain.
Detection and Identification of EtO-Induced Modifications of
HSA in EPO Formulations
Erythropoietin samples containing HSA that had been incu-
bated at 37◦C for 2 weeks were digested with trypsin and an-
alyzed by LC–MS. Samples that had been incubated in EtO-
sterilized plastic vials were compared with control samples that
had been incubated in unsterilized plastic vials as well as in
heat-depyrogenated glass vials. Figure 6 shows the HPLC–UV
chromatogram comparison for these samples. Two peaks are
shown to be significantly different in the EtO sterilized vial,
one at a RT of 131 min and another at 163 min. From the MS
analysis, the peak at 131 min has a mass of 1666.72 Da and
was speculated to be indicative of a 44 Da increase resulting
from EtO reaction with Met-328 of HSA peptide D324-R336.
Met-328 showed susceptibility to oxidation indicating that it is
solvent exposed on the surface of HSA (data not shown). Similar
to the previously described on Peg-GCSF results, the MS/MS
results for this EtO-modified peptide indicate a predominant
fragmentation pattern resulting in a loss of 92 Da because of
the loss of the modified side chain of methionine. Analysis of
lower abundance fragmentation products of mass spectrum re-
vealed fragments that confirmed the identity of the modified
peptide as D324-R336 of HSA (Fig. 7).
Close examination of the HPLC–UV chromatogram revealed
that the increase in the new peak at 163 min in the EtO-
sterilized plastic vial sample coincided with the decrease of
the adjacent peak eluting 1 min earlier. The native peak elut-
ing at 162 min was identified by MS/MS as A21-K41 of HSA
with a +58 Da modification of Cys-34 because of the reduction
and alkylation procedure performed prior to the tryptic digest
(Fig. 8). This peptide does not contain a methionine residue.
However, a previously published report indicated the possibil-
ity of cysteine reacting with EtO.1In addition, Cys-34 of HSA
is known to be the only free cysteine residue in HSA, with the
other 34 cysteine residues in HSA forming 17 disulfide bonds.14
DOI 10.1002/jps.24257 Chen et al., JOURNAL OF PHARMACEUTICAL SCIENCES
8RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
It was assumed that EtO adduct formation on Cys-34 would
prevent its alkylation during sample preparation and would
result in a −14 Da change in the expected mass of the Cys-
containing peptide. Comparing the full scan MS as well as the
MS/MS spectra revealed that the two peptides (RT: 162 and
163 min) are closely related but with a difference in mass of
14 Da. The native peptide with cysteine alkylation has a mass
of 2491 Da, whereas the new peak in the EtO sample has a mass
of 2477 Da. The MS/MS spectrum for the new peak is identi-
fied as a +44 Da modification of Cys-34 in the absence of the
+58 Da modifications from alkylation thus accounting for the
14 Da difference between the two peptides. The MS/MS frag-
mentation pattern of the EtO-modified cysteine peptide did not
show the side-chain loss pattern displayed by the EtO-modified
methionine peptides (data not shown).
Erythropoietin is somewhat unique because of the fact that
its formulation contains a second protein. HSA is added as a
stabilizer and is present at a significantly higher concentra-
tion relative to EPO and is therefore expected to be the greater
scavenger for side-chain modifiers. Thus, the relatively low con-
centration of EPO in the formulation, EtO-modified EPO could
not be detected in the tryptic peptide map performed in this
study.
DISCUSSION AND CONCLUSION
The Glu-C and tryptic peptide map analyses by LC–MS
played an important role for the characterization of the EtO–
methionine modifications in Peg-GCSF and HSA in this study.
We have closely examined the degradation of these proteins
in different EtO sterilized containers and found a characteris-
tic MS/MS fragmentation pattern for the methionine-modified
peptides. Although the modified peptide showed a mass in-
crease of 44 Da, its fragmentation mass spectra (MS/MS) often
contained a peak where the mass decreased by 92 Da relative to
the precursor ion. It appeared that the side chain of the methio-
nine residue was increased by 44 Da and was then lost during
the gas-phase fragmentation generating a total loss of 92 Da
relative to the modified peptide, which resulted in a net change
of −48 Da. We have found that an EtO-modified methionine side
chain has a unique fragmentation pattern under CID in that it
readily loses a C3H8OS group (92 Da). The loss of the side chain
and double-cleavage (side chain and peptide backbone) create
a fragmentation pattern that is difficult to identify for typical
software algorithms. The uncovered gas-phase fragmentation
pattern of peptides with methionine residues modified by EtO
should facilitate reliable and automated identification in future
analyses of this modification by LC–MS/MS in therapeutic pro-
teins.
Regarding the other biophysical and biochemical properties
of EtO adducts, the EtO modification of methionine residues in-
volved formation of a positively charged sulfonium compound
resulting from EtO reacting with the sulfur ion of methionine.
Windmueller et al.1reported in a model experiment that sul-
fonium derivatives of methionine possess different crystalliza-
tion ability and water solubility. In the present study, the EtO–
methionine modification species (EtO–methionine adducts) in
Peg-GCSF were eluted at the post peaks region on CEX–HPLC.
It was also found eluting in the prepeak region with less hy-
drophobicity than the oxidized methionine species on RP-HPLC
analysis of Peg-GCSF and HSA.
For the EtO-cysteine reaction, MS/MS of peptides containing
a modified cysteine were detected by tryptic peptide mapping
of the HSA. The modified peptide with a −14 Da difference
compared to the native peptide (A21-K41) was identified as
an EtO–cysteine modification. The −14 Da was the result of a
mass difference between alkylated-cysteine (+58 Da) and EtO-
cysteine (+44 Da).
Ethylene oxide modifications can alter the stability and
bioactivity of proteins and several published reports15 have
shown the impact of EtO-modified proteins on patient safety.
The possibility of EtO modifications of therapeutic proteins and
the potential impact should be considered during EtO steriliza-
tion processes development. Additional methods to confirm that
residual EtO have reached acceptable levels and the applica-
tion of these methods to understand the desorption kinetics of
EtO in a variety of materials would be advantageous for future
drug and container development.
ACKNOWLEDGMENTS
The authors would like to thank Gang Xiao for fruitful discus-
sions and support in performing LC–MS analyses, and Mike
Treuheit for careful review of the manuscript and valuable
suggestions.
REFERENCES
1. Windmueller HG, Ackerman CJ, Engel RW. 1958. Reaction of ethy-
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