IE41671B1 - Modified imune globulins and process for their preparation - Google Patents

Abstract

Amidated immunoglobulins suitable for intravenous administration are prepared by reacting immunoglobulins with a molar excess of a primary monoamine and a carbodiimide or its salt in weakly acidic to neutral aqueous solution. As a result of the amidation, the undesired complement binding by the immunoglobulins is prevented or at least substantially decreased without the antibody action of the immunoglobulins being impaired. The process can be applied to reduced immunoglobulins, in which at least one disulphide bond has been converted to two sulphydryl bonds.

Description

The invention relates to modified immune globulins, a process for preparing them and pharmaceutical compositions comprising them. It especially relates to chemically modified immune globulins which may be administered via the intravenous route. immune globulins prepared by fractionation from serum, especially from human serum, have the essential property of acting as antibodies agaihst antigens.

Immune globulin compositions have hitherto proved 10 suitable only for intramuscular administration. In the case of intravenous administration the recipients have shown a more or less marked reaction with anaphylactic effects. It is supposed that these secondary reactions are due to the fact that complement present in the serum is bound by the immune globulin administered. It is, however, desirable to administer immune globulin preparation via the intravenous route since the antibody activity is deployed faster via this route.

It has been attempted several times to modify immune globulins in such a way that their activity as antibodies is maintained, while at the same time the degree of complement binding is reduced to such an extent that the modified immune globulins can be used for intravenous administration. For example, immune globulin molecules may be modified by enzymatic degradation, so that the linking points for complement are split off, but the rest of the molecule is capable of binding antigens. Such an immune globulin has been administered intravenously with some success.

The reaetion of an immune globulin with an alkylating or acylating agent also leads to the formation of an immune globulin suitable for intraveneous administration. The reduction of the complement binding of an immune globulin may also be obtained by N-alkylation or benzylation.

Further, a process has been proposed according to which an immune globulin is partly split by reduction of intramolecular disulphide bonds and the sulphydrile groups thus formed are subsequently alkylated. In this process the original size of the molecule is maintained.

These processes are essentially based on the modification of the free amino groups or disulphide bonds of the immune globulin molecule.

Although these processes lead to products having reasonably satisfactory properties, there still remain problems for which improved solutions could be found, especially because the physical and chemical properties of the molecules are considerably modified by the processes described.

The present invention is based on the observation that immune globulins in which some carboxyl groups have been modified considerably change their binding behaviour with regard to complement without losing their efficiency as antibodies. Such modified immune globulins which bind complement to a smaller or even non-detectable extent, are suitable for use as medicaments for intravenous administration.

Thus, the present invention provides an amidated immune globulin and furthermore a process for preparing such an immune globulin, wherein an immune globulin is reacted with a carbodiimide or a salt thereof and a molar excess of a primary monoamine or a salt thereof in an aqueous solution having a pH within the range of from 3 to 7. The process is preferably carried out at o a temperature of from 5 to 50 C.

The molar .ratio of the amine to the immune globulin is preferably at least 50:1 and the ratio of carbodi imide to immune globulin is preferably at least 1:1.

We have found that the complement binding of the immune globulin is particularly low if the ratio of the carbodiimide to the immune globulin is within the range of from 5:1 to 20:1 i.e. this is the most preferred range.

(The degree of complement binding may be carried out, according to the method of A. Nowotny, Basic Exercises in Immuno-chemistry, page 160 et seq. (1969)).

As the starting material for the reaction of the invention there may be used an immune globulin fraction obtained from serum, plasma, another body liquid or an organ extract. Especially suitable are those fractions which are enriched with regard to immune globulins. A preferred method for preparing such a fraction is the . method according to Levy and Sober via chromatography on DEAE cellulose. Naturally, a pure immune globulin may be amidated according to the invention, but in practice a 100% pure immune globulin is rarely used at present, due to the expensive processes for purification The immune globulin is preferably of human origin.

The amine used is any primary monoamine, i.e. a compound of the general formula R-OTL wherein R represents a radical which according to known concepts does not have any marked antigenic activity, or a salt thereof.

Examples of suitable amines, are especially aliphatic amines, for example, methyl amine, ethyl amine and higher aliphatic amines, especially those having up to 10 carbon atoms. If an acidic aqueous solution is used in the process of the invention, the amine is generally present as the corresponding ammonium cation. According to the invention the preferred amines are those which carry one or more further functional groups, especially hydrophilic groups, for example, hydroxyl and/or acetal groups. Examples of such amines are ethanolamine, trishydroxymethylamino-methane and glucosamine, which have a favourable influence on the solubility of the reaction product in a physiologically tolerable aqueous medium.

As the process of the invention must be carried out under conditions under which the antibody activity of the immune globulin is not adversely affected, it is essential to carry out the reaction in known manner in the presence of a carbodiimide as activator. As the carbodiimide there may be used any representative of this clasB of compounds which is capable of having an activating effect on the formation of peptide bonds. Examples of such carbodiimides are l-ethyl-3-(3dimethylaminopropyl) - carbodiimide - hydrochloride I (EDC) and l-cyclohexyl-3-(2-morpholinoethyl) - carbodiimide - metho - £ - toluene-sulphonate (CMC). The carbodiimide is present as a salt if the aqueous solution is acidic, in the same way as is the amine. The carbodiimide is generally used as a salt as it is more stable and easier to handle in this form. In principle, a free carbodiimide may be used, but on dissolution in an acidic aqueous solution it will pass to the salt form.

The process of the invention for preparing an amidated immune globulin may also be carried out using an immune globulin/which at least one of the disulphide bonds present has been reduced to two sulphydrile groups The resulting amidated globulin has the same advantageo15 us properties as an amidated unmodified immune globulin.

For the reduction of disulphide bonds it is particularly advantageous to use dithiothreitol or dithioerythritol (Cleland's reagent). The disulphide bonds may also be reduced according to another known process with a reducing agent, for example, 2-mereaptoethanol or mercaptoethyl amine while using a high concentration of the reducing agent.

The reducing agents described by Cleland hnd having the following formulae HS· H Η H OH H ί Li i may be used for the reduction of an immune globulin.

The reduction of the disulphide bonds is preferr ably carried out in a slightly alkaline medium, advantageously with a concentration of reducing agent of 0.01 mol/litre and a molar ratio of globulin to reducing agent of from 2.5:1 to 50:1.

The amidated immune globulins of the invention, which have a sufficiently low degree of complement binding may be administered by intravenous route. They may be treated with a physiologically tolerable solvent or suspending agent to obtain a pharmaceutical preparation. Such a preparation advantageously in a form suitable for intravenous administration, may be made available in a liquid or freeze-dried form.

The following Examples illustrate the invention. Example 1: Preparation of the starting material. 22.1 litres of human serum obtained from spontaneously coagulated blood were passed for salt formation over a column equilibrated with 0.0175 molar sodium phosphate, pH 6.4, filled with Sephadex G—25 medium (Sephadex is a Trade Mark of Messrs.

Pharmacia for cross-linked dextran). With a passage photometer the absorption of the column eluate was measured at 280 nm. The first peak formed by the serum proteins was collected separately from the following low molecular portions and passed over a column rinsed with the above-mentioned phosphate buffer, of 15 kg of DEAE-cellulose with 1 mol equivalents/g of exchanger capacity. The column was rinsed afterwards with 1.5 column volumes of buffer. The immune globulins that - 8 had passed through the column were precipitated by the addition of solid ammonium sulphate to give a concentration of 2.2 moles per litre. Most of the supernatant liquid was siphoned off after the mixture had been allowed to stand for 24 hours, the rest was removed by centrifugation at 5,200 g. The residue obtained on centrifugation was freed from ammonium sulphate by dialysis against 0.1 molar NaCl solution. The volume of the dialysed immune globulin solution was made up with a NaCl solution to 2,000 ml. It contained 155 g of protein on the whole.

Amidation of the immune globulin. 1000 ml of the immune globulin solution obtained according to the process described above were dialysed for 24 hours while stirring against 1000 ml of 1 molar tris-(hydroxymethyl)aminomethane-(tris)-HCl buffer, pH 5.4, transferred to a glass vessel and mixed while stirring with 0.96 g 1-ethyl - 3 - (3 - dimethylaminopropyl) - carboxydiimide . HCl. The batch was stirred for 2 hours at room temperature.

The immune globulin amidated with tris-(hydroxymethyl) aminomethane was passed over a column containing 8 litres of Sephadex G—25 which had been previously rinsed with a solution having O.15 molar NaCl and 0.3 molar glycine as well as a pH value of 7.3. The optical density of the column eluate was measured at 280 nm with a passage photometer. The portion of the eluate containing the amidated immune globulin was collected and concentrated with an ultra-filter to a protein content of 5% (wv).

The following Table shows a comparison of the unmodified starting immune globulin with regard to complement bond and antibody activity: TABLE 1: Antibody specifity Complement German measles diphtheritis Tetanus binding titre IU / ml IU /ml starting immune globulin 22% 1:1024 >•0.5 <£1.0 >•1 ¢.2 amidated immune globulin 0% 1:1024 >0.5 xl.O >1 <£2 the evaluation of the complement binding was effected according to the method of Nowetny, A., Basic Exercises in Immunochemistry; Springer Verlag, 1969, page 160.

Example 2: 1000 ml of immune globulin solution obtained according to the method described in Example 1, and containing 75.5 g of protein were mixed with 890 ml of a solution containing 52.5 g of glucosamine . HCl and the pH value was adjusted to 6.0 with 2 molar NaOH. With stirring, 2.06 g of 1-cyclohexyl - 3 - (2 - morpholinyl4 - ethyl) - carbodiimide-metho-£-toluene sulphonate were added to the solution and stirring of the batch was continued. The reaction temperature was maintained at 25°C. The transfer of the amidated immune globulin into a solution containing 0,15 mole per litre Nacl and 0.3 mole per litre of glycine of pH 7.3 was carried out in the manner described in Example 1, but with a column containing 10 litres of Sephadex G—25. The concentra20 tion of 5% of protein was also effected as described in Example 1. The results of the evaluation of amida41671 ted immune globulin obtained according to Example 2 in comparison to those of the starting globulin are given in Table 2.

TABLE 2: Antibody specifity Complement German measles, diphthertis tetanus binding titre IU / ml IU / ml starting immune globulin 22% 1:1024 ?0.5 ^.1.0 >1 <2 amidated imune 1.5% 1:1024 ^0.5 <1.0 >1 <3 globulin Example 3: Reduction and amidation of the immune globulin.

The amidation of the immune globulin described in Example 1 and 2 was carried out in the same way with a reduced immune globulin. The reduction was carried out as follows: A solution of 3.3 g of immune globulin in 330 ml of 0.15 molar Nacl solution was adjusted to pH 8.2 with tris(hydroxymethyl)-aminomethane (Tris). To the immune globulin solution 15.3 mg of dithioerythrite (DTE) dis15 solved in 2 ml of water were added. After 60 minutes ml of a tris-HCl solution containing 5 g of Tris, of pH 1.0, were added while stirring. The pH value of the mixture was adjusted to 5.0 with HCl and amidated as described in Example 1 after addition of 82.2 mg of N20 ethyl-N'-(3-dimethylaminopropyl) - carbodiimide . HCl.

The reduction of the immune globulin may be carried out instead with dithiothreitol (DTT) under the same conditions.

Claims (5)

1. CIAIMS:1. An amidated immune globulin.

2. A process for preparing an amidated immune globulin wherein an immune globulin is reacted with a carbodiimide or a salt thereof and a molar excess of a primary monamine or a salt thereof in an aqueous solution having a pH within the range of from 3 to 7.

3. A process as claimed in claim 2,wherein the molar ratio of the amine to the immune globulin is at least 50:1 and the molar ratio of the carbodiimide to the immune globulin is at least 1:1.

4. A process as claimed in claim 2 or claim 3, wherein the molar ratio of the carbodiimide to the immune globulin is within the range of from 5:1 to 20:1. 5. A process as claimed in any one of claims 2 to 4, wherein the amine is an aliphatic monoamine having up to lo carbon atoms per molecule and which may be substituted, by one or more hydroxyl and/or acetal groups. 6. A process as claimed in claim 5, wherein the amine is ethanolamine, trishydroxymethylaminomethane or glucosamine. 7. A process as claimed in any one of claims 2 to 6, wherein the carbodiimide is 1 - ethyl 3 - (3dimethylaminopropyl) - carbodiimide hydrochloride or 1cyclohexyl - 3 - (2 - morpholino - ethyl) - carbodiimidemetho-p-toluene sulphonate. 8. A process as claimed in any one of claims 2 to 7, wherein the reaction is carried out at a temperature of from 5 to 50°C. 9. A process as claimed in any one of claims 2 to 8, wherein a human immune globulin is used. 10. A process as claimed in any orje of claims 2 to 9, wherein the immune globulin has had at least one disulphide bond converted into two sulphydrile groups with the aid of a reducing agent. 11. A process as claimed in claim 10, wherein the conversion of the disulphide bond into sulphydrile bonds is carried out with a reducing agent at a slightly alkaline pH-value. 12. A process as claimed in claim 11, wherein the conversion of the disulphide bond into sulphydrile bonds is carried out with a reducing agent at a slightly alkaline pH value with a concentration of the reducing agent of 0.01 mol/litre and a molar ratio of globulin to reducing agent of from 2.5:1 to 50:1. 13. A process as claimed in any one of claims 10 to 12, wherein the reducing agent is dithioerythrite or dithiothreitol. 14. A process as claimed in claim 2, whenever carried out substantially as described in any one of the Examples herein. 15. An amidated immune globulin whenever produced by a process as claimed in any one of claims 2 to 14. 16. An amidated immune globulin which is substantially as described in any one of the Examples herein. 17. A pharmaceutical preparation which comprises an amidated immune globulin as claimed in claim 1, claim 15 or claim 16, in admixture or conjunction with a pharmaceutically suitable carrier. 18. A pharmaceutical preparation as claimed in claim 17, in a form suitable for intravenous administration.

5. 19. A pharmaceutical preparation as claimed in claim 17 or claim 18, in freeze-dried form.