SI7912259A8 - Process for production of antigens which contain immunoglobulin preparation for intravenose use. - Google Patents

Description

IMMUNO ”Aktiengesellschaft fiir chemisch-medizinische Produkte

Method for preparing an antibody-containing immunoglobulin preparation for intravenous administration

Technical field of the invention

A 61K 37/06.

The present invention relates to the field of pharmacy and diagnostics, and more specifically to a process for the preparation of a novel, antibody-containing immunoglobulin preparation for intravenous administration, which can be administered in high doses intravenously without adverse reactions.

A technical problem

There was a need to find a new, technologically advanced process for the preparation of a novel, antibody-containing immunoglobulin preparation for intravenous administration, without disturbing enciraatic or chemical degradation, in good yields and with such quality that. it can be administered in high doses intravenously without adverse reactions.

The state of the art

After getting over the infection, the human body is generally protected from another infection by the same agent. Such protection can also be achieved by vaccination; · builds on cellular and humoral immunity. While cellular immunity carriers are lymphocytes, they induce a variety of specific antibodies to humoral immunity. Antibodies are high molecular weight proteins with a globulin character that occur in the blood. The complete formation of humoral immunity may take the first two decades of life and is weakest especially during the 6 and 24 months of life. It has been known for a long time (1952 Bruton) that antibodies may be lacking in certain patients (antibody deficiency syndrome), most of which are known to be familiar with the frequency. Such patients suffer from various infections, which are often recurrent and may even be life-threatening. Similar antibody deficiency states can not only occur genetically, but can also be acquired and occur mainly as selective or partial antibody deficiency syndrome (AMS).

As such patients cannot produce the missing antibodies, they need to be sufficiently fed in the treatment or prevention of infections. The effect of such substitution is limited in time and the duration of the effect depends on the biological one

- 3 half-lives of antibodies delivered. Antibodies formed in the body have a biological half-life of 3 to 4 weeks.

The preparation of antibody-containing immunoglobulins is known, inter alia, by fractionation of human blood plasma by e.g. Gbhn Methods (J. L. Oncley, M. Melin, D. A. Richert, J. W. Cameron, and P. M. Gross, Jr. The Separation of Antibodies, Isoagglutinins, Prothrombin, Plasminogen, and -Lipoprotein Into Subfractions of Human Plasma, J. Am. Chem. Soc., Volume 71, page 541 (1949) with alcohol precipitation, These preparations are suitable for intramuscular applications but not for intravenous applications, as they produce by-products with negative properties during the preparation process. sometimes even short-lived but very potent intolerance reactions during injection, such as lowering blood pressure, which can cause life-threatening conditions.To obtain portable immunoglobulins for intravenous injection, the immunoglobulins have so far been enzymatically degraded or chemically modified. and the therapeutic effect or biological quality of the immunoglobulin in the rectum has worsened, which not only reduced the immunoglobulin molecules due to the enzyme Thus, the resulting vaccine products also competitively inhibited the antigen / antibody reaction to be achieved and / or the binding of the antigen / antibody complexes to Fc receptors. The second flaw of such altered immunoglobulins, however, is the shortened biological half-life.

A further drawback of chemically altered immune globulins is the emergence of new antigenic specificities that, in the recurrent

- 4 administration in certain circumstances may lead to additional intolerance reactions.

A further process, belonging to the prior art (DE-OS 26 06 118), which sought to prepare gamma-globulin suitable for intravenous injection, involves stepwise purification of the plasma protein suspension with polyethylene glycol and complete precipitation with polyethylene glycol. Purification steps with polyethylene glycol are preferably carried out at a low ion concentration, which they consider essential to prevent protein denaturation. However, the fractionation at extremely low ionic concentration is, as they found out, wrapped and the products obtained are not without the occurrence of intolerance.

Finally, the process (AT-PS 344 833), by which gamaglobulin preparations from the plasma fraction are obtained for intravenous administration by precipitating the anticomplementary portion of gamaglobulin with PEG (polyethylene glycol) in the presence of hydroxyethyl starch, is also included in the prior art; however, it has also been shown that such purification is not sufficient to allow the finished product to be administered intravenously without adverse reactions.

Description of solution to a technical problem with implementation examples

However, it has now been found that a new antibody-containing, immunoglobulin preparation for intravenous administration is prepared in which the immunoglobulin-containing fraction is subjected to a first purification step prior to precipitation with polyethylene glycol by treatment with an aqueous solution of a polyvalent inorganic acid salt, in particular ammonium sulfate, in particular of the following purification steps with polyethylene glycol in the presence of a soluble carbohydrate or protein-free polyol, i.e. a polyol with at least three hydroxyl groups, then precipitate the immunoglobulin from which the protein impurities have been removed from the remaining solution with water-soluble polymers and then in a known manner we complete the pharmaceutical preparation.

It is advantageous to obtain an immunoglobulin-containing fraction from human blood plasma by the known Cohn method or method. by the modified Cohn method by ethanol precipitation and the fraction containing ethanol removed, preferably by dialysis.

According to a preferred embodiment of the process according to the invention, three cleaning steps are used, working in

- 6 ρΗ 7.0 to 8.0 Immunoglobulin-containing precipitate, after which this precipitate is obtained, dissolved in water and ammonium sulphate is removed from the solution, preferably by dialysis, and then the remaining immunoglobulin-containing solution in the third purification step in the presence of a saccharide, a polysaccharide or polysaccharide hydrolyzate and subjected to treatment with polyethylene glycol at pH 4.9 to 8.0, preferably 5.8 to 6.4, after which the precipitate formed is discarded and discarded, and the remaining "aztopine in the third cleaning step is subjected to re-treatment with polyethylene glycol at pH 6 , 4 to 7, θ, then the recovered precipitate is removed and discarded, and the immunoglobulin, which is in the remaining solution and no longer contains Noxious contaminants, is precipitated at ρΗ 7, θ to 7.5 with a polymeric precipitant and processed into a pharmaceutical preparation.

In this combination of measures, polyethylene glycol with a molecular weight of 2000 to 6000 in an amount of 60 to 90 g / l at a temperature of 0 to 40 ° C can be used as a precipitation agent in the third purification step. Polyethylene glycol with a molecular weight of 2000 to 6000 as a precipitant can also be used in the fourth purification step, preferably in an amount of 70 to 100 g / l, also at a temperature of 0 to 40 ° 0.

- 7 Of the soluble carbohydrates, respectively. in particular, saccharides, namely monosaccharides, such as ginkgo, fructose, mannose, galactose, or disaccharides, such as sucrose, lactose, maltose, or oligo-, polysaccharides, have proved to be suitable. otherwise in an amount of 1 to 55 weights. %, preferably 5 to 20 weights.

% with respect to solution. As soon as the immunoglobulins are purified as described above, they can be obtained from the solution ^ by precipitation with water-soluble polymers. Copolymers of ethylene oxide and polyoxypropylene (market name BASF “PLURONIC) have acted as such, delivering dextran, polyvinyl alcohol, polyvinylpyrrolidone and the like. Polyethylene glycol can also be used as a precipitant for pure immunoglobulin by increasing the concentration of polyethylene glycol in the remaining solution ^ to above 150 g / l. The precipitated immunoglobulin is then converted into a pharmaceutical preparation using known methods.

The conversion of the purified immunoglobulin into a pharmaceutical preparation is advantageously carried out by dissolution in pyrogen-free water, with further dialysis, ultrafiltration or gel filtration, adjusting the irounglobulin concentration to 5 to 200 g / l, preferably 5θ to 160 g / l and adjusting the ionic strength using sodium chloride to 0.005 to 0.3, preferably 0.1 to 0.2.

Plasma proteins contained in the finished product are at least 80% made up of bulk immunoglobulins with a sedimentation constant of 8 7.0 and a biological half-life of 21 to 28 days. The anticomplementary activity of the product corresponds to the value at which at least 35 horns of protein are required to neutralize the ΟΉ ^ θ unit. Furthermore, the compositions according to the invention are characterized in that they contain less than 5% protein components with a molecular weight of more than 160,000.

The process of the invention is explained in more detail by the following examples:

- 9 EXAMPLE 1

Adjust human blood plasma to pH 7.0 to 7.2 and maintain at -2 ° C. To the solution was added 8 weights. % ethanol, precipitating a precipitate containing essentially fibrinogen. After settling this precipitate, the ethanol concentration is increased to 25 weights. % and reduce the temperature to -6 ° C. The separated precipitate consisting essentially of crude immunoglobulin was extracted with phosphate-acetate buffer and Ί2 weight added. % ethanol at a pH of 5.5 P ^ and “2 ° C.

The precipitate (containing a-and β-globulin) was discarded and the ethanol concentration of the top layer (supernatant) was raised to a pH of 7.2 and a temperature of -dO ° C to 25 weights. % to precipitate immunoglobulin. The thus prepared pasty immunoglobulin fraction of the present invention is further treated as follows:

kg of the immunoglobulin paste was mixed with stirring in 2 l 0.9% sodium chloride solution and dialyzed. The protein concentration was then adjusted to 20 g / l as well as the ionic strength to 0.15, and at a pH of 6.25 176 g / l of ammonium sulphate was added, after which the precipitate containing the undesirable impurities was discarded and further ammonium was added to the top layer. sulfate until a concentration of 275 g / l is reached and the pile values are adjusted

7.2. The isolated immunoglobulin-containing precipitate is dissolved in water and dialyzed against tap water. Then a solution of 80 g / l polyethylene glycol in the presence of 150 g glucose / l at 6.0 and ionic strength 0.15 * is added. 'The precipitate is discarded and at pH

6.5 to 6.6 increase the concentration of polyethylene glycol to 95 g / l · Discard the precipitated precipitate.

Now increase the polyethylene glycol content of the solution at a pH of 7.2 to 180 g / l to precipitate the impurity-free immunoglobulin. The product is centrifuged and the polyethylene glycol present is removed by washing with 5 times.

EXAMPLE 2 kg Immunoglobulin Paste: In the same manner as described in Example 1, ir. is treated, except that in the second purification step, fructose in the amount of 150 g / l is added instead of glucose.

EXAMPLE 5 kg of immunoglobulin paste in the same manner as described in the example. 1, is obtained and treated, except that at the second purification step, sucrose in the amount of 150 g / l is added instead of glucose.

EXAMPLE 4

0.5 kg of immunoglobulin paste obtained in the same manner as described in Example 1 from human blood plasma was dissolved in a 6 1 0.9% solution of sodium chloride under stirring and ethhenol was removed by ultrafiltration from the solution. The protein concentration of the remaining solution was adjusted to 20 g / l ionic strength at 0.15, then added at a pH of 6.7 to 119.5 g / l disodium hydrogen phosphate.

The precipitate is discarded and the top layer is adjusted with sodium hydroxide to pH.

7.2, whereupon a precipitate is formed which is discarded. - 'A solution of 275 g / l ammonium sulfate is then added. The recovered immunoglobulin-containing precipitate was dissolved in water and dialyzed to remove inorganic salts. 87.5 g / l of polyethylene glycol and 15θ g / l of glucose were added to the solution and the pH was adjusted to 6.0. Discard the precipitate, 's', - increase the value of the remaining solution to 6.6 and increase the concentration of the solution to 95 g of polyethylene glycol / l by further addition of polyethylene glycol. Discard the precipitate recovered,. We now increase the polyethylene glycol content of the solution at a pH of 7.2 to 15 ° g / l, thereby precipitating the impurity-free immunoglobulin. The product was centrifuged as in Example 1 and the polyethylene glycol was removed by washing.

EXAMPLE p kg of iraunglobulin in the same manner as described in Example 1 is obtained and treated except for the final precipitation of impurity-free immunoglobulin by the addition of 68 g / l Pluronic F 68 (ethylene oxide copolymer with polyoxypropylene) maintaining a pH of 7.2.

- 12 The following Table I lists the anticomplementary activity of immunoglobulin prepared according to Examples 1 to 5, the values being determined according to E.A. Rabat and M.M.

Mayer ”Experimental Immunochemistrj (Thomas, Springfield 1961) oz. Public Health Monograph no. 74: Standardized diagnostic complement fixation method and adaptation to microtest (Washington, 1965).

Table I

Immunoglobulin prepared according to Example 1 Example 2 Example 3 Example 4 Example 5

Anticomplementary activity

96 mg 95 mg 101 mg 55 mg 104 JS £ L

The preparations made according to Examples 1 to 5 contain more than SO% 7 S-components listed in Table II. The determination is carried out in an analytical ultracentrifuge within the meaning of EuropSisches Arzneibuch, zv. Il, Deutscher Apotheker Verlag, Stuttgart 1975, determining a 1% solution (V / V) in phosphate buffer solution with a pH of 6.0 to 8.0 and an ionic strength of at least 0.2.

- 13 TABLE II

Imunglobulin, prepare yen after

Example 1

Example 2

Example 3 Example 4 Example 5

S-components

95.5% 95.5% 98.4% 88.0% 98.2%

For further determination, the Na-dodecyl sulfate-polyacrylamide-gel electrophoresis immunoglobulins obtained in Examples 1 to 5 were tested [SDS-PAGE, K. Veber and M. Osborn,

J. Biol. Chem. 244. 4406 (I969)] y whereby a breakdown of the resulting proteins by their molecular weight is obtained. In the immunoglobulins obtained according to the invention according to Examples 1 to 5 (Samples 1 to 5)>, the width bands at molecular weights of 150,000 are shown in the accompanying diagram.

In contrast, specimen 6 with pepein is an encyro-degraded preparation, and sample 7 with plasmin is an enzymatically-degraded preparation, in which the width bands at lower molecular weights are 105,000 oz. 5θ 000. Sample 8 shows the molecular weight distribution in a chemically modified preparation, where relatively viscous are present. molecular de14 lies above 160,000 as well as proportions with lower molecular weights of about 000.

According to the invention, the preparations made are compared in terms of their effect of affecting non-blood pressure with a known blood pressure active agent made by the Cohn method. Comparative experiments were performed on narcotic dogs. The vena jugularis externa of the dogs was prepared at the lower edge of the mandible, and the two vein branches were connected to the catheter. Furthermore, we freely prepared the carotis artery and tethered the catheter. Blood pressure was measured electromanometrically via an arterial catheter; a narcotic was introduced through both other catheters and the immunoglobulin preparation was compared, while maintaining the volume indicated. Blood pressure values were monitored for 60 minutes after the test substance was administered, with systolic and diastolic blood pressure values recorded each time and a median recorded. The doses administered were each Ι5θ mg / kg. The results are illustrated in the following table:

Blood pressure in mra / Hg (mean)

on to the beginning PO 5 min by 10 min PO 20 min by 30 min PO 60 min preparation, 100 made in 94 100 100 98 98 of the invention known preparation 100 43 58 58 70 90

An indication of the best way to make economic use of the invention

Adjust human blood plasma to pH 7.0 to

7.2 and maintained at -2 ° C. To the solution was added 8 wt% ethanol, precipitating a precipitate containing essentially fibrinogen. After settling this precipitate, the ethanol concentration was increased to 25% by weight and the temperature was reduced to -6 ° C. The precipitated precipitate, consisting essentially of crude immunoglobulin, was extracted with phosphate-acetate buffer and ethanol was added by weight at a pH of 5.3 at -2 ° C. The precipitate (containing alpha and beta-globulin) is discarded and the ethanol concentration of the top layer (supernatant) is increased at pH

7.2 and -10 ° C at 25% by weight, thereby precipitating the immunoglobulin. The thus prepared pasty immunoglobulin fraction of the present invention is further treated as follows:

kg of the immunoglobulin paste was mixed with stirring in 2 1 0.9% sodium chloride solution and dialyzed. The protein concentration was then adjusted to 20 g / l as well as the ionic strength to 0.15 and at a pH of 6.25 176 g / l of ammonium sulphate (first purification step) was added, after which a precipitate containing the undesirable impurities was discarded and t

further ammonium sulfate was added to the layer until a concentration of 275 g / l was reached and the pH was adjusted to 7.2. The isolated immunoglobulin-containing precipitate is dissolved in water and dialyzed against tap water. Then a solution of 80 g / l of polyethylene glycol is added in the presence of 150 g of glucose / l at a pH of 6.0 and an ionic strength of 0.15 (second purification step). The precipitate recovered is discarded (third stage).

We now increase the polyethylene glycol content of the solution at a pH of 7.2 to 180 g / l, thereby precipitating the impurity-free immunoglobulin. The product was centrifuged and the polyethylene glycol present was removed by washing 5 times.

For

IMMUNO ”Aktiengesellschaft fur chemisch-medizinische Products:

Claims (10)

PATENTHI APPLICATIONS What is claimed is: 1. A method for preparing an antibody-containing immunoglobulin preparation for intravenous administration in which human blood plasma is fractionated and an immunoglobulin-containing fraction is collected, characterized in that the immunoglobulin-containing fraction in aqueous medium is subjected to repeated treatment with uncleaned non-destructive proteins . is subjected in the first purification step to an aqueous solution of a salt of multivalent inorganic acid, in particular ammonium sulphate, and at least one of the following purification steps is carried out with polyethylene glycol in the presence of a soluble carbohydrate or protein-free polyol, i.e. polyol with at least three hydroxyl groups, then precipitate the immunoglobulin from which the protein impurities have been removed from the remaining solution with water-soluble polymers and then, in a known manner, complete the pharmaceutical preparation. Process according to claim 1, characterized in that the immunoglobulin-containing fraction of human blood plasma by Cohn's method is obtained by ethanol precipitation, and the ethanol-containing fraction is removed prior to step treatment with precipitation agents, preferably by dialysis. -10 The method of claim 1, wherein the immunoglobulin. the first-stage containing fraction is subjected to treatment with an ammonium sulfate solution having a content of 83 to 241 g / l (15 to 40% APB-saturation), preferably 145 to 208 g / l (25 to 35% saturated with ammonium sulfate) at pH 4, 5 to 8.0, preferably 5.9 to 6.5 "the precipitate is decanted and discarded and the remaining solution precipitated in the next precipitate stage, subjected to the second stage by further treatment with a solution of ammonium sulfate containing 241 to 343 g / l (40 to 55% saturation), preferably 268 to 289 g / l (44 to 47% saturation with ammonium sulfate) at pH 7, θ to 8.0, an immunoglobulin-containing precipitate, after which this precipitate is obtained, dissolved in water and removed ammonium sulphate from a solution, preferably by dialysis, followed by the remaining immunoglobulin-containing solution in the third purification step in the presence of saccharide, polysaccharide or polysaccharide hydrolyzate and subjected to treatment with polyethylene glycol at a pile value of 4.9 to 8.0, preferably 5.8 to 6.4, the resulting precipitate is then discarded and discarded and the remaining solution in the third cleaning step subjected to re-treatment with polyethylene glycol at 6.4 to 7.0, then the recovered precipitate is discarded and discarded, and immunoglobulin, which with If it is present in the remaining solution and no longer contains harmful contaminants, the precipitate is pH 7.0 to 7.5 with a polymeric precipitant and is processed into a pharmaceutical preparation. - / If- A process according to claim 3, characterized in that monosaccharides - such as glucose, fructose, mannose, galactose, or disaccharides, such as sucrose, lactose, maltose, or oligo - are used as saccharides. polysaccharides in an amount of 1 to 35 weights. %, preferably 5 to 20 weights. %. Process according to claim 1, characterized in that the immunoglobulin from which the protein impurities have been removed is precipitated by the addition of dextran, polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and Method according to claims 1 to 7, characterized in that the purified immunoglobulin is processed into a pharmaceutical preparation by dissolving in pyrogen-free water, by further dialysis, ultrafiltration or partial filtration, adjusting the immunoglobulin concentration to 5 to 200 g / 1 , preferably 50 άο ^ βΟ g / l and setting the ionic strength by means of sodium chloride to 0.005 to 0.3, preferably 0.1 to 0.2. An immunoglobulin preparation for intravenous administration comprising an antibody prepared according to one or more of the preceding claims, characterized in that it contains at least 80% of native immunoglobulin with a sedimentation constant of S of 7.0 and a biological half-life of 21 to 28 days and has anticomplementary activity corresponding to which at least 35 mg of protein is required to neutralize the ΟΉ ^ θ unit. 10. The composition of claim 9, which contains less than 5% protein components with a molecular weight of more than 160,000.