DK167844B1 - PARENTALLY SUBMITTED BLOOD PRODUCTS, PROCEDURES FOR THE MANUFACTURING OF THEM, AND THEIR USE - Google Patents
PARENTALLY SUBMITTED BLOOD PRODUCTS, PROCEDURES FOR THE MANUFACTURING OF THEM, AND THEIR USE Download PDFInfo
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Description
i DK 167844 B1in DK 167844 B1
Opfindelsen angår parenteralt indgivelige blodprodukter af blodplasma, en fremgangsmåde til fremstilling heraf samt deres anvendelse.The invention relates to parenterally deliverable blood plasma blood products, a process for their preparation and their use.
Kendte fremgangsmåder til fremstillingen af menneskelige 5 blodprodukter omfatter udover fraktioneringsforanstaltninger til renfremstillingen af det pågældende produkt også fremgangsmåder til fjernelse af kontaminerende stoffer, der kan fremkalde bivirkninger. Eksempler herpå er fremgangsmåder til fjernelse af vasoaktive stoffer (EP 0 120 835, EP 0 122 909) 10 samt til inaktivering af de i sådanne præparater eventuelt forekommende vira (EP 0 159 311, US-PS 4.297.344).Known methods for the preparation of human blood products, in addition to fractionation measures for the purification of the product in question, also include methods for removing contaminants that may cause side effects. Examples are methods for removing vasoactive substances (EP 0 120 835, EP 0 122 909) and for inactivating any viruses present in such preparations (EP 0 159 311, U.S. Patent 4,297,344).
Hos disse kendte fremgangsmåder blev der imidlertid ikke taget hensyn til problemerne med en mulig immunmodulerende bivirkning ved produkterne. De første henvisninger til en mu-15 lig terapimedieret immunmodulation giver undersøgelser, der blev gennemført med hæmofilipatienter. Disse patienter har en forøget tilbøjelighed til bestemte infektioner af bakteriel og viral art (jvf. f.eks. A.C. Beddall et al., J. Chem. Pathol. 1985 38, 1163-1165; Håmophilie, K. Lechner fra "Hand-20 buch der inneren Medizin", side 143, bind II/9, 1985, Springer Verlag; P.H. Levin et al., Health of the Intensively Treated Hemophiliac, With Special Reference to Abnormal Liver Chemistries and Splenomegaly, 1977, 50, 1-9; B.A. McVerry et al., J. Clin. Pathol. 1979 32, 377-381). Desuden tyder defek-25 ter hos de fra disse patienters perifere blod isolerede lymfocytter på forstyrrelser af immunsystemet. Som eksempler herpå kan nævnes forskydningen af forholdet mellem T-hjælper-og T-suppressor- celler (C.M. Kessler et al. 1983, Lancet, 991-992; P. Saidi et al., 1983, New Engl. J. Med. 308, 1291-30 1292), formindskelsen af den "naturlige dræber"-celleaktivi tet (M.M. Lederman et al., N. Engl. J. Med. 1983 308, 79-82) samt formindskelsen af cirkulerende mononukleare phagocyters antigenpræsenterende kapacitet (J.W. Mannhalter et al., Clin. Immunol. Immunopathol. 1986 38, 390-397).However, in these known methods, the problems of a possible immunomodulatory side effect of the products were not taken into account. The first references to a possible therapy-mediated immune modulation provide studies that were conducted with hemophilia patients. These patients have an increased propensity for certain bacterial and viral infections (cf., e.g., AC Beddall et al., J. Chem. Pathol. 1985 38, 1163-1165; Håmophilie, K. Lechner of "Hand-20 Book of Inner Medicine ", page 143, Volume II / 9, 1985, Springer Verlag; PH Levin et al., Health of the Intensively Treated Hemophiliac, With Special Reference to Abnormal Liver Chemistries and Splenomegaly, 1977, 50, 1-9; BA McVerry et al., J. Clin. Pathol. 1979 32, 377-381). In addition, defects in the lymphocytes isolated from these patients' peripheral blood suggest disorders of the immune system. Examples include the shift of the ratio of T helper and T suppressor cells (CM Kessler et al. 1983, Lancet, 991-992; P. Saidi et al. 1983, New Engl. J. Med. 308 , 1291-30 1292), the diminution of the "natural killer" cell activity (MM Lederman et al., N. Engl. J. Med. 1983 308, 79-82), and the diminution of the antigen presenting capacity of circulating mononuclear phagocytes (JW Mannhalter et al., Clin. Immunol. Immunopathol. 1986 38, 390-397).
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Ved hjælp af opfindelsen løses de beskrevne problemer ved tilvejebringelse af et parenteralt indgiveligt blodprodukt, som i det væsentlige er fri for immunmodulerende komponenter, udtrykt ved manglen på eller udeblivelse af en nedmodulering 5 af Fc-receptorer i leukocytters membran med det forbehold, at ekspressionen af receptorer for Fc-stykket af immunglobulin G i monocytters membran efter interaktion med blodproduktpræpa-ratet ikke er reduceret eller højst er reduceret 30%.By means of the invention, the described problems are solved by providing a parenterally deliverable blood product which is essentially free of immunomodulatory components, expressed by the absence or absence of a down-modulation of Fc receptors in the leukocyte membrane, provided that the expression of receptors for the Fc fragment of immunoglobulin G in the membrane of monocytes after interaction with the blood product preparation are not reduced or at most reduced 30%.
Herunder forstås, at ekspressionen af receptorer for Fc-styk-10 ket af immunglobulin G i Fc-receptorpositive leukocytters membran, f.eks. monocytter efter interaktion med et blodpro-duktpræparat, ikke eller ikke signifikant er reduceret, dvs. højst 30%. Ekspressionen af Fc-receptorerne i monocytters membran bestemmes ved hjælp af disse cellers evne til at til-15 knytte erythrocytter, som er ladet med immunglobulin G. Resultaterne udtrykkes som % rosetdannende celler (% RFZ). Bestemmelsesmetoden for nedmoduleringen af Fc-receptorerne er kendt og f.eks. beskrevet i litteraturstederne J.W. Mannhal-ter et al., Clinical Immunology and Immunopathology 1986 38, 20 390-397; A. Berken et al., J. Exp. Med. 1966 123, 119-144 og C.S. Scott, Clin. exp. Immunol. 1979 38, 300-305.It is understood that the expression of receptors for the Fc portion of immunoglobulin G in the membrane of Fc receptor positive leukocytes, e.g. monocytes after interaction with a blood product preparation, not or not significantly reduced, i. not more than 30%. The expression of Fc receptors in the membrane of monocytes is determined by the ability of these cells to associate erythrocytes loaded with immunoglobulin G. Results are expressed as% rosette forming cells (% RFZ). The method of determining the down-modulation of the Fc receptors is known and e.g. described in the literature sites J.W. Mannhalter et al., Clinical Immunology and Immunopathology 1986 38, 20 390-397; A. Berken et al., J. Exp. With. 1966 123, 119-144 and C.S. Scott, Clin. exp. Immunol. 1979 38, 300-305.
Ekspressionen af receptorer for Fc-stykket af immunglobulin G (Fc-receptorer) i monocytters membran er en vigtig forudsætning for en upåklagelig immunforsvarsfunktion. Disse recepto-25 rer letter ikke blot phagocytosen af opsoniserede patogener (Contemporary Topics in Immunobiology, bind 14, Regulation of Leukocyte Function, R. Snyderman, ed., Plenum Press New York og London 1971); en interaktion hos disse receptorer med deres ligander udløser også en række signaler, der er af betyd-30 ning for immunforsvarets regelmæssige forløb (induktionen af dannelsen af oxygenradikaler (R.B. Jonston et al., J. Exp.The expression of receptors for the Fc piece of immunoglobulin G (Fc receptors) in the membrane of monocytes is an important prerequisite for an impeccable immune defense function. These receptors not only facilitate the phagocytosis of opsonized pathogens (Contemporary Topics in Immunobiology, Volume 14, Regulation of Leukocyte Function, R. Snyderman, ed., Plenum Press New York and London 1971); an interaction of these receptors with their ligands also triggers a series of signals that are of importance to the regular course of the immune system (the induction of oxygen radical formation (R.B. Jonston et al., J. Exp.
Med. 1976 143, 1551-1556; D.N. Rush. et al., Cellular Immunology 1984 87, 252-258 og S.D. Wright, J. Exp. Med. 1983 158, 2016-2023), aktivering af T-celler (J.A. Griffin et al., J.With. 1976 143, 1551-1556; D.N Rush. et al., Cellular Immunology 1984 87, 252-258 and S.D. Wright, J. Exp. With. 1983 158, 2016-2023), activation of T cells (J. A. Griffin et al., J.
35 Exp. Med. 1979 150, 653-675 og F.M. Griffin et al., J. Immu- DK 167844 B1 3 nol. 1980 125, 844-49), antigenpræsentation (J.W. Mannhalter et al., Clin. Immunol. Immunophathol. 1986 39, 491-503)). En nedmodulering af F-receptorerne har derfor betydningsfulde kliniske konsekvenser og kunne bidrage til en forøgelse af 5 infektionstilbøjeligheden hos patienter, der var behandlet med blodprodukter.Exp. With. 1979 150, 653-675 and F.M. Griffin et al., J. Immunol. 1980 125, 844-49), antigen presentation (J. W. Mannhalter et al., Clin. Immunol. Immunophathol. 1986 39, 491-503)). Therefore, down-modulation of the F receptors has significant clinical consequences and could contribute to an increase in the incidence of infection in patients treated with blood products.
Blodprodukterne ifølge opfindelsen, som i det væsentlige er fri for immunmodulerende komponenter, kan opnås ved en mole-kylarsigtning af blodprodukterne, nemlig en gelpermeations-10 kromatografi (gelfiltrering) og/eller - ved behandling af blodprodukterne med et molekylarsigteag-tigt bæremateriale, hvortil selektivt adsorberes immunmodulerende komponenter, eller - ved filtrering af produkterne over et filter med en pore 15 diameter, som er lig med eller mindre end 10 nm, hvorved filtreringen yderligere kan understøttes ved adsorptions virkning.The blood products of the invention, which are essentially free of immunomodulatory components, can be obtained by molecular screening of the blood products, namely a gel permeation chromatography (gel filtration) and / or - by treating the blood products with a molecular weight carrier, selectively immunomodulatory components are adsorbed, or - by filtration of the products over a pore with a diameter 15 equal to or less than 10 nm, thereby allowing the filtration to be further supported by the effect of adsorption.
Ved gelpermeationskromatografi (gelfiltrering) adskilles molekylerne efter molekylvægt. Gelen består af mikroskopisk små 20 kugler, der indeholder porer af en bestemt størrelse. Molekyler, som er større end disse porer, kan ikke trænge ind i gelen og elueres først. Mindre molekyler kan trænge ind i gel-porerne og tilbageholdes derfor og vandrer dermed langsommere. På denne måde kan der i en proteinblanding opnås en opde-25 ling af de enkelte proteiner.By gel permeation chromatography (gel filtration), the molecules are separated by molecular weight. The gel consists of microscopically small 20 spheres containing pores of a certain size. Molecules larger than these pores cannot penetrate the gel and elute first. Smaller molecules can penetrate the gel pores and are therefore retained and thus migrate more slowly. In this way, in a protein mixture, a breakdown of the individual proteins can be obtained.
En foretrukken udførelsesform for den selektive adsorbering af immunmodulerende komponenter består i, at blodprodukterne behandles med et adsorptionsmateriale, bestående af et inaktivt bæremateriale, såsom sepharose, hvortil er bundet immo-30 biliserede ligander, især proteiner af bakteriel oprindelse, såsom protein A fra stafylokokker eller protein G eller protein M fra streptokokker.A preferred embodiment of the selective adsorption of immunomodulatory components consists in treating the blood products with an adsorbent consisting of an inert carrier such as sepharose to which are attached immobilized ligands, especially proteins of bacterial origin, such as protein A from staphylococci or protein G or protein M from streptococci.
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En yderligere fremgangsmåde af denne slags består i, at blodprodukterne behandles med et adsorptionsmateriale, bestående af et inaktivt bæremateriale, såsom sepharose, hvortil er bundet antistoffer, som er blevet opnået ved immunisering med 5 immunmodulerende stoffer.A further method of this kind consists in treating the blood products with an adsorbent composed of an inert carrier such as sepharose to which are bound antibodies obtained by immunization with 5 immunomodulatory agents.
Ved filtreringsfremgangsmåden, som især egner sig til fremstillingen af gammagiobulinpræparater, opdeles partiklerne efter deres størrelse. Ved den her anvendte fremgangsmåde anvendes specielle filtermembraner med meget lille porestør-10 relse (10 nm eller mindre). Partikler, som er større end disse porer, tilbageholdes indenfor filtreringsprocessens rammer af membranen. På denne måde kan der fra en proteinblanding fraskilles store (højmolekylære) partikler.In the filtration process, which is particularly suitable for the preparation of gamma giobulin preparations, the particles are divided according to their size. In the method used here, special filter membranes with very small pore size (10 nm or less) are used. Particles larger than these pores are retained within the membrane filtration process. In this way, large (high molecular weight) particles can be separated from a protein mixture.
Blodprodukterne ifølge opfindelsen egner sig med fordel til 15 anvendelse ved behandling af arvede og erhvervede koagulationsforstyrrelser, til behandling af primære og sekundære immundefekter samt til behandling af autoimmun-, immunkompleks-og infektionssygdomme. 1 de følgende eksempler belyses egenskaberne hos blodproduk-20 terne ifølge opfindelsen, deres fremstilling og bestemmelsen af eventuelt tilstedeværende nedmodulering over for blodpro-duktfrie kontrolmedier.The blood products of the invention are advantageously suitable for use in the treatment of inherited and acquired coagulation disorders, in the treatment of primary and secondary immune defects, and in the treatment of autoimmune, immune complex and infectious diseases. In the following examples, the properties of the blood products according to the invention, their preparation and the determination of any down-modulation present against blood product-free control media are illustrated.
EKSEMPEL 1EXAMPLE 1
Der fremstilles et faktor VIII-præparat efter den i EP 0 127 25 603 beskrevne metode. Dette præparat blev underkastet en af finitetskromatografi med immobiliseret protein A efter "batch"- metoden. Derved opløstes et lyofiliseret faktor VI11-præparat, der indeholdt 500 IE faktor VIII, i 20 ml destilleret vand, der var tilsat 10 IE konserveringsmiddel-30 frit heparin (Immuno AG, Wien) , og dialyseredes ved stuetemperatur natten over mod 2 1 af en citratpuffer (0,024 M natriumcitrat, 0,120 M NaCl, indstillet på pH-værdi 7,2 med DK 167844 B1 5 HC1, tilsat 10 IE/ml konserveringsmiddel frit heparin, i det følgende kort angivet med "citrat pH 1,2". 8 ml af dialysatet (svarende til ca. 200 IE faktor VIII) blev udtaget og blandet med 2 ml af en protein A-sepharosesuspension (50% suspension 5 [vol/vol] i citrat pH 7,2, protein A-sepharose blev forækvi-libreret i citrat pH 7,2) . Derefter inkuberedes i 4 timer ved stuetemperatur under lejlighedsvis omrystning. Efter afslutningen af inkubationstiden fracentrifugeredes protein A-sepharose (5 minutter, 700 x g, stuetemperatur) , den ovenståen-10 de opløsning blev afpipetteret, og gelen blev vasket endnu tre gange med 10 ml citrat pH 7,2. De enkelte centrifugerings supernat anter forenedes, sterilfiltreredes, indstilledes på 2 IE faktor VIII/ml og opbevaredes ved 4°C indtil undersøgelsen.A factor VIII preparation is prepared by the method described in EP 0 127 25 603. This preparation was subjected to finite chromatography with immobilized protein A by the "batch" method. Thereby, a lyophilized factor VI11 preparation containing 500 IU of factor VIII was dissolved in 20 ml of distilled water added with 10 IU of preservative-free heparin (Immuno AG, Vienna) and dialyzed at room temperature overnight against 2 L of citrate buffer (0.024 M sodium citrate, 0.120 M NaCl, adjusted to pH 7.2 with DK 167844 B1 5 HCl, added 10 IU / ml preservative free heparin, hereinafter referred to as "citrate pH 1.2". 8 ml of the dialysate (corresponding to about 200 IU factor VIII) was taken and mixed with 2 ml of a protein A-sepharose suspension (50% suspension 5 [v / v] in citrate pH 7.2, protein A-sepharose was pre-equilibrated in citrate pH 7.2), then incubated for 4 hours at room temperature with occasional shaking. After completion of the incubation period, protein A-sepharose (5 minutes, 700 xg, room temperature) was centrifuged, the above solution was pipetted and the gel was washed three more times with 10 ml of citrate pH 7 The supernatants of the individual centrifuges were combined, sterile filtered, adjusted to 2 IU factor VIII / ml and stored at 4 ° C until examination.
15 Det på den beskrevne måde fremstillede og ifølge opfindelsen behandlede faktor VHI-præparat blev som beskrevet i det følgende afprøvet for sin immunmodulerende aktivitet.The factor VHI preparation prepared and treated in accordance with the invention was tested for its immunomodulatory activity as described below.
Mononukleare celler fra perifert blod isoleredes ved densitetsgradientcentrifugering (J.W. Mannhaltr et al., Clin. Im-20 munol. Immunopathol. 1986 38, 390-397). Derved blev 8 ml af en densitetsgradient (Lymphoprep, Nyegaard & Co. , Oslo, Norge) belagt med 20-25 ml hepariniseret blod og centrifugeret i 35 minutter ved stuetemperatur med 400 x g. De mononukleare celler, som havde samlet sig i interfasen mellem plasma og 25 gradient, blev frasuget, vasket tre gange med fysiologisk kogsaltopløsning og suspenderet i RPMI-1640-medium, der indeholdt 15% samlet, varmeinaktiveret (30 minutter, 56°C) AB-se-rum samt penicillin (100 IE/ml) , streptomycin (100 μg/ml) og L-glutamin (2 mM) (RPMI-suppl.) .Peripheral blood mononuclear cells were isolated by density gradient centrifugation (J. W. Mannhaltr et al., Clin. Immunol. Immunopathol. 1986 38, 390-397). Thereby, 8 ml of a density gradient (Lymphoprep, Nyegaard & Co., Oslo, Norway) was coated with 20-25 ml of heparinized blood and centrifuged for 35 minutes at room temperature with 400 x g. The mononuclear cells that had collected in the interphase plasma and 25 gradient, were aspirated, washed three times with physiological saline and suspended in RPMI-1640 medium containing 15% total heat-inactivated (30 minutes, 56 ° C) AB serum and penicillin (100 IU / ml ), streptomycin (100 μg / ml) and L-glutamine (2 mM) (RPMI supplement).
30 Isoleringen af monocytterne fra den mononukleare cellepopulation foregik ved adhærens. Monocytter har i modsætning til andre mononukleare blodceller den egenskab, at tillejre sig på glas- eller plastoverflader. Til isoleringen af monocytterne indstilles mononukleare celler på en koncentration på 1 6The isolation of the monocytes from the mononuclear cell population was by adherence. Unlike other mononuclear blood cells, monocytes have the property of adhering to glass or plastic surfaces. For the isolation of the monocytes, mononuclear cells are adjusted to a concentration of 1 6
UIV ΙΟ/ΟΗ-Η- D IUIV ΙΟ / ΟΗ-Η- D I
x 107 celler per ml RPMI-suppl. 2 ml af denne suspension pippetteres derefter over i plastvævskulturskåle (Makroplatte TC, Greiner & Sdhne, Kremsmiinster, Østrig) og inkuberes i 3 timer i et C02-inkubationsskab (5% C02, mere end 95% luftfug-5 tighed) ved 37°C. Derefter afpippeteres de ikke-adhærerede celler. De tillejrede celler (monocytter) vaskes tre gange med fysiologisk kogesaltopløsning, tilsættes RPMI-suppl. og opbevares i C02-inkubationsskab indtil den videre anvendelse.x 107 cells per ml RPMI suppl. 2 ml of this suspension is then pipetted into plastic tissue culture dishes (Macroplate TC, Greiner & Sdhne, Kremsmiinster, Austria) and incubated for 3 hours in a CO 2 incubation cabinet (5% CO 2, more than 95% humidity) at 37 ° C . The non-adherent cells are then pipped. The embedded cells (monocytes) are washed three times with physiological boiling salt solution, RPMI supplement is added. and stored in CO 2 incubation cabinets for further use.
Ekspressionen af Fc-receptorer i monocytters membran blev på-10 vist ved tillej ring af okseerythrocyter, som var ladet med IgG (A. Berken et al., Exp. Med. 1966 123, 119-144). Okseblod blev aftaget i Al s ever-opløsning og vasket flere gange i phosphatpufferkogsaltopløsning, pH 7,2-7,4 (PBS). Derefter indstilledes erythrocytkoncentrationen på 2% i PBS. Belæsnin-15 gen af erythrocyteme med immunglobulin G foregik ved inkubation med en subagglu tiner ende dosis af et anti-okseerythro-cytantistof (IgG-fraktion: 0,167 mg/ml PBS, inkubationstid 1 time, 37°C) (C.S. Scott, Clin. exp. Immunol. 1979 38, 300-305) . Efter afslutningen af inkubationstiden fjernedes frit 20 IgG ved tre ganges vask i PBS og de med antistof ladede erythrocyter blev indstillet på en koncentration på 0,5% i PBS, der indeholdt 0,2% bovint serumalbumin (PBS-BSA). Denne erythrocytsuspension kunne nu anvendes til påvisning af Fc-receptorerne i monocytmembranen. 1 2 3 4 5 6 7 8 9 10 11The expression of Fc receptors in the membrane of monocytes was demonstrated by the attachment of bovine erythrocytes charged with IgG (A. Berken et al., Exp. Med. 1966 123, 119-144). Beef blood was collected in Al s ever solution and washed several times in phosphate buffer brine, pH 7.2-7.4 (PBS). Then the erythrocyte concentration was adjusted to 2% in PBS. The immunoglobulin G erythrocytes were loaded by incubation with a subagglutinic end dose of an antioxidant erythrocyte antibody (IgG fraction: 0.167 mg / ml PBS, incubation time 1 hour, 37 ° C) (CS Scott, Clin. Exp. Immunol. 1979 38, 300-305). After the end of the incubation period, 20 IgG was removed freely by washing three times in PBS and the erythrocytes loaded with antibody were adjusted to a concentration of 0.5% in PBS containing 0.2% bovine serum albumin (PBS-BSA). This erythrocyte suspension could now be used to detect the Fc receptors in the monocyte membrane. 1 2 3 4 5 6 7 8 9 10 11
Dertil løsnedes først de ved tillej ring til plastvævskultur 2 skåle isolerede monocytter forsigtigt ved hjælp af en gummi 3 skraber fra vævskulturskålen og indstilledes på en koncentra 4 tion på 2-3 x 106 celler/ml PBS-BSA. 100 μΐ af denne monocyt- 5 suspension blandedes derpå med 100 μΐ af den ovenfor beskrev- 6 ne erythrocyt suspens ion og centrifugeredes ved 4°C i 10 mi 7 nutter med 120 x g. Efter 1 times inkubation ved 0°C gensus 8 penderedes de pelleterede celler forsigtigt og procentsatsen 9 af monocytter, som havde tillej ret erythrocytter, bestemtes 10 ved hjælp af et fasekontrastmikroskop. Der undersøgtes mindst 11 200 monocytter for deres evne til at tilknytte erythrocytter, DK 167844 Bl 7 som var belæsset med IgG. Resultaterne er angivet som % ro-setdannende celler (% RFZ) eller som tillej ringsindeks (AI).To this end, the monocytes isolated gently by a rubber 3 scraper from the tissue culture dish were carefully loosened by attaching to plastic tissue culture 2 dishes and adjusted to a concentration of 2-3 x 10 6 cells / ml PBS-BSA. 100 μΐ of this monocyte suspension was then mixed with 100 μΐ of the above-described 6 erythrocyte suspension ion and centrifuged at 4 ° C for 10 ml 7 nuts with 120 x g. After 1 hour incubation at 0 ° C, g the pelleted cells gently and the percentage 9 of monocytes that had the right erythrocytes was determined 10 by a phase contrast microscope. At least 11,200 monocytes were examined for their ability to associate erythrocytes, DK 167844 B1, which were loaded with IgG. The results are given as% rosette forming cells (% RFZ) or as placement index (AI).
En monocyt betegnes som roset, når den har tillej ret eller tilknyttet mindst tre erythrocytter. Med tillej ringsindekset 5 angives det gennemsnitlige antal erythrocytter, som er til-lejret per monocyt.A monocyte is referred to as rosette when it has a right or associated at least three erythrocytes. The placement index 5 indicates the average number of erythrocytes stored per monocyte.
Afprøvningen af faktor VIII-præparatet foregik ved, at umiddelbart efter tillej ringen af monocytterne til plastvævskulturskålene tilsattes cellerne faktor VIII (2/IE ml) og inku-10 beredes i 1 time ved 37°C i C02-inkubationskab. Efter afslutningen af inkubationstiden vaskedes de adhærerede monocytter fire gange med fysiologisk kogsaltopløsning, tilsattes RPMI-suppl. og inkuberedes i 16 timer i C02-inkubationskab ved 37°C. Denne inkubationstid er nødvendig for at de i blodpro-15 dukterne indeholdte forurenende stoffer kan udøve deres immunmodul erende virkning. Efter afslutningen af denne 16 timers inkubationstid bestemtes ekspressionen af Fc-receptorerne i monocytmembranen efter den ovenfor beskrevne metode.The testing of the factor VIII preparation was carried out by adding the factor VIII (2 / IU ml) cells immediately after attaching the monocytes to the plastic tissue culture dishes and incubating for 1 hour at 37 ° C in the CO 2 incubation cabinet. After the end of the incubation period, the adhered monocytes were washed four times with physiological saline solution, RPMI supplement was added. and incubated for 16 hours in CO 2 incubation cabinet at 37 ° C. This incubation time is necessary for the contaminants contained in the blood products to exert their immune modulatory activity. At the end of this 16 hour incubation period, expression of the Fc receptors in the monocyte membrane was determined by the method described above.
Som kontrol inkuberedes monocytter umiddelbart efter tillej -20 ringen med RPMI-1640-medium, men uden faktor VIII i 1 time og behandledes desuden som beskrevet ovenfor.As a control, monocytes were immediately incubated with the RPMI-1640 medium immediately after the preparation, but without factor VIII for 1 hour and further treated as described above.
Resultaterne af Fc-receptorekspressionen er angivet som % rosetdannende celler (% RFZ) samt som tillejringsindeks (AI).The results of Fc receptor expression are given as% rosette-forming cells (% RFZ) as well as as index of attachment (AI).
25 Inkubation af Faktor VIII- % RFZ(a) AI(b) monocytterne i aktivitet nærværelse af RPMI-medium - 81±2 4,42+0,05 30 (kontrol)Incubation of Factor VIII-% RFZ (a) AI (b) Monocytes in Activity Presence of RPMI Medium - 81 ± 2 4.42 + 0.05 30 (Control)
Faktor VIII 2 IE/ml 20±3 0,79±0,06 (udgangsprodukt) UK K l 8Factor VIII 2 IU / ml 20 ± 3 0.79 ± 0.06 (starting product) UK K l 8
Faktor VIII 2 IE/ml 75+4 3,78±0,15 (ifølge opfindelsen behandlet med protien A-sepharose) 5 ------------------------------------------------------------- a) % af monocytterne, hvortil er lejret tre eller flere med IgG belæssede erythrocytter.Factor VIII 2 IU / ml 75 + 4 3.78 ± 0.15 (according to the invention treated with protien A-Sepharose) 5 ----------------------- -------------------------------------- a)% of the monocytes to which three or more are enclosed with IgG loaded erythrocytes.
b) gennemsnitlige antal tillejrede erythrocytter per mono-cyt. Til beregningen af AI anvendtes alle talte monocyt-10 ter (såvel de, hvortil ikke er tillej ret nogen erythro cytter som alle med en eller flere erythrocytter tillej- ret) .b) average number of erythrocytes embedded per monocyte. For the calculation of AI, all spoken monocytes were used (both those for which no erythrocyte is present and all with one or more erythrocytes).
Deraf fremgår, at faktor VIII-udgangsproduktet bevirker en nedmodulering af Fc-receptorekspressionen fra 81% RFZ til 20% 15 RFZ. I modsætning hertil andrager procentsatsen af rosetdannende celler 75% efter interaktion med monocytteme med det ifølge opfindelsen behandlede faktor VIII-præparat. Differensen mellem 81% RFZ i kontrollen og 75% RFZ efter interaktio-nen med det ifølge opfindelsen behandlede faktor VHI-præpa-20 rat er ikke signifikant (Students t-test for parvise stikprøver) . Dette betyder, at det ifølge opfindelsen behandlede faktor VIII-produkt er fri for immunmodulerende komponenter, da RFZ-værdien efter monocyttemes interaktion med det ifølge opfindelsen behandlede produkt ikke adskiller sig fra kon-25 trolværdien (% RFZ ved monocytter uden faktor Vlll-behand-ling) .It can be seen that the factor VIII starting product causes a down-modulation of Fc receptor expression from 81% RFZ to 20% RFZ. In contrast, the percentage of rosette-forming cells amounts to 75% after interaction with the monocytes with the factor VIII preparation treated according to the invention. The difference between 81% RFZ in the control and 75% RFZ after the interaction with the inventive factor VHI preparation is not significant (Student's t-test for paired samples). This means that the factor VIII product treated according to the invention is free of immunomodulatory components since, after interaction of the monocytes with the product treated according to the invention, the RFZ value does not differ from the control value (% RFZ in monocytes without factor VIII treatment). ling).
EKSEMPEL 2 500 IE af et efter den af Suokela H. et al. i Vox. Sang. 1977 33, 37-50 beskrevne metode fremstillede og lyofiliserede fak-30 tor IX-præparat opløstes i 20 ml destilleret vand og dialyseredes natten over mod 2 1 af en citratpuffer (citrat pH 7,2, den nøjagtige sammensætning af denne puffer er beskrevet i eksempel 1) ved 4°C. 8 ml (svarende til ca. 200 IE faktor IX) DK 167844 B1 9 udtoges og underkastedes en affinitetskromatografi på protein A-sepharose i en søjleprocedure. Derved pumpedes de 8 ml faktor IX-præparater med en gennemstrømningshastighed på 0,1 ml per minut over en protein A-sepharosesøjle (søjle C 10/10 fra 5 Pharmacia, 1 ml gelvolumen) som i forvejen var blevet ækvili-breret med 50 ml citrat pH 7,2. Den gennem søjlen pumpede faktor IX indstillededes til 2 IE/ml, sterilfiltreredes og opbevaredes ved 4°C indtil afprøvning. Afprøvningen af den immun-modulerende aktivitet hos udgangspræparatet samt hos det på 10 den beskrevne måde ifølge opfindelsen faktor IX-præparat foregik som angivet i eksempel 1. Resultaterne af disse undersøgelser er sammenfattet i den efterfølgende tabel.Example 2 500 IU of one after that of Suokela H. et al. in Vox. Song. 1977 33, 37-50 described method prepared and lyophilized Factor 30 preparation was dissolved in 20 ml of distilled water and dialyzed overnight against 2 L of a citrate buffer (citrate pH 7.2, the exact composition of this buffer is described in Example 1) at 4 ° C. 8 ml (corresponding to approximately 200 IU of factor IX) was taken and subjected to an affinity chromatography on protein A-Sepharose in a column procedure. Thereby, the 8 ml of factor IX preparations were pumped at a flow rate of 0.1 ml per minute over a protein A-Sepharose column (column C 10/10 from 5 Pharmacia, 1 ml gel volume) which had already been equilibrated with 50 ml. citrate pH 7.2. The factor IX pumped through the column was adjusted to 2 IU / ml, sterile filtered and stored at 4 ° C until testing. The test of the immunomodulatory activity of the starting composition and of the factor IX preparation described in the invention in accordance with the invention were carried out as in Example 1. The results of these studies are summarized in the following table.
Inkubation af Faktor IX- % RFZ^ AIIncubation of Factor IX-% RFZ ^ AI
15 monocytterne i aktivitet nærværelse af RPMI-medium - 75±1 4,09±0,04 (kontrol) 20 Faktor IX 2 IE/ml 42±3 2,31±0,07 (udgangsprodukt)15 Monocytes in Activity Presence of RPMI Medium - 75 ± 1 4.09 ± 0.04 (Control) 20 Factor IX 2 IU / ml 42 ± 3 2.31 ± 0.07 (Starting Product)
Faktor IX behand- 2 IE/ml 70+3 4,00±0,08 let ifølge opfindelsen 25 ------------------------------------------------------------- a) % af rosetdannende celler, dvs. % talte monocytter, hvor til er lejret tre eller flere med IgG belæssede erythro cytter.Factor IX treats 2 IU / ml 70 + 3 4.00 ± 0.08 readily according to the invention 25 ---------------------------- --------------------------------- a)% of rosette-forming cells, i.e. % spoke monocytes to which are stored three or more IgG loaded erythro cytes.
b) tillej ringsindeks, dvs. gennemsnitlige antal tillejrede 30 erythrocytter per monocyt.(b) index of placement, ie. average number of nested 30 erythrocytes per monocyte.
Af resultaterne fra denne tabel ses, at en interaktion mellem faktor IX med monocytter førte til en forminskelse af Fc-receptorekspressionen fra 75% RFZ (kontrol) til 42% RFZ (efter 10From the results of this table, it is seen that an interaction of factor IX with monocytes led to a decrease in Fc receptor expression from 75% RFZ (control) to 42% RFZ (after 10
Ulv ΙΌ/ΟΗ-Η- D IWolf ΙΌ / ΟΗ-Η- D I
interaktion med faktor IX) . Efter monocyttemes interaktion med det ifølge opfindelsen behandlede faktor IX-produkt androg Fc-receptorekspressionen dog 70% RFZ. Forskellen mellem 75% RFZ i kontrollen og 70% RFZ efter interaktion med den 5 ifølge opfindelsen behandlede faktor IX er ikke signifikant (Students t-test for parvise stikprøver) og det betyder, at de ifølge opfindelsen behandlede faktor IX-produkter er fri for immunmodulerende egenskaber.interaction with factor IX). However, following the interaction of the monocytes with the factor IX product treated according to the invention, Fc receptor expression was 70% RFZ. The difference between 75% RFZ in the control and 70% RFZ after interaction with the factor IX treated according to the invention is not significant (Student's t-test for paired samples) and this means that the factor IX products treated according to the invention are free of immunomodulatory properties.
EKSEMPEL 3 10 Et lyofiliseret FEIBA-præparat (500 IE, Immuno AG) opløstes i 20 ml destilleret vand og dialyseredes som beskrevet i eksemplerne 1 og 2 for faktor VIII henholdsvis faktor IX, mod citrat pH 7,2. Fjernelsen af de immunregulerende stoffer foregik med adsorptionskromatografi med protein A-sepharose efter 15 søjleproceduren. Den nøjagtige metode var som beskrevet i eksempel 2 for faktor IX. Afprøvningen af de immunmodul erende egenskaber hos udgangsproduktet samt hos det ifølge opfindelsen behandlede FEIBA-præparat foregik som beskrevet i eksempel 1. Resultaterne af disse undersøgelser, der er sammenfat-20 tet i den følgende tabel, viser, at behandlingen ifølge opfindelsen med immobiliseret protein A fjerner de immunmodule-rende aktiviteter fra FEIBA.EXAMPLE 3 A lyophilized FEIBA preparation (500 IU, Immuno AG) was dissolved in 20 ml of distilled water and dialyzed as described in Examples 1 and 2 for Factor VIII and Factor IX, respectively, against citrate pH 7.2. The immunoregulatory substances were removed by adsorption protein chromatography with protein A-Sepharose after the column procedure. The exact method was as described in Example 2 for factor IX. The testing of the immunomodulatory properties of the starting product as well as of the FEIBA treated according to the invention was carried out as described in Example 1. The results of these studies summarized in the following table show that the treatment according to the invention with immobilized protein A removes the immune modulating activities from FEIBA.
Inkubation af FEIBA- % RFZAIIncubation of FEIBA-% RFZAI
25 monocytterne i aktivitet nærværelse af RPMI-medium - 75±1 4,09±0,04 (kontrol) 30 FEIBA 2 IE/ml 34+4 1,89±0,15 (udgangsprodukt) DK 167844 B1 11 FEIBA (efter behand- 2 IE/ml 73+1 3,94+0,0525 monocytes in activity presence of RPMI medium - 75 ± 1 4.09 ± 0.04 (control) 30 FEIBA 2 IU / ml 34 + 4 1.89 ± 0.15 (starting product) DK 167844 B1 11 FEIBA (after treatment - 2 IU / ml 73 + 1 3.94 + 0.05
ling ifølge opfindelsen med immobiliseret protein Aaccording to the invention with immobilized protein A
5 ------------------------------------------------------------- a) % af rosetdannende celler, dvs. % monocytter, hvortil er lejret tre eller flere med IgG belæssede erythrocytter.5 ------------------------------------------------- ------------ a)% of rosette-forming cells, i.e. % of monocytes to which are stored three or more IgG-loaded erythrocytes.
b) tillejringsindeks, dvs. gennemsnitlige antal tillejrede erythrocytter per monocyt.(b) index of indexing, ie. average number of nested erythrocytes per monocyte.
10 Det kan fastslås, at hos alle undersøgte koagulationsfaktorpræparater (faktor VIII, faktor IX, FEIBA) fjernedes de immunmodul er ende egenskaber ved behandling med immobiliseret protein A såvel efter "batch"-metoden som efter søjlemetoden.It can be seen that in all coagulation factor preparations studied (Factor VIII, Factor IX, FEIBA), the immune module was removed end properties of treatment with immobilized protein A both by the batch method and by the column method.
EKSEMPEL 4 15 Et til intramuskulær anvendelse egnet immunglobulin-G-præpa-rat (i.m. IgG) fremstilledes ved ethanolfraktionering (H.F. Deutsch et al., J. Biol. Chem. 1946 164, 109ff). Dette præparat underkastedes en gelpermeationskromatografi. Dertil fyldtes en separationssøjle (K 50/60, Pharmacia) med 750 ml kro-20 matografigel (Sephacryl S 300, Pharmacia) og ækvilibreredes med en phosphatpuffer (20 mM KH2P04, 0,15 M NaCl, 0,02% NaN3, pH 8,0). Denne søjle belæssedes derefter med 300 mg i.m. IgG (i 10 ml phosphatpuffer) og eluerededes ved en gennemstrømningshastighed på 1 ml per minut. Eluaterne opfangededes i 11 25 ml fraktioner og proteinindholdet bestemtes ved hjælp af en UV-detektor ved 280 nm. Derved opnåedes elueringskurver, som vist på tegningen. De med betegnede fraktioner forenedes og afprøvededes for immunmodulerende aktivitet, som beskrevet i eksempel 1. Resultaterne af disse undersøgelser fremgår af 30 den følgende tabel, hvoraf det ses, at denne behandling har været tilstrækkelig til at gøre immunglobulin-G-præparatet fri for immunmodulerende komponenter. RFZ-værdien, der opnåe- UK 10/34-4 b l 12 des efter monocytternes interaktion med det ifølge opfindelsen behandlede i.m. IgG, stemmer i det væsentlige overens med kontrolværdien.Example 4 An immunoglobulin G preparation (i.m. IgG) suitable for intramuscular use was prepared by ethanol fractionation (H.F. Deutsch et al., J. Biol. Chem. 1946 164, 109ff). This preparation was subjected to gel permeation chromatography. To this was added a separation column (K 50/60, Pharmacia) with 750 ml chromatography gel (Sephacryl S 300, Pharmacia) and equilibrated with a phosphate buffer (20 mM KH2 PO4, 0.15 M NaCl, 0.02% NaN3, pH 8 , 0). This column was then loaded with 300 mg i.m. IgG (in 10 ml of phosphate buffer) and eluted at a flow rate of 1 ml per minute. The eluates were captured in 11 25 ml fractions and the protein content was determined by a UV detector at 280 nm. Thereby elution curves were obtained, as shown in the drawing. The designated fractions were pooled and tested for immunomodulatory activity, as described in Example 1. The results of these studies are shown in the following table, which shows that this treatment has been sufficient to render the immunoglobulin G preparation free of immunomodulatory components. . The RFZ value obtained in UK 10 / 34-4 b l 12 after the interaction of the monocytes with the invention treated i.m. IgG, essentially corresponds to the control value.
5 Inkubation af i.m. IgG % RFZ 1 AI2 monocytteme i mg/ml nærværelse af RPMI-medium - 84±3 7,11+0,22 10 (kontrol) i.m. IgG 10,8 20+2 1,97±0,07 (udgangsmateriale) i.m. IgG (efter be- 11,3 64+2 5,94±0,04 handling ifølge op-15 findelsen ved gelper-meationskromatografi % af rosetdannende celler, dvs. monocytter, hvortil er lejret tre eller flere med IgG belæssede erythrocytter.5 Incubation of i.m. The IgG% RFZ 1 AI2 monocytes in mg / ml presence of RPMI medium - 84 ± 3 7.11 + 0.22 10 (control) i.m. IgG 10.8 20 + 2 1.97 ± 0.07 (starting material) i.m. IgG (after 11.3 64 + 2 5.94 ± 0.04 action according to the invention by gel permeation chromatography% of rosette-forming cells, i.e., monocytes, to which are stored three or more IgG-loaded erythrocytes.
20 b) tillej ringsindeks, dvs. gennemsnitlige antal tillejrede erythrocytter per monocyt.B) index of placement, ie. average number of nested erythrocytes per monocyte.
2 EKSEMPEL 5EXAMPLE 5
Et som i eksempel 4 beskrevet fremstillet immungiobulin G-præparat (i.m. IgG) indstilledes på en koncentration på 50 25 mg/ml i phosphatpufret kogsaltopløsning (PBS). Efter passende forklaring over egnede filtre blev præparatet sendt gennem et specielt filter med meget lille porestørrelse (Sartorius SM 11318, porestørrelse 10 nm). Dertil blev filtermembranen lagt i en egnet filterholder og der sendtes f.eks. 20 ml af i.m.An immunogiobulin G preparation (i.m. IgG) prepared as described in Example 4 was adjusted to a concentration of 50 25 mg / ml in phosphate buffered saline (PBS). After appropriate explanation of suitable filters, the composition was passed through a very small pore size filter (Sartorius SM 11318, pore size 10 nm). In addition, the filter membrane was placed in a suitable filter holder and e.g. 20 ml of i.m.
30 IgGopløsningen gennem filteret ved et tryk på 1 til 1,5 bar. Filtratet blev derefter undersøgt som beskrevet i eksempel 1 for eventuelle immunmodulerende egenskaber. De i den følgende DK 167844 B1 13 tabel angivne resultater viser, at denne behandling fjerner hovedparten af præparatets immunmodulerende egenskaber. Mens en behandling af monocytterne med i.m. IgG-udgangsproduktet bevirker en nedmodulering af Fc-receptorekspressionen fra 79% 5 til 21%, andrager Fc-receptorekspressionen af monocytterne efter interaktion med det ifølge opfindelsen behandlede produkt 67%. Den ifølge opfindelsen behandlede i.m. IgG-fraktion er altså i det væsentlige fri for immunmodulerende egenskaber.30 IgG solution through the filter at a pressure of 1 to 1.5 bar. The filtrate was then tested as described in Example 1 for any immunomodulatory properties. The results set forth in the following table show that this treatment removes most of the immunomodulatory properties of the preparation. While treating the monocytes with i.m. The IgG starting product causes a down-modulation of the Fc receptor expression from 79% to 21%, and the Fc receptor expression of the monocytes after interaction with the product treated according to the invention is 67%. The i.m. Thus, IgG fraction is essentially free of immunomodulatory properties.
10 -------------------------------------------------------------10 ------------------------------------------------- ------------
Inkubation af monocytterne i.m. IgG % RFZ^ i nærværelse af mg/ml RPMI-medium (kontrol) - 79+3 15 i.m. IgG (udgangsmateriale) 10,0 21±3 i.m. IgG (efter behandling ifølge 10,0 67+2 opfindelsen ved filtrering gennem specielle filtre med en porestørrelse på 10 nm) 20 ------------------------------------------------------------- a) % af rosetdannende celler, dvs. monocytter, hvortil er lejret tre eller flere med IgG belæssede erythrocytter.Incubation of the monocytes i.m. IgG% RFZ + in the presence of mg / ml RPMI medium (control) - 79 ± 3 i.m. IgG (starting material) 10.0 21 ± 3 i.m. IgG (after treatment according to the 10.0 67 + 2 invention by filtration through special filters having a pore size of 10 nm) 20 ------------------------- ------------------------------------ a)% of rosette-forming cells, i.e. monocytes to which are stored three or more IgG-loaded erythrocytes.
EKSEMPEL 6EXAMPLE 6
Der fremstilledes et faktor VIII-præparat efter den i EP-A 0 25 127 603 beskrevne metode. Dette præparat underkastedes en af finitetskromatografi med immobiliserede, mod den immunmodule-rende komponent rettede antistoffer.A factor VIII preparation was prepared according to the method described in EP-A 0 25 127 603. This preparation was subjected to finite chromatography with immobilized antibodies directed against the immunomodulatory component.
For at fremstille de til affinitetskromatografien nødvendige antistoffer isoleredes først den immunmodulerende komponent 30 fra et som ovenfor beskrevet faktor VIII-præparat. Dette opnåedes med en kombination af affinitetskromatografi og søjlekromatografi. Dertil opløses 2500 IE faktor VIII i 100 ml 14To prepare the antibodies necessary for affinity chromatography, the immunomodulatory component 30 was first isolated from a factor VIII preparation as described above. This was achieved with a combination of affinity chromatography and column chromatography. In addition, 2500 IU of factor VIII is dissolved in 100 ml of 14
UIV ΙΌ/ΟΗ-Η- D IUIV ΙΌ / ΟΗ-Η- D I
destilleret vand og dialyseredes i 24 timer mod citrat pHdistilled water and dialyzed for 24 hours against citrate pH
7,2. Dette produkt blev derefter tilført til en søjle, som var fyldt med immobiliseret protein A (protein A-sepharose, 12 ml gelvolumen, søjle C 10/20, Pharmacia, Sverige) og cir-5 kuleret natten over ved stuetemperatur og en gennemstrømningshastighed på 0,5 ml/minut. Derefter blev søjlen vasket med 100 ml citrat pH 7,2 (gennemstrømningshastighed 0,5 ml/-minut). Det til protein A-sepharose bundne materiale eluere-des derefter med en sur citratpuffer (50 mM trinatriumcitrat, 10 50 nM citronsyre, pH 3,0, i det følgende kort benævnt "Citrat pH 3,0") og straks efter elueringen neutraliseret med 1 M natriumphosphatpuffer, pH 8,5. Efter dialyse mod PBS underkastedes dette materiale en molekylsigtekromatografi. Dertil anbragtes det dialyserede protein A-eluat på en Superase 15 Prep-Grade søjle (HR 16/50) og kromatograferedes med en gennemstrømningshastighed på 0,5 ml/minut. Fraktionerne med en molekylvægt på mere end 400 kD indeholdt den immunmodulerende komponent, hvilket blev fastslået ved det allerede beskrevne rosetforsøg. Disse fraktioner samledes og anvendtes til frem-20 stilling af antistoffer i forsøgsdyr. 150 ^g af den immunmodulerende komponent (i 200 μΐ PBS) blandedes med 200 μΐ komplet Freund's adjuvans, og en kanin immuniseredes dermed subkutant på to steder. På den 21. og 28. dag efter den første immunisering gennemførtes på samme måde en booster-immunise-25 ring. Fra den 35. dag udtoges i 6 uger med en uges afstand ca. 30 ml blod, hvorfra der fremstilledes serum. Derefter forenedes serumprøverne fra de forskellige blodudtagelsesdage og derudfra isoleredes immungiobulin-G-fraktionen.7.2. This product was then fed to a column loaded with immobilized protein A (protein A-sepharose, 12 ml gel volume, column C 10/20, Pharmacia, Sweden) and circulated overnight at room temperature and a flow rate of 0 , 5 ml / minute. Then, the column was washed with 100 ml citrate pH 7.2 (flow rate 0.5 ml / minute). The protein bound to protein A-Sepharose was then eluted with an acidic citrate buffer (50 mM trisodium citrate, 10 50 nM citric acid, pH 3.0, hereinafter briefly referred to as "Citrate pH 3.0") and immediately neutralized with elution. 1 M sodium phosphate buffer, pH 8.5. After dialysis against PBS, this material was subjected to molecular sieve chromatography. In addition, the dialyzed protein A eluate was placed on a Superase 15 Prep-Grade column (HR 16/50) and chromatographed at a flow rate of 0.5 ml / minute. The fractions with a molecular weight greater than 400 kD contained the immunomodulatory component, which was established by the rosette experiment already described. These fractions were pooled and used to prepare antibodies in test animals. 150 µg of the immunomodulatory component (in 200 μΐ PBS) was mixed with 200 μΐ complete Freund's adjuvant, thus a rabbit was immunized subcutaneously at two sites. On the 21st and 28th days after the first immunization, a booster immunization was similarly performed. From the 35th day, for 6 weeks at a distance of one week, approx. 30 ml of blood from which serum was prepared. Then, the serum samples from the different blood collection days were pooled and from there the immunogiobulin G fraction was isolated.
Dertil blev der til serumet sat ammoniumsulfat indtil 35% 30 mætning, og der omrørtes i 25 minutter ved stuetemperatur og centrifugeredes derefter 800 x g i 15 minutter. Bundfaldet blev gensuspenderet i til 33% mættet ammoniumsulfat, omrørt i 15 minutter og fornyet fracentrifugeret. Det derved opnåede bundfald blev opløst i PBS, der indeholdt 0,1% natriumazid og 35 underkastet en molekylsigtekromatografi ved hjælp af en Superase Prep-Grade søjle (HR 16/50, gennemstrømningshastighed DK 167844 B1 15 0,5 ml/minut). Fraktionerne i molekylvægtsintervallet fra 150 kD (altså de immunglobulin-G-holdige fraktioner) samledes og dialyseredes mod 0,1 M natriumhydrogencarbonat, 0,5 M natri-umchlorid, pH 8,5 (= koblingspuffer).To this, ammonium sulfate was added to the serum until 35% saturation and stirred for 25 minutes at room temperature and then centrifuged at 800 x g for 15 minutes. The precipitate was resuspended in to 33% saturated ammonium sulfate, stirred for 15 minutes, and centrifuged again. The precipitate thus obtained was dissolved in PBS containing 0.1% sodium azide and subjected to a molecular sieve chromatography by a Superase Prep-Grade column (HR 16/50, flow rate DK 167844 B1 0.5 ml / minute). The fractions in the molecular weight range of 150 kD (ie, the immunoglobulin G-containing fractions) were pooled and dialyzed against 0.1 M sodium bicarbonate, 0.5 M sodium chloride, pH 8.5 (= coupling buffer).
5 De således opnåede antistoffer immobiliseredes derefter ved kobling til cyanogenbromid(CNBr)-aktiveret sepharose (forskrift fra firmaet Pharmacia). Dertil blev 4 g CNBr-aktiveret sepharose 4B (firma Pharmacia, Sverige) kvældet i 1 mM saltsyre og vasket på en glas filtertragt med 500 ml 1 mM saltsy-10 re. 10 ml af gelen blandedes med 40 ml af det antistof, som befandt sig i koblingspufferen (svarende til 90 mg protein) og inkuberedes 2 timer ved stuetemperatur ved lejlighedsvis omrystning. Derefter fracentrifugeredes (800 x g, 15 minutter) , vaskedes ved gensuspension i koblingspuffer og efter-15 følgende centrifugering, og gelmaterialet suspenderes i 50 ml blokeringspuffer (= koblingspuffer + 1 M ethanolamin). Efter en inkubation på 2 timer ved stuetemperatur vaskedes gelmaterialet, nemlig først i 100 ml 0,1 M Tris, 0,5 M natrium-chlorid, pH 8,0 og derefter i 100 ml 0,1 M iseddikesyre, 0,5 20 M natriumchlorid, pH 4,0. pH 8 - pH 4-vasketrinnene blev gentaget tre gange (gelvasken gennemførtes ved gensuspension og fracentrifugering). Tilsidst suspenderedes gelmaterialet i 20 ml PBS + 10 IE heparin/ml. En analyse viste, at per ml gel var bundet 9 mg af de mod den immunmodulerende komponent ret-25 tede antistoffer.The antibodies thus obtained were then immobilized by coupling to cyanogen bromide (CNBr)-activated sepharose (Pharmacia specification). In addition, 4 g of CNBr-activated sepharose 4B (company Pharmacia, Sweden) was swollen in 1 mM hydrochloric acid and washed on a glass filter funnel with 500 ml of 1 mM hydrochloric acid. 10 ml of the gel was mixed with 40 ml of the antibody contained in the coupling buffer (corresponding to 90 mg of protein) and incubated for 2 hours at room temperature with occasional shaking. Then, it was centrifuged (800 x g, 15 minutes), washed by resuspension in coupling buffer and subsequent centrifugation, and the gel material suspended in 50 ml blocking buffer (= coupling buffer + 1 M ethanolamine). After a 2 hour incubation at room temperature, the gel material was washed, firstly in 100 ml of 0.1 M Tris, 0.5 M sodium chloride, pH 8.0 and then in 100 ml of 0.1 M glacial acetic acid, 0.5 20 M sodium chloride, pH 4.0. pH 8 - The pH 4 washing steps were repeated three times (gel washing was performed by resuspension and fracentrifugation). Finally, the gel material was suspended in 20 ml PBS + 10 IU heparin / ml. An analysis showed that per ml of gel was bound 9 mg of the antibodies directed against the immunomodulatory component.
4,5 ml af denne gel blev pakket i en kromatografisøjle (C 10/10, Pharmacia, Sverige) og skyllet i rækkefølge med 30 ml 3% bovint serumalbumin (i PBS), 50 ml 50 mM trinatriumcitrat, 50 mM citronsyre (pH 3,0) og til slut med 200 ml PBS (inde-30 holdende 10 IE heparin/ml heparin) (gennemstrømningshastighed 0,5/minut). Dette gelmateriale blev derefter anvendt til fra-skillelse af den immunmodulerende komponent.4.5 ml of this gel was packed in a chromatography column (C 10/10, Pharmacia, Sweden) and rinsed sequentially with 30 ml of 3% bovine serum albumin (in PBS), 50 ml of 50 mM trisodium citrate, 50 mM citric acid (pH 3 , 0) and finally with 200 ml PBS (containing 30 IU heparin / ml heparin) (flow rate 0.5 / minute). This gel material was then used to separate the immunomodulatory component.
Denne fraskillelse kan gennemføres såvel efter "batch"- som efter søjlemetoden. I eksemplet beskrives fraskillelsen af UK lb/844 di 16 den immunmodulerende komponent fra et faktor VI11-præparat, gennemført efter søjlemetoden. Efter samme princip kan den immunmodulerende komponent imidlertid også fjernes fra faktor IX- og FEIBA-præparater.This separation can be carried out both by "batch" and by the column method. In the example, the separation of UK lb / 844 di 16 describes the immunomodulatory component of a factor VI11 preparation performed by the column method. However, following the same principle, the immunomodulatory component can also be removed from factor IX and FEIBA preparations.
5 I eksemplet opløstes 500 IE af et som beskrevet ovenfor fremstillet faktor VIII-præparat i 20 ml destilleret vand og dialyseredes to gange i 24 timer mod hver gang en liter PBS, der indeholdet 10 IE/ml heparin. Derefter indstilledes koncentrationen af faktor VI11-præparatet med den samme puffer på 10 10 IE faktor VIII per ml. 100 IE af dette materiale blev derefter anbragt på den ovenfor beskrevne separationssøjle (sepha-rose, hvortil var blevet koblet antistoffer mod den immunmodulerende komponent) og cirkuleret i 16 timer ved stuetemperatur med en gennemstrømningshastighed på 0,5 ml/minut over 15 søjlen. Det ikke til søjlen bundne materiale blev derefter indstillet på 2 IE faktor VIII/ml og afprøvet som beskrevet i eksempel 1 ved hjælp af rosetforsøget for immunmodulerende aktivitet. Resultaterne af disse undersøgelser er sammenfattet i den følgende tabel.In the example, 500 IU of a factor VIII preparation prepared as described above was dissolved in 20 ml of distilled water and dialyzed twice for 24 hours against each liter of PBS containing 10 IU / ml of heparin. Then, the concentration of the factor VI11 preparation was adjusted with the same buffer of 10 10 IU factor VIII per ml. 100 IU of this material was then placed on the separation column described above (separa- ble to which antibodies had been coupled to the immunomodulatory component) and circulated for 16 hours at room temperature at a flow rate of 0.5 ml / minute over the column. The non-column material was then adjusted to 2 IU factor VIII / ml and tested as described in Example 1 using the rosette test for immunomodulatory activity. The results of these studies are summarized in the following table.
20 -------------------------------------------------------------20 ------------------------------------------------- ------------
Inkubation af mono- Faktor VIII- % RFZ^ AI^) cytterne i nærværelse af aktivitet RPMI-medium (kontrol) - 81+3 6,06±1,76 25 Faktor VIII 2 IE/ml 31+5 1,36±0,13 (udgangsprodukt)Incubation of mono- Factor VIII-% RFZ ^ AI ^) cytes in the presence of activity RPMI medium (control) - 81 + 3 6.06 ± 1.76 Factor VIII 2 IU / ml 31 + 5 1.36 ± 0 , 13 (starting product)
Faktor VIII 2 IE/ml 80±6 4,79±1,45 (behandlet ifølge opfindelsen) 30 ------------------------------------------------------------- a) % rosetdannende celler, dvs. % af de talte celler, hvortil er lejret tre eller flere med IgG belæssede erythrocytter.Factor VIII 2 IU / ml 80 ± 6 4.79 ± 1.45 (treated according to the invention) 30 ---------------------------- --------------------------------- a)% rosette-forming cells, i.e. % of the counted cells to which are stored three or more IgG-loaded erythrocytes.
Claims (5)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT29987 | 1987-02-12 | ||
| AT0029987A AT388501B (en) | 1987-02-12 | 1987-02-12 | METHOD FOR PRODUCING PARENTERALLY AVAILABLE BLOOD PRODUCTS |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DK73888D0 DK73888D0 (en) | 1988-02-12 |
| DK73888A DK73888A (en) | 1988-08-13 |
| DK167844B1 true DK167844B1 (en) | 1993-12-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DK073888A DK167844B1 (en) | 1987-02-12 | 1988-02-12 | PARENTALLY SUBMITTED BLOOD PRODUCTS, PROCEDURES FOR THE MANUFACTURING OF THEM, AND THEIR USE |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0278948B1 (en) |
| JP (1) | JPS63203622A (en) |
| AT (1) | AT388501B (en) |
| CA (1) | CA1306943C (en) |
| DE (1) | DE3869990D1 (en) |
| DK (1) | DK167844B1 (en) |
| ES (1) | ES2036720T3 (en) |
| FI (1) | FI93516C (en) |
| HU (1) | HU210810A9 (en) |
| NO (1) | NO175668C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AT399818B (en) * | 1992-04-24 | 1995-07-25 | Immuno Ag | METHOD FOR PRODUCING A HIGH PURIFIED VIRUS-SAFE FACTOR VIII PREPARATION |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1430461A (en) * | 1972-07-13 | 1976-03-31 | Research Corp | Purification of blood |
| DE3270830D1 (en) * | 1981-01-30 | 1986-06-05 | Centocor Inc | Immunoassay for multi-determinant antigens |
| JPH0686480B2 (en) * | 1983-02-21 | 1994-11-02 | 雪印乳業株式会社 | Monoclonal antibody for erythropoietin production |
| AT383739B (en) * | 1983-03-16 | 1987-08-10 | Immuno Ag | METHOD FOR INACTIVATING SUBSTANCES CAUSING INCOMPATIBILITY REACTIONS IN IMMUNALLOBULIN BLOOD FRACTIONS |
-
1987
- 1987-02-12 AT AT0029987A patent/AT388501B/en not_active IP Right Cessation
-
1988
- 1988-02-02 CA CA000557971A patent/CA1306943C/en not_active Expired - Lifetime
- 1988-02-08 ES ES198888890028T patent/ES2036720T3/en not_active Expired - Lifetime
- 1988-02-08 DE DE8888890028T patent/DE3869990D1/en not_active Expired - Fee Related
- 1988-02-08 EP EP88890028A patent/EP0278948B1/en not_active Expired - Lifetime
- 1988-02-11 NO NO880613A patent/NO175668C/en not_active IP Right Cessation
- 1988-02-12 DK DK073888A patent/DK167844B1/en not_active IP Right Cessation
- 1988-02-12 JP JP63031695A patent/JPS63203622A/en active Pending
- 1988-02-12 FI FI880684A patent/FI93516C/en not_active IP Right Cessation
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1995
- 1995-04-10 HU HU95P/P00100P patent/HU210810A9/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63203622A (en) | 1988-08-23 |
| NO880613L (en) | 1988-08-15 |
| HU210810A9 (en) | 1995-08-28 |
| EP0278948A1 (en) | 1988-08-17 |
| FI880684A0 (en) | 1988-02-12 |
| DK73888A (en) | 1988-08-13 |
| AT388501B (en) | 1989-07-25 |
| FI93516C (en) | 1995-04-25 |
| CA1306943C (en) | 1992-09-01 |
| DE3869990D1 (en) | 1992-05-21 |
| FI93516B (en) | 1995-01-13 |
| EP0278948B1 (en) | 1992-04-15 |
| DK73888D0 (en) | 1988-02-12 |
| ATA29987A (en) | 1988-12-15 |
| ES2036720T3 (en) | 1993-06-01 |
| NO175668C (en) | 1994-11-16 |
| NO175668B (en) | 1994-08-08 |
| FI880684A (en) | 1988-08-13 |
| NO880613D0 (en) | 1988-02-11 |
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