CA2175202C - A method for stabilizing protein c or activated protein c and the stabilized composition obtained by said method - Google Patents

Abstract

The present invention is directed to a method for stabilizing protein C or activated protein C and a preparation obtained by said method. Said method and preparation being applicable during procedures such as isolation and purification, lyophilization, heating, etc. or when stored. The method comprises adding to a salt buffer containing protein C or activated protein C and sodium ion at least one amino acid, and further adding either one or a combination of albumin and a non-ionic surfactant.

Description

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 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

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

- 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

- 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

_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.

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 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) 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~

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

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 20 99.2 A

Claims (5)

Claims:

1. A method for stabilizing protein C or activated protein C
which comprises adding, to a salt buffer containing protein C
or activated protein C for a final concentration of 100 to 2500 µ/ml and sodium ion for a final concentration of 50 to 200 mM, at least one amino acid for a final concentration of 5 to 100 mM, and further adding either one or a combination of albumin for a final concentration of 0.5 to 10% (W/V) and a non-ionic surfactant for a final concentration of 0.0005 to 0.1% (W/V).

2. The method for stabilizing protein C or activated protein C of claim 1 wherein said amino acid is selected from naturally occurring amino acids.

3. The method for stabilizing protein C or activated protein C of claim 2 wherein said amino acid constituting a protein is selected from glycine, alanine, lysine, arginine, aspartic acid and glutamic acid.

4. An aqueous buffer solution containing protein C or activated protein C or both which contains 100 to 2500 µ/ml of protein C or activated protein C or both, 50 to 200 mM of sodium ion, 5 to 100 mM of an amino acid, and further either one or a combination of 0.5 to 10% (W/V) of albumin and 0.0005 to 0.1% (W/V) of a non-ionic surfactant.

5. A composition containing protein C or activated protein C
or both which contains 1 × 10 5 to 2.5 × 10 6 U of protein C or activated protein C or both, 50 to 200 mg of sodium ion, 5 to 100 millimoles of an amino acid, and further either one or a combination of 5 to 100 g of albumin and 0.005 to 1 g of a non-ionic surfactant.