CS266252B1 - Process for preparing blood mixed preparate on base igg +iga+igm - Google Patents

Abstract

A method of preparing a blood mixed immunoglobulin IgG + IgA + 4 IgA + XgM for i.m. submission is a manufacturing process intended to produce therapeutic or a diagnostic preparation which may be used in the field of clinical immunology, medicine pharmacy and biology. Production process makes it possible to produce a mixed immunoglobulin IgG + IgA + XgM preparation, which can be administered intramuscularly. Bielkovmný a material containing the purified mixture immunoglobulins based on IgG + IgA + IgM plasma or serum defined antibacterial, antitoxic or antiviral antibody activity is suspended under aseptic conditions in such an amount distilled pyrogen-free water at room temperature 0 ° C to + 16 ° C to give a concentration value protein was found to be in the range of 10 to 150 grams per liter, immunoglobulin solution for example by filtration and separation low molecular weight compounds, expressed in 50,000 molecular weight, is converted gel filtration, dialysis, ultrafiltration or diafiltration by elution or with an aqueous chloride solution up to 30 grams per liter at a temperature of 0 ° C to + 6 ° C with a pH in the range of 4.0 to 8.0, wherein the immunobacterial solution based on IgG + IgA + IgM, low molecular weight compounds, are sterilized, for example filtration, radiation sterilization or by other methods, it is optionally concentrated ultrafiltration, freeze-drying, by vacuum distillation or other methods, it is filled, lyophilized and if necessary undergoes more processing.

Description

2 CS 266 252 B1

BACKGROUND OF THE INVENTION The present invention relates to a method for the preparation of a mixed immunoglobulin preparation based on IgG + + Ig? + IgM, from normal or hyperimmune plasma, or from normal or retroplacental or hypertensive serum, which is intended for therapeutic purposes via the i.m. applications

However, in the case of the ballast proteins of the mixture of IgG + IqA + IgM immunoglobulins, I I was used, I I vano I (hi hw I rk II. 11, I iiclii i. 1, 1; I I II.: I ... I I ....... ol I I I.

Stand. 4, 86, 1970; Rchwick II. G.: Vox Sang. 23, 112, 1972), boric acid (ududilL., Audran P., Steinbuch M.: Vox Sang. 23, 165, 1972) and caprylic acid (Blatrix G.,

Israel J., Reinert Ph. , Grisceli G., Steinbuch M.: 1, and Nouvelle presse Medicale 5, 1,193, 1976). Current technological practice, on the one hand, separates a mixed immunoglobulin preparation based on IgG + IgA + IgM from a mixture of other blood proteins, but on the other side into a solution of IgG and IgA + IgM low-molecular inorganic or organic compounds used for protein precipitation. The technological process can also concentrate some of the compounds with biochemical-physiological activity in the mixed immunoglobulin preparation solution, i.e. enrich with respect to their concentration in the starting material, from which the IgG + IgA + IgM immunoglobulins are isolated. The presence of an excess of inorganic salts or salts of organic acids reduces the usability of the injectable solution and, together with the presence of low molecular weight compounds in the immunoglobulin solution, creates the prerequisites for the undesirable interaction of these compounds with the IgG + IgA + IgM molecules, especially when stored at low temperatures. or storage conditions in the form of a therapeutic preparation.

The IgG + IgA + IgM mixed immunoglobulin preparation prepared according to the previously described technology has a proven hypotensive and anticomplementary effect which is also for the quality of the preparation for i.m. inappropriate. Administration of an intramuscular preparation of these pathogens may compromise the patient's blood pressure as a result of the anti-complementary activity and weaken its non-specific defense.

To remove the anti-complementary gamma globulin activity prepared from the plasma or serum protein mixture, a pH adjustment of 4.0 at 24 hours incubation at 37 ° C was used (Barandun S., Kistler P., Jeunet F., Isliker H .: Vox Sang 7, 157, 1962).

The decrease in anticomplementary gamma globulin activity was noted after the addition of activated charcoal (Steinbuch, Audran, R., Amouch, P., Blatrix, Vox. Sang. 13, 103, 1967), and tricalcium phosphate (Steinbuch, Audran, R.). , Amouch P., Blatrix C .: Rev. Med Tourssuppl No 3, 79, 1968) and after the addition of albumin (Auerswald W., Kiesewetter E.:Wien. Med. Wschr. 115, 690, 1965).

Reduction of the anti-complementary activity of the gamma globulin solution by proteolytic cleavage was performed by pepsin (Schultze, E., Schwick G .: Dtsch. Med. Wschr. 87, 1 643, 1962) and plasmin (Sgouris JT: Vox Sang. 13, 71) , 196?).

The anti-complementary activity of the gamma globulin molecule can be reduced by its chemical reduction by beta mercaptoethanol (Wiederman G., Miescher PA, Franklin EC: Proc. Soc. Exp. Biol. Med. 113, 609, 1963), modified by beta propiolactone (Stephan W .: Z. wedge.

Chem. 7, 282, 1969; Stephan W .; Vox Sang. 28, 422, 1975) by reduction with dithioerythritol (Isenman DE, Dorrington KJ, Painter RH: J. Immun. 114, 1726, 1975), by sulfonation (Masuho Y., Tomibe K, Matsuzawa K, Ohtsu A .: Vox Sang., 32, 175, 1977), by amidation (Schmidtergerger R.: US Patent No. 4,118,379 - 1978), by reduction with dithiothreitol and alkylation with iodoacetamide (Fernandes P., Lundblad JL: Vox. Sang 39, 101, 1980) with reduction with dithiothreito in combination with dithiorythritol followed by alkylation with iodacptamide butbomethyl iodide, acrylonitrile, benzyl bromide and ethylene oxide (Schroeder DD, Tankersley, L., Lundblad JL: Vox Sang. 40, 373, 1981) . CS 266 252 Bl * 3

The anti-complementary activity of the mixed immunoglobulin preparation is related, on the one hand, to the polymer of IgG + IgA + IgM polymers, which can be formed in the process of technological isolation and is probably due to the interaction of some low-molecular substances with immunoglobulin molecules. The use of an aqueous solution of sodium chloride in distilled pyrogen-free water at a concentration of up to 30 grams per liter guarantees the polymeric inorganic water. m tv vi ««: i <ik ti j. <11 ι Η, 11 'ii ii t .-) i i l l υ π> t <> </ι / ηΙζη I ιίι ku Hr,> ni ói · t 1 li I from the colloid of the tok t nyc mud, which then no longer cause the anticomplementary activity in the lyophilization process.

Anti-complementary activity was tested based on loss of complement activity.

The residual known value of complement activity after the addition of a known amount of a lyophilized mixed immunoglobulin preparation based on IgG + IgA + IgM was determined by the method of Kabat and Mayer (Rabat E. A., Mayer, M.i Experimental Immunochemistry of CSAV, Prague 1965 p. 180). When the higher polymers are not present in the mixed immunoglobulin solution, a reduction in anticomplementary activity was determined in 10 experimental lots after lyophilization of 80-95% compared to the same material in which the separation of low molecular weight substances was not realized. When polymers were present, a reduction in anticomplementary activity was determined by only 35 to 45% compared to the same material in which the separation of low molecular weight substances was not realized.

The hypotensive effect was tested on the basis of the drop in blood pressure of the rabbit by a direct blood method according to CSL 3 (CSL 3, Volume I, Issue III, Avicenum Prague p. 149), with 2 ml of mixed immunoglobulin base preparation being administered to a rabbit weighing 2 to 3 kg IgG + IgA + IgM per kg body weight. The protein content of the mixed immunoglobulin preparation applied is 15 grams per 1000 ml of solution. For the preparations prepared according to the proposed procedure, the hypotensive activity did not appear to be as pronounced as documented by the fact that out of the 10 experimental lots prepared according to the present invention, a decrease in systolic blood pressure was determined by a maximum of 4 to 8% compared to the same material in the same dose without separation of low molecular weight compounds where a decrease in systolic pressure from 20 to 30% was determined.

SUMMARY OF THE INVENTION It is an object of the present invention to eliminate ballast non-immunoglobulin impurities having a lower molecular weight in relation to IgG, IgA and IgM. Boundary values of about 50,000 molecular weight may be considered.

The removal of the ballast non-immunoglobulin impurities can be accomplished by: A. Gel filtration by B. Dialyza C. Ultrafiltration D. Diafiltration A. Currently, there are a number of commercial materials for gel filtration on baseextranslated gels, designated Sephadex, polyacrylamide gels, under the name Biogel, hydroxyalkyl methacrylate gels, designated Spheron, polystyrene gels, under the designation Styragel, polyvinyl acetate gels, under the designation Marko-gel, porous silicates under the designation Porasil, and cutting glasses under the designation Bioglas, which have graded pore sizes in gel particles and allow gel filtration in relatively wide molecular weight ranges. B. Dialyza has been re-applying in the last decade, in particular, in the manufacture of a medicament as a technological method of separating those mixtures of natural or synthetic substances whose molecules have a substantially different weight. At present, there are a number of commercial dialysis membranes, either in the form of a planar membrane or in the form of a pipe of varying diameter and pore size based on regenerated cellulose or its derivatives, optionally based on collodion and also on the basis of vinil polymers. C. CS 266 252 B1 C. The separation of low molecular weight compounds into a molecular weight of 50,000 can also be accomplished by ultrafiltration, which can be realized in the form of hollow threads while the immunoglobulin solution flows under pressure up to 800 kP inside the thread membrane and permeates low molecular weight through the pores in the thread membrane wall. in the aqueous solution to the collection vessel. It is possible to realize also in the form of a cassette system where, in a planar arrangement, the diaphragm or the plate is made of plastic with defined sizes. The mixed immunoglobulin solution under a pressure of up to 800 kP flows, for example, over the membrane and through the pores in the wall of a planar membrane or plastic dock, to transfer relatively low-molecular weight substances in an aqueous solution to a collection vessel. D. Removal of relatively low molecular weight compounds up to 50,000 molecular weights can also be accomplished by diafiltration, which is a combination of dialysis and ultrafiltration. Diafiltration can be accomplished both as hollow threads while gamma globulin solution flows under 200 kP inside the yarn membrane and through pores in the stenenite the membranes penetrate the real low molecular weight substances from the outer side of the solution solution, which releases the relatively low molecular weight substances from the yarn membrane to the waste.

Diafiltration can also be carried out in the form of a cassette system where, in a planar arrangement, a replaceable membrane falls on a plastic plate with defined size. The IgG + IgA + IgM immunoglobulin solution at moderate pressure up to 200 kP flows, for example, through the membrane, and through the pores in the plane of the planar membrane or porous wall, the relatively low molecular weight flows into the underflow flow membrane that flushes the relatively low molecular weight into the waste.

Protein material containing purified IgG + IgA + IgM is suspended under aseptic conditions in an amount of pyrogen-free distilled water at 0 ° C to + 6 ° C so that the protein concentration is between 10 and 150 grams per liter, with optimum 20 to 40 grams per liter, the mixed immunoglobulin solution is screened for example by filtration and the separation of low molecular weight compounds is carried out in an aqueous sodium chloride solution at a concentration of up to 30 grams per liter by elution or dilution. 0 to 9.0 grams of liter, at a temperature of 0 ° C to + 6 ° C with a pH in the range of 4.0 to 8.0 The low molecular weight immunoglobulin solution is sterilized by, for example, filtration, radiation sterilization or other methods, as required. is concentrated by ultrafiltration, freeze-drying, vacuum distillation or other methods U is a, in the case of lyophilized and subjected to further potřebysa spracovanju the final product.

The invention is further illustrated by the following examples, which are not intended to be limiting or exhausted. EXAMPLES Example 1 The starting material may be a mixed immunoglobulin preparation paste of IgG + IgA + IgM base obtained by, for example, the method of Pejaudier et al. (Pejaudier L., Audran R., Steinbuch M .: Vox Sang. 23, 165, 1972). The proteinaceous material precipitated in the mixed immunoglobulins is dissolved in an amount of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C, so that the concentration of the protein is between 10 and 150 gram liter, with an optimum of 20 to 40 grams per liter. The dissolution is accelerated by stirring. After dissolution, the IgG + IgA + IgM-based immunoglobulin solution is excreted, for example, by filtration. Under technological conditions, the above conditions are generally maintained when 1 kg of mixed immunoglobulin protein paste is dissolved in 2000 ml of pyrogen-free CS 266 252 B1 5 distilled water at 0 ° C to + 6 ° C. The pH of the resulting immunoglobulin solution is in the range of 4.0 to 8.0, with an optimum of 7.0, and the protein concentration is in the range of 20 to 40 qram per liter. The solution thus prepared is excreted, for example, by E-filtration and low-molecular-weight compounds from IgG + IgA + IgM-based immunoglobulin solutions. Gel filtration by B. DiaJyz C. Ultrafiltration D. Diafiltration A. Removal of low molecular weight compounds is carried out by elution gel chromatography distilled water in which sodium chloride was dissolved to a concentration of 30 gram liter at 0 ° C to 46 ° C at pH 4.0 up to 8.0 with an exclusion limit of gel expressed by molecular weight of up to 50,000, and the use of these materials in optimum grain flow ensuring technological flow. The optimal concentration of sodium chloride is 3.0 to 9.0 grams per liter and optimally ”pH is 7.0

Let the mixed solution of IgG + IgA + IgM immunoglobulins be sucked into the column filled with the swollen gel material at a rate such that the flow rate of the lower column surface is between 0.01 and 3.0 ml per minute per 1 cm. The amount of immunoglobulin solution loaded on the column should not exceed 25-30% of the volume of the gel portion of the column. The activities can be monitored according to the degree of separation of the ethyl alcohol, chloroform and ninhydrin-positive substances from the immunoglobulin solution. If there is no perfect separation, it is necessary to reduce the content of the mixed mixed immunoglobulin solution to the column and slow down the flow. After separation of the low molecular weight compounds from the immunoglobulin mixed solution, this is further processed. B. Sterile mixed immunoglobulin solution is carefully sieved into sterile dialysis tubing, which is closed with a knot prior to sieving at the bottom, and the top is closed with a loop of knot being filled with a pipeline to the hook so that the mixed immunoglobulin solution in the dialysis tubing was contacted with a flowing aqueous solution of sodium chloride distilled and hydrogenated at a concentration of up to 30 grams per liter, with an optimum of 3.0 to 9.0 grams per liter, at 0 ° C to + 6 ° C, at pH 4.0 to 8.0, with an optimum pH of 7.0, wherein the size of the dialysis membrane pores should be large enough to allow separation of relatively low molecular weight compounds expressed as molecular weight up to 50,000.

The end of dialysis is indicated by a negative reaction to ethanol, chloroform, and ninhydrinosites in the flowing dialysis fluid as well as a negative reaction to ethanol chloroform in the mixed immunoglobulin solution. Upon completion of the dialysis, the dialysis tubing is mixed from the hook with the mixed immunoglobulin solution, carefully removed, and the IgG + IgA + IgM solution is sheared into the collection vessel and further processed. C. Removal of relatively low molecular weight compounds up to a molecular weight of 50,000 can be accomplished by ultrafiltration, wherein the relatively low molecular weight solution in solution after flow through the membrane flows into a collection vessel.

Ultrafiltration can be carried out either in the form of hollow yarns or in the form of a cassette system where docks or membranes with defined pore sizes are used.

Since the immunoglobulin solution is concentrated by ultrafiltration, it is carefully diluted with an aqueous sodium chloride solution up to 30 grams per liter, with an optimum of 3.0 to 9.0 grams per liter, at 0 ° C to +6. ° C, at pH 4.0 to 8.0, 6 CS 266 252 B1 with an optimum pH of 7.0 and this concentration of the flood volume is until now the presence of chloroform and ethanol positive. for example, to half the volume and dilute the response of the immunoglobulin solution to

The sterilization of the drawing apparatus is carried out by steam or chemical grade. In the case of water / water, the water is poured into the water / water stream and then washed away by sterile sterile washing of formalin. distilled water. D. Removal of relatively low molecular weight compounds up to 50,000 molecular weight can be accomplished by diafiltration, while the relative low molecular weight substances are transferred to a sodium chloride solution in distilled, pyrogen-free water, at a concentration of 30 grams per liter, with an optimum of 3.0 to 9.0 grams per liter at temperature 0 ° C to + 6 ° C, at a pH of 4.0 to 8.0 with an optimum at pH 1.0, which releases the relatively low molecular weight material to waste.

Diafiltration can be carried out either in the form of hollow yarns or in the form of a cassette system where plastic or diaphragm with defined sizes are used.

Since the mixed immunoglobulin solution is concentrated in the diafiltration, dilution is carried out with an aqueous solution of sodium chloride which is used to add filtration and this concentration, for example, to half the volume and dilution to the water volume until the reaction of the mixed immunoglobulin preparation is based on IgG + IgA + IgM for presence chloroform and ethanol positive. The mixed immunoglobulin solution is poured into a collection vessel and further processed.

Sterilization of the diafiltration device is carried out by steam or chemical passage, for example by flushing the apparatus with an aqueous solution of formalin at a concentration of up to 50 grams liter followed by washing the formalin with sterile distilled water. The chloroform assay is performed by a Fujiwar reaction (Fujiwara: Sitzber. Naturw. Ges.

Rostock 6, 33, 1916) either in the original arrangement or in later applications by Setaa Schultze (Seto TA, Schultze MO: Anal. Chem. 28, 1 625, 1956), Hildebrecht (et al., CD: Anal. Chem. 29, 1 037, 1957), Friedman and Cooper / Friedman PJ,

Cooper J.R.: Anal. Chem. 30, 1774 (1958).

Ninhydrin is positively determined by Ruhemanna (Ruhemann S .: J. Chem.

Soc. 97, 1438, 1910). The determination of ethanol is carried out by Agulhon reaction (Agulhon H .:

Bull. soc. chim. France (4), 9, 881, 1911) either in its original layout or in newer applications by Kellett (Kellett EG: Analyst 62, 728, 1937) or Webb (Webb DA: Sci. Why Roy Dublin. Soc. 21, 281) , 1936).

The low-molecular weight compound immunoglobulin solution is sterilized by, for example, filtration, radiation sterilization, or other methods, concentrated by ultrafiltration, freeze-drying, vacuum distillation, or other methods, dissolved, lyophilized, and subjected to further processing if necessary. Example 2 The starting material may be a proteinaceous paste of a mixed immunoglobulin preparation of the base IgG + IgA + IgM or protein solution, which were also isolated by other methods. The protein paste or protein solution is processed in a similar manner to Example 1 except that the criterion for removing low molecular weight mixed immunoglobulin solutions is not related to the presence of alcohol or chloroform, but to the presence of a precipitating agent that has been used for precipitation or immobilization.

Claims (4)

OBJECT OF THE INVENTION A method for the preparation of a blood mixture of an immunoglobulin preparation based on XgG + IgA + i. For example, the clay content of purified IgG? g + + IgM is suspended under aseptic conditions in such a quantity of &quot; pyrogen-free water of temperature &quot;. 1.i, i = 10 to 150 grams nu I j lei, zimmny i iiiuikx. ) I / ubiline / locus is divided into 1 and 1 by low molecular weight and excreted at low molecular weight, expressed as molecular weight up to 0,000, then low molecular weight IgG + IgA and IgM-based solutions are removed. the compounds are sterilized, for example, by filtration, radiation sterilization, or other methods, optionally concentrated by ultrafiltration, freeze-drying, vacuum distillation, or other methods, filled, lyophilized, and, if necessary, subjected to further processing to the final product. 2. A method according to claim 1, characterized in that the separation of the low molecular weight substances is carried out by elution or dilution with a sodium chloride solution in distilled and dehydrated water at a concentration of up to 30 grams per liter at a temperature of 0 ° C to + 6 ° C at a pH of 4.0 to 8, 0. 3. The process of claim 1 wherein the separation of low molecular weight compounds, expressed by molecular weight up to 50,000, is carried out, for example, by gel filtration, dialysis, ultrafiltration and diafiltration. 4. The method of claim 1, wherein normal or hyperimmune plasma, or normally, retroplacental or hyperimmune serum with defined antibacterial, is used as the material from which the IgG + IgA + IgM mixed immunoglobulin preparation fraction is recovered. antitoxic or antiviral antibody activity. 4