epoetin delta, Dynepo

Analytical Biochemistry 383 (2008) 243–254
Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio
Structural analysis of the glycosylation of gene-activated erythropoietin
(epoetin delta, Dynepo)
Esther Llop a, Ricardo Gutiérrez-Gallego a,b,*, Jordi Segura a,b, Joaquim Mallorquí a, José A. Pascual a,b
a
b
Bioanalysis Research Group, Neuropsycho-pharmacology Program, IMIM–Hospital del Mar, Barcelona Biomedical Research Park (PRBB), 08003 Barcelona, Spain
Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park (PRBB), 08003 Barcelona, Spain
a r t i c l e
i n f o
Article history:
Received 16 June 2008
Available online 3 September 2008
Keywords:
Erythropoietin
Epoetin delta
Glycosylation
2-DE
Mass spectrometry
MALDI
Sialic acid
a b s t r a c t
Recently, a novel recombinant human erythropoietin (epoetin delta, Dynepo) has been marketed in the
European Union for the treatment of chronic kidney disease, cancer patients receiving chemotherapy,
and so forth. Epoetin delta is engineered in cultures of the human fibrosarcoma cell line HT-1080 by
homologous recombination and ‘‘gene activation.” Unlike recombinant erythropoietins produced in other
mammalian cells, epoetin delta is supposed to have a human-type glycosylation profile. However, the isoelectric focusing profile of epoetin delta differs from that of endogenous erythropoietin (both urinary and
plasmatic). In this work, structural and quantitative analysis of the O- and N-glycans of epoetin delta was
performed and compared with glycosylation from recombinant erythropoietin produced in Chinese hamster ovary (CHO) cells. From the comparison, significant differences in the sialylation of O-glycans were
found. Furthermore, the N-glycan analysis indicated a lower heterogeneity from epoetin delta when compared with its CHO homologue, being predominantly tetraantennary without N-acetyllactosamine
repeats in the former. The sialic acid characterization revealed the absence of N-glycolylneuraminic acid.
The overall sugar profiles of both glycoproteins appeared to be significantly different and could be useful
for maintaining pharmaceutical quality control, detecting the misuse of erythropoietin in sports, and
establishing new avenues to link glycosylation with biological activity of glycoproteins.
Ó 2008 Published by Elsevier Inc.
Human erythropoietin (EPO)1 was the first hematopoietic
growth factor to be cloned, and the recombinant molecule became
available as a drug in 1988 [1]. Recombinant erythropoietin (rEPO)
and analogues are frequently used in the treatment of anemia in
chronic kidney disease, AIDS, prior autologous blood transfusions,
* Corresponding author. Address: Department of Experimental and Health
Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park (PRBB),
08003 Barcelona, Spain. Fax: +34 93 316 04 67.
E-mail address: [email protected] (R. Gutiérrez-Gallego).
1
Abbreviations used: EPO, erythropoietin; rEPO, recombinant erythropoietin; rEPO
d, epoetin delta; CHO, Chinese hamster ovary; BHK, baby hamster kidney; CMAH,
CMP-N-acetylneuraminic acid hydroxylase; IEF, isoelectric focusing; PNGase F,
peptide-N4-(acetyl–glucosaminyl)-asparagine amidase F; DHB, 2,5-dihydroxybenzoic
acid; sinapinic acid, 3,5-dimethoxy-4-hydroxycinnamic acid; IAA, iodoacetamide; 2AB, 2-aminobenzamide; NaBH3CN, sodium cyanoborohydride; DMSO, dimethyl
sulfoxide; DTT, dithiothreitol; SDS, sodium dodecyl sulfate; TEMED, N,N,N,N’-tetramethylethylenediamine; DMB, 1,2-diamino-4,5-methylene dioxybenzene; TFA,
trifluoroacetic acid; UPLC–ESI–TOF, ultra-performance liquid chromatography–electrospray ionization–time-of-flight; MS, mass spectrometry; HPLC–FLD, highperformance liquid chromatography–fluorescence detection; 2-DE, two-dimensional
electrophoresis; PVDF, polyvinylidene fluoride; mAb, monoclonal antibody; PAGE,
polyacrylamide gel electrophoresis; MALDI, matrix-assisted laser desorption/ionization; WAX, weak anion exchange; NP, normal phase; GU, glucose units; LacNAc,
N-acetyllactosamine; Neu5Gc, N-glycolylneuraminic acid; Neu5Ac, N-acetylneuraminic acid; RAAM, reagent array analysis method; ELISA, enzyme-linked immunosorbent assay.
0003-2697/$ - see front matter Ó 2008 Published by Elsevier Inc.
doi:10.1016/j.ab.2008.08.027
and many other malignancies [2]. In sports, the misuse of these substances has also been demonstrated [3,4]. With the key patents for
rEPO expired, the market has opened for biosimilar recombinant
products from the same cell line with identical or improved pharmacokinetic properties and less immunogenic response [5]. Also, completely new products are produced, for example, epoetin delta (rEPO
d, Dynepo) from Shire Pharmaceuticals. The latter is homologously
expressed by gene activation in a human fibrosarcoma cell line
(HT-1080) [6]. rEPO d, as well as other epoetins, has the same 165amino-acid polypeptide chain as the naturally occurring human
forms (urinary and plasmatic). However, from a biochemical viewpoint, there is an interesting diversity in their posttranslational modification, most likely in glycosylation. Like endogenous EPO, epoetins
contain one O-linked oligosaccharide (Ser126) and three N-linked
oligosaccharides (Asn24, -38, and -83) [7,8], and these account for
approximately 40% of the total molecular weight ( 30 kDa). The
biosynthesis of glycans is species, tissue, and cell type dependent,
but the culture conditions may also contribute to the so-called
microheterogeneity, resulting in a large diversity of glycan structures [9–17]. Chinese hamster ovary (CHO) and baby hamster kidney
(BHK) cells are the hosts used for the production of rEPO pharmaceuticals. The enzymatic endowment of these frequently used host cells
is similar to that of human cells. However, some human tissue-spe-
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(IAA), bovine serum albumin, bovine fetuin, and trypsin obtained
from bovine pancreas (EC 3.4.21.4) were purchased from Sigma
(Barcelona, Spain). 2-Aminobenzamide (2-AB), sodium cyanoborohydride (NaBH3CN), and dimethyl sulfoxide (DMSO) were obtained
from Fluka (Barcelona, Spain). Dithiothreitol (DTT) was obtained
from GE Healthcare (Cerdanyola, Spain). Sialic acid reference panel
was obtained from Glyco (Ely, UK). Acrylamide-bis (97:3, w/w), silver nitrate, and sodium dodecyl sulfate (SDS) were obtained from
Merck (Barcelona, Spain). N,N,N,N’-Tetramethylethylenediamine
(TEMED) and ammonium persulfate were obtained from Bio-Rad
(el Prat de Llobregat, Spain). GELoader tips were purchased from
Eppendorf (Madrid, Spain), Quartz microfiber filters QMA were obtained from Whatman (Maidstone, UK). All other chemicals were of
the highest purity commercially available.
Sialic acid analysis
Fig. 1. IEF profiles obtained for analysis of reference preparations of endogenous
urinary EPO (uEPO, international reference preparation from National Institute for
Biological Standards and Control, UK), recombinant erythropoietins (rEPO d, epoetin
delta, Dynepo, Shire Pharmaceuticals, UK), rEPO a/b (biological reference preparation of the European Pharmacopoeia containing epoetin alpha and epoetin beta in
equal amounts), and new erythropoiesis-stimulating protein (NESP, darbepoetin
alpha, Aranesp, Amgen, USA). The different areas and identification of bands
established by doping control laboratories are indicated.
cific terminal carbohydrate motifs are not synthesized by BHK and
CHO cells because they lack the proper sugar-transferring enzymes
[18] (e.g., a2-6 sialyltransferase, a1-3/4 fucosyltransferase, bisecting
N-acetylglucosamine transferase). On the contrary, these cells contain the enzyme responsible for N-glycolylneuraminic acid synthesis
(CMP-N-acetylneuraminic acid hydroxylase [CMAH]) that is absent
in humans [19]. The isoelectric focusing (IEF) analysis of rEPO displays a profile in which more acidic isoforms can be observed with
respect to CHO-derived marketed rEPOs, albeit not the most acidic
as isoforms present in endogenous EPO (urinary or plasmatic) [20–
22] (Fig. 1). These different IEF profiles suggest that the glycosylation
could be different. Even though several articles have been published
on the gene expression of rEPO d and its use in medicine [23,24], details on the glycosylation affecting the biological activity have not
yet been reported. In the current article, we describe the glycosylation of rEPO d as compared with CHO cell-derived epoetins.
Materials and methods
Materials
Recombinant human EPO (rEPO produced in CHO cells) was obtained from the European Pharmacopoeia Commission (rEPO a/b,
Salisbury, UK), and epoetin delta (rEPO produced in human fibrosarcoma HT-1080 cells) was obtained from Shire Pharmaceuticals
(rEPO d, Dynepo, Hampshire, UK). Recombinant peptide-N4-(acetyl-b-glucosaminyl)-asparagine amidase F (PNGase F, EC 3.1.27.5),
recombinant b-1,4-galactosidase (EC 3.2.1.23), recombinant b1-RN-acetyl-glucosaminidase (EC 3.2.1.97), and recombinant a23,6,8,9-sialidase (EC 3.2.1.18) were purchased from Calbiochem
(La Jolla, CA, USA). a2-3 Sialidase (EC 3.2.18) was obtained from
Takara Biotechnology (Shiga, Japan). Carbograph graphitized carbon ultraclean columns (150 mg) were purchased from Alltech
(Deerfield, IL, USA). 2,5-Dihydroxybenzoic acid (DHB), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), iodoacetamide
For the study of the sialic acid heterogeneity, sialic acids were
released from the carbohydrate chains and derivatized with 1,2diamino-4,5-methylene dioxybenzene (DMB) [25]. Briefly, hydrolysis of 1 lg of each sample was performed, incubating samples
with trifluoroacetic acid (TFA) for 1 h at 50 °C. Next, a 7 mM
DMB solution in 1.4 M aqueous acetic acid containing 18 mM sodium hydrosulfite and 1 M b-mercaptoethanol was added. The
mixture was kept for 2 h at 50 °C. Fluorescently labeled residues
were analyzed by reversed-phase chromatography on a capillary
column (Zorbax SB-C18, 150 0.3 mm, 3.5 lm) using MeCN/H2O
(20:80) as eluent (mobile phase) and a flow rate of 4 ll/min. Chromatographic analyses were performed on an Agilent 1100 series
capillary instrument equipped with a Jasco micro21FP capillary
fluorescence detector (kex = 373 nm, kem = 448 nm).
DMB derivatives of sialic acids were also analyzed by ultra-performance liquid chromatography–electrospray ionization–time-offlight (UPLC–ESI–TOF) mass spectrometry (MS) [26] using an
Acquity ultra-performance liquid chromatograph (Waters) coupled
to an LCT premier XE ESI–TOF instrument (Micromass). The procedure for sialic acid hydrolysis was similar to that described above
but used 2 M aqueous acetic acid solution for 3 h at 80 °C instead
of TFA [27]. The derivatization protocol was identical, but sample
amounts prepared for LC–ESI–TOF were 10 times higher (10 lg)
than in high-performance liquid chromatography–fluorescence
detection (HPLC–FLD) analysis due to the lower sensitivity of the
former technique. LC analyses were performed at a flow rate of
0.2 ml/min using an Acquity UPLC BEH C18 column
(100 2.1 mm, 1.7 lm). Derivatized sialic acids were eluted isocratically employing MeOH/MeCN/H2O (7:9:84) for 5 min, and
then with a 1-min gradient the running buffer was changed to
MeOH/MeCN/H2O (7:25:68), maintained for 1 min, returned to initial conditions, and stabilized for 1 min before the next injection.
Mass spectra were acquired in negative ion mode over a mass
range-to-charge (m/z) ratio of 50 to 650. The capillary voltage
was set at 2800 V (negative), the desolvation temperature was
set at 250 to 300 °C, and the desolvation gas flow was set at
300 L/h. The TOF tube voltage was kept at 7200 (reflectron at
1800 V) with a pusher setting of 900 V (pusher offset at 0.93). Recorded data were processed using MassLynx software (version 4.1,
Waters).
Glycoform characterization
First dimension (IEF)
IEF was performed as described previously by Lasne and
coworkers [3]. In brief, rEPO d and rEPO a/b samples (sample
amounts loaded ranged from 0.3 ng for chemiluminescence detection to 5 lg for two-dimensional electrophoresis [2-DE] and silver
staining) were focused in a polyacrylamide IEF gel with a pH gra-
Glycosylation of Dynepo / E. Llop et al. / Anal. Biochem. 383 (2008) 243–254
dient 2 to 6. The samples were focused at a constant power of 1 W/
cm of the gel length at 8 °C until 3600 Vh in a Multiphor II Electrophoresis system (Amersham Pharmacia). After focusing, proteins
were blotted (0.8 mA/cm2 gel, 30 min) to a polyvinylidene fluoride
(PVDF) membrane and then blocked and incubated with a specific
mouse anti-human EPO monoclonal antibody (mAb). The mAb was
then reblotted (0.8 mA/cm2 gel, 10 min) to a second PVDF membrane using 0.7% acetic acid as transfer buffer [28]. After blocking,
the second PVDF membrane was incubated with biotinylated goat
anti-mouse IgG (H+L) and finally treated with streptavidin/horseradish peroxidase. The primary antibody–secondary antibody–
streptavidin/horseradish peroxidase complex was detected by the
addition of the peroxidase substrate. The chemiluminescence light
produced was detected using a FujiFilm CCD camera (LAS-1000).
Second dimension (2-DE)
After IEF, the strip of interest was excised and placed directly on
top of an SDS–PAGE (polyacrylamide gel electrophoresis) gel. Electrophoresis was performed using standard methods on a Bio-Rad
Mini-Protean III system (7 10-cm minigels). In the experiments,
10% acrylamide gels of 1 mm thickness were used. Gels were run at
150 V constant voltages in 25 mM Tris/190 mM glycine/0.1% SDS at
4 °C. Glycoproteins on gels were visualized by silver staining as described previously by Shevchenko and coworkers [29].
Enzymatic release of N-glycans
The in-gel digestion procedure followed the method described
previously by Llop and coworkers [30]. Briefly, protein bands were
excised from the gel, cut into small pieces, washed with water,
shrunk in MeCN for 10 min, and dried in a vacuum centrifuge. Then
proteins contained in gel pieces were reduced with DTT and alkylated with IAA. For deglycosylation of the protein, 50 mM sodium
phosphate buffer (pH 7.3) containing PNGase F (1 IU) was added
to the glycoprotein in solution or gel pieces sample, and the mixture was incubated at 37 °C for 24 h. In the case of gel samples,
the supernatant containing the released glycans was separated
for structural analysis, whereas gel pieces were submitted to a
tryptic digestion for protein identification.
245
of 1 lg of rEPO d and rEPO a/b. Samples were incubated at 37 °C for
24 h. Subsequently, samples were purified with POROS R2 resin
and eluted in 80% MeCN and 0.1% TFA. An aliquot (10 ng) was
mixed with sinapinic acid matrix and analyzed by matrix-assisted
laser desorption/ionization (MALDI)–TOF MS, and the other was
lyophilized for glycan profiling.
Exoglycosidase sequencing and reagent array analysis method
N-Glycans, generated via solution or in-gel digestions, were
submitted to simultaneous exoglycosidase digestions with a23,6,8,9 sialidase, b1-4 galactosidase, and b1-R-N-acetylglucosaminidase in 50 mM sodium phosphate buffer (pH 6.0) for 16 h at
37 °C [33]. After digestion, samples were filtered over 5-kDa filters,
lyophilized, resuspended in 100 ll of bidistilled water for HPLC
(weak anion exchange [WAX] after sialidase digestion and normal
phase [NP] after all other treatments) and MALDI–TOF MS analysis.
Glycan profiling
WAX HPLC
WAX HPLC of 2-AB N-linked glycans was carried out using an
Agilent 1090 HPLC device equipped with a fluorescence detector
(1100 Agilent fluorescence module, excitation k= 330 nm, emission
k = 420 nm). Glycan profiling before and after sialidase treatment
was performed on a Vydac 301 VHP column (7.5 50 mm) with
the following gradient conditions: solvent A was 20% MeCN in
water, and solvent B was 20% MeCN in 500 mM ammonium acetate
(pH 4.4). Initial conditions were 100% A at a flow rate of 0.4 ml/
min. Following injection, samples were eluted by a linear gradient
Glycan purification
Liberated glycans (either in solution or from gel pieces) were desalted with graphitized carbon columns [31]. Glycans were eluted
with 50 ll of 80% MeCN and 0.1% TFA and were frozen at 80 °C,
lyophilized, and dried at 60 °C in a vacuum oven for 30 min.
Fluorescence labeling
Oligosaccharide samples were derivatized with 2-AB as described previously by Bigge and coworkers [32]. A freshly prepared
solution of 0.35 M 2-AB in 500 ll of DMSO/acetic acid (70:30, v/v)
containing 1 M NaCNBH3 was prepared. A 10-ll aliquot was added
to each dried oligosaccharide sample, and the mixture was incubated 4 30 min at 60 °C with intermediate shaking. To eliminate
the excess of 2-AB, labeled samples were applied to Whatman
QMA paper, allowed to dry, and washed with 5 ml of MeCN. Carbohydrates were eluted with 1.8 ml of water and lyophilized.
Enzymatic digestions
Desialylation
Sialidase digestions were performed in 20 ll of sodium acetate
buffer (pH 5.5) in the case of a 2-3 sialidase and in 50 mM sodium
phosphate buffer (pH 6.0) in the case of a 2-3,6,8,9 sialidase. In
both cases, 20 mU of enzyme was added for complete desialylation
Fig. 2. Molecular weight determination of rEPO d and rEPO a/b by MALDI–TOF MS.
(A) Spectra of glycoproteins. (B) Spectra after sialidase treatment. After desialylation, the peak pattern reveals the glycan distribution in terms of antennas and
LacNAc repeating units.
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Glycosylation of Dynepo / E. Llop et al. / Anal. Biochem. 383 (2008) 243–254
of 100 to 0% B over 5 min, followed by a linear gradient of 0 to 100%
B over the next 35 min, returning to the start conditions over the
next 15 min. The total running time was 60 min. The column was
calibrated with 2-AB-labeled N-glycans from bovine fetuin [34].
NP HPLC
NP HPLC analyses were performed on a capillary system used
for sialic acid analyses. Fluorescence detector parameters were
the ones recommended for 2-AB tag as in WAX analyses. NP profiling was carried out on a TSK gel Amide-80 column (0.5 150 mm)
using the following gradient conditions: solvent A was 10% 50 mM
ammonium formate (pH 4.4) in 90% MeCN, solvent B was 90%
50 mM ammonium formate (pH 4.4) in 10% MeCN, and the flow
rate was 15 ll/min. Following injection, samples were eluted by
a linear gradient of 20 to 55% B over 100 min, followed by a linear
gradient of 55 to 100% B over the next 5 min. The column was
eluted using 100% B for 2 min and subsequently was reequilibrated
in 20% B before injection of the next sample. The system was calibrated in glucose units (GU) using a 2-AB-labeled dextran hydrolysate [35]. The total running time was 125 min.
Fig. 3. Comparison of O-glycoforms from rEPO d and rEPO a/b by MALDI–TOF MS (A) and ESI–TOF MS (B).
, N-acetylgalactosamine; j, galactose; D, sialic acid.
Fig. 4. (A) HPLC–FLD pattern of fluorescent DMB derivatives of sialic acids from rEPO d and rEPO a/b in comparison with the standard sialic acid (SA) mixture included in the
lower panel. The identification of the numbered species is given in panel B. The insert is a zoom of the chromatograms where the presence of N-glycolylneuraminic acid can
be appreciated in the rEPO a/b only. (B) Extracted ion chromatograms of LC–MS analysis of the DMB-derivatized sialic acid residues derived from rEPO d (left) and rEPO a/b
(right). The total ion chromatogram (TIC) is included at the bottom.
Glycosylation of Dynepo / E. Llop et al. / Anal. Biochem. 383 (2008) 243–254
MALDI–TOF MS analyses
Samples (proteins or carbohydrates) were dissolved in water at
varying concentrations. Aliquots were mixed with the corresponding matrix solution, and less than 1 ll of these preparations was
applied to the polished stainless-steel target and allowed to dry
at room temperature. A solution of sinapinic acid (10 mg/ml) in
MeCN/H2O/TFA (50:50:0.1, v/v/v) was chosen for protein analyses,
and a solution of DHB (10 mg/ml) in MeCN/H2O (50:50, v/v) was
chosen for N-glycan analyses. Experiments were carried out on a
Voyager-DE STR Biospectrometry workstation (Applied Biosystems) equipped with an N2 laser (337 nm). Typically, spectra of
sialylated N-glycans were acquired in linear mode with negative
polarity, and spectra of neutral N-glycans were acquired in reflectron mode with positive polarity. External calibration of the spectrometer was performed using a mixture of 2-AB-labeled glucose
oligomers in the positive ion mode and 2-AB-derivatized fetuin
N-glycans in the negative mode. Recorded data were processed
with Data Explorer software (Applied Biosystems).
LC–ESI–TOF MS analyses
Positive ion mode LC–ESI–TOF MS analyses of O-glycoproteins,
after de-N-glycosylation, were performed using the instrument de-
Fig. 5. WAX HPLC profiles of 2-AB-labeled glycans from rEPO d (upper panel), rEPO
a/b (center panel), and Fetuin used as standard (lower panel). The regions
corresponding to neutral (N) glycans and mono-, di-, tri-, and tetra-charged glycans
(1, 2, 3, and 4, respectively) are indicated with gray columns. 2-AB indicates the
excess of derivatization reagent. Structures were tentatively assigned based on MS
data (vide infra). h, fucose; d, N-acetylglucosamine; , mannose; j, galactose; D,
sialic acid. The structural assignments for rEPO a/b are based on NMR data. The
structural assignments for rEPO d have been done by analogy to those of rEPO a/b
and are indicative only.
247
scribed earlier in the ‘‘Sialic Acid Analyses” section. An aliquot of
1 ll from 0.3 lg/ll sample solution (EPO d and rEPO a/b) was analyzed in flow injection configuration at a flow rate of 0.05 ml/min
using a mobile phase of H2O/MeCN/FA (95:5:0.1, v/v/v). Mass spectra were acquired in positive ion mode for 3 min, covering the
range of m/z 500 to 1500. The capillary voltage was set at 3000
V, the desolvation temperature was set at 350 °C, and the desolvation gas flow was set at 400 L/h.
Results
Glycoprotein analysis
Intact rEPO d was analyzed by MALDI–TOF MS to determine the
molecular mass and estimate the microheterogeneity of the glycoprotein. The average molecular weight was 29.75 kDa for rEPO d, a
value slightly higher than the reference rEPO a/b (29.39 kDa). A
comparison of the Gaussian distributions of the MS peaks and their
widths at baseline suggests that rEPO d is less heterogeneous than
rEPO a/b (Fig. 2A). When the molecular weight was measured after
a2-R sialidase digestion (Fig. 2B), the signal became better resolved, reflecting the number of antennas and/or N-acetyllactosamine (LacNAc) repeats present in each glycan. The pattern of
peaks separated by 365 Da consisted of 3 peaks in rEPO d, whereas
this phenomenon was more abundant (11 peaks) in rEPO a/b, indicating more structural variety. At this stage, the most intense peak
in the mass spectrum of both preparations could be explained by
the peptide backbone plus 3 tetraantennary core-fucosylated Nglycans and 1 core–1 O-glycan. Interestingly, glycoforms containing tri- and diantennary N-glycans are negligible for rEPO d,
whereas they constitute an important part of the N-glycans in rEPO
a/b. The mass difference between each glycoprotein before and
after sialidase digestion allows estimating the average sialic acid
content. For rEPO d, the mass shift of approximately 3.5 kDa indicates the loss of an average of approximately 12.02 sialic acid residues; for rEPO a/b, the mass difference was 3.3 kDa,
corresponding to approximately 11.53 sialic acid residues. This
higher sialic acid content of rEPO d may justify the presence of
more acidic bands in the IEF profile (Fig. 1). MS analysis after complete removal of all N-glycans corroborated that rEPO d contains
only 1 O-glycan and allowed studying the microheterogeneity contained within this glycan. The MALDI–TOF analysis (Fig. 3A)
showed that 3 main O-glycoforms could be distinguished bearing
no sialic acid residue (m/z = 18,616), 1 sialic acid residue (m/z =
18,907), or 2 sialic acid residues (m/z = 19,198), as indicated by
mass increments of 291. Although heterogeneity was caused
mainly by a variable degree of sialylation of the O-glycan structures, non-O-glycosylated molecules could also be detected (m/z =
18,249), although the peak intensity was very low. To corroborate
the nature of these differences, the O-glycoprotein was analyzed
after sialidase treatment, showing the disappearance of sialylated
O-glycans and the increment of nonsialylated peak accounting for
up to 98% of the total area (data not shown). Although the use of
MALDI–TOF MS reflects the complexity present in the O-glycan,
the fact that part of the heterogeneity was caused by fragmentation
of sialic acids cannot be excluded. Then a milder ionization technique (LC–ESI–TOF) was used to minimize losses of sialic acids, giving more feasible quantitative results (Fig. 2B). Under these
conditions, mass values and relative abundances obtained by MALDI–TOF were confirmed. Also by this technique, rEPO d presented
quantitative differences in sialylation of the O-glycans compared
with rEPO a/b. The disialylated peak was the only one detected in
rEPO d, accounting for the 100% of the signal. For rEPO a/b, both species (di- and monosialylated O-glycans) could be clearly detected,
accounting for 56.36 and 43.64%, respectively. This result suggests
that even under mild MALDI conditions, a certain degree of
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desialylation cannot be avoided [36,37]. Hence, in this article, MALDI–TOF MS is proposed to describe the heterogeneity contained in
the glycoprotein and ESI–TOF MS is employed for more accurate
quantification.
Sialic acid analysis
Analysis by reversed-phase HPLC of the fluorescently tagged
sialic acids released by mild acid hydrolysis allowed determining
the percentage of N-glycolylneuraminic acid (Neu5Gc), N-acetylneuraminic acid (Neu5Ac), and other variants (Fig. 4A). Identification was carried out by comparing retention times with reference
compounds [38]. Neu5Ac turned out to be the major constituent
in both recombinant preparations, accounting for 98.1 mol% in
rEPO d and 87.8 mol% in rEPO a/b. In addition, O-acetyl modifications could also be identified, although they were far less abundant
in rEPO d; Neu5,9Ac2 accounted for 1.9 mol% in rEPO-d, whereas it
accounted for up to 7.9 mol% in rEPO a/b. Also, Neu5,7Ac2 (1.2
mol%) and Neu5,7(8),9Ac3 (1.7 mol%) could be detected, but in
rEPO a/b only. Nevertheless, identification of the exact locations
of the O-acetyl linkage is somewhat cumbersome due to the ability
of these substituents to migrate from one hydroxyl to another
along the alkyl structure. Despite the care taken in sample handling to avoid migration of O-acetyl groups, the microheterogeneity observed in replicates could still be affected by the specific
batch of rEPO and/or sample treatment prior to analysis. Finally,
an interesting feature in sialic acid speciation is the potential presence of N-glycolylneuraminic acid (Neu5Gc). As expected, rEPO d
produced in human cells (devoid of the corresponding hydroxylase) did not contain any Neu5Gc (limit of detection of 6 fmol
[i.e., 0.3 mol%] in these experiments), whereas rEPO a/b had
approximately 1.3 mol% Neu5Gc. To corroborate these results, sialic acids were analyzed simultaneously by UPLC–ESI–TOF (Fig. 4B).
Confirming what was seen by fluorimetric detection, rEPO d
showed a detectable peak at the trace corresponding to m/z
424.141 (protonated pseudomolecular ion of Neu5Ac) but none
at m/z 440.13, characteristic of Neu5Gc. On the contrary, the analyses of rEPO a/b showed a clear peak at the trace m/z 440.13, confirming the presence of Neu5Gc. Furthermore, besides the most
abundant peak of Neu5Ac (at m/z 424.14), other species with acetyl
groups at different positions were also found—Neu5,7(9)Ac2 (m/z
466.14) and Neu5,7,9Ac3 (m/z 508.15).
N-Glycan analysis
Peptide mapping of rEPO d and rEPO a/b after de-N-glycosylation yielded no structural differences between the two (data not
shown). At this point, the study was focused on the N-glycans released from the proteins in an attempt to identify and quantify
all structures present first in the total mixture and then in each
individual glycoform after 2-DE gel separation. WAX carbohydrate
profiling, standardized against fetuin N-glycans, was chosen to
analyze the charge state of N-glycans (Fig. 5). For rEPO d, 3 major
peaks were observed, showing a much less heterogeneous profile
than rEPO a/b. The single peak of tetra-charged structures of rEPO
d accounted for 44.28%, tri-charged accounted for 37.69%, and dicharged accounted for 16.81%. Although it was supposed to contain
a low proportion of mono-charged entities, they could not be
quantified because of the 2-AB excess. Neutral structures were
nearly absent (1.2%). For rEPO a/b, once the areas of the 2 peaks
accounting for tetrasialylated N-glycans were summed, they
yielded 47.85%. The 5 different peaks of tri-charged species contributed 36.85%, and another 4 peaks accounted for 12.63%, of disialylated N-glycans. Monosialylated and neutral N-glycans were
minorities, accounting for 0.76 and 1.90%, respectively. Percentages of N-glycans with different degrees of sialylation together
with percentages of each sialylated O-glycoform allowed calculating the average number of sialic acids. The equation applied is as
follows:
4 %N4SA þ 3 %N3SA þ 2 %N2SA þ 1 %N1SA
12
4 100
2 %O2SA þ 1 %O1SA
þ
2;
2 100
where% N4SA stands for the percentage of N-glycans tetrasialylated,% N3SA stands for the percentage of N-glycans trisialylated, and
so forth. The equivalent is also included for O-glycans. The denominator for N-glycans (4 100) normalizes the result and then is
multiplied by 12, the maximum number of sialic acids potentially
present in all 3 glycans (4 each). For O-glycans the normalization
factor is then 2 100 and the result is multiplied by 2.
To compute the average number of sialic acids in the entire glycoprotein, the sialic acid content of the O-glycan was calculated
from the O-glycoprotein MS analysis and that of the N-glycans
was calculated from the WAX analysis of the derivatized N-glycans.
Fig. 6. Negative ion mode MALDI mass spectra of 2-AB-labeled glycans from rEPO d (top) and rEPO a/b (bottom). The depicted structures indicate all possible isomers. h,
fucose; d, N-acetylglucosamine; , mannose; j, galactose; D, sialic acid. The structural assignments for rEPO a/b are based on NMR data. The structural assignments for rEPO
d have been done by analogy to those of rEPO a/b and are indicative only.
Glycosylation of Dynepo / E. Llop et al. / Anal. Biochem. 383 (2008) 243–254
249
The value obtained for rEPO d was 11.72 sialic acids, whereas that
obtained for rEPO a/b was 11.39 sialic acids.
Further structural information of the N-glycan composition was
obtained from MALDI–TOF MS analyses of the 2-AB-labeled structures. Profiles obtained for rEPO d (Fig. 6) displayed mainly fucosylated tetraantennary complex type N-glycans containing up to 4
sialic acid residues, some triantennary N-glycans containing up
to 3 sialic acids, and no diantennary N-glycans. These structures
are also present in rEPO a/b, but in this case diantennary N-glycans
were also present. Furthermore, in the latter, a relatively higher
content of triantennary N-glycans and other structures with up
to 2 LacNAc repeats was observed. NP HPLC profiling of the same
structures was conducted following the observations of the different charge profiles. The individual chromatograms represent the
structural heterogeneity present in the N-glycans of each sample.
From a comparison between both recombinant pharmaceuticals,
it becomes evident that rEPO d (Fig. 7, upper panel) is a more
homogeneous preparation. The rEPO d profile showed 5 peaks in
a retention time interval of 10 to 12 GU. Conversely, rEPO a/b
yielded much greater heterogeneity, with at least 13 distinctive
structures that elute at GU values between 8 and 14. Structures
at GU values greater than 12 are those potentially indicating the
presence of glycans larger than standard tetraantennary structures
(i.e., presence of LacNAc repeats). These structures, as seen by MS,
were minor in rEPO d and abundant in rEPO a/b. To corroborate the
initial structural assignments, both exoglycosidase and the reagent
array analysis method (RAAM) were conducted in conjunction
with HPLC and MALDI–TOF MS analyses. The HPLC profile for rEPO
d after desialylation basically showed 3 structures; core-fucosylated tetraantennary N-glycan (81.07% of the area), core-fucosylated
triantennary N-glycan (12.81% of the area), and core-fucosylated
tetraantennary N-glycan containing a single LacNAc repeat (6.12%
of the area). The equivalent treatment for rEPO a/b yielded a profile
where, apart from those structures, core-fucosylated diantennary
N-glycan and core-fucosylated tetraantennary N-glycan containing
2 LacNAc repeats were also seen (Fig. 7, lower panel). Mass spectra
obtained for these samples (Fig. 8) corroborated the presence of
the structures assigned to the NP HPLC peaks. With the aim of
determining the linkage type present in the sialic acid residues,
desialylation was conducted also with an a2-3 sialidase. The mass
spectra after both this treatment and that from complete desialylation yielded the same results, suggesting that all sialic acids are a23 linked. Next, in RAAM analysis, the b-1,4-galactosidase digestion
evidenced the presence of LacNAc repeats (Fig. 7) a very small proportion of tri- and tetraantennary glycans containing 1 LacNAc in
rEPO d but more abundant peaks corresponding to tri- and tetraantennary glycans containing 1 and 2 LacNAc repeats in rEPO a/b. Finally, after the complete digestion, the only detectable structure
was the common core-fucosylated pentasaccharide.
Gel-separated glycoforms
On separation by IEF, according to the isoelectric point, rEPO d
was resolved in up to 9 glycoforms. Most of the isoforms are also
present in rEPO a/b, but rEPO d extends its profile toward more
acidic pH values, with 2 isoforms being exclusively present in rEPO
d (Fig. 1).
To characterize individual rEPO d glycoforms, with particular
interest in the two more acidic bands, 2-DE (IEF 2-6 for the first
dimension and 10% SDS gel for the second dimension) was performed combined with MALDI–TOF MS analysis of the glycans obtained from the excised bands [30]. Patterns for both rEPO d and
rEPO a/b obtained after 2-DE gel separation and silver staining
are shown at the top of both panels in Fig. 9. Bands were numbered
according to their acidity (for rEPO a/b: from 1 to 7 in order of
increasing pI; for rEPO d: the same series from 1 to 7 plus addi-
Fig. 7. NP HPLC profiles of 2-AB-labeled glycans from rEPO d (A) and rEPO a/b (B).
The top profile shows an undigested pool of glycans followed by a series of
exoglycosidase digestions in the lower panels. The structural assignments for rEPO
d follow those of rEPO a/b that are based on NMR data but should be considered as
indicative only.
tional more acidic bands a and b following nomenclature of the
World Anti-Doping Agency) [39]. Purified N-glycans, labeled with
2-AB and analyzed by MALDI–TOF MS as described above due to
this technique, allowed the structural identification of the glycans
250
Glycosylation of Dynepo / E. Llop et al. / Anal. Biochem. 383 (2008) 243–254
Fig. 8. MALDI mass spectra of rEPO d before exoglycosidase digestions (upper panel, negative ion mode) and after exoglycosidase digestions (lower panels, positive ion
mode). The structural assignments for rEPO d follow those of rEPO a/b that are based on NMR data but should be considered as indicative only.
contained in individual bands. Mass spectra of purified N-glycans
from the least abundant bands (7 and b for rEPO d) could not be obtained (Fig. 9). However, the results clearly show the trend toward
a higher degree of sialylation following the decreasing pI values of
the IEF band. Because rEPO d posseses much less heterogeneity,
this variation resides mainly on the sialylation degree of tetraantennary chains, showing a progressive increase of tetra-charged
structures and a progressive decrease of mono-charged species
with increasing acidity of the band (Fig. 9A). Although rEPO a/b
also showed this trend (Fig. 9B), the larger structural heterogeneity
in this product (the presence of di- and triantennary structures and
the existence of LacNAc repeats) rendered a less pronounced phenomenon in individual structures. Overall, these results confirmed
that sialic acid residues are the sole charges present in the glycans.
The presence of other charged residues such as sulfates described
for other human glycoproteins were not observed despite the fact
that rEPO d is produced in human cells.
Discussion
Like most glycoproteins, rEPO d and rEPO a/b are also heterogeneous with respect to the glycosylation, resulting in different isoforms. Although it is extremely difficult to establish the precise
contribution of individual glycoforms to the overall activity, toxicity, and (in some cases) immunogenicity of these biopharmaceuticals, analysis of their glycosylation pattern is of utmost importance
in attempting such understanding in addition to guaranteeing drug
quality and efficacy [40]. Furthermore, the differential structural
elements could be useful for antidoping purposes through targeted
analyses.
On comparison of the structural features of the recombinant
and gene-activated EPOs, the first surprising observation came
from the molecular weights of both glycoproteins. The slightly
higher molecular weight found by MALDI–TOF MS for rEPO d when
compared with rEPO a/b contrasted with the molecular weight
determination by SDS–PAGE [41]. In this technique, rEPO a/b migrates slower, indicating higher hydrodynamic volume. MS and
HPLC analyses performed in this study suggest LacNAc repeating
units as being mainly responsible for this effect. This phenomenon
was analyzed in-depth, comparing migration of both rEPOs in a 2DE gel. Glycoforms from rEPO a/b are observed as a ‘‘train” of
bands that differ not only in pI but also in apparent molecular mass
(i.e., diagonality). The latter effect is not observed in urinary EPO
[42], and here we have demonstrated that it is also much less pronounced in rEPO d. With the obtained knowledge of the structural
differences between these two recombinant homologues, the diagonality in 2-DE and the band broadness in SDS–PAGE can be attributed predominantly to the presence of LacNAc repeats, whereas
sialic acids play a much less prominent role than is assumed so
far. To validate this assumption, the average mass conferred to a
glycoform through the 3 N-glycans in a single band, both with
and without sialic acid residues, was calculated. In Fig. 10, a clearly
Glycosylation of Dynepo / E. Llop et al. / Anal. Biochem. 383 (2008) 243–254
different slope can be appreciated for the total glycans, and this is
fairly consistent with the diagonal trends observed in the 2-DE of
the intact glycoproteins. Without sialic acid residues, rEPO d shows
a nearly horizontal mass trend, as expected given that it contains
predominantly tetraantennary N-glycans, whereas rEPO a/b still
shows a diagonal trend albeit with a lower slope.
The difference in microheterogeneity was also observed from
the molecular weight analysis. The narrower peak width of the
rEPO d in the MALDI–TOF spectrum suggested lesser heterogeneity
251
in comparison with its CHO homologue. This effect was also evidenced in SDS–PAGE gels through a much sharper band for rEPO
d and in 2-DE analyses by a lower band dispersion (indicated in
Fig. 9). Structural analyses of glycans permitted justifying this differential behavior through the observation of predominantly tetraantennary N-glycans, a very low proportion of triantennary-type
structures, the absence of diantennary-type structures, and a low
number of LacNAc repeats in rEPO d. In summary, all glycan structures appear in a narrower mass range between 2561 and 3800 Da,
Fig. 9. Silver-stained 2-DE gels (upper panel) and negative ion mode MALDI mass spectra of 2-AB-labeled N-glycans excised from 2-DE bands in the lower panels. Left:
Analyses for rEPO d. Right: Analyses for rEPO a/b. Identification of bands in the 2-DE gels is according to the criteria employed by doping control laboratories (c for more
acidic, to 7 for more basic band). Dashed lines drawn in the gel indicate molecular weight dispersion of bands. In the mass spectra, the numbering corresponds to the band
annotation in the 2-DE gels. h, fucose; d, N-acetylglucosamine; , mannose; j, galactose; D, sialic acid. The structural assignments for rEPO d follow those of rEPO a/b that
are based on NMR data but should be considered as indicative only.
252
Glycosylation of Dynepo / E. Llop et al. / Anal. Biochem. 383 (2008) 243–254
Fig. 10. Trend lines of the cumulative mass of the N-glycans from the individual bands in the 2-DE gels based on MS data. A clear difference in slope can be visualized for the
intact glycans from rEPO d (lower slope) and rEPO a/b (higher slope), indicating a narrower mass distribution over the glycans for the former than for the latter. This slope
difference is further exemplified after desialylation; a near horizontal line is observed for rEPO d, whereas for rEPO a/b still preserves diagonality. MW, molecular weight; SA,
sialic acid.
whereas for rEPO a/b this range was much wider (2196–4530 Da).
Interestingly, the lower mass heterogeneity for rEPO d in comparison with rEPO a/b contrasts sharply with the respective IEF profiles. The number of isoforms observed for rEPO d was higher,
sharing with rEPO a/b the 7 least acidic bands but showing at least
2 more acidic glycoforms. Several studies have shown that different epoetin isoforms have different biological activities [43]. The
more basic isoforms, containing a lower number of sialic acids,
are associated with a shorter half-life, so special attention was paid
to these charge residues in the analyses. The number of sialic acids
was deduced from the mass shift of the glycoprotein before and
after sialidase treatment by MALDI–TOF MS. In this context, the
molecular weight determination will be affected depending on
the matrix used, the laser energy applied, the sodium and/or potassium adduct formation during the ionization process, and so forth
[44]. However, internal calibration permitted accurate mass determinations. As such, differential measurement allowed establishing
that rEPO d contains a mean of less than 1 additional sialic acid residue with respect to rEPO a/b, also taking into account the sialic
acid O-acetylation observed for rEPO a/b (vide infra). Sialic acids
associated specifically with the unique O-glycan (at Ser126) were
evaluated after the complete removal of the N-glycans by both
MALDI–TOF and LC–ESI–TOF MS. Results from MALDI–TOF MS revealed, albeit in different proportions, the existence of nonsialylated, monosialylated, and disialylated O-glycans for both EPOs,
whereas in the LC–ESI–TOF MS experiments only the former two
specimens were less prominent. It is known that sugar residues,
and especially the terminal sialic acids, are labile and may be partly
eliminated during the ionization process [37] and that the milder
ESI technique results in much less degradation. The disialylated
species was the only glycoform present in rEPO d (again more glycan homogeneity), whereas in rEPO a/b the monosialylated form
accounted for 43.6%. Thus, the more acidic bands of rEPO d in IEF
experiments can be justified, in part, by higher sialylated Oglycoforms.
The degree of sialylation of N-glycans is more difficult to determine due to the presence of the three N-glycosylation sites and the
higher number of possible sialic acid residues per N-glycan (from 0
to 4 theoretically). MS analyses of the N-glycans showed the higher
structural homology for rEPO d in terms of antenna distribution but
also higher variability in the number of sialic acids contained in a
tetraantennary-type structure. In contrast, N-glycan composition
of rEPO a/b showed more antenna variability (di- and triantennary-type structures and LacNAc repeats) but a higher proportion
of tetrasialylated structures. For the same reason as expressed
above, MALDI–TOF MS analyses were performed only for identification of structures, but more accurate quantification of sialic acids
was obtained from WAX analyses. Surprisingly, the average number of sialic acids from N-glycans seems to be similar for rEPO d
(9.73) and rEPO a/b (9.83). Hence, summing O- and N-glycan contributions of sialic acids, rEPO d contains 11.73 as a mean of the
glycoprotein, whereas rEPO a/b contains 11.39. Despite the two
additional acidic isoforms in rEPO d, its mean sialic acid content
is only slightly higher. This can be explained by the relative proportion of these bands and by the balancing effect of the more
basic isoforms (bands 6 and 7) that appear to be more abundant
in rEPO d.
In the evaluation of possible immunogenicity [45] of these biopharmaceuticals, monitoring sialic acids from rEPO d and rEPO a/b
is mandatory. It is well known that rEPOs produced in CHO cells
contain approximately 1.3% Neu5Gc [9]. This particular sialic acid
occurs frequently in animal cells but is absent in humans because
of an internal frame shift mutation in the CMP-Neu5Ac hydroxylase gene [46]. Because humans do not synthesize Neu5Gc, successive injections of Neu5Gc-containing products have been
suggested to produce allergy-like symptoms [47]. Even though
rEPO d is produced in a human cell line using gene activation technology, it must be tested for its sialic acid repertoire, including the
Neu5Gc content, and because of potential Neu5Gc uptake from the
culture medium [48]. Our sialic acid analysis revealed an absence
of this residue under the most sensitive conditions (limit of detection of 6 fmol for standard sialic acids). From the sialic acid analyses, rEPO d also showed a more homogeneous composition without
O-acetyl groups (up to 3) that are present in rEPO a/b. This phenomenon should be noted because the presence of O-acetylation
on sialic acids has been correlated with increased circulation time
due to impaired hepatic uptake [49]. The absence of O-acetylation
in the sialic acids of rEPO d would be compensated by a higher
number of sialic acids per mole of glycoprotein. Although it is
not yet known whether the structural glycosylation differences
Glycosylation of Dynepo / E. Llop et al. / Anal. Biochem. 383 (2008) 243–254
will lead to any therapeutic advantages of the use of rEPO d
over rEPO a/b, articles published recently suggest a similar half-life
to rEPO b [50] and rEPO a [23] but slightly lower adverse effects
than rEPO a [51].
Despite the fact that CHO cells glycosylate proteins in a manner
that is qualitatively similar to that in human cells, some human tissue-specific glycan structures are not synthesized by these cells because they lack the proper glycosyl transferases. In this study, glycan
motifs that are present in human cells but absent in CHO cells
(bisecting N-acetylglucosamine residues, sialic acids in a2-6, fucose
residues in a1-3/4, or the presence of charged residues other than
sialic acids [e.g., sulfates] in the glycans) were investigated. However, structural analyses following specific exoglycosidase digestions depicted in Fig. 8, as well as specific enzyme-linked
immunosorbent assay (ELISA) and Western analyses (data not
shown) for sialyl LewisX and sialyl LewisA, failed to demonstrate
the presence of any of these particular structural elements in rEPO d.
With the rigorous structural analysis of rEPO d completed, it can
be concluded that the more acidic bands present in IEF, when compared with other recombinant EPOs, are due to a higher degree of
sialylation. The maximum number of sialic acids possible in the
molecule of EPO is 14, and the mean number of these residues
for rEPO d is approximately 11.7. Thus, it can be speculated that
glycans migrating in the band depicted as b contain a maximum
of 14 sialic acids. An interesting reflection concerns the fact that
endogenous EPO and rEPOs have the same number of glycosylation
sites and that both should present partially overlapping isoform
profiles, with potentially more basic isoforms present in the case
of endogenous EPO because it is not enriched for high sialic acid
content. However, the IEF analysis (Fig. 1) shows additional nonoverlapping isoforms of urinary EPO appearing at more acidic pI
values. Thus, this pattern cannot be explained in terms of sialic
acid residues only and indicates that endogenous EPO contains
additional charges that have not been found in rEPO d despite its
human origin.
Acknowledgments
The authors thank the World Anti-Doping Agency (WADA) for
its financial support. Furthermore, we thank J. Joglar (Department
of Biological Organic Chemistry, IIQAB–CSIC) for his kind help with
the LC–ESI–TOF MS measurements and C. de Bolós (Municipal
Institute of Medical Research, IMIM–Hospital del Mar) for the sialyl
LewisX/A assays. Dynepo was kindly supplied by Christian Reichel
(Doping Control Laboratory, ARC Seibersdorf Research GmbH)
and Peter Hemmersbach (Hormone Laboratory, Aker University
Hospital).
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