Chemical Papers (2019) 73:1805–1811

https://doi.org/10.1007/s11696-019-00743-8

ORIGINAL PAPER

Adsorption of recombinant human erythropoietin and protein

impurities on a multimodal chromatography membrane
Tomáš Kurák1 · Tomáš Molnár1 · Milan Polakovič1 

Received: 23 January 2019 / Accepted: 8 March 2019 / Published online: 14 March 2019
© Institute of Chemistry, Slovak Academy of Sciences 2019

Abstract
Multimodal chromatography adsorbents are novel materials with promising properties for downstream processing of thera-
peutic glycoproteins. This work analyzes the properties of a salt-tolerant membrane, Sartobind STIC, for the purification of
recombinant human erythropoietin (rhEPO) produced by human embryonic kidney cells. Batch adsorption experiments were
carried out to determine equilibrium properties of the membrane as a function of pH and NaCl concentration. It was found
that the membrane adsorbs strongly both rhEPO and impurity proteins but the selectivity of binding is low independent of
process conditions. Since the membrane demonstrated strong salt tolerance, flow experiments were designed to elucidate
the effect of elution conditions on the bound proteins desorption. These experiments have revealed irreversible character
of protein adsorption on the multimodal adsorbent probably caused by protein unfolding on the hydrophobic groups of the
adsorbent. The recovery degree was especially low in case of rhEPO with the maximum at about 45% even when solutions
of kosmotropic agents were used as desorbents.

Keywords  Recombinant human erythropoietin · Multimodal chromatography · Membrane chromatography · Sartobind

STIC · Hydrophobic interaction · Ion exchange · Irreversible adsorption

Introduction The first commercial rhEPO, epoetin-α (Epoge), was

The glycoprotein hormone erythropoietin (EPO) is a 165-
amino acid erythropoiesis-stimulating glycoprotein which
is the main regulator of red blood cell formation. EPO is
mainly synthesized in kidneys (Lacombe and Mayeux 1998).
Certain diseases such as chronic kidney disease, rheumatoid
arthritis, AIDS or cancer cause the decrease of EPO below
normal level. Human EPO was first isolated from the urine
of anemic patients (Miyake et al. 1977), and its gene was
isolated a few years later (Lin et al. 1985). Very soon after-
wards, two groups succeeded independently in cloning the
EPO gene and expressing it in Chinese hamster ovary cells,
which enabled the development of recombinant human EPO
(rhEPO) as a drug (Powe et al. 1993).
* Milan Polakovič
milan.polakovic@stuba.sk
1
Department of Chemical and Biochemical Engineering,
Institute of Chemical and Environmental Engineering,
Faculty of Chemical and Food Technology, Slovak
University of Technology, Radlinského 9, 812 37 Bratislava,
Slovakia
developed by Amgen Inc. in 1980s (Kalantar-Zadeh 2017)
and it has been successfully used to restore normal produc-
tion of red blood cells (Spivak 2000; Jelkmann 2004). Other
α- and β-isoforms slightly differing in carbohydrate chains
and therewith also in the molecular weight were introduced
to the market in the following years. A typical value of
rhEPO molecular weight is about 34 kDa (Kasper 2003).
About 40% of the total rhEPO molecule mass of EPO is
formed by sialic acid groups. They influence rhEPO con-
formation and its other physical, chemical and biological
properties (Dordal et al. 1985; Wasley et al. 1991; Yama-
guchi et al. 1991).
The production of rhEPO has been performed exclusively
using mammalian cells. An advantage of this production is
that rhEPO is excreted into the cultivation broth. Cultiva-
tion media for the rhEPO production are rather complex;
therefore, unutilized substrates form one part of process
impurities. The second part of the impurities that has to be
removed during downstream processing is the by-products of
cell metabolism. They include mostly low-molecular com-
pounds or other proteins (Hu et al. 2004; Bandi et al. 2017).
Downstream processing starts with cell removal from the

13
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cultivation broth. Subsequent operations typically comprise
membrane separations where low-molecular compounds and
very large proteins are removed. The intermediate product
is, thus, a desalted concentrated protein solution.
Protein purification is, in general, carried out using chro-
matographic processes usually classified as capture, interme-
diate purification and polishing steps. Affinity, ion-exchange,
hydrophobic interaction, and size-exclusion chromatography
are the most common types of separation units employed in
rhEPO purification (Hu et al. 2004; Adamíkova et al. 2019).
The most frequently used type is anion-exchange chromatog-
raphy that is used both as a capture step and an intermediate
purification step (Hu et al. 2004; Bandi et al. 2017).
Besides conventional anion exchangers with a quater-
nary amine or DEAE functional group, multimodal anion
exchangers have recently been applied (Koticha et al. 2011;
Bandi et al. 2017). Their advantage is that they are salt tol-
erant; so, their application eliminates desalting steps from
the purification train. They maintain rather high adsorp-
tion capacity at the salt concentrations as high as 500 mM
(Champagne et al. 2013). Almost all commercial multimodal
anion exchangers for protein separation are in particle form.
An exception is Sartobind STIC, a novel membrane adsor-
bent based on a backbone from regenerated cellulose with
a primary amine ligand. It was designed to allow high flow
velocities without separation efficiency loss (Frau et al.
2013). A Sartobind STIC membrane has recently exhibited
very good performance in the purification of adenovirus type
5 and retro virus-like particles (Nestola et al. 2015).
The objective of this work was to investigate the separa-
tion potential of a multimodal salt-tolerant anion-exchange
membrane for the purification of rhEPO from a partially
purified cultivation broth free of microbial contaminants,
large biological macromolecules and low-molecular impuri-
ties. Batch adsorption experiments were designed to investi-
gate the effect of process conditions such as pH, buffer type,
and salt concentration on the rhEPO binding capacity and
selectivity of the membrane adsorbent. Finally, the adsorp-
tion/desorption experiments were carried out in a laboratory
membrane flow module to examine the elution conditions for
rhEPO separation from protein byproducts.
Experimental
Materials
Lyophilized rhEPO-α standard with the specific activity of
120,000 IU/mg was purchased from Jena Bioscience (Jena,
Germany). EX-CELL 293, a serum-free medium for HEK
293 cultivation, multiwell plate sealers, Tween 20, second-
ary antibody (goat anti-mouse IgG peroxidase conjugate),
o-phenylenediamine (OPD), bicinchoninic acid (BCA)
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Chemical Papers (2019) 73:1805–1811
solution and bovine serum albumin were purchased from
Sigma-Aldrich (St. Louis, USA). Fetal calf serum (FCS)
was obtained from HyClone (Logan, Utah, USA). Human
EPO antibody from mouse IgG2A, clone AE7A5, used as
the primary antibody was obtained from Bio-Rad (Hercules,
California, USA). Salts and other chemicals used for buffer
preparation were obtained from Mikrochem (Pezinok, Slo-
vakia). The rhEPO stock solution (concentration of 5 μg/
ml) was prepared in the phosphate-buffered saline (PBS, pH
7.2). Standard solutions were prepared by diluting the stock
solution with EX-CELL 293.
The used 96-well high-binding polystyrene microtiter
plates and well polystyrene microtiter plates were obtained
from Greiner Bio-One (Frickenhausen, Germany). Poly-
ethersulphone syringe filters with the pore size of 0.22 µm
were obtained from AZ Chrom (Bratislava, Slovakia). The
tangential flow filtration module Sartocon cassette with the
molecular weight cut-off (MWCO) of 10 kDa and the salt-
tolerant interaction chromatography membrane Sartobind
STIC were kindly donated by Sartorius Stedim Biotech
(Göttingen, Germany). The membrane porosity and thick-
ness were 0.8 and 0.275 mm, respectively. The thickness of
membranes was determined using a contact thickness gauge
from MTS Systems (Cary, NY, USA).
rhEPO production
rhEPO was produced by cultivation of human embryonic
kidney cells 293 (HEK 293) transfected with the eukary-
otic expression vector carrying the gene for EPO under
the control of a CMV promoter in EX-CELL 293 (Molnár
et al. 2019). The cells were removed from the cultivation
medium by centrifugation (3000g/20  min). The super-
natant was then microfiltered through a 0.45-µm filter to
remove solid particles. Ultrafiltration using TFF modules
with MWCO = 10 kDa was used to concentrate the filtrate
approximately tenfold. Finally, a buffer exchange step was
performed by diafiltration. The product was stored in a
50-mM citrate–phosphate buffer with pH 6. Total protein
concentration in the concentrate was 1.5 mg/ml.
Quantification of rhEPO by indirect ELISA
An indirect ELISA test for rhEPO determination was per-
formed using 96-well high-binding plates. Samples were
prepared by diluting the protein solution with EX-CELL
293 as a coating buffer. Samples or standard solutions were
added into the plate wells and incubated at 8 °C for 20–22 h.
Unbound proteins were removed by washing at ambient
temperature, which was also used in the subsequent steps.
These steps included several washing and rinsing, blocking
of plate-free binding sites, binding of the primary and sec-
ondary antibody, reaction with color reagent and absorbance
Chemical Papers (2019) 73:1805–1811 1807
measurements at 490 nm using a microplate reader ELx808
(BioTek, USA). Analysis details are given in our recent
paper (Molnár et al. 2019).
Total protein assays
Protein concentration in the samples was measured by the
standard BCA protocol (Sorensen 1986) using 96-well
plates. A calibration curve was prepared using BSA stand-
ards in the concentration range of 0.02–1 mg/ml. A 25-μl
sample was pipetted into each well and 200 μl of BCA work-
ing reagent (BCA and 0.4% C ­ uSO4 solutions in 50:1 ratio)
was added. Samples were incubated at 37 °C for 30 min.
Absorbance was measured at 562 nm. Since an interfer-
ence of l-arginine with a component of the BCA assay was
observed, the total protein concentration in arginine-contain-
ing samples was measured by the standard Bradford protocol
using 96-well plates. In this assay, 5 μl of the sample was
pipetted into each well and 250 μl of Bradford reagent was
added. After 5 min of incubation at ambient temperature,
absorbance was measured at 590 nm.
Batch experiments
Adsorption equilibrium experiments were carried out in the
batch mode. To investigate the influence of pH on rhEPO
adsorption, three different buffers with the concentration
of 20 mM were used; namely acetate (pH 5 and pH 5.5),
bis–tris (pH 6, pH 6.5 and pH 7) and tris buffer (pH 7.5, pH
8 and pH 9). The effect of salt concentration was examined
using NaCl in the concentration range from 0 to 500 mM.
Membrane samples with the frontal area of 1.57 cm2 and bed
volume (BV) of 0.0432 cm3 were first properly washed with
redistilled water to remove glycerol. They were then equili-
brated with a protein-free binding buffer. Excess amount of
the equilibration buffer was removed from the pre-wetted
membrane surface with laboratory filter paper. Membranes
prepared in this way were immersed into test tubes con-
taining 2 ml of the rhEPO concentrate. The total protein
and rhEPO concentrations in the rhEPO concentrate were:
c0PR = 1.59 ± 0.04  mg/ml and c0EPO = 0.093 ± 0.009  mg/
ml. The corresponding impurity protein concentration,
c0IMP = 1.50 ± 0.04 mg/ml, was determined as the difference
of the two aforementioned values.
The test tubes were sealed and stirred in horizontal posi-
tion at 24 °C for at least for 24 h to reach equilibrium. Equili-
brated solutions were filtered through PES syringe filters
(pore size of 0.22 µm). Final concentrations of total proteins
and rhEPO in the solution, cEPO and cPR , respectively, were
Eq Eq
measured as described above. The final impurity concen-
tration, cIMP , was calculated analogously as c0IMP . Several
Eq
adsorption characteristics were evaluated using the specified
concentrations.
rhEPO and impurity protein binding capacity, qEPO and
qIMP , respectively, were obtained from the following equation:
( eq )
Vsol c0i − ci
qi = , (1)
Vm
where subscript i represents rhEPO or impurity proteins,
Vsol , rhEPO concentrate volume, and Vm membrane volume.
The corresponding distribution coefficients, kEPO and kIMP ,
were then calculated from Eq. (2),
qi
ki = . (2)
Eq
ci
The relation between rhEPO and impurity proteins binding
was quantified through selectivity S (Eq. 3),
kEPO
S= . (3)
kIMP
Flow experiments
Flow experiments were carried out using a fast protein liquid
chromatography (FPLC) system ÄKTA Purifier (GE Health-
care, Uppsala, Sweden). UV absorbance detection was carried
out at the wavelength of 280 nm. The measurements were per-
formed for one-layer membrane configuration (68 mm2 fron-
tal area) using the membrane holder Swinnex 13 (Sigma, St.
Louis, Missouri, USA). Dead volume of the chromatographic
system was calculated from the residence time of a 2% ace-
tone. Prior to the measurements, the membranes were washed
with 50 BV of redistilled water and equilibrated with 50 BV
of the binding buffer (20 mM bis–tris buffer with pH 6). The
membrane bed was loaded with 1 ml of rhEPO concentrate.
Washing was performed with the binding buffer in the extent
of minimally 100 BV. Elution was carried out either using
NaCl gradient or in isocratic mode for different desorbents.
All experiments were conducted at the flow rate of 1 ml/min.
rhEPO and impurity protein recovery was calculated from the
following equation:
mADS
Ri = i
(100%), (4)
mDES
i
where mADS
i
 , mDES
i
represent mass of adsorbed and desorbed
rhEPO or impurity proteins. The masses were evaluated
from the balance of feed and outlet stream composition.
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Results and discussion
Separation of target protein from impurities by liquid chro-
matography is based on the principle that, under a given set
of conditions, individual species dissolved in a mobile phase
interact differently with an adsorbent. Affinity of individual
species, characterized by the distribution coefficient (Eq. 2),
changes with the process conditions such as pH, buffer type,
salt concentration. The investigation of the process condi-
tions effect on the separated compounds affinity is essential
to achieve good separation in a chromatographic column.
Selectivity (Eq. 3) is a thermodynamic measure of the adsor-
bent potential to separate target protein from impurities.
The selectivity of rhEPO with regard to the impurity pro-
teins was investigated as a function of pH and NaCl con-
centration for the multimodal anion-exchange membrane
Sartobind STIC. Batch adsorption experiments were carried
out for this purpose. Figure 1a shows values of distribution
coefficients of rhEPO and impurity proteins in the absence
of NaCl as a function of pH in the range of 5–9. The pH
lower limit was chosen based on our experience that signifi-
cant aggregation occurs at lower pH. The upper limit was
set due to the loss of biological activity of rhEPO at higher
values (Seong-Hun et al. 2013). All proteins bind strongly
in the whole pH range, as it follows from the distribution
coefficient values above 100.
A 500
Distribuon coefficient [-]
400
300
200
100
0
5 5.5 6 6.5
pH
B2
Selecvity [-]
1 centration was changed immediately to 2 M (Fig. 5). Unfor-
0 feed. Moreover, the balances of rhEPO and impurity proteins
5 5.5 6 6.5
pH
Fig. 1  Influence of pH on a the distribution coefficient of impurity
proteins (gray) and rhEPO (stripes) and b rhEPO selectivity
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Chemical Papers (2019) 73:1805–1811
The highest distribution coefficients (about 300–450)
were observed in the region of neutral pH. The satura-
tion binding capacity at this pH was around 90 mg/ml for
impurity proteins and around 4  mg/ml for rhEPO. The
data on rhEPO binding capacity are very rare (Adamíkova
et al. 2019). Dynamic binding capacity values from 0.35
to 2.5 mg/ml were reported only for three different anion
exchangers without providing details on the source of rhEPO
(Lourdes et al. 2015). Figure 1b shows that the rhEPO selec-
tivity values were lower than 1 below pH 6 and slightly
higher than 1 above this threshold. Nonetheless, the poor
selectivity and high affinity of both rhEPO and impurity
proteins imply that Sartobind STIC is not suitable for the
flow-through/frontal chromatography regime at these pro-
cess conditions and is, thus, not an efficient adsorbent for
the protein purification capture step.
Salt-tolerant adsorbents adsorb proteins in broad ranges
of salt concentrations and often show non-monotonous
dependence of adsorption capacity on salt concentration
(Frau et al. 2013). This opens space for seeking good selec-
tivity by salt elution. To evaluate the potential of Sartobind
STIC in the elution chromatography mode, the effect of
NaCl concentration was evaluated at pH 6–8. Figure 2 shows
that the distribution coefficients of both rhEPO and impurity
proteins decreased significantly with the salt concentration.
On the other hand, they are still much higher than in case of
conventional anion exchangers which do not bind proteins
at concentrations as high as 500 mM. The selectivity values
were around 1 at salt concentrations below 500 mM (Fig. 3).
A slight indication of the preferable binding of rhEPO was
observed at 500 mM. Since the values of distribution coef-
ficients were still around 50, higher salt concentrations had
to be considered to achieve efficient elution.
Application of higher NaCl concentrations was then
investigated in flow adsorption/desorption experiments.
Several elution strategies were designed. One such strategy
with NaCl stepwise gradient from 0 to 2 M is presented in
Fig. 4 where also the UV signal course corresponding to
7 7.5 8 9 the concentration of eluted proteins is shown. A significant
portion of proteins was eluted during washing (sample 1).
Each increase of salt concentration resulted in a jump in the
protein concentration followed by a gradual slow decrease
characteristic for favorable isotherms.
A single elution peak was observed when the NaCl con-
tunately, the expected separation effect was not achieved in
any of the experiments presented in Figs. 4 and 5. Figure 4b
documents that the rhEPO fraction was smaller than in the
7 7.5 8 9
showed that desorption was only partial when only 18% of
bound rhEPO and 76% of bound protein were desorbed. This
is partly surprising considering the relatively long elution
time and high NaCl concentration.
Chemical Papers (2019) 73:1805–1811 1809

A 500 A40 1
2

NaCl concentraon [mol]

Distribuon coefficient [-]

400

UV signal [mAU]
30
2
300
20 3 1
4
200
10 5 6 7 8 9 10 11

100
0 0
0 20 40 60 80
0
0 100 200 300 400 500 Retenon volume [ml]
NaCl concentraon [mM]
B7
6
B 500

rhEPO fracon [%]

5
Distribuon coefficient [-]

400
4

300 3
2
200
1

100 0
feed 1 2 3 4 5 6 7 8 9 10 11
Sample
0
0 100 200 300 400 500
NaCl concentraon [mM] Fig. 4  Flow separation experiment at pH 6 with NaCl stepwise gra-
dient elution and rhEPO concentrate loading of 1  ml. a Solid line
C 500 represents the UV absorbance, red-dashed line NaCl concentration
Distribuon coefficient [-]

and dotted rectangles denote the retention volumes of taken samples

400
which are numbered consecutively. b rhEPO fraction in individual
samples
300

200

200 2
100

NaCl concentraon [mol]

UV signal [mAU]

0 150
0 100 200 300 400 500
NaCl concentraon [mM]
100 1

Fig. 2  Distribution coefficients of impurity proteins (△) and rhEPO

50
(●) vs NaCl concentration. a pH 6, b pH 7, c pH 8

0 0
0 5 10 15 20 25 30
Retenon volume [ml]

Fig. 5  Flow separation experiment at pH 6 with 2  M NaCl elution

2
and rhEPO concentrate loading of 1 ml. Solid line represents the UV
absorbance and red-dashed line the NaCl concentration
Selecvity

1
Irreversible protein adsorption has rarely been reported
for ion exchangers (Hunter and Carta 2000). On the con-
trary, it is very common for hydrophobic interaction chro-
matography adsorbents. Millitzer et al. (2005) demonstrated
0 that one portion of the protein was bound reversibly and the
0 100 200 300 400 500
NaCl concentraon [mM] other one irreversibly. Ueberbacher et al. (2008) supported
the phenomenon of irreversible binding also by attenuated
Fig. 3  Combined effect of NaCl concentration and pH on rhEPO total reflectance Fourier transform infrared spectroscopy
selectivity. pH 6 (gray), pH 7 (stripes) and pH 8 (black) measurements of protein unfolding. Deitcher et al. (2010),

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Table 1  rhEPO and impurity protein recoveries in individual flow
experiments
Run Desorbent REPO [%] RIMP [%]
1 2 M NaCl, bis–tris 20 mM, pH 6 42 68
2 2 M NaCl, bis–tris 20 mM, pH 9 48 78
3 2 M NaCl, phosphate 20 mM, pH 6 20 68
4 2 M NaCl, acetate 20 mM, pH 5 47 75
5 2 M NaCl, acetate 200 mM, pH 5 45 69
6 1 M Arginine, bis–tris 20 mM, pH 6 43 73
7 2 M ­(NH4)2SO4, bis–tris 20 mM, pH 6 44 72
8 2 M ­(NH4)2SO4, bis–tris 200 mM, pH 6 45 63
however, used several techniques and declared that the irre-
versibility is often only apparent and slow protein folding
takes place. It is, therefore, reasonable to hypothesize that
hydrophobic interactions are also responsible for the irre-
versible binding of rhEPO and impurity proteins on a mul-
timodal membrane adsorbent.
The irreversible and slowly reversible adsorption is incon-
venient in protein purification. Since rhEPO was bound
enormously strongly and NaCl elution was found inefficient
(Table 1), the final part of this work dealt with the search of
suitable desorption conditions. Since protein unfolding is
affected by process conditions, the effect of pH 5–9, buffer
type (bis tris, phosphate, acetate) and kosmotropic agents
(ammonium sulfate and arginine) was examined (Table 1).
Flow experiments were carried out and the results of rhEPO
and impurity protein recovery are given in Table 1. The
results show that complete recovery was not achieved for
any of these components. The rhEPO recovery was usually
around 45% and impurity protein recovery was between
60 and 80%. Incomplete recovery of impurity proteins is
not a problem here, since the membrane adsorbents are not
reused. On the contrary, the significant loss of rhEPO makes
this multimodal adsorbent unsuitable for the purification of
rhEPO from the mammalian cell cultivation medium.
Conclusions
This work analyzes the potential of a multimodal anion-
exchange chromatography membrane for the purification
of a therapeutic glycoprotein, recombinant human erythro-
poietin, produced by human embryonic kidney cells. The
concentrate obtained from the cultivation medium using
membrane filtration processes is a complex protein mixture.
The objective of rhEPO purification is to separate the target
protein from protein impurities. In this work, the key quan-
tities defining the thermodynamics of protein binding were
the distribution coefficients of rhEPO and impurity proteins
and the rhEPO selectivity with respect to impurity proteins.
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Chemical Papers (2019) 73:1805–1811
These quantities were obtained from batch adsorption exper-
iments. It was found that both components had high distribu-
tion coefficients in the broad pH range applied. It was found
that Sartobind STIC does not have good rhEPO selectivity.
Low selectivity was obtained in further batch experiments
focused on the investigation of the effect of NaCl concentra-
tion. These experiments demonstrated strong salt tolerance
of the membrane adsorbent although the adsorption capac-
ity decreased monotonously with the NaCl concentration.
Nonetheless, it was evident that compared to common ion
exchangers used for protein purification, very high NaCl
concentration has to be used to elute bound proteins.
Flow experiments were then carried out to find suitable
elution conditions for rhEPO purification. The effect of the
chromatographic mode (isocratic and gradient), pH, and
buffer type was investigated. It was found that a major part
of rhEPO and a smaller part of impurity proteins were irre-
versibly bound to the multimodal adsorbent. This can be
explained by protein unfolding on the hydrophobic groups
of the adsorbent. Unfolded proteins were not recovered
even when a solution of kosmotropic agents, ammonium
sulfate and arginine was used as the desorbent. The incom-
plete recovery of rhEPO makes the investigated multimodal
adsorbent not suitable for process application. The observed
behavior can have more general implications for the applica-
tion of multimodal adsorbents in protein purification.
Acknowledgements  This work was supported by the Grants from the
Agency of the Ministry of Education, Science, Research and Sport of
the Slovak Republic for Structural Funds of EU (Grant number: ITMS
26240220071), the Slovak Research and Development Agency (Grant
number: APVV-14-0474), and the Slovak Grant Agency for Science
(Grant number: VEGA 1/0573/17). Dr. Mária Bartošová from the Insti-
tute of Virology of the Slovak Academy of Sciences is kindly acknowl-
edged for providing the supernatant of the HEK cultivation medium.
Compliance with ethical standards 
Conflict of interest  On behalf of all authors, the corresponding author
states that there is no conflict of interest.
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