Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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~ ne invention relates to the recovery of heparin
from ~nimal tissues.
Heparin has been available in medicine for a long
time as a naturally-occurring anticoagulant. Even in
modern medicine it is still indispensible and in~eed is
used to an ever increasing extent.
In xecent years, a number of processes have been
developed, aimed at isolating the active substance,
heparin, in an increasingly favourable manner from
heparin-containing animal tissue, such as the lungs,
liver and, morQ recently, especially from the intestinal
mucus (mucosa) of pigs, cattle and sheep (GB-PS 754 885,
DE~PS 1 228 241, US-PS 3 058 884, DE-PS 1 253 868).
Owing to the fact that they deteriorate easily, such
raw materials have the disadvantage of a limited stor-
ability and, although this can be prolonged by freezing,
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in addition to greater technical complexity this leads
to considerable increase in costs. Both the operatives
, ...
; and the residents in the adjoining neighbourhood are
not infrequently faced with unpleasant problems on
account of the nuisance caused by odours associated with
the processing.
In DE-PS 1 253 868 a few of the customary methods
of recovering heparin are su~marised. The animal tis-
sue, e.g. in_estinal mucus, is brought in the initial
stage into contact with a hot aqueous salt solution in
order to dissolve the heparin out of the cells. The
~ proportion of heparin in the resulting medium is extremely
- small.
We have now found that heparin is present in the brine pro-
duced in abbatoirs and plants for processing intestinal
mucus, This brine is formed when animal intestines, from
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which the int~stinal muc~is has pr~viously ~c!en rl~lnoved, are
s-teeped for preserVaiion and dehydration in ~sodlur~ chloride
solution, usllally at 0~C to room temperature and up to a
` maximum of 30C. A 3 to 5.6 molar sodium chloride solution
- may for example, be used~
The present invention provides a process for recovering
.:~
heparin from animal tissue, which comprises isolating
heparin from intestinal brine formed by treatment with aqueous
sodium chloride solution or solid sodium chloride in the
presence of water, of animal intestines from which intestinal
mucus has been removed. Usually the intestinal brine is
produced from pigs, cattle or sheep intestines.
The present invention also provides heparin which has
been recovered by the above process. The heparin may be
recovered, for example, in the form of sodium heparinate or
other physiologically tolerable salt of heparin.
The actual method of isolation of the starting material
used according to the invention, and optional subsequent
purification, may be carried out by a process known per se.
In view of DE-PS 1,253,868, it is to be regarded as
positively surprising that the brine produced when steeping
animal intestines in sodium chloride solution at low
temperature under gentle conditions contains relatively large
amounts of heparin. It was also unexpected that this
heparin is almost completely free from chemically allied
by-products. Thus the brine, previously regarded only as
a waste product polluting the environment, represents a new
source of raw material for heparin, now in short supply
owing to a scarcity of raw material. Owing to the absence of
impurities, the work-up to form crude heparin can be carried
out in an especially favourable manner, and the complicated
isolation methods (see US-PS 3,451,996 and GB-PS 1,221,784),
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accordin~ to the prior art are therefore unnecessary
Further, the quality of heparin obtained is very
much better than that of heparin preparations available
; ` on the market. Crude heparin isolat~d by known work--up
~ methods from the intestinal brine used according to the
. .
invention is already in such a state of purity that
subsequent purification yields heparin having far above
200 USP units per mg. The heparin preparations hitherto
available commercially generally have heparin qualities
of 150 USP units per mg, or, in a few cases, even 155
USP units per mg. One USP unit per mg is the specific
activity which is obtained from the U.S.P. (United
States Pharmacopoeia) assay, which measures the inhibi- -
tion of the formation of clots in preserved sheep's
~- p]asma. 1 USP unit corresponds approximately to 1.1
international units ~I.U.). USP prescribes, for
example, that heparin preparations (from intestinal
mucus) must contain at least 140 USP units. In spite
of numerous improvements, the activity values of com-
mercially available heparin preparations continue to be
unsatisfactory. L. W. Kananagh and L. B. Jaques
~ tArzneimittelforschung (Drug Research) 24, ~o. 12, 1942
"! (1974)] have succeeded in isolating heparin having a
maximum of 175 USP units per mg, but this has involved
repeated crystallisation of heparin in the form of its
barium salt and has only been performed on a laboratory
~cale.
Sodium chloride solutions having a sodium chloride
content of from 0.5 M to saturation are especially suit-
able for preserving and dehydrating animal intestines,
~ and are preferably used in the process of the invention.
; Especially preferred are those solutions of sodium
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chloride having from a 3-molar content of sodium chlor-
ide up to a saturation molar amount. The saturation
molar amount is the molar amount of dissolved sodium
chloride contained in a concentrated aqueous solution
at room temperature in an abbatoir. (A concentrated
aqueous sodium chloride solution has a strength of sub-
stantialiy 26.4 wt.%) A concentrated aqueous sodium
chloride solution may also be produced, for example, by
~ sprinkling the dripping wet intestines with sodium chloride.
Normally, the animal intestines, from which the mucus
has been removed, are treated with the cold sodium chloride
solutions for periods ranging from 2 hours to 3 days.
Usually, however, the treatment of the animal intestines
is complete after 3 to 24 hours. After the preserved
and dehydrated animal intestines have be2n r~moved from
the sodium chloride solution, an intestinal brine having
an unexpectedly high content of heparin is obtained.
Through the use according to the invention of such
intestinal brines as a raw material, heparin has also
been succ~ssfully isolated on an industrial scale in a
purity such that it is in the form of an almost colour-
less substance. Crude heparins available up to the
presen~ day from other raw materials have to be bleached
by expensive and wasteful methods (US-PS 3 179 566).
~oreover, since intestinal brine is a storable
almost odourless raw material that does not suffer any
loss in quality during long storage, chemical and other
preservation methods can be dispensed with.
The present invention also provides the use of
, 30 intestinal brine that has been obtained by steeping
animal intestines, from which the iniestinal mucus has
` ~ been removed, in aqueous sodium chloride solution as a source
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of heparin, and also to the isolation of heparln from
such sodi~n chloride solutions.
; The following Examples illustrate the invention.
In each case, the intestinal br~ne used was obtained by
steeping animal intestines, from which the mucus had
been removed, in aqueous sodium chloride solution of 26.4%
:
concentration at approximately 10C.
~xample 1
600 litres of methanol were added at room
temperature in the course of ~ hour, while stirring, to
600 litres of intestinal brine in an apparatus of 1500
litres capacity equipped with a stirrer. Stirring was
continued for a further ~ hour, after which the mixture
was allowed to stand for about one hour.
The turbid supernatant liquid was then decanted
into a second vessel, leaving behind the~ precipitated
salt cake. The suspension obtained was again decanted,
the supernatant liquid was discarded and the precipitate
isolated by centrifuging. After drying in vacuo, 1.44
kg of a product that has about 10 USP units per mg was
obtained. Up to about 45 % of it still consisted of
sodium chloride.
An aliquot of the crude heparin thus obtained, cor-
responding to about 425 USP units, or 45.2 g, was
extracted three times with 200 ml of 2 M sodium chloride
solution each time, stirring being carried out for one
hour at 60C in each case. The combined extracts were
diluted with water until the solution contained about
; 0.9 mole/litr~ of chloride ions. 200 ml of an anion-
exchanger (Lewatit CA 9249) in the chloride form we_e
added and stirring was carried out for one hour. The
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mixture was then suction filtered and washed ~ith about
200 ml of a 0.9 M sodium chloride solution. The ion-
exchal~ger was then stirred for five hours at 40C ~!ith
2 M sodium chloride solution, filtered orf ~Jith suction
and ~.~ashed with 2 M sodium chloride solution. The
` eluate was combined with the washing solution and pre-
- cipitated with 1.5 times the volume of methanGl. ~he
precipitate was centrifuged, washed first with a~o~t
50 ml of water-methanol (1 + 1.5 parts by volu~e) and
then with about 50 ml of methanol, and dried.
198 g of sodium heparinate having 195 USP units
per mg were obtained.
15.5 g of a sodium heparinate obtained in the a~ve
manner and having about 200 USP units per mg ~ere dis-
solved in 200 ml of 2 M sodium chloride solution. ~he
solution was filtered through a suction filter and the
residue washed first with 30 ml of 2 M sodium chloride
solution and then with about 250 ml of completely
desalinated water. The combined solutions were brought
to a c'nloride molarity of 0.9 using completely desalina-
ted ~ater. ~ne purification using Lewatit CA 9249 des-
cribed above was then repeated.
The combined elution and washing solution was pro-
cessed further as above. After washing and drying the
precipitate, 11.2 g of sodium heparinate, having an
activity of 251 USP units per mg, were obtained.
Example 2
In a manner similar to that described in GB-PS
754 885, 3 litres of intestinal brine were acidified
with acetic acid to a ~H of 3.1 to 3.2 (ab~ut 50 ml),
while stirring. After a few hours the mixture was
decanted and the resldue was centrifuged. T~.e
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pxecipitate was discarded, and the combined solutions
were neutxalised with sodium hydroxide solution. I
The same volume of methanol was then slowly added
while stirring. After 45 minutes the suspension waY
decanted from the precipitated sodium chloride. This
suspension was allowed to stand for several hours, the
supernatant liquid was decanted and the residue was
centrifuged. The precipitate was dried in vacuo.
3.43 g having 21 USP units per mg were obtained.
An aliquot of the crude heparin thus obtained, cor-
responding to about 425 000 USP~units, or 20.2 g, was
purified in a marner analogous to that of Example 1.
10.65 g of sodium heparinate, having an activity of 262
USP units per mg, were obtained.
Exam~le 3
3 litres of intestinal brine were diluted with 12
litres of water to a chloride molarity of about 0.85 M.
50 g of Xieselguhr and a solution of 6 g of benzethonium
chloride (= Hyamine 1622, Rohm & Haas) in 100 ml of water
were then added. Afier a few hours decantation was car-
ried out and the residue was filtered off with suction.
After being washed with water, the still moist precipi-
tate was extracted three times by the method of DE-PS
1 228 241 with 200 ml of a 2 M solution of sodium chlor-
ide each time. The combined extracts were precipitated
with 2 parts by volume of methanol. The isolated and
dried precipitate weighed 465 mg and had an activity of
155 USP units per mg.
Example 4
30 litres of intestinal brine were diluted to a
chloride molarity of about 0.85 M with 120 litres of
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water (cf. DE-PS 1 156 9~8). The mixture was then
acidified to a pTI of 3.2 with acetic acid (about 50 mi).
After several hours a precipitate had settled and was
centriuged after decanting the clear supernatant liquid.
After drying, 111 g of crude heparin having 6.5 USP
units per mg were obtained.
~n aliquot of the crude heparin thus obtained, cor-
- responding to about 42S 000 USP units, or 65.5 g, was
purified according to the method described in Example 1.
After the first purification stage over ion-exchanger,
196 g of sodium heparinate having 198 USP units per mg
. . . .
were obtained.
..
- ~ 15 g of a sodium heparinate obtained in the manner
described above)and having about 200 USP units per mg,
were dissolved in 200 ml of a 2N solution of sodium
chloride. The solution was filtered through a suction
filter, and the residue was washed first with 30 ml of
a 2~ solution of calcium chloride and then with about
250 ml of completely desalinated water. The combined
solutions were ~hen brought to a chloride molarity of
0.9 with completely desalinated water. Stirring, as
.
above, with or.e litre of Lewatit CA 92a9 was carried
out. The elution and washing were carried out with 2N
~; calcium chloride solution instead of with sodium chlor-
,~, .
ide solution.
Ihe combined elution and washing solution was fur-
ther worked up as above. After washing and drying the
precipitate, 10.7 g of calcium heparinate, having an
activity of 260 USP units per mg, were obtained.
Examnle 5
10 litres of intestinal brine wexe diluted with 40
litre~ of water to a chloride molarity of about 0.85 M.
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The solution was purnped (at the ~ate of about 3 litres
per hour) through a filter over a column of ion-
~xchanger, having a diameter of 26 r~m and a length of
380 mm. A medium basic macroporous anion-exchange resin
in the Cl -form (Lewatit CA 9249) was used. Other
anion-exchan~e resins may be used, such as Dowex 1-X-1
~i described in DE-PS 1 253 868 for example.
The resin was then removed from the column, washed
twice with 500 ml of an 0.9 M solution of sodium chlor-
l0.. ide, and eluted with 400 ml of a 2 M solution of sodium
chloride for 5 hours at ~0C, whi.le stirring. This
; solution was precipi'ated with 1.5 parts by volume of
methanol. The isolated and dried precipitate weighed
1.2 g and had 149 USP units per mg.
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