DK157367B - PROCEDURE FOR PREPARING INTRAVENOEST ADMINISTRATIVE HUMAN IMMUNOGLOBULIN - Google Patents

Description

DK 157367 B

The present invention relates to a method of preparing a novel intravenously administrable native, chemically unchanged, and not enzymatically degraded, stable immunoglobulin that shows no anticomplementary activity.

5 Human immunoglobulins play a significant role in the prophylaxis and treatment of infectious diseases and antibody deficiencies. Gamma globulins are used, for example, for the prophylaxis of infections of viral origin such as hepatitis, measles, rubella, mumps or rabies, or of bacterial origin such as tetanus, diphtheria or whooping cough. In 10 antibiotic-resistant infections caused, for example, by staphylococci, Escherichia coli, Pseudomonas, etc., gamma globulins are used therapeutically. Specific immunoglobulins (IgG) are also used for prophylaxis of rhesus incompatibility.

In addition, treatment with gamma globulins to protect 15 immunodeficient patients from infection suffering from agamma or hypogammaglobulinaemia is of particular importance.

However, the usual intramuscular administration has some major drawbacks: 1. Only a limited amount of up to 10 ml can be injected.

20 2. Resorption through the organism is greatly delayed. For example, R. Martin du Pan et al., Bfut 5, 1959, p. 104, observed that after 5 days, 35-40% of the immunoglobulin was still present at the injection site.

3. A large portion of the immunoglobulin is degraded at the injection site due to proteolysis. Cf.. S. Barandun et al., Vox Sanguirtis, 28, 1975, pp. 157-175.

In contrast, the intravenous injection or infusion rapidly leads to a high immunoglobulin blood level, as is necessary, for example, for the treatment of septic / toxic infections.

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However, for intravenous use, an immunoglobulin prepared by known fractionation methods from biod plasma, semen or placenta should not be used and is suitable for intramuscular use only. Cf.. Cohn et al., J. Amer. Chem. Soc. 68, 1946, p.

No. 5,459, A.J.L. Strauss et al., J. Immunol. 93, 1964, p. 24, A. Horejsi et al., Acta Med. Scand. 155, 1956, p. 65, A. Poison et al., Biochem.

Biophys. Acta 82, 1964 pp. 463.

In intravenous administration of lateral immunoglobulins, ca. 15-30% of patients have anaphylactoid incompatibility reactions (C.A. Janeway et al., New Engl. J. Med. 278, 1968 p. 919).

In immunodeficient patients who require a particularly high level of fGG administration, the number of these anaphylactoid incompatibilities lasts approx. 90%. There is currently no completely satisfactory explanation for these reactions. However, it is known that they are most likely to be triggered due to complement-binding IgG aggregates that can be frequently detected in the commercial immunogiobulin preparations.

For this reason, there is a direct relationship between the clinical incompatibility reactions, the amount of circulating complement and the anti-complement activity of the immunoglobulins determined in vitro. A major purpose of the preparation of intravenously administered immunoglobulins is therefore the removal of their anti-complementary activity (S. Barandun et al., Vox Sanguinis 7, 1962, pp. 157-174).

To reduce the anti-complementary activity of immunoglobulins (IgG), there are so far several possibilities:

It has been proposed to remove the IgG aggregates by ultracentrifugation or chromatographic separation. However, the compositions obtained by these methods are unstable and rapidly become again anti-complementary, probably as a result of reaggregation of the monomers (S.

30 Barandun et al., Vox Sanguinis 7, 1962, p. 157).

According to Patriotic Patent No. 359,640, the anti-complementary activity can be reduced by mixing with albumin or semen. However, the preparations obtained by this process can no longer be designated 3

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immunoglobulins, since in order to reduce the anticomplementary activity required, a large amount of aibumin or serum must be added so that the IgG proportion of the entire protein amount becomes too M11.

The removal of the IgG aggregates has also been attempted by adsorption 5 by activated charcoal (M. Steinbuch, Vox Sanguinis 13, 1967, p.

103), with starch, with silicates (German Publication No. 2,658,334), and by precipitation with polyethylene glycol (German Public Publication No. 2,751,717). Neither of these methods allow for complete removal of the anti-complementary activity.

In partial enzymatic degradation of immunoglobulin, preparations without complement binding activity are prepared, as proteolytic enzymes such as pepsin and plasmin preferably cleave or deposit the Fc portion of the IgG molecule, which portion contains the complement binding region of the molecule.

However, the major disadvantage of this method is the complete abrogation of the biological functions associated with the Fc portion of the IgG molecule, such as the cytophilic activity, opsonization or cytolysis (bacteriolysis) that all require an undamaged Fc. moiety that can bind to Fc receptors. In addition, the enzymatic cleavage of the immunoglobulin molecule Fab and FCab + fragments occurs, which is capable of binding the specific antigen but has only a very short biological half-life, namely 18-24 hours instead of 18 -22 days, as is the case with the entire IgG molecule.

Despite these major drawbacks, several enzyme-cleaved, intravenously administered immunoglobulin preparations are commercially available: French Patent No. 2,382M discloses pepsin-treated products, wherein the FCab 80%. Only 3-5% of the IgG molecules have resisted the proteolysis. These preparations have no anti-complementary activity and the FCab fragments are capable of neutralizing toxins and viruses.

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Plasmin-treated IgG preparations are described, for example, in German Publication No. 2,752,694. They contain approx. 30-40% intact IgG (subclasses 2 and 4) that are not proteolytically cleaved. The complement binding activity of this preparation is weak (20-50 mg / ml / 2CH5Q).

By means of acid treatment at pH 4 for 24 hours at 37 ° C, the anti-complementary activity of IgG according to, for example, S. Barandun et al.,

Vox Sanguinis 7, 1962, p. 157, is greatly reduced. The obtained preparations consist of 85-90% monomeric IgG and 10-15% IgG aggregates. The complement binding activity of these products is weak (50-70 10 mg / ml / 2CHgQ). However, their biological half-life is shortened to approx.

14 days and the preparations are not stable during storage and their anti-complementary activity increases again. A stable, lyophilized preparation prepared by this method has recently come on the market.

Many attempts have also been made and suggested to obtain intravenously useful IgG preparations through the action of reactive chemicals.

a) With β-propiolactone the complement receptors are blocked in the Fc portion of IgG. The products thus obtained no longer bind and complement 90% of monomers, but their biological half-life is reduced to 4-12 days. Cf..

European Patent Application No. 13,901, S. Barandun et al., Thesis Alfergy 9, Karger, Basel 1975, pp. 39-60.

b) By reducing and sulfonating the disulfide bridges of the IgG molecule, the anti-complementary activity of T. Yamanaka et al., 25 Vox Sanguinis 37, 1979, pp. 14-20 can also be greatly diminished.

c) By reduction and alkylation according to a proposal by D.D. Schroder et al., Vox Sanguinis 40, 1981, pp. 383-394, or by amidation according to German Publication No. 2,442,655, may be administered intravenously administerable IgG preparations.

Changes in the molecular structure and occurrence of new antigenic determinants in the IgG molecule cannot be ruled out with these chemical interventions.

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So far, no ideal immunoglobulin preparation is known for intravenous administration. Either the IgG molecules are modified by enzymatic degradation with loss of the properties dependent on the Fc fragment, or by chemical interference with alteration of the molecular structure, as the biological half-life is also usually shortened, or also when predominantly working with physical precautions, some anti-complementary activity in the IgG preparation remains.

The purpose of the present invention is to purify and treat immunoglobulin in such a way as to produce a stable product whose natural molecular structure remains intact, thus retaining the original antibody activity and having no anti-complementary activity detectable in vitro , which can therefore be used intravenously without risk, even in particularly vulnerable immunodeficient patients.

This is achieved in accordance with the present invention by a method consisting of a combination of three consecutive and consecutive steps, thereby reducing the anti-complement activity of the immunoglobulins at each step.

The first step consists in a fundamental separation of monomeric immunoglobulin G and its aggregates by an ion exchanger, selectively eluting the IgG monomers while the IgG aggregates remain bound on the ion exchanger. The eluted monomers bind only the complex weakly, but are highly unstable and quickly become anti-complementary again when not stabilized.

This stabilization is achieved in another step according to the known method described by S.L. Miekka et al., Vox Sanguinis 29, 1975, p. 101, by a weak acid treatment and / or addition of polyalkylene glycols, sugars and / or polyols, the addition of a polyalkylene glycol and sugar being mandatory.

Finally, the removal of the last traces of anticomplementary activity is achieved in a final step consisting of a selective adsorption to aluminum hydroxide.

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The order of these three steps is decisive for the result.

The immunoglobulin obtained by this method no longer has any anti-complementary activity and retains its original molecular structure and also unchanged antibody activity.

5 This high target can only be achieved by the combination according to the invention of the three process steps.

Thus, the present invention relates to a method for preparing intravenously administrable immunoglobulin for enhancing the immune system of the human organism, which method is characterized in that an anticomplementary immunoglobulin isolated by known methods is first bound to human plasma or placenta. an ion exchanger, the monomeric immunoglobulin selectively eluted therefrom with a 0.02-0.2 molar buffer solution, pH 4.0-5.5, is concentrated, essentially freed of the anti-complementary activity by treatment with a weak acid with a pH at 4.1-4.6 and optionally by incubation at 30-45 ° C for 1/4-1 hour, then diafiltered with a diluted buffer, pH 5.8-6.1 and / or stabilized by addition of a polyethylene glycol or polypropylene glycol and sucrose, lactose, maltose or mannose and / or sorbitol or mannitol, the addition of a polyalkylene glycol and sugar is mandatory and the last residues of anticomplete Finally, elemental activity is removed by adsorption to aluminum hydroxide.

In detail, the process consists in: a) placing an immunoglobulin isolated by known methods on a 25-ion exchanger, from which the monomeric immunoglobulin is eluted with a 0.02-0.2 molar buffer solution having a pH of approx. B) the substantially monomeric eluate is diafiltered after concentration, or incubated at 30-45 ° C for approx. 1 / 4-1 hour as a weakly acidic solution having a pH value of> 4 and <5 and diafiltered, 7

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the thus stabilized immunoglobulin solution is added a polyalkylene glycol, a sugar and optionally a polyol, and c) an aluminum hydroxide gel is finally added to which the last residues of anti-complementary activity are adsorbed, the adsorbent 5 is again separated, and the obtained, stabilized immunoglobulin ion is obtained. is processed into an intravenous injectable immunoglobulin preparation suitable for storage.

As starting material for the process of the invention, an immunoglobulin prepared by known fractionation processes from human plasma or human placenta is used, e.g., by an ethanol fractionation method (Cohn et al.), Precipitation with salts (Strauss et al.), Polyethylene glycols ( Poison et al.) Or an acridine derivative such as Rivanol® (Horejsi et al.) Or by a chromatography procedure. Placental immunoglobulin which can be used as starting material, e.g., according to H.L. Taylor et al., J. Amer. Chem.

Soc. 78, 1956 p. 1356, from the isotonic aqueous placental extract by fractional precipitation with 95% ethanol.

As starting material, immunoglobulin may be used from normal or hyperimmune human plasma or from the corresponding placenta.

20 For the treatment of infectious diseases and for prophylaxis, immunoglobulins with specific antibodies are required.

For such indications, plasma or placenta-derived immunoglobulins containing antibodies against certain viral or bacterial infections are used, e.g., antiviral antibodies against hepatitis, measles, rubella, mumps or rabies, or antibacterial antibodies to tetanus. , diphtheria, whooping cough, staphylococci, Escherichia coli, Pseudomonas, etc. For the treatment of Morbus haemolytic neonatorum, plasma or placenta immunoglobulins containing anti-D (Rho) antibodies are used.

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The natural immunoglobulin used as a starting material is used at a protein concentration of 20-40 mg / ml and a pH value of 4.0-5.5.

For example, a Cohn Fraction I and III, a final precipitate of venous or placental immunoglobulin or a lyophilisate at solution, or an optional even ethanol-containing immunoglobulin solution at the corresponding dilution and acid addition to pH 4.0 are adjusted.

This solution of IgG monomers, IgG dimers, IgG trimers and IgG polymers is separated by ion exchange (chromatographed).

As an ion exchanger, a cation exchanger is preferred. In particular, carboxymethyl celluloses (CMC) have been found to be suitable. The ion exchanger is first equilibrated with a 0.01-0.04 molar buffer solution of pH 5.0 4.0.

Under these conditions, all immunoglobulin is bound to the ion exchanger;

As buffer solution for equilibrating the ion exchanger and eluting the 15 IgG monomers, any biologically harmless buffer such as acetate buffer, phosphate buffer, citrate buffer or amino acid buffer can be used. Acetate buffer is preferred. The IgG monomers are eluted with a 0.02-0.2 molar buffer having a pH of 4.0-5.5, further containing 0.05-0.15 mol of sodium chloride.

The IgG aggregates and also the polymers as well as the trimmers and dimers remain bound to the ion exchanger and can later be released with a high-molar buffer.

The anticomplementary activity of the monomeric IgG fraction is weak, namely 60-80 mg / mI / 2CHgQ as compared to the starting material (0.3-1.0 mg / ml / 2CH 2 Q). The IgG monomer solution is concentrated and incubated at a pH of preferably 4.1-4.6 at 30-45 ° C for 1/4 to 1 hour. The incubation can be omitted when the starting material already has a relatively low anticomplementary activity.

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The immunoglobulin solution is now diafiltered against a buffer solution of pH 5.8-6.1, e.g., against a 0.005-0.015 granular phosphate buffer. Citrate, phthalate and amino acid buffers can also be used for this purpose. The IgG solution purified by the diafiltration is added for further stabilization a polyglycol, a sugar and optionally a polyol. As a polyglycol, 0.05-0.3% (w / v) of a polyethylene glycol (PEG 1000-6000) or a polypropylene glycol which sugars 3-7% (w / v) sucrose, lactose, maltose or mannose is preferably used, with Sucrose is usually preferred, such as polyol 0-7% (w / v) sorbitol 10 or mannitol. The obtained solution is adjusted to pH 6.4-6, β.

The third step is indispensable for removing the last traces of anti-complementary activity. Therefore, 5-20% (w / v) of aluminum hydroxide gel is added to the neutralized IgG solution. The suspension is stirred for 20 minutes to 2 hours at room temperature or 15 overnight at 4 ° C, then centrifuged at ca. 10,000 x g and sterile filtered. The filtrate is filled into ready-made containers, for example, vials or serum bottles, and then lyophilized.

The immunoglobulin thus prepared, after dissolving the lyophilate in distilled water or a 0.05 molar sodium chloride solution, has the following properties: 1. It no longer contains any measurable anticomplementary activity.

2. The proportion of monomeric immunoglobulin (IgG), as determined by gel chromatography, is between 85 and 95%. There are no fragments.

3. In the anti-human antiserum immunoelectrophoresis, only a single IgG precipitation line is visible. Cleavage products cannot be detected.

4. The distribution of IgG subclasses in normal semen is preserved.

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5. The activity of the antibody spectrum, calculated from the protein concentration, remains unchanged.

Comparison between anticomplementary activity expressed in (mg / ml / -2CH50) of commercial intravenously administered immunoglobulin 5 preparations and the natural, unchanged and non-degraded, stabilized immunoglobulin obtained by the invention:

The anticomplementary activity is expressed in mg protein per ml. ml needed to inhibit two units of complement. It is determined by 10% of the 50% hemolysis of sensitized multiple erythrocytes (mg / ml / - 2CHgQ) by that of M.M. Mayer, Experimental Immunochemistry, 2nd Edition, pp. 133-240, Charles C. Thomas, Springfield, as well as the U.S. Department of Health, Education and Welfare Public Health Monograph No. 74, "Standardized Diagnostic Complement Fixation Method and Adaptation to Micro Test" described standard procedure. The table is shown on page 11.

The immunoglobulin preparations prepared according to the invention were administered intravenously at doses of 2.5-10 g (pro dosi) to 48 patients. The preparations had a good tolerability, as no unexpected side effects could be observed. This trial included only patients who did not show any evidence of immunodeficiency. On the basis of these experiences, a clinical trial of patients with a congenital or later acquired immunodeficiency in whom the vital immunoglobulin therapy using 25 all previous preparations is associated with some risk is justified.

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Table

Anti-complementary ac-

IgG Preparation (mg / ml / 2CH 2 g) 5 - 16% Standard IgG 0.5 - 1.5 (for intramuscular use)

Upon adsorption, IgG purified 3-30 µg -Propiolactone-treated IgG 40-50 10 Acid-treated IgG 60-80

Plasma cleaved or partially degraded IgG 20 - 50

Pepsin-cleaved or partially degraded IgG no measurable activity

The IgG prepared by the process of the invention

(Example 1)

Starting material 0.7 CMC Monomer fraction 60 - 88

IgG preparation after stabilization 150 - 200 20 Final product (Example 1) no measurable activity

The advantage of the composition produced by the process of the invention over the known compositions is obvious.

EXAMPLE 1 25 g of lyophilized immunoglobulin (IgG) prepared according to the known COHN method # 9 from normal human plasma are dissolved in 90 ml of distilled water. The solution is adjusted after a clear filtration with 10% acetic acid to pH 4.6 and taken up in a 1 liter 0.02 molar acetate buffer of pH 4.6 pre-equilibrated 30 carboxymethyl cellulose column (CMC column) (7 cm long x 6 cm in diameter). The immunoglobulin is adsorbed to the ion exchanger and column 12

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wash with 500 ml of the above acetate buffer. The monomeric IgG is then eluted with 1.5 liters of 0.1 molar acetate buffer of pH 4.6 containing 8.18 g NaCl per ml. and then concentrated with an ultrafilter to 50 ml. This solution, which has a protein concentration of 70-80 mg / ml, is incubated at pH 4.1 for 30 minutes at 37 ° C in a water bath. In the diafiltration against 1 liter of phosphate buffer (0.015 M, pH 6.05), the pH is gradually adjusted to 5.8.

For stabilization, the product is added 0.3% (w / v) PEG 4000 and 7% (w / v) sucrose. The pH is adjusted to 6.4 with 0.1 N NaOH. After addition of 10% (w / w) aluminum hydroxide gel, the solution is stirred for 30 minutes at room temperature. The suspension is centrifuged for 20 minutes at 10,000 x g and the supernatant is sterile filtered. The preparation is sterilized in use containers and lyophilized. Hereby, much IgG solution is filled into the use vessels (ampoules, serum bottles, etc.) that, by dissolving the lyophilizate in 2x distilled water or 0.05M sodium chloride solution, a 5% protein (IgG) solution is obtained. suitable for intravenous injection or infusion.

EXAMPLE 2 6 g of lyophilized IgG is dissolved, filtered and purified over CMC in the manner described in Example 1. The monomer fraction is diafiltered after concentration without acid treatment directly against the 0.015 molar phosphate buffer, pH 6.05, until the pH of the solution becomes 5.8. After addition of 0.5% PEG 4000 and 3.5% of maltose and mannitol respectively to the solution, the pH is adjusted to 6.4 and mixed with 10% (w / w) aluminum hydroxide gel. The suspension is stirred for 15 hours at 4 ° C, centrifuged, filtered and further processed as described in Example 1.

EXAMPLE 3

As the starting material, the final precipitate from COHN procedure # 9 which still contains ethanol is used. The precipitate is dissolved in ice-cold water and further processed by the example described in Example 1

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13 method, however, the separation on the CMC column is carried out at 4 ° C. The other operations are performed as described in Example 1. Instead of sucrose, an equal amount of lactose is added.

The final product has the same advantageous properties as the final product 5 of Example 1.

EXAMPLE 4

As starting material, lyophilized IgG prepared by COHN method # 9 is used from human hyperimmune plasma with a high specific anti-tetanus titer. The starting material contains at 10% 10% protein concentration 2SO I.E. Te / ml. The aqueous solution of this lyophilizate containing 30 mg protein / ml is separated, purified and stabilized by the three-step procedure described in Example 1. The product is free of anti-complementary activity and contains as a 5% protein solution 150 I.E. tetanus / ml.

EXAMPLE 5

As starting material, IgG containing specific anti-D antibodies (150 µg / ml in a 10% protein solution) is used as described in Example 1. The final product contains as a 5% IgG solution 85 µg / ml specific anti-D antibodies and are free of 20 anti-complementary activity.

EXAMPLES 6-12

Analogously to the compound described in Examples 4 and 5, there are also intravenously optimally compatible compositions containing the following specific antibodies: 6. Anti-diphtheria antibodies, 7. Anti-hepatitis B antibodies,

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14 8. anti-rabid canine antibodies, 9. anti-rabies antibodies, 10. anti-rabies antibodies, 11. anti-measles antibodies and 5 12. anti-B. Pertussis antibodies.

These products retain - based on the protein content - their specific antibody activity.

EXAMPLES 13-22

Immunoglobulin fractions obtained from isotonic aqueous placental extracts by precipitation with 95% ethanol are adjusted to a protein concentration of 30 mg / ml and a pH of 5.0 and separated, purified and stabilized at Example 1 described method. The products are free of anti-complementary activity and are thus optimally compatible intravenously and contain the antibody spectrum present in the starting material.

13. IgG with nonspecific antibody spectrum, 14. IgG with anti-RIPD antibody, 15. IgG with anti-tetanus antibody, 16. IgG with anti-D antibody, 17. 17. IgG with anti-diphtheria antibody, 18. IgG with anti-hepatitis antibody, 19. IgG with anti-rabies antibody, 20. IgG with anti-rabies antibody, 21. IgG with anti-measles antibody and 22. 22. IgG with anti-B. Pertussis antibody.

Claims (3)

A method of producing intravenously-administered immunoglobulin for enhancing the immune response of the human organism, characterized in that an anti-complementary immunoglobulin isolated by known methods from human plasma or placenta is first bound to an ion exchanger, the monomeric immunoglobulin selectively. eluted therefrom with a 0.02-0.2 molar buffer solution, pH 4.0-5.5, concentrated, essentially freed of the anti-complementary activity by treatment with a weak acid having a pH of 4.1-4.6 and optionally, by incubation at 30-45 ° C for 1/4-1 hour, and then diafiltered with a dilute buffer, pH 5.8-6.1 and / or stabilized by the addition of a polyethylene glycol or polypropylene glycol and sucrose, lactose , maltose or mannose and / or sorbitol or mannitol, the addition of a polyalkylene glycol and sugar is mandatory and the last residual anti-complementary activity is finally removed by adsorption to aluminum ydroxid. Method according to claim 1, characterized in that immunoglobulin is used from normal or hyperimmune human plasma or from corresponding placenta. Method according to claim 2, characterized in that immunoglobulin containing antibody against certain viral or bacterial pathogens or anti-D antibody is used.