Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1999-02-01
2002-04-30
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C435S180000, C435S181000, C435S261000, C435S372000, C435S395000, C530S413000, C530S815000, C530S816000
Reexamination Certificate
active
06379952
ABSTRACT:
FIELD OF INVENTION
This invention relates to the use of azlactone-functional supports to provide cell selection.
BACKGROUND OF INVENTION
Rapidly expanding knowledge in the areas of molecular and cellular biology, immunology, and genetics has led to the identification and characterization of a variety of highly specialized subpopulations of cells within many types of tissues. For example, the identification of rare “stem cells” in such crucial tissues as the brain, islet cells of the pancreas, and liver have led to speculation that one day many diseased tissues may be treated by regeneration of healthy tissue following cell transplantation (Beardsley,
Scientific American,
June 1998, pp. 11-12). Such therapies have, in fact, already been achieved in areas such as cancer treatment. High doses of chemotherapy or radiation needed to destroy cancerous tissue often also destroy the patient's bone marrow and, effectively, his entire immune system. A transplant of hematopoietic stem cells, previously isolated from either marrow or peripheral blood, can rescue the patient by reconstituting the bone marrow and cells of the immune system (Donahue, et al.,
Blood,
1996, 87, pp. 1644-1653). Purified stem cells or other specific cell populations are also believed to be important for developing a variety of immunotherapies (e.g., AIDS treatments) and gene therapy.
To fuel research, and indeed clinical applications, in these and related areas, increasingly effective methods of separation and purification of various cell populations are required. Over the years, a variety of methods of cell separation/purification have been utilized. These separation techniques have depended upon various biological and biochemical, physical, or immunological characteristics of the cell population to be separated (Esser, in “Cell Separation Methods and Applications”, D. Recktenwald and A. Radbruch, Eds., Marcel Dekker, NY, N.Y., 1997, pp. 1-14). Immunoaffinity-based separations have shown considerable promise in terms of providing relatively pure preparations of specific cells. U.S. Pat. No. 5,035,994 (Civin) describes the use of a solid-phase linked monoclonal antibody which binds specifically to an antigen on human pluripotent lympho-hematopoietic stem cells to separate said stem cells from a suspension of marrow or blood cells. Pope, et al., (
Bioconjugate Chem.,
1993, 4, pp. 166-171) describe the use of bifunctional silane reagents to activate glass and cellulose solid supports. Goat anti-mouse antibodies are then covalently linked to the activated supports and the derivatized supports used to selectively deplete CD34+ or CD4+ mononuclear cells from peripheral blood samples. U.S. Pat. No. 5,215,927 (Berenson, et al.) describes the immunoselection of cells using an avidin-biotin recognition system. While these and other methods described in the art allow for selection and purification of selected cell populations, there is a continued need for new methods and materials that can provide those cell populations in improved purities and yields.
Azlactone-functional supports have been described to be quite useful for the immobilization of biologically active materials. For example, U.S. Pat. No. 5,403,902 (Heilmann, et al.) describes the preparation of particulate or beaded materials to which biomacromolecules such as proteins, antibodies, enzymes, etc. can be coupled. These materials are useful, for example, in the affinity chromatographic purification of proteins. U.S. Pat. Nos. 5,262,484, 5,292,514, 5,451,453, 5,486,358, and 5,510,421 all describe other azlactone-functional supports and materials and their uses. In none of these references has it been described or suggested that azlactone-functional supports might be useful for whole cell purification or selection.
PCT Patent Publication WO 94/00464 (Bitner et al.) describes the ability of azlactone-functional supports to separate proteinaceous materials from nonproteinaceous materials. This can be useful for separation and purification of biological materials. When the azlactone-functional support is contacted with a mixture of proteinaceous and nonproteinaceous materials, the proteinaceous materials react with and become coupled to the support, and the nonproteinaceous materials (e.g., nucleic acids) do not react with the support, but remain in solution. This publication does not describe the ability to separate whole cells from naturally-occurring biological fluids, but does disclose that the nonproteinaceous material retains biological activity for further processing after separation from the azlactone-functional support that has proteinaceous material coupled thereto.
PCT Patent Publication WO 94/22918 (Velander et al.) describes a method of derivatizing a porous support in a manner that distributes the ligand. Examples disclosed in the publication identify the covalent coupling of monoclonal antibodies and proteins for further biological separation processes.
U.S. Pat. No. 5,200,471 (Coleman et al.) describes a method of covalently coupling ligands to azlactone-functional supports, particularly with a method to increase the quality of the covalently coupled ligands, to retain high specific bound biological activity.
U.S. Pat. No. 5,561,097 (Gleason et al.) describes a method of covalently coupling small molecule ligands to azlactone-functional supports in a manner that can control the density and distribution of the ligands. This method is useful for the preparation of chromatographic supports.
SUMMARY OF INVENTION
A need exists for new materials and methods for the selection or purification of whole cells. It has now been found that azlactone-functional support materials, previously known to be useful for preparation of chromatographic supports for protein purification or for the preparation of covalently coupled ligands such as proteins, enzymes, and the like, can also serve as starting materials for the preparation of supports for whole cell selection and purification.
Briefly, one aspect of the invention provides a method for cell selection comprising the steps of (a) providing an azlactone-functional support, (b) derivatizing the azlactone-functional support with a substance that is biologically active towards a desired type of whole cell, wherein the substance is covalently coupled to the azlactone-functional support, (c) contacting the product of step (b) with a mixture containing the whole cells, (d) allowing the whole cells in the mixture to interact with and bind to the coupled biologically active substance, (e) removing a remainder of the mixture from the support, and (f) optionally, eluting the bound cells from the coupled biologically active substance to produce a purified collection of the whole cells.
“Support” means any article that is or can be made azlactone-functional. Acceptable supports for use in the present invention can vary widely within the scope of the invention. A support can be porous or nonporous, depending on preferred final use. A support can be continuous or non-continuous depending on ultimate desired usage. A support can be made of a variety of materials, including supports made of ceramic, glassy, metallic, or polymeric materials or combinations of materials. A support can be flexible or inflexible depending on ultimate desired usage.
“Azlactone-functional” means that a support has azlactone-functional groups on internal and/or external surfaces of such support. Thus, such reactive supports have an azlactone-functional group of the formula:
wherein:
R
1
and R
2
independently can be an alkyl group having 1 to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon atoms and 0 to 3 S, N, and nonperoxidic O heteroatoms, or R
1
and R
2
taken together with the carbon to which they are joined can form a carbocyclic ring containing 4 to 12 ring atoms, and n is an integer 0 or 1.
“Covalently coupled” means chemically attached by means of a covalent bond.
“Biologically active substance” means substances which, once co
Coleman Patrick L.
Rasmussen Jerald K.
3M Innovative Properties Company
Gram Christopher D.
Hornickel John H.
Naff David M.
Rogers James A.
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