Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 214–220

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Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Short communication

Comprehensive glycan analysis of twelve recombinant human

erythropoietin preparations from manufacturers in China and Japan
Ben Cowper a,∗,1 , Xiang Li b,1 , Lei Yu b , Yong Zhou b,∗ , W.H. Fan b , C.M. Rao b
a
National Institute for Biological Standards & Control (NIBSC), Blanche Lane, South Mimms, Hertfordshire, EN6 3QG, United Kingdom
b
National Institute for Food and Drug Control (NIFDC), Tiantan Xili No. 2, 100050, Dongcheng District, Beijing, China

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

Article history: Recombinant, human, erythropoietin (rhEPO) is a glycoprotein hormone which is prescribed throughout
Received 23 October 2017 the world to treat anaemia caused by chronic kidney disease or chemotherapy. rhEPO is at the forefront of
Received in revised form 16 February 2018 the recent emergence of biosimilar medicines, with numerous products now available worldwide. Due to
Accepted 20 February 2018
its complex glycosylation profile, which has a crucial influence upon biological activity, therapeutic rhEPO
Available online 24 February 2018
preparations must be closely monitored to ensure consistency, safety and efficacy. Here, we have com-
pared twelve rhEPO preparations from eleven manufacturers in China and one in Japan, measuring in vivo
Keywords:
biological activity and exploring its relationship with glycosylation through sialic acid content determi-
Erythropoietin
Glycosylation nation, isoform distribution via capillary electrophoresis (CE), O-glycan profiling, and N-glycan mapping
Biosimilars using a novel anion-exchange/hydrophilic interaction chromatography-mass spectrometry (AEX/HILIC-
Mixed mode chromatography MS) approach. We observed differences between glycosylation profiles, including the varying occurrence
Sialylation of sialic acid O-acetylation, extension of N-glycan antennae with N-acetyllactosamine units, and the distri-
bution of sialic acids across multi-antennary structures. The presence of unusually high levels of suspected
penta- and hexa-anionic N-glycans in several samples is consistent with elevated rhEPO isoform acidity,
which is reflected by slightly elevated in vivo bioactivities. This aside, the observed differences in glycosy-
lation profile do not appear to have a significant influence upon biological activity in mice. Nonetheless,
with the continued emergence of biosimilars, the study highlights the importance of monitoring glyco-
sylation profiles in biological medicines, in order to detect and account for divergence between products,
as well as the presence of unusual or unexpected glycans.
© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction
Erythropoietin (EPO) is a glycoprotein hormone which stim-
ulates red blood cell production (erythropoiesis). Recombinant
human EPO (rhEPO) preparations are commonly prescribed glob-
ally to treat anaemia caused by chronic kidney disease or
chemotherapy. The first therapeutic rhEPO preparation, epoetin
alfa, was licensed in the EU in 1988, and in the USA a year
Abbreviations: rhEPO, recombinant human erythropoietin; PEG, poly(ethylene)
glycol; AEX, anion-exchange; HILIC, hydrophilic interaction chromatography; MS,
mass spectrometry; CE, capillary electrophoresis; CRS, chemical reference sub-
stance; LC, liquid chromatography; PNGase F, peptide N-glycosidase F; MWCO,
molecular weight cut off; LacNAc/LN, N-acetyllactosamine; HPAEC-PAD, high
pH anion exchange chromatography with pulsed amperometric detection; CHO,
Chinese hamster ovary; NANA/Neu5Ac, N-acetylneuraminic acid; NGNA/Neu5Gc,
N-glycolylneuraminic acid; KDN, Ketodeoxynonulosonic acid.
∗ Corresponding authors.
E-mail addresses: ben.cowper@nibsc.org (B. Cowper), zhouyong@nifdc.org.cn
(Y. Zhou).
1
Ben Cowper and Xiang Li contributed equally to this work.
later. Numerous EPO products have since been licensed world-
wide, including the sub-types epoetin beta, delta, and theta, which
possess distinct glycosylation profiles [1,2], and second genera-
tion modified EPOs darbepoetin (hyperglycosylated) and Mircera
(PEGylated). Since the expiry of originator epoetin alfa patents a
number of biosimilars have also emerged in various markets [3–5]
giving rise to a complex global network of rhEPO products (Table 1).
At the present time, there are more than ten manufacturers of
licensed rhEPO products in China alone.
EPO contains three N-glycosylation sites (Asn-24, Asn-38, Asn-
83) and one O-glycosylation site (Ser-126). The production of
therapeutic EPO preparations through recombinant gene expres-
sion in mammalian cell culture gives rise to a complex pattern
of glycosylation, including a wide range of bi-, tri- and tetra-
antennary structures [6,7]. Glycan composition has a strong
influence over EPO pharmacokinetics, with removal of negatively-
charged branch-terminal sialic acids causing a marked decrease
in plasma half-life and abolishment of in vivo biological activity
[8,9], due to enhanced clearance by asialoglycoprotein receptors
in the liver [10]. The glycan profile is therefore a critical quality

https://doi.org/10.1016/j.jpba.2018.02.043
0731-7085/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.
0/).
 B. Cowper et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 214–220 215

Table 1
A summary of originator and regional biosimilar EPO products. 1 Epoetin theta was not statutorily registered as a biosimilar by the EMA, but is often clinically considered to be
a biosimilar of epoetin alfa [5,25]. 2 All EPO products in China were not registered as biosimilar or originator, and are not classified by INN. INN = International Non-proprietary
Name.

INN Type Region Brand names Comments

® ® ® ® ®
Epoetin alfa Originator Worldwide Eprex , Erypo , Epogen , Procrit , ESPO Same product, licensed under multiple
brand names.
® ® ®
Biosimilar EU Abseamed , Binocrit , Epoetin Alfa Hexal Same production technology transferred
between manufacturers.
® ® ® ®
India Ceriton , Epofer , Epofit , Eporec , Multiple products.
® ® ® ®
Epotin , Relipoietin , Repoitin , Wepox
®
Epoetin beta Originator Worldwide NeoRecormon
®
Epoetin delta Originator EU Dynepo No longer marketed.
®
Epoetin kappa Biosimilar Japan Epoetin alfa BS JCR Reference product is epoetin alfa.
® ®
Epoetin lambda Biosimilar Australia Aczicrit , Gradicrit Reference product is epoetin alfa.
® ®
Epoetin theta Originator1 EU Biopoin , Eporatio Same product, licensed under multiple
brand names.
® ®
Epoetin zeta Biosimilar EU Retacrit , Silapo Reference product is epoetin alfa.
®
Methoxy PEG epoetin beta Originator Worldwide Mircera
® ®
Darbepoeitin alfa Originator Worldwide Aranesp , Nesp Same product, licensed under multiple
brand names.
® ® ®
Biosimilar India Actorise , Cresp , Darbatitor , Multiple products.
® ® ® ®
- – China2 Epoetin , Sepo , Renogen , Eposino Multiple products.

attribute which is closely monitored in therapeutic rhEPO products.
Glycan composition is strongly influenced by cell culture process
variables [11], and so the growing number of global rhEPO man-
ufacturers places increased importance upon glycan analysis, to
ensure consistency, safety and efficacy of products and batches.
Previous studies have highlighted the differences in glycosylation
which are observed between rhEPO products [2,12–14], including
variances in O-acetylation of sialic acid and N-acetyllactosamine
(LacNAc) extension of glycan antennae.
Glycan profiles can be characterised using a variety of tech-
niques [15], including capillary electrophoresis (CE), isoelectric
focusing (IEF), and mass spectrometry (MS), of intact glycopro-
teins or fragmented glycopeptides. Detailed structural information
is also commonly obtained through chromatographic analysis of
enzymatically-released N-glycans, with a variety of approaches
available. Due to the crucial influence of anionic sialic acid over
rhEPO bioavailability, it can be useful to separate rhEPO N-glycans
on the basis of charge, enabling rapid identification of the relative
quantities of multi-sialylated structures. This can be achieved using
high performance anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD) [16], however the use of
high pH and salt conditions necessitate the use of a specialised
instrument, and prevent routine coupling to a mass spectrome-
ter for N-glycan identification. Alternatively released N-glycans can
be fluorescently-labelled for analysis using conventional HPLC(-
MS) instrumentation [15]. This usually involves anion exchange
(AEX) or hydrophilic interaction chromatography (HILIC), the lat-
ter being particularly useful due to its use of MS-compatible mobile
phases [14]. However HILIC provides a polarity-based separation,
and therefore structures with different charges are not always fully
segregated.
Here, we present a comprehensive glycan analysis of eleven
rhEPO preparations acquired from manufacturers in China, and one
additional sample from Japan. Samples were subjected to N-glycan
mapping, using a novel anion-exchange/hydrophilic interaction
chromatography-mass spectrometry (AEX/HILIC-MS) method. This
method conveniently allows for AEX-type charge-based N-glycan
separation under HILIC-type MS-compatible conditions, enabling
resolution and simple identification of individual structures. Intact
O-glycan characterisation, capillary electrophoresis (CE) and sialic
acid content determination were also performed, and the in vivo
bioactivity of each preparation was also measured, in order to
explore the relationship between bioactivity and rhEPO glycan
composition.
2. Materials & methods
2.1. Reagents
The recombinant EPO for physicochemical tests CRS was
acquired from the European Directorate for the Quality of
Medicines (EDQM, Strasbourg, France). This is a lyophilised
preparation containing approximately 100 ␮g erythropoietin, with
epoetin alfa and beta present in equal amounts. rhEPO prepara-
tions S01-S12 were generously donated to the National Institute
for Food and Drug Control (NIFDC), China, by eleven manufactur-
ers from China and one manufacturer from Japan. Samples were
anonymised and identified via randomly assigned codes S01–S12.
Samples S01, S02, S05, S06, S08, S09 and S10 were manufactured in
bioreactors. Samples S03, S04, S07, S11 and S12 were manufactured
using roller bottles. GlycoworksTM RapiFluor-MSTM reagents were
purchased from Waters (Milford, MA, USA). Ammonium formate,
urea, methylcellulose, N-acetylneuraminic acid (Neu5Ac, NANA)
and trifluoroacetic acid were purchased from Sigma-Aldrich (St
Louis, MI, USA). Neuraminidase was purchased from New England
Biolabs (Ipswich, MA, USA). Ketodeoxynonulosonic acid (KDN) was
purchased from Worthington Biochemical (Lakewood, NJ, USA). pI
markers 3.59 and 5.85 were purchased from Protein Simple (San
Jose, CA, USA). Pharmalytes 3–10 and 2.5-5 were purchased from
GE Healthcare (Chicago, IL, USA). Acetonitrile (OptimaTM , LC/MS
grade) was purchased from Fisher Scientific (Hampton, NH, USA).
2.2. N-glycan preparation
RapiFluor-MS (RFMS) labelled N-glycans were prepared using
Waters GlycoworksTM RapiFluor-MSTM kit reagents and pro-
tocols (Waters, Milford, MA, USA). rhEPO preparations were
buffer-exchanged into water and concentrated to 2 mg/ml using
10,000 MWCO spin concentrators (Millipore, Burlington, MA, USA).
Volumes of 7.5 ␮l were labelled with RFMS according to the man-
ufacturers’ instructions. N-glycans were purified using a Waters
HILIC ␮Elution plate, with elution in 3 × 30 ␮l 200 mM ammonium
formate in 5% (v/v) acetonitrile. Samples were then directly ana-
lysed via LC–MS.
2.3. Intact O-glycan preparation
N-deglycosylated rhEPO samples were prepared using Waters
GlycoWorksTM reagents (Waters, Milford, USA). A 4 ␮l volume of a
216 B. Cowper et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 214–220

5% RapiGestTM SF Surfactant solution (in Glycoworks Rapid Buffer)

N-glycan mapping, capillary electrophoresis (CE) and sialic acid content determination data for each rhEPO preparation. N-glycan mapping: relative peak areas (RPA%) of mono- (S1), bi- (S2), tri- (S3) and tetra- (S4) sialylated,
and penta- (−5) and hexa- (−6) anionic N-glycans, and corresponding Z-numbers. The conventional Z-number equation [21](Hermentin, et al., 1996) has been expanded to account for the presence of penta-anionic N-glycans;
Z = (S0%RPA x 0) + (S1%RPA × 1) + (S2%RPA x 2) + (S3%RPA × 3) + (S4%RPA x 4) + (−5 %RPA × 5). Asialylated N-glycans were not detected. Copies of individual chromatograms are included in Supplementary Information A. All
values reported are means derived from triplicate analysis, summarised in Supplementary Table 1. “-“ = not detected. Capillary electrophoresis: relative peak areas (RPA) for peaks corresponding to different pI ranges. Copies of
individual electropherograms are included in Supplementary Information B. All values reported are means derived from triplicate analysis, summarised in Supplementary Tables 2a-m. “–“ = not detected. Sialic acid content: all
was added to 10.2 ␮l of water and 5 ␮l of rhEPO (at 2 mg/ml in

Sialic acid
(mol/mol
water). Samples were incubated at 90 ◦ C for 3 mins, then allowed

EPO)
11.8
11.3

11.2
14.5

13.4
10.8
10.6
10.7

10.1
10.3
10.2
10.9

N.D
to cool, before addition of 0.8 ␮l Glycoworks Rapid PNGase F. Reac-
tions were incubated at 50 ◦ C for 5 mins, allowed to cool, and then
directly analysed via LC–MS.

>5.0

2.6

5.9
–
–
–
–
–
–
–
–

–
–
–
2.4. Liquid chromatography-mass spectrometry (LC–MS)

4.88–4.98
N-glycan and intact O-glycan samples were analysed via LC–MS
using a Waters Acquity H–Class Bio UPLC system equipped with a

10.0
1.6
7.1

7.7

7.7

3.8

6.3
Single Quadruple Detector (SQD2) (Waters, Milford, USA). RFMS-

–
–

–
labelled N-glycan separations were achieved by injection of 1 ␮l
sample volumes onto a GlycanPac AXH-1 column (2.1 × 150 mm,

4.65–4.75
1.9 ␮m; Thermo Fisher Scientific, Waltham, MA, USA) maintained
at 30 ◦ C. Gradient elution was employed using 100 mM ammo-

26.5

17.6
15.6

26.1

13.8
28.8
13.7
18.8
30.2

27.0
3.4

6.3
3.1
nium formate-acetonitrile-water (2:70:28 to 15:57:28, v/v/v, over
40 mins), at a flow rate of 0.4 ml/min. Fluorescence was monitored

4.45–4.54
at 265/425 (Ex/Em). Intact O-glycan separations were achieved
by injection of 1 ␮l sample volumes onto a Waters Acquity UPLC

38.7
28.8
47.1

32.4
21.7
24.7
39.5
32.6

26.7
23.0
23.0
30.6

30.1
Glycoprotein Amide 300 Å column (2.1 × 150 mm, 1.7 ␮m; Waters,
Milford, MA, USA) maintained at 45 ◦ C. A gradient was employed
using 0.1% trifluoroacetic acid-0.1% trifluoroacetic acid in acetoni-

4.24–4.32
trile (23:77 to 33:67, v/v, over 20 min), at a flow rate of 0.2 ml/min.
Fluorescence was monitored at 280/320 (Ex/Em). Mass spectrom-

23.1
21.4
24.5
26.9
36.6
22.9
37.5
34.5
17.9
29.1
23.3
36.4
26.1
etry data was acquired in ES+ mode, with a capillary voltage of
3 kV, cone voltage of 40 V, source temperature of 150 ◦ C, desolva-
tion temperature of 350 ◦ C and desolvation gas flow of 800 l/hr. Data

4.09–4.18
was acquired and analysed using Empower 3.1 software (Waters,

26.9

23.9
11.2
13.2

19.3
10.6

20.8

10.9
21.0

15.0
Milford, USA).

8.7

8.3

9.7
values reported are means derived from triplicate analysis, summarised in Supplementary Table 3. N.D = not determined.
2.5. Capillary electrophoresis (CE)

3.90–4.02
Capillary electrophoresis (RPA%)

A master mix (MM) was prepared fresh daily containing 4 M

10.9
1.5
1.9

8.5
1.6
2.9

4.9
3.3
1.8
1.4
1.2
2.0
5.0
urea, 0.35% methylcellulose and 4% Pharmalytes mixture (Pharma-
lytes 3–10:Pharmalytes 2.5-5, 1:3). rhEPO samples were prepared
using 100 ␮l of MM with 0.5 ␮l each of pI markers 3.59 and 5.85,
3.76–3.85

at a target concentration of 0.2 mg/ml. Samples were vortexed

briefly to ensure complete mixing and centrifuged at 10,000 rpm
1.5
2.4
1.6

4.9
2.6
0.5
–
–

–
–

–
–
–
for 3 min to remove air bubbles. CE was performed using an
iCE3 whole-capillary system, an FC-coated column cartridge, and
<3.7

pI markers 3.59 and 5.85 (ProteinSimple, CA, USA). The anolyte

1.0
–
–
–
–
–
–
–

–
–
–
–
–
and catholyte solutions were 80 mM phosphoric acid and 100 mM
sodium hydroxide respectively. Samples were focused for 1 min at
356
365
376

372
363
345

357

358
350
350

350

360

370
1.5 kV, followed by 6.5 min at 3 kV, and A280 images of the capillary
Z

were taken using iCE analysis software.

1.3

4.3
2.6
0.9
−6

2.6. Sialic acid content determination

–
–

–
–

–
–
–
–

rhEPO samples were diluted to approximately 0.33 mg/ml with

12.8

18.8

23.5
16.2
5.7
3.2

3.9

5.4
4.3

4.8

1.2
9.0

0.3
−5

sodium phosphate buffer (5 mM, pH 7.2). A 20 ␮l sample volume

was transferred to screw capped vials, along with 20 ␮l neu-
raminidase (1U/100 ␮l) and incubated for 1 h (±15 min) at 37 ◦ C.
55.9
49.4
67.6
52.6
53.1

43.8
42.2
53.6
57.3
74.9
64.4
54.0

70.3
S4

A 50 ␮l volume of ketodeoxynonulosonic acid (KDN) internal stan-

dard working solution (0.04 mM) and 70 ␮l of purified water was
N-glycan mapping (RPA%)

added to the digested sample. 5-acetylneuraminic acid (NANA) cali-

27.6
31.1

19.5
19.3
29.8
19.3
17.7
23.5
26.8
28.8
19.9
26.7
24.0
S3

bration standards were prepared by mixing 20, 40, 60, 80 and 100 ␮l
NANA standard solution (12 ␮g/ml, 95%) with 50 ␮l KDN working
solution (0.04 mM) and 90, 70, 50, 30 and 10 ␮l water respectively.
10.6
7.7
9.7
8.4
5.5
9.7
5.5
6.2
9.3

4.5
6.9
9.0
8.0
S2

Samples were analysed via high pH anion exchange chromatog-

raphy with pulsed amperometric detection (HPAEC-PAD), using
a Thermo ICS-5000 ion chromatography system equipped with
2.1

3.2

2.4

4.4
6.1
1.5
1.1
2.0

0.8

2.0
0.5

0.4
0.8
S1

an anion-exchange column (CarboPac PA-1) and guard column

(CarboPac PA-100) (Thermo Fisher Scientific, Waltham, MA, USA).
Table 2

Both the column and detector were maintained at 20 ◦ C. 20 ␮l

CRS
S01
S02
S03
S04
S05
S06
S07
S08
S09
S10
S11
S12

sample/standard volumes were injected. Isocratic elution was

 B. Cowper et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 214–220
Fig. 1. N-glycan mapping of released RFMS-labelled N-glycans from the Ph. Eur. erythropoietin for physicochemical tests CRS via AEX/HILIC. The approximate elution times of
mono-, bi-, tri- and tetra-sialylated, and penta-anionic oligosacchairdes are annotated, as are structures which were identified via mass spectrometry (see Supplementary
Information A).
performed using a mobile phase blend of sodium hydroxide
(0.5 M)-sodium acetate (1 M)-water (30:12.5:57.5, v/v/v) and a flow
rate of 1.0 ml/min. Sialic acid contents were determined by com-
parison with standardized NANA solutions.
2.7. In vivo bioassays
In vivo bioassays were carried out according to the in vivo
normocythaemic mouse bioassay protocol of the Chinese Phar-
macopeia [17]. Assays were calibrated using the Chinese national
standard for erythropoietin, which is itself calibrated against the
WHO International Standard for erythropoietin, recombinant for
bioassay, coded 11/170 [18]. Eight-week-old BALB/c mice were
allocated to standard and sample groups in a fully randomized
order, with five mice per treatment group. The standard and test
rhEPO sample were diluted to appropriate concentrations with
saline containing 0.1% (w/v) bovine serum albumin. A single dose
of 7.5, 15 or 30 IU rhEPO/0.2 ml per mouse was injected subcuta-
neously on day 1. On day 4, blood was taken from the orbital venous
sinus of each mouse and reticulocytes were counted using a R-500
Hematology Analyzer (Sysmex). Biological activity was calculated
by comparing test sample response to the standard using a parallel
line method.
This study was approved by the Ethics Committee of National
Institute for Food and Drug Control.
3. Results & discussion
3.1. N-glycan resolution and identification via AEX/HILIC-MS
N-glycan analysis was performed using “mixed-mode” [19]
anion-exchange/hydrophilic interaction (AEX/HILIC) chromatogra-
phy, enabling separation of released N-glycans on the basis of both
217
charge and hydrophilicity. Multi-sialylated structures are sequen-
tially separated into “clusters” in order of increasing negative
charge, akin to a conventional anion-exchange chromatography
method, which allows the relative quantities of multi-sialylated
structures to be determined (Table 2). Within each charge clus-
ter, N-glycans are further resolved according to their degree of
branching and the occurrence of O-acetylated sialic acid and N-
acetyllactosamine extensions (Fig. 1). In recent years sophisticated
mass spectrometric approaches have enabled direct detection
of these modifications in rhEPO samples [12,13]. However this
method provides a novel approach to chromatographic separation
of individual N-glycan structures which are fluorescently-labelled,
which is comparable to that achieved through alternative high pH
anion exchange chromatography with pulsed amperometric detec-
tion (HPAEC-PAD) methods [16]. A further advantage of AEX/HILIC
is that elution is achieved using a HILIC-type gradient, utilising
volatile mobile phase conditions, which allows for direct coupling
to a mass spectrometer and identification of individual N-glycan
structures.
Bi-, tri- and tetra-sialylated oligosaccharides were detected and
identified via mass spectrometry in all rhEPO samples (Supple-
mentary Information A). Although it was not possible to formally
identify mono-sialylated N-glycans via mass spectrometry, the
appearance of a peak cluster prior to the bi-sialylated N-glycans
in all samples is strongly indicative of their presence. Asialy-
lated/neutral N-glycans were not detected. The appearance of
additional peak clusters following the tetra-sialylated N-glycans
is suggestive of the presence of penta-anionic (visible in all sam-
ples) and hexa-anionic structures (visible in samples S03, S05, S08
and S09). Although it was not possible to formally identify these
peaks via mass spectrometry, due to the poor m/z signal intensity
of overlapping structures, they are likely to represent additionally
sialylated (e.g. penta-sialylated) and/or sulphated glycans [16].
218 B. Cowper et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 214–220
Fig. 2. The in vivo bioactivity (in International Units per milligram, IU/mg) of each rhEPO preparation (green) plotted alongside the relative peak areas (RPA%) of the peak
corresponding to isoforms with pI 3.90-4.02 during capillary electrophoresis (CE) (blue) and the peak cluster corresponding to penta-anionic oligosaccharides during N-glycan
mapping (red). %RPA values have been “normalised” to a 0–1 scale to facilitate overlaid plotting. (For interpretation of the references to colour in this figure legend, the reader
is referred to the web version of this article.)
3.2. Sialic acid content and distribution
The sialic acid content varies slightly between samples, with an
observed range of 10.1–14.5 mol sialic acid per mol EPO (Table 2);
all above the minimum of 10 mol/mol EPO specified in the Chinese
Pharmacopoeia (ChP) monograph for Recombinant Human Ery-
thropoietin Injection (CHO cell) [17] and European Pharmacopoeia
monograph for Erythropoietin concentrated solution [20]. However
capillary electrophoresis (CE) and N-glycan mapping data reveal
significant differences between samples in terms of the molecu-
lar distribution of sialic acid. For example, CE analysis shows that
samples S05 and S08 are enriched with acidic isoforms, including
elevated levels of a peak with pI ∼3.98 (8.5% and 10.9% relative
peak area, RPA, respectively, compared with ≤5% in the other sam-
ples) with S08 also possessing elevated acidic isoforms at pI ∼3.83
and ∼3.69 (Table 2). S05 and S08 are also deficient in more basic
isoforms, with a peak at pI 4.74 present at only 3.4 and 3.1% RPA
respectively, compared to >6% in the other samples. These findings
are supported by the N-glycan mapping data for S05 and S08, which
possess unusually high levels of penta-anionic oligosaccharides
(18.8 and 23.5% RPA respectively), and evidence of hexa-anionic
structures, which together will enhance the overall isoform acid-
ity of each rhEPO preparation. This observation is consistent with a
strong overall correlation between the relative peak areas of acidic
isoforms (pI < 4.0) via CE, and the presence of highly acidic penta-
/hexa-anionic N-glycans (Table 2 & Fig. 2). Additionally, samples
which are enriched with more basic isoforms (pI ≥ 4.65), such as
S02, S06, S09 and S11, generally possess reduced levels of penta-
anionic N-glycans along with elevated levels of less acidic mono-,
bi- and/or tri-sialylated N-glycans. Sample S09 is unique amongst
the twelve preparations, as it presents CE peaks at pI ∼3.76 and
∼5.08 (both 2.6% RPA) and therefore has a particularly wide iso-
form distribution, which is supported by the presence of both
elevated mono-sialylated (6.1% RPA) and penta-anionic (16.2% RPA)
N-glycans. Sample S03 is also unusual in possessing elevated penta-
and hexa-anionic N-glycans without significant enrichment of low
pI (<4.0) peaks via CE. It is likely that their presence is offset by the
relatively high levels of mono- and bi-sialylated structures, which
together contribute to a narrow, more neutral isoform distribution
via CE.
The presence of penta-anionic structures in rhEPO preparations
is not commonly addressed in the literature. For example, they
were not accounted for in the original proposed calculation of
“Z-number”, a hypothetical summarisation of the relative abun-
dances of multi-sialylated N-glycans [21]. However their presence
in small quantities is reported in some therapeutic rhEPO prod-
ucts [16,22,23], and they are detected here, at 1.2% RPA, in the Ph.
Eur. EPO for physicochemical tests CRS, a 50:50 mixture of originator
products epoetin alfa and beta (Fig. 1 & Table 2). This correlates
with the isoform distribution of the Ph. Eur. CRS, in which low
and high pI forms are relatively deficient and prevalent respec-
tively. With the exception of one sample, S12, all of the rhEPO
preparations analysed contain significantly elevated penta-anionic
N-glycans compared to the Ph. Eur. CRS. Hexa-anionic N-glycans
have yet to be reported in literature concerning therapeutic rhEPO
products, and are not detected in the Ph. Eur. CRS. However they
are suspected to be present in several samples here, likely due to
further sialylation or sulfation of penta-anionic structures. Nine
of the twelve rhEPO preparations also contain elevated mono-
sialylated N-glycans compared to the Ph. Eur. CRS, and there are
also notable differences observed in bi-, tri-, and tetra-sialylated
N-glycan levels in many samples. Overall there is a wide range
of N-glycan distributions across the sample set, which are not
necessarily reflected by their calculated Z-numbers (Table 2). For
example, the S09 Z-number is very similar to S01, S02 and S06,
but their multi-sialylated N-glycan distributions differ significantly.
Therefore, whilst the Z-number provides a convenient summary of
overall sialylation, it does not account for different sialic acid dis-
tributions and may not provide sufficiently in-depth information
to compare biosimilar rhEPO preparations.
Analysis of N-deglycosylated samples via HILIC-MS has also
enabled characterisation and comparison of intact O-glycosylation
(Supplementary Information C). Mono-, bi- and asialylated struc-
tures have been identified in all cases, and whilst their relative
quantities vary slightly between rhEPO preparations, none differ
significantly from the Ph. Eur. CRS. Considering the relatively small
influence of the single O-glycan over total rhEPO sialylation, it
is unlikely that the small differences observed have a significant
impact upon drug pharmacokinetics.
 B. Cowper et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 214–220 219

3.3. O-acetylated and N-acetyllactosamine extended N-glycans

The AEX/HILIC method developed for rhEPO N-glycan analysis

achieves resolution of individual structures within multi-sialylated
peak clusters, which are identifiable via mass spectrometry (Sup-
plementary Information A). Previous studies have demonstrated
that rhEPO N-glycans are susceptible to modification, most notably
through O-acetylation of sialic acid, extension of one or more
antennae with an additional N-acetyllactosamine (LacNAc, LN) dis-
accharide, and the presence of N-glycolyl- (Neu5Gc, NGNA) rather
than N-acetyl- (Neu5Ac, NANA) neuraminic acid (i.e. sialic acid)
[12,13]. N-glycans bearing Neu5Gc are believed to be present at low
levels (∼1% total sialic acid) in rhEPO preparations [11] and have
not been detected here, however multiple O-acetylated sialic acids
and/or anntennal LacNAc extensions are present in all samples.
Analysis of the Ph. Eur. CRS provides the N-glycan distribution
of a prototypical therapeutic rhEPO preparation (Fig. 1). In each
peak cluster the most prominent peak is the basic multi-antennary
core structure containing one sialic acid per antenna (i.e. S2NA2F,
S3NA3F, S4NA4F). Further structures with one or more asialylated
antennae (e.g. bi-sialylated tri-/tetra-antennary, S2NA3F/S2NA4F,
and tri-sialylated tetra-antennary, S3NA4F) are also detected in
reduced quantities. O-acetylated sialic acid is present on bi-, tri-
and tetra-sialylated N-glycans, with the latter bearing as many as
four O-acetyl modifications. LacNAc extensions are also detected
in each peak cluster; present on up to three antennae of tetra-
sialylated, tetra-anntennary N-glycans. When compared with the
Ph. Eur. CRS, the various rhEPO preparations possess a wide range
of N-glycan profiles (Fig. 3 & Supplementary Information A). Sam-
ples S02 and S04 most closely resemble the Ph. Eur. CRS, whereas
some samples contain comparatively increased O-acetylated sialic
acid (S01, S09, S10, S11), or LacNAc-extended structures (S03, S05,
S06, S09). Samples S07 and S12 are comparatively deficient in both
modifications, whilst S09 is completely deficient in O-acetylated
N-glycans. Numerous samples also contain elevated levels of bi-
/tri-sialylated structures with one or more additional asialylated
antennae (S04–06, S08–11).
The differences observed between the rhEPO preparations
in terms of antennarity, sialylation, O-acetylation and LacNAc-
extension likely derive from differences in their respective Fig. 3. Expanded AEX/HILIC chromatograms of selected rhEPO samples (S01, S02,
S06, S07 and S09), highlighting the differences observed between the glycosylation
manufacturing processes. Although all rhEPO preparations were
profiles of different samples. The lower chromatogram is annotated with the posi-
produced in Chinese hamster ovary (CHO) cells, small differences tions of the tri- (S3), and tetra- (S4) sialylated and penta-anionic (-5) peak clusters
or changes in pH, temperature, culture method or media can still as well as the tetra-sialylated, tetra-antennary N-glycan (S4NA4F) and the relative
have a significant influence over the glycosylation profile of a positions of its O-acetylated and LacNAc (N-acetyllactosamine)-extended deriva-
tives. Full-scale individual chromatograms for each rhEPO preparation are included
recombinant protein [11]. The twelve preparations were produced
in Supplementary Information A.
using a mixture of roller bottles and bioreactors, however there
are no particularly strong correlations between the system used
and resulting glycosylation profile. O-acetyl sialic acid and LacNAc- in mice. rhEPO in vivo bioassays are inherently variable; the ChP
extended structures are elevated and depleted in preparations monographs for rhEPO products state that potencies must be within
produced using either method. Penta-/hexa-anionic N-glycans are 80–140% of the stated potency [17], whilst the Ph. Eur. mono-
most prevalent in samples S05, S08 and S09, which were produced graph permits confidence intervals of 64% and 156% [20]. Within
using a bioreactor, however these structures are also visible in sam- these bounds, the bioactivities of the twelve rhEPO preparations
ple S03, which was produced in roller bottles. Therefore it appears can be interpreted as approximately equal. Nonetheless there does
that these factors are more strongly influenced by the precise com- appear to be a trend between in vivo bioactivity and the presence
position of the culture medium. of elevated levels of acidic N-glycans and isoforms (Fig. 2). Sam-
ples S05, S08 and S09 all possess unusually high levels of suspected
3.4. Trends in glycosylation and biological activity penta-anionic N-glycans. The increased acidity of these prepara-
tions will theoretically lead to reduced clearance in vivo, which is
The close relationship between rhEPO biological activity and indeed reflected by slightly elevated bioactivities. Otherwise, the
glycosylation has long been established [8–10]. The in vivo bioactiv- more discrete glycan modifications observed here, such as varia-
ity of each preparation has been determined here (Supplementary tions in sialic acid O-acetylation and LacNAc extension, do not give
Table 4), in order to evaluate the impact of the observed variation rise to any particular trends in biological activity. However it should
in glycosylation upon the rhEPO structure-activity relationship. be noted that investigations into biological activity in this study
Bioactivities ranged from 100,905–122,957 IU/mg, suggesting that were restricted to use of a mouse model in vivo bioassay from the
the significant divergences in N-glycan profiles of the twelve prepa- Chinese Pharmacopoeia rhEPO monograph, which does not allow
rations are not reflected in major differences in in vivo bioactivity for a particularly in-depth assessment of the pharmacology of the
220 B. Cowper et al. / Journal of Pharmaceutical and Biomedical Analysis 153 (2018) 214–220
various rhEPO preparations. Incidentally, during a recent study of
a proposed manufacturing change for a therapeutic rhEPO, it was
observed that increases in LacNAc-extended antennae led to ele-
vated potency in chronic kidney disease patients, suggesting that
this modification may in fact have wider pharmacological implica-
tions in humans [24].
Interestingly, samples S07 and S12 possess significantly larger
sialic acid contents than the other ten samples (Table 2) but this
is not reflected by elevated in vivo bioactivities. Both samples are
particularly enriched with tetra-sialylated N-glycans (>70% RPA),
but are also relatively deficient in penta-anionic structures and low
pI isoforms compared with the more bioactive samples S05, S08
and S09. Taken together, this suggests that these samples, in spite
of their elevated sialic acid contents, are not sufficiently equipped
to prolong in vivo circulation. This neatly demonstrates that sialic
acid content alone is not a major determinant of in vivo bioactivity
of rhEPO, and that the distribution of sialic acid, in particular the
maximal sialylation/negative charge of tetra-antennary structures,
is a critical attribute.
4. Conclusions
This study has utilised several complimentary analytical tech-
niques to demonstrate the breadth of glycosylation profiles
observed in licensed rhEPO products available in China and Japan.
This includes a novel AEX/HILIC-MS method, which conveniently
combines simultaneous charge- and polarity-based N-glycan sep-
aration (allowing rapid quantitation of multi-charged N-glycan
populations) with simple mass spectrometric identification of
individual structures. The twelve preparations exhibit significant
differences in terms of the occurrence of sialic acid O-acetylation,
extension of N-glycan antennae with N-acetyllactosamine units,
and the distribution of sialic acids across multi-antennary struc-
tures, including the suspected presence of penta- and hexa-anionic
(sialylated/sulfated) oligosaccharides in some cases. Aside from
slightly increased in vivo bioactivities in samples enriched with
acidic isoforms/N-glycans, the observed differences do not confer
significant effects upon biological activity in the mouse bioassay.
Nonetheless, the varied presence and scale of glycan modifica-
tions observed in this selection of rhEPO samples demonstrates
the importance of monitoring glycosylation in biological medicines,
particularly in the context of the growing biosimilars market, in
order to ensure continued safety and efficacy. CHO cells used to
produce rhEPO are known to contain potentially harmful glycan
epitopes, such as N-glycolyl sialic acid, which is immunogenic in
humans [11]. Close monitoring of glycosylation in new and exist-
ing rhEPO products, using methods such as those described in this
study, will hopefully ensure that significant occurrences of such
harmful moieties are avoided in the future.
Acknowledgements
This work was financially supported by grants from the National
Science and Technology Major Project (No. 2015ZX09501008-001).
The funder had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.jpba.2018.02.043.
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