JPS6155895B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing immunoglobulin derivatives with a high monomer content. More particularly, the present invention relates to a method for obtaining S-sulfonated immunoglobulins with a high monomer content from immunoglobulins containing aggregates.

Immunoglobulin has medical significance as a carrier of humoral immunity and has immunological activity against various pathogenic microorganisms. Therefore, by administering this immunoglobulin, it is possible to prevent and treat viral infections such as measles and viral hepatitis, as well as infections caused by antibiotic-resistant bacteria such as Staphylococcus aureus. For the prevention and treatment of these diseases, intravenous injection is preferable to intramuscular injection because of the possibility of large-dose administration and rapid efficacy.
However, when immunoglobulin fractionated and prepared from human plasma is administered intravenously, there is a problem in that it causes anaphylaxis-like side effects accompanied by a drop in blood pressure, chills and fever, dyspnea, headache, and the like. This is because immunoglobulin fractionated and collected from human plasma often has some of its molecules aggregated into large molecules (aggregates), and immunoglobulin gradually aggregates during the formulation process. These aggregates bind to complement components in the blood (anticomplement activity) and activate them, releasing biologically active factors such as anaphylatoxin-like substances and vascular permeability factors. It is.

Various methods have been proposed to eliminate or reduce the side effects caused by such aggregates, but one of the best methods is to react immunoglobulin with an oxidizing agent such as sodium sulfite and sodium tetrathionate to induce immunostimulation. There is a known method for obtaining intravenously injectable sulfonated immunoglobulin, which has reduced anti-complement activity while retaining various antibody activities, by cleaving the SS bonds between peptide chains of globulin and S-sulfonating it. (For example, see Japanese Patent Application Laid-open No. 1630/1983). In the S-sulfonation reaction of immunoglobulin, immunoglobulin aggregates are not removed, but the aggregates are also S-sulfonated, which reduces anti-complement activity and is thought to result in a safe preparation. However, although S-sulfonated immunoglobulin aggregates have a lower anti-complement value and are safer than the original immunoglobulin aggregates, they still have some complement-fixing ability.
If it is contained in large amounts, it may cause side effects. Therefore, the above method has no problems when immunoglobulin with relatively few aggregates is used as a raw material for the sulfonation reaction, but it does not have any problems when using immunoglobulin that is relatively free of aggregates as a raw material for the sulfonation reaction, but it is difficult to use industrially available immunoglobulin, such as Cohn's ethanol fractionation method. The immunoglobulin obtained by this process contains many aggregates, and if this is used as a raw material for the S-sulfonation reaction, the anti-complement activity will not necessarily be sufficiently reduced, and there will still be problems with safety during intravenous injection. There were flaws.

In order to obtain intravenously injectable S-sulfonated immunoglobulin without the above-mentioned drawbacks, it is necessary to remove aggregates or dissociate the S-sulfonated immunoglobulin into monomers as much as possible. However, in the case of the method of removing aggregates, many monomers are also removed along with the aggregates, resulting in a decrease in the amount of monomer that can be used in the end. When you think about it, this is a huge disadvantage. The above problems can be solved by adopting a method of dissociating aggregates into monomers. However, all conventionally known methods for dissociating ordinary immunoglobulin aggregates into monomers have the following drawbacks. For example, a method is known in which immunoglobulin containing aggregates is treated with an acidic aqueous solution of pH 4 to dissociate the aggregates into monomers, but this method returns the PH of the immunoglobulin solution to neutral after treatment. The monomers start aggregating again, and if left at PH4 for a long period of time, the immunoglobulin will undergo denaturation. A method is also known in which aggregates are dissociated into monomers using the proteolytic enzyme plasmin, but this method has the disadvantage that it takes several days to dissociate aggregates, and furthermore, plasmin is released after treatment. It is difficult to remove completely, and if left untreated, immunoglobulin monomers will also be degraded. In addition, by adding sugars such as glucose and fructose, neutral amino acids such as glycine and alanine, or neutral salts such as sodium chloride and potassium chloride to the immunoglobulin solution at relatively high concentrations,
A method is known in which the aggregates are gradually dissociated into monomers while preventing the monomers from re-agglomerating (Japanese Patent Application Laid-Open No. 1983-20124). for 2 to 30 days (37℃ at 37℃)
It takes up to 7 days) and is difficult to use industrially.

As a result of intensive research to resolve the drawbacks of the conventional techniques, the present inventors discovered that when immunoglobulin is S-sulfonated, by allowing a nitrogen-based organic compound with a dissociation index PKb of 7 or less to exist in the reaction system, The present invention was achieved based on the discovery that S-sulfonated immunoglobulin with less aggregates can be obtained.

That is, the present invention provides S-sulfonation of immunoglobulin containing aggregates with an oxidizing agent and a compound that generates sulfite ions in water. This is a method for producing S-sulfonated immunoglobulin with a high monomer content, which is characterized in that an organic compound is present in the reaction system.

The immunoglobulin used as a raw material in the present invention refers to an immunoglobulin mainly composed of γ-globulin obtained from serum, plasma, other body fluids, or organ extracts by a known method such as Cohn's ethanol fractionation method. . These are usually 20% by weight
Contains aggregates with a sedimentation constant of 9S or more.

The S-sulfonation reaction of immunoglobulins involves cleaving the interchain SS bonds of immunoglobulins to form S-sulfonation, and is generally carried out using an oxidizing agent and a compound that forms sulfite ions in water. Compounds that form sulfite ions in water include sulfites such as sodium sulfite, potassium sulfite and ammonium sulfite; bisulfites such as sodium bisulfite, potassium bisulfite and ammonium bisulfite; sodium pyrobisulfite and potassium pyrobisulfite. , pyrobisulfites such as ammonium pyrobisulfite, and the like. As an oxidizing agent, it is desirable to use one that has low reactivity with sulfite ions.
For example, compounds that can form polythionate ions with 3 to 6 sulfur atoms such as trithionate ions, tetrathionate ions, pentathionate ions, and hexathionate ions in water, and compounds that can form divalent copper ions in water. , a compound capable of forming iodobenzoic acid ions in water, or a molecular oxygen-containing gas such as air (in this case, it is desirable to add a catalytic amount of cysteine or 2-mercaptoethylamine). The amount of the compound capable of forming sulfite ions in water is 2 times or more, preferably 10 times or more by mole, relative to the interchain SS bonds of the immunoglobulin to be cleaved. In addition, the amount of oxidizing agent is determined by
The amount used is 1 mole or more, preferably 2 mole or more relative to the SS bond.

The nitrogen basic organic compound used in the present invention has a PKb of 7 or less. here
PKb is PKb=-logk [K=[BH ⁺ ]/[B][H ⁺ ], [B] is the concentration of basic compound, [H ⁺ ] is the concentration of hydrogen ion, [BH ⁺ ] is the conjugate Represents the concentration of acid. ] is the dissociation index of basic compounds defined by Specific examples of these include primary alkyl amines such as methylamine and ethylamine; secondary alkyl amines such as dimethylamine and diethylamine;
Tertiary alkyl amines such as trimethylamine and triethylamine; cyclic saturated amines such as piperidine and pyrrolidine; nitrogen-containing heterocyclic compounds such as imidazole, 2-methylimidazole, pyrazole and triazole; basic amino acids such as lysine, ornithine and arginine; Examples include amide derivatives of neutral amino acids such as leucinamide, glycinamide, alaninamide, lower alkyl esters, and amine derivatives of glucose such as D-glucosamine. Particularly preferred are arginine, leucinamide, and D-glucosamine. Such nitrogen-based organic compounds are used as they are or in the form of water-soluble salts such as hydrochloride and hydrobromide, which are added to the reaction system.

In the present invention, the nitrogen basic organic compound (or its water-soluble salt) is added to the immunoglobulin solution in an amount of 10 to 600% by weight, preferably 20 to 600% by weight based on the immunoglobulin, before sulfonating the immunoglobulin.
It is added and mixed at a ratio of 400% by weight. If the amount of the nitrogen basic organic compound is less than 10% by weight, the effect of the present invention will not be sufficiently obtained, and if it exceeds 600% by weight, even though the effect of the present invention can be obtained, it will not be economical or effective. This is not preferable because it is disadvantageous in terms of operation. The temperature during addition is 0 to 50°C, preferably 0 to 30°C.

In the present invention, the sulfonation reaction is carried out in water, and the pH during the reaction is preferably in the range of 5.0 to 9.0. The reaction temperature is selected in the range of 0 to 50°C, preferably 10 to 45°C. Temperatures of 50°C or higher are undesirable because immunoglobulin molecules are susceptible to protein denaturation.
At temperatures below 0°C, the reaction progresses extremely slowly and is therefore not suitable for industrial use. The reaction time is until most of the interchain SS bonds of the immunoglobulin are cleaved and S-sulfonation occurs, and although it varies depending on the amount of reagents, reaction temperature, etc., it is generally selected between 0.5 and 24 hours.

According to the present invention, an immunoglobulin in which the interchain SS bonds of immunoglobulins are cleaved and the SS bonds are converted to S-sulfonate groups can be obtained. In this product, most of the H chain to H chain and H chain to L chain of the immunoglobulin are cleaved, but the three-dimensional structure of the immunoglobulin is maintained due to non-covalent bonds such as hydrogen bonds between each chain. . Separation of the product after the sulfonation reaction is generally performed using purification methods such as dialysis, salting out, and column chromatography. For example, by dialysis against physiological saline after the sulfonation reaction, a physiological saline solution of the target substance can be obtained.

The S-sulfonated immunoglobulin obtained according to the present invention has fewer aggregates than the S-sulfonated immunoglobulin obtained in a system without the addition of a nitrogen-based organic compound with a PKb of 7 or less, and therefore has less aggregates. It is extremely safe for intravenous injection.

The present invention will be explained in detail with reference to Examples below. All percentages in the examples mean percentages by weight.

In the Examples, the measurement of the monomer content of S-sulfonated immunoglobulin, the measurement of anti-complement value, and the sodium dodecyl sulfate (SDS) disk electrophoresis were carried out by the following methods.

[Monomer content of S-sulfonated immunoglobulin]

5% solution of S-sulfonated immunoglobulin 0.3
The content of monomers (sedimentation constant 7S, molecular weight approximately 160,000) was determined by gel permeation analysis using ml. Sepharose CL-6B (manufactured by Pharmacia) was used as the gel, and a column with a diameter of 1.5 cm and a length of 80 cm was used. The solution flow rate was 0.17ml/min.

[Anti-complement value]

The method described in Kabat and Mayer, Experimental Immunochemistry, p. 225 (published in 1961) was followed.

[SDS disk electrophoresis]

The obtained sulfonated immunoglobulin was processed by the method of Weber and Osborn [J.Biol.Chem244, 4406
(1969)], and the amount of unreacted immunoglobulin was measured.

Example 1 Human immunoglobulin (fraction by Cohn's ethanol fractionation method, monomer content 76.2%, anti-complement titer 90% <, antibody titer: diphtheria 2.0 units/ml)
1.5 g of l-arginine hydrochloride was added and dissolved in 30 ml of the 10% solution. Add 0.5 g of sodium tetrathionate to this solution in 0.1 M phosphate buffer with PH7.2 salt.
PH of the solution and 0.82 g of sodium sulfite dissolved in ml
A solution of 7.2 dissolved in 8 ml of 0.1M phosphate buffer with sodium chloride was added, and S-sulfonation reaction was carried out at 37°C for 4.5 hours. After the reaction was completed, the reaction solution was ice-cooled and dialyzed against a 0.9% saline solution to obtain 42 ml of a 6.5% solution of S-sulfonated immunoglobulin.
The obtained S-sulfonated immunoglobulin had the following properties.

Monomer content: 85.6% Anti-complement titer: 9.0% Immunoglobulin (H ₂ L ₂ ) amount 0.8% (by SDS disc electrophoresis) Antibody titer: Diphtheria 2.0 units/ml Comparative example 1 Add l-arginine hydrochloride Otherwise, the reaction was carried out under the same conditions as in Example 1, and a 6.7% S-sulfonated immunoglobulin solution was used.
Obtained 40ml. The monomer content of the obtained S-sulfonated immunoglobulin was 77.1%, and the anti-complement value was
It was 28.0.

Example 2 The reaction was carried out in exactly the same manner as in Example 1, except that 1.5 g of l-ornithine hydrochloride was used instead of l-arginine hydrochloride, to obtain 40 ml of a 6.4% solution of S-sulfonated immunoglobulin. The monomer content of the S-sulfonated immunoglobulin obtained was 84.1%.

Example 3 The reaction was carried out in exactly the same manner as in Example 1, except that 1.5 g of l-lysine hydrochloride was used instead of l-arginine hydrochloride, and S-sulfonated immunoglobulin was
41 ml of 6.6% solution was obtained. The monomer content of the S-sulfonated immunoglobulin obtained was 83.2%.

Example 4 The reaction was carried out in exactly the same manner as in Example 1, except that l-leucineamide hydrochloride was used instead of l-arginine hydrochloride, and 43 ml of a 6.3% solution of S-sulfonated immunoglobulin was obtained. The monomer content of the S-sulfonated immunoglobulin obtained was 85.3%.

Example 5 The reaction was carried out in exactly the same manner as in Example 1 except that D-glucosamine hydrochloride was used instead of l-arginine hydrochloride, and S-sulfonated immunoglobulin was
42 ml of 6.6% solution was obtained. The monomer content of the S-sulfonated immunoglobulin obtained was 83.9%.

Example 6 1.5 g of l-arginine hydrochloride was added and dissolved in 30 ml of a 10% solution of human immunoglobulin (fraction obtained by Cohn's ethanol fractionation method). Add 0.4 g of sodium trithionate to this solution and add salt to it at pH 7.2.
A solution dissolved in 2 ml of 0.1 M phosphate buffer and a solution of 0.82 g of sodium sulfite dissolved in 8 ml of 0.1 M phosphate buffer with pH 7.2 sodium chloride were added, and the reaction was carried out at 37° C. for 4.5 hours. After the reaction is completed, the reaction solution is cooled on ice,
By dialysis against 0.9% saline solution,
45 ml of 6.0% solution of S-sulfonated immunoglobulin
I got it. The monomer content of the obtained S-sulfonated immunoglobulin was 86.2%, and the anti-complement value was 8.7%.

Claims (1)

[Scope of Claims] 1. When immunoglobulin containing aggregates is S-sulfonated with an oxidizing agent and a compound that generates sulfite ions in water, the immunoglobulin has a concentration of 10 to 600
1. A method for producing S-sulfonated immunoglobulin with a high monomer content, characterized in that a nitrogen-based organic compound having a weight percent dissociation index PKb of 7 or less is present in the reaction system. 2. The method for producing S-sulfonated immunoglobulin with a high monomer content according to claim 1, wherein the nitrogen basic organic compound is a basic amino acid. 3. The method for producing S-sulfonated immunoglobulin with a high monomer content according to claim 1, wherein the nitrogen basic organic compound is an amide derivative of a neutral amino acid. 4. The method for producing S-sulfonated immunoglobulin with a high monomer content according to claim 1, wherein the nitrogen basic organic compound is an amine derivative of glucose.