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Capture, concentration and quantitation of abnormal prion protein from biological fluids using depth filtrationRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidCapture, concentration and quantitation of abnormal prion protein from biological fluids using depth filtration description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070148685, Capture, concentration and quantitation of abnormal prion protein from biological fluids using depth filtration. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0001] Transmissible spongiform encephalopathies (TSEs) are a collection of neurodegenerative diseases characterized by progressive dementia, ataxia, amyloid plaque formation and spongiform degeneration in the central nervous system (CNS) (Prusiner, S. B., 1993, Dev. Biol. Stand. 80, 31-44). The causative agent in such diseases is now understood to be abnormal prion protein The fundamental event in TSEs such as Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) is cattle and scrapie in sheep is the conversion of the normal cellular prion protein PrP.sup.C, into a pathogenic isoform, PrP.sup.sc. Accumulation of PrP.sup.sc in the brain of prion-infected animals correlates with the rise in titer of infectious prions and is used as a diagnostic marker for prion diseases. In light of the threat of an interspecies transmission of BSE to humans, a large number of domestic animals must be tested for the presence of PrP.sup.sc in the brain or other suitable material. In the absence of covalent modifications that would allow a distinction between PrP.sup.sc and PrP.sup.C, PrP.sup.sc is routinely detected in Proteinase K (PK)-treated homogenates by Western blotting or enzyme-linked immunosorbent assay (ELISA) utilizing the fact that PrP.sup.sc but not PrP.sup.C is partially protease resistant. Notably, these currently available assays do not take advantage of the fact that PrP.sup.sc forms aggregates. It is now believed that formation of detergent-resistant PrP.sup.sc aggregates is a general biochemical property of PrP.sup.sc even for rare prion strains where PrP.sup.sc is sensitive to proteolytic digestion. This aggregation occurs when the prions are exposed to an aggregation aid for example including a complexing agent. [0002] The fatal human neurodegenerative disorder CJD has also been transmitted iatrogenically via a number of routes suggesting the possibility that the causative agent might also be transmitted via blood products. The identification of a new form of human TSE, named "variant" CJD (vCJD), confirmation of an association with the agent of bovine spongiform encephalopathy (BSE) and evidence that the distribution of the agent of vCJD in human tissues may differ from that of classical CJD suggests the existence of a theoretical risk that blood or blood products may transmit PrP.sup.sc (see Turner et al., Blood Reviews 1998; 12:255-68). [0003] A number of blood products are prepared for medical use from pooled donations of human plasma including normal and specific immunoglobulins, coagulation factor concentrates and solutions of albumin. There is currently considerable concern about the possibility that biopharmaceutical products from human or animal sources may transmit TSEs. Human plasma proteins for parenteral administration inherently carry a risk for disease transmission. Current technology for plasma screening and process steps for the removal or inactivation of viruses has greatly improved the safety of these products, see Burnouf T, et al., Blood Reviews 2000; 14:94-110, in this regard. However, suitable screening tests have not yet been developed for abnormal PrP.sup.sc, which are also extremely resistant to chemical and physical means of inactivation. To determine the probability of vCJD having been transmitted to patients by products derived from this plasma, it is necessary to determine the transmissibility of the PrP.sup.sc in clinically relevant circumstances, the extent to which procedures used for plasma fractionation were capable of eliminating the PrP.sup.sc from plasma products, and the extent to which the agent PrP.sup.sc can be detected in the biological product using available assays. [0004] Human plasma is obtained from whole blood following removal of the larger cellular fractions. Recent studies performed by the plasma fractionation industry have demonstrated that process steps used in the manufacture of human plasma products may reduce PrP.sup.sc (see Foster P., Trans. Med. 1999, 9:3-14; Lee D C et al., J. Virol. Methods 2000, 84:77-89; Foster P. et al., Vox Sang 2000, 78:86-95, and Lee D. et al., Transfusion 2001, 41:449-55.) These process steps include Cohn fractionation, depth filtration and chromatography. Foster et al. (Vox Sang, supra.) demonstrated that depth filtration was effective in removing significant amounts of abnormal prion protein (PrP.sup.SC) from both immunoglobulin and albumin. [0005] There is therefore a need to develop methods of capture and removal of the abnormal infective prions from animal or human derived medicinal products or food products which are effective yet do not substantially degrade and/or remove the biological activity or food value of the product. Due to the limitations of the current methods of detection and quantitation of abnormal prions, there is an unmet need in ability to concentrate to above detection limits and thereafter detect and accurately quantitate the abnormal prion protein (PrP.sup.SC) from the sample. [0006] The instant invention is based on the surprising discovery that depth filtration of aqueous liquids containing biological products, such as for example a biologically active protein, with one or more depth filters having a pore size less than six microns, is surprisingly effective in removing abnormal infective prion proteins. More particularly, these inventors have made the surprising discovery that depth filtration of aqueous liquids containing biological products, such as for example a biologically active protein, with one or more depth filters having a pore size less than six microns, after treatment with an aggregation aid, is surprisingly effective in removing abnormal infective prion proteins. [0007] The invention provides a method for the capture, removal, concentration and subsequent accurate quantitation of PrP.sup.sc associated with TSEs, when such TSEs are contained in biological or food products. [0008] In particular, the invention provides a method for said capture, removal, concentration and subsequent accurate quantitation of PrP.sup.sc associated with TSEs, in biologicals that have been treated with one or more aggregation aids which results in aggregation of the PrP.sup.sc such that the PrP.sup.sc will be captured in and on a filter. Any method that results in such aggregation may be employed as an aggregation aid as contemplated herein. In particular it has been found that solvents such as for example alcohols may be employed. In the methods of the invention, an aggregation aid such as a solvent liquid that has been admixed with the biological or food product is passed through a filter formed of a matrix of cellulose fiber impregnated with diatomaceous earth or similar filter material which may be coated with a cationic resin having an average pore diameter of the filter ranging from 0.1 micron to 6 micron. Typically the filter may be a single use disposable filter. [0009] In particular, the invention provides a method for the capture, removal, concentration and subsequent accurate quantitation of PrP.sup.sc associated with TSEs in biologicals that have been treated with one or more aggregation aids, for example solvent such as for example an alcohol, such as for example alcohol-fractionated immunoglobulin solutions, which comprises passing the solvent liquid containing the biological or food product through a depth filter formed of a matrix comprising solid particles of porous material and having a pore size providing a retention less than 6 .mu.m. Typically the filter will be a single use disposable filter. The treatment with the aggregation aid(s) may be accomplished with the one or more aids admixed together or used in series. [0010] By the terms "removal" or "capture" is meant the actual physical removal of the PrP.sup.sc from the liquid containing the desired protein. For practical purposes, the recovery of the desired protein in its original biological state should be substantially maintained at least to a level in excess of 50%, preferably 80%, more preferably >90%. [0011] Using the methods of the invention, removal of the abnormal infective prion protein may be achieved to an extent of at least 10.sup.2.5, 10.sup.3, preferably 10.sup.4, more particularly >10.sup.5. [0012] Aside from removal of the infective PrP.sup.sc from the biological or food product, the invention also relates to the elution from the one or more filters and subsequent concentration of the captured and eluted PrP.sup.sc using an elution buffer which may comprise, for example, hypertonic solutions such as for example high salt solutions so the PrP.sup.sc may be accurately quantitated using available assays. [0013] Thus, the instant invention provides for aggregation of prions followed by filtration for the purification of a biological or food solution, the elution of the prions from the filter and the concentration of the PrP.sup.sc so as to enable one skilled in the art to employ available assays to quantitate both total prion and PrP.sup.sc in a biological or food sample. The invention will further allow the rapid high-throughput testing of large numbers of samples for PrP.sup.sc. [0014] The invention also relates to the treated biological or food solution. [0015] Since the source of human plasma is whole blood following removal of the larger cellular fractions, we therefore, in order to simulate the state expected of a TSE agent in plasma for fractionation, herein used as an inoculum a fraction of scrapie-infected hamster brain from which intact cells and larger fragments had been removed. TSE diseases are believed to be transmitted either by protease-K-resistant, conformationally abnormal prion protein (PrP.sup.SC). We herein disclose an in vitro method of analysis to determine the distribution of hamster-adapted scrapie PrP.sup.sc as a marker for the partitioning behavior of vCJD. [0016] TSE agents are highly resistant to inactivation, therefore reduction of any product-associated risk will be dependent on the physical removal of infective material during product manufacture. Process technologies used in the manufacture of plasma products include the separation of proteins by precipitation and chromatography with resultant protein solutions being clarified and sterilized by depth and membrane filtration procedures, respectively. Some of these technologies by their modification with the methods of this invention, may be capable of removing TSE agents from a product stream. [0017] PrP protein was detected herein using a Western Blot with the monoclonal antibody 3F4 specific for hamster PrP. This antibody reacts with residues 109-112 PrP from only humans, hamsters and felines. Incubation with 3F4 antibody was at a concentration of 0.6 ug/ml for a minimum of 1 hour, after which excess antibody was washed away and the membranes incubated with a rabbit anti-mouse horseradish peroxidase conjugate (1:1000 dilution) for a minimum of 1 hour. After extensive washing with TTBS, the membranes were developed using enhanced chemiluminescence. [0018] In the manufacture of RhoGAm.RTM. RHO(D) Immune Globulin (Human) by this Assignee, PrP.sup.sc was removed to the limit of detection during depth filtration steps that are also used in the manufacture of immunoglobulins. [0019] Western blotting is a method used to identify and characterize PrP.sup.sc. The PrP.sup.sc is isolated by extraction and is differentiated by its partial resistance to proteinase K digestion. The PrP.sup.RES (PrP.sup.sc resistant to proteinase digestion) is identified by the migration positions of the glycosylation forms and fragments. The sensitivity of this assay is approximately 3 logs less sensitive than the infectivity assay. This sensitivity issue is partially overcome by centrifuging the enzyme digested preparation, removing the supernatant and resuspending the prion material in a smaller volume, resulting in a concentration of the prion material. We have shown that the prions can be easily concentrated by filtering them through a filter after treatment with an aggregation aid, and later collected in a small volume by elution. This technique can be used on a large scale to remove prions from a product stream. [0020] This procedure will have a major impact on the use of the Western blot and indeed any other prion detection assay, to determine the presence of PrP.sup.sc in a biological matrix. This invention allows the TSE material to be quantitatively concentrated quickly to allow for enhanced detection. When seeking to purify a biological, food or cosmetic solution of PrP.sup.sc, this invention has the advantage in the ease in which the biological, food or cosmetic solution filters through the large nominal pore size of the filter. [0021] The methods of the invention are useful for the treatment of biologicals, foods and cosmetics by removing, eluting and, further, quantitating PrP.sup.sc, and depending on the aggregation aid(s) employed, PrP.sup.C. Among the biologicals that can be so treated are blood and blood components such as whole blood, blood serum and plasma, urine, cerebrospinal fluid and blood-derived biological products such as antibodies and immunoglobulins. One such antibody is the IgG immunoglobulin known as monoclonal anti-D immunoglobulin or RhoGAM.RTM. Rho (D) Immune Globulin (Human). This polyclonal immunoglobulin is used in the prevention of hemolytic disease of newborn wherein the mother is injected with Rho(D) immunoglobulin of human origin. Such a product is RhoGAM.RTM., available from the assignee hereof, and it operates by preventing the unimmunized Rho (D) negative mother from responding to Rho (D) antigen present on red cells and `received` from an Rho(D) positive infant. Thus, by preventing anti-Rho (D) production by the mother, the subsequent Rho (D) positive infant of this mother is protected from hemolytic disease of the newborn. This successful product is currently produced by a Cohn alcohol fractionation type process. [0022] RhoGAM.RTM. Rho(D) Immune Globulin (Human) was the first successful prophylactic use of specific antibody to achieve antibody mediated immune suppression. RhoGAM.RTM. is an IgG immunoglobulin solution containing anti-Rho(D) at a dose of 300 micrograms of anti-D activity per dose. RhoGAM.RTM. can be given to the nonimmunized, Rho(D) negative pregnant woman at the appropriate time prevent future disease in her Rho(D) positive offspring. The disease is called hemolytic disease of the newborn or more specifically, Rh-erythroblastosis fetalis. [0023] A smaller dose of anti-Rho(D), MICRhoGAM.RTM. Rho(D) Immune Globulin (Human) Micro-Dose (50 micrograms of anti-Rho(D)) is also sold by the Assignee hereof for treatment of women who have abortions and miscarriages at twelve weeks gestation or earlier. While the full dose protects the recipient for up to 15 ml of Rho(D) positive red cells, the smaller dose provides protection up to 2.5 ml of Rho(D) positive red cells. RhoGAM.RTM. is used as antenatal prophylaxis at 26 to 28 weeks gestation. Other indications include threatened abortion at any stage of gestation with continuation of pregnancy, abortion or termination of pregnancy at or beyond 13 weeks gestation, abdominal trauma or genetic amniocentesis, chorionic villus sampling (CVS) and percutaneous umbilical blood sampling (PUBS). Continue reading about Capture, concentration and quantitation of abnormal prion protein from biological fluids using depth filtration... Full patent description for Capture, concentration and quantitation of abnormal prion protein from biological fluids using depth filtration Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Capture, concentration and quantitation of abnormal prion protein from biological fluids using depth filtration patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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