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This article was downloaded by: [University of Toronto Libraries]On: 06 January 2015, At: 22:46Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UK
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Purification and Biological
Activity of a Recombinant
Human Erythropoietin
Produced by Lymphoblastoid
CellsA. Ben Ghanem
a , J. J. Winchenne
a , C. Lopez
a , S.
Chrétiena , M. Dubarry
a , C. T. Craescu
b , J. P. Le
Caerc , N. Casadevall
d , P. Rouger
a , J. P. Cartron
e
& P. Lambin a
a Institut National de Transfusion Sanguine , Paris,
Franceb Institut Curie-Biologie , Orsay, France
c Institut A. Fessard CNRS Gif / Yvette , France
d Laboratoire d'Hématologie Hôpital R. Poincaré ,
Garches, Francee
INSERM , U 76, Paris, FrancePublished online: 24 Sep 2006.
To cite this article: A. Ben Ghanem , J. J. Winchenne , C. Lopez , S. Chrétien ,M. Dubarry , C. T. Craescu , J. P. Le Caer , N. Casadevall , P. Rouger , J. P. Cartron& P. Lambin (1994) Purification and Biological Activity of a Recombinant HumanErythropoietin Produced by Lymphoblastoid Cells, Preparative Biochemistry, 24:2,127-142, DOI: 10.1080/10826069408010087
To link to this article: http://dx.doi.org/10.1080/10826069408010087
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PREPARATIVE BIOCHEMISTRY,
24(2),
127-142
(1994)
PURIFICATION AND BIOLOGICAL ACTIVITY OF A RECOMBINANT
HUMAN ERYTHROPOIETIN PRODUCED BY LYMPHOBLASTOID CELLS
A. Ben Ghanem1,J.J. Winchennel, C. Loped,
S.
Chdtienl,
M.
Dubarryl,
C. T. Craescua, J.P.
Le
Caer3,N.Casadeval14, P. Rougerl, J.P. Cartron5
and P. Lambin
Institut National de Transfusion Sanguine Paris France
Institut Curie-Biologie Orsay France
3 Institut A. Fessard CNRS Gif / Yvette France
4 Laboratoire d'H6matologie H8pital R. Pohcad Garches France
5
INSERM U
76 Paris
France
ABSTRACT
A recombinant human erythropoietin (rH-EPO) was obtained from the culture
supernatantsof human B-lymphoblastoid cells transfected by the human EPO gene.
rH-EPO was purified by a two-step method based on immunoaffinity and ion
exchange chromatography. The fiist step
was
achieved by an anti-EPO monoclonal
antibody (Mab).
This
Mab, immobilized on Sepharose 4B, allowed a 410-fold
purification
of
the protein. The second step consisted of ion exchange
chromatographyon DEAE Sephacel. The combination of these two steps results in
a highly purified rH-EPO with a global yield of about 50 ; the specific activity of
the protein was 176,000
IU/A2go.
The NMR spectrum was characteristic
for
a
well structured, single-conformation protein. The purified protein
was
analyzed by
SDS-PAGE and isoelectric focusing. The biolo ical activity of purified rH-EPO
polycythemic mice and in vitrQ by
the
proliferative response of an EPO-dependent
cell
line. The purified protein expressed
in
lymphoblastoid cells of human origin
had the same biological activity as that of urinary EPO and rH-EPO produced in
other mammalian cells.
was measured in
V ~ V Q ,
by the incorporation of 58Fe into red blood cells (RBC) of
Correspondence:
Dr. Lambin, Institut National
de
Transfusion Sanguine,
6
rue
Alexandre Cabanel, 75015 Paris France.
127
Copyright
994 by Marcel Dekktr, Inc.
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128 GHANEM ET
AL.
INTRODUCT ON
Erythropoietin (EPO) is the main regulator
of
human erythropoiesis (reviewed
in l y 2 . Human EPO (H-EPO) consists of a
166
amino acid polypeptide chain
heavily glycosylated (one 0-linked and three N-linked carbohydrate chains) 3 ~ 4 .
The apparent molecular mass (Mr) of EPO measured by SDS-PAGE is about
34 kD.
The protein exerts its activity on erythroid progenitor cells by binding to
specific cell surface receptors132. Clinical trials in anemic patients with end-stage
renal disease have revealed that rH-EPO is a very effective agent capable of
relieving this anemia
5.
The gene
of
human EPO has been cloned and expressed
in
several eukaryotic cell lines, including Chinese hamster ovary cells (CHO), SV40
transformed African green monkey kidney cells (COS) and baby hamster kidney
cells
(BHK) 6-8.
T he protein was f i t purified from the urine
of
aplastic anemia
patients 9910. Since then, several techniques describing the purification of this
recombinant protein have been published
11 14.
Recently we were able to introduce the EPO gene in a B-lymphoblastoid cell
line
of
human origin l5. A clone of this cell line with high expression
of
EPO,
allowing for the first time the production of rH-EPO in a non-tumoral human cell
line, was selected. In a previous paper
16
we described the preparation
and
some
properties of several anti-EPO monoclonal antibodies (Mabs). In preliminary
experiments, one of them was found to be useful for immunopurification purposes.
We now describe a simple method to purify rH-EPO produced in non-tumoral
human lymphoblastoid cells by immunoaffinity using this Mab and ion exchange
chromatography. The biological activity of the purified protein was measured by in
viva
and
n
vitro techniques.
-RIALS AND METHODS
H-EPO,
rH-EPO was obtained from the human B-lymphoblastoid cell line, RPM I 1788
(ATCC no CCL156) that had been transfected with the expression vector
pTS39
15,This
vector contained the human EPO gene placed under the control
of
the enhancer and the major late promoter of Adenovirus 5. The selection
of
stable
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RECOMBINANT HUMAN ERYTHROPOIETIN
129
transformants was carried out by placing this gene under the control of the
dominant XGPRT gene. Subclone B7was chosen for its high expression level and
growth capacity. supernatants containing 1,900 IU/ml of rH-EPO were obtained
after 7 days of culture in foetal calf serum and Iscove s m odified Dulbecco s
medium (Gibco, New York, U.S.A.).
rH-E PO (Eprex) produced in CH O cells, with an activity of 2,000 IU/ml was
purchased from Cilag (Levallois, France).
Semipurified urinary H-EPO
was
obtained from Dr. J. Espada (Corrientes,
Argen tina).
l 2 5 j
H-EPQ
Purified rH-EPO
was
labelled w ith 1251
after
Sasaki
et
al 17. Tetrachloro-DL-
phenylglycouril (Iodogen) coated tubes were prepared as follows: 120 pl aliquots
of
a
solution of Iodogen (25 pg/ml) in chloroform were dried in the bottom of
polypropylene tubes under vacuum. Purified rH-EPO (10 pg) in
30
p1 Hepes
(Hydroxyethyl piperazine ethanesulfonic acid) 0.025
M
pH 7.2 and 15 p1
of
phosphate buffer 1
M
pH
7.2
were mixed with of 0.44 mCi (5 pl) 1251Na
(Amersham, Les Ulis, France). The reaction was carried out under stirring with a
Vortex mixer for one minute at room temperature. Proteins and reagents were
transferred onto a stopping tube containing potassium iodide
(50
p1 at 0.2 M),
thiosulfate (50 p1 at 0.02 M) nd 400 pl of phosphate buffered saline
PBS)
.The
solution
was
transferred onto a PD 10 column (Pharmacia, Uppsala, Sweden)
previously saturated with 1 ml of 5 bovine serum albumin BSA) in PBS and
equilibrated with
50
ml
of
PBS.
Labelled rH-EPO was recovered in the void
volume. The
specific
activity of the 1251-labelled rH-EPO was about 1,000
CVmmole.
Preparatlon and Purification of Antl-EPO MabS;
As described in a previous paper 16, three stable clones E14, E 73 and
D7
were
selected for their production of high affiity antibodies against rH-EPO.
Immunoglobulins were purified from the ascitic fluids obtained from these clones
by
immunoaffinity on
protein G Sepharose (Pharmacia)
as
follows:
1
mlof ascitic
fluid
was
diluted
in 4
i
0.01
M phosphate buffer pH 7.0 and 5 ml H2O. The
mixture was pumped onto a 5 ml column at a flow rate of 30 mlh. After extensive
washing
of
the column with the same buffer, IgG was eluted with 0.1 M glycine-
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130
GHANEM ET AL.
HC1 at pH 2.7 and a flow rate adjusted to 60 mllh .The eluted fraction was rapidly
brought to neutrality by the addition of 1M
Tris
pH 9. Between
5
and 12 mg of
monoclonal antibodies were obtained from 1 ml of ascitic fluid.
PreDaration of
an
Immunoaffinity C olumn;
80
mg of purified IgG from E l 4 ascitic fluid were dialyzed overnight at 4°C
against a hydrogeno-carbonate buffer (0.1 M NaHCO3 0.5 M NaCl pH 8.3).
Four g of CNB r-activated Sepharose 4B (Pharmacia) were extensively washed
with 1 mM HC1 (about
15
ml of swollen gel). The gel was poured into a column
and equilibrated with the same buffer just before the addition of E l 4 Mab.
Coupling
of
E l 4 M ab to the Sepharose 4B gel was performed overnight at 4°C
with stirring. After several washings, residual active groups were neutralized for
3
h at room temperature w ith 1 M ethanolamine pH 8.8 with rotative shaking. The
gel was finally washed 3 times with 30 ml carbonate buffer and then once with 30
mlO.1 M Glycine-HC1 pH 2.2. Glycine-HC1 was replaced by PBS containing 0.2
NaN 3 and the column was kept at 4°C.
Immunochem ical Assay fo rH-EPO:
rH-EP O was measured by a radioimm unoassay EPO COA TRIA)
(BioMCrieux, Marcy L Etoile, France). In brief, the technique used tw o anti-EPO
Mabs with high affmities for the E PO molecule. The first Mab was coated on the
surface of plastic tubes. Samples containing EPO were introduced into the tubes
simultaneously with the second Mab labelled with 1251. The mixture was incuba ted
for 3 h at room temperature. After
2
washes with distilled water, radioactivity
bound to the tubes was counted. The EPO concentration of each sample was
determined by interpolation from a calibration curve with 7 EPO standards ranging
from 0 to 800 mIU/ml.
Protein Assay
According to Davis et al.
1*
the extinction coefficient
of
H-EPO (whole
molecule) is 0.743 at 280 nm at
0.1
concentration.
N terminal sequencing of EPQ
EPO was submitted to twenty cycles of Edman degradation on a 470A
gas-
liquid phase sequencer (A pplied Biosystem s), (Foster U.S.A.) The protein was
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RECOMBIN
ANT
HUMAN
ERYTHROPOIETIN 131
loaded in solution on polyethylenimine pretreated glass fiber
filters.
Phenyl thio-
hydantoin amino acids were identifed using
a
120 A PTH analyzer (Applied
Biosystems)
.
NMR S~ec tra rH EPQ
NMR spectra were obtained on a
Varian
UNITY
500
NMR spectrometer at
310
K.
The water signal was eliminated by irradiation during the relaxation delay
(1.2s). 256 scans were usually necessary to obtain a good signal-to-noise ratio. A
Gaussian multiplication (lb= -2Hz, gb=
0.15)
prior to Fourier transformation was
used
to improve the spectral resolution. Chemical
shifts
are
expressed in parts per
million (ppm) from the signal of 2 2dmethyl-5-silapentane-5-sulfonate.
The sample for NMR spectroscopy (0.2 mM) was obtained by dissolving
the
lyophilized protein in phosphate buffer (10 mM), pH
6.5
containing 1 mM
NaN3 and
7
2H20
ElectroDho etic Tech-
Polyacrylamide gel electrophoresis
in
the presence of SDS (SDS-PAGE) was
performed according to the instructions of the manufacturer
(Pharmacia) n
thin
layer polyacrylamide gels. SDS and bromophenol blue were added to the samples,
with final concentrations of 1 and
0.1
mglml respectively. The samples were
allowed to migrate for about
75
min under
600 V, 50
mA and
30 W.
Silver staining
was performed after Heukeshoven and Demick 19.
Isoelectric focusing: rH-EPO and
PI
markers were submitted to polyacrylamide
gel isoelectric focusing (PAGIF) with 2.5% Phmalytes (pH range
3
to
10)
(Pharmacia) in the presence
5
M
urea.
Biological Activitv of rH-EPO;
The in
V~VQ
iological activity of rH-EPO was measured by the incorporation of
59Fe in polycythemic mouse red blood cells (RBC) 2o in the presence of different
amounts
of
purified rH-EPO and compared
to
the biological activity
of human
urinary EPO and rH-EPO produced in CHO cells.
Polycythemia was induced in
8
week old DBA2 mice by two injections of a
suspension of DBA2 RBC (hematocrit about 70 ) 1 mlon day 0, and
0.5
ml on
day 3. On day
5,0.2 ml
containing 0.5, 2
or
5 IU of human EPO (recombinant or
urinary) was administrated subcutaneously(5 mice for each dose). On day 7,
1
pCi
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132
GHANEM ET AL.
of 59Fe (Amersham) was injected intravenously into each mouse, and blood was
collected on day
10.
The radioactivity incorporated in
EU3C
was counted and the
percentage of radioactivity was calculated with respect to hematocrit and body
weight of each
animal.
Statistical analysis of the results was performed with Mann-
Whitney rank test.
The
in
vitrq biological activity of purified rH-EPO was measured with an assay
using the proliferative response of a human leukemia
cell
line (UT-7) to purified
rH-EPO. The UT-7 cell line
was
established
from
the bone marrow of a patient
with acute megakaryoblastic leukemia and has a strict dependency on either
interleukin 3 (11-3), granulocyte-macrophage colony-stimulating factor (GM-CSF)
or EPO
21, After depriving
UT-7
cells of growth factor for 5 h, increasing
amounts
of either lymphoblastoid or CHO rH-EPO from 0.0012 to 0.3 IU in a
volume of
100
pl of Iscove's modified Dulbecco's medium (Gibco, New York
U.S.A.)
with
10%
foetal calf serum were added to 100 pl of culture medium
containing about
50 000
UT-7 cells in a 96 well culture plate. After 48 h of culture
at 37 C, 0.5 pCi of 3H-thymidine (hersham) was added to each well. After a
further 24
h
incubation, cells were collected on glass fiber filters (Skatron) (Lab
System, Les
Ulis,
France) and washed with distilled water. Filters were transferred
into tubes and radioactivity corresponding
to
3H-thymidine incorporation was
counted in
a beta
counter.
a - E P O
Purif-
n
Immunopurif ication of rH EPO:
Aliquots of 500 ml of supernatants from the lymphoblastoid
cells,
containing on
average 352 IU/A280 (1,900 IU/ml) of rH-Em, were first centrifuged for 30 min
at 3,000 g and then filtered through GF/B and GF/D Whatman glass microfiber
filters (Maidstone,
U.K.).
The immunosorbent column (80 mg of Mab
E l 4
immobilized on 15 ml of CNBr activated Sepharose 4B)
was
pre-equilibrated with
0.01 M Tris, 0.15
M
NaCl buffer pH 7.5. Each aliquot
was
pumped into the
column overnight at 4°C with a flow rate of approximately
30
mYh.
The
immunosorbent column was successively and extensively washed with the Tris-
NaCl buffer and
0.1
M acetate buffer pH 4.5 at a
flow
rate
of 60
mvh. Elution of
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RECOMBINANT HU MAN ERYTHROPOIETIN
133
s
8
2.0
c 1
I
m
.e 1.5
B
E
3
1.0
0.5
0
10 530 560 610 620 560
Volume of elution
m l )
FIGURE 1
Purification of rH-EPO from culture supernatants by affinity chromatography with
a m onoclonal antibody specific for
H-EPO.
he protein was eluted with glycine-
HC1 buffer pH 2.2 (flow
rate
60
mVh).
6.9
x
105
IU
were purified with 80 mg of
Mabs immobilized on 15ml of Sepharose 4B.
rH-EPO was obtained with
30
ml 0.1
M
glycine-HC1 buffer
pH
2.2 (Fig.
1).
The
eluted material was apidly brought to pH 7.5 by the addition of 1 M
Tris
pH 9 and
then
it
was concentrated on
PMlO
membranes (Amicon, Danvers, Ireland). About
20
8
f rH-EPO loaded did not bind to the antibody;
it
was recovered
in
the flow
through fraction or eluted at pH 4.5 whereas 73 of that loaded were eluted at
pH
2.2.
The rH-EPO in this fraction had a specific activity of 144,000 U/A
280
(Table
I).
The column was used more than 20 times without noticeable loss
of
efficiency.
DEQE
SephacelChromatography:
The material of 6 purification cycles on the immunosorbent column,
(corresponding to 28.8 A280 units) was pooled and dialyzed overnight at 4°C
against acetate buffer (0.04 M acetic acid, 0.0025
M
CaC12
adjusted
to pH 4.5
with 1 M. NaOH). 53 ml of pre-swollen DEAE Sephacel gel (Pharmacia) were
equilibrated with
the
same buffer and poured
onto a
2.5 cm diameter column. The
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134 GHANEM ET AL.
0 200 300 400
Volume of elution
(
m )
FIGURE
2
Purification of rH -EPO by ion exchange chromatography: 28.8 A280 units of rH-
EPO
obtained by immunoaffmity were applied on a 53
ml
DEAE Sephacel column
equilibrated with acetate buffer. EPO was eluted with
0.1 M
NaCl
in
the same
buffer (flow
rate
150
mVh).
dialyzate was loaded on the column at 150
ml/h
and 4°C. After absorption, rH-
EPO was eluted from DEAE with the acetate buffer to which was added
0.1
M
NaCl (Fig. 2). The eluted fraction was brought to pH 7.5 by the addition of 1M
Tris. About
72
of the rH-E PO activity was recovered
in
the eluted fraction.
After these
two
purification steps, the specificactivity
of
rH-EPO was increased
by 502 fold. The total yield was about 53 (see Table I).
ElectroDhoretic A n a l v s i s - E P O F
Th e fractions resulting
from
the two purification steps were analyzed by SDS-
PAG E (Fig. 3). The
final
product showed
a
band corresponding to an apparent
Mr
of 36 kD. The purity of the protein, estimated by gel scanning (Sebia
Densitometer, Issy les M oulineaux, France) was higher than
98
.
When analyzed by PAG IF, the purified rH-EP O showed a micro-heterogeneity.
Five to six components with apparent PI between 4 and 5 were observed
in
the
used gradient (Fig. 4).
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RECOMBINANT HUMA N ERYTHROPOIETlN
Fraction
Total protein
A280
units)
135
Supernatant
of rH-EPO
producing
cells
16,200
TABLE
I
Purification of rH-EPO Produced in Lymphoblastoid Cells
Fraction eluted
at pH
2.2
(Immunoaffinity)
28.8
Flow Fraction
through eluted with
fraction 0.1MNaCl
DEAE)
10.8
17.0
Total IU
of rH-EPO
5.7 x
106
Yield
( ) 100
4 . 1 6 ~o6 .91 x 106
3.01
x
lo6
SpecificActivity
IU/A280)
Purification
Factor
352
1
144,000
410
73
21.9 1’72.3
Y E
52.7 Global
84,300 176,000
/
502
in^ of ourified rH-EPO
The following sequence
was
found: Aia
Pro
Pro Arg Leu Ile Cys Asp Ser Arg
Val
Leu Glu
Arg
Tyr Leu Leu Glu Ala Lys.
JVMR
S ~ e c t r a f rH
EPQ
Fig.
5
shows the 500 MHz NMR spectrum of erythropoietin
in 10
mM
phosphate buffer at 310K.The 1 D spectrum is charackristic for
a
well structured,
single-conformation protein. The most down field peaks (Fig. 5
A )
should
correspond to the indole
NE
rotons of the three Trp residues experiencing distinct
chemical environments. The NH esonances are mainly concentratedbetween 7.5
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RECOMBINANT HUMAN ERYTHROPOIETIN
PI
137
FIGURE4
Polyacrylamide gel isoelectric
focusing and Coomassie blue
staining of purified rH-EPO.
. .
In vitro BiologicalAcuvitv;
The proliferation of UT-7 cells induced by rH-EPO produced in lymphoblastoid
cells or
in
CHO
cells
depends on the quantities of EPO added in the cell cultures
and increases at the same rate (Fig.7).
In the first purification step, we used a monoclonal antibody obtained by
immunization of mouse with SDS-treated EPO. This Mab had a high affinity for
native and SDS-treated EPO 16.
Its
dissociation constant was about 0.3
nM
according to the measurements performed by radioimmunoprecipitation and by
Scatchard s analysis. In spite of this high affiity, elution
was
possible under mild
conditions
and
with a satisfactory
yield (75 ).
After this step, the specific activity
of rH-EPO
was
increased by about
400
fold.
Electrophoretic analysis of the eluates obtained by this first step, showed,
in addition to the native molecule (apparent Mr 36 kD), a small proportion of
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138
4.5 4.*0
3. 5
S. O
2. 5 2. 0 1. 5
l . O
0.5 0.0
6
GHANEM
ET
AL.
A
FIGURE 5
5
NMR Spectra
ofrH-EPO:
low field
A)
and
high
field
B) regions of
the 500 MHz
NMR spectrum
of
EPO
in phosphate
buffer, pH
6.5
at 3
10
K.
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RECOM BINANT HUM AN ERYTHROPOIETIN
139
1 10
H - E p o
I .U.)
FIGURE 6
Measurement of the in
V ~ V Q
biological activity in DBA2 mouse
of 3
human EPO
preparations: 59Fe incorporated
in
mouse
RE3C
is expressed versus the quantity
of
EPO injected
0.5, 2
or 5
IU).
Each
point
corresponds to the mean value
observed in
5
mice.
5
Tj 45
P o
g 35
E
30
2
25
2
15
10
s e . 5
0
0,o
1 0,l 1 10
rH-EPO
IN,)
FIGURE
7
*rH-EPO Lynpho
rH-EPO
CHO
Measurement of the in v m biological activity
of 2
rH-EPO. The proliferation of
an
EPO dependent ce ll
(UT-7)was
measured by the incorporation
of 3H-thymidine
after the addition
of
EPO in the culture medium .
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140
GHANEM ET AL.
contaminants most of them corresponding probably to different fragments of the
native EPO molecule. These contaminants were removed by DEAE
chromatography. With this second step, ,the specific activity increased from
144,000 IU/A 280 to 176,000 IU/A 280 corresponding respectively to 112,000
IU/mg and to 137,000 W/mg (when calculated the extinction coefficient given in
. This latter activity is
similar
to that of rH-EPO expressed in CHO cells which
has a potency of 129,000 IU/mg 22. We estimated, after silver staining of the
protein submitted to SDS-PAGE, that the proportion of impurities should be less
than 2 of the total protein.
PAGIF experiments indicated a molecular heterogeneity of the erythropoietin
sample. Nevertheless, as can be seen in the extreme regions of the
NMR
spectrum,
the well-separated resonances, corresponding to the protein protons, are
characteristic for a single conformation of the polypeptide chain. In addition its
N-
terminal amino-acid sequence 20 AA) is identical to the sequence already
published by
Lai
et
al.
3. Therefore the electrophoretic heterogeneity should be
associated with the glycosyl moiety rather thanwith the protein itself. The isoforms
separated by PAGIF corresponds probably to a heterogeneity
in
sialic acid
composition of triantenary or tetraantenary chains of the carbohydrates l inked to
the protein .
A similar
heterogeneity was observed with natural 23 or rH-EPO
produced in CHO ells z4
The homogeneity of EPO preparations observed after SDS-PAGE is in contrast
with the heterogeneity observed after PAGIF (Fig. 3 and 4). Size homogeneity is
usually considered as a good criterion for the purity of a protein. Charge
homogeneity is not observed in natural or recombinant forms of EPO. However,
the purification of a particular isoform could
be
useful to obtain crystals of
high
quality allowing the determination of the three-dimensional structure of EPO
molecule. This purification can
be
obtained by techniques such
as
preparative
isoelectric focusing in a multicompartment electrolyzer described by Wenisch et
al.25 or chromatofocusing.
It also appears that the biological activity in mice of the EPO obtained from
lymphoblastoid cells is similar to a rH-EPO produced in an other mammalian
cell
and to urinary H-EPO. Jnvivg the response to the injection of lymphoblastoid rH-
EPO into polycythemic mice is dose-dependent and not statistically different from
the response obtained with rH-EPO from CHO cells or with urinary H-EPO.
In vitra, the proliferative response of
an
EPO-dependent cell line to purified
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ET
AL.
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