JPS6138171B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing immunoglobulin derivatives.

Immunoglobulin has medical significance as a carrier of coagulant immunity and has immunological activity against various pathogenic microorganisms. Therefore, by administering this immunoglobulin, it is possible to prevent and treat viral susceptibility diseases such as measles and viral hepatitis, as well as infections caused by antibiotic-resistant bacteria such as Staphylococcus aureus.

However, the administration of immunoglobulin fractionated and prepared from human plasma is limited to intramuscular injection, and intramuscular injection only allows a portion of the immunoglobulin to penetrate into the body, resulting in a rapid effect and making it impossible to administer large amounts. It is. Therefore, an immunoglobulin that can be injected intravenously is desired.

Intravenous injection of common immunoglobulin causes a severe increase in body temperature and blood pressure. This tendency is particularly remarkable in patients with agammaglobulinemia. Reactions due to intravenous injection are due to aggregates of immunoglobulins generated during isolation. Aggregated immunoglobulins, like antigen-antibody complexes, bind complement and tissue. The resulting anaphylactic factors produced in the body cause a reaction. The prepared immunoglobulin was 100,000×
This reaction can be prevented by using the supernatant obtained by ultracentrifugation. However, the immunoglobulin thus obtained also gradually undergoes reaggregation.

Much research has been conducted with the aim of making intravenous immunoglobulin injections safe. In Switzerland, by treating immunoglobulin with PH4
FC portion (Immunoglobulin G, which accounts for a large proportion of immunoglobulins, is normally cleaved into two types when cleaved with an enzyme such as papain. Of these, the one containing the amino terminus is called Fab, and the one containing the carboxyl terminus is called FC. Furthermore, when cleaved with pepsin, a product with two slightly larger molecules of Fab bound together was obtained, which is called F(ab')2).Attempts have been made to temporarily denature F(ab')2 and use it for intravenous injection. [See S. Barandum et al; Vox Sang 7 , 157, (1962)]. However, this treatment is unsafe and sometimes has adverse reactions. Pepsin-treated immunoglobulin is a commercially available intravenous immunoglobulin [H. Koblet, etal; Vox Sang. 13 , 92,
(1967)]. This means that 60-80% of it is F(ab′)
2, it is rapidly decomposed in the body, and its half-life is several hours, several tenths of that of the untreated product [B. Jager et al; .Intern.
Med.) 119 , 60 (1967)]. It is also improved by using plasmin instead of pepsin, but it has the disadvantage that plasmin cannot be removed [LA Hanson, et al: Int. Arch.
31 , 380, (1967)].

In recent years, immunoglobulins mainly contain interchain S.
It has been proposed to selectively cleave the -S bond and to S-alkylate the cleaved sulfur atom to reduce the reaction and to utilize such immunoglobulin derivatives for intravenous injection.

For example, Stephenson's method is known as an attempt to obtain a polypeptide chain by cleaving the interchain SS bonds of immunoglobulin [ST
Stevenson: Biochem.J. 118 , 703, (1960)]. The method involves reductively cleaving interchain SS bonds in human immunoglobulin G in dithiothreitol, followed by S-alkylation with iodoacetamide. However, acetamide bound by this method is hardly present in the normal body, and there are many uncertainties in terms of safety. In addition, the protected S-
The alkyl moiety is dispersed within the body after administration, and there is little possibility that it will be eliminated within the body even after the concern of anaphylaxis is gone, and it is unlikely to recombine with immunoglobulin and return to the S-S bond. Therefore, there is a drawback that favorable effects cannot be expected.

Furthermore, attempts have been made to decompose and sulfonate disulfide bonds in immunoglobulins using compounds that generate tetrathionate ions and sulfite ions in water (Japanese Patent Laid-Open Publication No. 121421/1983, etc.). This not only reduces complement binding through sulfonation and increases safety during administration, but also
S-SO ^- ₃ generated by cleavage of disulfide bonds
The group gradually leaves the sulfo group in vivo and restores the original globulin molecule without losing antibody activity.
It is characterized by being a globulin in its natural state with FC activity. It is known that after in vivo administration of this sulfonated immunoglobulin, it takes approximately 24 hours for the sulfo group to be removed and most of the immunoglobulin to be restored to its original globulin molecule (J.Biochem.79 , 1377-1379 (1976)).

The present inventor has conducted intensive research to obtain an immunoglobulin derivative that is expected to be highly safe as a formulation, and that is expected to quickly restore itself in the body and have faster efficacy when administered intravenously. The present invention was achieved by discovering that S-thiosulfate derivatives of immunoglobulin satisfy the objects of the present invention.
That is, the present invention reacts immunoglobulin with an oxidizing agent and a compound capable of forming thiosulfate ion in water to cleave the interchain disulfide bonds of the immunoglobulin and convert the cleaved sulfur atom into S-
This is a novel thiosulfated (-S-S ₂ O ^- ₃ ) immunoglobulin derivative. This is suitably used as an injectable composition. Therefore, an object of the present invention is to provide a method for producing an immunoglobulin derivative that can be suitably used as an injectable composition.

Another object of the present invention is to provide a method for producing a novel immunoglobulin derivative that has a reduced anti-complement value, retains excellent antibody activity in the body for a long time, and is fast-acting.

Still another object of the present invention is to provide a novel immunoglobulin derivative that can be quickly restored to natural immunoglobulin in an animal body, and a method for producing the immunoglobulin derivative that can be suitably used for injectable compositions. It is in.

Furthermore, another purpose is the S
-Provides a method for producing a sulfonated immunoglobulin intermediate.

That is, by reacting the S-thiosulfated immunoglobulin obtained by the method of the present invention with a compound capable of forming sulfite ions in water, the sulfur atoms of the interchain disulfide bonds or the interchain and intrachain disulfide bonds are converted to S- S-sulfonated (-S-SO ^- ₃ ) immunoglobulin can be produced.

The immunoglobulin used in the present invention is an immunoglobulin that can be obtained by a conventional method such as Cohn's low-temperature ethanol method, and has a molecular weight of about 160,000.
It is an immunoglobulin whose main component is 7S fraction.

The immunoglobulin used in the reaction in the present invention is prepared by dissolving the immunoglobulin in an aqueous solution of a neutral salt or an aqueous solution containing a stabilizer such as glycine and a solubilizing agent. The immunoglobulin concentration of the solution used for the reaction is 1 to 15 g/100 c.c., more preferably 5 to 10 g/100 c.c.

The method of the present invention is preferably carried out underwater. However, in some cases, the reaction may be carried out in a water-soluble medium or in a mixed medium of these and water, as long as the reaction of the present invention is not adversely affected.

The method of the present invention involves reacting an immunoglobulin as described above with (A) an oxidizing agent and (B) a compound capable of forming thiosulfate ions in water to remove the interchain disulfide bonds of the immunoglobulin or the interchain and chain bonds. S-thiosulfation of the sulfur atom of the internal disulfide bond (-S-S ₂ O ^- ₃ )
It is done by doing.

Examples of the oxidizing agent in (A) above include tetrathionic acid and alkali metal salts of tetrathionic acid such as sodium tetrathionate and potassium tetrathionate, compounds that can form tetrathionate ions in water such as ammonium tetrathionate, or compounds that can form tetrathionate ions in water such as ammonium tetrathionate; Also used are alkali metal salts of trithionic acid such as sodium trithionate and potassium trithionate, compounds capable of forming trithionate ions in water such as ammonium trithionate, and divalent copper ions. As the above-mentioned compound B which can form thiosulfate ions in water, sodium thiosulfate, sodium hydrogen thiosulfate, etc. are suitable. Of course, the material is not limited to these, and any material that can form thiosulfate ions in water may be used.

The amounts of the above-mentioned oxidizing agent and thiosulfate ion are at least twice the molar amount, preferably at least 10 times the amount of the interchain disulfide bond to be cleaved in the immunoglobulin. These thiosulfation reaction reagents are preferably dissolved in water or a buffer solution in advance and used as the reaction reagent of the present invention.

The reaction temperature is preferably 55°C or lower.
For example, a temperature of 30 to 55°C, particularly 40 to 50°C is suitable.

In the method of the present invention, in addition to the above reaction reagents, urea,
Although the reaction can be carried out by appropriately adding a protein denaturing agent such as guanidine, the absence of such a denaturing agent is rather preferable for the purposes of the present invention.

The reaction time varies depending on the amount of reagents, reaction temperature, and whether or not a denaturing agent such as urea is added. The pH of the reaction solution is approximately 4-10
A range of , particularly a range of 6 to 9, is preferred. There are no particular restrictions on the order of addition of the reaction reagents. After the reaction, a new immunoglobulin derivative can be produced by dialyzing the reaction mixture in, for example, physiological saline or an appropriate buffer, or by precipitating the immunoglobulin derivative with an appropriate salt and separating it from the reaction reagent. Furthermore, when heavy metal ions such as copper are used as an oxidizing agent, they may not be sufficiently removed by dialysis; in this case, they can be removed to 0.02Mole or less by using a compound that forms a metal chelate. . Further, purification by commonly used serum protein purification methods such as salting out, chromatography, or adsorption separation using poorly soluble salts can also be performed following the method of the present invention. It is even more convenient because things are removed.

The S-thiosulfated immunoglobulin derivatives obtained by the method of the present invention are highly safe, and as shown in the examples later, thiosulfate ions are quickly released in vivo and restored to natural immunoglobulins. do.
Furthermore, this product retains various antibody activities, has a reduced anti-complement value, and can be injected intravenously.

The S-thiosulfated immunoglobulin derivative obtained by the method of the present invention can also be used as an intermediate for S-sulfonated immunoglobulin derivatives. That is, the S-thiosulfated immunoglobulin obtained by the method of the present invention is further treated with sulfite in an amount twice or more molar relative to the thiosulfated disulfide bond according to a method separately proposed by the present inventors, for example,
S-sulfonated immunoglobulin derivatives can be easily obtained by reacting at a reaction temperature of 5 to 50°C. In this manner, S-sulfonated immunoglobulin derivatives can be easily produced.

For example, sodium thiosulfate used in the method of the present invention is used in producing S-sulfonated immunoglobulin derivatives. For example, it has higher storage stability than sodium sulfite, which makes quality control easier and industrially advantageous.

The method of the present invention will be described below in detail with reference to Examples, but the present invention is not limited thereto.

Example 1 PH of 9.3 g of sodium thiosulfate dissolved in 100 ml of 225% glycine solution containing 15 g of human immunoglobulin
50 ml of a phosphate buffered saline solution with a pH of 7.6 and 50 ml of a phosphate buffered saline solution with a pH of 7.6 in which 5.8 g of sodium tetrathionate had been dissolved were sequentially added, and the mixture was reacted at 50° C. for 5 hours. After the reaction is complete, the reaction reagent is reduced to 0.1mM in physiological saline.
200 ml of S-thiosulfated immunoglobulin at a concentration of 7.5% was obtained by dialysis until the concentration reached the following.

Anti-complement value of 5% solution of this [Kabat and
Mayer, “Experimental Immuno
According to the measurement method described in “Chemistry” P225 (61)]
_CH50 was 36.0%. The anti-complement value _CH50 of the raw human immunoglobulin used was 92%.
The sodium dodecyl sulfate disk electrophoresis (abbreviated as SDS disk electrophoresis) pattern of this product is as shown in the attached drawing, and there are some
H ₂ L ₂ chains are left, but H and L chains can be generated,
It was confirmed that the interchain SS bond was broken and thiosulfated. In addition, the diphtheria antibody titer of this product was 0.8 units/ml, which matched that of the raw material human immunoglobulin.

Example 2 10 ml of physiological saline in which 2.5 g of sodium thiosulfate was dissolved and 10 ml of physiological saline in which 0.77 g of sodium tetrathionate were dissolved were added sequentially to 50 ml of 2.25% glycine solution containing 5 g of human immunoglobulin, and incubated at 48°C for 7 days. Time reacted. After the reaction was completed, the reaction reagent was removed by dialysis against physiological saline to obtain 70 ml of S-thiosulfated immunoglobulin with a concentration of 7%. Next, add saturated sodium sulfate to this S-thiosulfated immunoglobulin aqueous solution for 13 hours.
ml, the resulting precipitate was centrifuged, and salting out purification was performed to obtain S-thiosulfated human with a 5% solution having an OD ₄₅₀ (absorbance at 450 mu) of 0.050 and an anti-complement value CH ₅₀ of 14%. Immunoglobulin was obtained.

Example 3 The 5% solution of S-thiosulfated immunoglobulin obtained in Example 1 was intravenously administered to rabbits, and blood was collected at 3 and 15 hours after administration, as described in the literature [J.
After treatment and separation according to Biochem. 79 , 1377-1379 (1976), Petersen and Dorrington of the method
We investigated the speed of restoration to the original globulin in vivo by SDS disk electrophoresis, but the SDS
The disk electrophoresis pattern was as shown in the figure, and was almost completely restored in 15 hours.

Example 4 50 ml of phosphate buffered saline containing 5 g of sodium thiosulfate and 50 ml of phosphate buffered saline containing 40 mg of copper sulfate were sequentially added to 100 ml of a 2.25% glycine solution containing 10 g of human immunoglobulin, and the mixture was incubated at 47°C. It reacted for 6 hours. After the reaction was completed, the reagent was removed by dialysis with physiological saline.

Next, in order to remove some residual copper () ions, 0.3% ethylenediaminetetraacetic acid was added, and an excess of saturated sodium sulfate aqueous solution was added to precipitate and separate the immunoglobulin derivative, which was dissolved in physiological saline. 200 ml of a 5% concentration S-thiosulfated immunoglobulin solution was obtained. This thing at concentration
The OD ₄₅₀ was 0.062 and the anti-complement titer CH ₅₀ was 9.7%.
Moreover, the copper() ion content was 0.01mMo or less.

[Brief explanation of the drawing]

Figure 1 of the accompanying drawings is an SD disc electrophoresis pattern of the immunoglobulin derivative obtained in Example 1 of the present invention. The figure shows Example 3 of the present invention.
These are the results of an experiment in which the immunoglobulin derivative of the present invention was intravenously injected into rabbits, and the subsequent SDS disk electrophoresis patterns are shown; a, b, and c are the results before and 3 hours after administration, respectively. and SDS disc electrophoresis pattern 15 hours after administration. Furthermore, in the figures and figures, H ₂ L ₂ represents immunoglobulin. In addition, H ₂ L represents one L chain removed from the two heavy chains (H) and two light chains (L) that make up immunoglobulin due to cleavage of the interchain SS bond. There is.
Similarly, _H2 refers to a substance that undergoes a similar cleavage and consists of two H chains, HL refers to a substance that consists of one H chain and one L chain, H refers to one H chain, and L A chain represents one L chain (the cleaved interchain SS bond is converted to -S-S ₂ O ₃ -).

Claims (1)

[Claims] 1. Reacting the immunoglobulin with an oxidizing agent and a compound capable of forming thiosulfate ions in water to cleave the interchain disulfide bonds of the immunoglobulin and convert the cleaved sulfur atom into S-thiosulfate (-S- _S2O ^- ₃ ) A method for producing an immunoglobulin derivative.