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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

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7/25/2019 10.1080@10826069408010087


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-



7/25/2019 10.1080@10826069408010087


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



7/25/2019 10.1080@10826069408010087


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



7/25/2019 10.1080@10826069408010087


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



7/25/2019 10.1080@10826069408010087


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).



7/25/2019 10.1080@10826069408010087


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



7/25/2019 10.1080@10826069408010087


7/25/2019 10.1080@10826069408010087


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



7/25/2019 10.1080@10826069408010087


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.



7/25/2019 10.1080@10826069408010087


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 .



7/25/2019 10.1080@10826069408010087


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



7/25/2019 10.1080@10826069408010087


7/25/2019 10.1080@10826069408010087


142 GHANEM

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