CS195693B2 - Method of preparing derivatives of immunoglobulin - Google Patents

Description

The invention relates to a process for the preparation of novel immunoglobulin derivatives suitable for aqueous injection preparations containing these globulin derivatives.

Along with the current advances in the field of protein chemistry, the dependence of immunoglobulin structure on its function, which plays a very important role for humoral immunity against tumors, has been clarified.

Studies have shown that immunoglobulin can be divided into five subtypes, called IgC, IgA, IgM, IgD, and IgE, with at least 70% by weight of IgC.

Immunoglobulins with the exception of IgM consist mainly of the four peptide chains, and contain some interchain and intra-chain disulfide bonds. For example, IgC, the most typical of immunoglobulins, can be fractionated by papain digestion into two fragments, the "antigen-binding" Fab and the complement-binding "Fc" and tissue. Furthermore, it has been found that IgC consists of two polypeptide heavy chains (H chains) and two light poly-peptide chains (L chains) that are tightly bound by disulfide bonds and non-covalent bonds to form a protein molecule of about 160,000 molecular weight, and that the molecule contains, on average, about 4.5 interchain di195893 sulfide bonds and about 12 intra-chain disulfide bonds, see B, Frangione and C. Milstein, J. Mol. Biol. 33, 893 (1968).

Based on previous empirically established facts, numerous studies have been conducted with regard to the use of immunoglobulins for intravenous injection. Immunoglobulin utilization has taken place rapidly after the end of Cohn fractionation, see E. C. Cohn et al., J. Amer. Chem. Soc. 88, 459 (1946).

Cotin and coworkers fractionated large amounts of human plasma by ethanol precipitation. Fraction II is predominantly IgC, containing minor amounts of IgA and IgM, and has activity against antibodies of various microorganisms causing infectious diseases. Therefore, administration of Fraction II appears to be effective in the prophylaxis and therapy of diseases caused by viruses, such as measles, viral hepatitis, as well as infectious diseases caused by antibiotic-resistant microorganisms such as Streptococcus aureus.

Intramuscular injections are the only means of administering such fractionated and modified globulin. However, intramuscular injection only results in partial osmosis of the injected liquid into the body, and the effect is not immediate. Intramuscular injection also does not allow the administration of larger amounts of fluid. Thus, it is highly desirable to prepare an immunoglobulin suitable for intravenous injection.

Intravenous injections of known immunoglohulins cause severe pyrogenic and cardiovascular reactions, which are particularly noticeable in χ-globulinized patients. These adverse reactions in intravenous injections are conditioned and caused by immunoglobulin aggregates contained in the fractionated immunoglobulin. Immunoglobulin aggregates bind to complement and tissue, like the antigen-antibody complex, in an undesirable reaction caused by anaphylactic factor produced in the body. The adverse reaction can be avoided by using supernatant which has been separated from the treated immunoglobulin by a half-centrifugation of 100,000 g, but nevertheless there is a slow re-aggregation in the treated immunoglobulin.

Numerous studies have been done to prepare truly appropriate intravenous immunoglobulin injections. In Switzerland, it has been attempted to process immunoglobulin at pH 4 to temporarily denature the Fc portion, and the adjusted portion is suitable for intravenous injection, see S. Barandum et al., Vox Sang I, 157 (1962). However, the repair is imperfect and undesirable reactions can still be observed occasionally.

Pepsin-modified immunoglobulin is commercially available for intravenous injections, see H. Koblet, S. Barandum and H. Diggelman, Vox Sang. 13, 92 (1967). Since 60 to 80% of the modified immunoglobulin consists of the F (ab ') 2 fragment, the injection liquid breaks down in vivo rapidly, as evidenced by its half-life of several hours, which is one tenth to several tenths with respect to untreated immunoglobulin, see B. Jager, Arch. Intern. Copper. 119, 60 (1967). Even better injectable fluids can be obtained by replacing pepsin with plasmin, but this procedure has the drawback that plasmin cannot be removed at a later stage, see L.A. Hanson & B.G. Johnson, Int. Sheet. Allergy 31: 380 (1967).

More recently, it is proposed to cleave selectively mainly the interchain disulfide bonds of an immunoglobulin and alkylate the formed sulfur atoms and to use the immunoglobulin derivatives thus obtained, which are characterized by a reduction in adverse reactions in intravenous injection.

For example, as one of the attempts to cleave the interchain disulfide bonds of an immunoglobulin to obtain polypeptide chains, the procedure described by S. T. Stevenson et al., Biochem. J. 118: 703 (1960); characterized in that it is reductively cleaved by interchain disulfide bonds of human immunoglibulin G by dithiothreitol followed by alkylation of the sulfur atoms in iodoacetamide. However, this process is a two-step process and, in addition, alkylated polypeptides are obtained as a product. It is also not easy to convert the S-moieties of the thiol group. Therefore, it is also difficult to obtain a suitable reagent on a suitable carrier such as a suitable polymer using an active thiol in this manner.

For the procedure described by Franek, see jColl. Czechoslov. Chem. Commus. 29, 1401. (1964), depends on the reaction of porcine immunoglobulin with sodium sulfate and copper ions to form S-sulfonated polypeptides.

However, when reproducing the experiments described by Frannek et al., The following drawbacks have been found:

(1) the denatured polypeptides obtained contain copper or copper ions extremely difficult to remove to substantially unrecoverable; (ii) denatured polypeptides are characterized by rapidly decreasing antigen binding, although they may have the desired low anti-complement activity, (III) and the original immunoglobulin conformation is disrupted; modified as seen from the measurement of optical activity, as evidenced by the control results below.

The fact that it is virtually impossible to remove minor residual copper from the extended-polypeptide products of Frank et al. Means that the product obtained is thermally unstable, prone to aggregate formation and is apparently physiologically undesirable for intravenous injection, cf. DA Derbe, Arthritis and Rheumatism 17, 85 (1974).

SUMMARY OF THE INVENTION It is an object of the present invention to provide novel immunoglobulin derivatives containing neither copper nor copper ions, selectively cleaving interchain disulfide bonds linking two polypeptide heavy chains (H chains) and two polypeptide light chains (L chains) in the immunoglobulin, or cleaving in addition to said interchain disulfide bonds a portion of intra-chain disulfide bonds, and by cleavage, exposed sulfur atoms are sulfonated (S-sulfonated) ·; The invention also relates to the preparation of aqueous preparations for injection containing new immunoglobulin derivatives.

The novel immunoglobulin derivatives have reduced anti-complement efficacy while maintaining extremely high antibody efficacy in the human body. for a longer period of time they exhibit low undesirable properties and excellent antibody activity with the possibility of reconstructing native immunoglobulin in vivo.

The invention relates to a process for the preparation of novel immunoglobulin derivatives of uniform quality and stability by selective cleavage of essentially only interchain disulfide bonds linking the immunoglobulin heavy chains (H chains) and light chains (L chains) with sulfonating sulfur atoms.

According to the process of the invention, novel immunoglohulin derivatives are produced at β to 5 interchain disulfide bonds, or inter-chain and intra-chain linkages of native immunoglobulin, cleaved to form sulfur atoms which are sulfonated to form a grouping of the formula SS-SOs.

Accordingly, the present invention provides a method of preparation; characterized by cleaving the interchain and intrachain disulfide bonds of an immunoglobulin and sulfonating the formed sulfur atoms by reacting with a compound capable of cleaving the tetrathionate ions and with a compound capable of cleaving sulfite ions simultaneously or sequentially in any order, in aqueous a pH of 3.5 to 10, and in the absence of oxidizing metal ions, such as flushed ions, at a temperature of 10 to 50 ° C, wherein the tetrathionate and sulphite ions in the reaction medium are at a molar concentration of 2- and 100-fold. immunoglobulin binding.

In particular, the novel immunoglobulin derivatives of the present invention are characterized in that they selectively cleave on average about 3 to 4.5, in particular about 4.5, of the substantially interchain disulfide bonds of native itoglobulin globulin, and so on. by cleavage of the exposed sulfur atoms, the process is carried out essentially in the absence of oxidizing metal ions such as copper ions.

The novel immunoglupulinine derivatives prepared by the process according to the invention are characterized in that:. ...

1. are produced by cleavage on average 3 to 5 interchain or 3 to 5. 5 interchain and intra-chain disulfide bonds of native immunoglobulin by sulfination by cleavage of the resulting sulfur atoms to form groups of the formula —S — SOs;

2. The immunoglobulin derivative contains at least 1.0 limit. units / ml of anti-diphtheria titer at a concentration of 10.0% by weight.

Water-soluble preparations useful in the manufacture of injectable solutions may be prepared from the immunoglobulin derivatives of the invention by mixing with non-toxic solvents. Also by dissolving the soluble formulation. in water, aqueous formulations for intravenous infection may be prepared. ;

Particularly preferred immunoglohulin derivatives of the invention are those having an average of substantially 4.5 cleaved disulfide bonds of the native immunoglobulin on average, and the resulting sulfur atoms (substantially on average 9 atoms) are sulfonated. These preferred immunoglobulin derivatives are formed by cleavage essentially of only the interchain disulfide bonds linking the H-chain and L-chain of the native immunoglobulin, and the resulting sulfur atoms are sulfonated.

The novel immunoglobulin derivatives of the invention can be prepared by reacting a native immunoglobulin with (A) a compound capable of forming tetrathionate ions and a (B) compound capable of forming sulfite ions in water so that I cleaves on average 3-5 interchain disulfide bonds, not -, inter-chain and intra-chain disulfide bonds of native immunoglobulin, and the resulting sulfur atoms are sulfonated (S-SOs). First, the predominantly interchain disulfide bond of the native immunoglobulin can be cleaved.

. Therefore, immunoglobulin derivatives of the invention wherein on average 3 to 4.5 intramolecular disulfide bonds of native immunoglobulin are cleaved and the resulting sulfur atoms are sulfonated can be considered to be products where essentially only interchain disulfide bonds to the immunoglobulin are cleaved, and the resulting sulfur atoms were sulfonated.

The process according to the invention is preferably carried out in water, but the medium used is by no means limited to water and any other aqueous medium can be used as long as it has no adverse effect on the reactions of the invention. .

In accordance with the invention, the following are treated:. a native immunoglobulin (A) a compound from which tetrathionate ions may be formed; and (B) a compound from which sulphite ions may be formed, in water. such that, on average, 3 to 4.5 interchain disulfide bonds are cleaved, or in addition to substantially interchain, fewer intra-chain double bonds, with the number of intramblecular disulfide bonds cleaved at a maximum of 5, and substantially all of the resulting sulfur atoms are sulfonate.

as compound (A) from which tetrathionate ions can be formed in water, tetrathionic acid, its corresponding alkali metal salts, such as sodium, potassium and the like, as well as other salts of said acid, if they are soluble in water, for example an ammonium salt, which is preferred, and any other substance can be used as long as it results in the formation of tetrathionate ion in water.

Typical examples of compounds (B) from which sulphite ions can be formed in water include sulfuric acid, sodium or potassium sulphite, sodium sulphite, but any other compound can be reused when sulphite ions are formed in the water.

Compound (A), from which tetrathionate ions are formed in water, acts as an oxidizing agent. On the other hand, compound (B), from which sulfite ions are formed in water, acts as a reducing agent as well as an S-sulfonating agent.

The amount of sulphite and tetrathionate ions in each of the two examples should be such that it corresponds to at least a 2 molar ratio, converted to interchain, or interchain and intrachain disulfide

195B93 binding of native globulin to be cleaved. It can also be 100-fold (mplarly) with respect to the bonds to be cleaved, or even multiple.

It is preferred to dissolve compounds (A) and (B), especially (BJ) in water or buffer solution before being added to the reaction system of the invention.

The reaction according to the invention can be described by cleavage of the disulfide bonds of native immunoglobulin by sulfite ions, one of the sulfur atoms being converted to the S-sulfonate group (—S — SO3) and the other to a thiol group (—SHJ, and in each case 2 of the resulting thiol groups are oxidized with tetrathionate ions to form a disulfide bond, or the thiol group can react with tetrathionate ions to form S-thiosulfate (· - S-S2O3), and this group reacts with sulfite ions. repeat.

As a result of the described reaction, the interchain disulfide bonds of native immunoglobulin are cleaved and the sulfur atoms thus formed are in both cases converted to S-sulfonate groups (-S-SO3J in both heavy and light chains).

The process according to the invention is carried out / preferably at a temperature of at most 50 ° C, in particular in the range of 10 to 50 ° C, in particular 15 to 48 ° C, and of course it is possible to carry out the process at lower temperatures.

Usually, at temperatures above 50 ° C, especially above 55 ° C, native immunoglobulin can be denatured, and intra-chain disulfide bonds are cleaved. So similar high temperatures should be avoided.

Rather, the present process avoids the presence of protein breakers, such as urea or guanidine, used in S-sulfitolysis of insulin or ribonuclease, see J. L. Bailey et al., J. Biol. Chem. 234, 1733 (1959). However, if such substances are used, they may not exceed 2.0 moles per mole of native immunoglobulin.

The reaction time varies depending on the type of immunoglobulin, the amount of reagent, the reaction temperature, and the presence of a denaturant such as urea and the like. Preferably, the pH in the reaction liquid is about 3.5 to 10, especially 6 to 9. At pH values below 3.5, intra-chain disulfide bonds in the Fc portion of the native immunoglobulin are more likely to be cleaved. Beyond pH values above 10, the globulin protein is denatured, which is associated with undesirable consequences such as inhibiting or reducing anti-complement efficacy and reducing antigen binding activity.

The procedure in which to. adding the individual components in the reaction according to the invention is not critical.

After the reaction, the reaction mixture is dialyzed against water and a suitable buffer solution, such as aqueous sodium chloride solution containing 0.01 M phosphate buffer (pH .8).

7.4J and 2.5% glycine, and immunoglobulin derivatives of the invention can then be obtained.

Moreover, the novel immunoglobulin derivatives of the invention can be further subdivided into. L-chains and H-chains (light and heavy chains) by column chromatography using a packed column of a suitable resin, for example a molecular sieve based on zextran cross-linked to a three-dimensional structure, useful for filtering gels termed Sefadex G-75 using solvents such as 1 M propionic acid solution.

However, the process according to the invention is by no means limited to the above-mentioned and specific conditions.

Every process is within the scope of the invention,

1- (4), (4), (4), (4), (4), (4), (4), (4), (4), and (B) sulphite ions in water, as described above, and cleaving predominantly interchained. disulfide bonds of native immunoglobulin and sulfonation with sulfur atoms released by cleavage.

The number of S-sulfonate groups (-S-SO3) in the novel immunoglobulin derivatives of the invention can be determined, for example, by carrying out a similar procedure using radioactive sodium sulfate and determining the S-sulfonate groups labeled ⁵⁵ S in the resulting immunoglobulin derivative by liquid scintillation counting. shown in the examples.

Half of the average number of labeled S-sulfonate groups per molecule of immunoglobulin derivative thus determined corresponds to the average number of cleaved II -ramolecular disulfide bonds in the parent native immunoglobulin.

On the other hand, immunoglobulin derivatives containing an average of about 9-S-sulfonate groups per molecule, i.e., groups formed by cleavage on average

4.5 disulfide bonds of native immuno. globulin by sulfonating the resulting sulfur atoms of the invention, fractionating to H and L chains using, for example, a sephadex G-75 or G-200 column, preferably in the presence of less urea or guanidine, and using a propionic acid solution as described above .

Any combination of reaction conditions may be used in the method of the invention as long as they are suitable for preparing immunoglobulin derivatives containing an average of 9 S-sulfonate groups per molecule that can be further fractionated into H and L chains using the column chromatography described above. Once such a combination of reaction conditions has been empirically determined, the number of cleaved disulfide bonds in native globulin can be appropriately controlled by adjusting the reaction time to the selected combination of reaction conditions.

The process according to the invention is explained in more detail in the appended non-limiting examples and in the accompanying drawings. . ' Giant. 1 expresses the relationship between the reaction time, plotted in hours on the x-axis, and the number of groups —S — SO3 ”contained in the reaction product; this number is expressed in moles / mol of immunoglobulin Ig. Use of sodium tetrathionate marked with white circles, use of divalent copper compounds for oxidation marked with black circles. The arrow indicates the addition of urea.

Giant. 2 expresses the relationship between the reaction time (indicated in hours, on the x-axis) and the anticomplement activity (indicated in percent on the y-axis) of the immunoglobulin derivative obtained by oxidation with both the hatriumtetrathic and the bivalent copper compounds. The arrow again shows the addition of urea.

Giant. 3 shows the relationship between the reaction time (indicated on the x-axis in hours) and the anti-diphtheria titer (on the y-axis in international units / ml) of the obtained immunoglobulin derivatives. The empty bars correspond to the oxidation with sodium tetra-thionate, while dashed with divalent copper compounds. The vertical line at the end of each column shows the deviation from the anti-diphtheria titer. The arrow indicates the addition of urea.

Giant. 4 shows the relationship between the reaction time indicated on pse x in hours and the optical rotation limit of [α] _D ²⁵ ° C of the obtained immunoglobulin derivatives. The reaction is carried out using a 2.5% immunoglobulin solution (similar to FIGS. 5 and 6). Oxidation by sodium tetrathionate. Marked by white wheels, divalent copper compounds by black wheels. The arrow again indicates the addition of urea.

Giant. 5 shows the haze change in heat treatment of immunoglobulin derivatives with a change in reaction time. The time in hours is indicated on the x-axis; on the y-axis, the OD of ₆₅ nm at 65 nm, which is the turbidity index.

' Giant. 6 shows the relationship between the reaction time indicated in hours on the x-axis and the copper content indicated in µg / ml on the y-axis in the obtained immunoglobulin derivatives.

Giant. 7 shows the results of dialysis in urea solution and chromatography of the reaction product. The x-axis shows the fraction number, the y-axis the OD ₂₈ o at 280 μιη.

Giant. 8 the upper part shows the relationship between the OD ₂₈ o plotted on the left axis y and the radioactivity expressed in dpm (decrease / min) plotted on the right Y-axis fractions plotted on the x-axis divided by dialysis treated urea and chromatographed;

the lower part shows the molar fraction of the S-SOs groups, as calculated by the decrease in radioactivity according to the diagram; on the y-axis the molar fraction of the groups "S" SOj "is indicated, on the x-axis the fraction number.

Fig. 9 shows the optical density (on the y-axis) of each fraction (on the x-axis) separated by S-sulfonated immunoglobulin chromatography in connection with Fig. 7; however, urea treatment is not carried out. - ’

Giant. Figure 10 shows the results of an in vivo anti-complement assay of S-sulfonated globulin. The black circles correspond to the β-sulfonated-globulin, the white salt, Λ the human immunoglobulin used for the control. The x-axis indicates the time in minutes after the intravehous guinea pig injection, and the y-axis indicates the complement titer.

Giant. 11 shows the in vivo stability of S-sulfonated globulin. The x-axis shows the number of days after intravenous injection of the rabbit, and the y-axis shows the titer indicating passive hemagglutination. The curve jj is valid for the business (ab‘j2, the curve -υ-φ- © is for S-sulfated anti-BS A-DNP-immunoglobulin and curve-Δ-jk-for intact BSA-DNP-immunoglobulin.

In the procedure used in the examples, the following reagents were used:

Iminoglobulin (human) I: 15% human immunoglobulin solution for intramuscular injection in buffered saline (neutral pH) containing 2.5% glycine (product of the Chems-Sero-Therapeutical Research Institute);

χ-globulin Fr (human), II (human immunoglobulin II), Nutritional Biochemieals Corporation (USA);

Sodium tetrathionic acid, prepared immediately before use by the reaction of sodium thiosulfate (Na 2 SO 2. 5 H 2 O) and iodine in aqueous ethanol, sodium sulfite (Komagna Kagaku Yakuhin, Co), sodium sulfite (NazSO 3), labeled ³⁵ S ⁺ (New England Nuclear, USA).

<. Tris HCl buffer, i.e. tris (hydroxymethyl) aryinomethane (Nakarai Kagaku Yakuhin Co), spec. Product for biochemical studies from pH reduced by addition of 1 N aqueous hydrogen chloride solution.

Antibody potency and complementary potency are measured as follows:

Measurement of antidiphtheria titer:

Reaction: a sensitized passive haemagglutination reaction is used (Note 1). Blood cells: human red blood cells (note 2) treated with formaldehyde are exposed to bis-diazobenzidine (note 3) and sensitized with diphtheria toxin.

Control: Standard Antibody on Diphtheria (NIH, Japan), (Note 1) NIH (Japan) method, see B. Murata, No. 21, Tokyo Vetianian & Animal Husbandry (1975), (Note.

2), see W. T. Butler, J. Immunol. 90, 663 (1963), (note 3), J. Dordon, B. Rose, A. H. Sehon, J. Exp. Copper. 108, 37 (1958).

, Measurement of measles, rubella and mumps antibodies (Notes 4 to 9):

Reaction: Microplate procedure except for mumps where the tube procedure is used.

9 5 8 9 4

Antigen: Hemagglutinating antigens [Toshiba Chemicals].

Control serum: Standard antiserum (Toshiba Chemicals), except mumps using standard antiserum (NIH, Japan). 'Note 4: haemagglutination inhibition, mumps test by microtiter method: NIH, Japan, note. 5: Sequence for the measurement of human immunoglobulin as an antibody activity against mumps: NIH, Japan, note. 6: T. Toyama, Clinical Examinatlons 16, no. 7: "An Introductlon to Virus Empirical Science", edited by NIH Japan Alumi. pub., Maruzen Publishing House (1973), note. R: ..Recnmmendert Rpeclficatlon for Mlcroblological Reagents, US Dept. of HEW, April, 1965, no. 9: Mumps HI Řeaction Techniques, NIH, Japan.

Measurement of anti-complement activity:

The method described by Kabat & Mayer in "Experimental Irnmunochemistry", p. 225 (1961) is used. A 1% solution of an immunoglobulin solution containing guinea pig serum, 20 CH 50 / ral, is added to 5 ml of GVB ⁺⁺ with heating at 37 ° C for an hour, and the anticomplete consumed is determined as described above. The level of anti-complement activity is given by the percentage of consumption over 20 CH 50 / mi.

Example 1

Correlation of reaction conditions with the number of cleaved disulfide bonds

Situation Τ

To 51 mg of the above human immunoglobulin II is added 36 mg of ³⁵ S-labeled sodium sulfite and 22 mg of sodium tetrathlonate and an aqueous solution of 0.1 M tris-HCl buffer (pH 8.2) to give a 5.0 ml sample, which was heated to 45 ° C, and the next reaction was carried out at this temperature. Immediately after mixing, 0.5 mL of the reaction mixture and additional 0.5 mL samples were taken over 30 minutes, 1 hour, 2 hours, followed by 4 hours, 7.4 hours and 24 hours of the reaction. Each sample was mixed with 10 ml of a 50% saturated ammonium sulfate solution under ice cooling, allowed to stand for 15 minutes under cooling in January, and then the precipitate formed was centrifuged. After washing with 50% saturated ammonium sulfate solution, the radioactivity of the sample is measured using a liquid scintillation counter, and the number of S-SO 2 groups is calculated.

Situation II

The reaction is started under the same conditions as in situation T, even if sampling during the reaction. 4 hours after the start of the reaction, an aqueous 10 M urea solution is added to the reaction mixture so that the urea concentration in the reaction mixture is 4 M.Za5 and 6 hours of reaction, i.e. an hour, , and this is treated similarly to the samples from situation I.

Situation III

Reaction a-treatment of samples as in situation I, provided that the reaction is carried out at 0 ° C,

Situation IV

Reaction and treatment of samples as in situation I, provided that the reaction is carried out at 25 ° C.

Situation.

Reaction and treatment of samples as in situation I, except that sodium tetrathionate (Na 2 SO 4 · 2 H 2 O) is not used.

The results of the reactions performed in situations I, II, III, IV and V are summarized in Table 1 below.

TABLE 1

Correlation of reaction conditions with the number of -S-SO3 groups formed

Reaction time, hours 0. 0.5

Reaction situation

I (45 ° C) 0 6.1

II (45 ° G, after 4 hours of urea addition) - -

III (0 ° C), δ 0.5

IV (25 ° C) · 0.2 0.2 1.6

V (without StOs. 2 HzO) 0 0.5

The number of cleaved disulfide bonds corresponds to half the number of -S-SO3 groupings.

This can be deduced from the results of the reactions summarized in Table 1. If this is done ρο-

1 2 4 5 . -6 7.4 24 9.0 8.9 9.4 - - 8.9 10.4 .— - 10.9 13.3 - 1.6 2.5 2.9 - ' -. 3.4 . 5.0 4.1 5.0 4,5 - - 7.0 . 8.5 0.8 1.1 0.8 - - 4,5 stup in the presence of tetrathiortate and sulfite

of sodium sulfide at 45 ° C, then, in situation I, about 3 disulfide bonds are cleaved, yielding about 4.5 within 1 to 7 hours of reaction, and an average of about 5.2 over 24 hours of reaction.

If the reaction is carried out in the same manner, but at a temperature of 25 ° C (situation IV), the number of cleaved disulfide bonds decreases, with the number of cleaved bonds reaching an average of about 4.3 per 24 hours of reaction. When operating at 0 ° C (situation III), the average number of disulfide bond cleavages is further reduced. However, without the presence of sodium tetrathionate (situation V), the disulfide bond cleavage is not smooth. If urea is added to the reaction system (situation II), then the average number of disulfide bond cleavage increases sharply after the urea addition.

Example 1 a d 2

Comparison of deterioration in physical properties when sodium tetrathionate or copper ions are added as oxidizing agent.

Situation

To a mixture of 2.0 g of human immunoglobulin II as defined above, 1.42 g of sodium sulfite and 0.86 g of sodium tetrathionate Na 2 SiO 4. 2 H 2 O was added 0.1 M Tris-HCl buffer (pH 8.2) to make up the volume of the reaction mixture to 200 mL and allowed to react at an initial temperature of 45 ° C. Immediately after the start of the reaction, and then after 30 minutes, hour, two and four, a sample of 15 ml of the reaction liquid is taken, and immediately after the fifth sampling, a solution of urea solution is added to the rest of the reaction liquid. a urea concentration of 4M is reached, and the reaction is continued. After 5 and 6 hours, the reaction, i.e. an hour and 2 after the urea was added, a sample of 22.5 ml of the reaction liquid was taken and 22.5 ml of ice-cooled saturated ammonium sulfate was added, and the reaction mixture was allowed to stand. 30 minutes under ice cooling. The resulting precipitate is washed after centrifugation with a 50% saturated ammonium sulfate solution and dialyzed for 24 hours against a buffered sodium chloride solution of pH 7.4. Virking tube. After dialysis, taking the samples required for the Specific Analysis, dilute the samples with buffered saline pH 7.4 containing 2.5% glycine and analyze.

Situation II

The reaction and work-up is repeated as in situation I except that sodium tetrathionate is Na2SO4. 2 H 2 O replaces 50 mg of copper sulfate pentahydrate, and the reaction is completed in 4 hours.

Solutions of the various immunoglobulin derivatives thus obtained are used for the following tests and measurements:

a) Correlation of reactions with time and number of groups —S — SO3

The number of groups —S — SO3 in the samples oxidized by the tetrathionate NazSiOg. 2 H 2 O is applied (see both 1) in the form of white circles (Oj together with similar data obtained in situations I and II of Example 1. Data on other copper ion oxidized samples in a similar embodiment and treatment as in situation I of Example 1 with the change that sodium tetrathionate has been replaced by 50 mg of copper pentahydrate are deposited in solid black circles in Figure 1.

bj Correlation of reaction time with anti-complement efficacy

The anti-complement efficacy of the samples obtained by oxidation with sodium tetrathionate Na 2 S 2 Cl 2 H 2 O for the aforesaid Example 7, as determined by the procedure described above, are also depicted in Fig. 2. From Figure 2, it can be deduced that the anti-complement efficacy of all samples is reduced to a maximum of 30% per 30 minutes reaction time.

c) Correlation of reaction time with anti-diphtheria titer

For the same sample as used in b) above, the anti-diphtheria titer is measured three times using the above procedure. The deviations and mean values are shown in FIG. 3. From the graph it can be seen that for the samples oxidized by cuprous ions the titre is not higher than 0.3 limit. units per 30 minutes reaction. _

d) Correlation of reaction time and optical rotation

For the same samples used to measure anti-complement activity ad b) (protein concentration 2.5%), the optical rotation is measured, the results being shown in Fig. 4. From the results it can be seen that the presence of copper ions causes a rapidly progressing denaturation during the reaction, and that the presence of urea promotes the course of denaturation, as evidenced by the noticeable increase in optical rotation.

The optical rotation was measured in this case at 25 ° C in a 1 cm cuvette.

ej Correlation of reaction time with turbidity after heat treatment

The same samples used to measure the anti-complement efficacy of b) are heated in air at 50 ° C at a concentration of 2.5% for 4 hours, the results being shown in Figure 5. As can be seen from Figure 5, already the presence of very small amounts. The copper ion causes a deterioration in the heat stability and increases the tendency to form agglomerates.

f] Correlation of reaction time with copper content

194 6 93

The same samples as those used in the measurement of the anti-complement activity of b) are used to determine the copper ion content by atomic absorption analysis, and the results are shown in Figure 6. From the results, it can be assessed that using copper ions to remove copper ions.

g) Division of heavy chains, from light chains

The same sample, taken after two hours of reaction in situation I for sample 2, was dialyzed for 24 hours using 1N propionic acid solution containing 6M urea, and then chromatographed on a G 200 sephadex column (1.5 cm). 7. The first peak on the left side of the graph indicates the H2L2 strings not yet separated, the second peak corresponds to the H chain, and the third peak to the L chain. that the sample was split into chains H and L.

Summarizing the results shown in Figures 1 to 7, the following conclusions can be drawn: -, ·

As can be seen from the results of situation I of Example 1, when the present process is carried out in the presence of tetrathionate and sulphite ions for any period of from 1 to 4 hours, the average number of disulfide bonds cleaved is about 4.5, and the sulfur atoms formed. se

S-sulfonate (average number of S-sulfonate groups is approximately 9) to form S-sulfonated immunoglobulin (Fig. 1), which is divisible into H and L chains using, for example, column chromatography on Sephex G 200 (Fig. 7), and it has essentially the same optical rotation values as the native immunoglobulin (Fig. 4).

From the above results, it is safe to assume that new immunoglobulin derivatives. they are essentially formed by cleavage of the interchain disulfide bonds of only native immunoglobulin and S-sulfonation by cleavage of the released sulfur atoms, given the fact that the number of interchain disulfide bonds in the native immunoglobulin is on average 4.5.

Such immunoglobulin derivatives reduce anti-complement efficacy to a maximum of 10%, a value substantially lower than that of native immunoglobulin (Fig. 2), but at the same time such immunoglobulin derivatives exhibit an anti-diphtheria titer not substantially lower than they were for native immunoglobulin, as is evident from the nearly identical activity of antigen binding. as shown in Figure 3, at a concentration of 10% by weight, the immunoglobulin derivative retains an antigen binding activity of at least 1.0. Units / ml.

2. S-sulfonated immunoglobulin derivatives resulting from less than or wavelength cleavage than an average of 4.5 disulfide bonds of native 16 immunoglobulin, for example an average of at least 3 and at most 5 for S-sulfonation of sulfur atoms, are characterized by slightly less anti-complement efficacy and less binding antigen compared to the preferred immunoglobulin derivatives described above in ad 1. (Figures 2 and 3), but still exhibit substantially improved and complementary efficacy compared to native immunoglobulin.

3. If, however, the tetra-thionate ions are replaced with copper ions as the oxidizing agent, the resulting immunoglobulin derivatives contain copper ions, albeit at a very low concentration, which tends to increase, with increasing reaction time (Fig. 6). Also, the number of cleaved disulfides that bind native immunoglobulin increases with longer reaction time (Figure 1), and as a result, it is difficult to produce the subject immunoglobulin derivatives with cleavage of an average of substantially 4.5 disulfide bonds of native immunoglobulin while maintaining stability.

Furthermore, the immunoglobulin derivatives obtained by the use of copper ions appear to decrease - the anti-complement efficacy (Fig. 2), but at the same time their anti-diphtheria titer decreases suspiciously compared to the native immunoglobulin titer. Fig. 3). Again, as soon as the reaction time exceeds 1 hour, the immunoglobulin derivatives appear to have an optical rotation different from that of the native immunoglobulin, which means that structural changes in the proteins underlying the constitution have occurred.

4. Similar changes in optical rotation can also be seen with immunoglobulin derivatives prepared in accordance with the present process but with the addition of urea during the reaction. For this reason, it is recommended not to add urea, guanidine and the like to the reaction system.

Example 3

Selective cleavage of the interchain disulfide bonds in an aqueous solution containing 20 mg of human immunoglobulin I, 80 mg of labeled sodium sulfite ^M S-Na2SO2 ⁺ (8.22.10 ¹¹ dpm / mol) and 40 mg of sodium tetrathlonate Na2S40e. The 2 H 2 O was adjusted to pH 7.2 by addition of acetic acid, and the reaction mixture was allowed to react at 39 ° C for 2 hours. Thereafter, the reaction liquid is dialyzed against 5 liters of water for 24 hours, and urea and propionic acid are slowly added until a final concentration of 4 M and possibly 1 N is reached. A 0.1 ml sample is taken from the solution prior to loading onto the chromatography column. on the left-hand ordinate of FIG. 8. The residue is chromatographed on a Sefadex G200 column (1.5 cm diameter)

193693

18 (height 100 cm) equilibrium using 1 N propionic acid solution containing 4 M urea.

The radioactivity of the samples is measured at OD _28Q for each fraction using a liquid scintillation counter, the results being shown in Figure 8.

Example 1 a d 4

Fractionation of heavy and light chains

To an aqueous solution containing 100 mg of human χ-globulin II, 0.4 g of sodium sulfite and 0.2 g of sodium tetrathionate Na2'S4Q6.2H2O, was added 1 N hydrogen chloride solution by adjusting the pH to 9.0 The reaction is allowed to proceed at room temperature for 43 hours. After the reaction, less insoluble materials are removed by centrifugation, and the reaction liquid is then dialyzed using a Visking tube against 2.5 liters of 5 ° C water for 15 hours, followed by 2.5 liters of 1 N aqueous solution. propionic acid at 5 ° C for. 28 hours. The dialysate is chromatographed on a sephadex G75 column (3.5 x 111 cm) using 1 N propionic acid solution to fractionate the polypeptide chains. As shown in Figure 9, there are 3 peaks found to be unfractionated S-sulfonated immunoglobulin, S-sulfonated H-chains, and taking into account molecular weights.

S-sulfonated L-chains. Identification was performed by immunoassays using the Ouchterlon method and further by electrophoresis as well as chemical analysis such as amino acid analysis. content of Kexos and the like.

The following conclusions can be drawn from the results in Figure 8:

1. Since it was found that the first peak corresponds to the unfractionated S-sulfonated immunoglobulin, the second peak. The S-sulfonated H chain and the third peak of the S-sulfonated L chain, both by immunological and chemical procedures, referring to Fig. 9, from left to right, are certainly logical to assume that similar 3 peaks and Figure 8 are from the left transport attributable to unfractionated S-sulfonated immunoglobulin, S-sulfonated H chain and S-sulfonated L chain. The differences between the chromatographic shapes are due to the presence of urea in the reaction system.

2. Referring to Fig. 8, the molar concentration can be calculated from the amount of protein in the L-chain constituting the third peak, and from the molar concentration and the level of radioactivity it has been found that the L-chains constituting the third peak contain 1.0 groups S — SOs ⁺ . In the same way it was calculated that the number of groups

The S-SO 3 + in the H chain of the second peak is 3.4, in the fractionated 'S-sulfonated immunoglobulin 8.8, and in the mixture prior to loading the chromatography column 8.6. As already mentioned, a deaulfide bond binds the L chain and the H chain, while on average 2.5 disulfide bonds are between the two H chains. Therefore, it contains human immunoglobulin. on average 4.5 interchain di; sulfide bonds.

3. According to the result of the chromatogram shown in FIG.

. 8, the largest portion of S-sulfonated immunoglobulin, containing an average of 8.6 S-SOs ⁺ groups, can be divided into H chains and L chains. It can therefore reasonably be assumed that selective interchain disulfide bond cleavage occurred while intra-chain disulfide bonds remained basically intact.

Example 5

One embodiment of the reaction procedure

133 ml of human χ-globulin I are dissolved in 1667 ml of 0.06 M phosphate buffer (pH

8.2 y containing 2.5% glycine. A solution of 7.0 g of sodium sulfite in 100 ml of the same buffer and 4.4 g of sodium tetrathionate was added to the solution. 2 H 2 O, dissolved in 100 ml of the same buffer, and the reaction is carried out for 3 hours at 45 ° C. The reaction liquid was then dialyzed for 6 hours against 20 liters of buffered sodium chloride solution using. hollow fiber dialyser (b / HFU-1 ultrafilter). The external dialysis fluid is changed every 2 hours. The dialysate is concentrated to 180 ml using the same filter and 50 g of glycine are added.

The solution is sterile filtered using a 0.25 µm membrane, and a 5.0 ml volume is filled with 10 ml ampoules which are lyophilized. The anti-complement efficacy of the 50% sterile GH 50 solution was 4.3%. Example 6. Comparison of Antibody Activity to Antigens in Experiments Using Tetrathi Sodium or Copper Ion As Oxidizing Agents.

Situation

Add 67 ml of human γ-globulin I. 7.1 g of sodium sulfite and 0.43 g of sodium tetrathionate dihydrate and 0.1 M tris-HCl buffer containing 2.5% glycine, to a volume of 1 liter / After 3 hours of reaction at 45 ° C, the reaction liquid is dialysed Using a b / HFU-1 ultrafilter, concentrate the dialysate to a volume of 200 ml, filter sterile with a 0.25 µm mesh filter, lyophilize with the addition of 2.5 g of glycine, and dilute to 100 ml with water. 5.0% solution. The antibody potency of the sample solution is shown in Table 2.

Situation II

The reaction and processing under situation I is repeated as described above with that of 193693

20, the reaction temperature and the reaction time. Sodium NaaSáCte. 2 HaO used 0.16 g retention at 25 ° C and 24 hours. Copper sulphate tahydrate.

For solutions of immunoglobulin derivatives, '/.. Situation III 1 obtained using previous situations I to; <III, the ria antibody activity is different

The reaction and processing of the I-site is opained by the antigens with the results contained therein, with the change that instead of tetrathlonate in Table 2.

TABLE 2 Antibody activities on various antigens

Antigen Diphtheria; jedn./ml

Reaction situation unchanged 0.8

I (Na 2 SO 4, 45 ° C / 3 h) .0.8

Π (NazSdúe, z5 “u, .24 hours) -

III (Cu ²⁺ , 45 ° C, 3 h) below 0.1

The following closures can be derived from the data in Table 2 above:

1. When using tetrathionate (situation I), the diphtheria titre of S-sulfonated immunoglobulin is substantially the same as that of native immunoglobulin, but when copper ions are used (situation III), the immunaglobulin derivative shows almost no efficacy.

In view of the measles titer, the S-sulfonated immunoglobulin derivatives obtained by oxidation with tetrathionate (situations I and II) retain more than 60% of the activity of native immunoglobulin, but when using copper ion oxidation · ((situation III) the efficacy falls below 30%). '%.

From these facts, it can be deduced that, when using copper ions, the reduction in antibody activity is greater than when tetrathionite is used.

Example 7

In vivo anti-complement assay using S-sulfonated globulins.

Using a procedure similar to situation I of Example 6, S-sulfonated human immunoglobulin was prepared.

ml of 5% buffered sodium chloride solution containing 2.5% glycine pH 7.4, 3 ml of buffered sodium chloride solution as a control and 1 ml of 15ψο native human immunoglobulin (ie untreated) in buffered chloride solution. Sodium containing 2.5% glycine, also as a control, is administered intravenously to female guinea pigs weighing 200-300 g, and the serum complement level of the test subjects is determined. is measured using 1.0 ml of serum, extracted in each case by cardiac puncture at regular intervals.

The results are shown in Table 10. From Figure 10, a decrease in serum complement levels was observed in

Measles Rubella Mumps / dies relatively relatively

6.7 17jL 11

5.0 170 -

4.4-10 under 2 test subjects following intravenous injection of native human immunoglobulin, which was known. zero with similar administration of human S-sulfonated immunoglobulin. It has also been observed that guinea pigs occasionally shake after injecting, which does not occur when injected with S-sulfonates. immunoglobulins to guinea pigs. From these findings, it can be safely inferred that the immunoglobulin derivative of the invention is free of adverse reactions in vivo. Example 8

Durability of S-sulfonated immunoglobulins in rabbits in vivo, and new formation of disulfide bonds.

(a) Preparation of rabbit antibody for bovine serum-DNP albumin

Bovine serum albumin is treated with dinitrofluorophenol to form a substance hereinafter referred to as BSA-DNP. This is injected into rabbits (male, white, New Zealand) on hind paws together with Freun's complete adjuvant followed by immunization. Rabbits are drained from the carotid artery 30 days after injection. The antiserum thus obtained is treated as known per se by precipitation with a 45% saturated ammonium sulfate solution to obtain rabbit anti BSA-DNP-immunoglobulin.

(b) Preparation of samples:

Situation 1 '

Anti-BSA-DNP-immunoglobulin is. hydrolyzes at 37 ° C for 24 hours together with 1% by weight, calculated on pepsin immunoglobulin, according to the A. Nisonoff procedure, see Arch. Biochem. Biophys. 89, 230 (1960). The hydrolyzate is chromatographed on a Se195693: 21 fadex G 150 column to give a. thus fragment F (ab ‘) 2.

Situation 2

250 mg of anti-BSA-DNP-immunoglobulin, 180 mg of sodium sulfite and 79 mg of sodium tetrathionate (Na 2 SiO 6 2H 2 O) are dissolved in 25 ml of 0.1 M tris-HCl containing 2.5% glycine (pH 8.2), and the solution is allowed to react for 4 hours at 37 ° C. The reaction liquid was dialysed for 24 hours against a buffered sodium chloride solution (pH 7.4) to form rabbit S-sulfonated immunoglobulin.

Situation 3

The BSA-DNP-immunoglobulin specified above is used as such.

(c) Reverse speed and durability measurements. '

The samples prepared as described above are in each case treated into 5% solutions.

. Samples of the solutions were injected intravenously into male rabbits (New Zealand, White, Sample 1: 3 rabbits, Sample 2: 3 rabbits, and Sample 3: 2 rabbits) at a dose of up to 3 ml. Blood is drained from the test animals at predetermined intervals, and their plasma is extracted. Anti ESA-DNP activity, plasma is determined by a passive hemagglutination reaction, see T. Matsuhashi, Chemistry & Biology 9, 396 (1971) where BSA-DNP is fixed to sheep red blood cells using glutaraldehyde, and a microtiter method using the results shown in Fig. 11.

As can be clearly seen from Fig. 11, the inverse rate in vivo is extremely short for commercially available F (ab ‘) 2, which means a short duration of pharmaceutical efficacy. In contrast, S-sulfonated immunoglobulins exhibit approximately the same half-life compared to native immunoglobulin, indicating a sustained pharmaceutical effect.

(d) Measurement of hemolytic activity of antibodies

The specified native anti-BSA-DNP-immunoglobulin is injected intravenously into rabbits, and their plasma is collected 10 minutes after injection, which is diluted with 100 µm parts following reaction with guinea pig complement and red blood cells of sheep on which BSA-DNP is adsorbed by tannin acid. for one hour at 37 ° C. The intact antibody in situation 3 causes 100% hemolysis, while the F (ab ‘) 2 according to situation 1 and the S-sulfonated sample of situation 2 have no haemolytic activity.

Taking into account the plasma collected from rabbits 24 hours after injection, the product from the situation causes 2100% hemolysis in tests similar to those described above. ..

This phenomenon is likely due to the conversion of S-sulfonated immunoglobulin into native immunoglobulin in vivo, for example by reducing the grouping of —S — SOs to —SH groups to re-form disulfide bonds.

(e) Measurement of in vivo reduction of labeled concentration:

mg of rabbit immunoglobulin, 21 mg of labeled sodium sulfite ³⁵ S-Na2S.O3- and 13 mg of sodium tetrathionate Na2SO2.2H2O are dissolved in 3 ml of 0.1 M tris-HCl containing

2.5% glycine (pH 8.2), and the reaction was allowed to proceed for 3 hours at 45 ° C. The reaction liquid is dialysed for 24 hours against a buffered solution of sodium chloride at pH 7.4, and is thus prepared. ⁵ -S-tagged rabbit

S-sulfonated immunoglobulin.

The above sample, diluted to a concentration of 5%, is injected intravenously into two rabbits (white race, New Zealand, male) using 1.5 ml each, and the rabbit plasma is collected at predetermined intervals of 0.5 ml each. on a rabbit. Plasma radioactivity is measured using a liquid scintillation counter with results that. are given in Table 3 below.

',. TABLE 3

Remaining radioactivity in the blood

Blood drain time after intravenous injection Remaining radioactivity in blood, 15 minutes rabbit A rabbit B 100% 100% hours 64 64 hours 34 36 hours 9.5. 8.0.

hours 2,2 1,0 hours 0,7 0

5 6 33

From the above results; it can be deduced that the cleavage of —S — SO3 ⁺ groups of S-sulfonoyanized immunoglobulins in the blood occurs.

As exemplified by the above examples of the process of the invention, it is possible to selectively cleave the disulfide bonds linking the H-chain and the L-chain in the native immunoglobulin, whereupon the resulting sulfur atoms can be S-sulfonated. Additionally, the number of cleavages can be controlled by cleaving an average of 3 to 5, about 4.5 interchain disulfide bonds of native immunoglobulin, whereupon the sulfur atoms released by the cleavage are S-sulfonated.

These novel immunoglohulin derivatives of the present invention exhibit anti-complement efficacy considerably lower compared to native immunoglobulins, as exemplified in the examples, and consequently cause significantly less adverse analytical reactions. Thus, these derivatives can be injected not only intramuscularly, but also intravenously. In addition, these novel immunoglohulin derivatives of the present invention are characterized by nearly equivalent antigen binding activity compared to native immunoglobulins, and therefore exhibit extremely excellent binding of various antigens.

It is already known that aggregates can be removed from immunoglobulin prepared by fractionation, i.e. adsorption or sedimentation on activated carbon or polymeric substances. However, these modified immunoglobulins are still very dense, adverse reactions that occur in the recipient of the injections. Furthermore, re-aggregation occurs again upon storage of the immunoglobulins so treated. Thus, the product still appears to be imperfect for the safe use of injectable formulations, see L.A. Hanson & B. G. Johanson, Intern. Sheet. Allergy 31: 380 (1967).

. As mentioned and exemplified, the immunoglohulin derivatives of the present invention are by far less undesirable reactions and excel in this regard, see Fig. 2.

Also, the immunoglohulin derivatives of the invention may be retained. The efficacy of binding the antigens for a longer period of time, as seen in Example 8, and furthermore, the S-sulfonate groups in the derivatives switch back in vivo to disulfide bonds while the sulfonate groups are removed from the body. Thus, the derivatives of the invention are subsequently converted to the native immunoglobulin. Therefore, the S-sulfonated immunoglobulin of the invention also has obvious advantages over the known S-alkylated immunoglohulins.

As already explained, it is also clear that the novel immunoglohulin derivatives of the invention are useful as pharmacologically active ingredients not only by intramuscular injection but also by intravenous injection.

Water-soluble formulations suitable for injectable liquids when mixed with solubilizing agents such as glycine, sodium phosphate, sodium citric acid, sodium chloride and the like can be prepared from the novel immunoglobulin derivatives of the invention at appropriate concentrations, by weight, 1 to 20%, preferably 2 to 10, so that intravenous injectable preparations are obtained.

It will be appreciated that the S-sulfonated immunoglohulins of the invention are not only useful in medicine, but can also be used in the treatment of animals such as cattle, horses, pigs, sheep, dogs. As immunoglobulin derivatives in the treatment of animals, the native immunoglobulins obtained from the corresponding animals are preferably S-sulfonated according to the process of the invention.

Claims (2)

SUBJECT A process for the preparation of immunoglobulin derivatives comprising cleaving the interchain and intrachain disulfide bonds of an immunoglobulin and sulfonating the formed sulfur atoms by reacting with a compound capable of cleaving the tetrathionate ions and a compound capable of cleaving the sulfite ions simultaneously or sequentially in any order, in aqueous at pH 3.5 to 10 and in the absence of oxidizing metal ions such as copper. ions, at a temperature of 10 to 50 ° C, wherein the tetrathionate VYNALEZU. And sulfite ions are 2 to 10 times molar in the reaction medium with respect to the number of disulfide bonds of the immunoglobulin. 2. A method according to claim 1, wherein the tetrathionate ion cleavage compound is tetrathionic acid or sodium, potassium or ammonium tetrathionate, and the sulfite ion cleavage compound is sulfuric acid, sodium sulfite or sodium bisulfite or potassium sulfite.