AU648823B2

AU648823B2 - Antimicrobial preservation of plasma

Info

Publication number: AU648823B2
Application number: AU85118/91A
Authority: AU
Inventors: Edward Shanbrom
Original assignee: Individual
Current assignee: Individual
Priority date: 1990-09-04
Filing date: 1991-09-03
Publication date: 1994-05-05
Anticipated expiration: 2011-09-03
Also published as: AU8511891A; CA2072749A1; JPH05502241A; EP0498871A1; EP0498871A4; WO1992004061A1

Description

ANTIMICROBIAL PRESERVATION OF PLASMA

Background of the Invention This invention relates to the treatment and preservation of blood plasma and plasma derivatives and to the protection of technicians, nurses and physicians and of the ultimate recipient patient from infection by plasma-borne microbes.

Definitions The following terms, which are used throughout the specification, will be used and understood to have the meaning stated unless another or different meaning is specified or clear from the context. Tissue cultures. Tissue 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 tissue cultures. An 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, standards and samples. Laboratory reagents and standards, as used in this specification and the claims, means reagents and standards produced from or comprising human or animal fluids.

Examples of such products are control sera and blood plasma chemistry controls.

Donor. 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 plasma is obtained for any purpose, and such term will be used to refer even to an unwilling donor. In the application of the invention to animals, the donor may be a mammal or the fetus of a mammal.

Povidone (USP) is used in the sense that it is used in the U.S. Pharmacopeia to identify polyvinyl pyrrolidone suitable for use in physiolog¬ ically acceptable solutions. Molecular Iodine Compound. The term "molecular iodine compound" is used in this patent to mean and include molecular iodine, I, diatomic iodine, I₂, or a compound or a mixture of compounds which either comprises iodine available in molecular form, typically as diatomic I₂, or which reacts with or in the presence of the sample to produce such iodine. Povidone-iodine is the principal example of such compounds. Povidone-iodine. Povidone-iodine is a complex of molecular iodine with polyvinyl pyrrolidone. Povidone-iodine complexes of the type under consideration have been described in the literature and are marketed by The Purdue-Frederick Co. When percent concentrations are referred to in connection with povidone-iodine, the percentage refers to the percent of povidone-iodine by weight, based upon the weight of the solution or material to which the povidone-iodine is added. Thus, a 1 weight percent (^w/o) solution of povidone-iodine indicates that enough povidone-iodine has been dissolved to result in a concentration of l^w/o povidone-iodine giving a concentration of 1000 ppm I₂. In most instances, povidone-iodine is added as a solution, e.g. 10% solution in water, pH about 1.5, but it can be added as a powder or otherwise. Povidone-iodine powder contains approximately 85% PVP, 10 % I₂ and 5%Iodide. A 10% solution of this powder contains 1 % free, available iodine. (Gershenfeld, Am. J. Surgery 94, 938 (1957)). The ratio of polyvinyl pyrrolidone to iodine in the povidone-iodine product used in the experiments referred to hereinafter is 8.5 parts of povidone-iodine per 1 part of active iodine. The product also contains about 0.5 parts of inactive iodine as iodide. Typical stock solutions are 10% (10,000 ppm I₂), 5% (5,000 ppm I₂) and 1 % (100 ppm I₂). In those instances in which a povidone to iodine ratio of higher than about 8.5 to 1 is referred to, additional povidone (polyvinyl pyrrolidone) is added to increase the PVP to I₂ ratio. The concentration of povidone-iodine in such compositions means the concentration of povidone-iodine added as 8:5 to 1 PVP to I₂ povidone-iodine, i.e. 1000 ppm I₂.

Nutrient. Nutrient, as used here, refers to materials which are derived in whole or in part from animal sources, such as fetal bovine serum (FBS), or which may become contaminated from other sources with pathogenic microbes or toxin- or pyrogen-producing microbes. Nutrient is distinct from tissue culture medium in that nutrient should be free of propagating microor¬ ganisms and, when incorporated in added to a tissue culture medium, provides nutritional components, e.g. minerals, amino acids, etc., to support propaga¬ tion of desired cellular components with which the medium is inoculated or which are maintained in the medium.

Viable. Viable as used herein refers to cells or microorganisms which are capable of replicating or reproducing such as, for example, tissue, bacteria, protozoa, etc, and virus which under favorable circumstances are capable of self-propagation. Those who deal with blood plasma, and the components thereof, e.g. sera, cryoprecipitate, etc., risk infection from pathogens borne in 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 furnace.

The risk of contracting disease is especially great in the handling of pooled plasma. All plasma pools contain infectious microbes and all who work with pooled plasma eventually contract one or more diseases from the plasma. Every technician who has long-term exposure to pooled plasma in the early stages of processing, e.g. during the initial pooling, spin down, etc. will eventually become infected with hepatitis. The risk of illness from pathogens borne in pooled plasma and the certainty that one will eventually become infected and, at least, become a carrier of pathogens is a long-standing problem in the blood banking industry for which no suitable solution has been forthcoming.

The risk to health care personnel 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 plasma and fluid sampling and handling apparatus, are exposed include hepatitis and human immuno¬ deficiency virus (HIV) as well as a large number of less life-threatening viruses.

Another organism which may contaminate blood plasma and blood plasma products or fractions and which presents a serious risk is the bacteria Yersinia enrerocolirica. It surpasses Shigella and rivals 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., Transfitsion 30, 3, p.207 (1990). Y. enterocolitica and other bacteria which propagate at relatively low temperatures, e.g. Staphylococcus epidermis and Legionella pne monophilia, present, potentially, a serious threat in blood plasma products. Bacterial infections are a continuing concern to blood banking industry.

Indeed, a national surveillance system for transfusion-associated bacterial infections has been called for, Editorial, Transfitsion 30, 3, p. 193 (1990).

Cytomegalovirus (CMV), probably the most ubiquitous of the pathogenic microorganisms found in animal fluids and tissues. CMV is frequently associated with, and may be a causative or contributing factor in, life threatening disease in individuals with suppressed immune systems, and can be a principal causative factor in pneumonia, neurological disorders, febrile illness, ocular disease and hepatitis. CMV infection is a serious limiting factor in the transfusion and plasma from one individual to another. Recipients of plasma run a serious risk of CMV infectious disease, the risk being multiplied where the immune system of the recipient is suppressed to prevent rejection of the foreign organ or cells, or where immunosuppression is present from natural causes.

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. Three subfamilies are of particular importance. The alpha subfamily includes HV 1 (herpes virus simplex 1) which causes cold sores, fever blisters, eye and brain infections, HV 2 (herpes virus simplex 2) which cause genital ulceration, and HV 3 (HV varicella zoster) which causes chicken pox, shingles and brain infections. The beta subfamily includes HV 5, the principal member of which is CMV discussed above. The gamma subfamily includes HV 4 (Epstein-Barr) which cause infectious mononucleosis and is involved in Burkitt's lymphoma and nasopharyngeal carcinoma.

In addition to the risk of transmitting infectious disease via blood plasma, the growth of bacteria in blood plasma products at various stages of production and processing introduces pyrogens into the blood plasma component or product which must be removed before the product can be used in therapy.

Nutrient for tissue culture media always contains pathogenic organisms which, without removal or treatment, destroy or greatly impair the value of the nutrient in tissue cultures. 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 use of elemental iodine as an antiseptic dates back to 1839. It is used today for various medicinal purposes. The combination of iodine with various solubilizing polymers led to a class of new compositions known as iodophors, which dominate the market once satisfied by simple alcoholic or aqueous iodine solutions. The iodine complexes with either nonionic surfactants, eg, polyethylene glycol mono(nonylphenyl)ether, or poly(vinyl- pyrrolidone) (PVP). The complexes function by rapidly liberating free iodine in water solutions. They exhibit good activity against bacteria, molds, yeasts, protozoa, and many viruses; indeed, of all antiseptic preparations suitable for direct use on humans and animals and upon tissues, only povidone-iodine is capable of killing all classes of pathogens: gram-positive and gram-negative bacteria, mycobacteria, fungi, yeasts, viruses and protozoa. Most bacteria are killed within 15 to 30 seconds of contact. These iodophors are generally nontoxic, nonirritating, non-sensitizing, and noncorrosive to most metals (except silver and iron alloys).

Iodine and iodine-containing compounds and preparations are employed extensively in medicine, eg, as antiseptics, as drugs administered in different combinations in the prophylaxis and treatment of certain diseases, and as therapeutic agents in various thyroid dyscrasias and other abnormalities. Iodine is a highly reactive substance combining with proteins partly by chemical reaction and partly by adsorption. Therefore its antimicrobial action is subject to substantial impairment in the presence of organic matter such as serum, blood, urine, milk, etc. However, where there is no such interference, non- selective microbicidal action is intense and rapid. A saturated aqueous solution of iodine exhibits anti-bacterial properties. However, owing to the low solubility of iodine in water (33 mg/100 ml at 25 °C. ), reaction with bacteria or with extraneous organic matter rapidly depletes the solution of its active content. Iodide ion is often added to increase solubility of iodine in water. This increase takes place by the formation of triiodide, I₂ + I^" = I₃ ^". An aqueous solution of iodine and iodide at a Ph of less than 8 contains mainly free diatomic iodine 12 and the triiodide I₃ ^". The ratio of 12 and I₃- depends upon the concentration of iodide. Medicinal povidone-iodine preparations include aerosol sprays, gauze pads, lubricating gels, creams, solutions, douche preparations, suppositories, gargles, perineal wash solutions, shampoos, and skin cleansers and scrubs. Povidone-iodine preparation are applied topically to the skin and to membranes, e.g. vaginal membranes, and in infected wounds and surgical incisions. An important solubilizing agent and carrier for iodine is polyvinyl pyrrolidone (PVP), one grade of which is identified as povidone USP. Povidone-iodine (PVP-iodine), is widely used externally on humans as an antiseptic. Such products are marketed as Betadine™ and Isodine™. Povidone- iodine products and the preparation of such products are described in U.S. Patents 2,707,701 , 2,826,532, and 2,900,305 to Hosmer and Siggia, assigned to GAF Corporation and in a number of GAF Corporation publications; see, e.g. Tableting with Povidone USP (1981) and PVP Polyvinylpyrrolidone (1982). Povidone-iodine powder contains approximately 85% PVP, 10 % l₂ and 5%Iodide. A 10% solution of this powder contains 1 % free, available iodine. (Gershenfeld, Am. J. Surgery 94, 938 (1957)).

Under ordinary conditions, PVP is stable as a solid and in solution. The single most attractive property of PVP is its binding capability. This property has permitted utilization in numerous commercial applications. Small quantities of PVP stabilize aqueous emulsions (qv) and suspensions, apparently by its absorption as a thin layer on the surface of individual colloidal particles. The single most widely studied and best characterized PVP complex is that of PVP-iodine. For example, hydrogen triiodide forms a complex with PVP that is so stable that there is no appreciable vapor pressure. It is superior to tincture of iodine as a germicide. It is well-known that the efficacy of iodine and povidone-iodine as a disinfectant is still 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 plasma, 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 whole 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. Ued. 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 ^12SI 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, J. of Immunol. 81, 1265 (1981) reported that povidone-iodine was absorbed in serum albumin and it is known that povidone-iodine is bound to albumin but it has been discovered that the antimicrobial activity of povidone-iodine is not destroyed by albumin binding. Whether the activity remains because the albumin povidone-iodine is active or whether povidone-iodine and/or iodine are released from the albumin-povidone- iodine complex has not been determined. The teachings of the prior art suggest that neither elemental (diatomic) iodine, l₃, nor complexed iodine, e.g. PVP-I,, would be an effective and reliable biocide in blood plasma or plasma derivatives.

Various medical and blood plasma 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 IMMUNOCHEMIS-

TRY, The American Association for Clinical Chemistry; MEDICINE, Vols. 1 - 2, Scientific American, New York; CARE of the SURGICAL PAΗENT, Vols 1 - 2, Scientific American, New York; CURRENT PROTOCOLS IN MOLECU¬ LAR BIOLOGY, Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons, New York.

Summary of the Invention

This invention is embodied in, inter alia, a method of treating patients with blood plasma comprising the steps of collecting plasma from a donor, and thereafter infusing the plasma into the patient to be treated. The improvement of this invention comprises the additional steps of mixing the plasma with molecular iodine compound to result in a concentration from 0.1^w/o to 2 o,

(100 to 2000 ppm I₂) the preferred range being from about 0.257o to about

0.5^w/o (250 to 500 ppm I₂), and allowing contact of said plasma with said molecular iodine compound for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microbes. The time of contact is not critical; however, longer contact times, e.g. up to 24 hours or more result in increased biocidal effect. Generally, however, effective biocidal action can be obtained in a short period of from one-half to two minutes contact.

This invention is embodied in, inter alia, a method treating plasma- containing nutrient for culture media to prevent the propagation of microbes in tissue culture media. The invention comprises the steps of mixing said plasma constituent of the nutrient, either neat or after mixing with other nutrient components, with molecular iodine compound to result in a concentra¬ tion of from 0.17o to 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm I₂), and allowing contact of said nutrient with said molecular iodine compound for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microorganisms and thereafter infusing the nutrient into a tissue culture medium.

This invention is embodied in, inter alia, an article of commerce, namely nutrient for use in tissue culture media. The nutrient is serum- containing liquid containing iodine introduced as a povidone-iodine in a concentration of from 0.17o to 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm I₂), sufficient to inactivate or destroy infective pathogenic microorganisms therein, said nutrient being free of both viable and viable microorganisms. This invention is embodied in, inter alia, a method of controlling a cell line comprising adding povidone-iodine to the tissue culture nutrient which supports cell replication in a concentration of from 0.17o to 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm I₂), based on the nutrient sufficient to arrest or inhibit the propagation of the cell line but insufficient to kill the cell line and harvesting the composition of interest after such composi¬ tion has been expressed by the cell line.

This invention is embodied in, inter alia, a method of purifying plasma by contacting the liquid to be purified into contact with solid povidone-iodine having sufficient surface area to expose the liquid to sufficient iodine on such surface to kill pathogenic organisms therein, and removing the liquid from contact with the solid povidone-iodine. The method may further comprise reacting the surface of the solid povidone-iodine with iodine between uses to regenerate the iodine content thereof.

This invention is embodied in, inter alia, a method of treating a patient which comprises the step of collecting plasma from the patient or another, preserving the plasma by refrigeration or freezing, warming and, if the plasma is frozen, thawing the plasma, and infusing the biological material into the patient. The plasma is treated with molecular iodine compound to result in a concentration from 0.17o to 27o, the preferred range being from about 0.257o to about 0.5^w/o, and allowing contact of said plasma with said molecular iodine compound for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microbes. The mixing of the plasma with povidone-iodine is carried out in three sub-steps, namely, first, introducing povidone-iodine into the plasma in a concentration of from about 0.1 to 27o, the preferred range being from about 0.257o to about 0.57o; second, maintaining the plasma in contact with the povidone-iodine for a period of about one-half minute or longer; and, third, again introducing povidone-iodine into the plasma in a concentration of from about 0.17o to 27o, the preferred range being from about 0.257o to about 0.57o. Mixing of the plasma with povidone-iodine before refrigerating for storage and/or freezing is preferred, but this step can be carried out at low temperatures, e.g. about 4°C. to 6°C, or after thawing. This invention is embodied in, inter alia, a method of disinfecting plasma derivatives comprising treating plasma before separation of the compo¬ nents thereof with povidone-iodine into the plasma in a concentration of from about 0.17o to 27o, the preferred range being from about 0.257o to about 0.57o, preparing the derivative of the plasma from the preceding step and treating the derivative from the next preceding step with povidone-iodine into the plasma derivative in a concentration of from about 0.17o to 27o, the preferred range being from about 0.257o to about 0.57o.

In one preferred embodiment, a method of treating patients with plasma comprises the steps of collecting plasma from a donor, and thereafter infusing the plasma into the patient to be treated, with the improvement of mixing the plasma with povidone-iodine with added povidone to give a povidone to iodine ratio of at least about 12: 1, preferably from about 15: 1 to 30: 1 and optionally up to about 60: 1 , sufficient to result in an iodine concentration of from about 0.17o to about 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm l₂), and allowing contact of said plasma with said povidone-iodine with added povidone to give a povidone to iodine ratio of at least about 12:1, preferably from about 15: 1 to 30: 1 and optionally up to about 60: 1, for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microbes in the plasma before infusing the plasma into the patient. In another embodiment, nutrient for culture media is treated to prevent the propagation of microbes in tissue culture media by mixing said nutrient with povidone-iodine with added povidone to give a povidone to iodine ratio of at least about 12 to 1 , preferably from 17: 1 to 30:1, in a concentration to produce a povidone-iodine concentration of from about 0.17o to about 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm I₂), and allowing contact of said nutrient with said povidone-iodine for at least about one-half minute sufficient to inactivate or destroy infective pathogenic microorganisms to thereby produce a nutrient which is free of both viable cells and viable microorganisms. In another embodiment, the invention is an article of commerce consisting essentially of nutrient free of viable cells and viable microbes for use in tissue culture media consisting essentially of serum-containing liquid containing iodine introduced as a povidone-iodine with added povidone to give a povidone to iodine ratio of at least about 12 to 1, preferably from 15:1 to 30: 1 , in an amount sufficient to produce a povidone-iodine concentration of from about 0.1 o to about 27o, sufficient to inactivate or destroy infective pathogenic microorganisms therein, said nutrient being free of both viable cells and viable microorganisms. The povidone-iodine is preferably introduced sufficient to produce a povidone-iodine concentration of from about 0.257o to about 0.57o. The invention is also embodied in a method of controlling a cell line by adding povidone-iodine with added povidone to give a povidone-iodine ratio of at least about 12: 1, preferably from about 15: 1 to 30: 1 and optionally up to about 60: 1, sufficient to protect the cell line from destruction, to the tissue culture nutrient that supports cell line replication in a concentration to produce a povidone-iodine concentration of from about 0.17o to about 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm I₂), based on the media, sufficient to arrest or inhibit the propagation of the cell line but insufficient to kill the cell line and insufficient to prevent the cell line from expressing a composition and harvesting said composition after such composition has been expressed by the cell line.

Another embodiment of the invention is a method of disinfecting plasma derivatives comprising (a) treating plasma before separation of the components thereof with povidone-iodine with added povidone to give a povidone to iodine ratio of at least about 12 to 1 , preferably from 15: 1 to 30: 1 , in an amount sufficient to provide from about 0.17o to about 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm I₂), povidone-iodine in the plasma, (b) preparing a derivative of the plasma from step (a), and (c) treating the derivative from step (a) with povidone-iodine to provide from about 0.17o to about 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm l₂), povidone-iodine in the derivative.

A method of separation of plasma factors by alcohol fractionation in which the improvement comprises the addition of povidone-iodine to the plasma before fractionation, the povidone-iodine with added povidone to give a povidone to iodine ratio in the plasma of at least about 12 to 1, preferably from 15: 1 to 30: 1 , in concentrations to provide from about 0.017o to about 27o, preferably from about 0.257o to 0.57o (250 to 500 ppm l₂), povidone- iodine to give higher yields and shaφer differentiation is also disclosed. The concentration in the plasma is preferably from about 0.257o to about 0.57o. An improvement in a method of separation of plasma fractions by cryoprecipitation comprising the addition of povidone-iodine to the plasma before cryoprecipitation, the povidone-iodine with added povidone to give a povidone to iodine ratio in the plasma of at least about 12 to 1, preferably from 15: 1 to 30:1 , in concentrations to provide from about 0.017o to about 27o povidone-iodine to give higher yields and shaφer differentiation is also disclosed. The concentration in the plasma is preferably from about 0.257o to about 0.57o.

Povidone' s known value as a plasma expander becomes an element in a greatly improved method of preparing plasma products which are also free of pathogenic organisms. Plasma-derived products which are used in diagnostic tests without altering the test results, with the single exception of an increase in iodine, may be prepared from plasma or plasma derivatives treated as described.

Description of the Preferred Embodiments A number of non-limiting exemplary embodiments of the present invention are given hereinafter, it being clearly stated that these are simply examples and are not limiting as to the scope of the inventive concept.

Quite suφrisingly, it has been discovered that molecular iodine compound, e.g. povidone-iodine, in the plasma in a concentration of from about 0.17o to 27o, the preferred range being from about 0.257o to about 0.57o, do not alter the results of conventional diagnostic tests nor cause significant hemolysis or other change in plasma. In generally, conventional diagnostic procedures may be followed without alteration to accommodate to the presence of the iodine. Povidone-iodine is an effective preservative solution, used as described above, for plasma preparations used as laboratory standards.

Plasma is be treated in accordance with this invention by introducing molecular iodine, e.g. povidone-iodine, into the plasma in a concentration of from about 0.17o to 27o, the preferred range being from about 0.257o to about 0.57o in the plasma. Preferably, in all embodiments, the povidone- iodine has a povidone to iodine ratio of at least about 12: 1, preferably from about 15: 1 to 30: 1 and optionally up to about 60: 1. A 0.25^w/o concentration, for example, in plasma provides a total kill of bacteria, virus and other pathogenic organisms. Most suφrisingly, the microbe kill is effective before the iodine reacts with other reducing agents in the plasma to the extent of reducing or eliminating the microbial activity of iodine, and without altering the essential characteristics of plasma. Indeed, the addition of povidone-iodine renders the cryoprecipitation and subsequent separations, e.g. alcohol separa¬ tion of fibrinogen, fibronectin and the factors, e.g. Factor VIII, more complete, with a cleaner separation and a higher yield of factors, etc.

Pooled plasma processes are an important and essential part of the production of many plasma products, particularly those components and fractions which are found in low concentration in the plasma. The risk of infection to workers and, potentially to users of the product, increases as the number of donor-samples are pooled. Inevitably, every large lot of pooled plasma carries a significant microbial contaminant load and presents a serious risk of infection to workers and technicians who are exposed to such products.

Pathogenic microbes in plasma and plasma products can be eliminated, without interference with other plasma treatment and processing procedures, by adding molecular iodine, e.g. povidone-iodine, into the plasma in a concentration of from about 0.17o to 27o, the preferred range being from about 0.257o to about 0.57o, preferably early in the process of collecting and pooling the plasma.

Substantial amounts of plasma in or from bags from individual donors and, sometimes, pooled plasma are frozen and preserved as fresh frozen plasma (FFP) for use in emergencies and in surgeries to replace lost blood. The industry and surgeons recognize a great risk in using FFP but large quantities, over one million units per year, of FFP are used because safe alternatives are not available. The addition of povidone-iodine into the plasma in a concentra¬ tion of from about 0.17o to 27o, the preferred range being from about 0.257o to about 0.57o to such plasma, preferably before freezing, renders an otherwise risk-burdened product perfectly safe. One result of this invention is that very large quantities of plasma, millions of pints, which heretofore has been too unsafe for use as FFP can now be used to save lives without risk of infection.

In plasma and serum, 6+ and 7+ log kills, i.e. total kills, of VSV. Tables I and II demonstrate the effectiveness of the methods described here in whole blood and in plasma.

Table I Kill of Vesicular Stomatitis Virus in Whole Blood and Washed

LOG KILL IN »

Whole Blood Whole Blood Whole Blood Whole Blood Whole Blood Rinsed RBC Rinsed RBC Rinsed RBC Rinsed RBC Rinsed RBC Whole Blood

Whole Blood Whole Blood Whole Blood

POSITIVE CONTROL Whole blood heated to 45°c. Total logs of VSV

POSITIVE CONTROL Whole blood Total logs of VSV

TABLE II - KILL OF VSV IN PLASMA WITH PVP AND PVP-I

LOG KILL IN > > > 1 HOUR 24 HOURS

1

2

24 1 % PVP K-26-28 + 0.25% PVP-I

TABLE II - CONTINUED

o-

A very important advantage results from the use of povidone-iodine in the treatment of plasma to kill bacteria and virus in that the PVP becomes an effective plasma expander. Thus, both the quality and the quantity of the plasma product is very substantially increased as compared with previous methods of treatment.

An interesting discovery was made respecting polyvinyl pyrrolidone, alone, in the course of studying the present invention. It was discovered that polyvinyl pyrrolidone alone was capable of killing up to 5 logs of virus in body fluids. Whether or not there is a synergism vis-a-vis this anti-viricidal activity and the activity of iodine has not yet been determined.

Cryoprecipitate may be produced from individual donor plasma or from pooled plasma. The risks of infection attendant to the preparation and use of cryoprecipitate-derived products are substantially eliminated by the present invention. The cryoprecipitate may, in effect, be treated by the treatment of the whole plasma or the plasma, or both, from which the cryoprecipitate is derived. In lieu of, or in addition to, such pre-treatment, the cryoprecipitate may be treated to kill bacteria and virus using tlk present invention. Molecular iodine, e.g. povidone-iodine, is introduced in a concentration of from about 0.17o to 27o, the preferred range being from about 0.257o to about 0.57o, into the plasma sufficient to kill the bacteria and virus. The plasma is then frozen. When the resulting cryoprecipitate is thawed there is an increase in the yield of cryoprecipitate, fibrinogen, fibronectin and Factor VIII, and the product is free of microbes.

Higher concentrations of povidone-iodine, e.g. greater than about 17o or 27o up to as high as about 57o, or an increase in the povidone to a povidone to iodine ratio of 15 to 1 or more, preferably about 17: 1 to 30: 1, can cause the precipitation of Factor VIII and thus present a valuable process for the production of Factor VIII in high yield and which is free of microbic contamination.

Likewise, other plasma fractions and plasmas from other species may be treated in a similar manner with povidone-iodine.

Plasma factors which are separated by alcohol fractionation, e.g. Factor VIII, are separated in higher yield and shaφer differentiation when the alcohol- containing fractionation liquid contains povidone-iodine. In addition, of course, the resulting fraction is free of microbic contamination. Infective pathogenic microorganisms are inactivated when molecular iodine compound is added to fetal bovine serum (FBS) or to nutrient containing FBS. The povidone-iodine is introduced into the FBS or the FBS-containing nutrient, or nutrient containing other serum, in an amount sufficient to result in a povidone-iodine concentration of from about 0.17o to 27o, the preferred range being from about 0.257α to about 0.57o. After a period of time, the molecular iodine compound is absorbed and does not interfere with culture growth after the nutrient is sold and shipped as an article of commerce and used in tissue or tissue culture media.

Povidone-iodine does not interfere with antibody function and, thus, can be used effective as a sterilizer for monoclonal antibodies and other genetically engineered products which result from processes which introduce the risk of infection by bacteria or virus.

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

Claims

WHAT IS CLAIMED IS:

1. The use of povidone-iodine for the manufacture of plasma or plasma-derived therapeutic composition for treating patients that require infusion of plasma or plasma-derived compositions, povidone-iodine being added to plasma or plasma-derived composition sufficient to result in a povidone-iodine concentration of from 0.17o to 27o and allow to remain in contact of therewith at least one-half minute sufficient to inactivate or destroy infective pathogenic microbes in the plasma.

2. The manufacture of Claim 1 wherein the povidone-iodine concentration in the plasma is from 0.257o to 0.57o

3. The manufacture of Claim 2 wherein the povidone to iodine ratio is at least 15 to 1.

4. The manufacture of Claim 1 wherein the povidone to iodine ratio is at least 15 to 1.

5. A method treating nutrient for culture media to prevent the propagation of microbes in tissue culture media comprising the steps of: mixing said nutrient with povidone-iodine in a concentration to produce a povidone- iodine concentration of from 0.17o to 27o and allowing contact of said nutrient with said povidone-iodine for at least one-half minute sufficient to inactivate or destroy infective pathogenic microorganisms to thereby produce a nutrient which is free of viable cells and viable microorganisms.

6. The method of Claim 5 wherein the povidone is added to give a povidone to iodine ratio of at least 12: 1.

7. Nutrient for use in tissue culture media comprising: serum- containing liquid containing iodine introduced as a povidone-iodine in an amount to produce a povidone-iodine concentration of from 0.17o to 27o sufficient to inactivate or destroy infective pathogenic microorganisms therein, said nutrient being free of both viable and viable microorganisms.

8. The method of Claim 7 wherein the povidone-iodine was introduced to produce a povidone-iodine concentration in the nutrient of at least 0.257o.

9. The method of controlling a cell line comprising adding povidone- iodine to the tissue culture nutrient that supports cell line replication in a concentration to produce a povidone-iodine concentration of from 0.17o to 27o, based on the media, sufficient to arrest or inhibit the propagation of the cell line but insufficient to kill the cell line and insufficient to prevent the cell line from expressing a composition and harvesting said composition produced by the cell line after such composition has been expressed by the cell line.

10. A method of disinfecting plasma derivatives comprising the steps of: (a) treating plasma before separation of the components thereof with povidone-iodine to provide from 0.17o to 27o povidone-iodine in the plasma;

(b) preparing a derivative of the plasma from step (a); and

(c) treating the derivative from step (a) with povidone-iodine to provide from 0.17o to 27o povidone-iodine in the derivative.

11. In the method of separation of plasma factors by alcohol fraction¬ ation, the improvement comprising the addition of povidone-iodine to the plasma before fractionation in concentrations to provide from 0.017o to 0.57o povidone-iodine in the plasma to give higher yields and shaφer differentiation.

12. A method treating nutrient for culture media to prevent the propagation of microbes in tissue culture media comprising the steps of: mixing said nutrient with povidone-iodine with added povidone to give a povidone to iodine ratio of at least 12 to 1 in a concentration to produce a povidone-iodine concentration of from 0.17o to 27o and allowing contact of said nutrient with said povidone-iodine for at least one half minute sufficient to inactivate or destroy infective pathogenic microorganisms to thereby produce a nutrient which is free of both viable cells and viable microorganisms.

13. An article of commerce consisting essentially of nutrient free of viable cells and viable microbes for use in tissue culture media consisting essentially of serum-containing liquid containing iodine introduced as a povidone-iodine with added povidone to give a povidone to iodine ratio of at least 12 to 1 in an amount sufficient to produce a povidone-iodine concentra¬ tion of from O. P/o to 27o sufficient to inactivate or destroy infective pathogenic microorganisms therein, said nutrient being free of both viable cells and viable microorganisms.

14. The article of commerce of Claim 13 wherein the povidone to iodine ratio is at least 15 to 1 and wherein the povidone-iodine is added to give an iodine concentration of from 250 ppm to 500 ppm.

15. A method of controlling a cell line comprising adding povidone- iodine with added povidone to give a povidone-iodine ratio of at least 12 to 1, sufficient to protect the cell line from destruction, to the tissue culture nutrient that supports cell line replication in a concentration to produce a povidone- iodine concentration of from 0.17o to 27o, based on the media, sufficient to arrest or inhibit the propagation of the cell line but insufficient to kill the cell line and insufficient to prevent the cell line from expressing a composition and harvesting said composition after such composition has been expressed by the cell line.

16. A method of disinfecting plasma derivatives comprising the steps of:

(a) treating plasma before separation of the components thereof with povidone-iodine with added povidone to give a povidone to povidone- iodine ratio of at least 12 to 1 in an amount sufficient to provide from 0.17o to 27o povidone-iodine in the plasma;

(b) preparing a derivative of the plasma from step (a); and

17. In a method of separation of plasma factors by alcohol fraction¬ ation, the improvement comprising the addition of povidone-iodine to the plasma before fractionation, the povidone-iodine with added povidone to give a povidone to iodine ratio in the plasma of at least 12 to 1 , in concentrations to provide from 0.017o to 27o povidone-iodine to give higher yields and shaφer differentiation.

18. The method of Claim 23 wherein the concentration in the plasma of povidone-iodine is from 0.257o to 0.57o.

19. In a method of separation of plasma fractions by cryoprecipitation, the improvement comprising the addition of povidone-iodine to the plasma before cryoprecipitation, the povidone-iodine with added povidone to give a povidone to iodine ratio in the plasma of at least 12 to 1, in concentrations to provide from 0.17o to 27o povidone-iodine to give higher yields and sharper differentiation.

20. The method of Claim 19 wherein the concentration of povidone- iodine in the plasma is from 0.257o to 0.57o.