EP0591483A1 - Albumin-iodine preservation of blood, tissues and biological fluids - Google Patents

Abstract

The invention relates to the treatment and preservation of blood, blood derivatives and other tissues, fluids and biological cells with ALB-I and then to the quenching of the iodine oxidation potential in ALB-I in order to kill pathogens without destroying biological tissue, fluids and cells.

Description

ALBUMIN-IODINE PRESERVATION OF BLOOD, TISSUES AND BIOLOGICAL FLUIDS

Field of the Invention This invention relates to the treatment and preservation of blood and blood derivatives, the treatment and preparation of other body tissues and cells, the treatment and preparation of tissue cultures and tissue culture products, and the preparation of laboratory reagents, standards and samples. According to the invention, a new composition, albumin-iodine, is used in the treatment of treating biological materials. Thereafter a physiologically compatible reducing agent such as an ascorbate salt, as an additive or on a solid support, e.g. in a bed or filter of solid albumin,, may be used to remove the last traces of oxidizing iodine. Delipidated, non-stabilized albumin, i.e. without caprylate or other caps on reactive positions, is the greatly preferred albumin_^ for use in preparing the compositions for use in this invention. This invention may, thus, be used to kill or inactivate virus, bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorganisms and to remove all oxidizing iodine.

The treatment and preparation of human blood, tissues, etc. and of the blood, tissues, etc. of other animals are contemplated. In general, the field of this invention lies in medicine and veterinary practice; most examples being related to the practice of medicine for the benefit of human patients, use in analogous fields of veterinary medicine to the extent applicable being within the scope of the invention.

Background of the Invention Iodine was officially recognized by the Pharmacopeia of the United

States in 1930, also as tincture iodine (tincture of iodine) and linimentum iodi (liniment of iodine). Clinicians and microbiologists described a great number of experimental data and clinical applications, which can be found in numerous surveys. Despite the successes that have been achieved with iodine, it was ascertained early that it also possesses properties unsuitable for practical application, including, for example, the fact that iodine has an unpleasant odor. In addition, it stains the skin with an intensive yellow-brownish color, causes blue stains in the laundry in the presence of starch, and combines with iron and other metals, its solutions are not stable, it irritates animal tissue, and is a poison. The adverse side effects of iodine, its painfiilness on open wounds and the possibility of allergic reactions in the past 100 years led to the production of a great many iodine compounds (and iodine preparations), with the aim of avoiding these incompatibilities without a significant loss of germicidal efficiency. In this connection, the iodophors finally succeeded as near-ideal forms of application. A new iodine complex that has all or most of the advantages of the iodophors, e.g. povidone-iodine is described in this application. Although exact details about the killing of a living cell by the I₂ molecule (or one of the reaction products occurring in aqueous solution) are not known, it can be assumed that iodine reacts:

(1) With basic N-H functions that are parts of some amino acids (e.g., lysine, histidine, arginine) and the bases of nucleotides (adenine, cytosine, and guanine) forming the corresponding

N-iododerivatives. By this reaction, important positions for hydrogen bonding are blocked, and a lethal disorder of the protein structure may occur.

(2) Oxidizing the S-H group of the amino acid cysteine, through which the connections of protein chains by disulfide (-S-S-) bridges, as an important factor in the synthesis of proteins, are lost.

(3) With the phenolic group of the amino acid tyrosine, forming mono- or diiodo-derivatives. In this case, the bulk of the iodine atom(s) in the ortho position may cause a form of steric hindrance in the hydrogen bonding of the phenolic OH group.

(4) With the carbon-carbon double bond (C=C) of the unsaturated fatty acids. This could lead to a change in the physical properties of the lipids and membrane immobilization. Iodine - polymer complexes, e.g., with poly(vinyipyrroIidone) (PVP), and complexes of iodine with nonionic surfactants, eg, polyethylene glycol mono(nonylphenyl) ether have been used with considerable success. However, use in direct contact with fragile biological materials has been limited because either the killing power of iodine is dissipated in the biological material or damages the biological material.

Povidone iodine is capable, in certain circumstances, of killing all classes of pathogens encountered in nosocomial infections: gram-positive and gram-negative bacteria, mycobacteria, fungi, yeasts, viruses and protozoa.

Most bacteria are killed within 15 to 30 seconds of contact. It is believed that albumin-iodine has a comparable killing spectrum.

Iodine is consumed by proteinaceous substrates and its efficacy as a disinfectant is reduced at certain antiseptic applications. This is due to a reducing effect of the material to be disinfected which leads to the conversion of iodine into non-bactericidal iodide. Thus, not only the reservoir of available iodine is diminished but also the equilibrium of triiodide is influenced as well. Both of these effects cause a decrease in the proportion of free molecular iodine, the actual anti-microbial agent. When povidone-iodine preparations are contaminated with liquid substrata (e.g. blood, etc.) there is, in addition, the dilution effect characteristic of povidone-iodine systems which causes an increase in the equilibrium concentration of free molecular iodine. To what extent the latter effect compensates for the other two effects depends on the content of reducing substances. Thus with full blood, a strong decrease of the concentration of free molecular iodine occurs, while, in the presence of plasma, it remains practically unchanged. Durmaz, et al, Mikrobiyol. Bui. 22 (3), 1988 (abstract); Gottardi W, Hyg. Med. 12 (4). 1987. 150-154. Nutrient broth and plasma had little inactivating activity but 1 g hemoglobin inactivated 50 mg of free I; experiments with ¹²⁵I showed that uptake of I by [human] red cells occurred rapidly. Optimal antimicrobial effects in clinical use should be achieved in relatively blood-free situations. Povidone iodine produced a potent and sometimes persistent bactericidal effect towards bacteria on healthy skin. Lacey, R. W., / Appl Bacteriol 46 (3). 1979. 443-450. The bactericidal activity of dilute povidone-iodine solutions is inversely proportional to the concentration of the povidone-iodine solutions and is inhibited to the greatest extent by blood, followed by pus, fat and glove powder. Zamora J L; Surgery (St Louis) 98 (1). 1985. 25-29; Zamora, Am. J. Surgery, 151, p. 400 (1986); see also, Waheed Sheikh, Current Therapeutic Research 40, No. 6, 1096 (1986). Van Den Broek, et al, Antimicrobial Agents and Chemotherapy, 1982, 593-597, suggests that povidone-iodine is bound to cell wall proteins leaving little for interaction with microorganisms in the liquid phase (See, also, Abdullah, et al., Arzneim.-Forsch./Drug Res. 31 (I), Nr. 5, 828). Ninneman et al, /. of

Immunol 81, 1265 (1981) reported that povidone-iodine was absorbed in serum albumin and it is know that povidone-iodine is bound to albumin but it has been, discovered that the antibiotic activity of povidone-iodine is not destroyed by albumin bounding. Whether the activity remains because the albumin povidone-iodine is active or whether povidone-iodine and/or I₂ are released from the albumin-povidone-iodine complex. It is expected that albumin-iodine will have similar limitations; however, data to that effect are not available.

Iodine is used widely in human medicine is the disinfection of skin, (e.g., the preoperative preparations of the skin, the surgical disinfection of hands, the disinfection of the perineum prior to delivery, and the disinfection of the skin prior to infections and transfusions). Iodine preparations are also used for therapeutic purposes, e.g., the treatment of infected and burned skin but is a strong irritant. Iodophors, and albumin-iodine of this invention, largely overcome the irritation. Iodine has also been used for the disinfection of medical equipment, such as catgut, catheters, knife blades, ampules, plastic items, rubber goods, brushes, multiple-dose vials, and thermometers. The use of iodine as an aerial disinfectant has been advocated since 1926, and experiments on the disinfection of air have been carried out, mainly during World War II. Aerial disinfection of air-raid shelters with iodine vapors as a prophylactic measure against influenza has been recommended and a "relatively tolerable" concentration of 0.1 mg ft³ (3.5 mg m³) was found to be sufficient for a rapid kill of freshly sprayed salivary organisms. Obviously, one is aware of the danger that iodine vapors pose to the respiratory organs, documented by the fact that the maximum allowed concentration of iodine comes to 1.0 mg m³'

The use of "oxidizing iodine" including "compounds incorporating molecules of oxidizing iodine" e.g. absorbed or grafted on a purified vegetable carbon, as blood-contacting reagents having bactericidal and bacteriostatic action are mentioned in passing in connection with an autotransfuser device in U.S. Patent 4,898,572, Surugue nee Lasnier, et al but without any explanation or elucidation. Iodine is, thus, an excellent, prompt, effective microbicide with a broad range of action that includes almost all of the important health-related microorganisms, such as enteric bacteria, enteric viruses, bacterial viruses, and protozoan cysts, if the sometimes severe limitations inherent in its use are overcome. Mycobacteria and the spores of bacilli and clostridia can also be killed by iodine. Furthermore, iodine also exhibits a fungicidal and trichomonacidal activity. As to be expected, varying amounts of iodine are necessary to achieve complete disinfection of the different classes or organisms. Within the same class, however, the published data on the disinfecting effect of iodine correspond only to a small extent. In particular, the published killing time os spores and viruses are widely disparate.

Various authors have tried to summarize the disinfecting properties of iodine and the other halogens by reviewing the literature and analyzing the existing data. The most important conclusions are:

(1) A standard destruction (i.e., a 99.999% kill in 10 minutes at 25o C) of enteric bacteria, amoebic cysts, and enteric viruses requires I₂ residuals of 0.2, 3.5, and 14.6 ppm, respectively.

(2) On a weight basis, iodine can inactivate viruses more completely over a wide range of water quality than other halogens. (3) In the presence of organic and inorganic nitrogenous substances, iodine is the cysticide of choice because it does not produce side reactions that interfere with its disinfecting properties.

(4) Iodine would require the smallest mg L dosage compared to chlorine or bromine to "break any water" to provide a free residual.

(5) I₂ is 2 to 3 times as cysticidal and 6 times as sporicidal as HOI, while HOI is at least 40 times as virucidal as I-,. This behavior is explained on the one hand by the higher diffusibility of molecular iodine through the cell walls of cysts and spores and on the other hand by the higher oxidizing power of HOI.

Gottardi, W. Iodine and Iodine Compounds in DISINFECTION, STERIUZATION, AND PRESERVATION, Third Edition, Block, Seymour

S., Ed., Lea & Febiger, Philadelphia, 1983, and the references cited therein provide more details respecting the background discussed above.

The major constituent of plasma is albumin whose primary role is that of osmotic regulation; it is responsible for 75-80% of the osmotic pressure of plasma. Albumin also serves important roles in the transport of small molecules such as drugs. An important feature which segregates albumin from other colloids as well as crystalloids is its unique ability to bind reversibly with both anions and cations; hence, albumin can transport a number of substances including fatty acids, hormones, enzymes, dyes, trace metals, and drugs. Substances which are toxic in the unbound or free state are generally not toxic when bound to albumin. This binding properly also enables albumin to regulate the extracellular concentration of numerous endogenous as well as exogenously administered substances.

Albumin in general has three types of binding sites (one for acidic, one for basic, and one for neutral compounds), and it plays a critical role in the binding and transport of lipid and lipid-soluble material. Albumin binds with and transports many administered drugs. Because of the phenomenon of mutual displacement of similar type substances, adverse drug interactions may occur. This phenomenon may have important ramifications during disease states such as sepsis, burn injury, and circulatory shock due to a number of etiologies, especially in conjunction with treatment with drugs which may be toxic at high concentrations.

Human serum albumin is believed to be a scavenger of oxygen-free radicals, an important phenomenon which also extends to scavenging of radicals required for lipid peroxidation.

Albumin is critical for the transport of numerous compounds, especially non-water soluble ones. It binds with iron and lipids and other potentially toxic substances, e.g., bilirubin. Thus albumin acts as a buffer to prevent increases in potentially cytotoxic endogenous lipid-soluble substances by binding with, and thus limiting, increases in plasma and interstitial fluid concentrations of these substances.

In addition to displacement of an albumin-bound drug by another, endogenous substances may also alter significantly the unbound or "free" plasma and interstitial fluid concentration of a drug. For example, as the concentration of bilirubin increases in certain disease states, a drug which occupies the same binding site as bilirubin will be displaced by the bilirubin, and the plasma concentration of the free drug will increase, possibly to toxic levels. Also, as the plasma concentration of albumin decreases, the plasma and interstitial fluid concentration of the unbound (free) drug will increase. Plasma albumin concentration is usually decreased to varying degrees in disease states such as sepsis, burn injury, and circulatory shock. Resuscita¬ tion with large volumes of non-albumin colloid or crystalloid solutions will further decrease an already low albumin concentration and may consequently further limit the ability of albumin to modulate the free concentration and transport of toxic substances or drugs administered for therapeutic purposes.

Another feature of albumin is its inhibitory effect on pathologic platelet aggregation, which may be due to a greater affinity of arachidonic acid for albumin than for platelet-generated cyclooxygenase. It has also been demonstrated that albumin enhances the inhibition of factor Xa by anti- thrombin-III (AT-III).

In addition to the well-known role of albumin in generating colloid osmotic pressure, it also may protect the lung and other organs from edema by preserving microvascular integrity.

Albumin has been used as an emulsion stabilizer oil-and-water emulsion injectable medical preparations, e.g. fluorbiprofen, Mizushima et al, U.S. Patent 4,613,505, Sept. 23, 1966; as a binding molecule for tryptophan, Pollack, U.S. Patent No. 4,650,789, Mar. 17, 1987; with chemical modification as complexing agents for cholesterol derivatives, Arakawa, U.S. Patent No.

4,442,037, Apr. 10, 1984; as conjugates with enzyme chemically linked to an antibody, Poznansky, U.S. Patent No. 4,749,570, June 7, 1988; and as chemically coupled conjugates of leukotrienes, Young, et al, U.S. Patent No. 4,767,745, Aug. 30, 1988.

Human serum albumin is a remarkable protein which performs numerous tasks critical to maintenance of the milieu interieur. The best known functions of albumin involve regulation of transvascular fluid flux and hence, intra and extravascular fluid volumes and transport of lipid and lipid- soluble substances. However, it is also involved in a number of other vital functions, some of which have only recently been suggested and perhaps others which are as yet unrecognized. Among recognized unique features of albumin are: a) binding, and hence, inactivation of toxic products; b) regulation of the plasma and interstitial fluid concentrations of endogenous and exogenously administered substances and drugs; c) involvement in anticoagulation; d) maintenance of microvascular permeability to protein; and e) scavenging of free radicals and prevention of lipid peroxidation. This latter property may prove to be critically important, particularly in inflammatory disease states in which free radicals are thought to be a major culprit in direct damage due to tissue oxidation and indirect tissue damage due to inactivation of important antiproteinases such as α^-PI and AT-IIL See UNIQUE FEATURES OF ALBUMIN: A BRIEF REVIEW, Thomas E. Emerson, Jr., Ph.D.,CπttcflZ Care Medicine, Vol. 17, No. 7 (1989). Procedures for large-scale, i.e. at least one unit of blood, fractionation of whole blood into its component cell types and plasma, and methods for the preparation of isolated plasma protein fractions from plasma are well-known. The principles of enhanced sedimentation, e.g. centrifugation, and adhesion can be used to separate anticoagulated whole blood into platelet concentrate, leukocyte concentrate, packed red cells or leukocyte-poor packed red cells, and platelet-rich or platelet-poor plasma. Techniques based upon these principles are available for the selective isolation of platelets (plateletpheresis), leukocytes (leukapheresis), and plasma (plasmapheresis), and for the separation of different leukocyte cell types (granulocytes, lymphocytes, and moπocytes).

Methods are known for the fractionation of plasma into the commonly used therapeutic plasma protein preparations: albumin; antihemophilic factor; fibrinogen; immune serum globulins (both normal and specific); plasma protein fraction; and prothrombin complex. The most commonly used fractionation procedures involve the techniques of cold ethanol or polyethylene glycol precipitation, heat denaturation, and ion-exchange chromatography. The plasma proteins serve a wide variety of functions in the human organism. Their roles in the maintenance of blood volume and other physical characteristics of blood, such as viscosity, are extremely important because blood, in order to perform any of its numerous functions, must be a rapidly circulating medium. If the volume of plasma falls, the pumping action of the heart is strained and there is increased resistance to flow owing to the increased concentration of the red cells relative to the plasma. The blood volume depends on the balance between the hydrostatic pressure of the blood in the capillaries, which tends to expel liquid from the blood into the tissues, and the osmotic pressure (owing to the plasma proteins), which tends to draw liquid back into the blood. The major contribution to the osmotic pressure of plasma is from albumin because of its concentration and properties.

Albumin comprises more than 50% of the plasma proteins by weight. It has a relatively low molecular weight and a high net negative charge at physiological pH. Albumin solutions have relatively low viscosity because of the spherical shape of the molecule. The major impetus for the development of fractionation methods was the need to provide large amounts of a blood volume expander for the treatment of battlefield injuries during World War II. A product was desired that would provide the required oncotic action, not require refrigeration, and be free from the transmission of disease. Human albumin was found to be the most acceptable therapeutic fraction. A fractionation method was developed during the 1940s by a group headed by Cohn at the Harvard Medical School. The procedures were scaled up at the Harvard pilot plant and made available to commercial laboratories under contract to the U.S. Navy to provide blood derivatives for the Armed Forces. The methods developed during this period, with some modifications, are still the most popular methods for the preparation of albumin and ISG.

Cohn and co-workers described methods for the separation and purification of the protein and lipoprotein components of human plasma. In each of these methods there was an initial separation of the protein components of plasma into a small number of fractions in which the major components are separated, and then into a large number of subtractions into which these components are further concentrated and purified. The methods involved lowering the solubility of proteins by reducing the dielectric constant of the solution by the addition of ethanol. Thus separations could be carried out in the range of low ionic strengths at which the interactions of proteins with electrolytes differ from each other markedly. The protein to be separated must have a high solubility when most other components of the system have low solubilities, or the converse.

The plasma used in the development of these methods was obtained from blood collected into sodium citrate anticoagulant. Acetate and carbonate buffer systems were used to adjust pH and ionic strength. Precipitation was carried out at the lowest convenient ethanol concentration and temperature, and at the optimum pH and ionic strength for each separation.

Although the fractional precipitation methods described above were found to be adequate for the purpose of producing large amounts of therapeutic concentrates of plasma proteins, a new procedure took advantage of the increased stability of proteins in the solid state. All of the proteins are rapidly precipitated by a combination of the effects of ethanol and zinc ion. Separations from the solid state are made by fractional extraction. Specific metal-protein interactions favor the separation of undenatured proteins by reducing the extremes of pH and ethanol concentration. The lowest pH used is 5.5 and the highest ethanol concentration is 19%. This method, in its entirety, has never been put into large-scale use. The use of 95% ethanol reduces the amount of ethanol required and reduces the volume of solutions to be processed.

For many therapeutic indications a preparation called plasma protein fraction (PPF) is used interchangeably with albumin. PPF is albumin in a slightly less pure form than the albumin produced by the methods described above. The contaminants are ALPHA - and BETA -globulins and salts. PPF can be produced by eliminating precipitation IV4 and precipitating fractions IV4 and V in a single step. If this is done, a filtration of supernatant phase IV1 is required. PPF is more economical to produce than albumin and can be recovered in higher yield. All immune serum globulin (ISG) for therapeutic use is prepared from large pools of plasma from many donors so that the final product will contain a broad spectrum of antibodies.

Alternative methods for the production of albumin and ISG are also known. An economical method for the preparation of albumin involves heat denaturation of the nonalbumin components of plasma. In this method, plasma or serum is heated to 70°C. in the presence of caprylate ions, under which conditions the globulins and fibrinogen become denatured. The caprylate serves to stabilize the albumin against thermal denaturation. By manipulation of pH, all the denatured proteins are precipitated and removed, leaving albumin in solution.

A modified method for the production of albumin by the heat denaturation of the nonalbumin components has also been developed. This method allows for the separation of the coagulation factors and ISG, if they are desired, whereas the isolation of albumin can begin at any step. The albumin produced is further concentrated by polyethylene glycol precipitation or ultrafiltration. Several methods have been used for the preparation of heat stable plasma fractions rich in albumin, to be used as plasma volume expanders.

Zinc complexes have also been used for fractionation, a fraction obtained by desalting plasma with ion-exchange resins and thus precipitating euglobulins, has been described and fractionation scheme using polyphosphate as a precipitant has also been used. None of these methods yield a fraction with a sufficiently high albumin content to meet regulations of the FDA for albumin or plasma protein fraction (PPF). Polyethylene glycol (PEG) has become a very popular protein precipitant. It acts by concentrating the protein component in the inter-PEG spaces by a displacement mechanism. Plasma fractionation schemes using precipitants other than ethanol or

PEG for the isolation of albumin and ISG have been developed and used successfully, primarily in Europe. Ethyl ether has been used as a precipitant in England. Rivanol and ammonium sulfate have been used in Germany and, in France, placental blood is fractionated with the use of chloroform, trichloroacetic acid, and ethanol as precipitants. Recently, Pluronic polyols (BASF Wyandotte Corp) and solid-phase maleic anhydride polyelectrolytes have been used successfully on an experimental scale. Adsorption chromatography has been used for the purification of ISG.

A large-scale method for the production of albumin utilizing PEG, adsorption chromatography, and gel chromatography has recently been developed. Continuous preparative electrophoresis, polarization chromatography, isotachopheresis and isoelectric focusing are all promising techniques for the large-scale purification of plasma proteins.

Hazards of viral hepatitis or pyrogen contamination of plasma fractions do exist, and precautions must be taken to minimize these risks. Indeed, the major hazard in producing fractions from large pools of plasma is the transmission of virus, the most serious, being hepatitis. This is a danger both for the recipient of the fractions and for the workers in fractionation plants.

It has been shown that fractionation workers, particularly those engaged in the preparation of plasma pools, are at high risk of developing hepatitis B. The high risk products are fibrinogen, AHF, and prothrombin complex. The low risk products are ISG, PPF, and albumin. The lack of infectivity of PPF and albumin is attributable to heating the final products at 60°C. for 10 hours.

Another hazard of plasma fractionation is the partial denaturation of some fractions such as ISG, caused by the fractionation methods. These denatured proteins may have toxic effects or may be immunogenic in the recipients. Among these undesirable side effects is the significant degree of loss of biological competence and the loss or blockage of many binding sites on albumin are lost by the inherent denaturation resulting from this pasteurization or heating process. According to present technology, the disadvantages of denaturation are more than compensated for by the increased stability and potency of concentrated fractions, but there remains a great need for more biologically competent albumin which is free of the hazards and risks of virus infections.

Those who deal with blood and other invasively obtained body fluid samples risk infection from the samples. Those at risk include the doctor, nurse or clinical technician who takes the sample, the technicians who handle the sample and who use the sample in conducting analyses and tests, those who handle the sampling and testing equipment and apparatus, and the entire chain of individuals who attend to the disposal of sampling apparatus and the like, from the individuals who pick up the used apparatus through those who ultimately dispose of the apparatus, usually in specially designed high temperature furnaces. The risk is substantial, as evidenced by the fact that nearly all health care professionals with long experience carry the Epstein- Barr virus (EBV) and/or cytomegalovirus (CMV), the latter being probably the most ubiquitous of the pathogenic viruses. Other pathogenic viruses to which health care workers, and those who handle blood and fluid sampling and handling apparatus, are exposed include hepatitis and human immunodeficiency virus (HIV) as well as a large number of less life- threatening viruses. Another organism which is frequently present in blood and blood products or fractions and which presents a serious risk in certain procedures is the bacteria Yersinia enterocolitica which is become a serious contaminant, surpassing Salmonella and Campylobacter as a cause of acute bacterial gastroenteritis. A significant increase in transfusion related infections of Y. enterocolitica has been reported, Tipple, et al., Transfusion 30, 3, p.207 (1990).

Y. enterocolitica and other bacteria which propagate at relatively low temperatures, e.g. Staphylococcus epidermidis and Legionella pneumophila, present, potentially, a serious threat in blood products.

It is generally recognized that proteinaceous materials destroy the biocidal effectiveness of iodine and iodophors such as PVP-I. This factor has been considered a major impediment to the use of iodine and iodophors in the presence of large amounts of biological materials. Albumin has been identified as having and extremely high capability of de-activating the biocidal power of iodine and iodophors. For example, Batts W N; Landolt M L; Winton J R, Appl Environ Microbiol 57 (5). 1991, 1379-1385, reported the results of using iodine in fishery waters to kill virus that iodine efficacy decreased when proteinaceous material was added to the water. Bovine serum albumin blocked iodine inactivation of the virus more effectively than did equal concentrations of fetal bovine serum or river sediment. Batts, et al, also noted that sodium thiosulfate effectively neutralized free iodine.

In addition to the risk of transmitting infectious disease via blood or blood products, the growth of bacteria in blood and blood products at various stages of production and processing introduces pyrogens into the blood component or product which must be removed before the product can be used in therapy. Introduction of molecular iodine, e.g. povidone-I₂, at an early stage in processing of blood products greatly reduces or eliminates the pyrogen-load of the ultimate product or fraction. Protozoa give rise to many diseases, some of great medical and economic importance. Examples of such protozoa are the genus Plasmodium, e.g. P. falciparum, P. malariae, P. ovale and P. vivax, which causes malaria, Trypanosoma, which causes Chagas' disease, and Leishmania, which cause a variety of leishmaniasis. The method of this invention is effective in eliminating these causative organisms in blood and blood products.

Generally, this invention is applicable to the treatment of donated blood and products produced from blood, tissues and fluids for inactivating virus, bacteria, chlamydia, rickettsia, mycoplasma and other potentially pathogenic microorganisms. Among the important potential pathogens to which this invention is applicable is cytomegalovirus (CMV), probably the most ubiquitous of the pathogenic microorganisms found in animal fluids and tissues and herpesviruses generally. Herpesviruses, of which CMV is a member, represent a very large group of viruses which are responsible for, or involved in, cold sores, shingles, a venereal disease, mononucleosis, eye infections, birth defects and probably several cancers. The present invention is also useful in preventing the transmission of human immunodeficiency virus (HIV). While testing has made blood products safer than it was a decade ago, the complete elimination of HIV contaminated blood and blood products has not been possible using present knowledge and technology.

As used here, the term "blood" means whole blood and blood fractions, components, and products of blood, unless "whole blood" or a specific blood derivative, e.g. a blood fraction, component or product of blood is stated. Thus, the term "blood" may apply to whole blood at the time of collection or a blood derivative at any stage in processing, as indicated by context. Blood derivatives mean blood components such as blood cell concentrates (red blood cells, platelets, etc.), plasma, and serum and products and factors prepared from blood such as albumin and the blood factors. Body tissues and cells means any tissue(s), organ(s) or cells or fluids which contain tissue(s), organ(s) or cells of animal origin. Thus, in a broad sense, body tissues and cells include blood and the cellular components of blood; however, for the most part, simply for clarity in presentation, blood is treated as a separate application of the invention. Examples of body tissues and cells include sperm, bone marrow, kidneys, cornea, heart valves, tendons, ligaments, skin, homograft or xenograft implants and prosthesis generally. Tissue and cell cultures means cells and tissues grown or enhanced in culture media and the culture media per se, but not including nutrients intended for use in cell cultures. Examples of a cultured tissue is cultured skin tissue for use in burn victims, cells and cellular products prepared by standard biological and/or genetic engineering techniques are other examples of tissue cultures. Laboratory reagents and standards, as used in this specification and the claims, means reagents and standards produced from or comprising human or animal fluids, cells or tissues. Examples of such products are red blood cell panel utilized for typing blood, control sera and chemistry controls. Samples of tissues and fluids to be tested include samples of blood, urine, sputum, cell smears, etc. While the term "donor" is not usually applied to the individual from whom such samples are acquired, that term, "donor" will be used here in a more general sense to include the individual from whom any blood, tissue, cells or fluid is obtained for any purpose, and such term will be used to refer even to an unwilling donor.

If a tissue is explanted into the culture media for the purpose of propagating its cells, the procedure is called tissue culture whereas the explanting of individual cells into culture media would be called cell culture; however, both procedures are often referred to by the term "tissue culture" procedures without differentiation, unless the distinction is critical for some ancillary reason. This general usage of the term is employed here. Tissue cultured cells are extremely fragile in many ways, having exacting requirements not only as to nutrients but also to the amount and type of resident organisms which can be tolerated, and culture media are highly susceptible to bacterial and/or viral infection. The treatment of the preparation and handling of sperm, both human and animal, is fraught with risk of infection. Sperm is quarantined for several months and the donor's health is followed to assure that the donor is not infected with a pathogenic microbe. In the case HIV and hepatitis, for example, and many other diseases, the donor may carry the disease-causing organism for months or years without showing any symptoms of the disease.

It would be an important step forward to be able to inactivate or destroy pathogenic microbes to prevent infection of the artificial insemination recipient.

It is, generally, impossible to define with precision the exact materials required to propagate a given cell line and, therefore, it is common practice to use media based upon or containing serum and to add nutrient serum as needed during the cell propagation. Bovine serum from adult animals may be suitable in some instances, but fetal bovine serum (FBS) (sometimes referred to as fetal calf serum (FCS)) is required for the safe propagation of many cell lines, and where high purity is critical. Even the use of FBS is not, however, a guarantee of freedom from infective agents. Indeed, every lot of commercially produced FBS is contaminated with infectious bovine viral diarrhea (BVD) virus and infections with infectious bovine rhinotracheitis (IBR), parainfluenza 3 (PI 3) are, extremely common. At best, pools of raw serum probably contain at least 10⁴ infectious BVD virus particles per milliliter. Serum filtration is a common step in reducing the load of infectious organisms in serum, but serum quality can be damaged by filtration if significant amounts of serum components are adsorbed to the filters or if macromolecules are sheared. Shearing of macromolecules during filtration occurs generally when tangential flow filtration is used and turbulence develops. It is currently very difficult to obtain reliable results on the removal of BVD viruses from serum using filtration.

The presence of adventitious viruses in cell cultures is well recognized, and when the cultures are of primate origin there are serious hazards for the production of human viral vaccines. This is one reason for the increasing use of bovine cell cultures. These cultures, however, are not free from viral contamination. Calf kidney (CK) and calf testis (CT) cells were often infected by non cytopathic mucosal disease virus (MDV): the cells seemed morphologically healthy, but nearly all showed fluorescence with BVD antiserum and rabbit anti-bovine conjugate.

The risks of infection from whole blood are well-known. One of the great tragedies of modern medicine is the infection of many patients, most frequently hemophiliacs who require frequent blood transfusions, with HIV.

The purification of the nation's and the world's whole blood for transfusion would constitute a monumental step forward in the history of medicine. The risks of infection from red blood cell concentrates is similar to comparable risks associated with whole blood. The teachings of the prior art suggest that neither elemental

(diatomic) iodine nor complexed iodine would be an effective and reliable biocide in a fluid or in a body, e.g. blood, packed or concentrated cells, organs, etc. in which massive amounts of protein are be available to react with the iodine. Various medical and blood handling procedures are referred to hereinafter. These are all well-known procedures and steps in these procedures are fully described in the literature. The following references are provided for general background and as sources for detailed reference to the literature as to specific procedures: TECHNICAL MANUAL of the American Association of Blood Bankers, 9th Ed. (1985); HLA

TECHNIQUES FOR BLOOD BANKERS, American Association of Blood Bankers (1984); Developments in Biological Standardization, Vols. 1 - 57, S. Karger, Basel; CLINICAL IMMUNOCHEMISTRY, The American Association for Clinical Chemistry; MEDICINE, Vols. 1 - 2, Scientific American, New York; Care of the SURGICAL PATIENT, Vols 1 - 2, Scientific American, New York; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley- Interscience, John Wiley & Sons, New York. Summary of the Invention Albumin is known to possess a cytophylactic effect upon blood cells, e.g., red blood cells, and upon other cells and tissues. It is also know and that albumin is particularly effective reducing or totally inhibiting the biocidal power of iodine.

It has now been discovered, in spite of the known fact that albumin has many binding sites for a large variety of molecules, including iodine generally destroys the biocidal power of iodine, that albumin when reacted with sufficient iodine forms a complex somewhat analogous to the previously known iodophors that has the advantages of the best known of the iodophors, povidone iodine, and has significant advantages over povidone iodine. Albumin iodine, abbreviated hereinafter as "ALB-I", is defined for purposes of this invention as a composition of matter consisting essentially of albumin and iodine, the iodine approximately saturating the binding sites on the albumin. A significant portion of the iodine is available for contact and reaction with biological materials and in an amount such that when the ALB-I is dissolved in aqueous solution there exists an equilibrium condition ALB-I ^^~ ALB + I, wherein oxidizing iodine is in solution in an amount of at least about 0.0001 wt percent. The invention, thus encompasses, a process of preparing ALB-I comprising reacting substantially pure albumin, preferrably unsterilized, unstabilized and delipidated albumin, with sufficient iodine-containing reagent to substantially saturate all binding sites thereon

Albumin iodine, "ALB-I", is useful in process in which povidone iodine is useful. In general, a liquid composition that contains bacteria, virus, or other pathogenic organisms can be sterilized by passing it in contact with ALB-I. Thereafter, if it is desired to assure total iodine removal, the solution can be passed into contact with albumin that is less than saturated with iodine, preferably having no more than a trace of iodine or another iodine absorbing material, such as cross-linked povidone to remove the excess iodine. Likewise a reducing agent such as a reducing sugar, ascorbate, sodium sulfite, etc., may be added to eliminate the last traces of oxidizing iodine. Reducing sugars, ascorbic acid (Vitamin C) and its salts, and sodium sulfite are well-known, readily available reducing agents that are physiologically acceptable. However, any physiologically acceptable reducing agents may be used. This invention comprises the use of ALB-I for the manufacture of a medicament consisting essentially of blood cells in plasma or another carrier liquid for the treatment of disorders wherein the patient requires the transfusion of blood cells, the ALB-I being added in an amount in excess of that required to kill or inactivate all microbes therein comprising from 0.1^w/o to 5^w/o of the medicament. Transfusion, transplantation and sperm-containing compositions are similarly prepared.

The invention is embodied in a method of disinfecting biological materials. The steps of the method include treating biological material before separation of the components thereof with ALB-I to provide from a concentration of 0.1^w/o to 5 o ALB-I in said material before separation of the components thereof. A derivative of the material resulting from the preceding step is prepared and, optionally, also treated with ALB-I to provide from 0.1^w/o to 5^w/o ALB-I in the derivative. Also optionally the derivative may be treated by addition of a physiologically acceptable reducing agent or contact with cross-linked PVP to reduce or remove residual iodine. These methods are applicable, for example, to whole blood, plasma, tissue, culture nutrient, sperm cells, packed red blood cells and cell-bearing liquids or non-cell-bearing biological liquids.

The invention also includes drug delivery material comprising blood cell concentrate wherein the cell walls of the cells have been opened by treatment with from 0.1^w/o to 5^w/o ALB-I, a drug has been introduced into the cells through passages produced by the ALB-I treatment, the cell walls have been sealed by heating the ceDs to from 42 to 48 °C. and the resulting material optionally having been treated by addition of a physiologically acceptable reducing agent or contact with cross-linked PVP to reduce or remove residual iodine.

Also included in the invention is the improved method of treating patients with plasma comprising the steps of collecting plasma from a donor, and thereafter infusing the plasma into the patient to be treated, of mixing the plasma with ALB-I sufficient to resulting a ALB-I a concentration of from about 0.1^w/o to about 5^w/o, and allowing contact of said plasma with said ALB-I for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microbes in the plasma and optionally thereafter removing oxidizing iodine from the resulting mixture by passing said mixture into intimate contact with cross-linked PVP or albumin or adding a physiologically acceptable reducing agent. A method of separation of plasma factors by alcohol fractionation is also contemplated. ALB-I is added to plasma before fractionation in concentrations to provide from about l^w/o to about 10^w/o ALB-I in the plasma to give higher yields and sharper differentiation, and optionally thereafter removing oxidizing iodine from the fraction by passing said fraction into intimate contact with cross-linked albumin or adding a physiologically acceptable reducing agent

An apparatus for treatment of liquid to kill microbes is also provided. The apparatus is in the form of a liquid container having, in use an upper reservoir portion for holding said Uquid and a lower elutriation portion for recovering liquid and structure defining first and second beds of particulate matter, the first bed comprising substantially insoluble ALB- I and the second bed consisting essentially of substantially insoluble PVP or albumin; the beds being so formed and configured as to permit the passage of the Uquid therethrough in intimate contact with the surfaces of the particles forming the respective beds. The first bed may be cross- linked PVP. The apparatus may comprising a third layer between the first and second layers, the third layer comprising substantiaUy insoluble PVP hydrogen peroxide particulate matter. The apparatus may contain a layer of particulate matter comprising an iσdiπe reducing^{^} agent. A layer of soluble ALB-I may be provided on the first layer in the Uquid reservoir.

AU or only part of the layers, after the first and second layers, may be provided. One method of sterilizing an implantable tissue in accordance with this invention comprises placing tissue that is physiologically acceptable for implantation into a human patient into a vacuum chamber, evacuating the chamber and maintaining a vacuum on the chamber for a period long enough to extract at least about one-half of the unbound water originally present in said tissue, and introducing into the vacuum chamber a solution of ALB-I for thereby reconstituting into the tissue said solution in place of the water that was vacuum extracted. Optionally, iodine may be removed by washing or reconstituting the tissue with a reducing agent such as ascorbic acid or a salt thereof or sodium sulfite, for example.

Brief Description of the Drawings Figure 1 depicts an apparatus for contacting a Uquid material with ALB-I and with either or both of (a) an iodine absorbing material and/or (b) an iodine reducing material, and for providing other materials for processing biological Uquids, in particular, according to this invention.

Figure 2 depicts, largely schematicaUy, an apparatus for treating soUd tissue samples.

Description of the Preferred Embodiments Albumin-iodine, abbreviated hereinafter as ALB-I. is prepared beginning with substantiaUy pure albumin. The albumin may have traces of other proteins and biological materials of course, and the term "pure" is used in the sense commonly used in reference to biologically isolates that inherently contain some biologicals other than the principal constituent. The degree of purity required is dependent upon the intended use of the albumin of ALB-I and the presence of trace amounts of other materials is not per se detrimental to the present invention. Non- denatured, non-pasteurized albumin free of stabilizers such as caprylate and fatty acids, etc., is preferred. Indeed, the time consuming and expensive steps required in the normal preparation of albumin to kill microbes is essentially surplus or irrelevant since all microbes will be killed by the iodine. Iodine in any form may be reacted with the albumin using any convenient technique or apparatus. It has found convenient to use soUd povidone-iodine as the iodine source because this material provides a convenient source of iodine, has high iodine content and does not add other chemicals or constituents to the resulting ALB-I. A bed of soUd povidone-iodine, cross-linked povidone reacted with iodine or povidone that has been cross-linked by reaction with iodine that binds to the povidone during cross-Unking, or any other soUd povidone iodine may be used. A solution of albumin is caused to pass through a bed or column of soUd povidone iodine where it becomes approximately saturated with iodine. Observations during formation of ALB-I and after the ALB-I has been formed leads to the conclusion that there are, generaUy, two sets of binding sites on albumin, one of binds iodine virtuaUy irreversibly and one of which holds the iodine reversibly, and apparently more on the surface of the molecule. It is not suggested, however, that all binding sites within one of the general types are the same, as it is known that albumin has many binding sites presumably having a great diversity of binding strength for various molecules. Approximately iodine saturated ALB-I is described has albumin in which aU of the irreversible iodine binding sites have been blocked and where a majority, approximately, of the reversible binding sites have iodine bound thereto. The use of ALB-I, optionally followed by treatment with a physiologicaUy acceptable reducing agent for the manufacture of a medicament is contemplated by this invention. Such a medicament may, for example, consist essentially of blood ceUs in plasma or another carrier Uquid. Such medicaments may be used for the treatment of disorders wherein the patient requires the transfusion of blood ceUs. ALB-I is added in an amount in excess of that required to kiU or inactivate all microbes is added. ALB-I may comprise, for example, from about 0.01 to 10 weight percent, preferably from 0.1 /o to 5^w/o of the medicament. The ALB-I is allowed to remain in contact with the blood ceUs or plasma, or other biological material being prepared to be a medicament, for a period of at least about a half a minute sufficient to kill the microbes, but not long enough to denature or otherwise injure the biological material. UsuaUy, contact of under an hour is preferred. Accordingly, the contact times wiU be referred to as from one-half minute to one hour with the caveat that longer contact is not necessary or beneficial and may result in injury to the biological, but would, nevertheless, be within the scope of the invention. The reducing agent is then added in an amount to reduce substantiaUy all iodine. The maximum amount of reducing agent required is easily calculated. The actual amount normaUy required, to which a safety margin amount will be added, is determined by an iodine assay on typical batches using known, routine procedures. A second treatment as described may be performed to assure total sterilization, if desired.

Likewise, a second similar treatment may be performed on a product or fraction of the initial biological material treated as described above.

The use of ALB-I and a physiologically acceptable iodine absorbent material, e.g.,solid albumin or cross-linked povidone, for the manufacture of a medicament is contemplated by this invention. Such a medicament may, for example, consist essentially of blood ceUs in plasma or another carrier Uquid. Such medicaments may be used for the treatment of disorders wherein the patient requires the transfusion of blood ceUs. Either simultaneously therewith, or afterward, ALB-I in an amount in excess of that required to kill or inactivate all microbes is added. ALB-I may comprise, for example, from about 0.01 to 10 weight percent, preferably from 0.1^w/o to 5^w/o of the medicament. The ALB-I is allowed to remain in contact with the blood ceUs or plasma, or other biological material being prepared to be a medicament, for a period of at least about a half a minute sufficient to kill the microbes, but not long enough to denature or otherwise injure the biological material. UsuaUy, contact of under an hour is preferred. Accordingly, the contact times will be referred to as from one-half minute to one hour with the caveat that longer contact is not necessary or beneficial and may result in injury to the biological, but would, nevertheless, be within the scope of the invention. The mixture resulting from the above is then contacted with an iodine absorbing reagent such as cross-linked PVP, or albumin, to remove the iodine. If desired, a reducing agent may thereafter be added in an amount to reduce any iodine that may not have been absorbed. The contact with the iodine absorbing material is preferably accomplished by passing the material undergoing treatment through a layer, i.e. a bed or filter, of soUd, substantiaUy insoluble albumin. A second treatment as described may be performed to assure total sterilization, if desired. Likewise, a second simUar treatment may be performed on a product or fraction of the initial biological material treated as described above.

In a siπular manner, the "addition" of a reducing agent to the material undergoing treatment may be accomplished by passing the material through a layer of substantiaUy insoluble material that has active reducing sites thereon or equilibrates with the Uquid material undergoing treatment to partiaUy dissolve into such Uquid, or make readily available in said Uquid (as by sweUing, for example) reducing moieties. A bed of beads or fibers, for example, that expose on the surface thereof reducing sugar moieties may be used very conveniently.

Reference is made to Figure 1 of the drawing for a better understanding of the invention in one form. Figure 1 depicts an apparatus for contacting a Uquid material with ALB-I and with either or both of (a) an iodine absorbing material and/or (b) an iodine reducing material, and for providing other materials for processing biological Uquids, in particular, according to this invention. The apparatus, being shown and described in a generaUy schematic fashion, may be in any of many configurations. The only significant structure, insofar as this invention relates is to the arrangement of the layers

The apparatus 10 may be viewed as a filter funnel or a column. As those in the art understand, the difference between a filter and a column is often insignificant in that both "filter" a Uquid and both cause the Uquid to contact soUd material. A filter may, indeed must, remove only part of the material. For example, either a filter or a column may let small ceUs or particles pass but retain larger ceUs, or it may permit only Uquid and extremely small particles pass. The apparatus comprises cyUndrical portion 12 that, in part, defines a reservoir portion. The reservoir may be large or very small as desired. The apparatus, in the configuration depicted comprises a second, smaller cyUndrical tube portion 14 and a conical transition zone 16 connecting the two cyUndrical portions as is conventional in funnel manufacture. It is again emphasized, however, that it is immaterial whether the apparatus defines a reservoir and or funnel portion of any particular size or configuration.

The apparatus defines a first layer 20 and a second layer 22. The first layer is made up of substantiaUy insoluble ALB-I. This layer is described as being made up of particulate materials in that the use or particulates in one way or another is usuaUy involved. Particles of soUd, insoluble ALB-I, e.g. cross-linked ALB-I, in the form of a layer or bed of particles, either supported directly by a layer below or by way of another support, e.g. being bonded to or entrapped within a layer of fibers or particles, is contemplated. The first layer may also contain some soluble

ALB-I. A frit made of particles bound together adhesively, by heat or pressure would also be within the disclosure and invention. The ALB-I may be formed in situ by iodinating a layer of albumin or the layer may be made up of pre-synthesized ALB-I. The second layer is downstream of the first layer, i.e. the Uquid to be treated flows through the first layer and then the second layer. The second layer may comprise an insoluble iodine absorbent, e.g. cross-linked povidone, or insolublized albumin, or an iodine reducing agent, or a mixture of both, or be a multiple sub-layer structure with a sublayer of iodine absorbent first and then a sublayer of iodine reductant. Again, the layer may be a self-supporting frit or other structure or may be supported by a support or other layer.

The essential function of the apparatus is to cause a Uquid that is to be treated to pass, with or without ceUs or other particles therein, first through a layer of ALB-I and, thereafter, to contact such Uquid with absorbent to remove the iodine and/or reductant to reduce the iodine. Hence, the layers may be quite deep or quite thin, adjacent each other or spaced from each other, as is necessary or desirable to provide adequate contact of the Uquid with each of the layers or beds.

Such an apparatus is conveniently suited for the treatment of Uquid to kfll microbes in the Uquid. The Uquid container that is generaUy defined by the overaU apparatus in the simphfied, schematic example of

Figure 1, and has an upper or Uquid inflow reservoir portion for holding Uquid to be treated. This may be a very smaU reservoir or quite large. The reservoir may displaced from the beds or layers by a very large distance, though this is not generaUy beneficial. The apparatus has a lower or elutriation or recovery portion for recovering Uquid that has been treated. Between these portions, first and second beds of particulate matter are defined by suitable structure. The first bed or layer comprises substantiaUy insoluble ALB-I. The second bed consists essentiaUy of substantiaUy insoluble albumin, or other iodine absorbent, and/or iodine reducing agent. The beds are so formed and configured as to permit the passage of the Uquid therethrough in intimate contact with the surfaces of the particles forming the respective beds. The usual and most common iodine absorbent is cross-linked povidone.

The apparatus may desirably further comprise a third layer 24 between the first and second layers. The third layer comprises substantiaUy insoluble povidone hydrogen peroxide particulate matter. The presence of the third layer entraps and regenerates iodine and significantly increases the biocidal activity of iodine.

A fourth layer 26, which may be in the form of a sublayer within the second layer, comprising particulate iodine reducing agent may be provided downstream from the second layer to provide for the reduction of any residual iodine from I₂ to iodide, or, if reduction is earUer provided, to add a safety step to assure that all oxidizing iodine has been reduced.

In may appUcations, it may be desirable to provide a fifth layer 28 of soluble ALB-I on the first layer in the Uquid reservoir to permit the actual dissolution into the Uquid of substantial amounts of ALB-I and thereby provide a greater reservoir of more avaflable iodine to the Uquid. The fifth layer may also comprise soluble albumin, preferably low molecular weight (MW < 12,000 daltons) on the top of the first layer for being dissolved into the Uquid to be treated to provide a ceU protective milieu for the ceUs carried by the Uquid. In Uke manner, the fifth layer may comprise soluble ALB-I to provide both iodine and ceU protection. Preferably, at least about one-fourth of all the PVP in solution is low molecular weight PVP, i.e., MW < ~ 15,000.

The first and second layers are essential to the full and proper functioning of the apparatus. After those layers or beds, however, any number of additional layers or additives may be provided, so long as they do not interfere with the combined function of the first and second beds or layers.

AU of the layers just described may, conveniently but not necessarily, be supported by a layer 30 that may be a frit, a filter paper or a porous layer. The thickness of the beds may be the same or greatly different. It is a simple matter to calculate contact time in a column and to provide suitable beds of materials therein.

Any of the beds may be made up the active material, e.g. ALB-I, reducing sugar, etc., attached to carrier particles, such as ground glass, charcoal, ion exchange resin, ceUulose derivatives, etc. The particulate matter may, in a preferred form, consist essentially of particles having a diameter of from about 10 to about 100 microns, but any size that permits suitable flow rates and assures intimate contact may be used.

The use of ALB-I and a physiologically acceptable reducing agent for the manufacture of transfusion biological material from one human or mammal for transfusion of such material to another human or mammal, or the transplant or transfusion biological material is a part of this invention. The transfusion or transplant is disinfected with a ALB-I solution having concentration x>f from about 0.01 to 10 weight percent, preferably 0.1^w/o to 5^w/o, and thereafter treated with the reducing agent to reduce the residual iodine. Liquid materials may be treated in any suitable manner, such as has been described. Solid tissue samples may be treated simply be soaking, by infusing or by vacuum infusing. Figure 2 depicts, largely schematically, an apparatus for treating solid tissue samples. The apparatus comprises a chamber system 100 capable of withstanding the forces of a vacuum. In the merely exemplary form shown, a cyUnder 102 is closed at the respective ends by end covers 104 and 106, the end 106 being removable to gain access to the inside of the chamber. For example, a portion 108 of the end 106 may be slipped into the cylinder 102 and sealed using "O" rings, etc., to provide a vacuum tight seal. A vacuum line 110 through valve 112 and line 114 permits evacuation of the chamber. An input line 120, coupled to valve 122 and line 124 permits the introduction of liquid into the chamber. A platform 126, secured to the end 106, supports a tissue sample 130. The tissue sample is placed in the chamber, the chamber evacuated and then liquid is introduced, thereby substantially replacing water in the sample with the liquid introduced.

Implantable tissues may be treated to kill microbes, i.e. "sterilized" by placing tissue that is physiologically acceptable for implantation into a human patient into a vacuum chamber, evacuating the chamber and maintaining a vacuum for a period long enough to extract at least about one-half of the unbound water originally present in said tissue and then introducing into said vacuum chamber a solution of ALB-I for thereby reconstituting into the tissue said solution in place of the water that was vacuum extracted. The thus treated tissue may then be soaked in a solution of an physiologically acceptable iodine reducing agent. Alternatively, the chamber may again be evacuated to extract the ALB-I solution from the tissue and a solution of physiologically acceptable iodine reducing agent introduced into the vacuum chamber for saturating the tissue for reducing any residual iodine.

As a method of disinfecting blood derivatives, the invention may comprise treating blood before separation of the components thereof with ALB-I to provide from a concentration of from about 0.01 to 10 weight percent, preferably 0.1 /o to 5^w/o, ALB-I in the blood, preparing a derivative of the blood from step, treating the derivative with ALB-I to provide from about 0.01 to 10 weight percent, preferably 0.1^w/o to 5^w/o, iodine in the derivative thereafter treating the derivative by addition of a physiologically acceptable reducing agent or contact with cross-linked PVP to reduce or remove residual iodine.

It is beUeved that ALB-I opens pathways through the ceU waU which permits certain components of the ceU, e.g. potassium salts, to "leak" from the ceU. By the same mechanism, treatment of red blood ceUs with from one to about five percent iodine as ALB-I opens the ceUs to "inward leaking". Thus, compounds which have a virucidal or other effect in the ceU can be introduced into the ceU. ALB-I can, for example, be used as described to increase the uptake of antiviral compounds, e.g. carbenoxolone, AZT, etc., which, in turn, may prevent the replication of virus in the ceU. The net effect of this procedure is a biological synergism. A new drug delivery system involves the use of ALB-I to open pathways through the ceU waU of red blood ceUs. Red blood ceU concentrates are treated as described to open passageways into the ceU. The then permeable ceU is emersed in or treated with a drug which is to be delivered to the patient. The ceU waUs having passages therethrough permit the drug to enter into the ceU. Thereafter, the iodine may be removed and the ceU concentrate is heated to 42 - 48 °C to seal the ceU waUs. The concentrated ceUs are then infused into the patient where they carry out the normal function of such ceUs. These ceUs have a finite Ufe. As the ceUs age, they lyse, thereby releasing the drug directly into the blood stream where the drug can become effective.

ALB-I is Unked to hemoglobin to produce a hemoglobin product which as a greatly increased Ufe as an oxygen carrier when introduced into the blood circulation system. ALB-I-hemoglobin complex in water or saline solution, thus, constitutes a blood substitute which can be stored with minimal risk of microbial contamination and used as a blood extender in emergency situations such as may be found on the battlefield or in remote areas. Ratios of ALB-I to hemoglobin of from about 0.1:1.0 to about 1.0:0.1 are considered satisfactory. The fact that ALB-I binds very strongly to hemoglobin aUows for the development of a blood substitute. Final traces of iodine may be removed as described above using ascorbate, or any other suitable physiologicaUy acceptable reducing agent, or by passing the product through a bed or layer of cross-linked PVP.

Infective pathogenic microorganisms are beUeved to be inactivated when ALB-I is used in solution to perfuse tissues and organs after removal from the donor and before transplantation to the recipient. The perfusion solution contains molecular iodine compound in a concentration of from about 0.01 to 10 weight percent, preferably 0.1^w/o to about 5^w/o (100 to 5000 ppm I₂), preferably from about 0.25^w/o to about 2^w/o. After a period of time, most of the unreacted molecular iodine compound is washed away and any residual molecular iodine compound is absorbed into the protein or converted to inactive iodides, e.g. using ascorbate or other reducing agent as described, and does not significantly interfere with acceptance by the recipient.

Sperm-bearing solutions treated to be made freed of pathogenic microbes by washing and/or storing the sperm in a solution which contains a concentration of ALB-I in a concentration is from about 0.01 to 10 weight percent, preferably 0.1 /o to about 5 o, (100 to 5000 ppm I₂) and, preferably, sufficient to inactivate bacteria, viruses and other pathogenic organisms, and washing the sperm ceUs in the solution, optionally with a solution of a reducing agent compound.

ALB-I is considered to be effective in protecting the sperm ceUs from the spermicidal activity of iodine sufficient to permit the killing of pathogenic organisms whUe leaving viable, motile sperm ceUs suitable for artificial insemination. The washing is continued or repeated to assure that substantiaUy all of the seminal fluid is replaced with ALB-I solution. Other reagents such as are conventionally used in sperm treatment, storage and preparation, or for particular purposes may, of course, also be included in the infusion solution. If desired, residual iodine may be washed out and/or removed using ascorbate or other reducing agent and any suitable storage fluid, including solutions of polyvinyl pyrrolidone, may be used to store and handle the sperm ceUs.

The above appUcations in which the material to be purified is a Uquid or ceUs carried in a Uquid can be carried out by flowing the Uquid through a bed (e.g. the conventional filter structure of soUd particles on a porous or foraminous support) of soUd particles of ALB-I of suitable size or by contacting the Uquid and/or the ceUs in the Uquid with particles or a membrane or surface of sohd ALB-I. Where a bed of particles is used with a ceU-bearing Uquid, the particles must be large enough to permit intimate contact without entrapping or binding the ceUs. The Uquid may then be passed through a layer or in contact with soUd phase ALB-I to assure complete biocidal effect. Thereafter, the Uquid is passed through or into intimate contact with cross-linked PVP to absorb the molecular iodine from the Uquid. Finally, a reducing agent such as ascorbate may be added if considered necessary as a precaution.

In carrying out this facet of the invention, the Uquid or ceU-bearing Uquid is contacted with the soUd ALB-I. This may be done most efficiently, in most cases, by passing the Uquid through a settled or fluidized or packed bed of ALB-I particles; however, such approaches will not, ordinarily, be suitable for treating ceU-bearing Uquids. CeU-bearing

Uquids may be treated by mixing the particles in a container of the Uquid or passing the Uquid over a surface of the ALB-I material, e.g. over a multiple-plate array of sheets of such material. The ALB-I may be washed and the iodine content therein regenerated between uses. In general a solution of reducing agent , e.g. a reducing sugar (or mixtures of reducing sugars), ascorbic acid or ascorbate, a sulfite, e.g. sodium sulfite, etc. in which the agent is in a concentration of 0.001 to 1 percent is suitable and such is impUcit unless otherwise noted.

Industrial Application This invention finds appUcation in medicine and veterinary science.

Claims

WHAT IS CLAIMED IS:

1. The use of ALB-I for the manufacture of a medicament consisting essentiaUy of blood ceUs in plasma or another carrier Uquid for the treatment of disorders wherein the patient requires the transfusion of blood ceUs, the ALB-I being added in an amount in excess of that required to kiU or inactivate aU microbes therein comprising from 0.01^w/o to 10 /o of the medicament.

2. The use of ALB-I and a physiologicaUy acceptable reducing agent in the manufacture of sperm ceU-containing compositions for the induction of pregnancy in a female by inseminating the sperm ceUs into the female, the sperm ceUs being washed with ALB-I in water solution in a concentration of from 0.01^w/o to 107o, sufficient to kiU bacteria, viruses and other pathogenic micro-organisms but insufficient to inactivate the sperm ceUs and optionaUy adding said reducing agent in an amount to reduce substantiaUy aU of the iodine.

3. The use of ALB-I and a physiologically acceptable reducing agent for the manufacture of transfusion biological material from one human or mammal for transfusion of such material to another human or mammal, the transplant or transfusion biological material being disinfected with a ALB-I solution having concentration of from 0.0 l^w/o to 10^w/o and optionaUy thereafter adding said reducing agent to reduce the residual iodine.

4. A method of disinfecting biological fluid comprising blood plasma, said method comprising the steps of: (a) treating said biological fluid comprising blood plasma with albumin-iodine complex to provide from a concentration of 0.01^w/o to 107o albumin-iodine complex in said fluid; and

(b) adding a reducing agent to said fluid to remove residual iodine.

5. A drug deUvery material comprising blood ceU concentrate wherein the ceU waUs of the ceUs have been opened by treatment with from 0.⁰¹w/o to 107o ALB-I, a drug has been introduced into the ceUs through passages produced by the ALB-I treatment, the ceU waUs have been sealed by heating the ceUs to from 42 to 48 °C. and the resulting material optionally having been treated by addition of a physiologically acceptable reducing agent or contact with cross-linked PVP to reduce or remove residual iodine.

6. In the method of separation of plasma factors by alcohol fractionation, the improvement comprising the addition of ALB-I to the plasma before fractionation in concentrations to provide from about V/o to about 10^w/o ALB-I in the plasma to give higher yields and sharper differentiation, and optionally thereafter removing oxidizing iodine from the fraction by passing said fraction into intimate contact with cross-linked albumin or adding a physiologically acceptable reducing agent.

7. An apparatus for treatment of Uquid to kill microbes therein comprising a Uquid container having, in use an upper reservoir portion for holding said Uquid and a lower elutriation portion for recovering Uquid and structure defining first and second beds of particulate matter, the first bed comprising substantiaUy insoluble ALB-I and the second bed consisting essentially of substantiaUy insoluble PVP or albumin; the beds being so formed and configured as to permit the passage of the Uquid therethrough in intimate contact with the surfaces of the particles forming the respective beds.

8. The apparatus of Claim 7 wherein the substantiaUy insoluble PVP is cross-linked PVP.

9. The apparatus of Claim 7 further comprising an additional layer between the first and second layers, said additional layer comprising substantiaUy insoluble PVP hydrogen peroxide particulate matter.

10. The apparatus of Claim 7 further comprising an additional layer of particulate matter below the second layer, said additional layer comprising an iodine reducing agent.

11. The apparatus of Claim 7 further comprising an additional layer of soluble ALB-I on the first layer in the Uquid reservoir.

12. The apparatus of Claim 11 wherein the insoluble ALB-I particles of the first layer are physicaUy supported by a layer of fibrous material.

13. A method of sterilizing an implantable tissue comprising:

(a) placing tissue that is physiologicaUy acceptable for implantation into a human patient into a vacuum chamber;

(b) evacuating said chamber and maintaining a vacuum on said chamber for a period long enough to extract at least about one-half of the unbound water originally present in said tissue; and

(c) introducing into said vacuum chamber a solution of ALB-I for thereby reconstituting into the tissue said solution in place of the water that was vacuum extracted;

14. The method of Claim 13 further comprising the step of soaking the thus treated tissue in a solution of an physiologically acceptable iodine reducing agent.

15. The method of Claim 13 further comprising the foUowing steps after said step (c):

(d) evacuating the chamber to extract the ALB-I solution; and

(e) introducing a solution of physiologically acceptable iodine reducing agent into the vacuum chamber for saturating the tissue with said solution for reducing any residual iodine.

16. The process of preparing ALB-I comprising reacting substantiaUy pure albumin with sufficient iodine-containing reagent to substantiaUy saturate aU binding sites thereon.

17. The process of Claim 16 wherein the albumin is deUpidated albumin.

18. The process of Claim 16 wherein the albumin is unsterilized and has not been stablized.

19. A method of disinfecting biological fluid comprising blood plasma, said method comprising the steps of:

(a) treating said biological fluid comprising blood plasma with albumin-iodine complex to provide from a concentration of 0.017o to 10^w/o albumin-iodine complex in said fluid; and

(b) contacting said fluid with cross-linked povidone to remove residual iodine.

20. A method of disinfecting blood plasma comprising the steps of:

(a) treating blood plasma with albumin-iodine complex to provide from a concentration of 0.01^w/o to 10^w/o of said complex in said plasma;

(b) separating the blood plasma into constituents thereof; (c) treating at least one constituent separated from said plasma with albumin-iodine complex and optionally

(d) adding a reducing agent to said constituent.

21. A method of disinfecting blood plasma comprising the steps of: __ (a) treating blood plasma with albumin-iodine complex to provide from a concentration of 0.017o to 10^w/o of said complex in said plasma;

(b) separating the blood plasma into constituents thereof;

(c) treating at least one constituent separated from said plasma with albumin-iodine complex and optionally

(d) passing the constituent into contact with cross-linked povidone to remove iodine from said constituent.