DE2311333C2 - Process for the preparation of an intravenously injectable modified immune serum globulin - Google Patents

Description

(a) an aqueous 1-20% solution of unmodified Immune serum globulin at weakly alkaline pH with dithiothreitol or dithioerythritol in a concentration of less than 0.01 M and a molar ratio to protein from 2.5 to 50 in the reaction mixture is treated until an average of 2 to 4 Disulfide bonds per immune serum globulin molecule are reduced,

(b) an aqueous solution of this immune serum globulin reduced according to step (a) in the case of weak alkaline pH with at least 2 molar equivalents, based on the reducing agent,

an O-lower-alkylisourea,
Iodine acetamide,

of an N, N-DialkyI - «- haloacetamide,

of a halo vinegar ester,
a Λ-halopropionester,
a haloacetic acid,
an α-halopropionic acid,
of an a-haloacetonitrile,

an alkylenimine or
a phenacyl halide
is alkylated, and

(c) the modified immune serum globulin thus obtained from the non-proteinaceous ones Separated reaction products and reactants and, if necessary, concentrated will.

2. The method according to claim 1, characterized in that the reduction stage (a) with a Reducing agent / protein molar ratio of 4 to 6 and a pH of 7.2 to 9.0 is carried out.

3. The method according to claim 2, characterized in that the reduction stage (a) with a Reducing agent / protein molar ratio of 5 and a pH of 8.0 to 8.6 and as long as carried out until 5.5 to 8.2 SH groups per protein molecule are formed.

4. A pharmaceutical preparation containing as active ingredient that according to any one of claims 1 to 3 manufactured immune serum globulin in a pharmaceutically acceptable, for intravenous administration suitable aqueous carrier.

55

The invention relates to pharmaceutical preparations that are modified, intravenously injectable Immune serum globulin contain and refer to a process for the manufacture of such preparations, which are used in human therapy be used when administered intravenously.

Conventional gamma globulin supplements cannot be administered intravenously because such Administration elicits severe side reactions, especially in agammaglobulinaemic recipients.

It is assumed that these side reactions are based on a reduction in the serum complement content, which is possibly due to complement fixation by the administered gamma globulin (p. Barandun, et al. Vox Sang. 7, 157-174, 1962). As "Anti-complementary" ability of gamma globulin, The binding of complement is exacerbated as a result of denaturation that occurs during the Fractionation occurs, particularly due to aggregation into high molecular weight species The complement fixation mechanism of these aggregates appears to be similar to that of antigen / antibody complexes to be identical (D.M. Marcus, J. Immunol. 84, 273-284, 1960). If the aggregates by ultracentrifugation are removed at 100,000 χ G, a product is obtained which has only low anti-complement activity and is well tolerated by intravenous injection (Barandun et al, loc. cit).

Various attempts have been made to obtain gamma globulin for intravenous administration to make suitable. All experiments assume that it is necessary to increase its anti-complement activity to eliminate. The above-mentioned ultracentrifugation is technically out of the question, and in this way obtained product is believed to regain its anti-complement activity during storage. Treatment of gamma globulin with the enzyme pepsin at pH 4.0 causes proteolytic cleavage of the molecule to form a fragment with a molecular weight of about 100,000 which is a Has sedimentation coefficients of about 5S (A. Nisonoff et al., Science 132, 1770-1771, 1960). Though this fragment retains bivalent antibody activity and no longer has anti-complement activity and is therefore well tolerated and effective for intravenous administration (W. Baumgarten, Vox Sang. 13, 84, 1967), however, the therapeutic effect is unacceptably short-lived because the product quickly recovers is eliminated; it has a half-life of only 18 hours, which is unique to agammaglobulinemic Patient may be slightly longer compared to 19.8 days for unmodified gamma-glubulin (E. Merler et al., Vox Sang. 13,102,1967; B. Jager, Arch. Intern. Med. 119, 60, 1967). True, the greatly reduced half-life of pepsin-treated Gamma globulin is believed to be due in part to the drastic reduction in molecular size, however There is also evidence that the rate of catabolism of gamma globulin is specific Properties of the part of the molecule that is digested by pepsin depends (j. L. Fahey et a., J. Exper. Med., 118, 845-868, 1963). This part of the molecule remains intact in the method of the present invention. A Another disadvantage of the pepsin treatment is that the remaining pepsin is of animal origin and, especially with repeated administration, can cause antibody formation (C. Blatrix et al .. Presse Med. 77, 635-637, 1969). The human use of plasmin bypasses this difficulty and is the basis of another method for obtaining intravenous injectable gamma globulin.

Treatment of gamma globulin with human plasmin causes it to split into three Components with a molecular weight of about 50,000 (J.T. Sgouris, Vox. Sang. 13, 71, 1967). if If sufficiently small amounts of plasmin are used, only about 15% of the molecules are broken down.

while 85% remains as intact gamma globulin. The undigested, intact gamma globulin has only low anti-complement activity and could be administered intravenously without undesirable reactions occurring (J. Hinman et ah, Vox Sang. 13, 85, 1967). The material obtained in this way appears to retain its protective effect in vitro and in vivo (FK Fitzpatrick, Vox Sang. 13, 85, 1967). A disadvantage of this method is that the plasmin cannot be completely removed. Therefore, the degradation continues even if the material is stored ^{at 4 C C.}

It was also observed that an incubation of gamma globules at pH 4.0 and 37 ° C for different Period of time the anti-complement activity decreases to low levels. It is believed that this Result is due to a small amount of a serum enzyme present as an impurity in the Gamma globulin is included (Blatrix et al, loc. Cit). As was the case with the plasmin-treated gamma globulin also found with this "pH 4 gamma globulin" that its anti-complement activity in storage recovered to an unpredictable extent such that it is necessary, prior to administration to one To re-determine the anticomplement activity of patients (J. Malgras et al. Rev. Franc. Trans, 13, 173, 1970).

Both plasmin-treated gamma globulin (Hinman et al, loc. Cit) and pH 4.0 gamma-logulin (H. Koblet et al. Vox Sang. 13, 93, 1967; J. V. Wells et al, Austr. Ann. Med. 18, 271, 1969) have shorter half-lives in vivo than unmodified gamn aglobulin. So z. B. the half-life of pH 4.0 gamma globulin in normal patients is about 14 Days (Koblet et al, loc cit), while that of plasmin-treated material is 16 days (Merler et al, loc. cit.).

The Center National de Transfusion Sanguine (C.N.T.S.) in Paris has after careful fractionation and filtration of gamma globulin from selected fresh plasma an intravenously injectable gamma globulin obtained with low anti-complementativity (Blatrix et al, loc. cit .; ibid. Presse Med. 77, 159-161, 1969; M. Steinbuch et al. Vox Sang. 13, 103, 1967). Apparently this is not gamma globulin completely free of anti-complement activity, because it must be applied with great care, and with some patients experience reactions. To prevent such reactions, you can do the injection Cortisone can be given, however, which is obvious insufficient elimination of the anti-complement activity prevents widespread use.

The influence that the reduction of disulfide bonds of gamma globulin and the subsequent Reaction with iodoacetamide on the anti-complement activity has already been investigated (p. Barandun et al, loc. cit.). It was found that the treatment of a 7% solution of gamma globulin with 0.2 M gysteamine and then with 0.2 M iodoacetamide to an almost complete loss of anti-complement activity while treatment with cysteamine or iodoacetamide alone results in anti-complement activity not significantly reduced. Because of the toxicity of iodoacetamide, these studies were not transferred to intravenous injectable gamma globulin. A similar way could be shown (Wiederman et al, Proc. Soc. Exp. Biol. Med. 113, 609-613, 1963) that the treatment of gamma globulin with 0.1 M mercaptoethanol with subsequent, prolonged dialysis against 0.02 m iodoacetamide leads to a solution which, when mixed with an antigen, does not Complement fixed Both untreated and treated gamma globulin were found in these insensitive test series a'is free of anti-complement activity. Apparently no attempt was made to use an intravenously injectable gamma globulin to win after this procedure. It has long been known that mercaptoethanoi (J. B. Fleischman et al. Arch. Biochem. and Biophys. Soup 1, 174-180, 1962) and mercaptoethylamine (G. M. Edelman, J. Am. Chem. Soc. 81.3155, 1959) in high molar concentrations are able to reduce interchain disulfide bonds of gamma globulin. The disulfide bonds, which are more unstable compared to a mercaptan reduction seem to have something to do with the fixation of complement to have to do while the disulfide bonds, which are more resistant to reduction by mercaptan appear to be involved in the interaction with antigens (P. H. Schur et al, J. Exp. Med. 120,531, 1964). The disadvantages associated with using high (0.1-0.5 m) concentrations of mercaptans Reduction of interchain disulfide bonds linked in gamma globulin can be avoided when dithiothreitol (DTT) is used in moderate concentrations as a reducing agent. (W. W. Cleland, Biochem. 3, 480, 1964). Since DTT is irreversibly oxidized to form a stable six-membered ring, Almost stoichiometric amounts of this reducing agent can be used and a large excess can be used of mercaptan should be avoided. A substantially complete reduction in interchain disulfide bonds of human gamma globulin was obtained by exposing one to 0.0125 m DTT 2% gamma globulin solution (P. Cunewardena et al, Biochem. Journ. 99.8, 1966). From these attempts went however, it does not reveal the influence of such a reduction and the subsequent alkylation Iodine acetamide on the antibody levels.

Despite all these extensive attempts to modify immune serum globulin so that the anti-complement activity is eliminated, no modified immune serum globulin is commercially available which (a) is largely free of acute and latent anti-complement activity. (b) essentially biological Has half-life and antibody activity of unmodified immune serum globulin, and (c) free from non-proteinaceous reaction products and injectable intravenously, is essentially pure.

The aim of the present invention are pharmaceutical preparations for intravenous administration which contain a new type of intravenously injectable, largely pure, modified immune serum globulin, which is essentially free of acute and labile anti-complement activity and essentially the exhibits biological half-life and antibody activity of unmodified immune serum globulin.

This aim is achieved according to the claims. The preparation obtained according to the invention contains, in a pharmaceutically acceptable, aqueous vehicle suitable for intravenous administration, a novel, intravenously injectable, largely pure modified immune serum globulin, in which essentially all interchain disulfide bonds connecting heavy and light chains, and a total of up to about 4 Disulfide bonds by pairs of e.g. B. -S-CH ₂ CONH ₂ groups are replaced, where

the light chains are linked to the heavy chains by non-covalent bonds, so that the apparent molecular weight in non-dissociating solvents essentially the same as that of is unmodified [mmourum globulin.

The serum globulin obtained according to the invention is largely free of acute and labile anti-complement activity and has essentially the same biological half-life and antibody activity as unmodified immune serum globulin, as determined in serological tests.

The invention is explained in more detail by the drawing:

F i g. 1 is a graph showing the relationship between the number of blocked SH groups and the resulting anti-complement titer of the new, modified immune cream globulin; the implementations were changing Dithiothreitol concentrations and reaction times performed;

F i g. Fig. 2 is a graph showing the gel permeation chromatography obtained in Example 1 (b) used starting immune serum globulin obtained molecular size distribution with that of the new, modified immune serum globulin, according to the values summarized in the table will.

The new injectable modified immune serum globulin according to the present invention is thereby excellent that it is with respect to physical properties and biological functions, except the anti-complement activity, is unchanged, but that it is chemically replaced by the replacement largely all inter-chain and a total of up to about 4 disulfide bonds through the same number of Pairs of e.g. B. - S — CHiCONHj groups changed is, while the immune serum globulin molecule is otherwise not chemically changed. The reduction these specific disulfide bonds and subsequent alkylation of the mercapto groups formed to eliminate anti-complement activity. The reduction of the disulfide groups is a prerequisite for the elimination of the complementary activity, and the alkylation of the mercapto groups formed in the process prevents the recurrence of anti-complement activity. Since the selective reduction and alkylation reactions affect according to the invention located between the chains disulfide bonds and in the Leave disulfide bonds lying in chains essentially unaffected, the protein molecule in Fragments are broken down and the antibody capacity of the modified immune serum globulin remains largely obtained by adding the rest of the molecule '.rn remains essentially unchanged, the biological half-life of the modified immune serum globulin also remains received unchanged after its infusion into human recipients.

Source material

The starting material for the method according to the invention is unmodified, human immune serum globulin (ISG).

The term "immune serum globulin" is intended to denote the substance that is used in the literature sometimes also with gamma globulin, y-globulin, IgG and immunoglobulin G is called. It consists predominantly and preferably of at least about 85% of the 7S species of gamma globulin having a molecular weight of about 160,000. The rest are preferably 9S species with a molecular weight of about 300,000. Both the standardized immune and hyperimmune serum globulins, such as B. the tetanus and rabies immune serum globulins be used; the modified product is then immune or hyperimmune ISG. A suitable starting material for the invention

ίο procedure is z. B. Cohn's Fraction 11, Figs. 5 and 6 (Cohn, E. J., et al., J. Am. Chem. Soc. 68, 459, 1946; ibid. 71, 541, 1949).

As studies in the ultracentrifuge have shown, fraction II consists predominantly (about 85%) from 7S species (sedimentation constant = 7) or from gamma globulin with an average Molecular weight of 160,000. The remaining protein is essentially 9S material of molecular weight of about 300,000. The wet Fraction II paste (about 30% solids) is usually lyophilized, whereby dry ISG powder is obtained which is then dissolved and used for intramuscular injection as a 16.5% sterile solution is prepared. Both the wet Fraction II paste and the dry ISG powders (before or after dissolving in a suitable aqueous vehicle) are useful starting materials for the method according to the invention. Any gamma globulin obtained, which is essentially has the same composition of protein components as the Cohn fraction II can be used as Starting material can be used in the modification process according to the invention. Blood plasma too or whole serum may have anti-complement activity by this chemical modification process to be largely exempted.

The starting material used can be typical immune serum globulin or hyperimmune serum globulin be. The latter is known to be obtained from the plasma or serum which selected donors which have much higher titers of a specific antibody than the rest Population. Such donors have either recently been immunized with a specific vaccine, or they have recently recovered from an infection or disease. These high titer sera or plasmas are collected and subjected to the usual Cohn fractionation until fraction II is isolated can be. The antibody standards for hyperimmune serum globulins of the Division of Biological Standards (DBS) are based e.g. Currently on products that are to be administered intramuscularly and go from the Assume that a standard intramuscular dose of the constituted globulin (1 to 10 ml) is administered will. As the amount of antibody required to achieve a desired immunological response is, when administered intravenously, is much smaller, it is clear that the I.V. dose is considerably smaller than the The I.M. dose is that which produces the same serum antibody titer. Therefore the dose has to be intramuscular administered ISG and hyperimmune serum globulin may be higher than that required to maintain the To achieve serum antibody, which is achieved when globulin of the same antibody activity is intravenous is administered.

The moist pastes or lyophilized powders used as starting material are in a such amount of water preferably dissolved in an aqueous solution of glycine and NaCl that a

Protein solution at the desired concentration, usually I to 20%, e.g. B. 3, 5, 10 and 16.5%, preferably about 5%. arises. While unmodified ISG solutions are most persistent high concentrations, e.g., 16-18%, solutions of the new, modified ISG are surprising at significantly lower concentrations, e.g. B. 10% resistant. Glycine and NaCl, although not necessarily required, are preferably added to help stabilize the protein solution. That Glycine can be present in any concentration up to about 1.0 m. The NaCl solution is preferred about 0.075 molar. After the protein has dissolved, the solution is made by adding sodium hydroxide or another base to the desired weakly alkaline pH, e.g. B. to about 7.2 to 9, preferably about 8.0 to 8.6 and in particular about 8.0 to 8.2. If the solution at this point is not clear, it is preferably clarified before the reduction.

Reduction stage

In the first stage of the process according to the invention, the interchain disulfide bonds are Initial immune serum globulins reduced with dithiothreitol. This is done by a total of about 2.5 to about 4, preferably about 2.8 to 3.8 disulfide bonds with at least about 2.5, e.g. B. 2.5 to 50, preferably about 4 to 6 and especially about 5 to 6 molar equivalents of the reducing agent are reduced.

Instead of dithiothreitol (dithiodihydrobutane), its diustereomer, dithioerythritol, can be used in the framework of the present invention.

As shown in FIG. 1, acute and latent anti-complement activity can be achieved by selective reduction with subsequent alkylation of about three of the interchain disulfide bonds per molecule of ISG is eliminated will. The reduction and alkylation of about one disulfide bond has no noteworthy effect on the complement fixation activity of immune serum globulin. The average reduction of 2.5 Disulfide groups drastically reduce the anti-complement activity. However, to avoid the anti-complement in To eliminate substantially completely is usually the reduction of about 2.8 or more disulfide groups necessary. The reduction of three to four disulfide bonds is harmless, and with a reduction and alkylation to such an extent that the desired complete elimination of acute and latent anti-complement activity is always achieved without a reduction in the less reactive disulfide bonds created within the chains essential for biological activity are important. The reduction and alkylation of more than four disulfide bonds has no beneficial effect on the recovery of gamma globulin for intravenous administration and on the contrary leads to a partial or complete destruction of biological functions.

The desired selective reduction of essentially all interchain disulfide bonds is achieved Achieved by a combination of different measures, namely: Concentration of dithiothreitol in the reaction mixture, molar ratio of dithiothreitol to starting protein, as well as reaction time and temperature and pH value

The reduction step is carried out at room temperature or with gentle heating or cooling, e.g. B. at about 0-4O ⁰ C, preferably 20 to 35 ° C, but best at room temperature. At temperatures lower than room temperature, higher concentrations of dithiothreitol are required to achieve the same level of reduction. Indirect analysis has shown that at room temperature, pH 8.2 and 5% protein concentration, about 60% of the DTT (at 0.0015 m) reacted in 10 min and 65% in 60 min.

These figures show that the degree of conversion under given conditions with reference to pH and concentrations of protein and DTT are largely independent of the reaction time. The implementation can thus for any length of time, e.g. B. about 2 minutes to about 2 hours. Or longer. at Temperatures higher than room temperature should use an amount of dithiothreitoi that is suitable for the Achieving the desired degree of reduction corresponds to the theoretically required amount; becomes a If excess is used, the course of the reaction should be followed and the reaction carried out by destroying the excess reducing agent can be started as soon as the desired number of - SH groups has been formed.

The reduction is preferably carried out at a mildly alkaline pH, e.g. B. about 7.2 to 9, in particular performed at about 8 to 8.6. At lower pH values the reaction rate both decreases for the reducing agent as well as for the alkylating agent. Therefore are at pH values below 8 require longer reaction times and / or higher dithiothreitol concentrations. At significantly higher pH values, the immune serum globulin tends to denature. To get the desired To adjust pH, a physiologically acceptable buffer is preferably used because of subsequent complete removal is not critical. The dithiothreitol can be added to the protein solution as a solution or added to the protein solution as a solid. In a different but less preferred way The protein can be dissolved in the dithiothreitol solution or the protein and dithiothreitol can be dry mixed with each other and together in the aqueous reaction solvent to the desired protein concentrations, z. B. at least about 1%, preferably about 5 to 18%, are dissolved.

The course of the reaction can, if desired, be followed by adding the reaction mixture to unreacted Dithiothreitol is analyzed. When no more reducing agent is consumed, the implementation is completely.

The reaction can easily be carried out in air. This leads to the Auioxidaiion a smaller amount of dithiothreitol, however, this can be done by using a larger amount than that corresponds to the theoretical molar ratio, are compensated. If desired, the implementation can also be in a protective gas atmosphere, e.g. B. be carried out under nitrogen.

The molar ratio of dithiothreitol to protein used depends in part on the protein concentration in the reaction mixture, the pH, the reaction time, the reaction temperature and the reaction atmosphere away. At least one molar equivalent of DTT is theoretically required to have one disulfide bond per Reduce moles of protein and create two —SH groups. That is why the theoretical minimum amount is on Dithiothreitol, which is used to reduce any disulfide bond

is required, at least one molar equivalent. Since, as already mentioned above (see also Fig. 1), the Reduction of about 3 disulfide bonds and subsequent alkylation of the SH groups for elimination of anti-complement activity is required, even under optimal conditions, at least 3 Molecular equivalents of DTT necessary to largely eliminate anti-complement activity. In the invention Processes will be about 2.5 to 50 molar equivalents, preferably at least about 3, and usually less than 10, e.g. B. 4 to 6 and especially about 5 molar equivalents of dithiothreitol used to the desired reduction within a reasonable period of time under the preferred conditions In relation to protein concentration, pH and reaction temperature.

Assuming an average molecular weight 160 000, then the molarity of the preferred starting material employed 5% protein solution is about 3x10- ⁴ m. To the desired extent to achieve at disulfide should therefore the 5% protein solution is added as much dithiothreitol, that its concentration χ in this solution at about 5 ΙΟ ^"4 m to 25 χ 10- ^4, preferably at about 15 m χ ~ 10. ⁴

The reduction can be carried out in air, and in the examples below it is Reaction atmosphere. However, since some of the dithiothreitol is oxidized by the air, the applied Amount of dithiothreitol if the reduction is carried out under nitrogen or some other inert atmosphere carried out should be kept closer to the theoretical amount.

Since the reduction stage can be ended at any time by destroying the dithiothreitol still remaining in the reaction mixture, it is possible under most reaction conditions. To use dithiothreitol in a considerable excess over the amount required to achieve the desired degree of reduction; z. B. 5 to 1200% and preferably 10 to 100% excess can be used if the reaction is stopped as soon as the desired degree of reduction has been reached. When using a very large molar excess of dithiothreitol, preferably lower reaction temperatures, e.g. B. 0 to 5 ° C, and / or lower pH values, e.g. B. 7.2 to 7.8 adhered to in order to ensure the selective nature of the implementation. If the molarity of the dithiothreitol is below about 1.6 × 10 ^-3 m, the reduction of a 5% protein solution can usually extend over a period of up to 1 hour before the reduction of more than 3 disulfide groups per molecule has occurred. At about 1.6 to 1.8 × 10 ³ m, the reaction usually has to be completed within 30 minutes. At higher dithiothreitol concentrations, e.g. B. 2.0 to 2.5 × 10 ~ ³ m, the reaction should be ended after 5 to 15 minutes. These reaction times are correspondingly shortened at temperatures higher than room temperature and, conversely, lengthened at lower temperatures. Likewise, at protein concentrations other than 5% and pH values other than 8.0 to 8.6, an adjustment of the reaction time is necessary if an excess of dithiothreitol is used.

The above information assumes that the amount of dithiothreitol contained in the reaction mixture in excess over the reduction of an average of 4 disulfide groups per molecule, the according to the invention maximum number to be reduced, required amount is present. If desired, can the exact amount of DTT necessary for the preferential reduction of about 3 disulfide bonds, e.g. B. 2.8 to 3.8 Disulfide bonds, and thus to the formation of an average of 5.6 to 7.6 —SH groups per molecule the chosen reaction conditions is required, determined on a subset of the starting ISA will. In this case the extent of —SH group formation is essentially independent of the reduction time.

The dithiothreitol should usually m in the reaction mixture in a concentration of at least 5.0x 10- ⁴ and less than 1xlO- ² m, for example about 1.2 χ 10- ³ and preferably 1.2 to 1.8 χ 10- ³ m present when an approximately 5% protein solution is to be reduced in a short time at approximately room temperature. The amount of dithiothreitol required for such an initial molarity can be added all at once or continuously or in batches during the course of the reaction.

The relationship between DTT concentrations and reaction time is shown in FIG. 1, which shows the results of the reduction of a 5% solution of immune serum globulin with DTT at different concentrations in the reaction mixture: With 5% ISG, the reduction of the 2.5 disulfide bond, which is regarded as the minimum, can be reduced to 5 —SH groups per Molecule with a DTT molarity of less than about 1. OxIO- ³ ITi, ie about 1.3 times the theoretically required amount, regardless of the reaction time, cannot be achieved. In order to generate 6 - SH groups within one hour, a molarity of at least about 1.6 χ 10 " ³ m, ie about 1.67 times the theoretical amount, is required. For the upper curve of this figure, the number of during —SH groups formed after reduction were determined from the carboxymethylcysteine content of an acidic hydrolyzate of the chemically modified ISG.

During the acid hydrolysis, the cysteine residues alkylated with iodoacetamide are converted into carboxymethyloyl stones, so that the number of alkylated mercapto groups results from the amount of these residues. For the lower curve, ¹⁴ C-iodoacetamide was used to block the mercapto groups so that the number of alkylated groups could be determined by radioactive measurements. The carboxymethylcysteine values are somewhat higher and are presumably more exact due to the way the experiment was carried out.

The product of the reduction stage, namely reduced, but otherwise largely unmodified Immune serum globulin in which essentially all interchain disulfide bonds and up to a total of about 4 disulfide bonds. to —SH pairs. reduced is then, usually without an intermediate purification or separation step, in an alkylation step converted into a stable end product.

Alkylation stage

In the alkylation stage, the product of the reduction stage is alkylated, with the new, modified immune serum globulin according to the present invention being formed.In this stage, as much alkylating agent is used as is necessary to react with all of the unreacted dithiothreitol and all of the - Alkylate SH groups.
The preferred alkylating agent is iodoacetate

amide used.

This step is usually carried out in the reaction product mixture of the reduction step. If so desired however, the reduced protein can be isolated, e.g. B. by failures, and can be a new one aqueous solvent can be used.

In order to fully alkylate the product from the reduction step, at least two molar equivalents of alkylating agent, calculated on the dithiothreitol used in the reduction step, are usually required. Preferably a molar excess of 10% or more is used to ensure complete alkylation of all residual DTT and conversion of all protein SH groups to -S-CH ₂ CONH ₂ groups. If z. As dithiothreitol was used in an initial concentration of 15x10- ⁴ m in the reaction mixture, the preferred Anfangsmolarität of the alkylating agent is in the reaction mixture at about 33XiO ^"4. Higher molar excesses, for. Example, up to 20 molar equivalents, can be employed without damage , since excess alkylating agent is separated from the alkylated product after the alkylation step, e.g. by precipitation of the protein or by dialysis.

The reaction conditions are essentially the same as used in the reduction step will; with the exception that usually somewhat longer reaction times, e.g. B. 1 to 2 hours. Applied will. The alkylating agent, e.g. B. Iodoacetamide can be added as a solid or as a solution. As in the reduction stage, the course of the reaction can be followed, in this case by determining the free —SH groups. Because the presence of free —SH groups in the protein molecule causes the reappearance of anti-complement activity, the reaction product should be as free as possible be from —SH groups, d. In other words, these should be present at most in traces. The perfect alkylation of —SH groups also prevents subsequent aggregation of gamma globulin molecules.

Although the alkylation of the —SH groups formed by the reduction of the interchain disulfide bonds to form pairs of —SCH ₂ CON H2 groups in the process according to the invention is preferably carried out with iodoacetamide, the —SH groups can also be blocked by the reduced ISG is alkylated with other alkylating agents to give a modified ISG having essentially the same physical and biological properties as that in which broken interchain disulfide bonds are replaced with pairs of -SCH2CONH2 groups. In these modified ISGs, essentially all of the interchain disulfide bonds are replaced with pairs of other -SR groups, and this protein has an apparent molecular weight substantially equal to that of unmodified serum globulin; it is largely free of anti-complement activity, possesses —SR groups, which are chemically stable and preclude the reestablishment of disulfide bonds, and has essentially the same biological half-life and antibody activity as unmodified immune serum globulin. The radical R in the thioether group can be a lower alkyl radical, such as. B.

Methyl, ethyl or propyl
be; a lower Ν, Ν-dialkylcarbamidoalkyl radical, such as N, N-diethylcarbamidoalkyl (-CH ₂ CONAIh ₂ );
a lower alkoxycarbonylalkyl radical, such as. B.

Ethoxycarbonylmethyl (-CH ₂ CO ₂ C ₂ H ₅ ) or

Ethoxycarbonylethyl (-CH (CH ₃ ) CO ₂ C ₂ H ₅ );
a lower carboxyalkyl radical, such as. B.

Carboxymethyl (-CH ₂ CO ₂ H),

Carboxyethyl (-CH (CH ₃ ) CO ₂ H);
a lower cyanoalkyl radical, such as. B.

-CH ₂ CN;
a lower / I-aminoalkyl radical, such as

z. B.-CH ₂ CH ₂ NH ₂ ,
or a lower Berszoylalkylrest such. B.

-CH ₂ COC ₆ H ₅ .

The alkylation of the —SH groups to —SH groups, in which R is a lower alkyl radical, can be achieved by removing the reduced immune serum globulin e.g. B. with O-methylisourea under weakly alkaline Conditions is treated, whereby - SCHj groups form. With the expression "lower alkyl or Alkoxy radicals are intended to denote radicals with 1 to 4 carbon atoms. Other substituted alkyl thioethers can be formed using the appropriate N.N-dialkyl-ot-haloacetamide, e.g. B.

ClCH ₂ CON (C ₂ Hs) ₂ ,
Haloacetesterjoc-Halopropionester, such as B.

JCH ₂ CO ₂ C ₂ H ₅

BrCH-CO ₂ C ₂ H ₅
CH ₃

Haloacetic acid; «-Halopropionic acid, such as B.

JCH ₂ CO ₂ H, BrCH (CHj) HCO ₂ H;
Haloacetonitrile, such as B.

JCH ₂ CN;
Alkylenimine such as B.

CH ₂ \

-CH ₂ ;

NH

Phenylacyl halide. such as B.

Phenylacyl chloride.

The alkylation reactions are carried out under weakly alkaline conditions.

Separation of modified ISG

In order to obtain a pharmaceutically acceptable product suitable for intravenous administration it is necessary to separate the new, modified ISG from the non-proteinaceous reaction products and participants. This can be done in a number of ways, e.g. B, by ion exchange chromatography. Gel permeation chromatography, ultracentrifugation or preferably by dialysis or by precipitation of the modified ISG, e.g. B. by means of a water-miscible organic solvent, such as. Methanol, ethanol, etc. If a solution of the modified ISG is obtained that is too dilute, ultracentrifugation can also be used to concentrate the solution to the desired protein concentration.
In a preferred mode of operation, particularly after diluting the protein solution with water to a protein concentration of about 1.5%, the modified immune serum globulin is precipitated with ethanol under approximately the same conditions as in the

known Cohn process the precipitate II is obtained from the above II. This precipitation is collected and washed, preferably at least twice with ethanol buffer solution at pH and temperature conditions as well as ethanol concentrations at which the protein will not recover dissolves, but all detectable amounts of residual reactants and non-proteinaceous Products are removed. The material is then dried in a conventional manner, e.g. B. after the Method used to dry the Cohn fraction II and then kept sterile after re-establishment of the desired protein concentration in a pharmaceutically acceptable, carrier suitable for intravenous administration to be administered intravenously.

In another preferred mode of operation, a concentrated, e.g. B. 15 to 18% ISG solution as Starting material is used and the product from the alkylation step is dialyzed until it is free of non-proteinaceous Components of the reaction product is. The modified ISG obtained can be sterile filtered and sterile be filled into containers, e.g. in 10 ml ampoules, to be used in this form or to be lyophilized. In a different way and in particular to get a To ensure complete removal of the reagents, the dialyzed solution can be used as described above, further processed, namely by precipitating the modified ISG, washing the precipitate and Readjustment of the desired protein concentration in a pharmaceutically acceptable carrier. Will If an excessively diluted solution of the modified ISG is obtained, ultracentrifugation can be used to concentrate the solution to the desired protein concentration.

The new immune serum globulin

The new gamma globulin according to the present invention is largely free from acute and latent Anti-complement activity. Its molecular weight is essentially unchanged. It sediments in the Ultracentrifuge mainly with 7S, with about 10 to 15% are present as 9S components, which is also the case with the unmodified gamma globulin in the Cohn fractionation the case is. His recording of gel filtration chromatography on polyacrylamide beads (Bio-Gel P-300) in Tris buffer (pH 8.0) shows a larger peak that is 80 to 90% of the total protein at a molecular weight of 160,000, mil few or no small sized fragments. However, gel chromatography shows in conditions promoting dissociation, e.g. B. in 1M acetic acid, a significant amount of light Chains (or light chain dimers), from which one can see that the disulfide bonds that make the light Link chains to the heavy chain dimers, cleaved by the process of the invention have been. As these disulfide bonds subdivide the heavy chains near that area of which one assumes that it contains the site of the complement fixation ("pivot point" area), is to be assumed that the change in the molecular structure caused by the reduction and alkylation is responsible for the loss of acute and latent anti-complement activity is responsible. The antibody titer differs not significantly different from that of the starting unmodified gamma globulin; i.e., is normal or hyperimmune, like / .. B. with tetanus or rabies hyperimmunoglobulin, depending on the antibody titer of the unmodified starting globulin. The antibody molecules are bivalent, as> nan can see from their ability to precipitate with antigen. The quantitative Precipitation with tetanus toxoid shows that 65 to 80% of the precipitating antibodies for tetanus after the chemical modification of a tetanus immunoglobulin have been preserved. The new ISG owns passive protective effect against tetanus toxin, such as

ίο have shown experiments on laboratory animals. the Half-life of the new, chemically modified gamma globulin in humans and laboratory animals practically does not differ from that of unmodified gamma globulin.

In contrast to the intravenously injectable gamma globulin obtained by heat treatment at pH 4.0 or by limited plasmin treatment, the new ISG does not show any appreciable complement fixation, even when it has been deliberately aggregated by e.g. B. heated or acidified and then neutralized. A significant restoration of the Anrikomplemem activity does not occur even if it is stored in the form of a sterile solution suitable for intravenous administration for two years at 5 ° C.

It is true that the inter-chain disulfide bonds between the light and heavy chains in the The method according to the invention is split, but the light chains remain due to non-covalent bonding linked to the heavy chains so that the apparent molecular weight of the reduced and alkylated product remains essentially unchanged as by gel filtration and ultracentrifugation was found in non-dissociating solvents. That is, however, the interchain disulfide bond can be recognized by dissolving the new product in 1M acetic acid, with the light Dissociate chains from the rest of the molecule. Chromatograph the resulting solution and analyze it the U.V. absorption of the part of the eluate which contains the light and heavy chains is separated, then the light fraction is found to be about the theoretical amount (about 20%) to the total U.V. absorption of the new product contributes, which is proven. that essentially no interchain disulfide bonds have been retained in the new, chemically modified ISG. The new, modified ISG according to the invention has approximately the same or only a slightly lower antibody content like the starting ISG.

A new feature of the modified ISG according to the invention is the lack of proteolytic activity. It is known that some samples of ISG form fragments on storage. This fragmentation is due to a proteolytic digestion by an enzyme contained as an impurity, of which it is usually assumed that it is plasmin. Such fragmentation is undesirable because it causes a decrease in the active antibody content in solution. The method according to the invention drastically reduces the proteolytic activity in ISG to indefinitely small values or at most Trace amounts. Further experiments have shown that chemically modified gamma globulin contains 0.004 casein units / ml (a measure of non-specific proteolytic activity) compared to 0.03 casein units / ml in the starting material. The method according to the invention thus eliminates proteolytic activi-

tat in gamma globulin, which increases product stability.

It is also known that heating ISG causes aggregation of molecules to proteins of higher molecular weight and greatly increases the anti-complement titer, e.g. B. from 16 to over 500. The method according to the invention eliminates this increased anti-complement activity and brings it back to values close to zero

Intravenous administration

The modified ISG according to the present invention is primarily for intravenous administration thought. The invention therefore preferably relates to pharmaceutical preparations consisting of a intravenously injectable solution of the modified ISG according to the invention in a pharmaceutical acceptable carrier suitable for intravenous administration exist. The modified ISG can present in these solutions in any concentration, either immediately or after dilution, z. B. with isotonic saline solution, is suitable for IV administration and e.g. 1 to 18%, preferably about 1 to 15%, in particular about 10% protein for immediate application and about 16% for may be a solution to be diluted before administration. These solutions are sterile and free from fine-grained fabrics. A variety of aqueous solvents can be used for the protein, such as z. B. water, buffered water, 0.4% saline solution, 0.3 molar glycerol solution, etc. The aqueous vehicles that conventionally used for unmodified ISG or hyperimmune serum can also be used, with the exception of materials that cause an undesirable reaction to I.V. application would cause. The modified ISG can be used alone or in combination with other blood products, z. B. whole blood, plasma, fibrinogen, clotting factor and albumin can be injected intravenously.

The preparations according to the invention are administered in the usual manner, e.g. B. in quantities that one produce a sufficient, pharmaceutical antibody titre. With a 16.5% protein solution, about 1 up to 25 ml the usual single dose. Subsequent doses are usually given within 24 to 72 hours administered depending on the severity and duration of the disease.

The invention is explained in more detail in the following examples. Raw materials for the chemical Modification procedures were prepared in the following way:

method

(1) A Cohn Fraction II paste. the about 30% Containing gammoglobulin, was suspended at -5 ° C in a glycine / saline buffer, with a Solution was obtained containing about 5% gamma globulin, 2.25% glycine and 0.45% sodium chloride and had a pH of about 6.5.

(2) Lyophilized Fraction II paste, its protein content consisted of approximately 100% gamma globulin, was stored at 25 ° C in a buffer with 2.25% Glycine and 0.45% table salt (pH = 6.8) dissolved, making the final solution about 5% protein contained.

(3) A commercially available. A 16.5% solution of immune serum globulin in glycine / saline buffer was diluted with additional buffer so that the final solution contained approximately 5% protein.
Fraction II paste, powder or solution were obtained by a Cohn fractionation of the collected plasma from normal human donors 5 or de? collected plasma from selected donors with a high titer of a specific antibody, e.g. B. Antibodies for tetanus, rabies, or hepatitis-related antigen.

The protein concentration was spectrophotometrically determined, with a specific extinction coefficient of

^ Ej * _m = 13.8 at 280 πΐμ

was used.

example 1

A solution containing 750 g of tetanus immunoglobulin and correspondingly (1) in 15 liters of glycine / saline buffer was warmed to 22 ° C and its pH was adjusted by adding ml of 1 η NaOH brought to 8.2. A solution of 3.47 g (0.0225 mol) of dithiothreitol in 23 ml of glycine / saline buffer was added so that the concentration of dithiothreitol in the reaction mixture was 0.0015 m fraud. The solution was stirred for 15 min, then a solution of 9.15 g (0.0494 mol) of iodoacetamide in ml glycine / saline buffer was added, so that the concentration of the iodoacetamide in the reaction mixture Was 0.0033 m. The pH of the solution was, if necessary, over the following 2 hours. kept at 8.1 to 8.2 by adding 1 η NaOH. The reaction mixture was then added by adding ml of a 1 η HCl solution adjusted to a pH of 6.80 and cooled to 5 ° C. The solution was processed using the following two methods:

(a) A 50 ml portion of the reaction mixture was added dialyzed against several changes of 2.25% glycine / 0.45% NaCl buffer (pH = 6.8). The dialysate was concentrated by ultracentrifugation to a volume of about 25 ml and sterile by filtered through a 0.22 micron membrane. The sterile solution is a material that can be used intravenously Injection in humans is suitable and contains 8.6% protein measured spectrophotometrically.

(b) The remainder of the reaction mixture (15 liters) was diluted with 19.2 liters of cold water. That Chemically modified gamma globulin was made from this solution by precipitation at 0 ° C with cold Ethanol under conditions similar to those of the Cohn method 9 separated and the gamma globulin paste was recovered by centrifugation. The wet paste was then washed with two 10 liter portions of glycine / saline buffer containing the Contained enough ethanol to prevent dissolution of the gamma globulin. Through this Wash removed any residual reactants present in the precipitated gamma globulin were included. The moist paste was then lyophilized, leaving 560 g of dry, chemically modified gamma globulin were obtained. A solution that contained approximately 10%, was prepared by placing 529 g of the powder in 4.5 liters of buffer (2.25% glycine, 0.45% table salt, pH 6.8). This solution was then sterile filtered, one for intravenous Application in humans suitable material was obtained. This solution of the new, modified

adorned ISG can ζ. B. sterile in 10 ml single dose ampoules are filled, which then in the usual way with a coated rubber stopper be locked.

Anti-complement activity was measured in a standard dilution series. 0.2 ml one in two Dilution of the test substance prepared in parallel series were 2h. at 37 ° C with two "full" Units of guinea pig complement incubated in 0.4 ml of saline solution (one full unit of complement is the amount that causes complete hemolysis of 0.4 ml of a 1% suspension of sensitized Sheep erythrocytes) The retained complement was then determined by adding 0.4 ml a 1% suspension of sensitized sheep erythrocytes were added and a further 30 min at 35 ° C was incubated. The extent of hemolysis was determined visually, and when anti-complement animals were found the strongest dilution of the test substance was taken which resulted in at least 50% hemolysis.

The starting material for Example 1 had an anti-complement titer of 1: 512 at 10% Protein concentration in this experiment. The chemically modified product, either obtained by method (a) or method (b) had an anti-complement titer less than 1: 1, i.e. less than 1: 1. i.e., in the No hemolysis was observed in the undiluted sample. The chemically modified one according to the invention The product essentially does not bind complement and is therefore largely free of anti-complement activity.

The product of Example 1 (b) was determined by amino acid _{analysis to contain 7.7 -S-CH 2} CON Hz groups per molecule of modified gamma globulin. This number was calculated from the results obtained with an acidic hydrolyzate of the product, using a molecular weight of 160,000 for gamma globulin and determining the number of carboxymethylcysteine residues per molecule of gamma globulin. The product of Example 1 (b) was examined for tetanus antibodies by the quantitative precipitin technique. It was found that 1 ml of a 10% solution of the product precipitated 163 Lf units of tetanus toxoid, while 1 ml of a 10% solution of the unmodified gamma globulin precipitated 198 Lf units. After chemical modification, 82% of the precipitating antibodies for tetanus remained unchanged.

The gel diffusion against tetanus toxoid and against group A Streptococcus antigen was essentially found the same antibody content for the chemically modified product as for the unmodified one Source material.

The molecular weight of the main component in immune serum globulin is determined by the chemical modification unaffected. In Fig. 2 is the recording of the gel filtration of the product of Example 1 (b) on Bio-Gel A-0.5 m compared with that of the unmodified starting material. This gel filtration was used carried out in a Tris buffer at pH 8.0. If the gel filtration is carried out in 1 M acetic acid, then the product of Example 1 (b) shows a peak representing about 17% of total protein and one Molecular weight is assigned which corresponds to that of the free light chains.

In an aqueous solution at pH 7, there is the product of Example 1 when it is in the Ultracentrifuge analyzed, from 83.7%, 6.68S, 14.5% 9.73S and 1.7% 12.1s. Fragments sedimenting at less than 6S were not observed

Example 2

A solution that contains 190 g of tetanus immunoglobulin in 3.54 Liter of glycine / saline buffer was prepared according to method 2. The solution was heated to 22 ° C heated and their pH by adding 45 ml 1 η NaOH adjusted to 8.35. This solution was mixed with 138 g (0.0090 mol) of dithiothreitoJ in 9 ml of glycine / saline buffer treated; the reduction lasted 10 minutes, after which the mixture became alkylated by adding 1.82 g (0.010MoI) iodoacetamide in 45 ml glycine / saline buffer. the pH of the reaction mixture was lowered to 6.75 by adding 50 ml of 1N HCl. These reduced and alkylated gamma globulin solution was prepared in a similar manner to example 1, method (b), described worked up. 156 g of lyophilized powder were obtained, in glycine / saline buffer dissolved and sterile filtered to obtain a 10% solution suitable for intravenous injection became. The product had an anti-complement titer of 1: 1 compared to that of the unmodified starting material Was 1: 512; both determinations were carried out on 10% protein solutions. After 18 Months of storage at 5 ° C., the chemically modified product had an anti-complement titer of 1: 2. In a repeat attempt after 19 months, the titer was 1: 1.

The amino acid analysis of the modified ISG gave 6.7 —SCH ₂ CONH ₂ residues to a molecular weight of 160,000 , 84S, 17% at 9.86S and 3% at 10-IIS in the ultracentrifuge.

The modified ISG was compared to the unmodified starting material for its ability to provide passive protection to guinea pigs which were subsequently infected with 100 MLD tetanus toxin. The end point of 50% passive protection was 7 ³ A days for the unmodified _{starting material and 8 V 2} days for the chemically modified product. These results are in agreement with the accepted endpoint of 9 days for human gamma globulin in guinea pigs.

The in vivo half-life of the modified ISG was determined to be 2.3 days in guinea pigs, compared to 2.5 days for the unmodified starting material.

In agammaglobulinaemic patients, the mean plasma half-life was modified ISG determined to be 22.4 days. The infusions were safe and well tolerated.

Example 3

To 6 ml of a 17.3% solution of rabies immunoglobulin in glycine / saline buffer a further 15 ml glycine / saline buffer were added to achieve a 5.04% Protein containing solution. 15 ml of this solution were warmed to room temperature and with 1 η NaOH adjusted to pH 8.2. The solution was stirred and with 0.10 ml of a 0.225 M solution of Dithiothreitol treated in glycine / saline buffer. After 10 minutes, 0.20 ml of a 0.25 ml solution of Iodoacetamide added in glycine / saline buffer. Of the

pH, which had fallen to 8.10, was increased by adding increased from 0.1 η NaOH to 8.25. After 2 hours, the reaction mixture was turned against glycine / saline buffer Dialyzed from pH 6.85 and filtered through a 0.22 micron filter iieril. The sterile, chemically modified ToHwut immune serum globulin solution contained spectrophotometrically found 3.3% protein.

This product was on its Schuu.ffekt in the Mouse examined in an in vivo test; this resulted in a numerically somewhat smaller titre of protective substances Antibodies against rabies virus (1: 1100) than unmodified rabies immunoglobulin (1: 2000). Because of the fluctuation peculiar to a biological experiment, this difference becomes insignificant viewed. The anti-complement activity (5% solution) of the modified ISG was negative, compared to a titer of 1:32 for the baseline ISG.

Examples 4 to 7

60 g fraction II paste were according to method 1 in a glycine / saline buffer to 422 ml of a solution with a protein content of 4.86%. These Solution was adjusted to pH 8.2 and divided into equal portions of 40 ml. Each sample was with Dithiothreitol reduced and alkylated with iodoacetamide, as indicated in the table. After being alkylated for the indicated length of time, each Sample against glycine / Kochsaiz buffer dialysed until it was free of non-proteinaceous reactants and reaction products. The samples were through Ultracentrifugation concentrated to a protein concentration of approximately 10% by sterile filtration a 0.22 micron filter was obtained as a sterile solution suitable for intravenous administration (Samples 4 to 7).

Each of the nine samples was tested for antibodies against Streptococcus antigen group A. Samples 2 to 8 contained approximately the same amount of antibody as untreated sample No. 1, while the antibody content of sample No. 9 was reduced to an indeterminate level. The samples were acid hydrolyzed and the hydrolyzates were subjected to amino acid analysis _{to determine the number of —SCH 2 CONH 2} groups per molecule of gamma globulin. The anti-complement activity of the samples was determined according to the method described in Example 1. The results of the anti-complement, antibody activity and amino acid determinations are summarized in the table:

at sample Dithio Reduction Iodoacetamide 30th Alky- anti- mole antibody antigen game No. threitol- time (min.) Concentr. lation complement- -SCH ₂ CONH ₂ for Strep. TVpA Concentr. (mM) time (min.) Titer Per (mM) 1.6 X 105 g

A. 1 - - - 40 11.0 - 1: 128 0 B. 2 ( ) .5 10 1.1 120 1: 128 3.00 C. 3 LO 10 2.2 35 120 1: 8 5.78 4th 4th .5 5 3.3 120 1: 2 7.50 5 5 .5 10 3.3 120 1: 1 7.40 6th 6th .5 60 3.3 120 <1: 1 7.27 7th 7th 1.5 10 3.3 30th <1: 1 8.13 D. 8th .5 10 - 1: 128 0 E. 9; j.O 60 120 <1: 1 8.13 *) Not definable.

These results show that a reduction of 10 min with 0.0015 M dithiothreitol and a subsequent alkylation of 2 h min 0.0033 M iodoacetamide (sample 5) leads to a product which is essentially free of anti-complement activity, 7 , Contains 4 - SCH _{2 CONH 2} groups per molecule of gamma globulin and does not reveal any decrease in the antibody content. A reduction of 60 min (sample 6) or 5 min (sample 4) or an alkylation of 30 min (sample 7) instead of 120 min delivers products that are largely identical to sample 5. However, the elimination of the anti-complement activity was imperfect when lower amounts of dithiothreitol were used. In sample no. 9, although the anti-complement activity was low (reduction with high DTT concentration for 60 min), the antibody function was destroyed

Example 8

A commercially available solution of gamma globulin containing 16.5% tetanus immunoglobulin was used in the manner described in Example 1 at room temperature with 0.0075 M dithiothreitol for the Reduced duration of 1 hour and alkylated with 0.00825 M iodoacetamide for 2 hours. That product obtained was dialyzed against glycine dilution until it was free of the non-proteinaceous Portions of the reaction product, sterile filtered and sterile filled into 10 ml ampules for human use were intended. The tetanus antibody levels and anti-complement levels were 127 Lf units. or 1: 256 for the untreated control sample and 110 Lf units, or 1: 4 for the chemically modified Rehearse. This method has the advantage that the modified globulin is in the desired final concentration of 16.5% is obtained and does not need to be concentrated for the final filling.

Example 9

By intravenous administration of 16.5 ml of a 10% solution of the modified ISG of Example 5 or 6 in sterile 0.3 M glycine / 0.45% NaCl buffer Agammaglobulinemic patients were assessed for their gamma globulin serum levels raised to at least the same level as that when administered intramuscularly from 10 ml of a 16.5% solution of unmodified ISG.

Example 10

By intravenous administration of 1 ml of a 16.5% solution of hyperimmune ISG (250 tetanus antibody units per ml of a 16.5% solution), which was modified according to Example 6, in 0.3 M glycine and solution containing 0.45% NaCl, at least the same immunological effect is achieved as in the

intramuscular administration of an equal dose of the hyperimmune ISG used as starting material (Hyper-Tet, Cutter-Laboratories, Berkeley).

The above examples can be repeated with similar success if the specifically mentioned Reactants and / or reaction conditions are exchanged for one another.

For this purpose 2 sheets of drawings

Claims (1)

Patent claims: 1. Process for the preparation of a modified immune serum globulin for intravenous administration, by reduction with dithiothreitol and subsequent alkylation, characterized in that that