Thrombin preparation

A process for preparing thrombin which comprises treating a mixture comprising prothrombin, factor Xa, factor Va, and phospholipids with calcium ions, at a pH of 6.0-7.0 is provided. In particular the pH of 6.0-7.0 may be generated by the addition of the calcium ions or by buffering the preparation to a pH of 6.0-7.0. Thrombin preparations so produced may be subjected to further purification and are particularly stable even when substantially free of exogenous stabilizing agents such as proteins, sugars, polyol and mixtures thereof, and may be subject to freeze-drying and a virus inactivation by heat treatment.

FIELD OF THE INVENTION 
The present invention relates to a novel process for the production of 
thrombin particularly human thrombin and to thrombin preparations capable 
of being produced in a freeze dried form, which may be heat-treated in 
order to inactivate any viruses present. 
BACKGROUND OF THE INVENTION 
Thrombin is the product of the activation of prothrombin by Factor Xa in 
plasma. It is a potent broadly specific serine proteinase that converts 
fibrinogen to fibrin and promotes fibrin cross-linking by activating 
Factor XIII. Amongst a number of other observed biological activities, 
thrombin also controls several feedback loops in the clotting cascade and 
induces the platelet release reaction (1, 2). 
Thrombin has been used as a topical haemostatic agent for many years. 
However, it is as a component of fibrin sealant (fibrin glue) that the 
clinical use of thrombin is likely to expand. Thrombin is used in fibrin 
sealant to convert fibrinogen to fibrin on a cut surface or within a graft 
and numerous surgical applications have been described in a wide range of 
surgical specialities (3, 4). 
Bovine thrombin is currently used widely as a topical haemostatic agent or 
as a component of commercial fibrin sealant products. While such thrombin 
products are biologically effective, they are associated with 
well-documented risk of allergic responses and induction of antibodies to 
the bovine thrombin or to impurities such as bovine factor V, usually 
after repeat use (5, 6 and 7). Ortel et al (8) recently concluded that 
such acquired coagulation factor inhibitors probably occur more commonly 
than is currently appreciated and although frequently clinically benign, 
these inhibitors may be associated with life-threatening haemorrhage. For 
this reason the development of a process to produce human thrombin 
suitable for use as a topical haemostat or for inclusion in a fibrin 
sealant product, has been sought. 
Intrinsically, thrombin is formed when prothrombin (Factor II) is converted 
by activated Factor X, activated a Factor V, phospholipid and calcium ions 
into thrombin. Conversion of prothrombin to thrombin can occur without 
some of the associated components, however, the rate of conversion is 
undesirably slow. 
There are three main in vitro prothrombin conversion methods known in the 
art. The first method relies on the use of thromboplastin. Prothrombin is 
converted to thrombin using thromboplastin preferably in the presence of 
calcium chloride. This is described in a number of patent specifications 
such as EP 0439156A and EP 0505604A. A disadvantage of this method is that 
the thromboplastin is usually a crude preparation which has been prepared 
from freshly homogenised brain, lung or intestinal tissue. This procedure 
is not appropriate for the preparation of human thrombin as the reagents, 
depending on their source, can carry the risk of virus or cross-species 
contamination. 
A second method utilises some components of snake venom to yield thrombin 
(9, 10, 11). However, it has been reported that some of the venoms do not 
cleave the same bonds within prothrombin, as the natural activator, Factor 
Xa (12). Thus, there may be dangerous implications should a 
non-physiological form of thrombin be used clinically. 
The third in vitro method is essentially the same as the intrinsic in vivo 
process, wherein prothrombin is converted to thrombin by activated Factor 
X, Factor V, phospholipid and calcium ions under near physiological 
conditions. This has been described, for instance in, EP 0528701, EP 
0378798 and U.S. Pat. No. 5,219,995. However, the thrombin produced is 
often unstable unless exogenous proteins, polyols and/or sugars are added 
to the thrombin to stabilise it. 
Since human thrombin is derived from plasma obtained from blood donations, 
there is a risk of contamination of the thrombin by any viruses present in 
the original blood donation. Thus, any human thrombin preparation designed 
for clinical use, should be subjected to a virus inactivation step, prior 
to use. 
Virus-inactivation by solvent-detergent treatment has been described 
previously (13). However, the thrombin preparation may need to be 
subjected to further purification steps in order to remove the 
solvent-detergent. Other workers have described the use of 
virus/inactivated prothrombin feedstocks, but have not described methods 
for virus/inactivation of the thrombin products prepared from them, for 
example EP 0378798 and EP 0543178. Terminal (e.g. a final step of a 
process) viral-inactivation of the product is viewed as probably the 
safest and most effective method of virus/inactivation, as it minimises 
the chance of recontamination. 
There is thus a requirement in the art to produce thrombin which is 
terminally virus/inactivated, especially by heat-treatment. 
Generally speaking the present invention is based on the surprising 
discovery that prothrombin can be converted to thrombin in good yield, 
under acidic conditions and that these acidic conditions promote the 
stability of the thrombin generated. 
SUMMARY OF THE INVENTION 
More specifically, a first aspect of the present invention provides a 
process for preparing thrombin which comprises treating a mixture 
comprising prothrombin, Factor Xa, Factor Va and phospholipids with 
calcium ions at a pH less than pH 7.0. 
Generally, the mixture comprising prothrombin, Factor Xa, Factor Va and 
phospholipids may be obtained from a supernatant of a cryoprecipitate 
(which is formed by freezing and thawing plasma) of human plasma. The 
mixture may be obtained by chromatographic purification of the supernatant 
of cryoprecipitated plasma, generally by anion-exchange chromatography. 
More particularly a DEAE-cellulose eluate of absorbed supernatant of 
cryoprecipitated plasma, which may be used for the production of clinical 
Factor IX concentrates, can serve as the mixture for thrombin production 
(14). The mixture may comprise additional clotting factors, such as, 
Factor X, Factor V, Factor IX, Factor IXa and trace amounts of thrombin. 
The prior art (e.g. EP 0378789 and EP 0528701) has previously taught the 
addition of low levels of calcium ions (5-25 mM) to a mixture comprising 
prothrombin, at or around physiological conditions (pH 7.0-7.3) and EP 
0528701 describes that the addition of higher levels of CaCl.sub.2 
inhibits the preparation of thrombin. It might be expected that conversion 
of prothrombin to thrombin would proceed best in conditions which approach 
those of physiological conditions. It is thus a surprising feature of the 
present invention that thrombin may be prepared in particularly good yield 
at a pH of less than pH 7.0. Preferably the pH is between pH 6.0-7.0 and 
more preferably between pH 6.4-6.6. Without wishing to be restricted to 
any postulated theories, it is thought that the pH of less than pH 7.0 
limits autodegradation of thrombin produced. 
Generally the pH of less than pH 7.0 may be generated by the addition of 
Ca.sup.2+ ions, (in particular CaCl.sub.2) at concentrations of 50 mM-90 
mM, more preferably 60 mM-80 mM and most preferably 65 mM-75 mM, to the 
mixture. 
Addition of CaCl.sub.2 in the ranges specified, further generally results 
in a drop in the pH of the mixture which may be sufficient to reach the 
required pH. 
Alternatively, the mixture may be buffered to between pH 6.0-7.0 or more 
preferably between pH 6.4-6.6 by any suitable buffer known to buffer in 
the required range, before adding Ca.sup.2+ ions to initiate the 
conversion of prothrombin to thrombin. Examples of suitable buffers 
include MES (2-N-Morpholino!ethanesulphonic acid); ACES 
(2-2-Amino-2-oxoethyl)amino!ethanesolphonic acid); BES 
(N,N-bis2-Hydroxyethyl!-2-aminoethanesulphonic acid); MOPS 
(3-N-Morpholino!propanesulphonic acid); TES 
(N-trisHydroxymethy!methyl-2-aminoethanesulphonic acid) and HEPES 
(N-2-Hydroyethyl!piperazine-N-2-ethanesulphonic acid) and the like. 
In order to convert substantially all the prothrombin to thrombin, the 
conversion should proceed for a period of time and at a suitable 
temperature to effect conversion. Typically the conversion should be 
allowed to proceed for 12-24 hours and more preferably for 16-20 hours. 
The conversion may proceed at room temperature, typically between 
18-25.degree. C. and does not require incubation at higher temperatures. 
Generally thrombin prepared in this manner has a thrombin clotting activity 
of between 4,000-9,000 U/ml and a specific activity of between 250-700 
U/mg. This is considerably higher than the activity of the thrombin 
prepared by the process described in EP 0528701 (clotting activity 
700-1,000 U/ml and a specific activity of 20-40 U/mg). 
Some unwanted insoluble material may be found in the thrombin preparation 
probably due to the generation of fibrin by the action of generated 
thrombin on any fibrinogen present as a contaminant in the original 
DEAE-cellulose eluate and of insoluble calcium phosphate. The unwanted 
insoluble material may be removed by centrifugation or by a filtering 
process. However, in some instances, the preparation is too viscous and so 
the thrombin preparation is preferably diluted to reduce the viscosity. A 
dilution of 1 volume of thrombin preparation with up to 3 volumes buffer, 
for example 3 volumes which can be any buffer suitable for use in the 
range of pH 6.0-7.0, is generally carried out. Typical buffers include 40 
mM sodium gluconate or 20 mM MES, both at pH 6.5. The diluted preparation 
may then be centrifuged or filtered to remove any insoluble material. 
Alternatively 20 mM citrate, pH 6.5 may be used as the diluting buffer. 
This may remove the need for centrifugation or filtering, possibly due to 
the solubilisation of insoluble calcium phosphate. 
The diluted thrombin preparation is suitable for immediate further 
processing, or may be stored at -40.degree. C. for at least six months 
without substantial loss of clotting activity. Alternatively, the diluted 
material may be formulated and freeze-dried as an intermediate purity 
preparation. 
A specific activity of the thrombin preparation of between 250 U/mg to 700 
U/mg is equivalent to a thrombin purity of between about 6%-17.5% based on 
a comparison to a specific activity of pure .alpha.-thrombin of 4,000 
U/mg. While this is sufficient in most clinical instances, it is possible 
to subject the thrombin preparation to further processing to yield a 
thrombin of higher purity. 
Further processing may comprise chromatographic purification of thrombin 
with an optional solvent/detergent virus inactivation step prior to 
chromatographic purification. A suitable solvent/detergent 
virus/inactivation step has been previously described in Edwards et al 
(13). 
Chromatographic purification is generally carried out by cation-exchange 
chromatography. Typically cation-exchange resins which may be employed 
include MONO-S (TRADEMARK), S-SEPHAROSE FF (TRADEMARK) and S-SEPHAROSE BIG 
BEADS (TRADEMARK), all methyl sulphonate strong cation-exchangers produced 
by Pharmacia Biotech, although other sulphonate gels or other 
cation-exchangers may be employed. The chromatography step serves to 
remove solvent and detergent, if a solvent/detergent virus inactivation 
step has been carried out and also serves to purify the thrombin 
preparation. 
Typically the thrombin preparation is bound to the cation-exchange 
chromatography resin and a purified thrombin is eluted using a suitable 
buffer with increased salt concentration. Examples of suitable buffers 
include 20 mM citrate pH 6.5, 20 mM MES pH 6.5 and 40 mM gluconic acid pH 
6.5. The pH of the buffer should preferably be in the range of pH 6.0-7.0 
and more preferably pH 6.4-6.6 in order to preserve the activity of the 
purified thrombin. Usually several salts are suitable for eluting with any 
given cation-exchange resin and typically these include NaCl. 
The concentration of eluted purified thrombin depends directly upon the 
amount bound to the resin, but typically concentrations of purified 
thrombin between 4,000-9,000 U/ml may be obtained. Even the lower range of 
these concentrations is adequate to allow suitable dilution with a 
formulation buffer, for subsequent freeze-drying. 
Purified thrombin may be frozen directly in elution buffer and stored for 
up to six months without substantial loss of thrombin activity. However, 
for ease of storage it is desirable that the intermediate purity thrombin 
and purified thrombin, be freeze-dried. 
Freeze-drying often results in a loss in activity of thrombin (intermediate 
purity thrombin and purified thrombin) and it is thus important to 
formulate the thrombin with a formulation buffer. This formulation buffer 
helps stabilise the thrombin during freeze-drying. Prior to formulating 
the thrombin, it is often desirable to centrifuge and/or filter the 
thrombin to remove insoluble material. 
The art has previously described that the addition of stabilising agents 
such as polyols, for instance, glycerol, mannitol and sorbitol; sugars 
such as sucrose and glucose and/or exogenous proteins such as albumin, to 
a thrombin preparation is desirable to improve the stability of a thrombin 
preparation, especially during freeze-drying. It is thus a surprising 
feature of the present invention that thrombin may be prepared, which is 
substantially stable without additional stabilising agents such as polyol, 
sugar, protein and mixtures thereof. 
Thus, in a further aspect the present invention provides a thrombin 
preparation, the preparation comprising thrombin substantially free of 
exogenous stabilising agents (such as protein, sugar, polyol and mixtures 
thereof) buffered at a pH of less than pH 7.0. 
Generally the thrombin preparation is freeze-dried and optionally 
heat-treated. Thus, in a still further aspect, the present invention 
provides a freeze-dried, optionally heat-treated, thrombin preparation, 
substantially free of exogenous stabilising agents, such as protein, sugar 
or polyol and mixtures thereof. 
Preferably the thrombin preparation is buffered to between pH 6.0-7.0, more 
preferably pH 6.4-6.6. This may be achieved by for instance 40 mM gluconic 
acid or 20 mM MES buffer in the suitable pH range. Preferably, the 
thrombin preparation further comprises citrate at a concentration of 10 
mM-30 mM, typically sodium citrate. More preferably the preparation 
further comprises sodium chloride at a concentration of between 100-250 mM 
for example 100-200 mM. A thrombin preparation comprising citrate and 
sodium chloride in addition to gluconate or MES has been found to be most 
stable to freeze-drying and optional heat-treatment. That is, the thrombin 
preparation retains a greatest percentage of clotting activity after 
freeze-drying and optional heat-treating. 
Freeze-drying is preferably carried out employing a two-stage freezing 
procedure. The frozen product is then primary dried at a shelf temperature 
of -20.degree. to -30.degree. C. and then secondary dried at a shelf 
temperature of +15.degree. to +30.degree. C. 
The freeze-dried thrombin preparation may then be heat-treated in order to 
inactivate any virus contaminants. Typically dry heat-treatment is carried 
out at temperatures of between 70.degree. C. to 100.degree. C. for up to 
96 hours. A particularly preferred heat-treatment is approximately 
80.degree. C. for around 72 hours. 
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
Embodiments of the present invention will now be described by way of 
Example, with reference to the attached Figures.

EXAMPLES SECTION 
Example 1 
Preparation of a mixture comprising prothrombin, Factor Xa, Factor Va and 
phospholipid by DEAE-cellulose 
450 liters of cryoprecipitate plasma was adjusted to pH 6.9.+-.0.05 and 
diluted with 150 liters of pyrogen free H.sub.2 O to a final volume of 600 
liters. 6 kg of DEAE-cellulose gel (DE-52 Whatman) was then added to the 
plasma/water solution and the resulting suspension mixed continuously for 
one hour to bind the clotting factors to the gel. The gel was then 
collected by centrifugation and the supernatant discarded. The gel was 
then resuspended in 30 mM citrate, 30 mM phosphate pH 6.9 buffer and the 
resulting suspension was poured into a chromatography column. The column 
was then packed by washing with 21 liters of the same buffer. The clotting 
Factors were then eluated from the column with an elution buffer of 30 mM 
citrate, 30 mM phosphate, 200 mM NaCl, pH 6.9. The eluate pool (3.1 
liters) was then filtered (0.45 .mu.m pore size) into sterile bottles and 
frozen. 
The eluate pool contains substantial amounts of prothrombin (Factor II) (at 
80 .mu.M and about 25% of the total protein). It also includes factors IX 
and X, activated and non-activated (at about 5 .mu.M), coagulant-active 
phospholipid and sufficient trace amounts of Factors V and VIII to support 
the physiological conversion of prothrombin to thrombin via the intrinsic 
clotting pathway. 
Example 2 
Preparation of intermediate purity thrombin 
Frozen DEAE-cellulose eluate (prepared according to Example 1) was thawed 
at room temperature or in a 37.degree. C. water bath. (Typical values of 
the eluate were as follows: conductivity=17 mS; pH=7.0; 30 mM citrate; 30 
mM phosphate; 200 mM sodium; 200 mM chloride; 15 mg/ml total protein and 
prothrombin 60 U/ml). 
1M CaCl.sub.2 solution was then added dropwise to the thawed eluate, with 
stirring, at a ratio of 75 ml CaCl.sub.2 to 1,000 ml eluate, at 20.degree. 
C. This resulted in a final calcium concentration of 70 mM and a drop in 
pH in the mixture to pH 6.4-6.6. The reaction was allowed to proceed with 
stirring overnight for 18 hours at 20.degree. C., to convert the 
prothrombin to thrombin. 
In 15 experiments, the thrombin clotting activity was 6,333.+-.1,146 U/ml 
(mean.+-.SD) and specific activity of 508.+-.110 U/mg (see FIG. 1). SDS 
PAGE indicated that effectively all the prothrombin band was converted 
into bands co-migrating with thrombin, by the end of the activation 
period. 
Thrombin clotting activity was measured by fibrinogen clotting time at room 
temperature with visual detection and duplicate samples. To 200 .mu.1 of 
human fibrinogen solution at 5 mg/ml in 50 mM tris-HCl 100 mM NaCl pH 7.5 
was added 100 .mu.l of standard (1-4 U/ml) or test solutions of thrombin 
diluted in 50 mM tris-HCl, 100 mM NaCl pH 7.5 supplemented with 100 mM 
CaCl.sub.2 and 0.1% w/v bovine serum albumin, whereupon time to subsequent 
clot formation was recorded. A standard curve was constructed by plotting 
log.sub.10 thrombin concentration (U/ml) against log.sub.10 clotting time 
(sec) using bovine thrombin standardised against the human alpha-thrombin 
standard 89/588. Thrombin clotting activity of test samples was derived by 
extrapolation from the standard curve (15). 
Example 3 
Effect of varying the pH of the reaction solution during activation 
Following the procedure described in Example 1, resulted in a mixture with 
a pH of 7.0-7.2. This immediately decreased to pH 6.5 on addition of 
CaCl.sub.2 to 70 mM. There was then a steady decrease in pH to 6.1-6.3 
during the conversion period (18 hours). The fall in pH was a requirement 
for the successful generation of thrombin of high activity. This was 
demonstrated by comparative experiments, where the pH of the solution was 
adjusted to pH 7.0 or pH 7.5 immediately after the addition of CaCl.sub.2. 
Here the final pH values at the end of the reaction period were pH 6.7 and 
pH 7.1 respectively and a much lower amount of thrombin activity was 
generated (see Table 1). 
TABLE 1 
______________________________________ 
pH of the mixture, 
pH of the mixture 
immediately after 
at the end of the 
Clotting 
CaCl.sub.2 addition 
conversion period 
activity 
Example (adjusted as necessary) 
(18 hours) (IU/ml) 
______________________________________ 
1 6.5 6.1 5716 
2 7.0 6.7 2012 
3 7.5 7.1 1493 
______________________________________ 
In a further experiment, the mixture was buffered (20 mM MES) to pH 6.5 
immediately after CaCl.sub.2 addition. This resulted in an additional 
small increase in conversion to thrombin, but the-increase in clotting 
activity was insignificant. 
Example 4 
Effects of varying the length of time or temperature employed for 
conversion of prothrombin to thrombin 
Studies were carried out to determine the optimum time course for the 
conversion of prothrombin to thrombin. A comparison of the amount of 
thrombin generated at 16 and 24 hours indicated that a plateau had been 
reached by 16 hours. 
An investigation was also carried out to determine the effect of incubation 
at 37.degree. C. for one hour prior to subsequent room temperature 
incubation, in view of a report that this step was necessary to obtain 
useful yields with this type of feedstock (European Patent Application No. 
92401889.8). It was found that while the initial rate of thrombin 
generation exceeded that obtained at room temperature, the final yield of 
thrombin was no better at 16 or 24 hours as compared to conversion at room 
temperature. 
Example 5 
Effects of varying calcium ion concentration on thrombin production 
The amount of thrombin generated at 24 hours with a range of added calcium 
ion concentrations (seven batches of DEAE-cellulose eluates) was 
determined (FIG. 2). It was found that the addition of 70 mM calcium 
consistently resulted in efficient conversion of prothrombin to thrombin. 
Example 6 
Viral inactivation by solvent/detergent 
Thrombin prepared according to Example 2 was mixed by stirring with 0.3% 
tri-(n-butyl) phosphate and a 1% solution of Tween 80 at a temperature of 
20.degree.-30.degree. C. for 6 to 24 hours. This was sufficient to 
inactivate any contaminating lipid-enveloped viruses. The 
solvent/detergent was removed by chromatography. 
Example 7 
Chromatographic purification of intermediate purity thrombin 
The chromatography step serves to remove solvent and detergent and to 
purify the intermediate purity thrombin. A 1.6 cm diameter chromatography 
column was packed with 10 ml of S-SEPHAROSE FF (TRADEMARK) at a linear 
flow rate of 2.2 cm/min (equivalent to 4.5 ml/min) using 40 mM gluconic 
acid, 20 mM MES, or 20 mM citrate all at pH 6.5. 100 ml of 
solvent/detergent treated thrombin according to Example 6 or intermediate 
purity thrombin according to Example 2, following a 1+3 dilution in 
equilibrating buffer was filtered at 0.45 .mu.m and applied to the column 
at the same flow rate. The column was then washed with equilibrating 
buffer until the absorbency at 280 nm returned to baseline and solvent or 
detergent were detectable only below acceptable low levels, in the column 
effluent (typically approximately 150 ml). Thrombin was then eluted from 
the column by washing with equilibrating buffer containing 0.5M NaCl. 
Purified thrombin was obtained in about 25 ml at a typical concentration 
of 4,000 U/ml and 2 mg/ml protein. 
The typical yield of purified thrombin after the chromatography step was 
88.+-.16%. This yield refers to a thrombin preparation which was not 
subjected to a solvent/detergent virus inactivation step as described in 
Example 6. 
Example 8 
Formulation, freeze-drying and terminal dry heat treatment 
Thrombin prepared according to Examples 2 or 6 was centrifuged at 3,000 rpm 
for 20 minutes at room temperature and then filtered through a Millpore 
prefilter (AP25) followed by a Whatman 0.2 .mu.m filter (Polydisc AS). The 
filtered solution was then diluted in a formulation buffer (40 mM gluconic 
acid or 20 mM MES, 20 mM trisodium citrate, 150 mM NaCl, pH 6.5) to a 
thrombin activity of 600 U/ml and dispensed in 2 ml lots into glass vials 
for freeze-drying. 
Freeze-drying was performed in a super-Modulyo (Edwards, Crawley) 
freeze-dryer with a freezing temperature setting of -45.degree. C., 
followed by a primary drying temperature setting of -25.degree. C. and a 
secondary drying temperature setting of +20.degree. C. 
The vials were then heat-treated to inactivate any contaminating viruses, 
at 80.degree. C. for 72 hours. 
Example 9 
Comparison of a stabilising effect on thrombin of various formulation 
buffers 
Thrombin was formulated in a variety of formulation buffers, in order to 
determine the optimum formulation buffer for stabilising thrombin during 
freeze-drying and subsequent heat-treatment (virus-inactivation). 
Intermediate purity thrombin prepared according to Example 2 and purified 
thrombin prepared according to Example 7, were diluted with various 
formulation buffers (as described in Table 2) to a thrombin concentration 
of 600 U/ml. The thrombin preparation was then freeze-dried according to 
Example 8 and a quantity of the freeze-dried thrombin was also subjected 
to a heat-treatment of 80.degree. C. for 72 hours. Thrombin clotting 
activity was determined, as previously described, to determine the 
percentage clotting activity that remained after freeze-drying and 
subsequent heat-treatment. The results are shown in Table 2. 
It can be seen from Table 2 that formulation buffers comprising 20 mM 
tris-HCL buffer at pH 7.2 with or without 20 mM trisodium citrate and/or 
150 mM sodium chloride, resulted in a recovery of thrombin clotting 
activity, after freeze-drying, of greater than 74%. However, large losses 
in activity were seen post-heat-treatment, particularly in the absence of 
trisodium citrate. The inclusion of sodium chloride in the formulation 
buffer gave rise to an intact plug of material, whereas without sodium 
chloride, the plug retracted and collapsed. 
Protein (e.g. Human albumin) can also be included in the formulation at 
concentrations of 0.5 g/l-10 g/l, to act as a bulking agent and improve 
plug structure and appearance. 
When the formulation buffer was made acidic by using gluconic acid or MES 
buffered at pH 6.5, recovery of thrombin clotting activity after dry 
heat-treatment was substantially improved. 
Long term stability was determined using the gluconic acid buffer 
formulation (see Table 2). These studies were performed by storing several 
vials at 4.degree. C. and 37.degree. C., after freeze-drying and heat 
treatment. No loss in thrombin clotting activity was observed over a six 
month period, when comparing the 37.degree. C. stored thrombin to the 
4.degree. C. stored thrombin. 
TABLE 2 
______________________________________ 
Recovery of clotting activity (%) 
Intermediate High purity 
Formulation purity thrombin 
thrombin 
buffer Post-FD Post-HT Post-FD 
Post-HT 
______________________________________ 
20 mM Tris-HCL 96 12 93 12 
pH 7.2 
20 mM Tris-HCL 92 56 91 56 
pH 7.2 + 20 mM 
trisodium 
citrate 
20 mM Tris-HCL 87 10 74 4 
pH 7.2 + 150 mM 
NaCl 
20 mM Tris-HCL 91 41 90 51 
pH 7.2 + 20 mM 
trisodium 
citrate + 
150 mM NaCl 
20 mM gluconic 100 99 93 85 
acid + 20 mM 
trisodium 
citrate + 
150 mM 
NaCl pH 6.5 
20 mM MES + 20 mM 
nd 97 nd 86 
trisodium 
citrate + 
150 mM NaCl 
pH 6.5 
______________________________________ 
FD = Freezedrying 
HT = Heattreatment of 80.degree. C. for 72 hours in vial 
nd = not done 
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