AQUEOUS PHARMACEUTICAL FORMULATIONS OF ERYTHROPOIETIN AND THE USE THEREOF

FORMULES PHARMACEUTIQUES AQUEUSES AYANT UNE ACTION ERYTHROPOIETIQUE, ET SON USAGE

English Abstract

Aqueous pharmaceutical formulations of proteins with
erythropoietin activity, especially of human native and
of recombinant human erythropoietin (EPO) are described.
Highly purified EPO without foreign proteins or other
customary stabilizers retains at least about 78% of its
original activity for at least 1 year at room temperature
in a physiologically tolerated aqueous phosphate buffer
which contains a physiologically tolerated alkali metal
halide but otherwise no stabilizing additives and has a
pH of 6 to 8. The products in this aqueous pharmaceutical
formulation are particularly suitable for subcutaneous or
intramuscular administration.

Claims

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THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An aqueous pharmaceutical formulation of purified
erythropoietin in a physiologically tolerated
aqueous buffer solution of pH 6 to 8 which contains
a physiologically tolerated alkali metal phosphate
and alkali metal halide, but otherwise no stabili-
zing additives.
2. A formulation as claimed in claim 1, wherein the
aqueous buffer solution is composed of 50 mM sodium
phosphate, 100 mM NaCl, pH 7.8.
3. A formulation as claimed in claim 1 or 2, wherein
the erythropoietin is human native or recombinant
erythropoietin.
4. A formulation as claimed in any of claims 1 to 3,
wherein the concentration of erythropoietin is
50 - 1000 µg per ml of buffer solution.
5. The use of an aqueous formulation of purified
erythropoietin as claimed in any of claims 1 to 4
for preparing injection products.
6. A process for preparing a stable pharmaceutical
formulation of erythropoietin as claimed in claim
1, which comprises dissolving erythropoietin with a
purity of ~ 99% in a physiologically tolerated
aqueous buffer solution of pH 6 to 8, said buffer
solution containing a physiologically tolerated
alkali metal phosphate and alkali metal halide, but
otherwise no stabilizing additives.

-7-
7. An aqueous pharmaceutical formulation as claimed in claim 1
and substantially as described herein.

Description

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BEHRINGWERRE ARTIENGESELLSCHAFT HOE 90/B 021 - Ma 787
Dr. LP/VO
Description
Aqueous pharmaceutical formulations of erythropoietin and
the use thereof
The invention relates to aqueous pharmaceutical formula-
tions of purified erythropoietin, in particular of hwnan
native and of recombinant human erythropoietin (rh EPO).
The formulations of the invention - without foreign
proteins, sugars, amino acids or other customary stabili-
zers - retain at least about 78~ of their original
activity for at least one year at a temperature of 4-8°C.
Erythropoietin (EPO) is a glycoprotein with 166 amino
acids, 3 glycosylation sites at amino-acid positions 24,
38 and 83 and a molecular weight of about 34,000. EPO can
either be isolated from natural sources such as human
urine (cf., for example, Miyake et al., J. Biol. Chem.,
vol. 252 (1977), 5558-5564) or be prepared by genetic
engineering processes (cf., for example, EP-A 0,148,605
and 0,267,678). Aqueous solutions of erythropoietin are
unstable at temperatures from about 3°C to room tempera-
ture.
Patients with kidney failure are unable to form EPO and
thus suffer from anemia. Attempts to compensate for this
undersupply of EPO by administering EPO and to reduce the
symptoms of anemia have already been successful. Further
clinical uses comprise administration of hEPO for iatro-
genic anemia after chemotherapy or radiotherapy of
malignant diseases.
A single dose of EPO amounts to only a few microgrammes.
Because of the short half-life after i.v. administration,
physiological plasma levels can best be achieved by
subcutaneous injection.
The pharmaceutical formulations of erythropoietin

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hitherto proposed or available contain added detergents
and/or proteins, sugars or polyalcohols, which are
intended, on the one hand, to stabilize EPO and, on the
other hand, to prevent adsorption of EPO onto the inner
wall of the storage container (i.e. the ampoule)
(EP-B-0,178,576; EP-A-0,178,665; G. Krystal et al., Blood
vol. 67 (1986), l, 71 - 79). Subcutaneous or i.m.
administration of EPO stabilized in this way results in
local inflammation with the formation of granulomas.
Highly pure EPO prepared by known processes
(EP-A-0,236,059; US-A 4,667,159; PCT/US 86/01342
{= WO 86/07594)) lost more than 25% of its activity
within one week on storage at 24°C. EP-B-0,178,576 also
discloses a solution of human erythropoietin, which has
been reductively methylated with l4C-formaldehyde, in PBS;
cf. Experiment 1 and 2.
The object on which the invention is based is to provide
aqueous pharmaceutical formulations of purified erythro-
poietin which are free of the customary stabilizing
additives and nevertheless have adequate stability at
temperatures from about 3°C to room temperature and are
suitable in particular for subcutaneous or intramuscular
administration.
It has been found, surprisingly, that purified erythro-
poietin (about 99% pure), especially purified native or
recombinant human erythropoietin, is stable for a lengthy
period at temperatures from 4 to 8°C in a physiologically
tolerated aqueous phosphate buffer of pH 6 to 8 which
contains a physiologically tolerated alkali metal halide,
but otherwise no stabilizing additives, in sealed test
tubes or glass ampoules.
Hence the invention relates to aqueous pharmaceutical
formulations of purified erythropoietin in a physiologi-
cally tolerated aqueous phosphate buffer of pH 6 to 8
which contains a physiologically tolerated alkali metal
halide, but otherwise no stabilizing additives. A buffer

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composed of 50 mM sodium phosphate, 100 mM NaCl, pH 7.8,
is particularly preferred. The concentration of erythro-
poietin in the aqueous buffer solution is 50 - 1000 ~g
per ml of buffer solution.
Examples of physiologically tolerated aqueous phosphate
buffers are sodium phosphate buffer and potassium phos-
phate buffer, preferably sodium phosphate buffer.
Examples of physiologically tolerated alkali metal
halides are sodium chloride and potassium chloride,
preferably sodium chloride. A preferred phosphate buffer
is a buffer containing 50 mM sodium phosphate, 100 mM
NaCl, pH 7.8. This buffer is also called PBS for brevity.
The aqueous solutions of erythropoietin in PBS are very
well suited for subcutaneous or intramuscular administra
tion.
The invention additionally relates to the use of the
aqueous pharmaceutical formulations for preparing in~ec-
tion products for subcutaneous or intramuscular adminis-
tration. Subcutaneous administration is particularly
preferred because, by contrast with the known formula-
tions stabilized With proteins, it causes no irritation
or inflammatory pain. This has emerged from clinical
trials.
The examples illustrate the invention.
xample 1
Purification of rh EPO
The purification of rh EPO started from serum-containing
or serum-free medium which was conditioned by rh EPO-
producing animal cells by the process described in
EP-A 0,267,678, page 6, line 45, to page 9, line 5. The
process comprises
(1) clarification, concentration and dialysis of the
culture medium,

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(2) ion exchange chromatography,
{3) preparative reverse phase HPLC and
{4) gel filtration chromatography.
For the gel filtration chromatography, the column was
equilibrated with PBS, i.e. 50 mM sodium phosphate
buffer, 100 mM NaCl, pH 7.8. With this buffer solution,
rh EPO was eluted in a single symmetrical peak {measure-
ment of the eluate at 280 nm). The resulting rh EPO was
at least 99% pure according to SDS-PAGE.
(5) Dilution
The combined rh EPO fractions from the gel filtration
step ( 4 ) contained 0 .1 - 0 . 8 mg of EPO per ml and were
dispensed in single doses of 100 ~g/ml using the sodium
phosphate/sodium chloride buffer (PBS) pH 7.8.
Example 2
Stability testing
The stability of the PBS solution of rh EPO obtained as
in Example 1 was tested after storage of the single doses
in sterile glass tubes, comparing with various stabili-
zers. The activity of rh EPO was measured by the incor-
poration of 3T into mouse spleen cells treated with
phenylhydrazine in a conventional manner. The data shown
in the Table which follows are related to the initial
activity of the employed rh EPO = 100%.

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(a) Storage at 4 - 8°C
Stabilizer Amount of stabilizer % activity
(wt./wt. relative to after 12
EPO) based on 100 ~g months
EPO/ml PBS
PBS alone - 7g,5
PBS + sorbitol 5000 51
PBS + glycerol 6150 0
PBS + HaemaccelR 100 68
(b) Storage at
37C (accelerated
test)
Stabilizer Amount of % Activ ity after months
stabilizer 1 2 3 6
PBS alone - 86.3 39 11.2 9.9
PBS + sorbitol 5000 33.5 10.75.1 0
PBS + HaemaccelR 100 42 14.86.0 5.1
Note: HaemaccelR is a degraded gelatin
The surprisingly better stabilization of rh EPO in PBS
(pH 7.8) without further addition of a stabilizer
is
evident Tables.
from the