CA1306943C - Blood products for parenteral administration - Google Patents
Blood products for parenteral administrationInfo
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- CA1306943C CA1306943C CA000557971A CA557971A CA1306943C CA 1306943 C CA1306943 C CA 1306943C CA 000557971 A CA000557971 A CA 000557971A CA 557971 A CA557971 A CA 557971A CA 1306943 C CA1306943 C CA 1306943C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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Abstract
ABSTRACT OF THE DISCLOSURE:
There are disclosed blood products for parenteral administration which are essentially free of immune-modulating components, and methods for their production. The absence of immune-modulating components is expressed by the lack or absence of down-modulation of Fc receptors in the membrane of leukocytes. The expression of the receptors for the Fc fragment of immunoglobulin G in the membrane of monocytes, after interaction with a blood product preparation, is to be reduced not at all or by 30 % at most. The blood products are obtained - by molecular sieving of the blood products by gel permeation chromatography (gel filtration), or - by treatment of the blood products with a molecular sieve-like carrier material on which immune-modulating components are selectively adsorbed, or - by filtration of the blood products through filters having pore diameters of equal to, or smaller than, 10 nm.
There are disclosed blood products for parenteral administration which are essentially free of immune-modulating components, and methods for their production. The absence of immune-modulating components is expressed by the lack or absence of down-modulation of Fc receptors in the membrane of leukocytes. The expression of the receptors for the Fc fragment of immunoglobulin G in the membrane of monocytes, after interaction with a blood product preparation, is to be reduced not at all or by 30 % at most. The blood products are obtained - by molecular sieving of the blood products by gel permeation chromatography (gel filtration), or - by treatment of the blood products with a molecular sieve-like carrier material on which immune-modulating components are selectively adsorbed, or - by filtration of the blood products through filters having pore diameters of equal to, or smaller than, 10 nm.
Description
~3~6~ ~3 The invention relates to blood products derived from blood plasma and destined for parenteral administration~ a method of producing such products, and their use.
Known methods for the production of human blood products include, in addition to fractionation measures for obtaining the respective product in the pure state, methods of removing contaminating substances, which may induce side effects. Examples thereof are methods of removing vasoactive substances (EP 0 120 835, EP 0 122 909) as well as of inactivating viruses possibly present in such preparations (EP 0 159 311, U.S. pat. 4,297,344~.
However, these known methods have not taken into account the problems of a possible immune-modulating side effect of the products. The first indications of a possible therap~-mediated immune modulation appeared in studies carried out in hemophiliacs. These patients show an increased susceptibility to certain infections of bacterial and viral origin (cf., e.g., A.C. Beddall et al., J. CIin. Pathol. 1985 38, 1163-1165; g.
Lechner~ H~mophilie,in "Handbuch der inneren Medizin", p. 143, Vol. II/9, 1985, Springer Verlag; P.H. Levine et al., Health of the Intensively Treated Hemophiliacl With Special Reference to Abnormal Liver Chemistrles and Splenomegaly, 1977, 50, 1-9; B.A. McVerry et al., J. Clin. Pathol., 1979, 32, 377-381). Furthermore, :.
~3~
defects of the lymphocytes isolated from the peripheral blood of such patients indicate disturbances of the immune system. Examples of this are changes in the ratio of T-helper to T-suppressor cells (C.M. Kessler et al., 1983, The Lancet 1983, 991-992; P. Saidi et al., N; Engl. J. Med. 1983 308, 1291-1292), the reduction of the natural killer cell activity (M.M.
Lederman et al., N. Engl. J. Med. 1983 308, 79-82) as - well as the decrease in the antigen presenting capacity of circulating mononuclear phagocytes (J.W. Mannhalter et al., Clin. Immunol.- Immunopathol., lg86 38, 390-397).
The invention solves the problems pointed out above by providing blood products for parenteral administration that are essentially free of immune-modulating components, as evidenced by the lack or absence of a down-modulation of Fc receptors in the membrane of leukocytes.
This means that the expression of receptors for the Fc fragment of immunoglobulin G in the membrane of Fc receptor positive leukocytes, e.g., monocytes, after interaction with a blood product preparation, is not reduced or is non-significantly reduced, i.e., by not more than 30 ~. The expression of Fc receptors in the membrane of monocytes is determined by the ability of these cells to attach immunoglobulin G-coated erythrocyt:es. The results are expressed as percentage 1i 3~36943 of rosette-forming cells (~ RFC). The technique for assessment of the down-modulation of Fc receptors is known and has been described, for example, in the following publications: J.W. Mannhalter et al., Clin.
Immunol. Immunopathol. 1986 38, 390-397; A. Berken et al., J. Exp. Med. 1966 123, 119~144, and C.S. Scott, Clin. Exp. Immunol. 1979 38, 300-305.
The expression of receptors for the Fc fragment of immunoglobulin G (Fc receptors) in the membrane of monocytes is an important -prerequisite for proper functioning o the immune defense sys*em. These receptors not only facilitate phagocytosis of opsonized pathogens (Contemporary Topics in Immunobiology, ~ol.
14, Regulation of ~eucocyte Function, R. Snyderman, ed., Plenum Press New York and London 1971);
interaction of these receptors with their ligands also triggers a number of signals that are relevant to the controlled course of the immune response ((induction of the formation of oxygen radicals (R. B. Jonston et al., J. Exp. Med. 1976, 143, 1551-1555; D.N. Rush et al., Cellular Immunol. 1984 87, 252-258 and S.D. Wright, J.
Exp. Med. 1983 158, 2016-2023), activation of T-cells (J.A. Griffin et al., J. Exp. Med. 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. Immunopathol. 1986 39, 491-503)). A down-modulation of Fc receptors, thus, has important .
~69i~3 clinical consequences and might contribute to an increase in the susceptibility to infections of patients treated with blood products.
The blood products according to the invention, which are essentially free of immune-modulating components, are obtained - by molecular sieving of the blood products, i.e., by gel permeation chromatography (gel filtration), and/or - by treatment of the blood products with a molecular sieve-like carrier material on which im~une-modulating components are selectively adsorbed, or - by filtration of the blood products through filters having pore diameters equal to or smaller than 10 nm, which filtration may be supported by the effect ~` of adsorption.
By gel permeation chromatography (gel filtration), the molecules are separated according to molecular weight. The gel consists of microscopically small particles with a defined pore size. Molecules that are larger than these pores cannot penetrate into the gel ¦ and are eluted first. Smaller molecules, however, are able to penetrate into the gel pores and thus migrate more slowly. In this way separation of individual proteins in a protein mixture can be achieved.
A preferred embodiment for the selective adsorption of immune-modulating components consists in ~3~6~'~3 that the blood products are treated with an adsorption material consisting of an inert carrier material, such as Sepharose, to which immobilized ligands, in particular proteins of bacterial origin, such as protein A of Staphylococci, protein G and protein M of Streptococci, are bound.
Another method of this kind consists in that the blood products are treated with an adsorptive material consisting of an inert carrier material, such as Sepharose, to which antibodies obtained by immunization with immune-modulating substances are bound.
With the filtration method, which is particularly suited to the production of gammaglobulin preparations, particles are separated according to their sizes. With the method used here, special filter membranes having very small pore sizes (10 nm or smaller) are used.
Particles that are larger than these pores are retained by the membrane in the course of the filtration procedure. In this manner, large ~high molecular) particlès can be separated from a protein mixture.
The blood products according to the invention are advantageously suited for use in the treatment of inherited and acquired coagulation disorders, in the treatment of primary and secondary immunodeficiencies, as well as in the treatment of autoimmune~ immune-complex and infectious diseases.
In the following examples, the properties of the 13Vti9-~3 blood products according to the invention, their production, and the assessment of the possible presence of Fc receptor down-modulation as compared to blood product-free control media are explained in more detail.
Example 1:
A Factor VIII preparation is produced according to the method described in EP 0 127 603. This preparation was subjected to affinity chromatography with immobilized protein A according to the batch method as follows. A lyophilized Factor VIII preparation containing 500 IU of Factor VIII was dissolved in 20 ml distilled water containing 10 IU of preservative-free heparin (Immuno AG, Vienna) and dialyzed against 2 liters of citrate buffer ~0.024 M sodium citrate, 0.120 M NaCl, adjusted to pH 7.2 with HCl, supplemented with IU/ml of preservative-free heparin, hereinafter referred to as "citrate pH 7.2") over night at room temperature. 8 ml of the dialyzed Factor VIII
preparation (corresponding to approximately 200 IU
Factor VIII) were mixed 2 ml of a protein A-Sepharose suspension (50 ~ suspension (v/v) in citrate pH 7.2;
; the protein-A Sepharose having been pre-equilibrated in ci~rate pH 7.2). Then this mixture was incubated, with occasional shaking, for 4 hours at room temperature.
At the end of the incubation period, the protein A-Sepharose was centrifuged (5 min, 700 x g, room 'T;~ rK
13~69 ~3 temperature), the supernatant was removed, and thP gel was washed three times with 10 ml citrate pH 7.2. The individual centrifugation supernatants were combined, sterile filtered, adjusted to 2 IU Factor VIII/ml and stored at 4 C until assaying.
The Factor VIII preparation produced in the manner described and treated in accordance with the invention was tested for its immune-modulating activity as ; described helow.
Mononuclear cells from peripheral blood were isolated by density gradient centrifugation (J.W.
Mannhalter et al., Clin. Immunol. Immunopathol. 1986 38, 390-397) in the follo~ing manner. 8 ml of a density gradient (Lymphoprep, Nyegaard & Co., Oslo, Norwav) were overlayered with 20 to 2S ml heparinized blood and centrLfuged for 35 min at 400 x g at room temperature.
The~mononuclear cells that had collected at the interphase of plasma and gradient were aspirated, washed three times with physiological saline, and suspended in RPMI-1640-medium containing 15 ~ pooled, thermo-inactivated (30 min, 56 C~ AB-serum as well as penicillin l100 IU/ml), streptomycin (100 ~g/ml) and L-glutamin (2 mM) (RPMI suppl.).
Monocytes were isolated from the mononuclear cell population by adherence. Unlike other mononuclear blood cells, monocytes characteristically adhere to glass or plastic surfaces. To isolate the monocytes, mononuclear ~3~9'~ -cells are adjusted to a concentration of 1 x 107 cells per ml RP~I suppl. 2 ml of this suspension are then pipetted into plastlc culture plates (Macroplate TC, Greiner & Sohne, Kremsmunster, Austria) and incubated at 37 C in a CO2 incubator (5 % CO2, over 95 %
humidity). At the end of this incubation period the non-adherent cells are aspirated and the adherent cell monolayer (monocytes) is washed three times with physiological saline. Then RPMI suppl. is added and the cells are stored in the CO2 incubator until further use.
The expression of Fc receptors in the membrane of the monocytes was detected by the adherence of IgG-coated bovine ~rythrocytes (A. Berken et al., J. Exp.
Med. 1966 123, 119-144). Bovine blood was drawn into Alsever solution and washed several times in phosphate-buffered saline, pH 7.2 to 7.4 (PBS). Then the erythrocyte concentration was adjusted to 2 % in PBS. Co~ting of the erythrocytes with immunoglobulin G was effected by incubation with a sub-agglutinating dose of an anti-bovine erythrocyte antibody (IgG fraction: 0.167 mg/ml PBS, incubation time 1 hour, 37C) (C.S. Scott, Clin.
Exp. Immunol. 1979 38, 300-305). At the end of the incubation period, free IgG was removed by washing three times in PBS, and the erythrocytes coated with the antibody were adjusted to a concentration of 0.5 ~
in PBS containing 0.2 % bovine serum albumin (PBS-BSA).
_ g _ ~3(3~;i9 13 This erythrocyte suspension could then be used to test for the presence of Fc receptors in the monocyte membrane.
To this end, the monocytes isolated by adherence to plastic tissue culture plates were first carefully scraped off the tissue culture plate by means of a rubber policeman and were then adjusted to a concentration of 2 - 3 x 10 cells/ml PBS-BSA. 100 ~1 of this monocyte suspension were mixed with 100 ~1 of the above described erythroc~te suspension and centrifuged at 120 x g at 4C for 10 min. After a one-hour incubation at 0C, the pelletized cells were carefully resuspended and the percentage of monocytes that had erythrocytes attached to them was determined by phase contrast microscopy. At least 200 monocytes were examined for their ability to form rosettes with IgG-coated erythrocytes. The results are expressed as percentage of rosette forming cells (% RFC) or as attachment index (AI). The term rosette designates a monocyte that has three or more erythrocytes attached to it. The attachment index is the average number of erythrocytes bound per monocyte.
Assessment of the immune-modulating activity of the Factor VIII preparation ~as carried out in the following mannPr. Immediately after adherence of the monocytes to the plastic tissue culture plates, the cells were incubated in the presence of Factor VIII (2 IU/ml) for one hour at 37C in the C02 incubator.
~3~69~3 Following this incubation, the adherent monocytes were washed four times with physiological saline, RPMI
suppl. was added, and the cells were incubated at 37C
in the CO2 incubator for 16 hours. This incubation period is necessary to enable the contaminants present in the blood products to exert their immune-modulating effect. Following this incubation, the expression of the Fc receptors in the monocyte membrane was determined according to the method described above.
As a control, monocytes were incubated in the presence of RPMI-1640 but without Factor VIII for one hour immediately after adherence and were then further treated in the manner described above.
The results with respect tQ Fc receptor expression are indicated as percentage of rosette-forming cells (~
RFC) and as attachment index ~AI).
13~36~3 ~3 Incubation of Factor VIII ~ RFC( ) AI(b) monocytes in the activity presence of ____________________________________________ ___ RPMI medium control~ - 81+2 4.42+0.05 , Factor VIII
lO (starting product) 2 IU/ml 20+3 0.79+0.06 Factor VIII
(treated acc. to the invention with 15 protein A-sepharose) 2 IU/ml 75+4 3.78+0.15 _______________ __,______ ______________ _____________ _____ I
(a) % monocytes having three or more IgG-coated erythro-attached.
(b~ Average number of erythrocytes attached per monocyte.
The AI was calculated using all the monocytes counted (those with no erythrocytes attached as well as those with one or more erythrocytes attached).
. ~
As can be seen, the initial Factor VIII product induces a down-modulation of Fc receptor expression from 81 % RFC to 20 % RFC. In contrast, the percentage ~L3~9~3 of rosette-forming cells is 75 % after intcraction of the monocytes with the Factor VIII preparation treated according to the invention. The differenc~ between 81 %
RFC in the controls and 75 % RFC after interaction with the Factor VIII preparation treated according to the invention is not significant (Student's t-test for paired samples~. This means that the Factor VIII product treated according to the invention is free from immune-modulating components, since, after interaction of the monocytes with.the product treated according to the invention, the RFC value of the treated.product does not differ from the control value (~ RFC for monocytes not treated with Factor VIII).
Example 2:
500 IU of a lyophilized ~actor IX preparation produced according to the method described by H. Suomela et al. in Vox Sang. 1977 33, 37-50 were dissolved in 20 ml of c~s~illed water and were dialyzed over night against 2 liters of a citrate huffer (citrate pH 7.2, the exact composition of this buffer is described in Example 1) at 4C. 8 ml of this preparation (corresponding to about 200 IU Factor IX) were removed and subjected to affinity chromatography on protein A-Sepharose by the column m~thod. In this procedure, the 8 ml of Factor IX
preparation were passed over a protein A-Sepharose column (c:olumn C 10/10 from Pharmacia, 1 ml gel volume) previous~y equilibrated with 50 ml citrate pH 7.2. The l~Q69 ~3 flow rate was 0.1 ml per min. The Factor IX eluting off the column was adjusted to 2 IU/ml, sterile filtered and stored at 4C until bei.ng assayed. Determination of the immune-modulating ac:tivi~y of the initial preparation as well as of the Factor IX preparation treated according to the invention in the manner described ~as carried out as described in Example 1.
The results of these assays are summarized in the following mable.
_________________________________________________________ _ Incubation of Factor IX % RFC(a) AI(b) monocytes in the activity presence of __ _______________ RPMI medium (control) - 75+1 4.09~0.04 Factor IX
(starting product~ 2 IU/ml 42+3 2.31+0.07 Factor IX
(treated acc. to the invention) 2 IU/ml 70+3 4.00+0.08 _________._____ ______________ _____________________________ (a) Percentage of rosette forming cells, i.e., %
monocytes having three or more IgG-coated erythrocytes attached.
~3~ 3 (b) Attachment index, i.e., average number of erythrocytes attached per monocyte.
From the results of this Table, it is apparent that an interaction of Factor IX with monocytes led to a decrease in Fc receptor expression from 75 % RFC
~controls) to 4. ~ RFC (after interaction with Factor IX). Yet after interaction of the monocytes with the Factor IX product treated according to the invention, the Fc receptor expression was 70 % RFC. The difference between 75 ~ RFC in the controls and 70 ~ RFC after interaction with the Factor IX treated according to the invention is not significant (Student's t-test for paired samples). This means that the Factor IX products treated according to the invention are free of immune-modulatlng properties.
Example 3:
A lyophilized FEIBA preparation (500 IU, Immuno AG) was dissolved in 20 ml distilled water and dialyzed against citrate pH 7.2 in the manner described in Examples 1 and 2 for Factor VIII and for Factor IX~
respectively. Removal of the immune-modulating substances was effected by adsorption chromatography with protein A-Sepharose according to the column techni~ue. The exact procedure was that described in Example 2 with respect to Factor IX. Assaying of the immune-modulating properties of the initial product as well as of the ~3Q6~3 FEIBA preparation treated according to the invention was carried out as described in Example 1. The results of these assays, which are summarized in the following Table, indicate that the treatment with immobilized protein A according to the invention eliminates the immune~modulating effects from FEIBA.
__________________________________________________________ Incubation of FEIB- % RFC( ) AI(b) monocytes in the activity presence of ______________________ ___________________________________ RPMI medium ~control) - 75+1 4.09~0.04 FEIBA
(starting product) 2 IU 34+4 1.~9~0.15 .
FEIBA
(treated acc. to the invention with immobilized protein A 2 IU 73+1 3.94+0.05 ----____ (a~ Percentage of rosette forming cells, i.e., monocytes having three or more IgG-coated erythrocytes attached.
(b) Attac:hment index, i.e., average number of erythrocytes attac:hed per monocyte.
: . ~
13Q~;9'~3 It can be noted that, with all the coagulation factor preparations investigated (Factor VIII, Factor IX, FEIBA), the immune-modulating properties are eliminated by treatment with immobilized protein A, both according to the batch method and according to the column technique.
Example 4: ¦
An immunoglobulin G preparation suited for intramuscular application (i.m. IgG) was produced by ethanol fractionation (H.F. Deutsch et al., J. Biol.
Chem. 1946 164, 109 cont.). This preparation was subjected to gel permeation chromatography in the following manner. A chromatography column (K 50/60, Pharmacia) was packed with 750 ml chromatography gel (Sephacryl S 300, Pharmacia~ and equilibrated wi~h a phosphate buffer (20 mM KH2PO4, 0.15 M NaCl, 0.02 %
NaN3, pH 8.0). Then 300 mg of i.m. IgG (in lQ ml phosphate buffer) were applied to the column and eluted at a flow rate of 1 ml/min. The eluates were collected in 11 ml fractions and the protein content was determined by means of a UV detector at 280 nm. Elution curves as illustrated in the figure resulted. The fractions - marked with ~-~ were combined and ~ested for their immune-modulating activity as described in Example 1.
The results of these assays are presented in the following Table, from which it can be seen that this treatment effectively rendered the immunoglobulin G
7-rAGl~ ,?2Ar/~ --17--13~i~i9'13 preparation free frcmimmune-modulating components. The RFC value obtained after interaction of the monocytes with the i.m. IgG treated according to the invention corresponds essentially to the control value.
_____ ____________________.______________________________ Incubation of i.m. IgG % RFC(a) AI(b) monocytes in themgJml presence of __ --__ __________________ RPMI medium (control) - 84+3 7.11+0.22 - i.m. IgG
(starting product) 10.8 20+2 1.97+0.07 i.m. IgG (after treatment acc. to the invention by gel permeatlon chrom.) 11.3 64+2 5.94+0.04 - -__-_____________________________ (a) Percentage of rosette forming cells, i.e., % of monocytes having three or more IgG-coated erythrocytes attached.
(b) Attachment index, i.e., average number of erythrocytes bound per monocyte.
130~9'~3 Example 5:
An immunoglobulin G preparation (i.m. IgG) produced as described in Example 4 was adjusted to a concentration of 50 mg/ml in phosphate buffered saline (PBS). After an appropriate pre-clarification over suitable filters, the preparation was passed through a special filter having a very small pore si~e (Sartorius SM 11318, pore size 10 nm). This was done by inserting the filter membrane ir~to a suitable filter holder and passing, for example, 20 ml of the i.m. IgG solution through the filter at a pressure of 1 to 1.5 bar. The filtrate was then tested for possible immune-modulating properties as described in Example 1. The results presented in the following Table show that this treatment almost completely eliminated the immune-modulating properties of this preparation. While treatment of the monocyte~ ~, with the initial i.m. IgG product causes down-modulation of the Fc receptor expression from 79 % to 21 %, the Fc ,~
receptor expression of the monocytes, after interaction with the product treated according to the invention, was 67 %. The i.m. IgG fraction treated according to the invention thus is essentially free of immune-modulating properties.
~r~de ~Ar~
13C~69'13 Incubation of monocytes in the i.m. IgG ~ RFC( ) presence of mg1ml S ______________ RPMI medium (control) - 79+3 i.m. IgG
(starting material) lO.0 21+3 i.m. IgG (after treat-ment acc. to the invention by filtration over special filters with a lS pore size of lO-nm lO.0 67~2 --________ ~a? Percentage of rosette forming cells, i.e., monocytes with three or more IgG-coated erythrocytes attached.
Example 6 A Factor VIII preparation was produced accordins to the method described in EP-A-0 127 603. This preparation was subjected to affinity chromatography with immobilized antibodies directed against the immune-modulating component.
In order to produce the antibodies required for affinity chromatography, the immune-modulating component ~3~69~3 was first isolated from a Factor VIII preparation produced as described above. This was done by a combination of affinity chromatography and column chromatography. For this purpose, 2500 IU of Factor VIII were dissolved in 100 ml distilled water and were dialyzed against citrate pE[ 7.2 for 24 hours. This product was then applied to a column filled with immobilized protein A (protein A-Sepharose, 12 ml gel volume, column C 10/20, Pharmacia, Sweden) and circulated over night at room temperature and at a flow rate of 0.5 ml/min. Subsequently, the column was washed with 100 ml citrate pH 7.2 (flow rate 0.5 ml/min). The material bound to protein A-Sepharose was then eluted with a citrate buffer (50 mM trisodium citrate, 50 mM
citric acid, pH 3.0; hereinafter referred to as "citra~e pH 3.0") and was neutralized with 1 M sodium phosphate buffer, pH 8.5, immediately upon elution. After dialysis ; against PBS, this material was subjected to molecular sieve chromatography in the following manner. The dialyzed protein A eluate was applied to a Superose 6 Prep Grade Column (HR 16/50) and was chromatographed at a flow rate of 0.5 ml/min. The fractions having a molecular weight of more than 400 kD contained the immune-modulating component, as was determined by the rosette test already described. These fractions were pooled and used for the production of antibodies in test animals.
Tr~d~-mo.~ 21 -13~169 ~3 150 ~g of the immune-modulating component (in 200 ~l PBS) were mixed with 200 ~l complete Freund's adjuvant and a rabbit was subcutaneously immunized at two spots. On the 21st and 28th days after the first immunization, a booster immunization was carried out in the same way. From the 35th day onward, about 30 ml blood were drawn at weekly intervals over a period of six weeks and serum was produced therefrom. Subsequently, the serum samples of the various blood sampling dates were combined and the immunoglobulin G fraction was isolated therefrom.
To isolate t IgG fraction, the serum was first mixed with ammonium sulfate till reaching a saturation of 35 %, the stirred at room temperature for 25 minutes and subsequently centrifuged at 800 x g for ~5 minutes.
The resulting precipitate was resuspended in 33 %
saturated ammonium sulfate, stirred for 15 minutes and re-centrifuged. The precipitate thus obtained was dissolved in PBS containing 0.1 % sodium azide and was subjected to molecular sieve chromatography by means of a Superose 6 Prep Grade Column (HR 16/50, flow rate 0.5 ml/min). The fractions in the molecular weight range of 150 kD (i.e., the fractions containing immunoglobulin G) were collected and dialyzed against 0.1 M sodium hydrogen carbonate, 0.5 M sodium chloride, pH 8.5 (=
coupling bu f f er).
The antibodies obtained by this procedure were l3~69 ~a then immobilized by coupling to cyanogen-bromide(CNBr)-activated Sepharose 4B according to the instructions of the manufacturer (Pharmacia, Sweden). 4 g of CNBr-activated Sepharose 4B were first swollen in 1 mM
hydrochloric acid; this was followed by washing with 500 ml of 1 mM hydrochloric acid on a Buchner funnel.
10 ml of this gel were mixed with 40 ml of the antibody present in the coupling buffer (correspondin~ to 90 mg protein) and incubated at room temperature for 2 hours, with oeeasional shaking. The gel material was then eentrifuged (800 x g, 15 min), washed by resuspension in the eoupling buffer and subse~uent centrifugation, and finally suspended in 50 mlof blocking buffer ~i.e.
coupling buffer + 1 M ethanolamine). After incubation for 2 hours at room temperature, the gel material was washed twiee, first in 100 ml 0.1 M Tris, 0.5 ~. sodium chloride, pH 8.0, and then in 100 ml 0.1 M acetic aeid, 0.5 M sodium chloride, p~ 4Ø The pH 8 - pH 4 washing steps were repeated three times. (Washing of the gel was performed by resuspension and centrifugation~.
Finally, the gel material was suspended in 20 ml PBS + 10 IU heparin/ml. An analysis proved that 9 mg of Lhe antibody directed against the immune-modulating component was bound per ml gel.
4.5 ml of this gel were packed into a chromatography column (C 10/10, Pharmacia, Sweden~ and rinsed eonseeutively with 30 ml 3 % bovine serum albumin (in 130~9'13 PBS), with 50 ml 50 mM trisodium citrate, 50 mM citric acid (pH 3.0) and finally with 200 ml PBS (containing 10 IU/ml heparin) (flow rate 0.5 ml/min). This gel material was then used to separate the immune-modulating component.
This separation can be performed by means of - either the batch method or the column method. In the Example, separation of the immune-modulatins component from a Factor VIII preparation by means of the column method-is described. According to the same principles, it is also possible to eliminate the immune-modulating component from Factor IX and FEIBA preparations.
In the Example, 500 IU of a Factor VIII preparation produced as described above were dissolved in 20 ml of distilled water and dialyzed twice for 24 hours against one liter PBS containing 10 IU heparin per ml. Then the concentration of the Factor VIII preparation was adjusted to 10 IU Factor VIII per ml with the same buffer. 100 IU of this material were then applied to the separation column described above (Sepharose, to which antibodies against the immune-modulating component had been coupled) and circulated over the column for 16 hours at room temperature at a flow rate of 0.5 ml/min.
The material not bound to the column was then adjusted to 2 IU Factor VIII/ml and was tested for its immune-modulating activity by means of the rosette test as described in Example 1. The results of these investigations - ~4 -13(~69 13 are summarized in the following Table.
________________.__________.________________ _____________ Incubation of Factor VIII % RFC( ) AI~ ) monocytes in the activity presence of __________________________________________________ ______ I
RPMI medium (control) - 81+3 6.06+1.76 Factor VIII
(starting product) 2 IU/ml 31+5 1.36+0.13 I
Factor VII
(treated acc. to 15 the invention) 2 IU/ml 80+6 4.79+1.45 ____________________.____________,________________________ (a) ~ rosette forming cells, i.e., % of monocytes having three or more IgG-coat~d ervthrocytes attached.
(b) Attachment index, i.e., average number of erythrocytes attached per monocyte.
The results illustrated in the above Tahle clearly reveal an immune-modulating activity of the initial Factor VIII product. An interaction of the monocytes with the initial product led to a decrease in the Fc receptor expression from 81+3 % RFC (controls) to ` 13Q69~3 31+5 % RFC (after interaction with Factor VIII). In ; contrast, an interaction o:E the monocytes with the Factor VIII product treated according to the invention does not cause a reduction of Fc receptors in the monocyte membrane (81+3 % RFC after interaction with control medium as opposed to 80+6 % RFC after interaction with the Factor VIII product treated according to the invention). The difference between 81+3 % RFC and 80+6 % RFC is statistically not significant (Student's t-test for paired samples), and this means that the Factor VIII product treated according *o the invention is free of immune-modulating properties.
Known methods for the production of human blood products include, in addition to fractionation measures for obtaining the respective product in the pure state, methods of removing contaminating substances, which may induce side effects. Examples thereof are methods of removing vasoactive substances (EP 0 120 835, EP 0 122 909) as well as of inactivating viruses possibly present in such preparations (EP 0 159 311, U.S. pat. 4,297,344~.
However, these known methods have not taken into account the problems of a possible immune-modulating side effect of the products. The first indications of a possible therap~-mediated immune modulation appeared in studies carried out in hemophiliacs. These patients show an increased susceptibility to certain infections of bacterial and viral origin (cf., e.g., A.C. Beddall et al., J. CIin. Pathol. 1985 38, 1163-1165; g.
Lechner~ H~mophilie,in "Handbuch der inneren Medizin", p. 143, Vol. II/9, 1985, Springer Verlag; P.H. Levine et al., Health of the Intensively Treated Hemophiliacl With Special Reference to Abnormal Liver Chemistrles and Splenomegaly, 1977, 50, 1-9; B.A. McVerry et al., J. Clin. Pathol., 1979, 32, 377-381). Furthermore, :.
~3~
defects of the lymphocytes isolated from the peripheral blood of such patients indicate disturbances of the immune system. Examples of this are changes in the ratio of T-helper to T-suppressor cells (C.M. Kessler et al., 1983, The Lancet 1983, 991-992; P. Saidi et al., N; Engl. J. Med. 1983 308, 1291-1292), the reduction of the natural killer cell activity (M.M.
Lederman et al., N. Engl. J. Med. 1983 308, 79-82) as - well as the decrease in the antigen presenting capacity of circulating mononuclear phagocytes (J.W. Mannhalter et al., Clin. Immunol.- Immunopathol., lg86 38, 390-397).
The invention solves the problems pointed out above by providing blood products for parenteral administration that are essentially free of immune-modulating components, as evidenced by the lack or absence of a down-modulation of Fc receptors in the membrane of leukocytes.
This means that the expression of receptors for the Fc fragment of immunoglobulin G in the membrane of Fc receptor positive leukocytes, e.g., monocytes, after interaction with a blood product preparation, is not reduced or is non-significantly reduced, i.e., by not more than 30 ~. The expression of Fc receptors in the membrane of monocytes is determined by the ability of these cells to attach immunoglobulin G-coated erythrocyt:es. The results are expressed as percentage 1i 3~36943 of rosette-forming cells (~ RFC). The technique for assessment of the down-modulation of Fc receptors is known and has been described, for example, in the following publications: J.W. Mannhalter et al., Clin.
Immunol. Immunopathol. 1986 38, 390-397; A. Berken et al., J. Exp. Med. 1966 123, 119~144, and C.S. Scott, Clin. Exp. Immunol. 1979 38, 300-305.
The expression of receptors for the Fc fragment of immunoglobulin G (Fc receptors) in the membrane of monocytes is an important -prerequisite for proper functioning o the immune defense sys*em. These receptors not only facilitate phagocytosis of opsonized pathogens (Contemporary Topics in Immunobiology, ~ol.
14, Regulation of ~eucocyte Function, R. Snyderman, ed., Plenum Press New York and London 1971);
interaction of these receptors with their ligands also triggers a number of signals that are relevant to the controlled course of the immune response ((induction of the formation of oxygen radicals (R. B. Jonston et al., J. Exp. Med. 1976, 143, 1551-1555; D.N. Rush et al., Cellular Immunol. 1984 87, 252-258 and S.D. Wright, J.
Exp. Med. 1983 158, 2016-2023), activation of T-cells (J.A. Griffin et al., J. Exp. Med. 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. Immunopathol. 1986 39, 491-503)). A down-modulation of Fc receptors, thus, has important .
~69i~3 clinical consequences and might contribute to an increase in the susceptibility to infections of patients treated with blood products.
The blood products according to the invention, which are essentially free of immune-modulating components, are obtained - by molecular sieving of the blood products, i.e., by gel permeation chromatography (gel filtration), and/or - by treatment of the blood products with a molecular sieve-like carrier material on which im~une-modulating components are selectively adsorbed, or - by filtration of the blood products through filters having pore diameters equal to or smaller than 10 nm, which filtration may be supported by the effect ~` of adsorption.
By gel permeation chromatography (gel filtration), the molecules are separated according to molecular weight. The gel consists of microscopically small particles with a defined pore size. Molecules that are larger than these pores cannot penetrate into the gel ¦ and are eluted first. Smaller molecules, however, are able to penetrate into the gel pores and thus migrate more slowly. In this way separation of individual proteins in a protein mixture can be achieved.
A preferred embodiment for the selective adsorption of immune-modulating components consists in ~3~6~'~3 that the blood products are treated with an adsorption material consisting of an inert carrier material, such as Sepharose, to which immobilized ligands, in particular proteins of bacterial origin, such as protein A of Staphylococci, protein G and protein M of Streptococci, are bound.
Another method of this kind consists in that the blood products are treated with an adsorptive material consisting of an inert carrier material, such as Sepharose, to which antibodies obtained by immunization with immune-modulating substances are bound.
With the filtration method, which is particularly suited to the production of gammaglobulin preparations, particles are separated according to their sizes. With the method used here, special filter membranes having very small pore sizes (10 nm or smaller) are used.
Particles that are larger than these pores are retained by the membrane in the course of the filtration procedure. In this manner, large ~high molecular) particlès can be separated from a protein mixture.
The blood products according to the invention are advantageously suited for use in the treatment of inherited and acquired coagulation disorders, in the treatment of primary and secondary immunodeficiencies, as well as in the treatment of autoimmune~ immune-complex and infectious diseases.
In the following examples, the properties of the 13Vti9-~3 blood products according to the invention, their production, and the assessment of the possible presence of Fc receptor down-modulation as compared to blood product-free control media are explained in more detail.
Example 1:
A Factor VIII preparation is produced according to the method described in EP 0 127 603. This preparation was subjected to affinity chromatography with immobilized protein A according to the batch method as follows. A lyophilized Factor VIII preparation containing 500 IU of Factor VIII was dissolved in 20 ml distilled water containing 10 IU of preservative-free heparin (Immuno AG, Vienna) and dialyzed against 2 liters of citrate buffer ~0.024 M sodium citrate, 0.120 M NaCl, adjusted to pH 7.2 with HCl, supplemented with IU/ml of preservative-free heparin, hereinafter referred to as "citrate pH 7.2") over night at room temperature. 8 ml of the dialyzed Factor VIII
preparation (corresponding to approximately 200 IU
Factor VIII) were mixed 2 ml of a protein A-Sepharose suspension (50 ~ suspension (v/v) in citrate pH 7.2;
; the protein-A Sepharose having been pre-equilibrated in ci~rate pH 7.2). Then this mixture was incubated, with occasional shaking, for 4 hours at room temperature.
At the end of the incubation period, the protein A-Sepharose was centrifuged (5 min, 700 x g, room 'T;~ rK
13~69 ~3 temperature), the supernatant was removed, and thP gel was washed three times with 10 ml citrate pH 7.2. The individual centrifugation supernatants were combined, sterile filtered, adjusted to 2 IU Factor VIII/ml and stored at 4 C until assaying.
The Factor VIII preparation produced in the manner described and treated in accordance with the invention was tested for its immune-modulating activity as ; described helow.
Mononuclear cells from peripheral blood were isolated by density gradient centrifugation (J.W.
Mannhalter et al., Clin. Immunol. Immunopathol. 1986 38, 390-397) in the follo~ing manner. 8 ml of a density gradient (Lymphoprep, Nyegaard & Co., Oslo, Norwav) were overlayered with 20 to 2S ml heparinized blood and centrLfuged for 35 min at 400 x g at room temperature.
The~mononuclear cells that had collected at the interphase of plasma and gradient were aspirated, washed three times with physiological saline, and suspended in RPMI-1640-medium containing 15 ~ pooled, thermo-inactivated (30 min, 56 C~ AB-serum as well as penicillin l100 IU/ml), streptomycin (100 ~g/ml) and L-glutamin (2 mM) (RPMI suppl.).
Monocytes were isolated from the mononuclear cell population by adherence. Unlike other mononuclear blood cells, monocytes characteristically adhere to glass or plastic surfaces. To isolate the monocytes, mononuclear ~3~9'~ -cells are adjusted to a concentration of 1 x 107 cells per ml RP~I suppl. 2 ml of this suspension are then pipetted into plastlc culture plates (Macroplate TC, Greiner & Sohne, Kremsmunster, Austria) and incubated at 37 C in a CO2 incubator (5 % CO2, over 95 %
humidity). At the end of this incubation period the non-adherent cells are aspirated and the adherent cell monolayer (monocytes) is washed three times with physiological saline. Then RPMI suppl. is added and the cells are stored in the CO2 incubator until further use.
The expression of Fc receptors in the membrane of the monocytes was detected by the adherence of IgG-coated bovine ~rythrocytes (A. Berken et al., J. Exp.
Med. 1966 123, 119-144). Bovine blood was drawn into Alsever solution and washed several times in phosphate-buffered saline, pH 7.2 to 7.4 (PBS). Then the erythrocyte concentration was adjusted to 2 % in PBS. Co~ting of the erythrocytes with immunoglobulin G was effected by incubation with a sub-agglutinating dose of an anti-bovine erythrocyte antibody (IgG fraction: 0.167 mg/ml PBS, incubation time 1 hour, 37C) (C.S. Scott, Clin.
Exp. Immunol. 1979 38, 300-305). At the end of the incubation period, free IgG was removed by washing three times in PBS, and the erythrocytes coated with the antibody were adjusted to a concentration of 0.5 ~
in PBS containing 0.2 % bovine serum albumin (PBS-BSA).
_ g _ ~3(3~;i9 13 This erythrocyte suspension could then be used to test for the presence of Fc receptors in the monocyte membrane.
To this end, the monocytes isolated by adherence to plastic tissue culture plates were first carefully scraped off the tissue culture plate by means of a rubber policeman and were then adjusted to a concentration of 2 - 3 x 10 cells/ml PBS-BSA. 100 ~1 of this monocyte suspension were mixed with 100 ~1 of the above described erythroc~te suspension and centrifuged at 120 x g at 4C for 10 min. After a one-hour incubation at 0C, the pelletized cells were carefully resuspended and the percentage of monocytes that had erythrocytes attached to them was determined by phase contrast microscopy. At least 200 monocytes were examined for their ability to form rosettes with IgG-coated erythrocytes. The results are expressed as percentage of rosette forming cells (% RFC) or as attachment index (AI). The term rosette designates a monocyte that has three or more erythrocytes attached to it. The attachment index is the average number of erythrocytes bound per monocyte.
Assessment of the immune-modulating activity of the Factor VIII preparation ~as carried out in the following mannPr. Immediately after adherence of the monocytes to the plastic tissue culture plates, the cells were incubated in the presence of Factor VIII (2 IU/ml) for one hour at 37C in the C02 incubator.
~3~69~3 Following this incubation, the adherent monocytes were washed four times with physiological saline, RPMI
suppl. was added, and the cells were incubated at 37C
in the CO2 incubator for 16 hours. This incubation period is necessary to enable the contaminants present in the blood products to exert their immune-modulating effect. Following this incubation, the expression of the Fc receptors in the monocyte membrane was determined according to the method described above.
As a control, monocytes were incubated in the presence of RPMI-1640 but without Factor VIII for one hour immediately after adherence and were then further treated in the manner described above.
The results with respect tQ Fc receptor expression are indicated as percentage of rosette-forming cells (~
RFC) and as attachment index ~AI).
13~36~3 ~3 Incubation of Factor VIII ~ RFC( ) AI(b) monocytes in the activity presence of ____________________________________________ ___ RPMI medium control~ - 81+2 4.42+0.05 , Factor VIII
lO (starting product) 2 IU/ml 20+3 0.79+0.06 Factor VIII
(treated acc. to the invention with 15 protein A-sepharose) 2 IU/ml 75+4 3.78+0.15 _______________ __,______ ______________ _____________ _____ I
(a) % monocytes having three or more IgG-coated erythro-attached.
(b~ Average number of erythrocytes attached per monocyte.
The AI was calculated using all the monocytes counted (those with no erythrocytes attached as well as those with one or more erythrocytes attached).
. ~
As can be seen, the initial Factor VIII product induces a down-modulation of Fc receptor expression from 81 % RFC to 20 % RFC. In contrast, the percentage ~L3~9~3 of rosette-forming cells is 75 % after intcraction of the monocytes with the Factor VIII preparation treated according to the invention. The differenc~ between 81 %
RFC in the controls and 75 % RFC after interaction with the Factor VIII preparation treated according to the invention is not significant (Student's t-test for paired samples~. This means that the Factor VIII product treated according to the invention is free from immune-modulating components, since, after interaction of the monocytes with.the product treated according to the invention, the RFC value of the treated.product does not differ from the control value (~ RFC for monocytes not treated with Factor VIII).
Example 2:
500 IU of a lyophilized ~actor IX preparation produced according to the method described by H. Suomela et al. in Vox Sang. 1977 33, 37-50 were dissolved in 20 ml of c~s~illed water and were dialyzed over night against 2 liters of a citrate huffer (citrate pH 7.2, the exact composition of this buffer is described in Example 1) at 4C. 8 ml of this preparation (corresponding to about 200 IU Factor IX) were removed and subjected to affinity chromatography on protein A-Sepharose by the column m~thod. In this procedure, the 8 ml of Factor IX
preparation were passed over a protein A-Sepharose column (c:olumn C 10/10 from Pharmacia, 1 ml gel volume) previous~y equilibrated with 50 ml citrate pH 7.2. The l~Q69 ~3 flow rate was 0.1 ml per min. The Factor IX eluting off the column was adjusted to 2 IU/ml, sterile filtered and stored at 4C until bei.ng assayed. Determination of the immune-modulating ac:tivi~y of the initial preparation as well as of the Factor IX preparation treated according to the invention in the manner described ~as carried out as described in Example 1.
The results of these assays are summarized in the following mable.
_________________________________________________________ _ Incubation of Factor IX % RFC(a) AI(b) monocytes in the activity presence of __ _______________ RPMI medium (control) - 75+1 4.09~0.04 Factor IX
(starting product~ 2 IU/ml 42+3 2.31+0.07 Factor IX
(treated acc. to the invention) 2 IU/ml 70+3 4.00+0.08 _________._____ ______________ _____________________________ (a) Percentage of rosette forming cells, i.e., %
monocytes having three or more IgG-coated erythrocytes attached.
~3~ 3 (b) Attachment index, i.e., average number of erythrocytes attached per monocyte.
From the results of this Table, it is apparent that an interaction of Factor IX with monocytes led to a decrease in Fc receptor expression from 75 % RFC
~controls) to 4. ~ RFC (after interaction with Factor IX). Yet after interaction of the monocytes with the Factor IX product treated according to the invention, the Fc receptor expression was 70 % RFC. The difference between 75 ~ RFC in the controls and 70 ~ RFC after interaction with the Factor IX treated according to the invention is not significant (Student's t-test for paired samples). This means that the Factor IX products treated according to the invention are free of immune-modulatlng properties.
Example 3:
A lyophilized FEIBA preparation (500 IU, Immuno AG) was dissolved in 20 ml distilled water and dialyzed against citrate pH 7.2 in the manner described in Examples 1 and 2 for Factor VIII and for Factor IX~
respectively. Removal of the immune-modulating substances was effected by adsorption chromatography with protein A-Sepharose according to the column techni~ue. The exact procedure was that described in Example 2 with respect to Factor IX. Assaying of the immune-modulating properties of the initial product as well as of the ~3Q6~3 FEIBA preparation treated according to the invention was carried out as described in Example 1. The results of these assays, which are summarized in the following Table, indicate that the treatment with immobilized protein A according to the invention eliminates the immune~modulating effects from FEIBA.
__________________________________________________________ Incubation of FEIB- % RFC( ) AI(b) monocytes in the activity presence of ______________________ ___________________________________ RPMI medium ~control) - 75+1 4.09~0.04 FEIBA
(starting product) 2 IU 34+4 1.~9~0.15 .
FEIBA
(treated acc. to the invention with immobilized protein A 2 IU 73+1 3.94+0.05 ----____ (a~ Percentage of rosette forming cells, i.e., monocytes having three or more IgG-coated erythrocytes attached.
(b) Attac:hment index, i.e., average number of erythrocytes attac:hed per monocyte.
: . ~
13Q~;9'~3 It can be noted that, with all the coagulation factor preparations investigated (Factor VIII, Factor IX, FEIBA), the immune-modulating properties are eliminated by treatment with immobilized protein A, both according to the batch method and according to the column technique.
Example 4: ¦
An immunoglobulin G preparation suited for intramuscular application (i.m. IgG) was produced by ethanol fractionation (H.F. Deutsch et al., J. Biol.
Chem. 1946 164, 109 cont.). This preparation was subjected to gel permeation chromatography in the following manner. A chromatography column (K 50/60, Pharmacia) was packed with 750 ml chromatography gel (Sephacryl S 300, Pharmacia~ and equilibrated wi~h a phosphate buffer (20 mM KH2PO4, 0.15 M NaCl, 0.02 %
NaN3, pH 8.0). Then 300 mg of i.m. IgG (in lQ ml phosphate buffer) were applied to the column and eluted at a flow rate of 1 ml/min. The eluates were collected in 11 ml fractions and the protein content was determined by means of a UV detector at 280 nm. Elution curves as illustrated in the figure resulted. The fractions - marked with ~-~ were combined and ~ested for their immune-modulating activity as described in Example 1.
The results of these assays are presented in the following Table, from which it can be seen that this treatment effectively rendered the immunoglobulin G
7-rAGl~ ,?2Ar/~ --17--13~i~i9'13 preparation free frcmimmune-modulating components. The RFC value obtained after interaction of the monocytes with the i.m. IgG treated according to the invention corresponds essentially to the control value.
_____ ____________________.______________________________ Incubation of i.m. IgG % RFC(a) AI(b) monocytes in themgJml presence of __ --__ __________________ RPMI medium (control) - 84+3 7.11+0.22 - i.m. IgG
(starting product) 10.8 20+2 1.97+0.07 i.m. IgG (after treatment acc. to the invention by gel permeatlon chrom.) 11.3 64+2 5.94+0.04 - -__-_____________________________ (a) Percentage of rosette forming cells, i.e., % of monocytes having three or more IgG-coated erythrocytes attached.
(b) Attachment index, i.e., average number of erythrocytes bound per monocyte.
130~9'~3 Example 5:
An immunoglobulin G preparation (i.m. IgG) produced as described in Example 4 was adjusted to a concentration of 50 mg/ml in phosphate buffered saline (PBS). After an appropriate pre-clarification over suitable filters, the preparation was passed through a special filter having a very small pore si~e (Sartorius SM 11318, pore size 10 nm). This was done by inserting the filter membrane ir~to a suitable filter holder and passing, for example, 20 ml of the i.m. IgG solution through the filter at a pressure of 1 to 1.5 bar. The filtrate was then tested for possible immune-modulating properties as described in Example 1. The results presented in the following Table show that this treatment almost completely eliminated the immune-modulating properties of this preparation. While treatment of the monocyte~ ~, with the initial i.m. IgG product causes down-modulation of the Fc receptor expression from 79 % to 21 %, the Fc ,~
receptor expression of the monocytes, after interaction with the product treated according to the invention, was 67 %. The i.m. IgG fraction treated according to the invention thus is essentially free of immune-modulating properties.
~r~de ~Ar~
13C~69'13 Incubation of monocytes in the i.m. IgG ~ RFC( ) presence of mg1ml S ______________ RPMI medium (control) - 79+3 i.m. IgG
(starting material) lO.0 21+3 i.m. IgG (after treat-ment acc. to the invention by filtration over special filters with a lS pore size of lO-nm lO.0 67~2 --________ ~a? Percentage of rosette forming cells, i.e., monocytes with three or more IgG-coated erythrocytes attached.
Example 6 A Factor VIII preparation was produced accordins to the method described in EP-A-0 127 603. This preparation was subjected to affinity chromatography with immobilized antibodies directed against the immune-modulating component.
In order to produce the antibodies required for affinity chromatography, the immune-modulating component ~3~69~3 was first isolated from a Factor VIII preparation produced as described above. This was done by a combination of affinity chromatography and column chromatography. For this purpose, 2500 IU of Factor VIII were dissolved in 100 ml distilled water and were dialyzed against citrate pE[ 7.2 for 24 hours. This product was then applied to a column filled with immobilized protein A (protein A-Sepharose, 12 ml gel volume, column C 10/20, Pharmacia, Sweden) and circulated over night at room temperature and at a flow rate of 0.5 ml/min. Subsequently, the column was washed with 100 ml citrate pH 7.2 (flow rate 0.5 ml/min). The material bound to protein A-Sepharose was then eluted with a citrate buffer (50 mM trisodium citrate, 50 mM
citric acid, pH 3.0; hereinafter referred to as "citra~e pH 3.0") and was neutralized with 1 M sodium phosphate buffer, pH 8.5, immediately upon elution. After dialysis ; against PBS, this material was subjected to molecular sieve chromatography in the following manner. The dialyzed protein A eluate was applied to a Superose 6 Prep Grade Column (HR 16/50) and was chromatographed at a flow rate of 0.5 ml/min. The fractions having a molecular weight of more than 400 kD contained the immune-modulating component, as was determined by the rosette test already described. These fractions were pooled and used for the production of antibodies in test animals.
Tr~d~-mo.~ 21 -13~169 ~3 150 ~g of the immune-modulating component (in 200 ~l PBS) were mixed with 200 ~l complete Freund's adjuvant and a rabbit was subcutaneously immunized at two spots. On the 21st and 28th days after the first immunization, a booster immunization was carried out in the same way. From the 35th day onward, about 30 ml blood were drawn at weekly intervals over a period of six weeks and serum was produced therefrom. Subsequently, the serum samples of the various blood sampling dates were combined and the immunoglobulin G fraction was isolated therefrom.
To isolate t IgG fraction, the serum was first mixed with ammonium sulfate till reaching a saturation of 35 %, the stirred at room temperature for 25 minutes and subsequently centrifuged at 800 x g for ~5 minutes.
The resulting precipitate was resuspended in 33 %
saturated ammonium sulfate, stirred for 15 minutes and re-centrifuged. The precipitate thus obtained was dissolved in PBS containing 0.1 % sodium azide and was subjected to molecular sieve chromatography by means of a Superose 6 Prep Grade Column (HR 16/50, flow rate 0.5 ml/min). The fractions in the molecular weight range of 150 kD (i.e., the fractions containing immunoglobulin G) were collected and dialyzed against 0.1 M sodium hydrogen carbonate, 0.5 M sodium chloride, pH 8.5 (=
coupling bu f f er).
The antibodies obtained by this procedure were l3~69 ~a then immobilized by coupling to cyanogen-bromide(CNBr)-activated Sepharose 4B according to the instructions of the manufacturer (Pharmacia, Sweden). 4 g of CNBr-activated Sepharose 4B were first swollen in 1 mM
hydrochloric acid; this was followed by washing with 500 ml of 1 mM hydrochloric acid on a Buchner funnel.
10 ml of this gel were mixed with 40 ml of the antibody present in the coupling buffer (correspondin~ to 90 mg protein) and incubated at room temperature for 2 hours, with oeeasional shaking. The gel material was then eentrifuged (800 x g, 15 min), washed by resuspension in the eoupling buffer and subse~uent centrifugation, and finally suspended in 50 mlof blocking buffer ~i.e.
coupling buffer + 1 M ethanolamine). After incubation for 2 hours at room temperature, the gel material was washed twiee, first in 100 ml 0.1 M Tris, 0.5 ~. sodium chloride, pH 8.0, and then in 100 ml 0.1 M acetic aeid, 0.5 M sodium chloride, p~ 4Ø The pH 8 - pH 4 washing steps were repeated three times. (Washing of the gel was performed by resuspension and centrifugation~.
Finally, the gel material was suspended in 20 ml PBS + 10 IU heparin/ml. An analysis proved that 9 mg of Lhe antibody directed against the immune-modulating component was bound per ml gel.
4.5 ml of this gel were packed into a chromatography column (C 10/10, Pharmacia, Sweden~ and rinsed eonseeutively with 30 ml 3 % bovine serum albumin (in 130~9'13 PBS), with 50 ml 50 mM trisodium citrate, 50 mM citric acid (pH 3.0) and finally with 200 ml PBS (containing 10 IU/ml heparin) (flow rate 0.5 ml/min). This gel material was then used to separate the immune-modulating component.
This separation can be performed by means of - either the batch method or the column method. In the Example, separation of the immune-modulatins component from a Factor VIII preparation by means of the column method-is described. According to the same principles, it is also possible to eliminate the immune-modulating component from Factor IX and FEIBA preparations.
In the Example, 500 IU of a Factor VIII preparation produced as described above were dissolved in 20 ml of distilled water and dialyzed twice for 24 hours against one liter PBS containing 10 IU heparin per ml. Then the concentration of the Factor VIII preparation was adjusted to 10 IU Factor VIII per ml with the same buffer. 100 IU of this material were then applied to the separation column described above (Sepharose, to which antibodies against the immune-modulating component had been coupled) and circulated over the column for 16 hours at room temperature at a flow rate of 0.5 ml/min.
The material not bound to the column was then adjusted to 2 IU Factor VIII/ml and was tested for its immune-modulating activity by means of the rosette test as described in Example 1. The results of these investigations - ~4 -13(~69 13 are summarized in the following Table.
________________.__________.________________ _____________ Incubation of Factor VIII % RFC( ) AI~ ) monocytes in the activity presence of __________________________________________________ ______ I
RPMI medium (control) - 81+3 6.06+1.76 Factor VIII
(starting product) 2 IU/ml 31+5 1.36+0.13 I
Factor VII
(treated acc. to 15 the invention) 2 IU/ml 80+6 4.79+1.45 ____________________.____________,________________________ (a) ~ rosette forming cells, i.e., % of monocytes having three or more IgG-coat~d ervthrocytes attached.
(b) Attachment index, i.e., average number of erythrocytes attached per monocyte.
The results illustrated in the above Tahle clearly reveal an immune-modulating activity of the initial Factor VIII product. An interaction of the monocytes with the initial product led to a decrease in the Fc receptor expression from 81+3 % RFC (controls) to ` 13Q69~3 31+5 % RFC (after interaction with Factor VIII). In ; contrast, an interaction o:E the monocytes with the Factor VIII product treated according to the invention does not cause a reduction of Fc receptors in the monocyte membrane (81+3 % RFC after interaction with control medium as opposed to 80+6 % RFC after interaction with the Factor VIII product treated according to the invention). The difference between 81+3 % RFC and 80+6 % RFC is statistically not significant (Student's t-test for paired samples), and this means that the Factor VIII product treated according *o the invention is free of immune-modulating properties.
Claims (16)
1. A blood product comprising blood coagulation factor or immunoglobulin preparation derived from blood plasma and destined for parenteral administration, which blood product is devoid of an immune-modulating component that down-modulates Fc receptor in a membrane of monocyte to such an extent that expression of a receptor for Fc fragment of immunoglobulin G in the membrane of the monocyte, after interacting with the blood product, is not reduced or is reduced by not more than 30%.
2. The blood product as set forth in claim 1, which is obtained by treating the blood product containing the immune-modulating component in one of the following process:
(a) molecular sieving by gel permeation chromatography (gel filtration);
(b) treatment with a molecular sieve-like carrier material capable of selectively adsorbing the immune-modulating component; and (c) filtration over a filter having a pore diameter of not more than 10nm.
(a) molecular sieving by gel permeation chromatography (gel filtration);
(b) treatment with a molecular sieve-like carrier material capable of selectively adsorbing the immune-modulating component; and (c) filtration over a filter having a pore diameter of not more than 10nm.
3. A method of producing blood products derived from blood plasma and destined for parenteral administration, which blood products are essentially free of immune-modulating components, as is shown by the lack or absence of down-modulation of Fc receptors in the membrane of leukocytes, which method comprises treating said blood products with an adsorptive material composed of an inert carrier material to which immobilized ligands are bound.
4. A method as set forth in claim 3, wherein said inert carrier material is Sepharose.
5. A method as set forth in claim 3, wherein said immobilized ligands are proteins of bacterial origin.
6. A method as set forth in claim 5, wherein said proteins of bacterial origin are selected from the group consisting of protein A of Staphylococci, protein G and protein M of Streptococci.
7. A method of producing blood products derived from blood plasma and destined for parenteral administration, which blood products are essentially free of immune-modulating components, as is shown by the lack or absence of down-modulation of Fc receptors in the membrane of leukocytes, wherein said blood products are treated with an adsorptive material composed of an inert carrier material to which antibodies obtained by immunization with immune-modulating substances are bound.
8. A method as set forth in claim 7, wherein said inert carrier material is Sepharose.
9. The use of blood products derived from blood plasma and destined for parenteral administration, which blood products are essentially free of immune-modulating components, as is shown by the lack or absence of down-modulation of Fc receptors in the membrane of leukocytes, for the production of preparations to be used for the treatment of inherited and acquired coagulation disorders, for the treatment of primary and secondary immunodeficiencies, and for the treatment of autoimmune, immune-complex and infectious diseases.
10. The blood product according to claim 1 which is a blood coagulation factor obtained by treating a blood product with an inert carrier material capable of selectively adsorbing immune-modulating components, the inert carrier material being protein A of Staphylococci, protein G or protein M of Streptococci or an antibody obtained by immunization with an immune-modulating substance.
11. The blood product of claim 10 wherein the blood coagulation factor is factor VIII.
12. The blood product of claim 10 wherein the blood coagulation factor is factor IX.
13. The blood product of claim 10 wherein the blood coagulation factor is FEIBA.
14. The blood product as set forth in claim 1 which is an immunoglobulin preparation obtained by gel permeation chromatography.
15. The blood product of claim 1 which is an immunoglobulin preparation obtained by gel permeation chromatography with Sephaeryl S-300.
16. The blood product according to claim 1 which is an immunoglobulin preparation obtained by filtration of a blood product over a filter having a pore diameter of not more than 10 nm.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0029987A AT388501B (en) | 1987-02-12 | 1987-02-12 | METHOD FOR PRODUCING PARENTERALLY AVAILABLE BLOOD PRODUCTS |
| ATA299/87 | 1987-02-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1306943C true CA1306943C (en) | 1992-09-01 |
Family
ID=3486861
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000557971A Expired - Lifetime CA1306943C (en) | 1987-02-12 | 1988-02-02 | Blood products for parenteral administration |
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 |
| EP0071635B1 (en) * | 1981-01-30 | 1986-04-30 | 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 EP EP88890028A patent/EP0278948B1/en not_active Expired - Lifetime
- 1988-02-08 DE DE8888890028T patent/DE3869990D1/en not_active Expired - Fee Related
- 1988-02-11 NO NO880613A patent/NO175668C/en not_active IP Right Cessation
- 1988-02-12 FI FI880684A patent/FI93516C/en not_active IP Right Cessation
- 1988-02-12 JP JP63031695A patent/JPS63203622A/en active Pending
- 1988-02-12 DK DK073888A patent/DK167844B1/en not_active IP Right Cessation
-
1995
- 1995-04-10 HU HU95P/P00100P patent/HU210810A9/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| DE3869990D1 (en) | 1992-05-21 |
| DK167844B1 (en) | 1993-12-27 |
| DK73888A (en) | 1988-08-13 |
| FI93516C (en) | 1995-04-25 |
| ATA29987A (en) | 1988-12-15 |
| HU210810A9 (en) | 1995-08-28 |
| FI880684A (en) | 1988-08-13 |
| AT388501B (en) | 1989-07-25 |
| NO175668C (en) | 1994-11-16 |
| EP0278948A1 (en) | 1988-08-17 |
| FI93516B (en) | 1995-01-13 |
| NO880613D0 (en) | 1988-02-11 |
| DK73888D0 (en) | 1988-02-12 |
| FI880684A0 (en) | 1988-02-12 |
| JPS63203622A (en) | 1988-08-23 |
| NO880613L (en) | 1988-08-15 |
| ES2036720T3 (en) | 1993-06-01 |
| EP0278948B1 (en) | 1992-04-15 |
| NO175668B (en) | 1994-08-08 |
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| MKLA | Lapsed |