Note: Descriptions are shown in the official language in which they were submitted.
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ULTRA-PU~E THROMBIN PREP~RATION
BACKGROUND
Thrombin, a proteolytic enzyme, i~ es~antial for
hemo~ta~i~. It i9 a principle reagent in the formation
~of blood clot~ via fibrin production. Due to its
effactivene-~ a~ a clotting aid, thrombin and its
preparations are useful during surgical procedure~ to
control bleeding. While dry thrombin i9 available,
liquid preparation~ are generally preferred due to
handling and time con~ideration~.
Until now, there have been no highly ~table, clear
liquid thrombin preparations which are both ~torage
stable and ready for u~e during ~urgery. Thi~ is
becau~e thro~bin, when di~solved in water or ~aline,
rapidly loses it3 activity due to denaturation and
autoly3i3 of the thrombin protei~.
T~E XNVENTION
The pre~ent in~ention iq directed to a ~ovel
modification of a~proce3~ for the preparatio~ of
thro~bin of ultra-pure quality in 301ution. Thi~
qolution i~ completely clear and free o~ turbidity, an~
ha~ a characteristic of hi~h clotting activity, le~
inactive protain, and a high Qpecific activity~ more
than any thrombin product hereto describ~d.
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-2- 2 ~ ~ ~s~ ~
The novelty o* the presen~ proce s, which achieves
the goal of a clear thrombin solution, is the unique
combination of a whole host of steps in a particular
sequence.
First of all, the common thrombin, in circulating
blood, exists in an inactive form called prcthrombin and
a factor called thromboplastin is required in order to
convert prothrombin to thrombin. The present invention
does not involve the usual use of bovine lung extract as
a source of thromboplastin but uses an isolated, highly
purified thromboplastin, as described hereinafter. This
process eliminates a significant amount of contaminati~g
proteins which are the probable ~ource of impuritie~ and
turbidity often seen in a final product.
The prothrombin to thrombin conversion mixture,
following the usual centrifugation step, is passed
through an anion exchange chromatogxaphy column,
affording an eluted material which is still turbid.
This material is then, in the present invention, frozen
and then thawed, followed by centrifugation to remove
most of the turbidity. Removal of this turbidity
improves the solution flow thxough the second stage
cation-exchange column chromatography procedure.
Following the second passage of the solution
through a cation e~changer, the i.mprovement in this step
compriseQ eluting the material through thi~ col D by a
normal flow from top to bottom with a salt gradient
rather than a standard sodium chloride ~olution.
The results of the~e modifications have provided
the isolation of a~water-clear, ultra-pure thrombin, the
specific activity being much ~uperior to any pro~uct
available on the market, as shown in Table 1.
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TABLE 1. Comparison Between Thrombostat~, Thrombinar~,
and Ultra-Pure Thrombin
Thrombostat~a Thrombinar~ Ultra-
Pure
Clotting activityC2164 1372 7820
(U/mL)
Protein (mg/n~) 11.56 0.82 0.82
Specific activity 187 1663 9500
(U/mg)
.
a Parke-Davis
b Armour
c Determined by a modified NIH method hereinafter
described.
Accordingly, the present inven-tion concerns, in its
broadest aspects:
I. An ultra-pure, clear, colorless thrombin
solution having specific activity from 4000 to
11,000 Units/mg protein.
II. An ultra-pure, clear, colorless tbrombin
solution prepared by reacting prothrombin with purified
thromboplastin and treating the resulting thrombin, after
centrifugation, by eluting the supernatant through an
anion-exchange agaxose column; freezing, then thawing up to
about 25C, the desired eluant fractions; centrifuging and
eluting the supernatant through a cation-exchange agarose
column with a salt gradient in a buffer.
III. A process for preparing an ultra-pure, clear,
colorless thrombin solution comprising:
reacting prothrombin with purified thromboplastin;
centrifuging the suspension; eluting the supernatant
~hrough an anion-exchange agarose column with buffer;
freezirlg then thawing up to about 25C the desired eluant
fractions; centrifuging and eluting the supernatant through
a cation-exchange agarose column with a salt gradient in a
buffer.
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A DVANTAGE S
The thrombin compositions and methods of the
invention have several advantages over conventional
preparations and methods for assisting in blood clotting.
Unlike powdered preparations, the compositions of the
instant invention require no reconstitution prior to use.
Thus, measuring, mixing, sterilizing, etc. o~ one or more
component(s) or container(s) are not important
considerations. The instant clear preparations can be used
without preparation before final use because of the absence
of particles which cause the turbidity in former liquid
preparations.
Furthermore, the stability of the instant thrombin~
containing materials is such that the need for stock
inventories and/or rotation of products is largely
eliminated. Unlike most saline or water-solutions of
thrombin, which are stable for only about one week at ~C,
the instant preparations are designed to be stable at
normal refrigeration temperatures (i.e., about 4~C) and at
room temperature (i.e., about 25C) for 6 months or more.
It is known that high concentrations of glycerol,
sucrose, and other polyols can stabilize proteins in
solution. In the case of thrombin, it is known that a
glycerol concentration of 67~ can greatly stabilize a
l,000 ~/mL thrombin solution. However, use of high
glycerol concentrations is not practical in the large scale
manufacture of a sterile thrombin solution because of the
high viscosity of such a preparation. The instant
compositioDs, which may contain 30% or less, o~ glycerol
avoid these problems.
Other advantages and aspects of the invention will
become apparent from a consideration of the ~ollowing
description of the invention.
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BRIEF DESCRIPTION OF THE DR~WING
Figure 1 is a graph showing the purification of
thrombin by way of the prvfile of elution of the CM-
Sepharose column using the salt gradient of 0.1 to 1.0 M
sodium chloride solution as described in the e~ample.
DETAI~ED DESCRIPTION OF THE PR~FERRED EMBODIMENTS
Isolation of Thromboplastin From Lunq Tissue
Bovine/veal lung tissue is trimmed to remove fatty
tissue, cut into small pieces, and homogenized in a tissue
homogenizer with 25-50 mM phosphate buffer p~ 6.2-6.7,
preferably pH 6.5 containing 0.5-1.0% polysorbate 80. The
surfactant aids in the extraction of the enzyme. Other
nonionic surfactants can be used, e.g., fatty acid esters
of polyoxyethylene sorbitan, e.g., Tween~, ICI;
polyoxyalkylyne fatty acid esters, e.g., MYRJ~, ICIi
polyoxyethylene fatty ethers, e.g., Brij~, ICIi
polyoxypropylene-polyethylene ethers, e.g., Pluronic~,
BASF; sucrose mono esters; and Triton X-100. The
homogenate is centrifuged at 12-14K RPM and the supernatant
is collected. The pH of the supernatant is adjusted to pH
5.2-5.7, preferably pH 5.5 using drops of 10% acetic acid
and centrifuged at 12-14K ~PM. The supernatant from this
step is loaded on a conventional cation exchange
crosslinked agarose column, e.g., CM~Sepharose~ (other
cationic resins can be used, e.g., CM-Sephade~) and eluted
with 25-50 mM phosphate buffer as described above. The
eluate is monitored at 280 nM. The activity of
thromboplastin in t~e eluate is also monitored indirectly
by first converting prothrombin to thrombin and the latter
activity is then determined by measuring clotting activity
using a fibrometer.
The initial fractions eluting from ~he column form
part of the fixst peak which contains thromboplastin.
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Fractions eluting later which are part of the second peak
contain nonactive extraneous proteins. This puri.fied
thromboplastin solution is opalescent without any haze or
turbidity. This fraction can be clarified further by a
freeze/thaw and centrifugation cycle without any loss of
prothrombin converting activity.
Preparation, Isolation, and Purificat1on of Ultra-Pure
Thrombin
Assay for Thrombin ~ctivity
Thrombin clotting activity was determined using a
modified NIH method. The solutions used consisted of:
a) Imidazole buffer, stock, (IBS) made by dissolving 1.72 g
imidazole in 90 mL of 0.1 N hydrochloric acid and then made
to 100 mL with distilled water (final pH should be about
7.2). b) PEG/IBS solution made by dilu~ing I~S, 58.8 mL;
sodium chloride, 9.0 g; and polyethylene glycol (PEG,
molecular weight 8000), 5 g to 1000 mL with water. Normal
human plasma was used as a source of fibrinogen and was
diluted with 0.154M sodium chloride (1:1) prior to use.
NIH thrombin was used as a standard and was diluted with
polyethylene glycol (8000)/imidazole buffered saline
(PEG/IBS) to give 5 U/mL. Clotting assay was performed
using a fibrometer. Diluted plasma (200 ~L) was incubated
at 37~C for 3 minutes, then standard thrombin (100 ~L) was
added and the clotting time (in seconds) was recorded (14
to 15 seconds). Thrombin-containing unknown sample was
diluted with PEG/IBS to give clotting time values higher
and lower than the standard clotting time value by about
5 seconds. Enzyme activity was calculated as follows:
( ~ (A-B) (C-D) ] IB) g = units/mL
-- - D) ~
A: high dilution factor of unknown thrombin sample
B: low dilution factor of unknown thrombin sample
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C: average clotting time of standard thrombin
D: average clottiny time of low dilution of unknown
thro~bin sample
E: average clotting time of high dilution of unknown
thrombin sample
g: units of standard thrombin in test mixture divided
by volume of standard thrombin in test mixture
(0.5/0.1)
Enzyme activity is expressed as Units/mL (up to 8000 U/mL)
and as Units/mg protein (up to 10,000 U/mg protein).
Purified or partially purified bovine plasma
prothromhin is reacted with purified thromboplastin in the
presence of 10-40 mM calcium chloride solution at a
temperature between lO and 25C for 15-~5 minutes as
described under Example B, "Isolation of Ultra-Pure
Thrombin". The amount of thromboplastin activity is two to
three times that of prothrombin at pH 6.5-7Ø The
thrombin produced by this reaction is further purified as
follows. The resulting protein suspension is centrifuged
(12K RPM) in a refrigerated centrifuge (2-lO~C) to
separate insoluble nonactive proteins. The supernatant is
loaded on a weak anion-exchange column (DEAE-Sepharose~,
DEAE-Sephadex~, DE-52~). The column is eluted with
25-50 mM phosphate buffer (pH 6.5) containing 0.1 M sodium
chloride (2-10C). The eluant is monitored at 2~0 nM.
Fractions with high UV absorbance are further checked for
thrombin clotting activity (using a ~ibrometer). These
pooled fractions containing thrombin are turbid and are
clarified by freezing the suspension overnight~ followed by
thawing (<25C) and centrifugation or filtration. The
thrombin-containing pool is loaded on a cation-exchange
column (CM-Sepharose~, CM-Sephadex~) and eluted using a
salt gradient (0.1 ~ to 1 M sodium chloride in 25-50 mM
phosphate buf~er~ pH 6.5).
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The eluant is monitored at 2~0 nM and the fractions
containing thrombin activity (as determined by the
fibrometer) are pooled (Figure l). The fractions
containing ultra-pure thrombin are water clear and may have
about 8000 U/mL of activity. The purity of ultra-pure
thrombin is determined by reversed-phase HPLC,
polyacrylamide gel electrophoresis, and isoelectric
focusing.
Protein content of the thrombin fraction was
determined using the Bradford assay (Bradford, M., Anal.
Biochem., 72:248, 1976) and the protein reagent made by
BioRad (Richmond, CA). Specific activity of the
preparation was calculated by dividing the amount of enzyme
units per unit volume into the amount of proteins per same
unit volume.
The exceptionally high specific activity of thrombin
made by the present invention is attributed to the
following:
1. The use of a freshly harvested prothrombin yields
a thrombin product of high specific activity. Also,
inactive thrombin can co-elute with active thrombin and
this can result in decreased specific activity of the final
product. Therefore, all the isolation steps need to be
carried out at a temperature between about 2 to 7C.
Furthermore, solutions were not allowed to standr even
refrigerated, for extended periods of time prior to use.
Free7ing at about -10 to ~20C was adequate to protect the
products.
2. The use of highly purified thromboplastin as
describecl in the present invention diminishes the extent o~
contaminants or the presence of nonspecific proteins in the
final thrombin preparation, hence increasing the specific
activity.
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The Thrombin_Preparations
The preparations ma~e in accordance with the
invention must contain, in a liquid medium, ultra-pure
thrombin, and one or more buffers. They may contain
saline, and other substances cor~ventionally employed in
protein preparations.
While the term "preparatio~s" is employed, it should
be noted that Applicants contemplate all types of
formulations in which thrombin, in substantially
solubili7ed form, is present in combination with one or
more glycols and buffers.
When a liquid formulation is made, it is generally
preferred that the solvent(s) or other diluent(s) employed
have a suitable miscibility with thrombin such that
production standards, e.g., uniformity of thrombin
concentration from batch to batch, can be readily met.
The thrombin employed is an ultra-pure thrombin
obtained by the process of the present invention.
This thrombin solution is, if desired, then mixed
with glycerol containing either acetate buffex or phosphate
buffer and saline, in order to prepare a stabilized
solution.
Thrombin is known to be soluble in physiological
saline -- i.e., a solution containing about 0.9% NaCl in
water. However, other saline solutions are contemplated as
useful herein. Furthermore, the replacement of all or part
of the NaCl in such solutions with one or more other
suitable salts is contemplated.
Water is a preferred medium for the preparations of
the invention. However, the use of one or more other
diluents which do not adversely affect the solubility
and/or stability of thrombin in the subject preparations is
desirable.
One such diluent is glycerol. Other useful polyols
include mannitol, sorbitol, sucrose, glucose, and the like~
Mixtures are operable. Glycerol is highly preferred.
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The glycerol or other polyol ingredient(s) will be
employed at a total concentration of from about 10 to about
40 wt. %, preferably 20 to 30 wt. % based on total
composition weight.
Unless stated otherwise, all quantities recited are
weight percentages based on total compositions weight.
Suitable buffer systems are those whose aqueous
solutions will maintain pH of the final thrombin solution
between about 5.0 and about 8.0, with a preferred pH range
of about 5.5 to about 6.5. It is highly preferred that
when a phosphate buffer is used the final pH of the
preparation be about 6.0 to about 6.5 and when an acetate
buffer is used, the final pEI be about 5Ø
pH measurements are made using an ordinary pH meter
with a combination electrode.
Useful buffer systems include 2cetate, phosphate,
succinate, bicarbonate, imidazole, TRIS, and the
zwitterionic buffers described by N. E. Good and S. Izawa,
in Methods in Enzymol, 24, Part B, 53 (1972); and
W. F. Ferguson, ~. I. Braunschweiger, W. R. Braunschweiger,
J. R. Smith, J. McCormick, C. C. Wasmann, N. P. Jarvis,
D. H. Bell, and N. E. Good in Anal. Biochem 104, 300
(1980). These disclosures are hereby incorporated by
reference.
Suitable reagents for use in the instant buffer
systems include MES, ACES, BES, MOPS, TES, HEPES, and the
like. Phosphate should only be used when calcium ion i9
absent or in the presence of EDTA. Mixtures of such
reagents can be employed. If mixed buffers are used, the
final p~ should be suitably adjusted.
Buffers contai~ning phosphate ion and acetate ions are
preferred. Mixtures are operable.
The buffers will be present in the buffer solution,
along with water and/or other suitable diluent~s) at total
concentrations of about 0.01 M to about 0.2 M, preferably
about 0.025 M to about 0.10 M.
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The use of various other conventional additives,
e.g., antio:~idants, colorants, surfactants, and the like,
is also contemplated. Glutathione may be employed as an
optional ingredient. Amino acids may be employed as
optional ingredients, but their presence must not be in
such ~uantities as to interfere with the stabilizing action
of the polyol and buffer components on the purified
thrombin. In general, it is preferred that they be used in
only minute quantities at concentrations of O . 5% or less,
if at all.
Hemostats
Hemostatic materials, such as GELFOAM~, SURGICEL~,
and AVICEL~, and collagen, which are presently used alone
or in combination witb thrombin powder or thrombin in
saline, can be effectively used with the stabili7ed
thrombin formulations of the present invention using a
variety of techniques. Preferably, the stabilized solution
is absorbed onto the hemostatic agent and the pad is
freeze-dried and packaged in a sterile manner.
Antimicrobial or antibiotic agents can also be
incorporated into such pads, especially for use on burn
patients, where prevention o~ infection is critical.
One type of bandage suitable in the preparation of
coagulants in accordance with the invention is set forth in
U.S. Patent 4,363,319, the disclosure of which iq hereby
incorporated by reference.
The following is illustrative of the preparation of
an ultra-pure thrombin solution.
EXAMPLE
A. Isolation of Thrombo~lastin:
100 g Veal lung was homogenized in 200 mL, 25 mM
sodium phosphate buffer, pH 6.5, containing 0.5%
polysorbate 80 and centrifuged at 12R RPM for 20 minutes at
5C. The supernatant was collected and the pH adjusted to
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-12-
5.72 using 10% acetic acid, and let stand in the
refrigerator for 1 hour. The mixture was centrifuged for
20 minutes as above and the supernatant collected, 160 mL
of the supernatant was loaded on a CM-Sepharose~ column
(30 x 20.5 cm), which was saturated with 25 mM sodium
phosphate buffer, pH 6.5, and eluted using the same buffer.
Fractions of 230 drops were collected (about 13 mL) and the
optical density at 280 nM of the fractions was measured.
Tube No. OD at 280 nM
. . _
1 clear 0.0
2 clear 0.0
3 clear 0.0
4 opalescent 1.369
opalescent 3.751
6 opalescent 3.828
7 opalescent 0.451
8 yellowish 3.427
9 yellowish 3.993
10 reddish 3.513
11 reddish 4.060
12 reddish 4.071
13 reddish 4.055
14 reddish 4.073
15 reddish 3.944
16 reddish 3.723
Pool 1 fractions 4-6
Pool 2 fractions 7-9
The pools were assayed for thromboplast.in activity by
converting prothrombin to thrombin as follows:
Mix prothrombin 1.0 mL
saline 0.5 mL
pool fract. 0.1 mL
CaC12 ~0.3 M) 100 ~L incubated the mixture
at 25C for 25 minytes, centrifuged at 5C at 13K for
10 minutes and assayed for thrombin clotting activity using
a fibrometer.
-13-
FractionClottinq time (seconds)
Pool 1 10.9
Pool 2 9.9
The two pools contained significant ~hromboplastin
(prothrombin converting) activity. The first pool was used
for the conversion of prothrombin to thrombin since it has
less color. The second pool, however, could be utilized
also.
B. Isolation-o-f Ul-~=L9~ ~D~~b~
The conversion mix was made of:
Prothrombin 97 mL
saline 40 mL
CaC12 (0.3 M) 10 mL
Thromboplastin 15 mL
(Pool 1)
incubated at 25C for 30 minutes and centrifuged at 13K for
20 minutes at 5C, and collected the supernatant.
C. DEAE-Sepharose Column Chromato~raphv
Equilibrated the column (30 x 2.5 cm) with 25 mM
sodium phosphate buffer pH 6.5 containing 0.02% sodium
azide. Sodium azide was used as a bacteriostatic agent,
however, this agent cannot be used during the isolation of
thromboplastin since it causes browning of the red
hemoglobin. Other common bacteriostatic agents can be
substituted and are preferred. These include phenols and
substituted phenols, chlorobutobenzyl alcohol, benzalkonium
chloride, benzethonium chloride~ thimerosal, and
phenylmercuric nitrate.
Loaded 115 mL of the converted preparation on the
column and eluted using the same buffer containing 0.1 M
sodium chloride. Collected fractions of 230 drops and
assayed for thrombin clotting activity.
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-14-
Fractions 7~48 showed significant thrombin clotting
activity; they were pooled (510 mL) and the material
appeared turbid. The pooled material was kept in a plastic
bag and frozen overnight. The ma~erial was thawed,
centrifuged at 13K for 20 minutes at 5C and the
supernatant collected.
D. CM~Sepharose Column ChromatoqraphY
Equilibrated the column (30 x 2.5 cm) with 25 mM
sodium phosphate buffer, pH 6.5 containing 0.02~ sodium
azide and 0.1 M sodium chloride.
Loaded 493 mL of the previous supernatant from the
DEAE-sepharose column step.
Eluted the column with a salt gradient rnade of 225 mL
of the buffer containing 0.02~ sodium azide and 0.'l M
sodium chloride and 225 m~ buffer containing 0.0~% sodium
azide and l.0 M sodium chloride. Fractions were collected
as described above and assayed for thrombin clotting
activity using a fibrometer.
Fractions 15-21 contained significant activity and
were pooled (volume of 90 mL). The pooled material was
then assayed for thrombin acti~ity and showed clotting
activity of 8~13 U/mL.
This pooled fraction was also assayed for protein
content using the Bradford method and Bio-Rad protein assay
kit. The pooled material contained 0.82 mg/mL protein.
Final specific activity = 8213/0.82 = 10015 U/mg
protein.
