Method for the purification of erythropoietin and erythropoietin compositions

A method for purifying erythropoietin is described. The method comprises treating partially purifying erythropoietin by reverse phase high performance liquid chromatography to obtain homogeneous erythropoietin having a molecular weight of about 34,000 daltons on SDS PAGE and moving a single peak on reverse phase HPLC. The homogeneous erythropoietin protein preferably has a specific activity of at least 120,000 IU, more preferably at least 160,000 IU per absorbance unit at 280 nm.

FIELD OF THE INVENTION 
The present invention is directed to the purification of erythropoietin and 
to compositions comprising highly purified erythropoietin. 
BACKGROUND OF THE INVENTION 
Erythropoietin (hereinafter EPO) is a circulating glycoprotein, which 
stimulates erythrocyte formation in higher organisms. See, Carnot et al., 
Compt. Rend., 143:384 (1906). As such, EPO is sometimes referred to as an 
erythropoiesis stimulating factor. 
The life of human erythrocytes is about 120 days. Thus, about 1/120 of the 
total erythrocytes are destroyed daily in the reticulo-endothelial system. 
Concurrently, a relatively constant number of erythrocytes are produced 
daily to maintain the level of erythrocytes at all times (Guyton, Textbook 
of Medical Physiology, pp 56-60, W.B. Saunders Co., Philadelphia (1976)). 
Erythrocytes are produced by the maturation and differentiation of the 
erythroblasts in bone marrow, and EPO is a factor which acts on less 
differentiated cells and induces their differentiation to erythrocytes 
(Guyton, supra). 
EPO is a promising therapeutic agent for the clinical treatment of anemia 
or, in particular, renal anemia. Unfortunately, the use of EPO is not yet 
common in practical therapy due to its low availability. 
For EPO to be used as a therapeutic agent, consideration should be given to 
possible antigenicity problems, and it is therefore preferable that EPO be 
prepared from a raw material of human origin. For example, human blood or 
urine from patients suffering from aplastic anemia or like diseases who 
excrete large amounts of EPO may be employed. These raw materials however, 
are in limited supply. See, for example, White et al., Rec. Progr. Horm. 
Res., 16:219 (1960); Espada et al., Biochem. Med., 3:475 (1970); Fisher, 
Pharmacol. Rev., 24:459 (1972) and Gordon, Vitam. Horm. (N.Y.) 31:105 
(1973), the disclosures of which are incorporated herein by reference. 
The preparation of EPO products has generally been via the concentration 
and purification of urine from patients exhibiting high EPO levels, such 
as those suffering from aplastic anemia and like diseases. See for 
example, U.S. Pat. Nos. 4,397,840, 4,303,650 and 3,865,801 the disclosures 
of which are incorporated herein by reference. The limited supply of such 
urine is an obstacle to the practical use of EPO, and thus it is highly 
desirable to prepare EPO products from the urine of healthy humans. A 
problem in the use of urine from healthy humans is the low content of EPO 
therein in comparison with that from anemic patients. In addition, the 
urine of healty individuals contains certain inhibiting factors which act 
against erthropoiesis in sufficiently high concentration so that a 
satisfactory therapeutic effect would be obtained from EPO derived 
therefrom only following significant purification. 
EPO can also be recovered from sheep blood plasma, and the separation of 
EPO from such blood plasma has provided satisfactorily potent and stable 
water-soluble preparations. See, Goldwasser, Control Cellular Dif. 
Develop., Part A; pp 487-494, Alan R. Liss, Inc., N.Y., (1981), which is 
incorporated herein by reference. Sheep EPO would, however, be expected to 
be antigenic in humans. 
Thus, while EPO is a desirable therapeutic agent, conventional isolation 
and purification techniques, used with natural supply sources, are 
inadequate for the mass production of this compound. 
Sugimoto et al., in U.S. Pat. No. 4,377,513 describe one method for the 
mass production of EPO comprising the in vivo multiplications of human 
lymphoblastoid cells, including Namalwa, BALL-1, NALL-1, TALL-1 and JBL. 
For therapeutic use of EPO, it is necessary to purify the EPO protein to 
homogeneity. 
SUMMARY OF THE INVENTION 
EPO has been purified from the urine of patients with aplastic anemia by 
methods described by Miyake et al., J. Biol. Chem., 252:5558 (1977). It 
had been thought that EPO purified by this method was homogeneous because 
it moved as a single band during electrophoresus. It has now been 
discovered that this seemingly homogeneous EPO composition produced by the 
method of Mayaki et al. is composed of several polypeptide components 
ranging from 30,000 to 70,000 daltons. 
In accord with the present invention a homogeneous EPO protein composition 
is prepared by treating partially purified EPO by reverse phase high 
performance liquid chromatography and eluting the EPO protein in 
preferably an acetonitrile gradient to purify the EPO protein to 
homogeneity. In accord with this invention pure EPO protein is produced 
having a molecular weight of about 34,000 daltons on SDS PAGE and moving 
as a single peak on reverse phase HPLC. The EPO preferably has a specific 
activity of at least 120,000 International Units (IU) per absorbance unit 
at 280 nanometers, and more preferably 160,000 IU.

DETAILED DESCRIPTION OF THE INVENTION 
In accord with the present invention, it was found essential to treat 
purified EPO compositions by reverse phase high performance liquid 
chromatography in order to obtain homogeneous EPO protein. 
EPO can be obtained from several sources which include from the blood or 
urine of patients suffereing from asplastic anemia (natural sources) or 
from expression of genetically engineered vectors introduced into 
organisms for production of EPO by cell culture and fermentation 
processes. 
No matter what the source of EPO is, the EPO protein must be purified to 
homogeneity for therapeutic use. The crude EPO compositions obtained from 
natural sources or from expression in transformed cells containing 
genetically engineered DNA vectors can be partially purified by a variety 
of techniques. Preferably, the method for purifying EPO that was described 
by Miyake et al. in J. Biol. Chem., 252:5558 (1977) is used. The method of 
Miyake et al. comprises deactivating any proteolytic enzymes by treating 
the crude EPO preparations with phenol p-aminosalicylate. Such proteases 
can also be deactivated by other means such as by heating, which is 
presently preferred. The purification steps described by Miyake et al. 
include ethanol precipitation, DEAE-agarose fractionation, 
sulfopropyl-Sephadex chromatography, gel filtration and hydroxylapatite 
chromatography. Of course other equivalent procedures could be substituted 
for the steps of Miyake et al in order to obtain a "purified" EPO 
composition preferably having a specific EPO activity of at least about 
50,000, preferably at least about to 80,000 IU per absorbance unit at 280 
nm. 
The "purified" EPO composition has been found to be non-homogeneous. Thus, 
in accord with the present invention, the "purified" EPO composition is 
further treated by reverse phase high performance liquid chromatography 
(RPHPLC) to obtain a homogeneous EPO protein composition having a 
molecular weight of about 34,000 daltons when analyzed by sodium dodecyl 
sulfate (SDS) polyacrylamide gel electrophoresus (PAGE). Preferably, the 
reverse phase HPLC is conducted on a C-4 Vydac column using an eluant 
consisting of a 0 to 95% acetonitrile gradient in 0.01 to 1.0%, preferably 
0.1% trifluoroacetic acid over a period of about 100 minutes. Other 
equivalent combinations of columns and eluants can also be used. 
When purified in accord with the present invention, EPO compositions having 
a specific activity of at least 120,000 IU, preferably 160,000 IU, per 
absorbance unit at 280 nm are obtained. 
An "absorbance unit", as used herein, is approximately 1mg protein per ml. 
The amino acid sequence of an EPO protein derived from a human source, 
including its secretory leader sequence is illustrated in FIG. 2. The 
mature EPO protein begins with the "Ala" residue identified by the arabic 
numeral "1". The secretory leader sequence is the polypeptide sequence 
preceding the mature EPO protein beginning with the "MET" residue 
identified by the numeral "-27". Typically, the EPO protein having the 
secretory leader sequence is expressed in a cell capable of processing the 
protein to eliminate the leader sequence and secrete the mature protein 
into the media. The mature EPO protein may be expressed by recombinant 
means without using DNA coding for the secretory leader sequence and, in 
such case, may have a "Met" residue immediately preceding the "Ala" 
residue beginning the mature EPO protein (sometimes referred to as 
Met-EPO). 
The biologically active EPO produced from natural sources or by the 
procaryotic or eucaryotic expression of cloned EPO genes and purified in 
accord with the present invention can be used for the in vivo treatment of 
mammalian species by physicians and/or veterinarians. The amount of active 
ingredient will, of course, depend upon the severity of the condition 
being treated, the route of administration chosen, and the specific 
activity of the active EPO, and ultimately will be decided by the 
attending physician or veterinarian. Such amount of active EPO was 
determined by the attending physician is also referred to herein as an 
"EPO treatment effective" amount. For example, in the treatment of induced 
hypoproliferative anemia associated with chronic renal failure in sheep, 
an effective daily amount of EPO was found to be 10 Units/kg for from 15 
to 40 days. See Eschbach et al., J. Clin. Invest., 74: 434 (1984). 
The active EPO may be administered by any route appropriate to the 
condition being treated. Preferably, the EPO is injected into the 
bloodstream of the mammal being treated. It will be readily appreciated by 
those skilled in the art that the preferred route will vary with the 
condition being treated. 
While it is possible for the active EPO to be administered as the pure or 
substantially pure compound, it is preferable to present it as a 
pharmaceutical formulation or preparation. 
The formulations of the present invention, both for veterinary and for 
human use, comprise an active EPO protein, as above described, together 
with one or more pharmaceutically acceptable carriers therefor and 
optionally other therapeutic ingredients. The carrier(s) must be 
"acceptable" in the sense of being compatible with the other ingredients 
of the formulation and not deleterious to the recipient thereof. Desirably 
the formulation should not include oxidizing agents and other substances 
with which peptides are known to be incompatible. The formulations may 
conveniently be presented in unit dosage form and may be prepared by any 
of the methods well known in the art of pharmacy. All methods include the 
step of bringing into association the active ingredient with the carrier 
which constitutes one or more accessory ingredients. In general, the 
formulations are prepared by uniformly and intimately bringing into 
association the active ingredient with liquid carriers or finely divided 
solid carriers or both, and then, if necessary, shaping the product into 
the desired formulation. 
Formulations suitable for perenteral administration conveniently comprise 
sterile aqueous solutions of the active ingredient with solutions which 
are preferably isotonic with the blood of the recipient. Such formulations 
may be conveniently prepared by dissolving solid active ingredient in 
water to produce an aqueous solution, and rendering said solution sterile 
may be presented in unit or multi-dose containers, for example sealed 
ampoules or vials. 
The term "EPO protein" includes the 1-methionine derivative of EPO protein 
(Met-EPO) and allelic variations of EPO protein. The mature EPO protein 
illustrated by the sequence in FIG. 2 begins with the sequence 
Ala.Pro.Pro.Arg . . . the beginning of which is depicted by the number "1" 
in FIG. 2. The Met-EPO would begin with the sequence Met.Ala.Pro.Pro.Arg. 
. . 
The following examples are provided to aid in the understanding of the 
present invention, the true scope of which is set forth in the appended 
claims. It is understood that modifications can be made in the procedures 
set forth, without departing from the spirit of the invention. All 
temperatures are expressed in degrees Celsius and are uncorrected. The 
symbol for micron or micro, e.g., microliter, micromole, etc., is "u", 
e.g., ul, um, etc. 
EXAMPLE 1: PURIFICATION OF ERYTHROPOIETIN 
Crude erythropoietin preparations were concentrated by dialysis and 
proteolytic enzymes deactivated when necesary by heat treatment at 
80.degree. C. for 5 minutes. The crude preparation concentrates were then 
purified by the method described by Miyake et al., (1977) supra (the 
disclosure of which is hereby incorporated by reference) as follows. 
A. Ethanol Precipitation 
Batches containing about 100,000 IU of EPO activity at concentations of 
about 50 to 100 IU per absorbance unit at 280 nm were diluted to 50 ml 
with phosphate buffered solution (PBS) at 4.degree. C. 12.5 ml of 10 M 
LiCl were added. Absolute ethanol 62.5 ml) at 4.degree. was added slowly 
with stirring, which was continued for 30 min. after the addition was 
complete. After the flocculent precipitate had been allowed to settle for 
10 min. it was removed by centrifugation at 21,000.times.g for 10 min at 
-15.degree.. The pellet was washed three times with 10 ml of 50% ethanol, 
1 M LiCl and the supernatants were pooled. The washed precipitate was 
dissolved in 20 ml of PBS, yielding a turbid solution (50% precipitate). 
Sixty-seven milliliters of absolute ethanol were added slowly to the 
combined supernatants: stirring was continued for 30 min. and settling for 
15 min. The precipitate was collected as before and washed twice with 10 
ml of 65% ethanol, 0.7 M LiCl and the supernatants were pooled. The washed 
precipitate was dissolved in 20 ml of PBS (65% precipitate). 
The the pooled supernatants, 96 ml of ethanol were added slowly and 
stirring was continued for 30 min. after which the precipitate was allowed 
to settle for 14 hr. at 4.degree.. The precipitate was washed twice with 
10 ml of 75% ethanol, 0.5 M LiCl, the supernatants were pooled, and the 
precipitate was dissolved in 20 ml of PBS (75% precipitate). 
The combined supernatant was brought to 90% ethanol by addition of 540 ml 
of absolute alcohol, stirred for 30 min. and stored at -20.degree. for 48 
hr. before the precipitate was collected, dissolved in 50 ml of cold water 
and immediately frozen. 
B. DEAE-Agarose Fractionation 
The solution, in water, of a 90% ethanol precipitate was concentrated to 
about 5 ml on an Amicon UM-10 ultrafilter, then brought to 25 ml with 
0.01M Tris, pH 7.0, and a 50- ul aliquot was removed. The DEAE-agarose, 
100 to 200 mesh, was degassed under reduced pressure, suspended in 0.01M 
Tris, pH 7.0, and packed into a column 9.2.times.2.5 cm in diameter (bed 
volume, 45 ml). The gel was washed with 1.5 liters of 0.01M Tris, pH 6.9; 
the ratio of absorbance units added to bed volume (ml) was 6.65. The 
sample was added to the column over a period of 40 min. and 150-drop 
fractions were collected. the column was washed with 211 ml of 0.01M Tris, 
pH 7, and then eluted with the following buffers: 366 ml of 0.01M Tris, pH 
7.0; 5 mM CaCl.sub.2, 270 ml of 0.01 Tris, pH 7.0; 17 mM CaCl.sub.2 ; 194 
ml of 0.01M Tris, pH 7.0; 3 mM CaCl.sub.2 ; and 65 ml of 0.1M CaCl.sub.2. 
From this point on in the fractionation calcium was added to all buffers 
except those used with hydroxylapatite columns because there were 
inconsistent results and appreciable losses of activity when buffers 
without calcium were used. For the next step in purification, eluates from 
DEAE agarose columns were selected that had significant quantities of EPO 
activity. 
C. Sulfopropyl-Sephadex Chromatography 
The eluates (17 mM CaCl.sub.2) from DEAE-agarose columns were desalted and 
concentrated on a UM-10 ultrafilter and then dialyzed against 2 liters of 
5 mM CaCl.sub.2. pH 7.5 overnight. In the sample run described below, 30 
ml of dialyzed solution were brought to pH 4.50 by dropwise addition of 
0.1M HCl: the small amount of precipitate formed was removed by 
centrifugation and washed with 5 ml of 5 mM CaCl.sub.2, pH 4.5. The wash, 
pooled with the supernatant, was applied to a sulfopropyl-Sephadex column 
(15.0.times.2.5 cm in diameter, bed volume, 78.3 ml) which had been 
equilibrated with 5 mM CaCl.sub.2, pH 4.50. The absorbance units to bed 
volumn (ml) ratio was 2.47. A low value for this ratio is preferred for 
optimal fractionation on sulfopropyl-Sephadex: for example, if the 
absorbance unit to bed volume ratio was greater than 10, almost all of the 
activity was found in the effluent fraction. The following buffers were 
used in developing the column. Input was: 5 mM calcium acetate, pH 4.50, 
specific conductivity-1.075 umho cm.sup.-1. Eluting buffers were: 7.5 mM 
calcium acetate, pH 4.70, specific conductivity 1,500 umho cm.sup.-1 : 15 
mM calcium acetate, pH 5.25, specific conductivity=2,100 umho cm.sup.-1 : 
15 mM calcium acetate, 0.01 m Tris, pH 7.24, specific conductivity=11,500 
umho cm.sup.-1. the column was run at 0.4 ml/min. at 4.degree., and 
200-drop fractions were collected. After a reading was taken at 280 nm and 
the appropriate pools were made, the solutions were neutralized (within 1 
hr. after elution) and aliquots were removed for assay and stored at 
-20.degree.. 
D. Gel Filtration 
The 12.5 and 15 mM calcium acetate eluates from the sulfopropyl-Sephadex 
column separations were run in two separate batches on the same gel 
column. The pools were concentrated on Amicon UM-2 ultrafilters to about 5 
ml and equilibrated with 10 mM CaCl.sub.2, 10 mM Tris, pH 6.87, before 
application to the column. The Sephadex G-100 gel was degassed under 
reduced pressure and equlibrated with the same buffer before the column 
was poured. The column (100.times.2.5 cm diameter) was calibrated with 
markers of known molecular size before being used for the erythropooietin 
fractions. The void volume was 135 ml; bovine serum albumin monomer eluted 
at 224 ml. ovalbumin at 258 ml, and cytochrome at 368 ml. The sample was 
added to the bottom of the column, as was the buffer which was passed 
through the column at 21 to 22 ml by means of a Mariotte bottle with a 42 
cm hydrostatic head. Each fraction collected was 4.1 ml (120 drops), and 
pools were made. The pools were concentrated by ultrafiltration and 
aliquots were assayed. 
E. Hydroxylapatite Chromatography 
Hydroxylapatite was packed under unit gravity into a column (6.1.times.1.5 
cm diameter) and washed with 500 ml of water and then with 400 ml of 0.5 
mM phosphate buffer, pH 7.1, conductivity=69 umho cm.sup.-1 (Buffer I), by 
use of a peristaltic pump which maintained the flow at 0.3 ml/min. After 
the buffer wash, the length of the column was 3.4 cm and the bed volume 
was 6.0 ml. the input sample was concentrated and desalted on an Amicon 
DM-5 ultrafilter by adding water to the concentrate and the wash of the 
filter were centrifuged at 6,000.times.g for 20 min. at 4.degree.. The 
small insoluble pellet was washed once with 0.5 mM phosphate, pH 7.1, and 
the wash was added to the supernatant. An aliquot for assay was removed 
and the remainder (22 ml) was added to the column. The ratio of absorbance 
units added to bed volume (ml) was 1.82. The input buffer was pumped 
through the column until the effluent A was less than 0.005 (149 ml) and 
the following elution schedule was carried out: Buffer II, 1 mM phosphate 
(pH 7.1, specific conductivity 131=umho cm.sup.-1, 150 ml (Fraction II)); 
Buffer III, 2 mM phosphate (pH 6.9, specific conductivity=270 umho 
cm.sup..sup.1, 220 ml (fractions IIIA and IIIB)); Buffer IV, 3 mM 
phosphate (pH 6.9, specific conductivity=402 umho cm.sup.1, 84 ml 
(Fraction IV)); Buffer V, 0.1M phosphate (pH 6.8, specific 
conductivity=9.6 umho cm.sup.-1, 134 ml (Fraction V)). 
Fractions containing EPO were concentrated by means of Amicon DM-5 
ultrafilter, an aliquot assayed and the concentrate stored frozen. The 
assay indicated a specific EPO activity of 83,000 IU per absorbance unit 
at 280 mm. 
F. Reverse Phase HPLC 
When analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis 
(SDS PAGE) according to Laemmli, U.K., Nature, 15:Vol. 22, No. 259 pp. 
680-685 (1970) this material from the hydroxylapatite column revealed 
several polypeptide components ranging from approximately 70,000 MW to 
30,000 MW with the major component at about 34,000 MW. This partially pure 
preparation of EPO which was obtained in volumes of up to 10 ml in 10 mM 
phosphate buffer pH 7.0 was subjected to RPHPLC as described below. 
The EPO preparation was concentrated 10-fold by partial lyophilization. 
Approximately 200 microliters of this concentrated material was injected 
onto a C-4 Vydac R-P HPLC column (25.times.0.45 cm, the Separations Group) 
and fractionated by reverse phase HPLC using the gradient conditions 
described in Table 1. Protein peaks were detected by UV absorption at 280 
nm. A typical elution profile of this fractionation process is shown in 
FIG. 1. The identity of EPO was confirmed by SDS PAGE and N-terminal amino 
acid sequence analysis of the various peaks observed after R-P HPLC. The 
peak that is underlined in FIG. 1 (19) elutes coincidentally with a 
reading of 53% B on the gradient maker (Beckman Instruments, model 421). 
This material runs as a single band of about 34,000 MW using SDS PAGE and 
yields a single amino terminal sequence of: Ala, Pro, Pro, Arg, Leu, Ile, 
Cys - as has been previously reported for human EPO. Only this R-P HPLC 
fraction of about 34,000 MW showed any significant biological activity in 
vitro. The EPO protein eluted by R-P HPLC is about twice as pure as the 
material eluted from the hydroxylapatite column (STEP E). 
TABLE 1 
______________________________________ 
Pump A 0.1% Trifluoracetic Acid (TFA) in water 
Pump B 95% Acetonitrile in 0.1% TFA in water 
Gradient Time (min.) 
% B Duration 
______________________________________ 
0 0 2 
2 25 3 
5 100 75 
90 0 3 
100 Reinject 
Flow 1 ml/min. 
______________________________________ 
EPO is quantified by either the 3H-thymidine assay (Krystal, Exp. Hematol. 
11:649-60 (1983)) or CFU-E assay (Bersch et al., In vitro Aspects of 
Erythropoiesis, M. J. Murphy (Ed.), New York: Springer-Verloz (1978)). 
EXAMPLE 2: PURIFICATION OF EPO 
COS-cell conditioned media (121) with EPO concentrations up to 200 ug/litre 
was concentrated to 600 ml using 10,000 molecular weight cutoff 
ultrafiltration membranes, such as a Millipore Pellican fitted with 5 sq. 
ft. of membrane. During the purification steps, immunologically active EPO 
was quantified by radioimmunoassay as described by Sherwood and 
Goldwasser, Blood 54:885-93 (1979). The antibody was provided by Dr. 
Judith Sherwood. The iodinated tracer was prepared from the homogeneous 
EPO produced in Example 1. The sensitivity of the assay is approximately 1 
ng/ml. The retentate from the ultrafiltration was diafiltered against 4 
ml. of 10 mM sodium phosphate buffered at pH7.0. The concentrated and 
diafiltered conditioned medi contained 2.5 mg of EPO in 380 mg of total 
protein. The EPO solution was further concentrated to 186 ml and the 
precipitated proteins were removed by centriguation at 110,000 xg for 30 
minutes. 
The supernatant which contained EPO (2.0 mg) was adjusted to pH5.5 with 50% 
acetic acid, allowed to stir at 4.degree. C. for 30 minutes and the 
precipitate removed by centrifugation (at 13,000xg for 30 min.). 
Carbonylmethyl Sepharose Chromatography 
The supernatant from the centrifugation (20ml) containing 200 ug of EPO (24 
mg total protein) was applied to a column packed with CM-Sepharose (20 ml) 
equilibratred in 10 mM sodium acetate pH5.5, washed with 40 ml of the same 
buffer. EPO which bound to the CM-Sepharose was eluted with a 100 ml 
gradient of NaU(0-1) in 10mM sodium phosphate pH5.5. The fractions 
containing EPO (total of 50 .mu.g in 2 mg of total proteins) were pooled 
and concentrated to 2 ml using Amicon YM10 ultrafiltration membrane. 
Reverse phase-HPLC 
The concentrated fractions from CM-Sepharose containing the EPO was further 
purified by reverse phase-HPLC using Vydac C-4 column. The EPO was applied 
onto the column equilibrated in 10% solvent B (Solvent A was 0.1% CF.sub.3 
CO.sub.2 H in water; solvent B was 0.1% CF.sub.3 CO.sub.2 H in CF.sub.3 
CN) at flow rate of 1 ml/min. The column was washed with 10%B for 10 
minutes and the EPO was eluted with a linear gradient of B (10-70% in 60 
minutes). The fractions containing EPO were pooled (.about.40 ug of EPO in 
120 ug of total proteins) and lyophilized. The lyophilized EPO was 
reconstituted in 0.1M Tris-HCl at pH7.5 containing 0.15M NaCl and 
rechromatographed on the reverse phase HPLC. The fractions containing the 
EPO were pooled and analyzed by SDS-polyacrylamide (10%) gel 
electrophoresis (Laemmli, U.K., supra). The pooled fractions of EPO 
contained 15.5 ug of EPO in 25 ug of total protein. 
This invention has been described in detail including the preferred 
embodiments thereof. However, it will be appreciated that those skilled in 
the art, upon consideration of this disclosure, may make modifications and 
improvements within the scope of this invention.