Human activated protein C and process for preparing same

A human Activated Protein C preparation with a high specific activity of 3500 U/mg or more and substantially free from thrombin or other proteases which can convert Protein C into Activated Protein C is provided. A process for preparing this human Activated Protein C, which involves, contacting a solution of human Activated Protein C, after activation of Protein C with thrombin or other activating protease, with a cation exchanger to allow for adsorption of both thrombin or another activating protease and Activated Protein C to the cation exchanger followed by elution of the human Activated Protein C alone.

CROSS-REFERENCE TO RELATED APPLICATIONS 
This is a 371 national stage application of PCT/JP94/01807, filed Oct. 27, 
1994. 
TECHNICAL FIELD OF THE INVENTION 
The present invention relates to a human Activated Protein C preparation 
having a high specific activity which is prepared by activation of human 
Protein C with thrombin or an equivalent protease, said Protein C being 
derived from plasma or prepared by using the genetic recombination 
technique. The present invention further relates to a method for 
activating human Protein C and a process for purifying human Activated 
Protein C a high purity. 
TECHNICAL BACKGROUND 
Protein C (hereinafter also abbreviated as "PC") is a kind of a vitamin K 
dependent protein synthesized in the liver and is an enzyme precursor 
having a molecular weight 62,000 consisting of two chains, i.e. L chain 
(molecular weight 21,000 ) and H chain (molecular weight 41,000 ). Protein 
C is partially degraded in vivo by a thrombin-thrombomodulin complex, 
thrombin bound to thrombomodulin occurring on the membrane surface of the 
vascular endothelial cells, and thereby a peptide comprising 12 amino 
acids is released from the amino terminal of H chain to form Activated 
Protein C (hereinafter also abbreviated as "APC"). APC is a kind of a 
serine protease which exhibits a strong anti-coagulant activity by 
specific degradation and inactivation of the blood coagulation Factor V 
and Factor VIII (primarily activated form Va, VIIIa) and promotes the 
release of a plasminogen activator from the vascular wall to accelerate 
the fibrinolytic system. Accordingly, APC is expected to be used as a 
therapeutic agent. 
APC itself is well known in the art and includes those obtained by in vitro 
activation of protein C with thrombin or thrombin-thrombomodulin complex, 
said protein C being derived from plasma or prepared by using the genetic 
recombination technique (Blood, 63, p.115-121 (1984 ); J. Clin. Invest., 
64, p.761-769 (1979); J. Clin. Invest., 79, p.918-925 (1987)); or those 
directly expressed as APC by the genetic recombination technique (Japanese 
Patent First Publication (Kokai) No. 61-205487, Japanese Patent First 
Publication (Kokai) No. 1-2338 and Japanese Patent First Publication 
(Kokai) No. 1-85084), and the like. 
However, for preparation of APC, especially in the case where protein C is 
fractionated from plasma and then activated to produce the desired 
protein, there are various problems need to be overcome in order to 
efficiently remove contaminating proteins having physico-chemical 
properties quite similar to APC and to highly purify APC so that APC 
having a desired high specific activity is obtained. For example, there 
still remain a number of problems to be solved with regard to efficient 
activation of Protein C into APC, subsequent removal of an activating 
agent, and purification of APC. 
Known methods for activation of Protein C include, for example, activation 
with trypsin, RVV-X, thrombin, thrombin-thrombomodulin, activation using 
gel wherein RVV-X is immobilized to cepharose, activation using gel 
wherein thrombin-thrombomodulin complex is immobilized and the like (J. 
Biol. Chem., 251, 3052-3056 (1976); Biochemistry, 15, 4893-4900 (1976); 
Biochemistry, 16, 5824-5831 (1977); J. Clin. Invest., 64, 761-769 (1977); 
Biochem. Biophys. Res. Commun., 94, 340-347 (1980); J. Clin. Invest., 77, 
416-425 (1986)). 
However, the methods mentioned hereinabove are not satisfactory in view of 
production of APC on industrial scale. For activation of a large amount of 
Protein C, it is preferable to activate Protein C at a high concentration 
with a small amount of an activating agent. The above methods also do not 
satisfy this requirement. 
As to purification of Activated Protein C after activation of Protein C, a 
method is known wherein APC is developed in an eluent fraction by 
SP-SEPHADEX chromatography and thereby thrombin added during activation of 
Protein C is adsorbed and removed (Biochemistry, 16, 5824-5831 (1977); J. 
Clin. Invest., 64, 761-769 (1979); J. Biol. Chem., 251, 3052-3056 (1976); 
Biochemistry, 20, 2156-2161 (1981)). However, it is hard to remove 
thrombin added during activation of Protein C with a cation exchanger to a 
clinically applicable level. Actually, the use of procedures for 
concentration of APC is indispensable after the procedure of the cation 
exchange treatment, and hence, autolysis of APC is unavoidable during 
concentration of APC. Accordingly, at present, an APC preparation having a 
high purity and high biological activity without contamination by various 
proteins has not yet been obtained. 
DISCLOSURE OF THE INVENTION 
The present invention has been made in order to solve the above-mentioned 
problems. The present inventors have earnestly studied, and as a result, 
have found that Protein C can be activated efficiently with a small amount 
of thrombin by using Protein C at a high concentration; that a human 
Activated Protein C preparation containing substantially no thrombin or 
equivalent protease and/or Protein C which has not been activated is 
obtained by contacting a solution containing Activated, Protein C after 
activation with a cation exchanger so that thrombin or an equivalent 
protease and Activated Protein C are adsorbed to the exchanger, and 
subsequently eluting Activated Protein C alone under a suitable salt 
concentration; surprisingly, the result human Activated Protein C 
preparation exhibits a much higher specific activity than that of human 
Activated Protein C obtained by the conventional method. An object of the 
present invention is to provide a human Activated Protein C preparation 
with a high specific activity substantially free from thrombin or an 
equivalent protease and/or Protein C which has not been activated and a 
process for preparing the same.

BEST MODE FOR CARRYING OUT THE INVENTION 
The human Activated Protein C preparation of the present invention shows 
specific activity of 3500 U/mg or more, the unit of specific activity 
being defined as an amount which prolongs twice an activated 
thromboplastin time (APTT) of normal human plasma, and is substantially 
free from thrombin or an equivalent protease and/or Protein C which has 
not been activated. In accordance with the process for preparing the 
preparation of the present invention, after activation of human Protein C 
with thrombin or an equivalent protease, a solution containing human 
Activated Protein C is contacted with a cation exchanger under condition 
of pH 5.0 to 6.0, an NaCl concentration of 80 mM or less, to allow for 
adsorption of thrombin and Activated Protein C to the exchanger, and then 
human Activated Protein C alone is eluted with a salt concentration of 0.1 
to 0.35M to solely recover a highly purified human Activated Protein C. 
For activation of Protein C in the above solution containing Protein C with 
thrombin or an equivalent protease, thrombin is added to a solution 
containing Protein C at a concentration of 0.5 to 8.0 mg/ml at a 
thrombin/Protein C ratio of 1 to 20 U/mg and activation is conducted under 
pH 6.0 to 8.0 to efficiently activate a large amount of Protein C. 
Subsequent to this step, procedures for removal of the activating agent 
and purification of APC are carried out wherein a solution containing APC 
after activation is contacted with a cation exchanger under condition of 
pH 5.0 to 6.0, NaCl concentration of 80 mM or less, to allow for 
adsorption of both thrombin and APC to the exchanger and then Activated 
Protein C alone is eluted therefrom with a salt concentration of 0.1 to 
0.35M. 
In accordance with the method for activating human Protein C of the present 
invention, it is possible to efficiently activate Protein C at a high 
concentration with a small amount of thrombin. This is advantageous in 
that degradation of Protein C during activation is decreased and the 
subsequent removal of thrombin becomes easier. Subsequent procedure for 
purification of APC provides a thrombin concentration in the eluent of 
0.001 U/ml or less, and hence, APC can be recovered quantitatively. 
Furthermore, a concentrated APC can be obtained since APC is eluted after 
adsorption thereof. In addition, the APC preparation recovered by the 
method of the present invention has a much higher specific activity than 
the preparations obtained by the conventional methods. 
"Specific activity" of the human Activated Protein C preparation as used 
herein means a ratio of APC activity to a whole amount of protein (mg). 
One Unit of APC activity is defined as an amount which prolongs twice an 
activated thromboplastin time (APTT) of normal human plasma. Accordingly, 
actual measurement of APC activity is conducted as follows: the APTT 
(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 
control (buffer) is determined to be the activity of APC of sample. 
The main component of the preparation of the present invention, APC, and 
the starting material of the process for preparation of the present 
invention, Protein C, may be derived from any source, but preferably from 
human plasma. 
The activation step in the above-mentioned procedures of the present 
invention is conducted as follows: thrombin is added to a solution 
containing Protein C at a concentration of 0.5 to 8.0 mg/ml at a ratio of 
thrombin/Protein C of 1 to 20 U/mg, and the reaction is carried out under 
condition of pH 6.0 to 8.0, a salt concentration of 0.1 to 0.15M, for 
example, at 37.degree. C. for 5 to 6 hours. 
The resultant reaction solution of the above activation procedure, after 
adjustment of pH or salt concentration as desired, is then subjected to 
treatment with a cation exchanger to purify APC. This treatment with a 
cation exchanger removes thrombin and further contaminating proteins such 
as Protein C which have not been activated. A cation exchanger as used 
herein may be any exchanger as far as it is an insoluble carrier having a 
cation-exchange group (e.g. sulfo group, carboxyl group), and includes a 
cation exchanger which is conventionally used in the art, for example, a 
cation exchange resin such as S-SEPHAROSE (trade name for a methyl 
sulfonate bound cation exchanger based on agarose), S-SEPHAROSE (trade 
name for sulfopropyl bound cation exchanger based on dextran), both 
manufactured by Pharmacia, SP-TOYOPEARL (trade name for sulfopropyl bound 
cation exchanger based on a hydrophilic vinyl polymer), TSK GEL SP-5PW 
(trade name), both manufactured by Toyo Soda K.K. Among these, SP-SEPHADEX 
and SP-TOYOPEARL are preferable. This step may be carried out either by a 
column process or a batch process. In view of the removal efficiency of 
contaminating proteins, a column process is preferable. 
The purification procedure of APC in accordance with the present invention 
is characteristic in that the reaction solution of activation is contacted 
with a cation exchanger to allow for adsorption of both thrombin and APC 
to the exchanger and then Activated Protein C alone is eluted therefrom 
under condition of a salt concentration 0.1 to 0.35M. The APC preparation 
prepared by such purification procedure is substantially free from 
thrombin or an equivalent protease and/or Protein C which has not been 
activated and has an extremely high specific activity of 3500 U/mg or 
more. 
Furthermore, the present inventors have also studied in order to eliminate 
defects of the conventional method in a series of steps for preparing APC 
having a high specific activity starting from a fraction of Protein C, and 
as a result, have found several useful improvements to further enhance the 
effects of the present invention. 
For purification of Protein C, there have been known a method wherein 
Protein C, after being purified by adsorption and precipitation with 
barium citrate, fractionation with ammonium sulfate, and DEAE-SEPHADEX 
(trade name for diethylaminoethyl bound anion exchanger based on dextran 
column chromatography, is further subjected to purification procedures 
such as preparative polyacrylamide gel electrophoresis, dextran sulfate 
agarose chromatography, etc. or a method for purification of Protein C by 
using a gel to which an antibody against Protein C is immobilized, and the 
like (J. Biol. Chem., 251, 355-363 (1976); J. Clin. Invest., 64, 761-769 
(1979); Blood, 54, 1272-(1979); FEBS LETTERS, 191, 75-81 (1985); J. Bio. 
Chem., 261, 11097-11105 (1986)). The above-mentioned methods, however, are 
suitable for the laboratory, but are not suited for industrial large scale 
purification when consideration is given to yield, working efficiency, 
etc. It is further noted that in case of purification with a gel to which 
an antibody against Protein C is immobilized, a strong chaotropic ion or 
acidic pH or a strong chelating agent such as EDTA for elution is 
utilized, but a trace amount of the antibody against Protein C is 
disadvantageously released from the gel and contained in the eluent. 
The present inventors have found that release of the antibody against 
Protein C from the gel is diminished by eluting Protein C with a citrate 
buffer solution in place of a strong chelating agent, EDTA. The obtained 
solution containing Protein C is then contacted with an anion exchanger 
under condition of pH 7.0 to 9.0 to allow for adsorption of Protein C to 
the exchanger, thereby removing a trace amount of the antibody against 
Protein C, followed by elution of Protein C under condition of a salt 
concentration of 0.3 to 1.0M. This procedure can remove almost all the 
antibody against Protein C. 
Based on the above findings, an improved process for preparing Activated 
Protein C on an industrial scale is provided in accordance with the 
present invention, said process comprises the following steps: 
(1) a solution containing human Protein C is contacted with an anion 
exchanger to allow for adsorption of Protein C to the exchanger, and 
thereafter Protein C is eluted to prepare a fraction of human Protein C 
having a Gla domain; 
(2) the solution containing human Protein C is contacted with an adsorbent, 
i.e. an insoluble carrier to which an antibody specifically recognizing 
Protein C bound with calcium ion is attached, in the presence of calcium 
to allow for adsorption of Protein C to the adsorbent, and then Protein C 
is eluted therefrom using a citrate buffer solution; 
(3) the solution containing Protein C is contacted with an anion exchanger 
to allow for adsorption of Protein C to the exchanger, and thereafter 
Protein C is eluted therefrom to remove the antibody against Protein C 
from the above solution containing Protein C; 
(4) human Protein C is activated with thrombin or an equivalent protease 
wherein thrombin is added to the solution containing Protein C at a ratio 
of thrombin/Protein C 1 to 20 U/mg under condition of pH 6.0 to 8.0; and 
then 
(5) the solution containing human Activated Protein C after activation is 
contacted with a cation exchanger under condition of pH 5.0 to 6.0, a salt 
concentration of 80 mM or less, to allow for adsorption of both thrombin 
and Activated Protein C to the exchanger, and then human Activated Protein 
C alone is eluted therefrom with a salt concentration of 0.1 to 0.35M and 
recovered. 
In the case of using purified Activated Protein C for therapy, especially 
when purified Activated Protein C is derived from human plasma, there is a 
risk of infection with viruses that may be present in source material 
(e.g. hepatitis virus, HIV, etc.), and hence, removal and inactivation of 
viruses is necessary. Protection from viral infection in case of blood 
preparations has been carried out by removal of viruses by screening of 
source material, membrane filtration, adsorption, column chromatography, 
precipitation, fractionation, or inactivation by a solvent detergent 
method, .beta.-propiolactone, heating, electromagnetic radiation, etc., or 
in combination thereof. However, it is difficult to remove and inactivate 
viruses without causing denaturation of a protein, decrease in 
physiological activity, decrease in yield, etc. The present inventors have 
found that removal and inactivation of viruses can effectively be 
conducted for Activated Protein C preparation by removal of viruses with a 
membrane filtration using a virus-removing membrane in combination with 
freeze-dry heating using as a stabilizing agent albumin at 0.5 to 10% 
(W/V). With such procedure, viruses can effectively be removed and 
inactivated without causing denaturation of Activated Protein C, decrease 
in physiological activity and yield. 
The present inventors have studied for developing an efficient process for 
purification and activation of Protein C and further a means for 
protection from viral infection for therapeutic use, and as a result, have 
found that the use of a citrate buffer solution for elution in 
purification of Protein C by affinity chromatography using an antibody 
against Protein C; activation of Protein C at a high concentration; and 
purification of Activated Protein C by adsorption to a cation exchanger 
are effective. Based on these purification procedures, and in combination 
with removal of viruses with a virus-removing membrane and viral 
inactivation by freeze-dry heating, the present inventors have established 
the technique to efficiently produce a large amount of Activated Protein C 
of a high purity for therapeutical application in high yield. 
The APC preparation of the present invention, in addition to its decreased 
level of contaminating proteins, is characteristic in that it shows a 
higher specific activity (APC activity/whole amount of protein (mg)) than 
those of APCs prepared by the conventional methods, for example, by the 
method disclosed in Biochemistry, 16, 5824-5831 (1977). In fact, the APC 
preparation of the present invention shows a specific activity 1.5 times 
to twice higher than that of the conventional preparations. The main 
reason why such increase in specific activity was observed remains still 
unclear, but it is estimated that removal of contaminating proteins 
(degradated products of APC, Protein C not being subjected to activation, 
coagulated proteins, etc.), which the conventional methods could not 
attain, may be one of main factors. 
The present invention is illustrated in more detail by means of the 
following examples, but should not be construed to be limited thereto. 
EXAMPLE 1 
1.1 Preparation of Activated Protein C preparation having a high specific 
activity 
A solution of Protein C preparation derived from plasma (8.7 mg/ml of 
Protein C, pH 7.5, 34.3 ms/cm of conductivity) was dialyzed against 20 mM 
citrate/0.1M sodium chloride buffer (pH 6.0) and then diluted to a 
concentration of 4 mg/ml of Protein C. To this solution was added human 
thrombin at a final concentration of 60 U/ml and the mixture was heated at 
37.degree. C. for 5 hours to activate Protein C. 
After activation, the solution after activation was diluted twice with 20 
mM citrate buffer (pH 6.0) and applied to a column of a cation exchanger 
(SP-TOYOPEARL) for removal of thrombin. The exchanger was washed well with 
20 mM citrate buffer (pH 6.0) containing 60 mM sodium chloride and then 
Activated Protein C was eluted with 20 mM citrate buffer (pH 6.0) 
containing 0.3M sodium chloride. Under this condition, Activated Protein C 
alone was eluted without elution of thrombin. A concentration of remaining 
thrombin was 0.001 U/ml or less. The obtained APC preparation showed a 
specific activity of 4750.9 U/mg. 
1.2 Study for condition of APC adsorption and elution in a cation 
chromatography after activation 
(1) Adsorption condition 
Using 20 mM citrate buffer, an adsorption condition of APC was examined 
within a range of pH 6.0 to 7.0 and a salt concentration of 0.0 to 0.15M 
which can be applicable in view of stability of APC. Adsorption of APC 
hardly occurred at a condition of pH 7.0 and a salt concentration of 0.1M. 
At a condition of pH 6.5 and a salt concentration of 0.1M, most of APC was 
eluted although it was partly adsorbed. At a condition of pH 6.0 and a 
salt concentration of 0.1M, most of APC was adsorbed onto the 
chromatographic carrier. 
Based on these results, a degree of APC adsorption was studied using a 
fixed pH 6.0 and various salt concentrations. At a salt concentration of 
0.15M, not only APC but also a part of thrombin used for activation were 
eluted, whereas at a salt concentration of 0.1M, most of APC was adsorbed 
but adsorption was not sufficient and a part of APC was eluted as 
mentioned above. Thus, a salt concentration was adjusted at 80 mM and 
thereby a suitable adsorption of APC to the chromatographic carrier was 
observed. 
(2) Elution condition 
After the chromatographic carrier was equilibrated with 20 mM citrate 
buffer (pH 6.0) containing 60 mM NaCl, a gradient elution was conducted at 
a concentration of 60 mM to 0.5M NaCl. Elution of APC started gradually at 
a concentration of around 0.1M NaCl, and at 0.35M or more, thrombin was 
also partially eluted. Accordingly, a preferable elution condition is a 
concentration of less than 0.35M NaCl. 
Reference Example 1 
Preparation of Activated Protein C preparation by the conventional method 
The above solution of Protein C preparation (8.7 mg/ml of Protein C, pH 
7.5, 34.3 ms/cm of conductivity) was dialyzed against 50 mM Tris-HCl/0.1M 
sodium chloride buffer (pH 8.0) and then diluted to a concentration of 0.7 
mg/ml of Protein C. To this solution was added thrombin at a final 
concentration of 10 U/ml and the mixture was heated at 37.degree. C. for 5 
hours to activate Protein C. After activation, the reaction solution was 
applied to a column of a cation exchanger (SP-TOYOPEARL), which was 
previously washed and equilibrated with 50 mM Tris-HCl/0.15M sodium 
chloride buffer (pH 8.0), and thereby thrombin was adsorbed and Activated 
Protein C was recovered in an elution fraction. A specific activity of 
Activated Protein C in this solution was 3259.6 U/mg. 
EXAMPLE 2 
Comparison of the preparation of the present invention with the 
conventional preparations 
The preparations of Example 1 and Reference Example 1 were compared for 
their specific activity and purity using electrophoresis, etc. 
2.1 Measurement of Activated Protein C activity: 
The activity of APC was measured herein in accordance with the following 
procedures. 
One unit of APC activity is defined as an amount of APC which prolongs 
twice an activated thromboplastin time (APTT; second) to double that of 
normal human plasma. Accordingly, the activity of APC is measured wherein 
APTT (second) is measured for a normal human plasma to which a diluted 
sample is added and the dilution at which the measured APTT value is twice 
as that of control (buffer) is determined and regarded as the activity of 
APC for samples. 
(Procedures) 
A sample was diluted with a veronal buffer containing 1% HSA to, for 
example, 400 times, 500 times, 800 times or 1000 times dilution. To each 
100 .mu.l of either control (buffer) or samples of each dilution were 
added 100 .mu.l of normal human plasma (e.g. Citrol I: Baxter Diagnostics 
Inc.) and 100 .mu.l of APTT reagent (e.g. Actin: Baxter Diagnostics Inc.) 
at 37.degree. C. successively with an interval of 15 seconds, the mixture 
is stirred, and after 2 minutes, 0.025M CaCl.sub.2 100 .mu.l is added and 
a coagulation time is measured. 
(Calculation of activity) 
A linear regression formula and a correlation coefficient of 10.sup.3 /X 
and Y are obtained from values of APTT (Y) at each dilution (X) of control 
and samples as follows: 
EQU Y=A(10.sup.3 /X)+B 
A value of X.sub.1 obtained from the following formula: 
EQU X.sub.1 =10.sup.3 {(Y.sub.1 -B)/A} 
wherein Y.sub.1 is a value twice that of APTT (second) of control, is 
regarded as the activity of APC (U/ml) for samples. 
(Measurement of protein) 
A concentration of Activated Protein C was measured based on measurement of 
absorbance A.sub.280, i.e. based on the estimation that A.sub.280 of APC 
at a concentration of 1% (W/V) (10 mg/ml) is 14.5 as estimated from an 
amino acid composition of APC (J. Clin. Invest., 64, 761-769 (1979)). 
Accordingly, a concentration of Activated Protein C is calculated by the 
following formula: 
EQU Concentration of Activated Protein C (mg/ml)=A.sub.280 as measured/1.45 
Based on the activity and the concentration of APC measured above, a 
specific activity of APC (U/mg) as used herein was calculated. 
2.2 Effect of the activation conditions on specific activity of APC 
Using samples just after activation, effect of difference in activation 
condition on a specific activity was studied. Measurement of APC activity 
after activation was conducted wherein each 1 U and 10 U of anti-thrombin 
and heparin was added to 1 ml of a sample before measurement and the 
mixture was heated at 37.degree. C. for 30 minutes to inactivate thrombin. 
The results are shown in Table 1. No effect on specific activity was 
observed for the activation condition of the present invention (pH 6.0, 
NaCl concentration 0.1M) as compared to the activation condition of the 
conventional method (pH 8.0, NaCl concentration 0.15M). 
TABLE 1 
______________________________________ 
Conventional method 
Present invention 
______________________________________ 
Concentration of 
0.72 1.38 
protein (mg/ml) 
Activity (U/ml) 
2069.9 4133.4 
Specific activity 
2885.9 3089.3 
(U/mg) 
______________________________________ 
2.3 Comparison of specific activity before and after treatment with cation 
chromatography 
Using various samples, a specific activity of APC before and after 
treatment with a cation chromatography in accordance with the present 
invention was compared. The results are summarized in Table 2. Increase in 
specific activity was barely observed in the conventional preparation. On 
the contrary, the preparations of the present invention showed specific 
activity of APC (U/mg) of more than 4500, specific activity of APC being 
increased by about 1.5 times after the chromatography treatment. The main 
reason why such increase in specific activity was observed remains still 
unclear, but it is estimated that removal of contaminating proteins 
(degraded products of APC, Protein C which has not been activated, 
coagulated proteins, etc.), which the conventional methods could not 
remove, may be one of main factors. 
TABLE 2 
______________________________________ 
Specific activity (U/mg) 
Rate of 
Before After increase 
Sample Chromatography (times) 
______________________________________ 
Invention 1 
3089.3 4750.9 1.54 
Invention 2 
3108.3 5750.5 1.85 
Invention 3 
3869.5 5107.8 1.32 
Conventional 
2885.9 3259.6 1.13 
preparation 
______________________________________ 
2.4 Comparison with electrophoresis before and after treatment with cation 
chromatography 
APC has been eluted at pH 8.0 in the procedure of a cation chromatography 
in the conventional method whereas, in the method of the present 
invention, APC is first adsorbed at pH 6.0 and then eluted. FIG. 1 shows 
results of SDS-PAGE in the conventional method and the method of the 
present invention. 
In accordance with the method of the present invention wherein APC is 
adsorbed at pH 6.0 and Protein C which has not been activated is eluted, 
fraction bands of those regions having higher or lower molecular weight 
than that of APC are removed. In addition, in comparison with the fraction 
eluted at pH 8.0, the fraction obtained by adsorption at pH 6.0 and a 
subsequent elution showed a sharper band of the corresponding APC, and 
hence, Protein C which has not been activated is possibly removed. On the 
other hand, although the eluted fraction obtained by the conventional 
method showed some quantitative decrease in fraction bands having higher 
or lower molecular weight than that of APC, these bands were not perfectly 
removed. 
2.5 Content of Protein C not being subjected to activation in APC 
preparation 
Decreased content of Protein C which has not been activated was considered 
to be one of the factors which led to an increase in specific activity of 
the APC preparation of the present invention having a high specific 
activity. Thus, a content of Protein C which has not been activated was 
measured for both the APC preparations of the present invention and the 
conventional preparations. Measurement was made by employing ELISA system 
using a monoclonal antibody specific for Protein C in accordance with the 
usual protocol. The results are shown in Table 3. 
TABLE 3 
______________________________________ 
Specific Content of Protein C 
activity of 
which has not 
Sample APC (U/mg) 
been activated 
______________________________________ 
Present 5750.5 0.47 
preparation 1 
Present 5107.8 1.22 
preparation 2 
Present 4457.1 0.36 
preparation 3 
Conventional 2661.7 5.41 
preparation 1 
Conventional 3286.2 5.83 
preparation 2 
Conventional 2470.4 3.93 
preparation 3 
______________________________________ 
The conventional preparations having a low specific activity had a content 
of 4 to 6% Protein C which has not been activated whereas the preparations 
of the present invention having a high specific activity had a content of 
as little as 0.4 to 1.2%. Although the conventional preparations do have a 
higher content of Protein C which has not been activated as compared to 
that of the preparations of the present invention, the content is very 
small, and hence, is not liable to exert a direct effect on specific 
activity of the preparations. In order to confirm this, to the preparation 
3 of the present invention having the smallest content of Protein C not 
being subjected to activation was added Protein C at a final concentration 
of 5% and the APC activity (APTT) was measured. As a result, no change was 
observed in APTT values as compared to the case without addition of 
Protein C (cf. Table 4). Accordingly, decrease in content of Protein C 
which has not been activated is not thought to be a main factor for 
increase in specific activity of APC as observed in the preparations of 
the present invention. 
TABLE 4 
______________________________________ 
Specific activity of APC (U/mg) 
Without addition 
With addition 
Sample of Protein C 
of 5% Protein C 
______________________________________ 
Present 5077.9 5173.7 
preparation 3 
Conventional 2726.0 2725.7 
preparation 3 
______________________________________ 
EXAMPLE 3 
Preparation of Activated Protein C preparation 
Industrial scale quanity of fresh frozen human plasma (100 L) was melted 
without heating and precipitates formed were separated by centrifugation. 
The obtained supernatant was added to an anion exchanger (DEAE-SEPHADEX 
A-50). The exchanger was well washed with 20 mM citrate buffer (pH 7.0) 
containing 0.1M sodium chloride and elution with 20 mM citrate buffer (pH 
7.0) containing 0.5M sodium chloride was carried out to elute a fraction 
of human Protein C having a Gla domain. 
To this solution was added 30 mM calcium chloride and then the mixture was 
applied to a column of affinity chromatography using an anti-Protein C 
antibody. The column was well washed with 50 mM Tris-HCl buffer (pH 8.0) 
containing 0.15M sodium chloride and 2 mM calcium chloride and thereafter 
Protein C was eluted with 20 mM citrate buffer (pH 6.0) containing 0.15M 
sodium chloride. 
This solution was adjusted to pH 8.0 with 0.1M sodium hydroxide and then 
added to a column of an anion exchanger (Q-SEPHAROSE Fast Flow a 
quaternary ammonium bound anion exchanger). The exchanger was well washed 
with 50 mM Tris-HCl buffer (pH 8.0) containing 0.15M sodium chloride and 
elution was conducted with 0.3M glycine buffer (pH 7.0) containing 0.4M 
sodium chloride. Protein C obtained in this step was confirmed to be a 
single band in SDS-PAGE. 
For activation, the solution of purified Protein C obtained by the above 
procedure was diluted with 20 mM citrate buffer (pH 6.0) to a 
concentration of 4 mg/ml of Protein C. To this solution was added thrombin 
at a final concentration of 60 U/ml and the mixture was heated at 
37.degree. C. for 5 hours to activate Protein C. 
After activation, for removal of thrombin, the solution after activation 
was twice diluted with 20 mM citrate buffer (pH 6.0) and applied to a 
column of a cation exchanger (SP-TOYOPEARL). The exchanger was well washed 
with 20 mM citrate buffer (pH 6.0) containing 60 mM sodium chloride and 
then Activated Protein C was eluted with 20 mM citrate buffer (pH 6.0) 
containing 0.35M sodium chloride. Under this condition, Activated Protein 
C alone was eluted without elution of thrombin and thereby thrombin is 
removed. The obtained solution of Activated Protein C was subjected to 
filtration with a virus-removing membrane (Planova 35N manufactured by 
Asahi Chemical Industries, K.K.). The resultant solution had 5392.7 U/mg 
of specific activity of Activated Protein C and 0.001 U/ml or less of a 
concentration of remaining thrombin. 
The filtrate was adjusted to 0.7% (W/V) sodium chloride, 0.5% (W/V) 
glycine, 0.6% (W/V) sodium citrate, 2.5% (W/V) human albumin and 600 U/ml 
of the activity of APC at a final concentration. The solution of Activated 
Protein C thus prepared was subjected to sterile filtration, 
freeze-drying, and then dry-heated at 65.degree. C. for 96 hours for 
inactivation of viruses to ultimately give Activated Protein C suitable 
for therapeutic application.