Note: Descriptions are shown in the official language in which they were submitted.
` `- 1079Z72
.
The present invention relates to a method ~or the
purification of heparin to produce heparin that is very pure and
possesses high specific activity.
Heparin has been used in medical treatment for several
decades. It is one of the best-known anticoagulants and in
extensive use, i.e., for the prevention of thrombosis. Heparin
is a sulfated polysaccaride which can be prepared from animal ~-
intestinal mucus or lungs by relatively complicated methods. The
heparin preparations used clinically today contain material with
a great range in molecular size, from about 5,000 to about 35,000.
The specific activity usually is about 130 units per mg Thus,
the commercial preparations available today are quite heterogenous.
The frequency of side effects in heparin therapy is rather low
but where cases occur, they often present severe problems. Some
of these side effects are probably caused by impurities in the ~-
heparin preparation.
There are, therefore, good arguments for producing -
purer, more specifically active heparin preparations than those
in current use. In connection with studies of antithrombin,
a co-factor of heparin, the possibilities of binding the protein
to a matrix were investigated. Surprisingly, it was found that
matrix-bound antithrombin could specifically bind the molecule
fraction of therapeutically used heparin that acts as a carrier
of the clinically valuable, thrombose-prophylactic effect of
heparin. With suitably selected adsorption and desorption
conditions, an extraordinarily pure heparin can be prepared.
The specific activlties obtained were 200 - 270 units/mg. versus
approximately 130 units/mg. of the starting material. The
distribution of molecular weights in the specifically purified
heparin was far more limited than in the primary material. In
addition to the heparin co-factor antithrombin, other heparin-
binding proteins can be used to advantage, such as inter-~-trypsin
'''' '
- 1 - ~ :.
'' ' ' ~, ' : ' :,
107927~:
,
inhibitor with a molecular weight of about 150,000, obtained
during isolation of coa~ulation factor IX (B-factor). With
matrix-bound antithrombin, the best quality of heparin was
obtained, with specific activities up to 270 units per mg. -;~
Thus, heparin with a very high degree of purity and
specific activity can be produced by the process of the present
invention which is a simple one and applicable for industrial
use for the preparation of heparin with greater specific activity
than is the case with existing preparations. The determination
of the specific activity of heparin has been made according
to Denson and Bonnar (1975, Brit J Haematol, 30, p. 139).
According to-the present invention therefore there is
provided a method for the preparation of heparin of high specific
activity, which comprises contacting heparin with an adsorbent
which is matrix-bound heparin-binding plasma proteins.
The following examples illustrate the invention:
Example 1
Purification of he~arin on matrix-bound ~ . Column ~rocedur~
The yel adsorbent was prepared as follows:
Activation
3 g. of BrCN was dissolved in 30 ml. of distilled
water. 50 ml. of settled agarose (Sepharose~ 4B, Pharmacia Fine
Chemicals, Uppsala, Sweden) was added to the BrCN-solution. The
mixture was cooled on an icebath while stirred. By adding ~ M NaOH,
pH was raised to 11.2 and kept constant for 10 minutes. The gel
was then washed with cold distilled water and 0.2 M NaHCO3.
Linkinq
200 mg. antithrombin purified according to Miller-
Andersson et al (1974, Thromb Res 5 pp 439-452) and dissolved in
50 ml. 0.2 M NaHCO3, ph 9.0 was added to the gel. The gel
protein suspension was gently stirred in room temperature over
niqht and then carefully washed with buffers of high ion strength
- 1079272
with alternately high and low pH values.
Purification of he~arin -
The gel adsorbent containing matrix-hound antithrombin
was packed in a column and equilibrated with 0.05 M Tris,
0.15 M NaCl, pH 7.4 buffer. 30b mg. heparin (Vitrum, specific
activity 130 units/mg.) was dissolved in 20 ml. 0.05 M Tris, B
0.15 M NaCl, pH 7.4 buffer, and pumped through the column. There-
after, the gel was washed with the abovementioned buffer and
then the adsorbed heparin was desorbed with a 0.05 r~ Tris, 1~.0 M
NaCl, pH 7.4 buffer. The salt was removed from the eluted
he~arin by gel filtration is distilled water in a column packed
with SephadexR G 25 (Pharmacia) and after that the heparin was
freeze-dried. The purified heparin had a specific activity
of 270 units/mg. Studies of the distribution of molecular weights
show that it has a considerably more limlted molecular weight
distribution than has the primary heparin. Carbon hydrate
analysis was performed with the carbazol-H2SO4 method.
Example 2
Purification of heparin on matrix-bound antithrombin. Batch
~rocedure
The antithrombin matrix was prepared according to
Example 1.
Purification of heparin
500 mg. heparin (Vitrum, specific activity 130 unitsjmg.)
was dissolved in 300 ml. 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer.
300 ml. of settled matrix-bound anti-thrombin was equilibrated
in the abovementioned buffer and suction-dried. The adsorbent :-
was added to the heparin solution which was then stirred in room
temperature for one hour. The gel was suction-dried on a glass
filter and washed with 10 x 200 ml. of a 0.05 M Tris 0.15 M NaCl,
pH 7.4 buffer. The adsorbed heparin was then desorbed from
the gel by 3 x 200 ml. 0.05 M Tris, 1.0 M NaCl, pH 7.4. buffer.
-- 3
- 10792~2 :-
The eluted heparin was concentrated by freeze-drying. The
remaining salt was removed by gel filtration on Sephadex ~, 25
(a trademark) in distilled water, and then the heparin was
freeze-dried. The specific activity of the purified heparin
was 250 units/mg.
Example 3
Purification of heparin on ~el adsorbent containin~ matrix-
__________________ ________ __.,.__________________.~________
bound inter-~-trypsin inhibitor
________________ _____________
Preparation of gel adsorbent
During the purificationof coagulation factor IX(~factor)
(L.-O. Andersson et al., Thromb Res 7 (1975), pp. 451-459),
the heparin-binding protein inter-~-trypsin inhibitor was
obtained. Its molecular weight is approximately 150,000. 250 mg.
of this protein was dissolved in 200 ml. of a 0.2 M NaHCO3, pH
9.0 buffer and then added to 50 ml. of settled SepharoseR 4B
activated with BrCN according to the procedure as described in
Example 1. The gel-protein suspension was gently stirred in
room temperature over night. Then the gel was washed as the
gel according to Example 1.
Purification of heparin:
The gel adsorbent containiny matrix-bound protein
; was packed in a column. 300 mg. heparin (Vitrum) was dissolved
in 20 ml. of a 0.05 M Tris, 0.15 M NaCl, pH 7.4 buffer and
pumped into the column. The gel was washed with the abovementioned
buffer and the adsorbed heparin was then desorbed with a 0.05 M
Tris, 1.0 M NaCl pH 7.4 buffer. The salt was removed from the
heparin by gel filtrationon Sephadex G25 in distilled water
and then the heparin was freeze-dried. The specific activity of
the purified heparin was 230 units/mg.
