Process for the preparation of injectable chondroitin polysulfate

The invention relates to a process for the preparation of an injectable chondroitin polysulfate, which is characterized in that PA0 A crude chondroitin polysulfate is depolymerized oxidatively, PA0 B the depolymerized chondroitin polysulfate is bleached, if necessary, with peracetic acid, PA0 C the product obtained after A or B is subjected once or twice to precipitation with methanol and/or ultrafiltration for fractionation with respect to molecular weight, PA0 D the product obtained after C is demineralized, if necessary, with cation exchangers, and PA0 E the product obtained after C or D is decolorized, if necessary, with active charcoal. The invention furthermore relates to a chondroitin polysulfate which can be prepared by the above process. The invention also relates to a chondroitin polysulfate with an acute toxicity (LD.sub.50), on intravenous administration, of >3,500 mg/kg in NMRI mice, of >3,000 mg/kg in Wistar rats, of >1,000 mg/kg in cats and of >1,000 mg/kg in dogs. Finally, the invention relates to a pharmaceutical composition for human and veterinary medicine, characterized in that it contains a chondroitin polysulfate which can be prepared according to the above process of a chondroitin polysulfate with the above characteristics.

Both the preparation of chondroitin polysulfate, in particular the 
sulfation of chondroitin sulfate, and various ways of treating chondroitin 
polysulfate crude products with the aim of purification have been 
described in the prior art (S. Bergstrom, Hoppe-Seyler's Z. f. physiol. 
Chemie 238, 163; 1936--Chargaff, F. W. Bancroft J. biol. Chem. 115, 149, 
155; 1936--Husemann et al. Z. Ges. Exp. Med. 114, 722; 1945--J. Piper Acta 
Pharm. Toxicol. 2, 317; 1946--P. Karrer, H. Koenig, E. Usteri, Helv. chim. 
acta 26, 1296; 1943--German Patent Specification 870,094--German Patent 
Specification 935,843--German Patent Specification 968,752--German 
Offenlegungsschrift 1,618,857--U.S. Pat. No. 3,454,560 and German 
Democratic Republic Patent Specification 136,572). However, none of these 
procedures leads to a satisfactory product. 
The toxicity, which is still too high in spite of technical advances, and 
other disadvantages of chondroitin polysulfates according to the prior art 
as yet prohibit risk-free therapeutic use of these substances in the form 
of an injection. 
The aim of the invention is therefore to obtain, in an economically 
advantageous manner, a highly pure chondroitin polysulfate in which the 
disadvantages of known chondroitin polysulfates are reduced to a minimum. 
It should be possible to use such a chondroitin polysulfate in the therapy 
of illnesses in humans and animals in any mode of administration. 
The above aim is achieved by a process, the essence of which can be seen 
from the nature and sequence of the process steps which follow: oxidative 
depolymerization of a crude chondroitin polysulfate corresponding to the 
prior art, if necessary bleaching with peracetic acid, isolation of a 
fraction with a mean molecular weight of 5,000-15,000 by precipitation 
with methanol and/or ultrafiltration, if necessary demineralization by 
means of ion exchangers and if necessary treatment with active charcoal. 
The invention thus relates to a process for the preparation of an 
injectable chondroitin polysulfate, which is characterized in that 
A crude chondroitin polysulfate is depolymerized oxidatively, 
B the depolymerized chondroitin polysulfate is bleached, if necessary, with 
peracetic acid, 
C the product obtained after A or B is subjected once or twice to 
precipitation with methanol and/or ultrafiltration for fractionation in 
respect of molecular weight, 
D the product obtained after C is demineralized, if necessary, with cation 
exchangers, and 
E the product obtained after C or D is decolorized, if necessary, with 
active charcoal. 
The processes known from the prior art, for example those such as are 
described in the above-mentioned literature references, can be used for 
obtaining the crude chondroitin polysulfate used as the starting material. 
Without restricting the invention to this method or to one of the 
abovementioned methods of preparing crude chondroitin polysulfate, it is 
possible to start from a product which is obtained by sulfation of 
chondroitin sulfate with pyridine and chlorosulfonic acid. Chondroitin 
polysulfates of widely varying coloration can be used. It is also possible 
to use high-molecular chondroitin polysulfates originating from stage C of 
the present process. 
The following reagents can be used to adjust the pH value in the process 
described below: acids such as inorganic acids, for example hydrochloric 
acid, and lower aliphatic carboxylic acids or peracids thereof, for 
example acetic acid or peracetic acid, and alkalis such as alkali metal 
hydroxide solutions, for example sodium hydroxide solution or potassium 
hydroxide solution. The individual steps of the process are preferably 
carried out under normal pressure. Unless otherwise indicated, the 
percentage data in the following text are "weight/volume" data. In the 
present process, methanol is given as the precipitating agent for 
intermediates and the end product. Other water-miscible organic liquids, 
such as, for example, ethanol, isopropanol, acetone, tetrahydrofuran and 
dioxane, are also similarly suitable. The intermediates produced in the 
individual steps of the process do not necessarily have to be isolated as 
dry substances. It is also possible to further process the solvent-moist 
masses without drying them. Salts in a concentration range of 0.1-10%, 
preferably 0.5-5%, are advantageously added during precipitation of the 
chondroitin polysulfate substances with water-miscible solvents. Salts 
which are particularly suitable are sodium or potassium chloride and 
sodium or potassium acetate. 
The conditions for stages A to E of the process according to the invention 
which are described in more detail in the following text (page 8 paragraph 
3 to page 16 paragraph 3) can be varied within a substantial range. 
Accordingly, the parameters given below for the individual stages are not 
to be understood as obligatory but only as an advantageous embodiment of 
the invention, so that the invention is not restricted to these 
parameters. 
Chondroitin polysulfate can be depolymerized with hydrogen peroxide in the 
absence of heavy metals. The depolymerization step can be carried out in 
aqueous solution under the following conditions: the concentration of the 
crude chondroitin polysulfate can be 2-35%, preferably 10-20%. The 
concentration of hydrogen peroxide in the reaction medium can be 0.3-15%, 
preferably 1-5%. The reaction temperature can be 20.degree.-100.degree. 
C., preferably 50.degree.-100.degree. C. The reaction time is in the range 
from 10 minutes to 24 hours, preferably from 30 to 200 minutes. The pH 
value can be kept constant at between 3 and 9 or can also remain 
unadjusted, and is preferably between 4.5 and 7. 
Heavy metals which are present can be blocked by complexing agents such as 
nitrilotriacetic acid, ethylenedinitrilotetraacetic acid, 
1,2-cyclohexylenedinitrilotetraacetic acid, diethylenetriaminepentaacetic 
acid, 3,6-dioxaoctamethylenedinitrilotetraacetic acid or mixtures thereof 
or alkali metal salts thereof. The concentration of the complexing agent 
in the reaction medium is kept between 0.001 and 0.5%, preferably between 
0.005 and 0.15%. When the reaction has ended, the reaction product can be 
precipitated with an excess of methanol, after addition of an alkali metal 
salt and either without cooling or after cooling, and isolated. 
The depolymerization of chondroitin polysulfate with hydrogen peroxide can 
also be carried out in the presence of heavy metal salts, under 
particularly mild conditions. The depolymerization is carried out, for 
example, as follows: the concentration of the crude chondroitin 
polysulfate can be 2-35%, preferably 15-30%. The concentration of hydrogen 
peroxide in the reaction solution can be 0.5-15%, preferably 1-5%. The 
reaction temperature can be in the range from -10.degree. C. to 
+80.degree. C., preferably from 0.degree. C. to 30.degree. C. The reaction 
time can be in the range from 5 minutes to 24 hours, preferably from 30 to 
500 minutes. 
The pH value can be between 3 and 8, preferably between 4.5 and 7.5. Heavy 
metal salts which have proved suitable are inorganic and organic salts of 
iron, cobalt, copper, manganese and vanadium. The metals of these salts 
are preferably in the following valency levels: iron and cobalt: di- and 
tri-valent, copper: mono- and di-valent, manganese: di-, tri-, tetra-, 
hexa- and hepta-valent, and vanadium: di- and penta-valent. The 
concentration of metal salt is adjusted to between 0.001 and 0.5%, 
preferably between 0.05 and 0.2%. After the degradation, the pH value is 
adjusted to within the range from 5 to 14, preferably from 6 to 10, and 
the depolymerized product is isolated by precipitation with methanol. 
The step of oxidative depolymerization of chondroitin polysulfate can be 
carried out by oxidative degradation of chondroitin polysulfate with 
peracetic acid in the presence of certain heavy metal salts with the same 
result as the above methods. The following conditions have been found to 
be those under which the reaction can be realized in an advantageous 
manner: the concentration of the crude chondroitin polysulfate can be 
5-35%, preferably 10-25%. The concentration of peracetic acid in the 
reaction solution can be 0.1-7%, preferably 0.5-2%. The heavy metal salt 
concentration can be within the range from 0.001 to 0.5%, preferably 0.005 
to 0.2%. The reaction temperature can be -10.degree. C. to +90.degree. C., 
preferably 0.degree. C.-30.degree. C. The reaction time can be within the 
range from 5 minutes to 24 hours, preferably from 15 minutes to 500 
minutes. The pH value can be 2-12, preferably 3-8. 
The reaction proceeds at a good rate, for example, with inorganic or 
organic salts of the following heavy metals: iron, cobalt, copper, 
manganese and vanadium. As regards the valency of the metals in these 
metal salts, the statements made above for depolymerization with hydrogen 
peroxide apply. It may be advisable to stop the depolymerization operation 
by means of a chelating agent, such as, for example, 
ethylenedinitrilotetraacetic acid or other metal-blocking agents, 
especially in the case of relatively short reaction times. The 
depolymerized chondroitin polysulfate can be precipitated with methanol, 
if necessary after addition of a neutral salt and adjustment of the pH 
value to within the range from 3 to 14, in particular from 6 to 10. 
Crude chondroitin polysulfates which differ greatly in purity and 
coloration are suitable for use in the present process. In spite of the 
depolymerization operation, which greatly lightens the color, it may be 
necessary to include a decoloration step or a bleaching step before 
further processing. Bleaching is carried out with peracetic acid. The 
following conditions have been found to be advantageous: the pH range can 
be from 2.5 to 14, preferably from 3 to 11. The peracetic acid 
concentration can be 0.1-10%, preferably 1-5%. The concentration of the 
crude chondroitin polysulfate can be 2-40%, preferably 5-25%. The reaction 
is carried out in the temperature range from 0.degree. to 100.degree. C., 
preferably from 15.degree. to 70.degree. C. The reaction time can be 
between 1 minute and 96 hours, in particular between 1 and 24 hours. 
According to a preferred embodiment, the peracetic acid treatment can be 
incorporated in the subsequent molecular fractionation process. As has 
already been mentioned above, the peracetic acid treatment can also be 
omitted completely. 
The molecular weight range from about 5,000 to 15,000 is fractionated out 
of the product obtained after the bleaching or depolymerization step by 
precipitation with methanol at various concentrations. 
For this step, the depolymerized and, if necessary, bleached chondroitin 
polysulfate can be treated in aqueous solution with methanol in the 
presence of a neutral salt as follows (1st precipitation with methanol): 
the concentration of the depolymerized chondroitin polysulfate in the 
aqueous solution can be 5-30%, preferably 5-20%. The pH value can be in 
the range from 2 to 10, preferably from 3.0 to 7.5. The neutral salt 
content can be 0.5-10%, preferably 1-5%. A preferred neutral salt is 
sodium chloride. The temperature can be in the range from 0.degree. to 
50.degree. C., preferably from 15.degree. to 30.degree. C. The amount of 
methanol can be 25-45% by volume, preferably 30-40% by volume, relative to 
the entire treatment medium. High-molecular and highly sulfated 
chondroitin polysulfate entities are precipitated, together with 
impurities, and can be removed by filtration or centrifugation. 
The methanol-containing solution which remains is brought to a methanol 
concentration of 60-85% by volume, preferably 75-80% by volume, relative 
to the entire treatment medium, by further addition of methanol (2nd 
precipitation with methanol), and a chondroitin polysulfate precipitate 
from which the very low-molecular and low-sulfated components are removed, 
together with impurities, is obtained; these components are not 
precipitated. 
The entire fractionation process with methanol or a part thereof can be 
replaced by ultrafiltration. For example, by ultrafiltration of aqueous 
solutions of depolymerized chondroitin polysulfate with membranes with an 
exclusion limit of 6,000 and above (for example membranes of cellulose 
acetate, polyamide or polysulfone), it is possible to retain the same 
first fraction as described above in the retained material. The 
concentration of the chondroitin polysulfate solution can be 1-10%, 
preferably 1-5%. The pH value of the solution can be 2-14, preferably 
5-10. The permeate can be freed from low-molecular chondroitin polysulfate 
components by precipitation with methanol as described above. 
This effect can also be achieved by a second subsequent ultrafiltration 
with membranes with an exclusion limit of 8,000 and below. The 
concentration of the chondroitin polysulfate solution can be 1 to 10%, 
preferably 1 to 5%. The pH value of the solution can be 2 to 14, 
preferably 5 to 10. 
The exclusion limit for substances of a certain molecular weight given by 
the manufacturers of ultrafiltration membranes in most cases does not 
apply to chondroitin polysulfates. The suitable membrane in an individual 
case must therefore be established by experiment, but experience shows 
that it is within the ranges indicated. 
The removal of salts, if necessary heavy metal ions, and impurities is 
achieved by treatment with a cation exchanger. The metal ions are bonded 
by exchanger resins in the protonated form and the free 
mucopolysaccharide-polysulfuric acid passes into the filtrate or eluate. 
The treatment can be carried out at a temperature from 0.degree. to 
50.degree. C., and advantageously at room temperature. It can be carried 
out by the batch method by stirring in the cation exchanger, or by the 
continuous or discontinuous column technique. Suitable ion exchanger 
resins are strongly acid products, such as, for example, Lewatit S 100, 
Dowex X 50 or Amberlite IR 120 in the protonated form. 
The batch method is carried out in aqueous solution with chondroitin 
polysulfate concentrations of 2-20%, in particular 5-15%. The amount of 
ion exchanger resin can be 2-10 parts by volume, in particular 3-8 parts 
by volume, relative to 1 part by weight of chondroitin polysulfate dry 
substance. The removal of salts can also be carried out under the same 
conditions using exchangers poured into columns. 
The strongly acid filtrate or eluate from the demineralization step is 
subjected to precipitation with 2.5-6 parts by volume, preferably 3-4 
parts by volume, of methanol in the presence of 0.5-5% of a neutral salt, 
in particular 0.5-3% of sodium chloride. During the precipitation from an 
acid medium, free sulfuric acid and low-molecular chondroitin polysulfate 
components as well as impurities remain in solution. After the 
chondroitinpolysulfuric acid precipitate has been separated off, it can be 
either reprecipitated from neutral solution or further used directly for 
the subsequent process step whilst still methanol-moist. 
Demineralization can be omitted if ultrafiltration has been carried out 
using an ultrafiltration membrane with a low exclusion limit (2nd 
ultrafiltration), in which case the retained material is further 
processed. 
For further lightening of the color and in order to achieve a particularly 
good color stability during heat sterilization of the pure chondroitin 
polysulfate, demineralization can be followed by a further purification 
step. 
Treatment of chondroitin polysulfate solutions with active charcoal and 
complete removal of the adsorbate gives a chondroitin polysulfate solution 
from which, after the pH has been adjusted, the product is precipitated 
with methanol in the presence of a neutral salt. The concentration of the 
aqueous chondroitin polysulfate solution to be treated is 5-25%, in 
particular 10-20%. Its pH value is 4-10, in particular 5-8. 0.1-3 parts by 
weight of active charcoal (relative to one part by weight of chondroitin 
polysulfate) are employed. The temperature can be 20.degree.-100.degree. 
C. The treatment time is 10-180 minutes. 
The active charcoal is separated off. Fine filtration through a membrane 
filter is then advisable, in order to obtain a completely charcoal-free 
solution. The pH value is adjusted to 6-10, in particular 7-8, and the end 
product is precipitated with methanol in the presence of a neutral salt, 
for example sodium chloride in a concentration of 0.5-5%, in particular 
1-2%. The final concentration of methanol is adjusted to 60-85% by volume, 
preferably 75-80% by volume. Examples of suitable active charcoals are 
Brilonit normal (Lurgi), active charcoal pure, Cat.No. 2183 (Merck), 
active charcoal pure, powder, Cat.No. 18003 (Riedel de Haen). Whether 
decoloration with active charcoal is carried out depends on the 
chondroitin polysulfate used as the starting material. 
It may be advantageous to carry out the precipitation of the purified 
chondroitin polysulfate with methanol in the presence of a complexing 
agent and/or sodium pyrosulfite. This measure is particularly expedient in 
the case of the end product. As a result thereof, injection solutions 
which contain chondroitin polysulfate and which, when stored, are free 
from turbidity and subsequently darken only unnoticeably remain. Examples 
of complexing agents which are used are nitrilotriacetic acid, 
ethylenedinitrilotetraacetic acid, 1,2-cyclohexylenedinitrilotetraacetic 
acid, diethylenetriaminepentaacetic acid and 
3,6-dioxaoctamethylenedinitrilotetraacetic acid, in concentrations of 
0.01-0.5%. Concentrations of 0.05-0.35% are preferably used. The 
concentration of the sodium pyrosulfite which may be used during the 
precipitation with methanol can be 0.01-0.5%. 
The chondroitin polysulfate described is of therapeutic significance in 
humans and animals. The substance has an anticoagulemic, thrombolytic, 
thromboprophylacetic (especially on the deep venous thromboses), 
antiatherosclerotic, antilipemic, antiinflammatory, antiexudative, 
antiarthrotic and virustatic action. 
A characteristic novel substance which is distinguished by a surprisingly 
low toxicity is obtained by the particular course and the nature of the 
treatments of the process according to the invention. 
TABLE 1 
______________________________________ 
Acute toxicity 
Animal species 
Administration 
LD.sub.50/7d 
______________________________________ 
NMRI mice i.v. &gt;3,500 mg/kg 
Wistar rats i.v. &gt;3,000 mg/kg 
Cats i.v. &gt;1,000 mg/kg 
Dogs i.v. &gt;1,000 mg/kg 
______________________________________ 
The invention thus also relates to a chondroitin polysulfate with an acute 
toxicity corresponding to a lethal dose, on intravenous administration, of 
&gt;3,500 mg/kg in NMRI mice, of &gt;3,000 mg/kg in Wistar rats and of &gt;1,000 
mg/kg in cats and dogs. A preferred product according to the invention has 
an acute toxicity corresponding to a lethal dose, on intravenous 
administration, of &gt;4,000 mg/kg in NMRI mice, of &gt;3,500 mg/kg in Wistar 
rats, of &gt;1,500 mg/kg in cats and of &gt;1,000 mg/kg in dogs. 
Chondroitin polysulfates with a LD.sub.50 (mice, i.v.) of only 1,000 mg/kg 
have so far been described in the literature. The process according to the 
invention thus permits the preparation of chondroitin polysulfate with a 
considerably wider therapeutic range. 
In particular, the chondroitin polysulfate prepared by the process 
according to the invention displays the following parameters: 
nitrogen: 1.65-2.00% 
sulfur: 12.5-14.5% 
hexuronic acid: 0.98-1.22 mmoles/g 
hexosamine: 0.96-1.24 mmoles/g 
Finally, the invention relates to a pharmaceutical composition, which is 
characterized in that it contains a chondroitin polysulfate which can be 
prepared by the process according to the invention or a chondroitin 
polysulfate as has been defined above. 
Experiments have shown that single dosages of between 20 and 250 mg, in 
particular between 30 and 150 mg, per individual in 0.2-2.0 ml, in 
particular 0.2-1.0 ml have a therapeutic action in humans and are well 
tolerated. Daily doses of up to 500 mg prove to be therapeutically 
advantageous. The injection can be intramuscular, intraarticular, 
subcutaneous or intravenous, without the doctor being tied specifically to 
only these types of injection. Therapeutically usable solutions of the 
active compound can be packed, for example, in ampoules, ready-to-use 
syringes, pierceable bottles, phials for injection guns and other primary 
means of packing appropriate for the injection. Other methods of 
administration are peroral, percutaneous, rectal, sublingual or buccal 
administration. The preferred dosage corresponds to that for 
administration by injection. 
The chondroitin polysulfate obtained according to the invention is also 
suitable for use in veterinary medicine, in which it shows the actions 
described. The dosage for dogs is between 0.1 and 5 mg/kg of body weight 
and day, preferably 0.5 to 1.5 mg/kg, in a volume of 0.1-10 ml, in 
particular 0.5-1.0 ml. In the case of horses, for example, single daily 
doses of 100-1,000 mg, preferably 150-500 mg, in 0.1-20 ml, in particular 
1-5 ml, prove to be suitable. The chondroitin polysulfate can be 
administered in the same manner as to humans. 
The following method was used to determine the mean molecular weight: 
A 20 mg sample is chromatographed into 2M sodium chloride solution on a 
Biogel P 10 column (50 g; H=100 cm, .phi.=2 cm). The chondroitin 
polysulfate is detected in the 10 ml fractions collected, by hexuronic 
acid determination in accordance with the method of Z. Dische: J.Biol. 
Chem. 167, 189 (1947) modified by J. D. Gregory: Arch, Biochem. Biophys. 
89, 157 (1960). Chondroitin polysulfate fractions in which the molecular 
weight has been determined by ultracentrifugation are used as standards. 
The mean molecular weight is calculated from the volume of eluate in which 
50% of the total amount of hexuronic acid found in the chromatogram is 
eluted.

The following examples illustrate the invention. 
EXAMPLE 1a 
(Stage A) 
7.5 kg of crude chondroitin polysulfate (obtainable according to German 
Patent Specification 870,094) are dissolved in 75 liters of hot water, and 
2.25 kg of sodium chloride and 2 ml of octan-1-ol are added. A solution of 
75 g of ethylenedinitrilotetraacetic acid in 750 ml of 1N sodium hydroxide 
solution is now added. The mixture is heated to 95.degree. C. and 7.5 
liters of 30% strength hydrogen peroxide solution are then stirred in. The 
temperature is kept between 90.degree. and 95.degree. C. for 100 minutes 
and the mixture is then cooled rapidly to room temperature. 
1.5 kg of sodium chloride are dissolved in the reaction mixture, 360 liters 
of methanol are stirred in and, after 30 minutes, the clear supernatant 
liquor is decanted off and the precipitate is filtered off over a suction 
filter. The material on the filter is washed with methanol and dried at a 
temperature below 60.degree. C. About 7 kg of depolymerized chondroitin 
polysulfate are obtained. 
EXAMPLE 1b 
(Stage A) 
7.5 kg of crude chondroitin polysulfate are dissolved in 22.5 liters of 
water, and first 1.5 kg of sodium chloride and then a solution of 37.5 g 
of copper-II sulfate pentahydrate in 750 ml of water are added. 3 liters 
of 30% strength hydrogen peroxide are added, with cooling, and the mixture 
is kept at 25.degree. C. for 5 hours. It is then adjusted to pH 10.5 with 
30% strength sodium hydroxide solution, precipitation is effected with 132 
liters of methanol, with stirring, the supernatant liquor is decanted off 
and the precipitate is filtered off with suction. The material on the 
filter is washed with methanol and dried at a temperature below 60.degree. 
C. About 6.8 kg of depolymerized chondroitin polysulfate are obtained. 
EXAMPLE 1c 
(Stage A) 
9 kg of crude chondroitin polysulfate are dissolved in 45 liters of water, 
a solution of 8.57 g of cobalt-II chloride hexahydrate in 1 liter of water 
is added and the mixture is made up to 67 liters with water. 1.35 liters 
of 40% strength peracetic acid are added at room temperature in the course 
of 2 hours, with continuous stirring. A solution of 27 g of disodium 
ethylenedinitrilotetraacetate dihydrate in 1 liter of water is then added. 
The pH is now adjusted to 11 with 30% strength sodium hydroxide solution, 
2.2 kg of sodium chloride are added and precipitation is carried out with 
290 liters of methanol, with stirring. After one hour, the supernatant 
liquor is decanted off, the precipitate is filtered off on a suction 
filter and the material on the filter is washed with methanol and dried at 
a temperature below 60.degree. C. About 8.4 kg of depolymerized 
chondroitin polysulfate are obtained. 
EXAMPLE 2 
(Stage B) 
8.0 kg of depolymerized chondroitin polysulfate are dissolved in 74 liters 
of water, the solution is mixed with 2 liters of 40% strength peracetic 
acid, the pH value is adjusted to 8 with 30% strength sodium hydroxide 
solution and the mixture is left to stand at room temperature for 24 
hours. 0.4 kg of sodium chloride is dissolved in the mixture and 
precipitation is carried out with 320 liters of methanol. The precipitate 
is filtered off, washed with methanol and dried. About 7.9 kg of white 
material are obtained. 
EXAMPLE 3a 
(Stage C) 
6.3 kg of depolymerized chondroitin polysulfate are dissolved in 27.1 
liters of hot water, 3.15 liters of 40% strength peracetic acid are added, 
the components are mixed and the mixture is left to stand for 4 hours. 
45.4 liters of water are then added, 2.1 kg of sodium chloride are 
dissolved in the mixture and the pH value is adjusted to 10.5 with 30% 
strength sodium hydroxide solution. In order to obtain a methanol 
concentration of 36.5% by volume in the mixture, 0.5748 liter of methanol 
per liter of solution are then stirred in, that is to say, for example 
48.6 liters of methanol. The precipitation mixture is left to stand 
overnight and the supernatant liquor is then decanted off and the 
semiliquid precipitate is centrifuged off. The clear supernatant liquor, 
for example 128.5 liters, is stirred with 2.175 liters of methanol per 
liter of solution, that is to say, for example, 279.5 liters of methanol, 
so that a final methanol concentration of 80% by volume is achieved. The 
precipitate is allowed to settle for 1 hour, the supernatant liquor is 
decanted off and the residual suspension is filtered with suction. The 
precipitate is washed with methanol and dried at a temperature below 
60.degree. C. About 5.6 kg of intermediate are obtained. 
EXAMPLE 3b 
(Stage C) 
2 kg of depolymerized chondroitin polysulfate are dissolved in 200 liters 
of water and the solution is filtered through a sterile filter and 
subjected to ultrafiltration under a pressure of 2.8 bars at 32.degree. C. 
using a plate ultrafiltration apparatus (membrane area: 2.25 m.sup.2, 
exclusion limit stated for the cellulose acetate membrane: molecular 
weight of 15,000). The solution is pumped round at a flow rate of 6.5 
m.sup.3 /hour. When the volume of retained material is about 20 liters, 
this material is subjected to ultrafiltration 3 times, after being mixed 
with 50 liters of water each time. 
The combined permeates are subjected to ultrafiltration using a cellulose 
acetate membrane with a stated molecular weight exclusion limit of 6,000, 
but otherwise under identical conditions to those above. The retained 
material is concentrated down to a concentration of 10%, sodium chloride 
is added up to a concentration of 0.5%, the pH value is adjusted to 7.5 
and precipitation is carried out with 4 parts by volume of methanol, with 
stirring. After washing the precipitate which has been filtered off with 
suction, it is dried. The resulting intermediate weighs 1.78 kg. 
EXAMPLE 4a 
(Stage D) 
5.4 kg of the intermediate from process step 3 are dissolved in 54 liters 
of water, 21.6 liters of strongly acid cation exchanger (for example 
Lewatit S 100) in the protonated form are stirred in and the mixture is 
stirred at room temperature for 1 hour. The cation exchanger is filtered 
off with suction and rinsed twice with 8.1 liters of water each time and 
the filtrates are combined. 2.19 kg of sodium chloride are added to the 
filtrate and precipitation is carried out with 292 liters of methanol, 
with stirring. The precipitation batch is left to stand overnight and the 
supernatant liquor is then decanted off, the suspension which remains is 
filtered with suction and the material on the filter is washed out 
thoroughly with methanol. The precipitate is dissolved in 43.2 liters of 
water, the pH value is adjusted to 7.5 with 30% strength sodium hydroxide 
solution, 700 g of sodium chloride are added and precipitation is carried 
out with 139.2 liters of methanol, with stirring. The clear supernatant 
liquor is decanted off, the residual suspension is filtered through a 
suction filter and the material on the filter is washed with methanol. 
After drying, 3.6 kg of demineralized chondroitin polysulfate are 
obtained. 
EXAMPLE 4b 
(Stage D) 
9 kg of the intermediate obtained after process step, or the still 
methanol-moist precipitate from this process step, are dissolved in an 
amount of water such that 90 liters of solution are obtained. This 
solution is allowed to run through an ion exchanger column filled with 36 
liters of the cation exchanger Amberlite IR 120 in the H.sup.+ form. The 
ratio of the height to which the column is filled to the diameter is, for 
example, about 2.5:1. When the substance solution has run through, the 
column is rinsed with 33.3 liters of water. 3.7 kg of sodium chloride are 
added to the combined filtrates containing the substance and precipitation 
is effected by addition of 492 liters of methanol. The precipitate is 
filtered off with suction and washed with methanol and the still 
solvent-moist precipitate is dissolved in pyrogen-free water to give a 
total volume of 72 liters. 1.15 kg of sodium chloride are added, the pH 
value is adjusted to 7.5 with 30% strength sodium hydroxide solution and 
precipitation is carried out with 219 liters of methanol. The precipitate 
is filtered off with suction, washed with methanol and dried. About 6 kg 
of demineralized chondroitin polysulfate are obtained. 
EXAMPLE 5 
(Stage E) 
20 kg of demineralized chondroitin polysulfate are dissolved in 190 liters 
of pyrogen-free water. The solution is heated to 90.degree. C. and 40 kg 
of active charcoal are added (Riedel de Haen No. 18003). The temperature 
is kept at 90.degree. C. for 60 minutes and the mixture is then filtered 
whilst still hot using a pressure filter. The charcoal residue is washed 
twice with 40 liters each time of pyrogen-free water, and the filtrates 
are combined. 3 kg of sodium chloride and 660 g of disodium 
ethylenedinitrilotetraacetate dihydrate are added. The pH value is 
adjusted to 7.5. The mixture is then filtered through a pre-washed 
membrane filter (pore diameter: 0.2 .mu.m). The completely clear solution 
is subjected to precipitation with 720 liters of methanol, with stirring. 
The precipitate is filtered off with suction, washed with methanol and 
dried at a temperature below 50.degree. C. About 18 kg of white, 
fine-particled, pure, injectable chondroitin polysulfate are obtained. 
Investigation by chemical analysis gives the following substance values: 
N: 1.77% 
S: 12.96% 
hexuronic acid: 1.08 mmoles/g 
hexosamine: 1.12 mmoles/g 
EXAMPLE 6 
(Preparation of an injection solution) 
3.25 kg, calculated relative to anhydrous substance, of pure chondroitin 
polysulfate and 0.325 kg of sodium chloride are dissolved in about 50 
liters of aqua pro iniectabilia, with stirring, and the solution is made 
up to 65 liters. After the pH value has been adjusted to 8.4 with N sodium 
hydroxide solution and the solution has been subjected to sterile 
filtration through a membrane filter, the solution is filled into ampoules 
under sterile precautions in a known manner. The sealed ampoules are 
exposed to heat treatment in a boiling water bath for 60 minutes.