CA2072749A1

CA2072749A1 - Antimicrobial preservation of plasma

Info

Publication number: CA2072749A1
Application number: CA002072749A
Authority: CA
Inventors: Edward Shanbrom
Original assignee: Individual
Current assignee: Individual
Priority date: 1990-09-04
Filing date: 1991-09-03
Publication date: 1992-03-05
Also published as: WO1992004061A1; EP0498871A1; JPH05502241A; AU648823B2; AU8511891A; EP0498871A4

Abstract

The treatment and preservation of plasma, plasma derivatives with povidone-iodine to kill pathogenic microbes without destroying the utility of the plasma is disclosed.

Description

- wO s2/04n61 PCr/US91/06243 2~727 i~

ANT~MlCROBlAL PRESEPcVAT~ON OF PLASMA
Backgrollnd of the Invent~o~
This invention relates to the treatment and preservation of blood plasma and plasma derivatives and to the pro~ection of technicians, nurses and 5physicians and of the ultimate recipient ,oalient from infection by plasma-borne microbes.
De~finirions 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.
10Tissue cultllres. 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 cultllres. An examples of a cultured tissueis cultured skin tissue for use in burn victims. Cells and cellular products prepared by standard biological andlor genetic engineering techniques are other 15exal les of tissue cultures.
Laboratory re~gent~, 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 ~ ~
20controls. i -I)onor. While the term "donor" is not usually applied to the individual from whom such samples are acquired, tha~ term, "donor" will be used here in a more general sense to include the individual ~rom whom any blood plasma is obtained for any purpose, and such term will be used to refer 25even to an unwilling donor. In the applica~ion of the invention to animals, the donor may be a mammal or the fetus of a mammal.
Povidone (USP) is used in the sense tha~ it is used in lhe U.S.
Pharmacopeia to identify polyvinyl pyrrolidone suitable for use in physiolog-icaliy acceptable solutions.
30Molecll1nr lodine fCompol~nd. Thei term "molecular iodine compound" is used in this paten~ to mean and include molecular iodine, I, :'. ' - wo 92/0406I PCr/~JS91/062q3 ~ ~ 7 9~
diatomic iodine, ~" or a compound or a mixture of compounds which either comprises iodine available in molecular form, typically as diatomic 12, or which reacts wilh or in the presence of the sample to produce ~uch iodine.
Povidone-iodine is the principal example of such compounds.
Povidone-lodine. 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 p~vidone-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 ]W/O povidone-iodine giving a concentration of 1000 ppm 1~. In most instances, povidone-iodine is added as a solution, e.~.
10% solution in water, pH about ~.5, but it can be added as a powder or otherwise. Povidone-iodine powdëF contains approximately 85% PVP, 10 %
12 and 5%1Odide. 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 I 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 12), 5% (5,000 ppm 12) and 1%
(100 ppm 1,). In those instances in which a povidone to iodine ratio of higher than about 8.5 to 1 is referred to, additionai povidone (polyviny1 pyrrolidone~
is added to increase the PVP to lz ratio. The concentration of povidone-iodine in such compositions means the concentration of povidone-iodine added as 8:5 to I PVP to !, povidone-iodine7 i.e. 1000 ppm 1~.
J Nll~rient. Nlltrient, as used here, refers to materials which are deri~ed in whole or in part from animal sources, such as fetal bovine serum (FBS), or which rnay become contaminated from other sources with pathogenic microbes or loxin- or pyrogen-producing microbes. Nutrient is distinct from WO ~2/0406~ PCr/US91/06243 3 2 ~ 7 ~
tissue culture medium in that nutrient should be free of propagating microor-ganisms and, when incorporated in added to a tissue cul~ure medium, provides nutritional components, e.g. minerals, amino acids, etc., ~o supp~rt propaga-tion of desired cellular components with which the medium is inoculated or 5 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, :issue, 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, cryo~.-ecipitate, 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 salnple and who use the sample in conducting analyses and tesls, those who handle the sampling and testing 15 equipment and apparatus, and the entire chain of individuals who attend to the disposal of sampling apparatus and ~he like, from the individuals who pick up the used apparatus through those who ultimately dispose of the apparatus, usually in specially desiF d high temperature furnace.
The risk of contracting disease is especially great in the handling of 20 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 s~ges of processing, e.g. during the initial pooling, spin down, etc. will eventually become infected with hepatitis. The risk of illness from pathogens 25 borne in pooled plasma and ~he certainly that one will eventually become infected and, at least, become a carrier of pathogens is a long-standing problemin the blood banking industry for which no suitable solu~ion has been forthcoming.
The risk to health care personnel is substantial, as evidenced by the ~act 30 that nearly all heal~h care professionals with long experience carry the Epstein-Barr virus (EBV) and/or cytomegalovirus (CMV), the latter being probahly the wo 92/04061 PCl'/US91/062~3 4 2 ~4 7 h ~J ~ ~
most ubiqui~olls of the pathogenic vinlses. Other pathogenic viruses to which health care worl;ers, and those who handle b100d plasma and fluid sampling and hand~ing apparatus, are exposed include hepa~itis and hum'àn immuno-deficiency virus (HIV) as well as a large number of less life-threatening 5 viruses.
Another organism which may contaminate blood plasma and blood plasma producls or fractions and which presents a serious risk is the bacteria Yersinia en~erocolitica. l t surpasses ~higella and rivals Salmonella and campyl~bac~L~r as a cause of acute bacterial gastroenteritis. A significant 10 increase in transfusion related infections of Y. emerocoli~ica has been reporte~, Tipple, et al., Tr~nsfusion 30, 3, p.207 (1990). Y. enterocoli~ica and other bacteri'a which propagate at relatively low temperatures, e.g. Staphylococcus epidermis and Le~ion~lla pn(~L~m~nopJ7ilia, present, potentially, a serious threat in blood plasma products.
lS Baclerial infections are a continuing concern to blood banking industry.
Indeed, a national surveillance-'system for transfusion-associated bacterial infections has been called for, Editorial, Trar~sion 30, 3, p. 193 (1990).
Cytomegalovims (CMV), probably the most ubiquitous of the pa~hogenic microorganisms found in animal fluids and lissues. CMV is 20 frequently associated with, and may be a causative or contributing factor in,life threalening 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. Recipien~s of 25 plasma run a serious risl; 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 30 group of vimses which are responsible for, or involved in, cold sores~ shingles, a venereal disease, mononllcleosis, eye infections, birth defects and probably . - .. . ., . ; ~ , .. : ~ . , .. ,.,,. . ,, ." , ~

WO g2/û4û61 PCT/US91/062~13 2 ~ ~ 7J ~
several cancers. Three subfamilies are of particular importance. The alpha subfamily includes HV I (herpes virus simplex 1) which causes cold sores, f~ 7r blisters, eye and brain infections, HV 2 (herpes virus simplex 2) which e genital ulceration, and HV 3 (HV varicella ~oster) which causes chicken 5 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 Iymphoma and nasopharyngeal carcinoma.
In addition to the risli of transmittin~ infectious disease via blood 10 plasma, the growth of bacteria in blood plasma products at various stages of production and processin~ introduces pyrogens into the blood plasma component or product which must be removed before the product can be used in therapy.
Nutrient for tissue cultllre media always contains pathogenic organisms 15 which, without removal or treatmen~, destroy or greatly impair the value of the nutrient in tissue cultures. It is, genërally, 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 ~0 animals may be suitable in some instances, but fetal bovine serum (FBS) (sometimes referred to as fetal calf semm (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 comn)ercially produced FBS is contaminated with infec~ious bovine 25 viral diarrhea (BVD) virus and infections with infectious bovine rhinotracheitis (IBR), parainfluenza 3 (Pl 3) are extremely common. At best, pools of raw serum probably contain at least 1 OJ infectious BVD virus particles per milliliter. Serum filtration is a common step in reducing the load of infectiousorganisms in serum, but serum quality can be damaged by filtration if 30 significant amounts of serum components are adsorbed to the filters or if macromolecules are sheared.- Shearing of macromolecules during filtration WO 92/04061 PCr/US91/06243 ~72 1 1~
occurs generaliy 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 ~o 1839. It is 5 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-10 pyrrolidone) (PVP). The complexes fllnction by rapidly liberating free iodinein water solutions. They exhibi~ good activity against bacteria, molds, yeasts, protozoa, and many vinlses; indeed, of all an~iseptic 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 15 bacteria, mycobacteria, fungi, yeasts, viruses and protozoa. Most bacteria are killed within 15 to 30 seconds ~f 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 20 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. Iodineis a highly reactive substance combining with proteins partly by chemical reaction and partly by adsorption. Therefore its antimicrobial action is subject2~ 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 solutionof iodine exhibits anti-bacterial properties. However, owing to the low solubility oî iodine in water (33 mg/100 ml at 25 C. ), reaction with bacteria 30 or with extraneous organic matter rapidly deple~es the solution of its activeconten~. Iodide-ion is often added to increase solubility of iodine in water.
.
, .. .. . .. . ., . . . ~ ... . . .

WO 92/04~61 PCr/US91/06243 7 2~7~
rhis increase takes place by the forr ion of trii~,dide, 12 + I = 13. An aqueous solulion of iodine and iodid~ at a Ph of less than 8 contains mainly free diatomic iodine 12 and the triiodide 13'. The ratio of 12 and 13- 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-ic;line (PVP-iodine), is widely used external]y on humans as an antiseptic. Such products are marketed as BetadineT" and Tsodin~". Povidone-iodine products and the preparation of such products are described in U.S.
Patents 2,707,701, 2,8~6,532, and 2,900,305 to Hosmer and Siggia, assigned to GAF Corporation and in a nuTnber of GAF Corporation publications; see, e.g. Tableting with Povidone USP (1981) and PVP Polyvinylpyrrolidone 2). Povidone-iodine powder contains approximately 85% PVP, 10 % 1 ar 5%1Odide. A 10~ solution of this powder contains 1% free, available iodlne. (Gershenfeld, Am. J. Surgery 94, 938 (1957)).
Under ordinary conditions, PVP is stable as a solid and in solution.
The single most altractive property of PVP is its binding capability. 'rhis property has permit~ed utilization in numerous commercial applications. Small quantities of PVP stabilize aqueous emulsions (qv) and suspensions, apparently by its absorption as a thin layer on Ihe surface of individual colloidal particles.
The single most widely studied and best character.ized PVP complex is tha~ 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.
lt 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 ':
, :.

- . WO.92/()406I pcr/us91/06243 PJ~ ~ ~

reducing effec~ of the material to be disinfected which lea~s to the conversion of i~dine inta non-bacte~icidal iodide. Thus, not only the resen~oir of availab]e iodine is diminished but also the equilibr~um of t~iodide i~ influenced as well.~oth of these effæt~ caus~ a decrease in the proportion of free molecular iodine,theactu~ mic~obiala~ent. Whenpovidone-iodineprepa~ion~are contarninated with liquid substrata (e.g. blood p~asma, etc.) there is, in addition, the ~ilution effect characteristic of povidon~iodine systems which causes an increa~ in the equilibrium concentration of frec molecular iodiDC.
To wh~t extcnt the latter effect compensates for the olher two cffects depeads on Lhe content of reducing substances. Thus with whole blood, a strong decrease of ~he concen~ration of free molecular io~ine occurs, while, ~n the presence of pl~sma, it remains practically unchanged. Durrn~z, e~ al, Mikrobiyol. Bul. 22 (3), 1988 (abstract); Gottardi W, ~, Med. i2 (4).
1987. 15~154. Nutricnt broth and plasma had little inactiva~n~ activity but 1~ 1 g hemoglobin inactivated S0 mg of fr~e ~; experiments wi~ 51 showed that uptakc of I by human r~d cells occu~ rapidly. Op~imal antimicrobial effects in clinical use should be achieved in rcla~ively blood-frce situa~ns. Povidone-io~ine prodtlced a potent and sometime~ persistent bactericidal effect towards bac~a on hcal~hy skin. Lacey, R. W. J Appl B~ctenol 46 (3). 1979.
443-450. The bactencidal aclivity of dilute povidon~iodi~le s~lutions is - inversely proportional to Ihe conc~ntration of the povidon~io~ine solutions and is inhibited to the greatcst extcnt by blo~l~ followc~ by pus, fat and glove powd~r. Zamora J L; S~r~ery (Sl Louis) 98 (1). 1985. 2S-29; Zamora, Ara.
J. Surgery, 151, p. 400 ~1986); see also, Waheed Sheikh, Curren ~r~ ~ic 2~ Research 40, No. 6, 1096 (1986). Van Den BT~I~ et al, Antimic~obial Agents and Chemothe~py, 1982, 593-S97, suggest~ that povidoo~iodine is bound lo ccll wall pro~cins leavin~ little for interac~on w,ith microo~gar~isms in the liquid phase (See, also, A~dullah, el al."~ncfm.~o~cl~./l;~r~ Res.
31 (~), Nr. 5, 828). Ninneman e~ al, J. of Imm~nol. 81, 12~5 ~1981) r~
that povidone ~odine was abso~cd in ærum albumin and it is ~nown that - povid~ne-iodine is bound t~ albumin but it has been discove~ that the . -; . .,.... ~. i .. ,. .. , . , ., . ,. , . . , , .. , .,.. ~ . . .... .. . .

- WO 92/04n6l PCr/US91/06243 2 ~3 7 n~ O ~ ~
antimicrobial activity of povidone-iodine is not destroyed by albumin binding.
Whether the activity remains because the albumin povidone-iodine is active or whe~her povidone-iodine and/or iodine are released from the albumin-povidone-iodine complex has no~ been determined.
The teachings of the prior art su~est that neither elemental (diatomic) ieiine, 13, nor complexed iodine, e.~ 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 sîeps in these procedures are fully described in the literature. The following references are provided forgeneral background and as sources for detailed reference to the literature as tospecific procedLlres: TECWNICAL MANIJAL of fhe Arnencan Assodanon of Blo~lBankers, 9lh Ed. (1985); HLA TECHNIQUESFORBLOODBA~S, .
American Associalion of Blood Bankers (1984); I:~evelopments in Biological Standardiza~ion, Vols. 1-57, S. Karger, Basel; CLINICALIMMUNOCHEMIS-T~Y, The American Association f~r Clinical Chemistry; MEDIC1NE, Vols. I -2, Scienîific American, New York; CARE ~If the SURGICAL PATIE~, Vols I - 2, Scientific American, New York; CVRRENI PROTOCOL5 INMOLEClJ-LAR BIOLOGY, Greene Publishing Associates and Wiley-lnterscience, lohn 2û Wiley & Sons, New York.
Summarv of the Invention This invention is embodied in, inter alia, a method of treating patienîs with blood plasma comprising the steps of collec~ing plasma from a donor, and thereafter infusing the plasma into Ihe patient to be treated. The improvement of this invention comprises îhe additional steps of mixing the plasma with molecular iodine compound lo resulî in a concentration from O.IWIO to 21 (lOO to 2000 ppm 1,) the preferred range being from about 0.25WJo to about 0.5W/o (~50 îo 500 ppm 1~), and allowing contact of said plasma with said molecular iodine compound for at least about one-half minute sufficient to - 30 inactivate or destroy infeclive pathogenic microbes. The time of contact is not critical; however, longer contact times, e.g. up to 24 hours or more result in .:

- WO 92/04061 Pcr/ussl/o6243 1 o ~ 3 7 ~ ~
increased biocidal effec~. Generally, however, effective biocidal actiQn can be obtained in a short period of from one-half to two minutes contact.
This invention is embodied in, inler 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 nulrienl, either ne~t or after mixing wilh other nulrient components, with moiecular iodine compound to result in a concentra-tion of from 0.1~/o to 2W/o, preferably from about 0.25~/o to 0.5W/o (250 to 500ppm 17), and allowing contact of said nutrient with said molecular iodine compound for at least about one-half minll~e sufficien~ to inactivate or destroyinfective 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 0.1~ to 2~1~; preferably from about 0.25W/o to O.S~/o (250 to 500 ppm 17), s~lffiCiellt 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 O.lW/o to 2W/o~ preferably from about 0.25'`/o to 0 5W/o (25~ to 500 ppm 17~, based on the nutrient sumcient to arrest or inhibit the propagation of the cell line but insufficient to kill ~he 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 me~hod 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 sufflcient iodine on suchsurface to kill pathogenic organisms therein, and removing the liquid from conlact with the solid povidone-iodine. The method may fur~her comprise .
,.

wo 92104061 PCr/US91/062q3 !
I l 2 S~ S~
reacting the surface of the solid povidone-iodine with iodine between uses to regenerate thP iodine content thereof.
This invention is embodied in, inter alia, a method of treating a patient which comprises th~ step of collectin~s plasma from the patient or another, preserving the plasma by refrigeraiion or freezing, warming and, if the plasma is frozen, thawing ~he plasma, and in~fusing the bioiogical material into the pa~ient. The plasma is treated with molecular iodine cornpound to result in a concentration from o ]W/o to 2W/o~ the preferred range being from about 0.25~/o to abont 0 5W/lu and allowing conlact of said plasma with said molecular iodine 10 compound for at leas~ about one-half minule 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 p1asma in a concentration of from abollt 0.1 to 2W/o. .e preferred ran~,e being from about 0.25W/o to about 0.5~/o; second, maintaining the plasma in 15 contact with the povidone-iodine for a period of about one-half minute or longer; and, third, again introd.ucrr~g povidone-iodine into the plasma in a concentration of from about 0.I~/O to 2W/o, the preferred range being from about 0.25Wlo to about 0.5~1~. Mixing of the plasma with povidone-iodine before refrigerating for storage and/or treezing is preferred, but this step can20 be carried out at low temperatures, e.g. about 4C. to 6C., or after thawing.
This invention is embodied inj inter alia, a method of disinfec~ing plasma derivatives comprising treating plasma before separation of the compo-nents thereof with povidone-iodine into the plasma in a concentration of from about O.l~io to 2~/o, the pre~erred range being from about 0.25~/o to about 25 0.5W/o~ prepar~ng the derivative of the plasrna from the preceding step and treating the de~ivative from the next preceding step with povidone-iodine into the plasma derivative in a concentration of from about 0.1W/o to 2Wlo, the preferred range being from about 0.25W/o ~o about 0.5W/o.
In one preferred embodiment, a method of treating patients with plasma 30 ~omprises 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 . ..

~VO 92~04061 Pcr/US91/06243 1 2 2 ~ ~ 2 7 ~ ~
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 oi from about Q. I~/o to about 2`'/o, preferably from about 0.25Wlo to 0.5W/o (250 to 500 ppm 12), 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 tisslle culture media by mixing said nutrient with povidone-iodine witlI 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 O.lW/o to about 2~/o, preferably from about 0.25~/o to 0.5~/o (250 to 500 ppm I.), and allowing contact of said nutrient with said p~vidone-iodine for at least abaut one-half minute sufficient tO inactivate or destroy infective pathogenic microorganisms to thereby produce a nutrien~ which is free of both viable cells and viable micruor~anisms.
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 cul~ure 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 amollnt sufficient to prod~lce a povidone-iodine concentration of from about 0.1~/o to abou~ 2W/o~ sufficient to inactivate or des~roy infective pathogenic microorganisms therein, said nutrient being free of both viable cellsand viable microorganisms. The povidone-iodine is preferably introduced sufficient to produce a povidone-iodine concentration of from about 0.25W/o ~o about 0.5W/o.

.:

WO g2/0406l PCr/US91/06243 13 ~ 7~
The invention is also embodied in a method of controlling a cell line by adding povidone-iodine with added poviclone to ~ . e 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 S cul~ure nutrient thal supports cell line replica~ion in a concentration to produce povidone-iodine concentration of from about 0 IWIO to about 2W/a~ preferably from about 0.25~/o to 0.5~1O (250 to 500 ppm 1~), based on the media, sufficient to arrest or inhibit the propagation of the cell line but insufficient to ~ill 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 ~he cell line.
Another embodiment of ~he inven~ion is a method of disinfecting plasma derivatives comprising (a) ~reating 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 b lW/o to about 2W/o~ preferably from about 0.25W/o to O SW/o (250 to 500 ppm 1~), 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 ~o provide from about 0. lW/o to about 2W/o~ preferably from about 0.25~V/o tO 0.5W/o (2~0 to 500 ppm 12), povi~ -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 piasma of at least about 12 to 1, preferably from lS: I to 30:1, in concentrations to provide from about O.OIW/o to about 2W/o~ preferably from about 0.25W/o to 0.5W/o (250 to 500 ppm I2), povidone-iodine to give higher yields and sharper differentiation is also disclosed. The concentration in the plasma is preferably from about 0.25Wlo to about 0.5~/o.
An improvement in a method of separation of plasma fractions by cryoprecipitatioQ comprising the addition of povidone-iodine ~o the plasma .

WO 92/~4061 PCrtUS91/06243 ~ ~ 7 ~

before cryoprecipitation, the povidone-iodine with added povidone !o 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 lo provide from abou~ O OlW/o to about 2Wlo povidone-iodine to give higher yields and sharper differen~iation is aiso 5 disclosed. The concentra~ion in the plasma is preferably from about 0.25W/o to about 0.5W/o.
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.
10 Plasma-derived products which are used in diagnostic tests without altering the test results, wilh the single exceptiol~ of an increase in iodine, may be prepared from plasma or plasma derivatives ~reated as described.
Descrip~ion of the Preferred Embodiments A number of non-limiting exemplary embodiments of the present 15 invention are given hereinafter, lt being clearly stated that these are simply examples and are not limiting-as to the scope of the inventive concept.
Quite surprisin&ly, it has been discovered that molecular iodine compound, e.g. povidone-iodine, in the plasma in a concentration of from about 0. IW/o to 2W/o~ the preferred range being from about 0.25wto to about 20 0.5W/o~ do not alter the results of conventional diagnostic tests nor cause significant hemolysis or other change in plasma. In generally, conventional dia~,nostic 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 25 standards~
Plasma is be treated in accordance with this invention by introducillg molecular iodine, e.g. povidone-iodine, into the plasma in a concentration of from about 0. IWIO to 2W/o~ the preferred range being from about 0.25~1o to about 0 5W/o in the plasma. Preferably, in all embodiments, the povidone-30 iodine has a povidone tO iodine ralio of at least about 12:1, preferably fromaboul 15:1 ~o 30:1 and optionally up to about 60:1. A 0.25W/o concentra~ion, wO s2/n4o6l PCr/VS91/06243 2 ~ 7 2 7 ~
for example, in plasma provl~les a tolal kill of bacteria, virus and other pathogenic organisms. Most s~lrprishlgly, lhe microbe kill is effeclive before the iodine reacts with other reducing a~ents in the plasma to the extent of reducing or elimina~ing the microbial activity of iodine, and without altering 5 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 Vlll, more complete, with a cleaner separation and a higher yield of factors, etc.
Pooled plasma processes are an important and essential part of the 10 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 15 risk of infection to wori;ers and tec!lnicians 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 0.1Wlo to 2W/o~ the preferred range being from about 0.2SW/o to 20 about 0.5W/o, preferably early in the process of collecting and pooling the plasma.
Substantial amounts of plasma in or from bags from individual donors and, some~in,~s, pooled plasma are frozen and preserved as fresh frozen plasma (FFP) for use in emergencies and in surgeries to replace lost blood. The 25 industry and surgeons recognize a great risk in using FFP but large quantities, over one million units per year, of F~P are used because safe altematives are not available. The addition of povidone-iodine into the plasma in a concentra-tion of from about 0. IW/o to 2A/o, the preferred range being from about 0.25W/oto about 0.5Wlo to such plasma, preferably before freezing, renders an 30 otherwise risk-burdened product perfeclly safe. One result of this invention is that- very large quantities of plasma, millions of pin~s, which heretofore has wo 92/04061 PCr/U~91/06243 ( been too unsafe for use as FFP can now be used to save lives without~k of infection .
In plasma and serum, 6~ and 7+ log kills, i.e. total kills, of VSV.
Tables I and 11 demonstrale the effectiveness of the methods described here in 5 whole blood and in plasma.

~`.'0 92/1~4061 PCr/l)S91/06243 !

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WO 92~'~)4061 P~/US9~/06243 ... .
1 8 2 3 7 ~, 7 ; i LOG KILL IN > > > I HOUR 24 ~OURS
3% PVP C-15~ 1.67 2.0 ~ 2% PVPC-I5 2.0 3.0 3 1% PVPC-IS 2.33 3.0 4 3 % PVP C- 15 + 0.25 ~o PVP-I- 5.33 8 -~
25~o PVP C-15 1 0.~5% PVP-I 5.0 8+

6 1 % PVP C-15 + 0.'75% PVP-I 4.67 8+

7 3% PVP C-15 + 0.105~ PVP-I 4.33 5.5 0 8 2% PVP C-15 + 0.~0% PVP-I 4.33 6.33 9 IS~o PVPC-IS + 0.10% PVP-I ~.33 5.33 10 3% PVP C-303 3-33 4-33 Il ~% PVP C-30 3.0 4-33 1~ 1% PVP C-30 3.33 5.0 13 3% PVP C-30 ~ o.~sc~ PVP-I 6.67 8+
14 ~% PVP C-30 + 0.~5~ PVP-I 7.33 8+
15 1!;'o PVPC-30 + 0.~5% PVP-I 8+ 8+
16 3% PVP C-30 t 0.10% PVP-I 4.5 6.67 17 7% PVPC-30 + 0.10% PVP-I 4.67 6.5 18 1% PVP C-30 + 0.10% PVP-I 5.0 6.33 19 3% PVP K-''6-;~8~ 3.67 5.67 i 20 25'o PVP K-~6-28 3.5 5.33 ..
21 1% PVP K-'~6-'~8 4.0 . 5.00 2~ 3~o PVP K-~6-~8 + 0.~5% PVP-I 8+ 8+
23 ~% PVP K-~6-~8 + 0.25% PVP-I 8+ 8+
24 1% PVPK--~5--18 + 0.'~5% PVP-I 8+ 8+
25 3% PVP K-26-~8 + 0.105~ PVP-I 5.0 8+

.
C-15 is polyvinyl pyrrolidone havin~ a molecular weight of 12,5û0 produced by GAF Corpora~ion.
2 3o PVP-I is povidone-iodine r)owder r~ro~luced by Purdue-Frederick Companywhich conlains approximately 85% PVP, 1() %1~ an~l 5%10dide.
3 C-30 is polyvinyl pyrrolidone ~rodu~e~ hy GAF Corporation having a molecular wei~ht of 50,(1()().
K-26-2~ is ~olyvinyl pyrroli~lone r roduce~ by GA~ ~orporation having a, molecular wei~ht of l~ctween 4(),()(1() ~n~l 5(),()()0.

.

WO 92/()4061 P~/US91/062q3 ~7~
Ig ~.
TABL~ CONTINUED
LOG KILL IN ~ > > ~B _4 HOURS

262% PVP K-'~6-~8 ~ 0.10% PVP-I 4.67 7.0 27 1% PVP K-~6-~8 + 0.10% PVP-I 4.5 6.5 28 0.25% PVP-I 4.67 6.0 29 0.10% PVP-I 2.33 3.67 CONTROL
Vin~s Pr~senl Vin~s Present ! HOUR _4 HOURS
30 POSITIVE CONTROL (VSV/SERUM) 8.0 LOGS 8.0 LOGS
"
A very important advantage reslllts from the use of povidone-iodine in the ~reatment of plasma lo kill bacteria and virus in that the PVP becomes an effective plasma expander. Thus, boîh the quality and the quantity of the plasma product is very substantially increased as compared with previous S methods of treatment.
An interesting discovery-was made respecting polyvinyl pyrrolidone, alone. in the course of studying the present invention. lt was discovered that poly~ ~nyl pyrrolidone alone was capable of killing up to S logs of virus in body fluids. Whether or not there is a synergism vis-a-vis this anti-viricidal activity 1() 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 atîendant to the prepara~ion and use of cryoprecipitate-derived products are substantially eliminated by Ihe present invention. The cryoprecipitate may, in effect, be treated by the treatment of 5 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 the present invention.
Molecular iodine, e.8. povidone-iodine, is introduced in a concentration of from about O.l~/o to 2W/o~ the preferred Mnge being from about 0.?5W/o to 20 about O.SW/o, into the plasma sufflcient to kill the bacteria and virus. The plasma is then fro2en. When the resulting cryoprecipitate is thawed there is .

wo 92/04061 PCr/US91/06243 3 7 ~
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 l~/o or 2~/o up to as high as about 5W/o, or an increase in the povidone to a s povidone to iodine ratio of 15 to I or more, preferably about 17:1 to 30:1, can cause the precipitation of Factor Vlll and thus present a valuable process for the production of Factor VTTl in high yield and which is free of microbic contamination.
Li~ewise, other plasma fractions and plasmas from other species may o be treated in a similar manner with povidone-iodine.
Plasma factors which are separated by alcohol fractionation, e.g. Factor Vlll? are separated in higher yield and sharper differentiation when the alcohol-containing fractionation liquid contains povidone-iodine. In addition, of course, the resulting fraction is free of microbic contamination.
s Infective pathogenic microorganisms are inactivated when molecular iodine compound is added to fetal b~vine serum ~FBS) or to nutrient containing FBS. The povidone-iodine is introduced into the FBS or the FE~S-containing nutrient, or nulrient containing other serum, in an amount sufflcient to result in a povidone-iodine concentration of from about 0. lW/o to 2~/o, the preferred ~o range being from about 0.25~/~, to about 0.5W/o. 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 2~ be used effective as a sterilizer for monoclonal antibodies and other genetically engineered proclucts which result from processes which introduce the risk of infection by bacteria or virus.
Industrial Application This invention finds application in medicine and veterinary sciencc.

. , .~ .
'~

Claims

WHAT IS CLAIMED IS:

1. The use of povidone-iodine having a povidone:iodine ratio of at least 15:1 for the manufacture of a medicament consisting essentially of a human blood plasma-containing composition for the treatment of disorders wherein the patient requires the transfusion of blood plasma, the povidone-iodine being added in an amount in excess of that required to kill or inactivate all microbes therein comprising from 0.1w/o to 2w/o of the medicament.

2. The manufacture of medicament of Claim 1 wherein povidone-iodine is introduced into blood plasma in a concentration of from 0.1w/o to 2w/o, the blood plasma is maintained in contact with the povidone-iodine for a period of at least one to two minutes and povidone-iodine is again introduced into the blood plasma such that the povidone-iodine comprises from 0.1w/o to 2w/o thereof.

3. 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 0.1w/o to 2w/o 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 microor-ganisms.

4. The method of Claim 3 wherein the povidone is added to give a povidone to iodine ratio of at least 15:1.

5. 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 0.1w/o to 2w/o sufficient to inactivate or destroy infective pathogenic microorganisms therein, said nutrient being free of both viable and viable microorganisms.

6. The method of Claim 5 wherein the povidone-iodine was introduced to produce a povidone-iodine concentration in the nutrient of at least 0.25w/o.

7. 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 0.1w/o to 2w/o, based on the media, sufficient to arrest or inhibit the propagation of the cell line but insufficient to kill the cell line and insuffi-cient 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.

8. A method of disinfecting human blood plasma derivatives comprising the steps of:
(a) treating human blood plasma before separation of the components thereof with povidone-iodine to provide from 0.1w/o to 2w/o povidone-iodine in the plasma;
(b) preparing a derivative of the human blood plasma from step (a); and (c) treating the derivative from step (a) with povidone-iodine to provide from 0.1w/o to 2w/o povidone-iodine in the derivative.

9. The method of separation of human blood plasma factors by alcohol fractionation characterized by the addition of povidone-iodine to the plasma before fractionation in concentrations to provide from 0.01w/o to 0.5w/o povidone-iodine in the plasma to give higher yields and sharper differentiation.

10. The method of treating human blood plasma comprising collecting plasma from a donor and characterized by the steps of thereafter mixing the plasma with povidone-iodine with added povidone to give a povidone to iodine ratio of at least 15:1 and optionally up to 60:1, sufficient to result in an iodine a concentration of from 0.1w/o to 2w/o, and allowing contact of said plasma with said povidone-iodine with added povidone to give a povidone to iodine ratio of at least 12 to 1 for at least one-half minute sufficient to inactivate or destroy infective pathogenic microbes in the plasma.

11. 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 0.1w/o to 2w/0 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.

12. 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 concentration of from 0.1w/o to 2w/o sufficient to inactivate or destroy infective pathogenic microorganisms therein, said nutrient being free of both viable cells and viable microorganisms.

13. The article of commerce of Claim 12 wherein the povidone to iodine ratio id at least 12 to 1 and wherein the povidone-iodine was added to give an iodine concentration of from 250 ppm to 500 ppm.

14. 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 0.1w/o to 2w/o, 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 linefrom expressing a composition and harvesting said composition after such composition has been expressed by the cell line.

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

(a) treating plasma before separation of the components hereof 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 0.1w/o to 2w/o 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 0.1w/o to 2w/o povidone-iodine in the derivative.

16. In a method of separation of plasma factors by alcohol fractionation, 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 0.01w/o to 2w/o povidone-iodine to give higher yields and sharper differentiation.

17. The method of Claim 16 wherein the concentration in the plasma of povidone-iodine is from 0.25w/o to 0.5w/o.

18. In a method of separation of plasma fractions by cryoprecipita-tion, 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 0.1w/o to 2w/o povidone-iodine to give hig-her yields and sharper differentiation.

19. The method of Claim 18 wherein the concentration of povidone-iodine in the plasma is from 0.25w/o to0.5w/o.