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  • B01J20/3206—Organic carriers, supports or substrates
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  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
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  • C07K1/14—Extraction; Separation; Purification
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  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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  • C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
  • C12H1/02—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages combined with removal of precipitate or added materials, e.g. adsorption material
  • C12H1/04—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages combined with removal of precipitate or added materials, e.g. adsorption material with the aid of ion-exchange material or inert clarification material, e.g. adsorption material
  • C12H1/0416—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages combined with removal of precipitate or added materials, e.g. adsorption material with the aid of ion-exchange material or inert clarification material, e.g. adsorption material with the aid of organic added material
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
• • A—HUMAN NECESSITIES
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Definitions

• This invention is concerned with the separation of lipin-containing particles from aqueous milieu. More specifically this invention relates to the removal of lipoprotein or glycolipid-containing vesicles from aqueous suspensions. This invention is particularly concerned with the general, nonspecific adsorption of microbes such as -bacteria, yeast, fungi and viruses from contaminated aqueous suspensions.
• Lipins are a group of compounds comprising fats and lipoids which are soluble in ether. They include fats, fatty oils, essential oils, waxes, sterols, phospholipids, glycolipids, sulfolipids, aminolipids, chromolipids, fatty acids and lipoproteins.
• lipins A great variety of biological structures contain lipins.
• particles such as animal viruses may contain lipids at up to about 50 per cent by weight.
• Chylomicons a major circulating lipid transport medium in higher animals, are essentially fat globules enveloped by a lipoprotein membrane. Animal cells, bacteria, yeast and fungi all contain varying proportions of lipins in their cell walls and protoplasm.
• Lipin vesicles Chylomicrons, liposomes, cellular microorganisms and animal cells are examples of lipin vesicles, a major class of lipin-containing particles with which this invention is concerned. Lipin vesicles are generically defined as substantially water insoluble
• particles ranging about from 250 to 10,000 ⁇ in mean diameter which are characterized by a lipin-containing membranous envelope enclosing a liquid interior.
• the contained liquid may consist almost entirely of lipid, as in the case of chylomicrons, or be relatively free of the substance, as in the case of microorganisms.
• sterile filters are generally porous membranes having an average pore diameter of about from 0.2- to 0.45 microns, ordinarily about 0.2 microns. These filters are capable of retaining most cellular microorganisms since the smallest bacterium is believed to be about 0.3 microns in diameter.
• the pharmaceutical industry has also employed membrane filters of this type to sterile filter products which cannot be chemically or thermally sterilized because of their lability. Examples of such products include insulin and human blood protein fractions such as factor VIII.
• Sterile filters approach the absolute in retaining particles greater than the stated pore size. However they are readily clogged by relatively small numbers of particles, particularly those which have a pore size close to the average pore size of the filter. Consequently, it is conventional to pass the liquid to be sterilized through a depth filter before contacting it with the sterile filter. These filters have a high capacity to retain particles throughout rather than by sieving only at the liquid-filter interface.
• Depth filters are fabricated from many materials including cellulose, polypropylene, diatomaceous earth and asbestos. Most of the depth filters trap particles by physical entrapment at points where two or more fibers or granules form a pore, alone or in combination with what usually are assumed to be London-van der Waal's attractive forces. Depth filters have the advantage of removing particles while retaining a high filter flux, i.e., a high flow rate of feedstock per unit of filtration area, even in the face of a particle load that would rapidly clog a sterile filter. However, depth filters are largely ineffective in removing particles in the 0.5 to 3 micron size range, at least when compared to sterile filters.
• sterile filters used in parenteral administration sets rarely have to deal with high levels of suspended particles, and thus clogging is not usually encountered.
• the flux of hydrocollodial solutions such as blood protein fractions through sterile filters is very low. This low flux is attributed to the affinity of the hydrocolloids for the filter surfaces, resulting in increased hydrocolloid binding by the filter and the formation of hydrocolloid concentration gradients upstream from the filter surface.
• Similar problems are encountered in industrial sterile filtration of the same products. Consequently, membrane filters used in conventional parenteral administration sets for large-volume, non-colloidal parenterals such as dextrose or protein hydrolysates in water have proven inadequate for the filtration of viscous hydrocolloid solutions such as factor VIII or albumin.
• an optical assay is defined as any analytical method in which the concentration or activity of an analyte is measured by a change in light as it is passed into the sample, and includes nephelometry and spectrophotometry in the main.
• Lipemic serum samples often contain chylomicrons in such concentrations that the serum appears milky, and even at lower chylomicron concentrations, light scattering particles in the sample will interfere. While such samples may be diluted to reduce the interference this also necessarily dilutes the analyte, thereby reducing sensitivity, and in any case the comparative effect of the chylomicrons is still significant relative to analyte concentration. Reagents such as detergents may be added to destroy the lipid suspension but may interfere in various assays (United Kingdom patent 1,542,982). Ultracentrifugation will remove the particles but requires costly equipment and is tedious to perform. A need therefore exists for a method and device to remove lipin particles from biological fluids to be assayed by optical methods.
• Immunoadsorbents have also been used in various affinity chromatography techniques for cell separation. See Cuatrecasas et al., "Ann. Rev. Biochem.” 40:275 (1971). In these techniques inert matrices are substituted with ligands. Animal cells expected to contain membrane receptor proteins for the ligands are contacted with the immobilized haptens.
• Tanny et al. "J. Parenteral Drug Association” 33(1):40-51 (1979) speculate that 0.45 and 0.20 micron cellulose triacetate membranes retain Pseudomonas diminuta by a combined adsorptive and sieve effect. Similarly, Tanny et al., advance the same hypothesis to account for losses in the titer of influenza vaccine passed through mixed cellulose esters, cellulose triacetate and acrylonitrile-vinyl chloride copolymer ["J. Parenteral Drug Association" 32(6):258-267 (1978)]. pertsovskaya et al., "biol.
• Nauki 14(3) :1005 (1971 ⁇ disclose that glass, methylene and amine-substituted glass, and films of polyamide, polysacrylate, cellulose triacetate, and polyethylene all adsorb different groups of bacteria to varying degrees. In some cases, e.g., with bacilli, no adsorption at all was observed. Gerson et al. in Immobilized Microbial Cells, K. Ven Katsubramanian, Editor pp 39-43 (1978) also report adsorbing various bacteria to surfaces.
• hydrophilic moieties having pendant hydrophobic groups and strong ionogenic groups avidly adhere to a great variety of lipin particles, including animal virions animal cells, bacteria, yeast, fungi, and chylomicrons. Accordingly, certain of the objects of this invention are achieved by contacting an aqueous suspension of lipin particles with noval compositions having the formula [(Y) e B] d Z wherein Y is a hydrophobic ligand, B is a strong ionogenic group, Z is a water insoluble carrier, e is an integer and d is greater than 2, and then separating the composition from the fluid.
• adsorbent compositions having the formula [(Y) e B] d Z wherein Y, Z, e, and d are as described above and B is a linking gcoup or bond, are capable of nonspecifically adsorbing aerosols of lipin particles or such particles from aqueous liquids containing proteins, ethanol or low ionic strength.
• compositions have been found useful in mammalian tissue culture and as a binding medium for the adsorption of lipin vesicles having active enzymes for use in enzyme reactors.
• the hydrophobic ligand Y is characterized broadly by its low solubility in water and its affinity for lipid solvents, primarily ether.
• Suitable hydrophobes are generally those in which, when the group BZ is replaced by methyl, the water solubility of the resulting compound at 20°C will be less than about 0.075 parts by weight of the compound per 100 parts by weight of water and its solubility in ether at the same temperature will be infinite.
• this hypothetical compound will be insoluble in water at 20°C. Its molecular weight will range from about 70 to 600, ordinarily from about 100 to 400.
• the pendant hydrophobe should roughly appear as a cylinder having average dimensions of from about 7 to 40 2 in length and from about 3 to 15
• A preferably from about 3 to 10 ⁇ , in diameter.
• the effect of the hydrophobe is generally less satisfactory as the diameter increases above from about 10 to 15oA, but the length is less material.
• hydrophobe-substituted adsorbents herein generally contain pendant hydrophobic ligands having the formula
• R is hydrogen, nitro, alkyl, alkyl ether, halogen, monocyclic aromatic hydrocarbon or a carbocycle system;
• A is a bond , monocyclic aromatic hydrocarbon or carbocycle system;
• b is an integer ;
• J is oxygen, sulfur or a bond; and n and y are zero or an integer; with the proviso that where A is a bond and R is hydrogen, nitro or halogen then the sum of n and y is an integer greater than 5.
• Suitable carbocycle systems for the hydrophobe adsorbents are multiple hydrocarbon ring systems which may be fused or bridged, contain from about 4 to 30 carbon atoms and be saturated or unsaturated. Preferably the systems will contain from about 6 to 20 carbon atoms and be either aromatic or fully saturated.
• Examples of suitable bridged systems are bicyclo [2.2.1] heptane, bicyclo [3.2.1] octane, bicyclo [1.1.0] butane and bicyclo [2.2.1] hept-2-ene.
• Suitable spiro systems are spiro [2.3]pentane and spiro [3.4]oct-1-ene.
• the fused systems may be ortho or peri, preferably ortho-such as naphthalene, indene, fluorene, anthracene and phenanthrene. Ortho fused systems having more than 3 rings, e.g., steroids such as cholesterol, may also be employed. Ring assemblies such as tercyclohexane and biphenyl are acceptable.
• the monocyclic aromatic hydrocarbons useful in or as the pendant hydrophobic ligand will contain generally about from 6 to 12 carbon atoms, preferably 6-8 and are most desirably phenyl.
• All R groups are preferably hydrogen, although substitution with nitro, alkyl, alkyl ether, halogen, monocyclic aromatic hydrocarbon or carbocycle systems is within the scope of this invention. Ordinarily, from about 1 to 3 R groups will be other than hydrogen, alkyl or alkyl ether, suitable halogens are fluorine, chlorine or bromine, preferably fluorine.
• the monocyclic aromatic hydrocarbons or the carbocycle systems are usually singly substituted at the pendant hydrophobe terminus, with a monocyclic aromatic hydrocarbon preferred over a carbocyclic system.
• the branched chain systems which result from the use of alkyl R groups are generally satisfactory where R is a short chain alkyl or alkyl ether, on the order of C 1 to C 6 .
• Group J is preferably a bond. If J is other than a bond then the oxygen either is preferred.
• n + y will range from 1 to about 20 in total, but each will tend to vary inversely with one another if x is oxygen or sulfur.
• the sum of n and y is usually from about 4 to 25, preferably 7 to 23 where A is a bond and R is H.
• the value for n + y preferably will be from about 1 to 10 if at least one R is an aromatic hydrocarbon or carbocycle system, and particularly when A is a bond and not a ring.
• J oxygen or sulfur
• n is usually greater than 2, particularly when no terminal R is an aromatic hydrocarbon or carbocycle system.
• A is a bond and R is hydrogen, nitro, or halogen, then the sum of n and y is greater than 5.
• Y groups designed e, will depend upon the nature of the linking group chosen. Generally, e will range from 1 to about 3, with one being preferred.
• the degree of major branching of the hydrophobic ligand is designed by b. In the preferred instance both b and e are 1 when A is a bond.
• A is an aromatic hydrocarbon or carbocycle system
• the designation b indicates the degree of substitution of the the aromatic hydrocarbon or carbocycle system. This degree of substitution is preferably low, with b ranging from about 1 to 4. This is particularly the case where an R group is an aromatic hydrocarbon or carbocycle system, or where n + y is greater than 5.
• the preferred hydrophobes are long chain, normal, secondary or tertiary alkyl such as n-hexyl, n-octyl, n-dodecyl, n-tetradecyl, or n-octadecyl.
• the degree of substitution of the hydrophilic macromolecule with pendant hydrophobic groups is represented by d, a minimum of 2 with a maximum dependent upon the characteristics desired in the adsorbent.
• the degree of substitution must be correlated with the size of the macromolecule Z, its hydrophilicity, i.e., the nature of the non-hydrophobic substituents of Z, the hydrophilicity of the linking groups B and the dimensions of the hydrophobe ligand.
• the adsorbent as a whole should be water wettable but not water soluble. Accordingly, d should not be so high as to render the material water repellent.
• the ratio of d to the number of hydrophilic groups that exist on the hydrophilic macromolecule at the pH, temperature and ionic conditions of adsorbent use i.e., the number of exposed polar groups
• Z is an organic
• hydrophilic polymer than d will range from about 0.5 to 0.1 times the number of monomer units constituting the polymer.
• the BZ moiety functions as a hydrophile.
• the linking groups B, the water insoluble carrier Z, or both B and Z contain hydrophilic groups which impart several desirable properties to the adsorbent.
• permeation of aqueous suspensions into adsorbent matrices is facilitated by the overall water wettability of the matrices.
• the adsorbent affinity for lipin particles is enhanced by the net hydrophilic character of the adsorbent when compared to entirely hydrophobic surfaces or those in which the hyrophobe is not pendant, i.e., branched from a hydrophilic matrix.
• the presence of hydrophilic groups lessens the nonspecific binding of low molecular weight lipophilic compounds such as drugs and dyes.
• the combined effect of ionic and hydrophobic binding enhances adsorption of lipin particles.
• Z is a water insoluble carrier. It need not be hydrophilic so long as the linking groups render it water wettable or swellable after substitution with the pendant hydrophobic ligands. Also, the carrier conceivably may be so strongly hydrophilic and of such a molecular weight that it is water soluble before substitution. This, however, is not preferred since separation of the adsorbent from suspension after it has bound lipin particles is not as efficient as with highly water insoluble hydrophilic carriers. It is preferred that the carrier be an organic polymer containing a high density of hydrophilic groups.
• the average molecular weight of the carrier which is desirable will vary widely as it depends upon a number of factors, including the carrier's hydrophilicity as reflected by the water adsorptive capacity of the hydrophobe-unsubstituted carrier, the extent of cross-linking within the carrier and the degree of hydrophobe substitution which is contemplated. For example, a suitable molecular weight for starch would be greater than that for cross-linked dextrans, which in turn would be greater than that needed for satisfactory performance with cellulose or nylon. Further, the average molecular weight needed for the carriers can be reduced as the degree of substitution by the pendant hydrophobes rises. Generally, the carrier will have a molecular weight of greater than about 1000, ordinarily from about 2000 to 2,000,000.
• a principal function of the carrier is to provide physical integrity for the adsorbent, e.g., as a formed article, fibrous mass, woven textile or a membrane. This is an important function where the adsorbent is to be used in a filtration mode.
• carriers which have been cross-linked for example, by bisepoxide, glutaric dialydehyde, divinylsulfone, dibromopropanol or epichlorohydrin, are useful because of their more rigid structure.
• the carriers should also be nonbiodegradable.
• materials such materials as glass, silica, diatomaceous earth, agarose, polyvinyl alcohol, polyvinyl pyrrolidone, cross-linked dextrans, polyacrylamide, polystyrene, styrenedivinylbenzene copolymers and nylon are preferred.
• nylon polyvinyl alcohol
• lower alkyl esters of cellulose polyvinyl pyrrolidone
• polyacrylamide polyanhydroglucose or polyacrolein.
• Nylon is most preferred for this purpose.
• polyolefins and inorganic carriers such as glass or diatomaceous earth are preferred because of their comparatively low cost.
• the carrier need not contain or be substituted with any hydrophilic groups at all if the linking groups bonding the pendant hydrophobe to the carrier are sufficiently numerous and hydrophilic to impart water wettability to the adsorbent.
• such an embodiment is not preferred.
• the hydrophobe is always pendant, which means that it is branched from the carrier as a side chain. This is critical.
• the linking group found intermediate the carrier and hydrophobe is, however, optional so long as the carrier in such cases is hydrophilic.
• the linking group may be dispensed with, i.e., be a bond, when the hydrophobe is bound directly to the carrier, for example, by copolymerization of a hydrophobe-substituted vinyl compound with a hydrophilic comonomer, or by radiation grafting.
• most of the convenient techniques for linking the hydrophobe to a carrier will deposit a hydrophilic residue between the hydrophobe and the carrier.
• linking groups include one or more of the groups oxo or thio ether, amido, ester, carboxyl, sulfonate, sulfone, imido, hydroxyl, thiourea, azo, silane, and amino (primary, tertiary or quaternary).
• the hydrophilic linking groups will range about from 5 to 50 A in length and have a molecular weight of about from 25 to 1000.
• the group is about 10
• Either the carrier or the linking group must be hydrophilic, but the nature of the groups which provide the hydrophilic nature may vary considerably. They are conveniently placed into three functional categories: substantially nonionogenic, weakly ionogenic and strongly ionogenic. The category used will largely depend upon the desired function for the adsorbent, i.e., the solvent and lipin particles expected to be encountered.
• Substantially nonionogenic substituents are defined for the purposes herein as those which have a pK of greater than about 12.
• substituent groups such as hydroxyl, amido, ester, ether or silane will fall in this category.
• Carriers and linking groups which contain or are composed of these groups are preferably used to adsorb lipin particles from protein-containing fractions, from solutions intended for therapeutic administration, or from alcoholic beverages. They are also preferred when the physical integrity of lipin vesicles is to be maximized, i.e., in enzyme reactors and tissue culture.
• the preferred embodiments are polyamide or polyhydroxylated carriers, e.g., cellulose, nylon or polyvinyl alcohol, bound to the hydrophobe through an ether linking group.
• the carrier or linking group may contain or be composed of weak or strong ionogenic substituents .
• Weak substituents have a pK of from about 2 to 12. These are usually carboxyl, phosphoryl, or primary, secondary or tertiary amino groups.
• Strong substituents have a pK of less than about 2. Examples are sulfonate or quaternary amino substituents. These substituents are particularly useful in water treatments because of a lipid structure biocidal effect that is similar to that of detergents in solution. It is preferred to use the hydroxide form of the quaternary amine as it will not contribute metal ions to the product.
• the acidic hydrophobic resins should be charged with pharmaceutically acceptable ions such as sodium or potassium.
• the location of the ionogenic groups is not critical. However, they are optimally substituted immediately adjacent the pendant hydrophobic group, i.e., within about 10 A*.
• sulfonyl, tertiary or quaternary amino or phosphoryl groups may be linked through one functionality to the carrier and then substituted at least once with a hydrophobic group.
• the hydrophobic group is preferably normal alkane of 6, 8, 10 or more carbon atoms, up to about 20 carbon atoms, so as to minimize steric hinderance of simultaneous hydrophobic and ionic bonding of the lipin particle.
• Such linking groups are preferably employed, with nonionogenic or weakly ionogenic hydrophilic carriers, or with hydrophobic carriers.
• Disulfide or thioesther linking groups are particularly useful because these groups may be cleaved, respectively, by reduction with diothiothreitol or by hydrolysis at pH 11.5 for 15 minutes. Thus, any adsorbed particles can be recovered for further use or for destruction and the adsorbent then regenerated by reforming the labile linkage with fresh hydrophobe.
• hydrophilic, pendant hydrophobe-substituted polymers may be adsorbed or covalently linked to other polymers. This enables the artisan to more carefully control the macroscopic character of any formed articles made from the adsorbents of this invention, in particular, to improve their hydraulic shear resistance. It also multiplies sites for binding the hydrophobes.
• dextran-substituted glass may be prepared and covalently linked to hydrophobic moeities in accordance with known techniques. In such cases the composite macromolecule functions as the carrier of this invention.
• tne populations be segregated into mosaics in the matrix rather than being substituted adjacent to one another on the same carrier. This is easily done by preparing differently charged matrices separately in finely divided form, followed by mixing.
• Suitable adsorbents which are contemplated are set forth below (d> 2).
• adsorbents may in general be made by known processes, principally either by copolymerization of hydrophobe-substituted monomers with hydrophilic monomers or by linking the hydrophobes to carriers which are hydrophilic, or which become so by virtue of the linking group residues which contain polar groups. It is preferred to use hydrophilic carriers and to accomplish the hydrophobe linking by use of mild and well-defined linking techniques such as the well-known carbodimide, cyanogen halide or bisoxirane linking techniques; the reactions are mild and well defined, and the products are stable. A myriad of other suitable synthetic procedures will be readily apparent to the artisen.
• adsorbents may be in the physical form of gels; porous films having single or multiple layers; hollow microspheres; solid particles; woven matrices; compressed, randomly aligned fibrous mats; fibrous plugs; or suspensions which may in turn be precipitated by floculating agents, collected on coarse filters or separated by centrifugation. Fibrous mats are preferred.
• the adsorbents are typically used by draining the suspension to be purified through an absorbent membrane, mat or column packed with particles or fibers of the adsorbent. This is preferred over the alternative of simply admixing suspension and adsorbent in bulk and then removing the adsorbent by filtration or centrifugation. Compressed mats, woven matrices or membranes are most suited to situations where the adsorbent will be stressed, while loose, randomly arranged fibrous masses are satisfactory for low pressure embodiments.
• the suspending fluid need not be an aqueous solution but may also be a gas such as air.
• aeorsols of anhydrous lipin particles, oil droplets or aqueous suspensions may be freed of the suspended matter by passage through the adsorbents described herein.
• the liquid suspension may also contain lipophilic proteins such as albumin or moderate concentrations of water-miscible organic solvents such as ethanol without significant adverse affect on adsorbent performance. Generally less than 30% v/v of organic solvent in water is acceptable.
• the particles to be separated from suspension may be oil droplets, oil-in-water emulsions, viruses, lipin vesicles such as cellular microorganisms, liposomes, animal cells (particularly blood cells) chylomicrons and mixtures of these particles.
• the nature and concentration of the particles are not critical but will influence the selection or particular hydrophobe adsorbents and the amounts thereof to be used.
• the anticipated particle size will bear on the average pore diameter selected for the adsorbent matrix. Generally, the average pore diameter should be from about 1.5 to 10 times the average diameter of the particles to be removed from suspension. If a mixture of particles is to be filtered the largest particle should determine the pore diameter. However, layered adsorbents having decreasing pore size may be employed satisfactorily.
• suspensions contacted with cation substituted adsorbents should be at a slightly basic pH, and vice versa for anion substituted adsorbents.
• adsorbent may be desirable to thoroughly elute the adsorbent with a prewash of solution having similar ionic content to the suspension to be purified, particularly if ion exchange phenomena are to be avoided during small scale preparative procedures for labile substances such as proteins, or where the ion exchange capacity of the adsorbent could be deleterious to or change the ionic composition of the final product, e.g., as in the case of parenteral salt solutions.
• Both the period of time for the suspension to remain in contact with the adsorbent and the comparative amounts of adsorbent and suspension will depend upon the comparative avidity of the adsorbent for the particles of interest in the particular suspending fluid used, the presence and rate of adherence of competitive lipin particles or lipophiles, the particle contamination load and the pore size and hydrophilicity of the adsorbent matrix.
• the dwell time and quantity of adsorbent will be unique to each procedure, both parameters are readily determinable by the artisan by simply varying the quantity of adsorbent and the contact time of the suspension to arrive at an optimal separation.
• the adsorbents may be used in conjunction with separate filters which act primarily by a sieving mechanism. For example, large, non-lipin particles may first be- removed from a crude, bacteria-containing suspension by passage through a conventional depth filter first, followed by the adsorbent described herein, and finally a 0.2 micron pore diameter filter. Thus, even though a sterile filter is used, one may employ a considerably smaller surface area than heretofore feasible because the bacterial load is reduced or eliminated by the adsorbent, thereby essentially relegating the sterile filter to an insurance role.
• Hydrophobe adsorbents may be employed to separate viruses and cellular microorganisms from drinking water, sewage effluent, parental solutions, pharmaceuticals, alcoholic and non-alcoholic beverages. They are useful in diagnostic assays which require the removal of cells or chylomicrons from test samples. They may be employed in synthetic procedures using microorganisms, for example, harvesting bacteria or viruses from suspension culture as well as aiding in the cultivation of tissue cultures. Finally, they are effective in removing lipin particles from aerosols. All cellular microorganisms are capable of adsorption to a lesser or greater degree by the hydrophobe adsorbents described herein.
• Examples include mycoplasma and organisms of the families Pseudomonadaceae, Micrococcaceae, Lactobacillacea, Corynebacteraceae, Achromobacteraceae, Enterobacteraceae, Parvobacteraceae, Spriochaetaceae and Treponemataceae.
• Animal viruses such as cytomegalovirus, herpes virus group, influenza and rubella are also bound, as are yeasts.
• adsorbents in sewage treatment are primarily to reduce rather than eliminate the potentially infectious bioburden.
• flat surfaced adsorbents or matrices having large pore sizes in the range of 100-750 microns are preferable because of the large bulk of suspended particles typically encountered; a proportion of these particles for example cellulose fibers, inorganic particles and the like, are relatively innocuous and need not be retained.
• the carrier and linking groups should not be susceptible to hydrolysis or other deleterious changes brought on by enzymes found in sewage; polyolefin carriers are preferred over polysaccharides for this reason.
• Hydrophobe adsorbents are effective in removing viruses and bacteria from drinking water. Hence, it is preferred to use adsorbents which carry strong ion exchange functions, particularly positively charged groups such as quaternary ammonium, and that the ion exchange function be situated adjacent to the hydrophobe.
• the hydrophobe is covalently bonded either directly into the ion exchange group or to an atom to which the ion exchange group also is bound.
• the carrier need not be hydrophilic but it is desirably substituted with weakly polar groups as described above. Hydroxy-substituted polymers are preferred.
• Hydrophobe adsorbents may also be used to entrap microorganisms in therapeutic solutions to be administered to animals, whether during manufacturing of the solutions or in their administration.
• These solutions are predominantly parenteral and peritoneal dialysis solutions containing salts, carbohydrate or proteins, for example, saline, amino acids solutions, KC1 solutions, 5% dextrose, and blood protein fractions such as antihemophilic factor, prothrombin complex, albumin, activated prothrombin complex, insulin, hemoglobin and plasma protein fraction.
• Antihemophilic factor (AHF) concentrates typically contain AHF at greater than 3 times the activity in normal plasma per unit weight of protein.
• Hydrophobe adsorbents are especially useful where the therapeutic solution is labile to conventional sterilizing agent such as heat and ethylene oxide, or contains a hydrocolloid.
• the former include some antibiotics, amino acid and carbohydrate mixtures, proteins and polypeptides, while the latter include proteins, dextrins and cellulose ethers.
• hydrophobe adsorbent and its physical conformation will primarily depend upon the therapeutic solution to be filtered. It is generally preferred to use adsorbents having substantially nonionogenic character—particularly with protein-containing parenterals. However, strongly ionic adsorbents containing physiologically acceptable ions are suitable for use with most therapeutic solutions where the adsorbent has been prewashed with a representative sterile aliquot of the solution, thereby neutralizing the ion exchange activity with respect to that particular solution. However, there may be certain instances where the ion exchange will be beneficial, for example, demineralization of carbohydrate solutions using amphoteric exchangers. Here ion exchange and hydrophobe adsorption activity can be advantageously combined into one processing step. Finally, the exposed polar groups of the adsorbent should carry the same charge as the net charge of the therapeutic solute.
• the intended mode of administration of the therapeutic solution-peritoneal, intravenous infusion, injection or oral-generally is not material to the selection of adsorbent at the manufacturing level.
• Table 1 adsorbents 1, 3, 8, and 16 are preferred, with the adsorbents 1 and 3 most preferred. They are most advantageously employed as membranes, woven fabrics or random, fibrous masses having an average pore diameter of about from 0.75 to 20 microns, preferably about from 1.5 to 10 microns.
• the same adsorbents may be used when administering the solutions to patients. This is conveniently accomplished by including the adsorbents in administration sets. These sets usually include (a) a conduit terminating at one end with a means for connecting the conduit to a container of the solution and at the other end with a means for entering the body of the animal and (b) a filter interposed in said conduit between said both means.
• the means for entering the body include needles, and venous or peritoneal catheters. Flow control devices and connectors for the multiple attachment of parenteral solution containers are frequently included in such sets.
• Hydrophobe adsorbents can be used in place of the filter or interposed between the filter and the solution container as an adjunct to the filter.
• the hydrophobe adsorbent is preferably the sole filter when protein-containing parenteral solutions such as antihemophilic factor are to be administered, because the average pore size may be increased to from about 2 to 20 microns from the usual 0.2 to 0.5 microns, thereby increasing the filter flux.
• air block formation is reduced by the hydrophobe adsorbents when compared to the wholly hydrophilic filters previously employed.
• the complex dual filters which have been proposed to ameliorate this problem may be replaced by unitary, hydrophobe absorbent filters.
• the capacity of the hydrophobe adsorbents for hepatitis is generally superfluous when treating blood protein-containing pharmaceuticals.
• the starting plasma has been screened for assayable hepatitis and, in the case of products such as albumin, heat treated to destroy the virus.
• Other parenteral solutions are free of hepatitis virus because no potentially infective substance is used as a starting material.
• such products are essentially free of assayable hepatitis it is desirable to remove other viruses that may be present and are not screened for, e.g., herpes and rhinoviruses.
• the hydrophobe adsorbents are also useful in the pasteurization of alcoholic beverages, primarily beer and wine. Such products are difficult to pasteurize in a manner which does not also deleteriously affect the beverage quality. Surprisingly, it has been found that ethanol concentrations in aqueous solutions of up to from about 0.5% to 30% do not significantly interfere with the capacity of the hydrophobe adsorbents to bind yeast and bacteria suspended in such solutions.
• beer and wine ordinarily are pasteurized by simply passing the fermentate through a matrix of hydrophobe adsorbent.
• the average pore diameter here will be larger than with filters having the primary task of removing bacterial because the yeast cells are comparatively larger.
• a suitable pore diameter ranges from about 3 to 20 microns.
• Ion exchange adsorbents may be used, keeping in mind the caveats expressed above regarding parenteral solutions. The same embodiments as were discussed above in connection with hydrocolloid solutions are satisfactory in pasteurizing alcoholic beverages.
• microbes as contaminants, where their removal is ordinarily followed by their destruction.
• the hydrophobe adsorbents also are extremely useful in recovering cells from suspension culture or mammalian cell culture.
• the adsorbents are most useful in the first embodiment.
• a microbe generally a bacterium
• a substrate solution is applied to a column of hydrophobe adsorbed organisms and product drawn off as column eluate.
• the solution contains no general growth factors such as carbohydrates or nitrogen sources.
• Products which may be manufactured by this technique or by fluidized bed fermentations include fructose, various amino acids, nucleotides, penicillins, and staphylococcal protein A.
• Surface adhering cells such as mammalian cell lines from disaggregated organs also may be cultured simply by agitating finely divided adsorbent in a nutrient medium inoculated with the cells. The cells are spearated from suspension by centrifuging or filtering the adsorbed cells. They may then be used in the same way as bacteria supra or in applications unique to. animal cells, e.g., artificial organs or in the synthesis of unique products such as antibodies by hybridoma cells and viruses for vaccine production.
• the adsorbed lipin particles also can be desorbed for their recovery or for regeneration of the adsorbent. This may be accomplished by (a) cleaving the linking group as described above, (b) introducing solvents having nonpolar groups in place of or as a substantial proportion of the eluting solvent, or (c) adding other lipin particles to displace the adsorbed materials.
• virions may be recovered for vaccine preparation or other uses by eluting the adsorbent with an aqueous solution of salt and a high concentration, i.e., greater than 30% v/v, of a lipophilic solvent such as ethylene glycol, acetone, ether or alkanol.
• adsorbent is facilitated if the solvent is also water soluble. Forty percent ethylene glycol in saline is preferred.
• a saline suspension of liposomes prepared in known fashion may be passed through the matrix and the virions recovered from the aqueous phase after removing the eluted liposomes, e.g., by centrifugation or extraction of the liposomes into an immiscible solvent.
• hydrophobe adsorbents greatly facilitate diagnostic assays for analytes in lipemic samples of plasma or serum.
• the opaque, milky appearance of such samples and the difficulty with their use in optical assays is largely a function of chylomicrons.
• the chylomicrons can be adsorbed simply by passing the sample through a matrix of hydrophobe adsorbent, preferably in conjunction with a serum skimmer.
• Serum skimmers are disclosed in U.S. patents 3,799,342 or 3,965,731. Their salient features are a filter for removing the insoluble constituents of serum and a chamber for collecting filtered serum or plasma. They are used to skim serum form collection tubes in which collected blood samples have been allowed to clot.
• These devices generally comprise a collection receptacle for filtered sample, a valve which permits the sample to flow into the collection receptacle but not in the opposite direction, a filter disposed on the opposite side of the valve from the receptacle and a flexible sealing member for engaging the inner surfaces of the test sample container.
• the device is used by pushing it into the test sample container, usually a collection tube containing clotted blood.
• the sealing member prevents passage of fluid between tne skimmer and the walls of the collection tube. Instead, the sample flows through the filter and one-way valve into the receptacle.
• the improvement of this invention comprises using a hydrophobe adsorbent in such devices as the filter, or as an element thereof.
• the entire filter is nonwoven plug or mat of fibrous hydrophobe adsorbent having an average pore diameter from about 15 to 100 microns.
• a large pore diameter of about 75 microns is considered optimal because the rapid passage of plasma or serum facilitated by such an open network reduces the adsorption of high density and low density lipoproteins. Smaller pore diameters are acceptable if lipoprotein assays are not contemplated.
• the sample may then be assayed for an analyte using a procedure in which chylomicrons ordinarily would interfere, for example, in optical assays as defined above.
• the hydrophobe adsorbents are particularly useful for screening aerosol-borne lipin particles from air.
• the maintenance of sanitary, or particle-free atmospheres in hospital operating rooms, sterile product manufacturing, and electronics assembly operations is paramount.
• Hydrophobe adsorbents are effective in screening oil droplets and microorganisms from the air supplies used in such environments, whether or not the aerosols contain water.
• the adsorbents may be employed with conventional passive filters to collect any non lipin materials.
• the adsorbents may be regenerated by washing thoroughly with a volatile organic solvent or by hydrolysis or reduction of linking groups as described above.
• adsorbent 1 Preparation of adsorbent 1, Table 1.
• 5 g of nylon wool (sold by the Fenwal Division of Travenol Laboratories for the affinity collection of granulocytes) was immersed in a 400 ml of an emulsion containing 350 ml distilled water and 50 ml of 1,4-butanediol diglycidyl ether and stirred for 2.45 hours. After decanting, but without rinsing, 100 ml octylamine were added and stirred for one hour. Both reactions were conducted at 22°C. A white , oily precipitate was eluted from the substituted nylon under extensive washing with distilled water. The product was soft and felt oily to the touch.
• a 20 ml syringe was packed to the 5 ml mark with adsorbent and rinsed exhaustively.
• the effect of an ethanol rinse was evaluated by rinsing a packed syringe with 30 ml ethanol followed by 30 ml of distilled water.
• the ability of the adsorbent to removed bacteria was assayed by adding 25-30 ml of contaminated normal serum albumin to the syringe and allowing the albumin to passively flow through the filter.
• This albumin was heavily infected with flora indigenous to the blood plasma fractionation facility from which it was obtained, primarily thought to be Pseudomonas sp. in populations greater than 10 organisms/ml.
• the last 5 ml were collected, diluted as indicated, and aliquot plated onto culture media. The results are shown in Table 2 below.
• Example 1 modified nylon was evaluated using additional suspending fluids and fully characterized contaminant organisms.
• the organisms designated in Table 3 below were seeded at the indicated populations into sterile phosphate buffered saline (PBS), 5% normal serum albumin (NSA), distilled water, pasteurized whole milk and beer (Coors). Plugs of modified and unmodified (control) nylon were placed into a 12 ml syringe and washed with 15 ml of sterile heart infusion broth. Three ml of each contaminated liquid were then passed through the syringes and the eluate bacterial population determined by promptly plating the eluate onto culture media in conventional fashion. The results are reported in Table 3.
• hydrophobe adsorbents are capable of multifold reduction or elimination of the microbial burden of a wide variety of contaminated liquids. This effect is accentuated by electrostatic or mechanical sieving by the nylon matrix as evidenced by the lowered populations in the control eluates.
• the small reduction in milk contamination is believed to be the result of cometitive binding of the hydrophobes by lipid vesicles and residual flora endogenous to the milk after pasteurization; an increased amount of absorbent would remove both of these components as well as added bacteria.
• Parallel experiments with a yeast, Saccharomyces cerevisiae also were conducted.
• the modified nylon was somewhat more effective than the control in removing the contaminants from PBS, NSA, water and beer, but with milk the control was effective as the modified nylon. This was attributed to the likelihood that the yeast was considerably larger than bacteria and predominately filamentous, therefore, mechanically retained by the nylon control.
• Mycoplasma prepared in known fashion and suspended in isotonic nutrient broth could be satisfactory absorbed upon passage through absorbent 16, Table 1, in the form of five layers of cotton cloth.
• Absorbent 4 prepared from hardwood sawdust and sandwiched at a thickness of 200 mm between two fine mesh stainless steel screens could remove endogenous microorganisms, primarily coliforms, from raw sewage.
• Acanthamoeba castellani a soil amoeba, could be removed from a growth medium containing 1.5% glucose and 1.5% proteose peptone by passage of the suspension through a cotton fiber plug of absorbent 9, Table 1.
• An aerosol of a saline suspension of E. coli produced by a household vaporizer could be screened from a 1:100 dilution in humidified air by blowing the aerosol at a rate of 1 cubic foot/min through a 5 x 15 cm column packed loosely with an absorbent 3, Table 1, in the form of modified nylon fibers.
• 5 ml of lipemic human serum could be clarified by the conventional use of an Accu-sep serum skimmer in which the filter was replaced by absorbent 4, Table 1, in the form of a matted cellulose fiber plug.
• Phospholipid vesicles were prepared according to the procedure of Batzri et at., "Biochem. Biophys. Acta” 298:1015-1019 (1973) using diacetyl phosphate to render the bilayer vesicles negatively charged.
• the vesicles could be absorbed by briefly mixing absorbent 11 of Table 1 with the suspension and centrifuging.
• This example is concerned with the continuous manufacture of streptokinase from immobilized streptococci.
• a 5 liter column was packed with polyacrylamide beads modified as absorbent 21, Table 1.
• Hemolytic streptococci were cultured as described in U.S. Patent 3,855,065 and a 1 liter inoculumn of log phase organisms introduced onto the column, followed by a steady flow of 75 ml/hour of a nutrient medium containing 8% corn steep liquor and 7% Cerelose in water.
• the column eluate was collected for 24 hours and the streptokinase recovered by precipitation with ammonium sulfate.
• the columns would function satisfactorily until microbial replication clogged the pores and restricted flow or until the column adsorptive capacity became overloaded and effluent break out occurred.
• the column lifetime may be extended by substituting saline for nutrient medium after 3 hours cultivation.
• Example 2 of U.S. Patent 3,821,086 is followed by the manufacture of fructose from glucose except that the Primafloc C-7 employed by the patentee to insolubilize the Arthrobacter species was substituted by absorbent 17, Table 1, as modified course hardwood sawdust.
• the absorbent could be recycled by reducing the absorbent with an aqueous solution of 0.05M dithiothreitol and 0.001M EDTA for 2 hours, washing the absorbent extensively with distilled water, and reacting the absorbent with dioctyldisulfide under oxidizing conditions regenerate the absorbent.
• Herpes virus type 1 strain hf was recovered from MA104 cell culture and the suspension absorbed upon passage of 5 ml of suspension through a 1 ml volume plug of either of the adsorbents employed in Example 1 or 2. Adsorption was evaluated by serially diluting the adsorbent eluate, inoculating monolayer MA104 cell cultures with the dilutions and observing for cytopathic effects.
• nylon fibers 1.5 g were packed into a 1-inch diameter filter holder, mixed overnight with about 15 ml of concentrated acetone solution of bis-epoxide. Then an equeous solution of linear dextran (average 200,000 D) at saturation is mixed with the activated fibers for 24 hours at pH 9. The product is rinsed with copious quantities of cold distilled water and brought to dryness with 50%, 70% and then 95% acetylnitrile followed by acetone. Then 100 mg cyanogen bromide in ice cold water (pH 9) is mixed slowly with the modified nylon and the pH maintained with NaOH. When no more NaOH was required, a saturated solution of n-octyl amine is mixed overnight with the activated matrix.

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