IL23466A - Blood purification - Google Patents

Description

'ΠΙΠΠ ΙΠ3 TJlill"! m PATENTS AND DESIGNS ORDINANCE SPECIFICATION BLOOD PURIFICATION I (we) RCKM & HAAS COMPANt, A CORPORATION ORGANIZED UNDER TH£ tAW8 OP THE S ATE OF DELAWARE* U.S.A., OF 222 WEST WASHINGTON SQUARE, PHILADELPHIA 5» PENNSYLVANIAj U*S,A. , do hereby declare the nature of this invention and in what manner the same is to be performed, to be particularly described and ascertained in and by the following statement: — This invention relates to the removal of cLieoaoo-causing viruses from blood, either during the process of collecting blood from donors or prior to the process of transfusing the blood into a human being.

More particularly, this invention relates to the removal of disease-causing viruses from blood that is to be stored in blood banks for future transfusions, or which is to be pooled and fractionated so as to isolate various products such as gamma OX globulin, plasma^ fibrinogen, -etey It has long been the practice to use whole blood to replace the loss of circulating blood in human beings, particularly those undergoing surgical operations. It has been known for many years that some risk is encountered in transfusing blood due to the possibility of transmitting disease-causing viruses. In the course of collecting, storing, and transfusing blood, there are no known methods for ascertaining whether or not some disease-causing viruses are present in the blood and there are no known methods for removing these viruses either during the collection or transfusion of the blood. It has been estimated that many thousands of patients become infected annually because of the infected blood that is used for transfusions. In many cases, those infected in this manner do not survive the disease. Although several diseases may be transmitted in this manner, the most prevalent one is virus hepatitis.

As blood donors are not always available when needed, methods have been developed for collecting, preserving, and storing whole blood at refrigeration temperatures, and the use of such stored blood in transfusions has become common. Such blood generally is useful for about twenty-one days.

Before whole blood can be stored, it must be collected in a manner so as to prevent its coagulation. Several methods are known for doing this. These include the use of chemical anticoagulants such as acid citrates and heparin, and the use of ion exchange resins. However, none of the prior blood collection and preservation methods is capable of removing viruses from blood, and thus the recipient of the blood donation is faced with the possibility of succumbing to a virus disease every time he or she receives a blood transfusion.

There are no known methods, except by means of the present invention, for avoiding or eliminating this problem.

According to the present invention, disease-causing viruses are removed from blood by a method which comprises contacting the blood with a mixture of at least one cation exchanger and at least one anion exchanger, the exchangers having a particle size in the range of about 297 to about 1190 microns.

Although stored whole blood develops increases in potassium content which may cause the patient to suffer serious complications, and this level of potassium therefore must be lowered, the level should generally not be permitted to go below the normal amount of about four railliequivalents per liter (meq./L.) or else the blood will tend to hemolyze and become unsuitable for transfusions.

According to a preferred embodiment of the invention this hemolysis is reduced or prevented by contacting the whole blood, as it is either collected from a donor or transfused into a patient, with a of mixture/exchangers in which the cation exchanger is partly in the sodium form and partly in the potassium form. This preferred embodiment may be carried out by following the teachings of U.S. Patent ,833»6 1 b using cation exchange resin buffered to a pH of 7·2-7·ί*» part of the resin being in the potassium salt form and the remainder being in the sodium salt form.

Instead of using one anion and one cation exchanger, there may be employed a total of three or four Σόη (exchangers. As the cation exchanger there may be used a strongly acidic or a weakly - i - there may be used a strongly basic or a weakly basic anion exchange resin. Illustrative of the strongly acidic cation exchangers is Amberlite 200, a macroreticular-structured, styrene-divinylbenzene copolymer which has been sulfonated. Illustrative of the weakly acidic cation exchangers is Amberlite IRC-50, which has carboxlic groups in the molecule and is prepared by suspension copolyraerizing a mixture of methacrylic acid and about 3 to 1C# divinylbenzene.

The strongly basic anion exchanger is typified by Amberlite IRA-½02, a styrene-divinylbenzene copolymer which has been chlororaethylated and subsequently aminated with trimethylamine. The weakly basic anion exchanger is typified by Amberlite IRA-68, a resin which is formed by reacting a polyamino compound containing at least one primary amino group with an insoluble, cross-linked copolymer of an ester of acrylic or methacrylic acid, divinylbenzene being the cross-linking agent. Amberlite 200, Amberlite IRC- 50, Amberlite JRA-OZ and Amberlite IRA-68 are all commercial products of the Rohm & Haas Company, Philadelphia, Pennsylvania.

Actually, all of the ion exchangers useful in the practice of the present invention are available from a number of sources. The sulfonated materials, for example, are well-known insoluble products which can exchange hydrogen for metal ions of soluble salts. They may be an insoluble phenolformaldehyde condensate having methylene sulfonic groups. They may likewise be styrene copolymers which are insolubilized by means of a cross-linking agent, such as divinylbenzene or trivinylbenzene, and which contain sulfonic groups in nuclear positions. Carbonaceous zeolites, prepared by sulfonation or sulfonation and oxidation of coals and lignites may be used, and the same applies to sulfonated condensed lignins. Details of the preparation of some sulfonated cation exchangers may be found in U.S. Patents Nos. 2t195»196; 2,228, 159; 2,228,160; 2,366,007, and 2, 97^-38. 23466/2 The quaternary ammonium resins are strongly basic materials which are capable of splitting salts, supplying hy-doxyl ions or other anions. They are insoluble quaternary ammonium compounds in which the anion is a hydroxyl ion or the anion of an exceedingly weak acid, such as carbonic acid, including the bicarbonate ion, or boric acid. The radicals attached to the quaternary nitrogen atom comprise one or more resin-forming groups,the other being lower alkyl, a ralkyl, aryl, cycloalkyl or hydroxy alkyl groups. The functional group of these anion materials may be represented as where X is a hydroxyl group or anion of a weak acid, R,l,t is a copolymer chain to which the nitrogen is attached, and R' , R' 1 , and R' ' · are lower alkyl, aralkyl, aryl, cycloalkyl or hydroxy-alkyl groups, such as methyl, ethyl, propyl, but l,benzyl, hy-droxyethyl or hydroxypropyl. Typical quaternary ammonium ion exchange resins are prepared from styrene by copolymerization ..with a cross-linking agent such as divinylbenzene, chloro-methylation of the copolymer, and reaction of chloromethyl groups with a tertiary amine, such as trine hylamine , butyl-dimethylamine or hydroxyethyldimethylamine . The chloride ion is replaced by the ion by treatment with a base such as sodium hydroxide. Styrene may also be cross-linked with methylene groups, halomethylated, and then quaternized. Details of the preparation of such quaternary ammonium anion exchange resins are available in US Patents 2,540,985; 2,591,573 and 2,614,099.

The carboxylic cation exchange resins are insoluble them to enter into copolymers or heteropolymers with polymerizable substances, including those which cause cross-linking. It is known, for instance, that maleic anhydride and styrene can be polymerized together and, when there is present an unsaturated material having at least two non-conjugated double bonds, an insoluble resin results.

The cross-linking material may be one such as divinj'lbenzene, trivinylbenzene, ethylene diacrylate, diallyl maleate or fumarate or Another source of carboxylic exchangers is based on the copolymerization of acrylic or methacrylic acid and a polyunsaturated polymerizable substance such as diallyl maleate or fumarate or itaconate, allyl acrylate, allyl methacrylate, diallyl ether, ethylene dimethacrylate^ divinylbenzene, ©r—bl.a liko. The copolymers or heteropolymers are formed in the conventional way with the aid oiL^catalyst, such as benzoyl peroxide, lauroyl peroxide, o tert.-perbenzoate^ tert.-butyHydroperoxide, e«(w. The resin, when formed, may be crushed to a fine powder. The insoluble carboxylic resins may also be formed by emulsion polymerization and then precipitated as fine particles. Acid anhydride groups are converted to carboxyl groups by treatment of the resins with an alkali or a strong acid. If alkali is used, the resulting salt form of the resin is readily converted to the acid form by washing it with acid. Details of the preparation of some carboxylic cation exchange resins may be found in U.S. Patents Nos. 2,3^0, 110; 2,3^0, 111 , and 2,597ι½37· Amino anion exchange resins are available from a number of sources. Phenols, aldehydes or ketones, and strongly basic amines can be condensed together by known methods to give insoluble resins which take up acids. Particularly useful resins of this sort are those made from polyphenylol compounds, such as di(hydroxyphenyl) methane or di(hydroxyphenyl) sulfone, formaldehyde, and polyalkylene- ol amines such as trieth lenetetramine or tetraethylene entamine.

Another type of anion exchange material is prepared by chloromethylating an insoluble styrene copolymer, such as one from styrene and divin lbenzene, and then reacting the chloromethylated product v/ith an amine having hydrogen on the amino nitrogen. Polyamines, such as diethylenetriamine^ tetraethylenepentaraine. anct tho Ί ak(^ are Particularly useful, although such amines as dimethylamine, diethylamine, methylamine, ethylamine^ ethanolaraine, and tho Iriko, also give very useful amine anion exchange products. Another type of anion exchange resin is prepared from urea or melamine, op- the lilco, formaldehyde, and a polyamiiae, such as one of those named above or such a combination which includes guanidine or biguanide. Yet another type is based on phenylenediamine. As is known, m-phenylenediamine and formaldehyde give insoluble resinous materials which have a good capacity for taking up acids. Other types of amino exchangers can be used. These anion exchange resins contain tertiary or secondary amine groups, or both, which have a good capacity for absorbing acids and yet do not have any marked tendency for splitting salts of strong bases and strong acids. Many of the amino anion exchange resins have been fully described in the art, e.g., U.S. Patents 2,59^ 57^ and 2,675,359.

Although various ion exchange resins have been employed previously for removing viruses from biological fluids, the form of the ion exchange resin and the conditions .of operation have been such that the processes could not be employed for removing viruses from blood without destroying the value of blood. In essence, those processes were primarily designed to recover, concentrate, and purify viruses growing in various biological fluids. The fluid was later discarded. This is not the case where blood is involved.

The particle size of the ion exchangers used in the method of the invention is most important. Should the particle size of the ion exchange resins be etc small as has been used previously for removing viruses, one will remove, by adsorption, the formed elements (red cells, platelets, and white cells) as well as the viruses.

On the other hand, if the particles are too large, an excessive amount of the viruses can get by them without being absorbed thereon. It has been found that, by the use of the particular particle size distribution specified earlier in this specification, one can remove viruses from blood without adversely affecting the value of the blood.

A preferred range of particle sizes is between No. 20 U.S. Standard Sieve (B O microns) and No. O U.S. Standard Sieve (kZQ microns). Ion exchange materials whose particle size is outside the range of 297 to about 1190 microns, i.e. materials passing through a No. 16 U.S. Standard Sieve and being retained on a No.

U.S. Standard Sieve, will not function to remove the viruses satisfactorily.

The following examples will illustrate the invention in more detail. Mesh sizes are given in terms of U.S. Standard Sieves, Example I (a) A sterile hemo-repellant plastic tube with a 100-mesh filter at the effluent end is filled with a well-mixed bed of sterile ion exchange resins consisting of about grams of Amberlite 200 in the sodium form, 3 grams of Amberlite 200 in the potassium form, and 20 grams of Amberlite ΤΒΑ-^ΟΣ in the chloride form. The particle size of the resins is in the 20-40 mesh range.

The resin bed is then thoroughly washed with a sterile physiological dextrose solution.

One-half liter of human blood, stored for 20 days, is taken from a blood bank and about 50 agglutination units per ml. of the influenza A virus is added to it. The blood is then passed through the bed of resin for a period of 1 hour. The treated blood is then given a hemotological examination and the presence of virus determined by hemagglutinin and infectivity tests. Typical results on blood tested in this manner show normal hemotology and the absence of the influenza virus. (b) The experiment of part (a) is repeated, using the identical conditions with the exception cf substituting 100-200 mesh resins instead of the 20-*t0 mesh resins. In this c se the analysis of the treated blood reveals complete removal of the virus; however, coagulation and adsorption of the formed elements occurs to such an extent that the treated blood is useless for transfusion purposes, (c) The experiment of art (a) is repeated, using the identical conditions except for the substitution of resins in the No. 60-100 ( 1^9-250 microns) sizes for the 20-½0 mesh resins.

The results are similar to those described in part (b) · (d) The experiment of part (a) is repeated, using the identical conditions except for the substitution of resins in the No. 10-1½ ( 1^10-2000 microns) sizes for the 20-^0 mesh resins.

The results are an abnormal hemotology and incomplete removal of the influenza virus. (e) The experiment of part (a) is repeated, using the identical conditions with but one exception. The particle size of the resins is in the 12-16 mesh range. Although the hemotology of the blood appears normal, there is an appreciable (35%) fraction of the original virus present.

Example II The experiment of Example I, part (a), is repeated, using the identical conditions except for the substitution of resins in the No. 16-50 (297-1 90 microns) sizes for the 20-*K) mesh resins.

The results are similar to those described in Example I, part (a).

Example III The experiment of Example I, part (a), is repeated, using the encephalitis (eastern equine) virus instead of the influenza virus. The results obtained reveal that the treated blood has normal hemotolog and is free of the virus.

Example IV The experiment of Example I, part (a), is repeated, using the identical conditions with two exceptions. The mumps virus is substituted for the influenza virus, and 2 grams of Amberlite IRC-50 and 2 grams of Amberlite IRA-68 are added to the mixture.

All the resins are in the 20-O mesh range. The results reveal that the treated blood is free of virus and the blood has normal heraotology. In this example, the pH of the treated blood is raised from 6,5 to 7·2· Example V The experiment of Example I, part (a), is repeated, using the identical conditions with two exceptions. The polion^elitis virus (Lansing strain) is substituted for the influenza virus and 20 grams of Amberlite IEA-93 in the chloride form is substituted for the Amberlite I A-½02. The results obtained reveal that the treated blood has normal hemotology and is free of the original virus present.

Claims (1)

1. NOW THE NATURE OP OUR IN THE TO WE THAT What we claim The method of removing viruses from blood which comprises contacting the blood with a mixture of at least one cation exchanger and at least one anion the exchangers having a particle size in the range of about 297 to about A method according to claim wherein the cation exchanger is partly in the sodium form and partly in the potassium A method according to claim 1 or the ion exchangers have a particle size in the range of about to about A method according to any one of the preceding wherein the ion exchangers are ion exchange A method according to claim 1 substantially as hereinbefore described with reference to any one of Examples I and II to Blood when purified by a method according to any one of claims THIS THE insufficientOCRQuality