Note: Descriptions are shown in the official language in which they were submitted.
21 75202
- 1 -
A METHOD FO:R STABILIZING PROTEIN C OR
ACTIVATED PROTEIN C AND THE STABILIZED COMPOSITION
OBTAINED BY SAID METHOD
The present inven~ion relates t:o a method for
stabilizing protein C' «r activated protein C which is
derived from plasma or is prepared using genetic
recombination techniques. More part.i.cularly, the present
invention relates to a method for stabilizing protein c. or
activated protein C why=_n it is stored or subjected to
procedures such as iso:Lation and purification,
lyophilization, treatmE~:nt by heating, etc. and to a
preparation stabilized :by said method.
l~ Protein C (herein<~fter also referred to as "PC") is a
kind of a vitamin K dependent protein, i.e. a protein
containing 'y-carboxyglutamic acid, and is activated to
activated protein C (hs~reinafter also referred to as "APC")
by thrombin in the pre:~ence of throrribomodulin present on the
surface of the vascular endothelial cell. Activated protein
C is a kind of a serins=_ protease and exhibits a strong anti-
coagulant activity by :inactivating ca-factors of the blood
coagulation system such as Factor Va (FVa) and Factor VIIIa
(FVIIIa) . It is also l~nown that act.i.vated protein C
2~ releases a plasminogen activator from the vascular wall to
accelerate the fibrino:Lytic system. Furthermore, it is
known that a defect in protein C causes severe thrombosis.
Thus, it has been established that activated protein C is
the most important factor which regulates the blood
2.~ coagulation and fibrinolytic system. Therefore, protein C
or activated protein C is expected t.o be exploited as a
novel anti-coagulating agent or profibrinolytic agent.
It has hitherto :been known that the amount of protein C
present in plasma or e:~pressed in a tissue culture system is
30 extremely low. Accordingly, in order to use protein C or
activated protein C as an anti-coagulant agent or a
'.
2'~ 75202
- 2 -
profibrinolytic agent widely and safely, isolation and
purification of protein C or activated protein C is
important. In addition, storage as a solution or in frozen
form for long periods of time, lyophilization or procedures
for inactivation of contaminating viruses such as heating
are indispensable to the process when protein C or activated
protein C is industria:Lly prepared on a large scale.
However, storage, freezing or freeze-drying, or heat
treatment of: a highly pure protein C' or activated protein C
1~ severely lowers the act=ivity thereof. There has also been
no report on the stabi:Lity of highly purified protein C or
activated protein C. iJnder such circumstances, it is
impossible to provide a highly pure protein C or activated
protein C efficiently and stably on an industrial scale.
1~ Under such circum:stances, the present inventors have
earnestly studied the :stability of protein C or activat=ed
protein C, and as a re:~ult, have found that the activity of
protein C or activated protein C can be maintained even
after storage for a significant period of time or after
20 procedures such as iso_Lation and pur.i.fication,
lyophilization, heating, etc. by adding, to protein C or
activated protein C, a salt buffer such as phosphate or
citrate buffer containing sodium ion supplemented with at
least one amino acid, <~nd further adding either one or a
25 combination of albumin and a non-ionic surfactant. The
present invention is based on these findings.
The present inveni~ion relates to a method for
stabilizing protein C or activated protein C which comprises
adding, to a salt buffer containing protein C or activated
30 protein C and sodium ion, at least one amino acid, and
further either one or a combination of albumin and a
non-ionic surfactant. More particularly, the present
invention relates to a method for stabilizing protein (. or
activated protein C wh=ich comprises dissolving protein C or
3!~ activated protein C in a salt buffer' such as phosphate or
citrate buffer containing sodium ion, and adding to said
buffer at least one amino acid which constitutes a protein,
A
-3- 2175202
e.g. glycine, alanine, lysine, arginine, aspartic acid,
glutamic acid, etc. anc3 a polypeptide having a
protein-stabilizing act=ivity such. as albumin, globulin, etc.
and, in a preferred embodiment, an optional non-ionic
surfactant, typically 'rween* 80 is added. The invention also
relates to preparation: stabilized by said method.
Protein C or activated protein C used herein
encompasses variants o:r derivatives thereof which
substantially has APC a~~tivity anal may be prepared by known
methods, for example, by preparing protein C by isolation
from human plasma or p=roduction utilizing the genetir_
recombination technique and then activating protein C; by
directly isolating APC from human blood; or by producing APC
by the use of genetic .recombination techniques, etc.
1!~ Activation of protein c. into APC can be carried out by any
known method, for example, by activation with thrombin
isolated from human or bovine blood, or by activation with
an equivalent protease, etc.
Production of APC derived from blood can be conducted,
for example, by actival:ing protein C', which is purified from
human plasma by affinii;y chramatography using an
anti-protein C antibody, with human thrombin, and purifying
the resulting activated protein C by ration chromatography
(Blood, 63, p. 115-121 (1984)); or by activating protein C
which is purified from human plasma by barium citrate
adsorption and elution, fractionation with ammonium sulfate,
DEAF-Sephadex* column chromatography, dextran sulfate
agarose chromatography and polyacrylamide gel
electrophoresis, etc. i=o produce APC' in accordance with the
method described by Ki:~iel (J. Clin. Invest., 64, p. 761-769
(1979)); or by activating a commercially available
blood-coagulating preparation containing protein C to
produce APC in accordance with th.e method described by
Taylor et al.. (J. Clin. Invest., 79, p. 918-925 (1987)), and
the like.
* Trade mark
21 X5202
- 4 -
Production of APC utilizing the genetic recombination
technique can be conducted, for example, in accordance with
the methods described :Ln Japanese Patent First Publication
(Kokai) No. 61-205487, .Japanese Patent First Publication
3 (Kokai) No. 1-2338 or Japanese Patent First PublicatiOT1
(Kokai) No. 1-85084, etc. A process for preparing the
starting material protein C or activated protein C as used
herein is not limited to the above-mentioned procedures.
The thus prepared starting material protein C or
activated protein C is isolated and purified by a
combination of usual biochemical procedures for isolation
and purification, including, for example, a salting-out with
ammonium sulfate, an ion-exchange chromatography with an ion
exchange resin, gel fi:Ltration, electrophoresis, etc.
When the degree of purification is increased by such
procedures, protein C or activated protein C is liable to
become unstable. Even a product having not as high a purity
also shows a decrease :Ln activity due to procedures such as
storage, freezing, lyophilization, heating, etc. The
present invention is p:.imarily aimed at the stabilization of
such protein C or activated protein C which became unstable
as the degree of purification increases. Protein C or
activated protein C to be stabilized in accordance with the
present invention may be either in the form of a solution or
a powder.
In the stabilizat_LOn method for protein C or activated
protein C of the present invention, a salt containing as a
stabilizing agent sodium ion at a concentration of
preferably 50 mM to 200 mM, at least one amino acid and a
polypeptide having a si=abilizing effect are added to a
buffer containing 100 to 2500 U/ml of protein C or activated
protein C. The salt and amino acid may each be used alone
or in a combination of two or more thereof. Preferred
buffers include, for e:~ample, sodium citrate, sodium
phosphate, and sodium :sulfate, etc. The amino acid is added
at a final concentrati<Jn of 0.005 M to 0.1 M, more
preferably G.Ol M to 0.05 M. A polypeptide having a
...
2~ X5202
- 5 -
stabilizing effect such as albumin or globulin is added at
an appropriate concentration which may be determined based
on common sense or from an economic point of view,
preferably at a concentration of 0.50 (W/V) to 10% (W/V).
The unit "% (W/V)" as used herein denotes an amount of a
solute dissolved in onf=_ liter of a solution, for example,
when 10 g of a solute :is dissolved in one liter of a
solution concentration is 1% (W/V). In a preferred
embodiment of the presf=_nt invention, a non-ionic surfactant
such as Tween 80 may o~~tionally be added at a concentration
of 0.0005% (W/V) to O.:Ls (W/V) to accelerate the
stabilization effect.
A typical embodimf=_nt of the present invention is an
aqueous buffer solution containing protein C or activat=ed
1.~ protein C, which comprises 100 to 2500 U/ml of protein C
and/or activated protean C, 50 to 2G0 mM of sodium ion, 5 to
100 mM of an amino acid, and further either one or a
combination of 0.5 to _LO% (W/V) of albumin and 0.0005 to
0.1% (W/V) c>f a non-ionic surfactant..
When a stabilizing agent is added to protein C or
activated protein C in the form of powder, it is used in
such an amount that thc= concentration of the stabilizing
agent reaches the abovf~-mentioned. range when said powder is
dissolved.
Thus, in another typical embodiment of the present
invention, a composition containing protein C or activated
protein C comprises 1 :~ 105 to 2.5 x 106 U of protein C
and/or activated protean C, 50 to 200 mg of sodium ion, 5 to
100 millimoles of an arn:ino acid, and further either one or a
combination of 5 to 10c) g of albumin and 0.005 to 1 g of a
non-ionic surfactant.
The manner of addition of these ingredients may not be
specified but includes various methods, for example, by
directly adding powdery materials of the present invention
to a buffer solution containing protein C or activated
protein C; or by previously dissolving said powdery
A
21 75202
- 6 -
materials in water or a suitable buffer and adding the
solution to the buffer solution containing protein C or
activated protein C; o:r by mixing said powdery materials
with a protein C- or activated protein C-containing powder.
3 Addition may be carried out either during the process of
isolation arid purificai~ion of said protein or the process
for producing a pharmaceutical preparation.
When a solution containing protein C or activated
protein C added with the stabilizing agent of the present
invention is stored, o:r subjected. to procedures such as
isolation and purificat=ion, or process for producing a
pharmaceutical preparation in the state of solution, it. is
preferably done at 0 to 30°C, more preferably at 0 to .LO°C.
When said solution is :stored in a freezed state, it is
1~ preferably done at a lower temperature than the freezing
point, more preferably at lower thar~ -20°C, or when it is
stored in a lyophilized state, it is preferably done at. room
temperature or lower. By using the solution containing
protein C or activated protein C incorporated with the
2~~ stabilizing agent of the present invention, the activity of
protein C or activated protein C cars be stably maintained
even during storage in the state of a solution, or in a
freezed or lyophilized state, or even in the treatment
thereof such as isolation and purification or process for
25 producing a pharmaceutical preparation.
The activity of AI?C was measured in accordance with the
following procedures.
One unit of APC activity is defined as the amount of
APC which prolongs by t:wo times the activated thromboplastin
30 time (APTT; second) of normal human plasma. Accordingly,
the activity of APC is measured wherein APTT in second: is
measured for normal human plasma to which a diluted sample
is added and the dilution at which the measured APTT value
is twice that of the control (buffer) is determined and
3'.~ regarded as the activit=y of APC for samples.
A
2~'~5202
_ 7 _
(Procedures)
A sample is diluted with a veronal buffer containing 1%
human serum albumin to, for example, 400 times, 500 times,
800 times or 1000 time; dilution. To each 100 ~1 of either
control (buffer) or samples of each dilution are added
100 ~.l of normal human plasma (e. g. Citrol* I: Baxter
Diagnostics Inc.) 100 'al and APTT reagent (e.g. Actin*:
Baxter Diagnostics Inc.) 100 ~1. a.t 37°C successively with an
interval of 15 seconds, the mixture is stirred, and after 2
minutes, 0.025 M CaClz 700 ~l is added and the coagulation
time is measured.
(Calculation of activity)
A linear regression formula and a correlation
coefficient of 103/X and Y are obtained from the APTT values
1~ (Y) at each dilution (:~) of the control and the samples as
follows:
Y = A (103/X) + B
A value of Xl obtG,ined from the following formula:
X1 = 103 ~ (Y1-B) /A~'
wherein Y1 is a value twice that of the APTT (second) of
control, is regarded a;~ the activity of APC (U/ml) for
samples.
The activity of p=rotein C wa.s measured using "Staclot*
Protein C" manufactured by Boehringer Mannheim.
The present invent=ion is i.llust.rated in more detail by
means of the following Examples but should not be construed
to be limited thereto.
Example 1
Effect of various counterions on stability of APC:
To a solution containing human activated protein C with
an activity of 500 U/m:L was added 2.5o human serum albumin
(hereinafter also abbrc=viated as "HSA";f. The solution was
then dialyzed against ~~ solution of Na citrate, Na phosphate
and Na sulfate (each 20 mM), containi.ng 0.7% NaCl and
* Trade mark
_A
21 75202
_8_
0.067 M glycine. After dialysis, each solution was left to
stand at 37°C for 24 hours and the activity was measured.
The results are shown :in Table 1. All the counterions
tested, Na citrate, Na phosphate and Na sulfate, showed a
similar satisfactory stability.
Table 1
Effect of various counterions on stability of APC
Rate of remaining activity (%)
Counterion (37°C, 24 hours)
Na citrate 20 mM 97.4
Na phosphate 20 mM 94.8
Na sulfate 20 mM 92.2
Examgle 2
Effect of amino acids can stability of APC:
To a solution coni_aining activated protein C with an
activity of 500 U/ml w<~s added 2.5% HSA. The solution was
then dialyzed against <~ solution of sodium citrate buffer
containing 0.7% NaCl and each 0.05 M of either glycine,
alanine, lysine, argin:ine, aspartic acid or glutamic acid.
After dialysis, each solution was left to stand at 37°C for
24 hours and the activity was measured. The results are
shown in Table 2. All of the above six amino acids
exhibited a high stabi_Lity without deteriorating the
stability of: APC.
Table 2
Effect of various amino acids on stability of APC
Amino acid Rate of remaining activity (o)
(0.50) (37°C, 24 hours)
No amino acid 86.1
Gly 97.7
Ala 97.7
Lys 107
Arg 102
Asp 108
Glu 10E~
A.
21 75202
- 9 -
Example 3
Effect of addition of 33SA:
Solutions containing human activated protein C (1'700
U/ml), 20 mM citrate, 0.7e NaCl and 0.067 M glycine with and
without addition of 2.5s HSA were prepared. The solutions
were then left to stand at 37°C and 4°C. The activity was
measured with the passage of time in ar_cordance with the
method described herein and a retention percent of activity
was obtained. The results are shown in Table 3.
Table 3
Retention percent of activity under respective
conditions (o)
At 37°C:
Retention time (hour
0 L 3 6 8 24
HSA (+) 100 - 99.2 - 101 97.4
HSA (-) 100 88.8 92.0 86.2 90.9 79.3
At 4°C:
Retention time (day)
0 1 3 5 7 14
HSA (+) 100 - 105 - - 98.1
HSA (-) 100 1:12 99.1 104 98.9 85.2
As will be understood from the results shown in
Table 3, the system without HSA cannot prevent a decrease in
the activity of APC whf°_n it is left standing. From these
results, it is concluded that 2.5o HSA is very effecti~ae in
stabilizing APC.
Example 4
Effect of HSA on stability of APC:
The data shown i.n Table 4 indicates that the activity
of APC is dependent on the concentration of HSA. To a
solution of activated ~arotein C 1500 U/ml) was added 0.5 to
A
2~ 75202
- 10 _
10.0% HSA. This solution was put in a storage vessel and
left to stand at 37°C. After 24 hours, a sample was taken
and the activity was measured. In the case where no HSA was
added, the activity wa;~ decreased. by about 20o whereas in
the case where HSA was added at a concentration ranging from
0.5 to 10.0%, almost no decrease in the activity was
observed and APC remained stable.
Table 4
Effect of HSA on ;stability of APC activity
Rate of remaining activity (%)
HSA (%) (37°C, 24 hours)
0.0 79.0
0.5 96.0
2.5 97.4
1~ 5.0 98.0
10.0 102
Example 5
Effect of Tween 80 on ;stability of APC:
To a solution containing human activated protein C (500
U/ml), 20 mM citrate, 0.7% NaCl and 0.067 M glycine was
added 0.0005% to 0.1% non-ionic surfactant, Tween 80 (t=rade
mark). This solution was put in a storage vessel and left
to stand at 37°C. AftE=r 24 hours, a sample was taken and
the activity was measured. The results are shown in
Table 5. In the case where Tween 80 was added, the activity
decreased by about 20% and the stabi.l.ity deteriorated
whereas in the case whE~re no Tween 80 was added at a
concentration ranging :From 0.0005 tc> O.lo, no change in the
activity was found and the solution maintained high
stability.
Table 5
Rate of remaining activity (%)
Tween 80 (%) (37°C, 24 hours)
No addition 79
0.0005 10I.
0.025 104
0.1 103
A
2~ 752p~
- 11 --
Example 6
Effect of NaCl on quality of lyophilized solid citrate:
An APC lyophilized preparation containing
APC 500 U/ml, HSA 2.50, Gly 0.067 M and Na citrate 20 mM was
prepared such that it contained sodium chloride at a
concentration ranging :E:rom 1 mM to 500 mM. Each lyophilized
preparation was stored at 60°C for a month and the
appearance quality of t=he solids was observed as shown in
Table 6.
Table ~6
Quality of lyophi:Lized preparation containing NaC1 at a
selected concentration
Na chloride c2uality solids
of
(mM ) Pre-storage After One month storage
1~ at 60C
1 White solid, shrunken Extre mely shrunken
10 White solid, a bit shrunken as pre-storage
Same
50 White porous lump Same as pre-storage
100 White porous lump Same as pre-storage
500 Glassy shrunk :Lump Same as pre-storage
As is clear from t=he results shown in Table 6, a solid
APC preparation is dif:Eicult to formulate into a
pharmaceutical preparation in the case of addition of sodium
chloride both at a hi.glzer and lower concentration. The
presence of sodium chloride at a concentration of 50 mM to
200 mM is considered tc~ contribute t.c> the stabilization of
the lyophilized preparation.
Example 7
APC activity in lyophi:Lized preparation:
A citrate buffer ;solution containing the stabiliz_Lng
agent of the present invention (0.70 NaCl, 0.067 M glycine
and 2.5a HSA) was prepared so that i.t contained the APC
activity at 100 to 2500 U/vial and, maintaining sterile
conditions, was divided into vials, which were lyophilized
A
21 75202
- 12 --
and sealed. Each vial was left t:o stand at 10°C, 15°C and
60°C and decrease in the activity was determined. The
results are shown in Table 7. The data show that the method
for stabilization of APC of the present invention is
effective in the state of lyophil.ization.
Table 7
Rate of remaining activity (%)
APC Time 10C 15C 60C
(U/vial)
100 30 months 100 101 -
500 30 months 9T 99 -
1.1 days 99
1000 30 mont~~s 99 97 -
2500 30 mont:~s 98 95 -
11 days 105
Example 8
Effect of repeated fret=_zing-melting on the stability of APC:
To a citrate buffer solution containing activated
protein C with an activity of 500 U/ml was added to the
stabilizing agent of the present invention (0.7o NaCI,
0.067 M glycine and 2.5o HSA). The resulting solution was
subjected to repetition (5 times, 10 times, 15 times and 20
times) of freezing (-80°C) and melting and the activity was
measured. The results are shown in Table 8. In spite of 20
repetitions of freezing-melting, the activity of APC showed
no significant change and remained stable.
Table .g
Repetition Rate of maintainincl activity (o)
3 ~3 5 1.02
10 95.1
15 106
20 99.2
A
