Liquid purification or separation – Processes – Using magnetic force
Reexamination Certificate
2002-01-11
2004-05-04
Reifsnyder, David A. (Department: 1723)
Liquid purification or separation
Processes
Using magnetic force
C210S800000, C210S222000, C252S06251C, C435S005000, C435S007200, C435S007320, C435S261000, C436S523000, C436S526000
Reexamination Certificate
active
06730230
ABSTRACT:
TECHNICAL FIELD
This invention relates to methods for separating and isolating pathogens from biological fluid samples such as blood and blood components by means of high density microparticles.
BACKGROUND
Whole blood includes cellular (erythrocytes or red blood cells, leukocytes or white blood cells, thrombocytes or platelets) along with non-cellular components (plasma). When blood is collected from a donor for use, the whole blood is typically separated by centrifugation into such components, which can then be used therapeutically, rather than administering whole blood, in order to maximize the clinical and economic utility of blood. The leukocytes present in whole blood are often carried during processing into each of the blood components. Leukocytes may transmit infectious agents, such as cell-associated viruses (e.g. cytomegalovirus or human immunodeficiency virus) or they may cause adverse immunological reactions, such as alloimmunization. For those reasons, leukocyte removal is often desirable and several methods have been developed to remove leukocytes without causing appreciable damage to the blood or blood component. See for example, Coulter, et al., U.S. Pat. No. 5,576,185 and Pall, et at., U.S. Pat. No. 5,229,012.
However, other pathogenic substances may still be present in whole blood or its various components, that can be harmful to a patient receiving such blood. This is of particular concern when the patient is immune compromised and more susceptible to pathogens that may be present in the blood. Such pathogens include viruses, protozoa and bacteria. In addition, more recently, concern has arisen over prions, which are protein agents believed to be capable of transmitting spongiform neuropathies such as Creutzfeld Jacob's Disease. Experimental evidence in animals suggests that these agents may be transmitted by blood transfusions (Houston et al., Lancet 356(9234): 999-1000, 2000), and concern over transmission of these agents has resulted in recall of some human blood-derived products.
Although substantial removal of some pathogens such as cell-associated viruses may occur during leukocyte removal, there is a continuing need to develop more quantitative and broader methods of removing such pathogens from whole human blood and blood components, while maintaining the integrity of the blood.
One method of eliminating pathogens is by inactivation, for example, by directly or indirectly inhibiting the virus's ability to replicate. Reichl, U.S. Pat. No. 5,633,349 describes the inactivation of prions, viruses and other infectious agents by treatment with a chaotropic agent such as urea or sodium thiocyanate. Use of a chaotropic agent for treatment of blood cells has the undesired consequence of destroying the therapeutic utility of the resulting cellular product. Miekka, et at., U.S. Pat. No. 6,106,773 relates to the use of an iodinated matrix to disinfect biological fluids by inactivating pathogens contained therein. Cook et at., PCT/US98/00532 describes the use of frangible compounds for chemical inactivation of pathogens by targeting nucleic acids. Other inactivation methods use photoactivation, which is a combination of a photochemical agent and light. Such agents include psoralens (Lin, et at., U.S. Pat. No. 5,459,030), methylene blue (Wolf, Jr., et at., U.S. Pat. No. 5,527,704) and phthallocyanines (Horowitz, et at., U.S. Pat. No. 5,637,451).
Inactivation is not uniformly successful in eliminating pathogens since some are not susceptible to inactivation under conditions that preserve the therapeutic or diagnostic usefulness of a biological fluid. The Hepatitis A virus is a small non-enveloped, blood borne virus that resists inactivation by detergents, heat and most small-molecule chemical and photochemical inactivating agents. Prions are another example of a pathogen that resists inactivation by almost all forms of sterilizing treatment, including heat, ionizing radiation, and chemical treatments. In particular, because prions lack nucleic acids and form an extraordinarily stable protein structure, they are generally resistant to practical methods of inactivation. For agents such as these, a removal method that also preserves the therapeutic or diagnostic utility of the biological fluid is clearly desirable.
Methods of removing pathogens also include physical separation techniques such as by filtration or chromatography. Wick, et at., U.S. Pat. No. 6,051,189 relates to the detection and extraction of submicron particles such as viruses and prions, by centrifligation and ultrafiltration. Gawry, et al., U.S. Pat. No. 5,808,011 describes a method of prion removal using an anion exchange chromatographic column under conditions that cause a gradient elution.
Physical separation techniques often use magnetic particles. For example, Giaever, et at., U.S. Pat. No. 3,970,518 describes the use of antibody-coated magnetic particles to separate select cells, bacteria or viruses from multi-cell, bacteria or virus populations. Magnetic particles are available in various sizes and can be either non-uniform (Josephson, U.S. Pat. No. 4,672,040) or very uniform (Homes, et at., U.S. Pat. No. 5,512,439). Magnetic particles are generally <4.5 &mgr;m in diameter and have a density of <1.8 g/cm
3
. The magnetic microspheres are intended to be maintained in suspension in the sample and consequently are designed not to settle by gravity.
Non-magnetic, physical separation methods have also been used to separate various cell components from samples of whole blood or bone marrow. Coulter, et at., U.S. Pat. No. 5,576,185, describes the use of reactant-coated, high density microparticles that separate under gravity, a mechanism that allows for separation of undesired cells without substantially physically damaging the blood cells. The advantages of high density microparticles over magnetic particles in the area of cell separation are well established. However, until now, no one has attempted to apply this technology for removal of cellular pathogens such as viruses, bacteria and non-cellular pathogens such as prions.
SUMMARY OF THE INVENTION
The invention provides a novel method for separating pathogens from a biological fluid sample. A plurality of high density microparticles (“HDM”) having a reactant such as an anti-pathogen antibody, bound thereto are mixed with the sample. The HDM, with the pathogen bound thereto, are allowed to differentially settle by gravity and the remaining sample is removed.
One aspect of the invention pertains to a method of removing at least one population of target pathogens from a biological fluid sample, comprising: providing a plurality of high density microparticles having bound thereto a reactant which specifically binds to the target pathogen, and having a density sufficient to provide differential gravity settling of the target pathogen from the sample; mixing a portion of the sample with the microparticles to bind the microparticles to the target pathogen; settling the microparticles with the bound pathogen in the sample to produce a supernatant substantially free from the bound pathogen, where the settling is accomplished primarily by gravity; and separating the microparticles bound to the pathogen from the supernatant.
DESCRIPTION OF THE INVENTION
Definitions
The term “high density microparticles” or “HDM” is used to mean particles having a density greater than that of the non-target materials present in the sample, so that the HDM are able to settle out of the sample by differential gravity, i.e., the HDM will settle more rapidly than the non-target materials. Typical “non-target” materials include red blood cells or white blood cells, platelets, plasma proteins and so forth. Clearly, the greater the differences in density between the HDM and the non-target materials present in the sample, the faster the differential settling will occur. Preferably the particles have a density of at least twice, more preferably 2 or 3 times the density of the non-target materials present in the sample. In particular, HDM preferably have a density grea
Cook David N.
Monroy Rodney L.
Miltenyi Biotec GmbH
Olstein Elliot M.
Reifsnyder David A.
Stauffer Raymond E.
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