JPH08231406A - Blood preparation - Google Patents

Abstract

PURPOSE: To obtain the subject preparation not causing an antigen-antibody reaction with erythrocytes in the body of a patient, and not causing the decrease of the erythrocytes, their lyses and the subsequent side effect, even when administered in a large amount, by removing blood type antibodies of A, B and C types. CONSTITUTION: This preparation is prepared by removing blood type antibodies of A, B and C types to <=1:1, respectively, expressed by antibody titers measured by a direct Coombs' method. The preparation is preferably obtained by a method comprising dissolving a blood preparation contaminated with the blood type antibodies in a solvent in a protein concentration of 1-10w/v%, and subsequently bringing the solution into contact with an immobilized human blood type substance produced by binding a human blood type substance comprising at least two kinds of blood type antibodies selected from the A, B and C types under conditions comprising a pH of 6-8 and an ion strength of 0.05-0.1M to a water-insoluble carrier. The human blood type substance is preferably heated at a temperature sufficient for the inactivation of viruses and not inactivating the blood type substances and the blood preparation.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a blood product in which all A, B and D blood group antibodies have been substantially removed.

[0002]

BACKGROUND ART Blood group antibodies are mixed in blood products such as immunoglobulin products and blood coagulation factor products. Therefore, when a large amount of blood product is administered, red blood cells and the administered blood group antibody cause an antigen-antibody reaction, which causes side effects such as red blood cell decrease. Therefore, it is desired to exclude blood group antibodies from these preparations.

Blood types are roughly divided into four groups, A, B, O and AB. In addition to these blood types, Rho (D) factor is known as an important blood type, and antibodies (blood group antibodies) to these blood group substances are present in plasma (or serum).

By the way, immunoglobulin (IgG) may be purified from plasma by a conventional alcohol fractionation method, ammonium sulfate fractionation method, ion exchanger treatment method, polyethylene glycol fractionation method or a combination thereof. Since blood group antibodies and other antibodies have similar physicochemical properties, it was not possible to obtain an immunoglobulin preparation containing no blood group antibody.

Further, since blood coagulation factor 9 and factor 8 preparations contain immunoglobulins together with these coagulation factors, blood group antibodies also exist together like the above-mentioned immunoglobulin preparations.

[0006] These immunoglobulin preparations and blood coagulation factor preparations are produced by mixing plasma collected from a large number of humans and separating them from each other.
B-type and D-type antibodies are contaminated. When a large amount of such a preparation is administered to a human, human red blood cells and the administered blood group antibody cause an antigen-antibody reaction, which causes side effects such as lysis of red blood cells and subsequent renal damage. Therefore, it is desirable to remove blood group antibodies from these formulations.

As a method for removing blood group antibodies, it is possible to carry out an antigen-antibody reaction using blood group antigens.
For example, it is a method in which human A type red blood cells and human B type red blood cells are added to a sample solution containing a blood group antibody, an antigen-antibody reaction is performed for a certain period of time, and then the red blood cells adsorbing the blood group antibody are removed by centrifugation. This method is suitable for treating a small amount, but is not suitable for industrial scale because a large amount of red blood cells are required and the used red blood cells cannot be reused.

Even if a large amount of red blood cells are available, infectious viruses such as hepatitis B virus and non-A non-B hepatitis virus are present on the surface or inside of the red blood cells. It is considered that the virus may be transferred to the drug product due to the above, which is a problem from the viewpoint of safety.

On the other hand, with the development of synthetic technology, it has become possible to synthesize a blood group antigen, which is then bound to silica gel powder or the like to form an insoluble blood group antigen, and the blood group antibody is adsorbed and removed. It became possible to do it. As such, Synsorb A (for absorption of type A antibody) and Synsolv B (for absorption of type B antibody) (both Chembiomed LT
D.) etc. are commercially available.

However, it is problematic to use it on an industrial scale because it is extremely expensive, because it is a synthetic antigen, it adsorbs only specific blood group antibodies, and its adsorption capacity is low. .

[0011]

DISCLOSURE OF THE INVENTION The object of the present invention is that it has a strong adsorptive power, is not infectious by viruses, and can be applied to the removal of all types of blood group antibodies, and is inexpensive and almost completely on an industrial scale. It is to provide a method for removing blood group antibodies.

[0012] Another object of the present invention is to provide a blood product in which all A, B and D blood group antibodies are substantially removed.

[0013]

[Means for Solving the Problems] The inventors of the present invention have conducted extensive research to solve these problems. As a result, blood group substances are treated by heat treatment sufficient to inactivate viruses. We found that contaminant proteins other than type substances were denatured and could be removed, and succeeded in obtaining blood group antigens in large quantities.

It has been found that the blood group substance is bound to an insoluble carrier to give an insoluble blood group antigen, which can be used to efficiently remove blood group antibodies from blood products.

The present invention has been completed based on such new findings, and is an insolubilized human blood obtained by binding at least two human blood group substances selected from A type, B type and D type to a water-insoluble carrier. From blood products characterized by treating blood products with a type substance
Type of blood group antibody removal.

The present invention also relates to a blood product in which all blood group antibodies of type A, type B and type D have been substantially removed, and specifically, blood group antibodies of type A, type B and type D. However, in the antibody titer display by the indirect Coombs method, both are 1:
It relates to a blood product removed to 1 or less.

Targets for Removal of Blood Group Antibodies In the present invention, the target for removing blood group antibodies, that is, blood products that contaminate blood group antibodies are all blood products that may be contaminated with human blood group antibodies, Examples include immunoglobulin preparations, various blood coagulation factor preparations, albumin preparations and the like. It is efficient to perform this treatment in the final step of isolation / purification of the blood product, but it may be performed at any stage of the isolation / purification if recovery rate and the like do not matter.

Preparation of Immobilized Human Blood Group Substance The immobilized human blood group substance is a human blood group substance immobilized on an insoluble carrier.

The human blood group substance used in the present invention is A type,
The immobilized human blood group substances used in the present invention, which are B type and D type blood group antigens, have at least two of these human blood group substances immobilized on an insoluble carrier. Human blood group substances can be prepared, for example, by collecting them from human red blood cells of A, B, AB and O types, placenta, and the like.

A known method may be used to prepare the human blood group substance. For example, it can be prepared by lysing human erythrocytes in a hypotonic solution, or sonicating and then purifying by a ammonium sulfate fractionation method or a PEG fractionation method. It can also be prepared from placenta by the same method.

[0021] The human blood group substance is preferably subjected to a heat treatment sufficient to inactivate the virus and not to inactivate the blood group substance and the blood product. For example, blood group substances are preferably dissolved in physiological saline and then 50-125.
The heat treatment is performed at 1 ° C. for 1 minute to 20 hours, preferably at 60 ° C. for 10 hours or at 95 to 121 ° C. for 2 to 30 minutes. Then, for example, centrifugation is performed to remove insoluble matter, and the mixture is dialyzed against distilled water to obtain a human blood group substance.

On the other hand, the insoluble carrier is water-insoluble such as agarose, cellulose, dextran, silica gel, glass powder, nylon, etc., and can be directly or indirectly fixed to human blood group substances, and achieves the object of the present invention. There is no particular limitation as long as it is possible.

Immobilization may be performed according to a method known per se.
For example, human blood group substances can be immobilized by the CNBr activation method for agarose and the like, and aminosilane formation method for silica gel and the like.

Treatment Method Usually, a blood product contaminated with blood group antibodies is first dissolved in a suitable solvent. The protein concentration at that time is 1 to 10 w /
v% is preferred. Furthermore, pH 5 to 10, preferably pH 6
-8, ionic strength 0.001-1M, preferably 0.0
Contact treatment with the immobilized human blood group substance under the condition of 5 to 0.1M. Examples of the contact treatment means include a batch method and a column method.

For example, in the batch method, 1 to 500 ml of a solution of the blood product is added to 1 ml of immobilized human blood group substance.
ml, preferably 1 to 100 ml, and the mixture is stirred at 0 to 15 ° C., preferably 0 to 4 ° C. for 20 minutes to 4 hours, preferably 30 minutes to 2 hours and then centrifuged (for example). 6000-8000 rpm, 10-30 minutes) and collect the supernatant. In the column method, a solution of the blood product is brought into contact with an immobilized human blood group substance to develop it, and a non-adsorbed fraction is collected.

[0026]

EXAMPLES The present invention will be described in detail below with reference to examples.

Example 1 A commercially available insoluble blood group antigen (blood group antigen / cellulose) obtained by binding purified human blood group substances (types A and B) to formyl cellulose (manufactured by Chisso Corporation) A, B, and D blood group antibodies were removed from the immunoglobulin for intravenous injection. For comparison, Synsolv A and Synsorb B were used.

The treatment conditions are human blood group substances, cellulose,
Synsorb A or Synsorb B was packed in a 0.5 ml volume column, and the intravenous immunoglobulin was allowed to flow down in a 30 ml cold room, and the IgG concentration of unadsorbed fraction (OD280nm)
And the blood group antibody titer was measured. Table 1 shows the results.

[0029]

[Table 1]

In Synsolv A, only the A-type antibody was adsorbed and in Synsorb B, only the B-type antibody was adsorbed and removed, whereas in the method of the present invention, A, B and D type antibodies were simultaneously removed. I
The yield of gG was good in all three cases. The antibody titer was measured by the indirect Coombs method (Br. J. Exp. Pathol 26: 255, 19
45) and D antibody were determined by the indirect Coombs method / sensitization method (method in which the amount of sample added is increased to increase the sensitivity).

Example 2 The same treatment as in Example 1 was carried out by the batch method using the factor 8 preparation. The results are shown in Table 2. However, A and D types were used as blood group substances.

[0032]

[Table 2]

Example 3 The same treatment as in Example 1 was carried out using the Factor 9 preparation. Table 3 shows the results. However, B and D types were used as blood group substances.

[0034]

[Table 3]

Reference Example 1 Preparation of type 1 substance The placenta extract was adjusted to pH 6.0, heat-treated at 95 to 100 ° C. for 30 minutes to remove insolubles, and then salted out with 100% saturated ammonium sulfate to obtain a precipitate. . The precipitate was dialyzed against distilled water and freeze-dried to obtain a prepared substance. Types A, B and D are
It is determined by a blood test in advance and prepared from blood having each antigen.

Preparation of water-insoluble substance Sepharose 4B having a particle size of 40 to 190 μm About 300 ml
Add 75 g of cyanogen bromide to the mixture and stir it for 4
The pH is adjusted to 11 with N-sodium hydroxide. During this period, the temperature was kept at 10 to 20 ° C. Keep stirring at pH 11 and continue stirring for about 15 minutes. Then, the reaction solution was filtered and the filter cake was washed with 0.1 M sodium hydrogen carbonate to obtain about 300 ml of activated Sepharose 4B. Part 6
After taking up ml and washing with PBS, 18 ml of PBS was added and suspended, and 5.2 ml of the 10 mg / ml solution of the type substance obtained above was added thereto and reacted for 3 hours to obtain a water-insoluble type substance. The water-insoluble substance was washed with PBS, packed in a column, and subjected to ordinary affinity chromatography.

[0037]

INDUSTRIAL APPLICABILITY In the blood product treated by the present invention, each blood group antibody intended to be removed is almost completely removed. In particular, when at least two types of A type, B type and D type are simultaneously immobilized, the blood group antibody can be almost completely removed. Moreover, the recovery rate of blood products by this treatment is 80
~ 100%. Moreover, the treatment of the present invention can be applied to a large amount of blood products. Therefore, the method of the present invention is extremely advantageous as a method for removing various blood group antibodies from blood products on an industrial scale.

Further, since the blood preparation of the present invention is substantially free of A, B and D blood group antibodies, even if a large amount of the preparation is administered, the blood preparation will remain in the patient's body. An antigen-antibody reaction is not elicited with erythrocytes, and it has effects such as reduction of erythrocytes, lysis of erythrocytes, and subsequent occurrence of side effects such as renal damage.

Claims (2)

[Claims] 1. A blood group antibody of type A, type B and type D
Blood products with antibody titer display by indirect Coombs method, all of which were removed to 1: 1 or less. 2. The blood product according to claim 1, which is an immunoglobulin product, a blood coagulation factor product or an albumin product.