JPS632936B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for removing a pyrogen, and more particularly, to a method for removing a pyrogen, and more particularly, to a pyrogen-containing solution, a cellulose sulfate ester or a cross-linked polysaccharide sulfate obtained by sulfuric esterification of cellulose or a cross-linked polysaccharide is disclosed. The present invention relates to a method for removing pyrogens by subjecting the compound to affinity chromatography.

Technical background of the invention and industrial fields of application In general, injectable drugs and the like contain pyrogens in their raw materials or during the manufacturing process, and their removal may require a great deal of effort.

Pyrogens are a general term for substances that cause an abnormal increase in body temperature in warm-blooded animals, even in minute amounts.
It is classified into endogenous and exogenous. However, most of what is commonly called pyrogens are derived from exogenous microorganisms, especially bacterial pyrogens.
In particular, phospholipid polysaccharide (lipopolysaccharide), which is a component of the capsule of Gram-negative rods, is considered to be the culprit, and its presence in injectable drugs is extremely undesirable. That is, a substance called lipopolysaccharide is mainly a bacterial component or metabolite of Gram-negative rods, and has a molecular weight of about 6,000 to several million. Such lipopolysaccharides are not decomposed by the sterilization methods applied to ordinary liquid pharmaceuticals, their base materials or buffer solutions, and their production equipment, and are
It is extremely difficult to remove such lipopolysaccharides from solutions containing more than 6000 polymeric substances.

Conventional technology Conventionally, in the production of injectable drugs containing polymeric substances with a molecular weight of approximately 6,000 or more, purified water from which pyrogens have been removed by distillation, reverse osmosis, or ultrafiltration methods, or dry heat treatment, has been used. Techniques are widely used to prevent the contamination of pyrogens by using equipment that decomposes pyrogens through sterilization, acid or alkali treatment, etc., but it is generally not known to actively remove pyrogens from drugs. . A method is known in which pyrogens are inactivated by adding a small amount of polymyxin B, which is a peptide antibiotic, but in this case, the undesirable situation that polymyxin B remains until the end cannot be avoided. In order to avoid the contamination of pyrogens, careful attention is required in process control, which requires great care and effort, and also increases manufacturing costs. Moreover, if for some reason a pyrogenic substance such as lipopolysaccharide were to be mixed into an injectable drug, the drug would have to be disposed of, which would be extremely uneconomical.

Purpose of the Invention The present invention overcomes these disadvantages, removes pyrogens from injectable drugs as much as possible, improves medical safety, and provides a method for manufacturing drugs extremely economically. It is something to do. In addition, it has become possible to remove not only pyrogenic substances that have been introduced from the outside, but also causative substances contained in microbial cells and cultures that are the raw materials for preparations.

That is, the present inventors have conducted various studies to find a method to easily and efficiently remove pyrogens from solutions containing undesirable pyrogens such as those mentioned above, and as a result, we have found that cellulose or cross-linked polysaccharide is Using cellulose sulfate ester gel or cross-linked polysaccharide sulfate ester gel obtained by sulfate esterification as a gel for affinity chromatography, we have demonstrated that pyrogens can be specifically separated and removed by treating a pyrogen-containing solution. This led to the completion of the present invention.

Structure and Effects of the Invention The present invention is applicable to the production of preparations containing substances with a molecular weight of about 6,000 or more as active ingredients, such as various blood products and vaccines, by using affinity chromatography from a solution containing a pyrogen. When specifically removing only pyrogens,
The gel for Affinity chromatography is characterized by using a cellulose sulfate ester gel or a cross-linked polysaccharide sulfate ester gel obtained by sulfuric esterification of cellulose or cross-linked polysaccharide having a degree of polymerization, and the gel has a specific A novel drug that specifically adsorbs useful active ingredients such as plasma proteins, coagulation system proteins, complement, enzymes, and various viral and bacterial antigens, but does not adsorb pyrogens such as lipopolysaccharide. Based on knowledge, it specifically removes only pyrogenic substances.

Examples of pyrogen-containing solutions to be used in the method of the present invention include blood-derived preparations such as antithrombin, blood coagulation factors, platelet factor 4 lipoprotein lipase, and complement components, hepatitis B vaccines, Japanese encephalitis vaccines, influenza vaccines, Various virus-derived preparations such as rabies vaccine, antigenic activities and physiologically active products derived from transformed animal cells or transformed microorganisms, component vaccines and antigenic activities and physiologically active products derived from cultures of Bordetella pertussis etc. This includes, but is not limited to, various injectable drugs such as enzyme preparations and other enzyme preparations, especially drugs whose active ingredients are polymeric substances with a molecular weight of approximately 6,000 or more, and many drugs containing pyrogens such as lipopolysaccharide. applies.

The cellulose sulfate ester used in the present invention is obtained by sulfate esterifying cellulose, but preferably crystalline cellulose or
It is preferable to sulfate cellulose consisting of crystalline and non-crystalline regions. In this case, the cellulose sulfate ester obtained retains the shape of the raw material, has excellent physical stability, and is suitable as a gel for affinity chromatography. These raw material celluloses are already commercially available, such as Cellulofine GC-15, Cellulofine GH-25, Cellulofine GC-
100, GC-200 (manufactured by Chitsuso Corporation), and Avicel (manufactured by Asahi Kasei Industries, Ltd.). The desired cellulose sulfate ester can be obtained by reacting these gels with chlorosulfonic acid, sulfuric anhydride, etc. in the presence of an organic solvent such as pyridine, and neutralizing the gel with an alkaline solution such as sodium hydroxide.

Crosslinked polysaccharide sulfate is a crosslinked polysaccharide obtained by crosslinking polysaccharide such as dextran, cellulose, agarose, etc. with a crosslinking agent such as epichlorohydrin, dichlorohydrin, dibromohydrin, or ethylene glycol bisepoxypropyl ether. It is obtained by sulfuric acid esterification. Cross-linked polysaccharides are already commercially available, such as Cephadex G-10 and G-10 as cross-linked dextran.
-25, G-50, G-100 (manufactured by Pharmacia), etc., and as cross-linked agarose, Cepharose CL
-2B, CL-4B, CL-6B (manufactured by Pharmacia), etc. Cellulofine is a cross-linked cellulose.
There are GCL-25, GCL-90 (manufactured by Chitsuso), etc.
By treating these gels with chlorosulfonic acid, sulfuric anhydride, or the like in the presence of an organic solvent such as pyridine, the desired crosslinked polysaccharide sulfate ester can be obtained.

To remove pyrogens by column chromatography using these cellulose sulfate esters or crosslinked polysaccharide sulfate esters, operations employed in ordinary column chromatography are applied. For example, a column is filled with the cellulose sulfate or crosslinked polysaccharide sulfate (preferably a gel formed into spherical particles),
Add this to a suitable buffer solution with a pH of 5 to 8 and an ionic strength of about 0.001 to 1.0, such as 0.01M salt added with 0.1M salt.
After equilibrating with phosphate buffer (PH7.2),
A pyrogen-containing solution is passed through the column, and the column is washed by passing 5 to 10 volumes of the sample through the buffer solution used for the above-mentioned equilibration. This is followed by a suitable pyrogen-free buffer whose ionic strength is greater than that of the buffer used for equilibration, such as 0.01 M phosphate buffer (PH 6-9) containing 0.6 M NaCl. By eluting the target substance, a pyrogen-free solution of the target substance can be obtained.

In addition, in the above operation, the buffer solution
Chromatography can also be performed by adding a suitable surfactant such as Tween 80, NP-40, Triton X-100, etc.

The method of the present invention can be applied not only to column chromatography but also to batch methods, and can be carried out with simple operations on an industrial scale, and does not require any special expensive reagents, and can be used to remove pyrogens at an extremely low cost. Can be separated and removed.

EXAMPLES Next, the present invention will be explained in more detail with reference to Preparation Examples and Examples.

Preparation Example 1 117 g of chlorosulfonic acid is added dropwise to 600 ml of pyridine at a temperature below 0°C and mixed. After the dripping is finished,
Heat the mixture to 65-70°C. 80 g of Avicel for chromatography (manufactured by Asahi Kasei Co., Ltd.), which is crystalline cellulose, is added to this and kept at 65 to 70° C. for 4 hours while stirring. After the reaction is complete, cool and neutralize by adding 10% aqueous sodium hydroxide solution. The gel is over-separated and thoroughly washed with 0.01M phosphate buffered saline to obtain a cellulose sulfate gel.

Preparation Example 2 117 g of chlorosulfonic acid is added dropwise to 600 ml of pyridine at a temperature below 0°C and mixed. After finishing dropping,
Heat the mixture to 65-70°C. Add 80g of Cellulofine GH-25 (manufactured by Chitsuso) to this,
Hold at 65-70°C for 3 hours with stirring. After the reaction is complete, cool and neutralize by adding 10% aqueous sodium hydroxide solution. The gel is over-separated and thoroughly washed with 0.01M phosphate buffered saline to obtain a cellulose sulfate gel.

Preparation Example 3 Add 11 ml of chlorosulfonic acid dropwise to 200 ml of pyridine at a temperature below 0°C and mix. After the dripping is finished,
Heat the mixture to 65-70°C. 7.5 g of Cephadex G-50 (manufactured by Pharmacia), which is an epichlorohydrin-crosslinked dextran, is added to the mixture, and the mixture is maintained at 65 to 70°C for 4 hours while stirring. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution. The gel is overseparated and washed thoroughly with 0.01M phosphate buffered saline to obtain cross-linked dextran sulfate.

Preparation Example 4 Pyridine prepared in the same manner as in Preparation Example 3 above.
7.5 g of dried cellulofine GCL-25 (manufactured by Chitsuso Corporation), which is a crosslinked cellulose gel, is added to 210 ml of the chlorosulfonic acid mixture, and the mixture is reacted at 65 to 70°C for 4 hours. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution. The gel is hyperseparated and thoroughly washed with 0.01M phosphate buffered saline to obtain 7.2 g of crosslinked cellulose sulfate.

Preparation Example 5 Pyridine prepared in the same manner as in Preparation Example 3 above.
Add 30 ml of pyridine inclusion of Cephalose CL-6B (manufactured by Pharmacia), a cross-linked agarose gel, to 210 ml of chlorsulfonic acid mixture, and heat at 65-70°C.
Allow to react for 4 hours. After the reaction is completed, it is cooled and neutralized by adding an aqueous sodium hydroxide solution. The gel is hyperseparated and thoroughly washed with 0.01M phosphate buffered saline to obtain 23 ml of cross-linked agarose sulfate.

Example 1 A cross-linked agarose sulfate gel obtained in the same manner as in Preparation Example 5 was placed in a column (26.4 mmφ x 182
0.01M phosphate buffer (PH7.2) containing 0.1M saline after sterilization by passing 0.1% formalin into
Equilibrate by passing the liquid through. This column contains purified
HBsAg in 0.01M phosphate buffered saline [HBsAg
1.0μg/ml, rabbit fever test 2 rabbits total 1.8℃, pregel endotoxin test (manufactured by Teikoku Kinki Co., Ltd.)
10 ⁴ ] Pour 60ml of liquid. Then, 10 of the column volume
After washing with twice the volume of 0.01M phosphate buffered saline (pregel endotoxin test: negative), elution was performed with pyrogen-free 0.01M phosphate buffer containing 0.6M saline (pregel endotoxin test: negative). Obtain 18.0 ml of elution fraction.

This elution fraction contained 3.27 μg/ml of HBs antigen, and the recovery rate was 98.1%. When this fraction was adjusted to 1.0 μg/ml of HBs antigen with pyrogen-free 0.01 M phosphate buffered saline, the rabbit fever test resulted in a total of 0.7°C for the two rabbits, and the pregel endotoxin test showed a negative value, indicating that the pyrogen content was 1.0 μg/ml. had been almost completely removed.

Example 2 Cellulose sulfate ester gel prepared in the same manner as in Preparation Example 2 was sterilized under high pressure, and then thoroughly washed with pyrogen-free 1/75M phosphate buffered saline (PH7.2) on a glass filter. , equilibrate.
To this, add 1000 ml of Bordetella pertussis culture supernatant (HA value 512, total 3.0°C for 2 rabbit fever test, pregel endotoxin test: 10 ¹⁵ or higher), stir thoroughly, and then aspirate the solution. Next, stir thoroughly with pyrogen-free 1/75M phosphate buffered saline solution, and then aspirate. This washing operation is repeated four times. Finally, after adding pyrogen-free 1.5M table salt,
Add 0.05M phosphate buffer (PH7.0), stir thoroughly to suspend, and then aspirate the solution to obtain 120 ml of eluate.

From this, almost 100% of Bordetella pertussis HA (hemagglutinin) was recovered, and this eluate had an HA value of 5120,
Pre-gel endotoxin test: 10 ² , two rabbit fever test, total temperature was 0.6°C, and almost all pyrogens were removed. The rabbit fever test and pregel endotoxin test were carried out by adjusting the eluate to 12.5 μg protein/ml with the above buffer.

The fever tests in Examples 1 and 2 were both conducted in accordance with the fever standards of the Biological Products Standards.

Claims (1)

[Claims] 1. A method for removing a pyrogen by subjecting a pyrogen-containing solution to affinity chromatography, characterized in that a cellulose sulfate ester or a cross-linked polysaccharide sulfate ester is used as a gel for chromatography. How to remove the substance. 2. The method according to item 1 above, wherein the pyrogen-containing solution is an injectable drug. 3. The method according to item 1, wherein the pyrogen-containing solution is passed through an affinity gel for chromatography to wash and remove the pyrogen without adsorbing the pyrogen. 4. The method according to item 1, wherein the cellulose sulfate is crystalline cellulose or a cellulose sulfate consisting of a crystalline region and an amorphous region. 5. The method according to item 1, wherein the crosslinked polysaccharide sulfate is one selected from crosslinked cellulose sulfate, crosslinked agarose sulfate, and crosslinked dextran sulfate. 6. The method according to item 5 above, wherein the crosslinked cellulose sulfate is epichlorohydrin crosslinked cellulose sulfate. 7. The method according to item 5 above, wherein the crosslinked agarose sulfate is epichlorohydrin crosslinked agarose sulfate. 8. The method according to item 5 above, wherein the crosslinked dextran sulfate is epichlorohydrin crosslinked dextran sulfate. 9. The method according to item 1 above, wherein the affinity chromatography method is column chromatography or batch method.