CS244306B1 - Blood gamma globulin preparation method with reduced hypotensive and anti complementary effect - Google Patents

Description

The invention relates to a method for the preparation of blood gamma globulin, from normal] or hyperimmune plasma, optionally from normal or retroplacental or hyperimmune serum, which is intended for therapeutic purposes.

Until now, the preparation of gamma globulin for therapeutic purposes has been carried out largely according to the criteria for intramuscular administration, i. j. a stop is placed on the yield, the electrophoretic purity and the content of substances with the ability to induce a pyretic reaction.

Ammonium sulfate and aluminum hydroxide were used to precipitate gamma globulin from a mixture of plasma or serum proteins (Schultze, E., Matheka HD: Behringwerke Mitt. 28, 9, 1954; Schultze, E., Schonenberger M., Matheka HD: Behringwerke Mitt. 26, 21, 1952], rivanol (Horejsi J., Smetana R .: Acta Med. Scand. 155, 65, 1956), sodium sulfate (Amiraian K., Leikhim E.] .: J. Immunol. 87, 301, 1961), aluminum chloride (Lewin J .: Therapie 9, 523, 1954), DEAE cellulose (Sober HA, Peterson EA: Fed. Proc. 17, 1116, 1958; Peterson EA, Sober HA: J. Am. Chem. Soc., 78, 756, 1956; Fahey, JL, Herbett, AP: J. Biol. Chem., 234, 2645, 1959; Stanworth DH; Nature 188, 156, 1960), caprylic acid (Steinbuch M., Audran R. : Rev. Franc D'etud Clin Biol., 14, 1054,

1969), Zn ⁺⁺ and Al ^{++ +} ions (Rejnek J., Skvaril F .: Coll. Czechoslov. Chem. Commun. 22, 1 489, 1957); Rejnek)., Škvařil F .: Coll. Czechloslov. Chem. Commun. 23, 773 (1958); Krauze, R., Naimski, K., Zakrewski, K., Acta Biochim. Pol. 8, 209, 1961), polymetaphosphate (Nitschman HS, Rickli R., Kistler P .: Vox Sang. 5, 232, 1960), ether (Pennell RB in Putnam FW / ed / The Plasma Proteins vol I Academic Press New York 1960 9) and ethanol (Cohn EJ, Gurd FNR, Surgenor DM, Barnes BA, Brown RK, Derouaux

G., Gillespie J.M., Kahnt F.W., Lever W. F., Liu C. H., Mittelman D., Mouton R. F., Schmid K., Uroma E., J. Am. Chem. Soc. 72, 465 (1950); Deutsch H.F., Gostling G.L., Alberty R.A., Williams J.W .; J. Biol. Chem. 164, 109 (1946); Oncley JL, Melin M, Rickert D A., Cameron J W., Gross P. M .: J. Am. Chem. Soc. 71, 541 (1949); Nitschmann

H. S., Kistler P., Lergier W., Helv. Chim. Acta 37, 866 (1954); Kistler P., Nitschman H.

S. Vox Sang. 7, 414 (1962); Taylor H. L. Bloom F. C., McCall K. B., Hyndman L. A., Anderson H. D., J. Am. Chem. Soc. 78, 1356 (1956).

Gamma globulin preparations prepared according to the hitherto valid criteria for intramuscular administration generally have a detectable histamine content, as well as a hypotensive and anticomplementary effect, which is unacceptable for intravenous formulations. Administration of an intramuscular preparation of these qualities intravenously may compromise the patient's blood pressure due to a sudden drop in blood pressure and, due to anticomplementary activity, weaken his non-specific immune defense.

Although the hypotensive effect of intramuscular administration does not have the same course as intravenous administration, it can be described, along with a high anti-complementary effect and a detectable histamine content, as undesirable for gamma globulin preparations intended for therapeutic purposes, whether intramuscular or intravenous.

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

A decrease in the anti-complementary gamma globulin activity was observed after the addition of activated carbon (Steinbuch, M., Audran, R., Amouch, P., Blatrix, C .: Vox Sang. 13, 103, 1967), after the addition of tricalcium phosphate (Steinbuch, M., Audran, Amouch, P., Blatrix C .: Rev. Med Tours, Suppl. No. 3, 79, 1968) and after the addition of albumin (Auerswald W., Kiesewetter E .: Wien. Med. Wschr. 115, 690, 1965) .

The reduction of the anti-complementary activity of the gamma globulin solution by proteolytic cleavage was realized by pepsin (Schultze, E., Schwick, G .: Dtsch. Med. Wschr. 87, 1643, 1962) and plasmin (Sgouris JT: Vox. Sang. 13, 71, 1967, Barandun S, Castel F, Makula MF, Morell

A., Pian R., Skvaril F .: Vox Sang. 28, 157 (1975).

The anti-complementary activity of the gamma globulin molecule can be reduced by its chemical reduction by beta mercaptoethanol (Wiedermann G., Miescher PA, Franklin EC: Proc. Soc. Exp. Biol. Med. 113, 609, 1963), by modification of beta propiolactone (Stephan W. Chem. 7, 282 (1969); Stephan W .: Vox Sang. 28, 422 (1975), with dithioerythritol reduction (Isenman DE, Dorrington KJ, Painter RH: J. Immun. 114, 1726, 1975). sulfonation (Masuho Y., Tomibe K., Matsuzawa K., Ohtsu A .: Vox Sang. 32, 175, 1977), amidation (Schmidtberger R .: US Patent No 4,118,379 (1978)), reduction with dithiothreitol and alkylation with iodoacetamide (Fernandes PM, Lundblad JL: Vox Sang. 39, 101, 1980) and reduction with dithiothreitol in combination with dithioerythritol followed by alkylation with iodoacetamide or methyl iodide, acrylonitrile, benzylbromide, optionally ethylene oxide, D JL: Vox Sang., 40, 373 (1981).

The anti-complementary activity of gamma globulin is related both to the formation of gamma globulin polymers, which may arise in the process of technological isolation, and also to the lyophilization process, due to the interaction of low molecular weight substances with gamma globulin molecules. The use of distilled pyrogen-free water in the separation of low molecular weight substances from gamma globulin results in precipitation of at least the higher gamma globulin polymers and, on the other hand, removal of low molecular weight substances, since their interaction with gamma globulin molecules cannot occur due to lyophilization. The use of an aqueous solution of sodium chloride, at a concentration of up to 30 g per liter, guarantees the stability required by the gamma globulin polymer in solution and only separates the low molecular weight substances that cause anti-complementary activity in the isolation process.

Anti-complement activity was tested for complement activity loss. The decrease in the known value of complement activity after addition of a known amount of a lyophilized gamma globulin preparation was determined by the method of Kabat and Mayer (Kabat E.A., Mayer, M.: Experimental Immunochemistry, Czechoslovak Academy of Sciences, Prague, 1965, p. 180).

The effect of distilled pyrogen-free water in the removal of anticomplementary activity can be documented in 10 experimental batches of gamma globulin, which was isolated by extraction from fraction III according to Cohn and Oncley. When distilled, pyrogen-free water was used alone, the anti-complementary activity of gamma globulin was reduced by 80-95% after lyophilization. The use of an aqueous solution of sodium chloride, using the same gamma globulin material, at a concentration of up to 30 grams per liter caused a decrease in the anti-complementary activity of gamma globulin by only 35 to 45% after lyophilization. If higher polymers present in gamma globulin solution are present, the results of testing for anti-complementary activity in a lyophilized gamma globulin preparation from which low molecular weight substances were removed using distilled pyrogen-free water or using an aqueous sodium chloride solution of up to 30 g per liter were practically the same.

The hypotensive effect was tested by decreasing rabbit blood pressure by a direct blood method according to CSL 3 (CSL 3, Volume I, Edition III., Avicenum Prague 1970, p. 149), with a 2 to 3 kg rabbit being administered 2 ml of gamma globulin solution per kg of weight. The gamma globulin content of the applied solution is 50 g per 1000 ml of solution. In the formulations prepared according to the proposed procedure, the hypotensive activity did not occur to such a significant intensity, as evidenced by the data that from a total of 10 experimental batches of the present invention a systolic blood pressure maximum decrease of 2-444306 was not 4 to 8% compared to the same material. at the same dose without the separation of low molecular weight compounds, where a decrease in systolic or blood pressure of 20 to 30% was determined.

The histamine content was tested by the Gadurn modification (Gadum J. H .: J. Physiol 8, 467, 1949) of the Magnuson method (Magnus R .: Pltlg. Arch. 102, 123, 1904). The preparations prepared according to the proposed procedure did not occur in guinea pig intestines, as evidenced by the data that from 10 experimental batches prepared according to the present invention, 0.0 micrograms of histamine per 100 grams of gamma globulin were found compared to the same material as lyophilized without prior separation of low molecular weight substances, where the histamine content was determined at a concentration of 20 to 40 ml programs per 100 grams of lyophilized gamma globulin.

Current technological practice, on the one hand, separates gamma globulin from a mixture of other blood proteins and, on the one hand, separates low molecular weight substances of inorganic or organic nature into the gamma globulin solution when separating and also reducing anticomplementary activity. The principle of removal of low molecular weight substances using dialysis, compared to 0.1 to 0.3% aqueous sodium chloride solution, was used for gamma globulin (Štěpánek I .: AO No. 202 180; Štěpánek I., Uhrík J .: AO 208 868 Stepanek I., Stachy A., Baria I., Banda I .: AO 204 168), as well as a mixed immunoglobulin preparation based on IgG + IgA + + IgM (Stepanek I., Uhrik J .: AO 208 867 j.

It is an object of the present invention to remove ballast negamaglobulin admixtures having a lower molecular weight relative to gamma globulin. weight. Data of about 50,000 molecular weight can be considered as limit values.

The starting material may be a Fraction II paste, isolated according to the Cohn and Oncley method, from normal or hyperimmune plasma, optionally from normal or retroplacental or hyperimmune serum. However, protein gamma globulin materials isolated using other methods may also be used.

The proteinaceous material is dissolved in an amount of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C such that the protein concentration value is in the range of 10-180 grams per liter, with an optimum of 60-100 grams per liter. Dissolution is accelerated by stirring. After dissolution, the gamma globulin solution becomes clear, for example by filtration.

In technological conditions, the aforementioned optimum conditions are generally maintained when dissolving 1 kg of gamma globulin paste in 2000 ml of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C. The pH of the thus prepared gamma globulin solution is generally in the range of 4.0 to 8.0 and the protein concentration in the range of 60 to 100 grams per liter. The gamma globulin solution thus prepared is clarified, for example by filtration, and the low molecular weight compounds up to a molecular weight of 50,000 are separated according to the method of the invention by ultrafiltration, diafiltration, gel filtration and dialysis, against distilled pyrogen-free water at 0 ° C to + 6 ° C, at pH 4.0 to 8.0. Further, the gamma globulin solution after sterilization of the low molecular weight compounds is sterilized, for example by filtration or other methods, and then concentrated, for example, by lyophilization or by means of membranes, vacuum distillation, freeze-drying or other methods and subjected, if necessary, to further processing.

The invention is further elucidated by means of exemplary embodiments in which its scope is neither limited nor exhausted.

Examples of design

Example 1

The starting material may be a Fraction II paste isolated by the Cohn and Oncley method from normal or hyperimmune plasma, optionally from normal or retroplacental, or hyperimmune serum with antibacterial, antitoxic or antiviral antibody activity. The protein gamma globulin material is dissolved in an amount of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C such that the protein concentration is in the range of 10-180 grams per liter, with an optimum of 60-100 grams per liter. Dissolution is accelerated by stirring. After dissolution, the gamma globulin solution becomes clear, for example by filtration.

In technological conditions, the above-mentioned optimum conditions are generally maintained when dissolving 1 kg of gamma globulin paste in 2000 ml of distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C. The pH of the gamma globulin solution is generally in the range of 4.0 to 8.0 and the protein concentration in the range of 60 to 100 grams per liter. The purified gamma globulin solution thus prepared is clarified, for example by filtration, and the low molecular weight compounds are removed from the gamma globulin solution:

A. Gel filtration

B. Dialysis

C. Ultrafiltration

D. Diafiltration

A.

There are currently a variety of commercial dextran gel filtration materials under the designation Sephadex, polyacrylamide gels, under the designation Biogel, hydroxyalkylmethacrylate gels, under the designation Spheron, polystyrene gels, under the designation Styragel, polyvinyl acetate gels, polyvinyl acetate gels, under the name Porasil, and porous glasses under the name Bloglas, which have graduated pore sizes in the gel particles and allow gel filtration through relatively wide molecular weight ranges.

Removal of low molecular weight compounds is accomplished by elution gel chromatography with distilled pyrogen-free water at a temperature of 0 ° C to + 6 ° C, at a pH of 4.0 to 8.0 with a gel exclusion limit of molecular weight up to 50,000. optimum grain size to ensure flow in technological conditions.

The gamma globulin solution is sucked into the column packed with the swollen gel material at a rate such that the flow through the bottom of the column is between 0.01 ml and 3.0 ml per minute per area of 1 cm ² . The amount of gamma globulin solution applied to the column should not exceed 25 to 30% by volume of the gel portion of the column. The separation efficiency can be monitored according to the degree of separation of the ethyl alcohol, chloroform and ninhydrin positive substances from the gamma globulin solution. If complete separation is not necessary, it is necessary to reduce the content of gamma globulin solution to be loaded per column and slow down. flow. After separation of the low molecular weight compounds from the gamma globulin solution, this is further processed.

B.

In the last decade, dialysis has been re-applied, especially in the manufacture of pharmaceuticals, as a technological method of separating those mixtures of natural or synthetic substances whose molecules have a significantly different mass.

There are now 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, possibly on a collodium basis and also on a vinyl polymer basis.

The sterile purified gamma globulin solution is carefully transferred to a sterile dialysis tubing, which is sealed with a knot before sifting at the bottom and, after sieving, the top is closed with a knot with a loop behind which the filled tubing is hung on a hook so positioned The gamma globulin in the dialysis pipeline was in contact with flowing distilled pyrogen-free water at 0 ° C to + 6 ° C at pH 4.0 to 8.0, the pore size in the dialysis membrane being large enough to allow separation of low molecular weight compounds a molecular weight of up to 50,000.

The end of dialysis is indicated by a negative reaction to ethanol, chloroform and ninhydrin positive substances in the overflowing dialysis fluid as well as a negative reaction to ethanol and chloroform in the gamma globulin solution. When dialysis is complete, the gamma globulin dialysis tubing is suspended from the hook, carefully removed and after cutting the gamma globulin solution is poured into a collection vessel and further processed.

C.

Separation of relatively low molecular weight compounds to a molecular weight of up to 50,000 can also be accomplished by ultrafiltration in the form of hollow threads, wherein the gamma globulin solution flows under a pressure of up to 800 kP inside the yarn membrane and permeates relatively low molecular weight through pores in the yarn membrane wall. substances in an aqueous solution into a collection vessel. Since the gamma globulin solution is concentrated by ultrafiltration, careful dilution with distilled pyrogen-free water is carried out, and this concentration, for example to half the volume and dilution to the original volume, is carried out until the gamma globulin solution reacts to chloroform and ethanol.

The ultrafiltration can also be carried out in the form of a cassette system with a replaceable membrane or a plastic plate with defined pore sizes in the planar configuration. The gamma globulin solution flows under a pressure of up to 800 kP, for example above the membrane, and through the pores in the wall of the planar membrane or plastic thatch, the relatively low molecular weight substances in the aqueous solution pass into the collection vessel. The gamma globulin solution is further processed if desired.

Sterilization of the ultrafiltration device is carried out or by steam or chemical means, for example by rinsing the apparatus with an aqueous solution of formalin at a concentration of up to 50 g per liter and subsequently washing the formalin with sterile distilled pyrogen-free water.

D.

Removal of relatively low molecular weight compounds to a molecular weight of 50,000 can also be accomplished by diafiltration, a combination of dialysis and ultrafiltration. The diafiltration may be carried out first, in the form of hollow strands, wherein the gamma globulin solution flows under a light pressure to 200 kPa within the thread of the membrane and through the pores in the wall of the thread of the membrane permeate the relatively low molecular weight substance to the outside of the flowing solution of distilled pyrogen-free water at 0 ^C C to + 6 ° C at a pH of 4.0 to 8.0, which low-molecular substances wash away from the yarn membrane into waste.

The diafiltration can also be realized in the form of a cassette system, in which there is or a replaceable membrane in the planar arrangement, or a plastic plate with a defined pore size. The gamma globulin solution flows under gentle pressure up to 200 kP, for example above the membrane and through the pores in the wall of a planar membrane or porous thatch, they actually pass through a relatively slow flow of relatively low molecular weight material into the flowing solution of distilled] pyrogen-free water. low molecular weight waste.

Since the gamma globulin solution is concentrated during diafiltration, a dilute dilution with distilled pyrogen-free water is carried out and this concentration, for example to half the volume and dilution to the original volume, is carried out as long as the gamma globulin solution is reacted to chloroform and ethanol. The gamma globulin solution is poured into a collection vessel and further processed.

Sterilization of the diafiltration device is carried out or by steam or chemical means, for example by rinsing the apparatus with an aqueous solution of formalin at a concentration of up to 50 g per liter and subsequently washing the formalin with sterile distilled water.

Evidence of chloroform is carried out by the Fujiwar reaction (Fujiwara: Sitzber. Naturw. Ges. Rostock 6, 33, 1916), whether in a flood pattern or in newer applications according to Seto and Schultze (Seto TA, Schultze MO: Anal. Chem. 28, 1). 625, 1956), Hildebrecht (Hildebrecht CD: Anal. Chem. 29, 1037, 1957), Friedman and Cooper (Friedman PJ, Cooper JR: Anal. Chem. 30, 1674, 1958).

Ninhydrin is positively determined by the method of Ruhemann (Ruhemann S .: J. Chem. Soc. 97, 1438, 1910). The determination of ethanol is carried out by the Agulhon reaction (Agulhon H .: Bull. Soc. Chim. France / 4 /, 9, 881, 1911), either in the original configuration or in more recent applications of the Kelletta stage (Kellett EG: Analyst 62, 728, 1937). ) or

Webba (Webb, D. A., Sci. Proc. Roy Dublin Soc. 21, 281, 1936).

The gamma globulin solution after separation of the low molecular weight substances is sterilized, for example by filtration or other methods, and then concentrated, for example, by lyophilization or by means of membranes, vacuum distillation, freeze-drying or other methods, and subjected to further processing if necessary.

The use of distilled pyrogen-free water compared to using 0.1% to 3.0% sodium chloride solution in distilled pyrogen-free water is more operational and technologically easier to implement. It makes it possible to use hot distilled pyrogen-free water which has escaped directly from the distillation apparatus, after cooling in a flow-through heat exchanger immediately for technological application, thereby minimizing the possibility of bacterial contamination. The use of a 0.1% to 3.0% solution of sodium chloride in distilled pyrogen-free water requires a container for its preparation and storage at elevated temperature, avoiding the possibility of contamination, as well as a device for homogenizing the solution. Smooth operation of the technological line using aqueous sodium chloride solution requires at least 2 such vessels.

Separation of low molecular weight substances with a molecular weight of up to 50,000 by ultrafiltration, diafiltration, gel filtration or dialysis, against a 0.1% to 3.0% sodium chloride solution in distilled pyrogen-free water, has also been successfully tested in the technological process scale.

Example 2

The starting material of the gamma globulin protein paste can also be isolated by other methods. The protein paste or protein solution is processed in a further procedure similar to that of case no. 1, except that the criterion for removal of low molecular weight compounds from gamma globulin solution is not related to the presence of alcohol or chloroform, but to the presence of a precipitant which has been used to form gamma globulin or forms a transient, degradable complex.

Claims (2)

1. A method for the preparation of blood gamma globulin with reduced hypotensive and anticomplementary effect comprising suspending the protein material containing purified gamma globulin under aseptic conditions in an amount of pyrogen-free distilled water at a temperature of 0 ° C to + 6 ° C to obtain a protein concentration value. found to be in the range of 10 to 180 g / l, the gamma globulin solution is clarified, for example by filtration, the separation of low molecular weight substances by ultrafiltration, diafiltration, gel filtration or dialysis, against a sodium chloride solution in distilled pyrogen-free water, then gamma globulin solution sterilized filtration and subsequent invention is concentrated, for example by lyophilization or by means of membranes, vacuum distillation, freeze-drying and, if necessary, further processing, characterized in that the separation of low-molecular-weight substances with a molecular weight of up to 50,000 is performed by ultrafiltration, diafiltration, gel filtration or dialysis, against distilled pyrogen-free water. 2. A method according to claim 1, wherein the proteinaceous material is normal or hyperimmune plasma, or normally, retroplacental or hyperimmune serum with defined antibacterial, antitoxic or antiviral antibody activity.