CS277564B6 - Method of preparing modified immunoglobuline preparation with low anti-complementar activity - Google Patents

Abstract

A method for preparing a modified immunoglobulin low anticomplementary activity is based on the principle of non-specific ballast sorption calcium phosphate, reducing the immunoglobulin dithio-DL-treitol, alkylating it iodoacetamide and separation of low molecular weight by dialysis or diafiltration. So prepared the modified immunoglobulin solution is stabilized adding maltose, glycine, optionally serum albumin. 5% modified solution immunoglobulin when prepared the conditions for apyrogenic work are maintained suitable for intravenous administration. He's low anti-complementary activity, does not contain high aggregate protein complexes capable of inducing adverse reaction has a half-life comparable to native immunoglobulin and its application is minimized risk of transmission of viral diseases.

Description

The present invention relates to a process for the preparation of a chemically modified immunoglobulin preparation with low anticomplementary activity, which is suitable for intravenous administration, by a method which improves quality, in particular disease.

Over the last two decades, there has been a growing effort to use the immunoglobulin fraction of blood plasma not only as an intramuscular preparation, but also to modify it so that it can be safely administered intravenously. This development is characterized by the desire to prepare a fully effective intravenous solution that does not cause adverse anaphylactic reactions in recipients and is safe for transmission of viral diseases. The task is to prepare an immunoglobulin solution which has very low anticomplementary activity, contains only a minimal amount of aggregated protein complexes and contains as few biologically active substances as possible, such as blood grouping substances, prekallikrein activity, gonadotropic hormone and the like. At the same time, the possibility of transmitting viral diseases must be ruled out. Over time, much experience has been gained in this field and there are a number of publications dealing with both methods for the preparation of intravenous preparations and their quality, as well as clinical verification of their efficacy and examples of application in various diseases. The longest known method of preparation is partial proteolytic cleavage, for example with pepsin / H. E. Schultze, G. Schwick: Dtsch.Med.Wschr. 87, 1643 (1962) /, / S.Barandun, P.Kistler, J.Jeunet, H.Istiker: Vox Sang. 7, 157 (1962) /, /S.Barandun: Biblthemat.No.17 (Kargel ·, Basel 1964), Die Gamma-globulin - Therapie /, /1.Banda a kolektiv: čs. author's certificate No. 239838 / or plasmin /J.T.Sqouris: Vox Sang. 13.71 (1967) /, /I.Biněva,L.Bazadžiev,B.Zacharia:Probl.Infect.Parazit.Dis. 12.84 (1985). Preparations of this type have minimal anticomplementary activity and do not contain aggregated pornylers (F. R. Seiler: Die gelben Hefte XXII (1), L (1982)). Some experts consider the immunoglobulin preparation prepared in this way to be the safest in terms of possible reactions in the recipient, while another group describes it as obsolete, especially due to very low biological half-life and possible presence of by-products of enzymatic cleavage /F.Škvařil, B.Roth Wicky, S.Barandum: Vox Sang. 38, 147 (1980) /, /J.Rómer, F.Škvařil: Vox Sang.42, 62 (1982) /, /B.M.Alving,J.S.Findayson (editors): Immunoglobulins, characteristics and uses of intravenous preparations. US Dep. of Helth, 1979 /, /F.R.Seiler,R.G. Geurses (editors): 7S-Immunoglobulin for intravenous use. Kargel 1982 /, /R.E.Schmidt,I.Stroehmann (editors): Immunoglobulin therapy Grundlagen und klinische Dwendung. Symposium in Bonn, Karger 1981 /, /R.A.Good (editor): Am.J.Med 76,3A (1984) Intra veil ·: as Immune Globulin and the Compromised Host /. Another type of intravenous preparation is a solution of unmodified, highly purified fully native immunoglobulin. This type of preparation is highly effective and has the same half-life as normal immunoglobulin. However, the use of the native type of such a preparation has certain drawbacks. The problem is to safely rule out all reactions in the recipient. This is because it becomes debatable to objectively define the boundary between an intramuscular and an intravenous preparation and thus to determine an acceptable degree of risk of the possible occurrence of adverse clinical reactions. In general, there is a principle requiring the maximum achievable electrophoretic purity of the immunoglobulin, the lowest possible content of aggregated proteins, a minimum of anticomplementary activity and a minimum content of biologically active substances. The risk of transmission of viral diseases in these preparations is eliminated by careful control of the starting material and by the inclusion of an ethanol precipitation step or other purification steps in the fractionation procedures /Η.H.Hoppe,T.Mester^.Henning, H.J.Krebs: Vox Sang. 25, 308 (1973) /, /C.J.Cunninghan: Biochem.Soc.Transs. 8, 178 (1980) /, /W.Scheider,D.Wolter,L.J.McCarty: Vox sang. 31, 141 (1976)], / A. D. Friesen, J. M. Bowmann, H. W. Price: J. Appl. Biochem. 3, 166 (1981) /, /J.Vaňák, A. Stejskal: Czechosl. Author's Certificate No. 220 725 /, /R.M.Condie: US Patent 4.130094 - 1979 /, /R.M.Condie: US Patent 4.296027 - 1981 /, / W. Guddat, K.Hillger: DD patent 208918 A 61 K - 39395 /, /1.Štěpánek: čs. author's certificate No. 255 356 /, / RSLane: Lancet 974 (1983) /, / AMLLewer and team: Lancet 1062 (1984) /, / HDOchs and team: Lancet 404 (1985) /, / MAWells and team : Transfusion 26 (2), 212 (1986) /, /H.Friedli, J. Morgethaler: Lancet 1215 (1985). The quality of intravenous undigested immunoglobulin is defined by a directive, for example the World Health Organization / WHO Guidelines The Collection, Fractionation, Quality, Control and Use of Blood and Blood Products, Geneva 1981, p. 50 / or a standard / Company standard PNY 31-147-82 / . When choosing the quality, it is necessary to keep in mind that the more tolerant the values of the monitored variables, the greater the risk of complications in the recipient.

A third compromise solution, which overcomes some of the disadvantages of the two types of intravenous immunoglobulin solutions mentioned above, is a third method of preparation based on the chemical modification of an immunoglobulin. The partial cleavage of the immunoglobulin molecules and the chemical blocking of the respective functional groups prevents the formation of aggregates capable of eliciting reactions in the recipient. At the same time, a partial reduction in anticomplementary activity is achieved. The biological half-life of the organism is comparable to that of native immunoglobulin, as the grafts are capable of recombination in vivo and in vitro under appropriate conditions. The content of active antibodies remains unchanged. Chemical modification of protein molecules eliminates biologically active substances, for example prekallikrein activity /P.M.Fernandes,J.L.Lundblad: Vox Sang. 39, 101 (1980)] and to deactivate any native viruses present (W. Stephan: Vox Sang. 20, 442 (1971) /, /A.M.Price,W.Stephan, B.Brotman: Vox Sang. 46 (2), 80 (1984) /, /W-.Stephan: Z.Klin.Chem. Klin.Biochem .: 7, 286 (1969) /, /D.D.Schroeder et al., Am.J.Med. 76 (3A), 33 (1984) /. One of the best known immunoglobulin modification procedures is the reaction with beta propiolactone [W. Stane: Beitr.Infusionther.Klin.Enahr. 11, 20 (1983)], immunoglobulin amidation (R) Schmidtberger: U.S. Pat. No. 4,118,779-1978, sulfitolytic cleavage (F. Frankek, O. M. Zorina: Coll. Czech. Chern.Commun. 32, 3229 (1967) /, / H. Muller: Open-laid Patent Gazette DE-AS 2658 334 - 1976 /, /Y.Masubo,Y.Tomibe,T.Watanabe, Y.Fukomoto: Vox Sang. 32, 200 (1977) /, /Y.Masubo,Y.Tomibe, Y.Fukomoto, T.Watanabe: Vox Sang. 32, 290 (1977) /, /P.Gronski, E.Kanzy, G.Luben: Die gelben Hefte 40 (1982) /, /Z.Olšovská, F.Franěk: Folia biologica 29 (4), 282 (1983) /, /Z.Olšovská, F.Franěk: Folia biologica 29 (6), 365 (1983) /, /P.Gronski,

T.Hofstaetter, E.J. Kanzy, G.Luben, F.R.Seiler: Vox Sang. 45, 144 (1985) /, / T. Hofstaetter, P. Gronski, E. J. Kanzy, H.U.Schorlemmer, F.R. Seiler: Vox Sang. 45, 155 (1983) /, /P.Gronski et al., Ric.Clin.Lab. 14 Suppl. 1, 7 (1984). A good method of modification is reduction followed by alkylation of [P.M. Fernandes, J. L. Lundblad: Vox Sang. 39, 101 (1980) /, D. D. Schroeder, D. T. Tankersley, J. L. Lunblod: Vox Sang. 40,373 (1981) /, /.D.D.Schroeder et al., Am.J.Med. 76 (3A), 33 (1984) /. The composition of the stabilizing solution has a significant effect on the quality of the final preparation, on its safety during clinical application and on the expiration time. Glycine in combination with a sugar, for example maltose or glucose in various concentrations, is most often used to stabilize intravenous immunoglobulin solutions. In some cases, a native immunoglobulin solution is used to protect its serum albumin [D. Gislason et al., Vox Sang. 34, 143 (1978) / or modified gelatins (T. Tomoto, K. Ikeda, T. Suzuki: Vox Sang. 51, 85 (1986).

Based on the results of our experiments, shown in the following table, and the data contained in the cited works, a process for the preparation of an intravenous immunoglobulin solution with low anticomplementary activity was proposed, as presented by the present invention.

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Overview of the results of the determination of anticomplementary activity in 5% immunoglobulin solutions prepared by various procedures.

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Note: a) - microassays according to Fulton and Dumbell, b) - assays according to PNY 31-147-82 c) - immunoglobulin contains approximately 10% of aggregated parts of proteins d) - range of determined values for more assays

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line Ovan -> i β Φ > 0 '> 1 β φ> 0 '> 1 β Φ> 0 '> 1 β φ> 0 '> · Β Φ> 0 '> 1 β φ ft β '> t 44 > 1 44 44 '> 1 44 44 '> 1 44 > Φ Ή Λ β H β • Η β • Η β • Η β Η β Ή • μ β 0 Φ Hive Φ Μ-Ι φ ΨΙ Φ ιμ Φ ιμ Φ Ψ4 > tn χφ r4 ft • H ft • Η ft • Η ft Ή ft Η ft • Η > tn > φ Ό > Φ > φ Ό > Φ Ό > Φ > Φ Ό > 1 0 0 + » 0 +> 0 +> 0 -μ 0 -μ 0 μ 0 44 . TABLE (cont Immunoglobulin solution immunization prepared in different ways 2+ sulfonated (SO 2) partially w g 1 xn > Φ β r — 1 Χφ • Η 0 μ φ ft + CN 'D Ο ω '> ϊ β φ > 0 β 0 U-I rd β ω g 1 > ω > φ β χφ Η 0 μ Φ ft '> 1 β Φ> 0 > 1 44 Φ Φ '> 1 β Φ> 0 44 β t Φ μ g 1 g Φ 0 +> • Η Φ μ +> 0 • Η Λ μ • Η Ό > ω > Φ β r — I ΧΦ • Η 0 μ φ ft g φ 0 -μ Η Φ μ -μ 0 • Η X! • μ • Η β Φ> 0 44 • Η m • μ Ό 0 g g 1 β β • Η g β Λ γΗ φ dP ΙΓ> Φ > ω > Φ β χφ • Η 0 μ φ ft β Ν φ χφ β > 1> Φ Ό 0 ft β Φ> Φ μ ft • Η> μ ft g 1 β β Ή g β X) φ Ή β Χφ Ό • Η> μ ft Λ Φ Λ > ϋ) > Φ β χφ • Η 0 μ φ ft β Ν Φ χφ β> 1> φ Ό 0 ft '> 1 β Φ> Φ μ ft • Η> μ ft g 1 β β • Η g β Λ Φ dP uO ω Ο • Η -μ> 1 0 Φ -μ 0 μ ft ft ω ω ω 'w * g φ β Ft ω ft Φ ft > 1 β Φ ft > φ μ > ω

The proposed process for the preparation of a modified immunoglobulin preparation with low anticomplementary activity, which is suitable for intravenous administration, based on a combination of known calcium phosphate preparation methods, chemical modification of immunoglobulin with DL-dithiothreitol and addition of maltose, glycine and serum albumin as stabilizing components. an aqueous solution containing 3 to 10 g of immunoglobulin, with an electrophoretic purity of at least 95%, is contacted with calcium phosphate for every 100 ml for 30 to 60 minutes, then the calcium phosphate is separated from the liquid, to the immunoglobulin solution thus obtained is added 1 , 4-dithio-DL-threitol in an amount of 23 to 38 mg for every 100 ml of solution and after stirring for 30 minutes, 50 to 100 mg of iodoacetamide for every 100 ml of solution is added, stirred for another 60 minutes, the immunoglobulin solution thus modified is dialyzed or diafiltered against a solution containing 0,2 to 0,4 g of sodium chloride per 100 ml, then adjust the co concentrate the proteins by diluting with sodium chloride solution of the indicated concentration, or, conversely, concentrate by ultrafiltration to contain 4,5 to 5,5 g of modified immunoglobulin per 100 ml of solution, then add the stabilizing components 1 to 5 g of maltose, 1 to 2,5 g glycine or 1 to 5 g of serum albumin for every 100 ml of solution, its pH is adjusted to 6.4 to 7.4 and sterile filtration is carried out.

The advantage of the proposed process for the preparation of modified immunoglobulin is that it allows the production of the necessary drug suitable for intravenous administration, which has low anticomplementary activity, does not contain aggregated protein, has a reduced content of biologically active substances, maximally reduces the risk of transmitting viral diseases, is clinically safe and has biologically safe. half-life comparable to native uncleaved immunoglobulin. The modified immunoglobulin solution prepared according to the proposed procedure retains the same antibody content as the starting material. The preparation is stable when tested for stability according to the guidelines of the World Health Organization and does not change its appearance and viscosity after heating at 58 ° C for four hours. The modified immunoglobulin solution is harmless in guinea pigs and mice.

The process for the preparation of a preparation with low anticomplementary activity is in principle based on a suitable combination of non-specific sorption of high molecular weight substances to calcium phosphate, chemical modification of the immunoglobulin molecule by reduction with dithiothreitol and alkylation with iodoacetamide. Unwanted low molecular weight substances are removed by dialysis or diafiltration. The solution for injection is suitably stabilized. The effect of calcium phosphate can be achieved in another suitable way, for example by using an ion exchanger or a sorbent with similar properties. According to the results of our experiments and the experience of other authors, performing chemical modification with diothiothreitol and iodoacetamide is more advantageous than modification by sulfitolysis. The inclusion of properly efficient dialysis or diafiltration achieves not only the removal of by-products of reduction and alkylation, but also the separation of other low molecular weight substances originally contained in the immunoglobulin starting solution by the order of four orders of magnitude. The combination of maltose and glycine with the addition of serum albumin proved to be the best stabilizing component. The presence of albumin increases the stability of the modified immunoglobulin, blocks even trace residues of reaction products, further reduces the risk of anaphylactic reactions and in clinical application helps to detoxify substances caused by infectious diseases in the recipient's body.

In terms of technological preparation, the proposed procedure is simple and does not require any special equipment. An advantage of the process is also the fact that it allows the preparation of the desired intravenous preparation, the therapeutic importance of which will undoubtedly increase further, from an immunoglobulin prepared in a manner suitable for intramuscular administration, for which interest is constantly declining.

Example 1 g of lyophilized immunoglobulin suitable for the preparation of an intramuscular preparation having at least 95% electrophoretic purity is dissolved in 200 ml of distilled pyrogen-free water and the pH of the solution is adjusted to 7.2 ± 0.2 with 0.2 M sodium hydroxide solution. or 0.2 M acetic acid. To the solution was added 40 ml of a thick calcium phosphate sediment prepared according to the following procedure. The resulting suspension was stirred for 60 minutes, then the phosphate was separated by filtration through K5 and EKS2 type layers. Next, 23 to 38 mg of 1,4-dithio-DL-threitol are added for every 100 ml of filtrate, and after stirring for 30 minutes, 55 to 100 mg of iodoacetamide are added. The solution was stirred for another 60 minutes. The reduced and alkylated immunoglobulin solution is then dialyzed three times repeatedly against a 20-fold volume of 0.2 to 0.4% sodium chloride solution or diafiltered under similar conditions. The dialyzed solution is diluted to a concentration of 5 ± 0.5% protein and added to each 100 ml of 1 to 5 g of maltose, 1 to 1.5 g of glycine and 1 to 5 g of serum albumin. A final check is performed, or the pH is adjusted to 6.4 to 7.4, the solution is sterile filtered and filled to the required doses. The entire procedure is performed at temperatures of 4 to 8 ° C and must be performed according to the principles of pyrogen-free operation. From 20 g of the starting amount of lyophilized immunoglobulin, 260 ml of a 5% solution having an anticomplementary activity of 0.06 CH ₅₀ / mg was prepared according to the above procedure. The solution was harmless when tested in mice and guinea pigs and had unchanged antistaphylococcal antibody content. The solution was stable when heated to 58 ° C for 4 hours.

Example 2 ‘

250 ml of a 3 to 8% solution of immunoglobulin, which is formed by dissolving the protein sediment in ethanol franking and has at least 95% electrophoretic purity, is filtered through a ΕΚ type filter pad. To the filtrate was added 40 ml of a thick calcium phosphate sediment prepared according to the following procedure. Calcium phosphate sorption, reduction and alkylation, dialysis, diafiltration and stabilizer composition are the same as in Example 1. If the protein concentration is lower after dialysis, the solution is concentrated by ultrafiltration to the desired concentration. The sterile filtered solution is filled into the required doses and dried by lyophilization.

Preparation of calcium phosphate:

Calcium phosphate is prepared by slowly mixing a 0.2M solution of calcium chloride and 0.2M medium sodium phosphate in an equimolar ratio with constant stirring. The resulting suspension is stirred for a further 20 to 30 minutes after precipitation and then washed by decantation with distilled water until the solution reacts with the presence of calcium or phosphate ions. The precipitate is allowed to settle at + 4 ° C for 16 to 24 hours, the aqueous phase is separated and the sediment is used for sorption or autoclaved for a further 24 hours. The whole work procedure must be performed according to the principles valid for pyrogen-free work.

Claims (1)

PATENT CLAIMS A process for the preparation of a modified immunoglobulin preparation with low anticomplementary activity, which is suitable for intravenous administration, using processes comprising the preparation of calcium phosphate, chemical modification of immunoglobulin with DL-dithiotreitol and addition of maltose, glycine and serum albumin as stabilizing components. containing 3 to 10 g of immunoglobulin, with an electrophoretic purity of at least 95%, is contacted with calcium phosphate for every 100 ml for 30 to 60 minutes, then the calcium phosphate is separated from the liquid, and 1,4-g of the immunoglobulin solution thus obtained is added. dithio-DL-treitol in an amount of 23 to 38 mg for every 100 ml of solution and after stirring for 30 minutes 50 to 100 mg of iodoacetamide for every 100 ml of solution is added, stirred for another 60 minutes, the immunoglobulin solution thus modified is dialyzed or diafiltered against containing 0.2 to 0.4 g of sodium chloride for every 100 ml, then the protein concentration is adjusted dilute with sodium chloride solution of the indicated concentration or, conversely, concentrate by ultrafiltration to contain 4,5 to 5,5 g of modified immunoglobulin per 100 ml of solution, then add the stabilizing components 1 to 5 g of maltose, 1 to 2,5 g of glycine or 1 to 5 g of serum albumin for every 100 ml of solution, its pH is adjusted to 6.4 to 7.4 and sterile filtration is carried out.