Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-04-10
2001-11-06
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S526000, C210S695000, C210S222000
Reexamination Certificate
active
06312910
ABSTRACT:
TECHNICAL FIELD
1. Field of the Invention
This invention relates an innovative method for quantitatively separating cells, chemicals, proteins, and other ligands, or other particles, using multistage, magnetically assisted separation technology, (“MAGSEP”). MAGSEP is extremely well suited to immunological research and analysis, pharmaceutical delivery, research and processing and other biomedical applications. Cell separation problems associated with clinical, animal, and plant research can be address using MAGSEP technology.
2. Description of the Prior Art
Almost all prior art in this field can be classified as magnetic filtration, that is, non-magnetic particles are separated from magnetic particles irrespective of their degree of magnetization. For example, Miltenyi et al., teaches that cells labeled with magnetic particles (paramagnetic, superparamagnetic or ferromagnetic) are trapped in a static tube or a flowing channel by a strong magnetic field gradient that causes them to be attracted to said tube or channel wall. Non-magnetic particles are sedimented or convected away, leaving magnetic particles captive until released from the field and collected at a later time. In U.S. Pat. No. 5,053,344, Zborowsky applies the term “magnetapheresis”—magnetic stopping, to a similar process. Liberti et al., in U.S. Pat. No. 4,795,698 teach that thin ferromagnetic pole pieces extending into a suspension of magnetic particles will attract them, and only the magnetic particles, to said pole pieces; non-magnetic particles are convected or sedimented away, the field is switched off releasing the trapped particles into suspension where they are collected as purified cells. In a chromatography-like approach, Ugelstad teaches that high field gradients can be established around beaded ferromagnetic media and fibres, thereby trapping cells labeled with magnetic particles. Other embodiments of these magnetic filtration devices have been patented previously as set forth in U.S. Pat. Nos. 4,795,698 and 5,053,344. All of these teach a similar, simple binary separation of magnetic from non-magnetic particles, and they utilize high-gradient magnetic fields.
Prior art that is closer to the field of the invention has been presented by Powers et al., who teach that a low-gradient magnetic field applied to a horizontally flowing suspension in a channel can trap magnetically labeled cells dynamically and hence potentially according to their level of magnetization by the adsorption of magnetic particles. This method has only been applied to binary separations, however. Winoto-Morbach et al. introduced the concept of “magnetophoretic mobility” implying an intrinsic parameter whereby particles could be separated according to their speed of migration in a magnetic field gradient. Mobility is the ratio of the velocity to the driving force. In an embodiment that exploits this concept, Zborowsky et al. in U.S. Pat. No. 5,968,820, measured magnetophoretic mobilities and in U.S. Pat. No. 5,974,901 teaches that a controlled laminar flow of a suspension of particles between large permanent magnet pole pieces results in the deflection of particles according to their magnetophoretic mobility. Said deflection can be exploited as a means of recovering particles according to their mobilities, or degree of magnetization. Reddy, et. al. (1995) and Zborowski, et al. (1995) have developed analytical methods for directly evaluating the magnetization of different magnetic particle types.
Competing alternative preparativetechnologies consist of different types of separation processes, including electrophoresis and centrifugation. Electrophoresis involves separating materials by passing them through an electric field with separation occurring based on the attractions of the cells to one particular charge, whether positive or negative. Many of the manufacturers in this market are dedicated solely to the manufacturing of electrophoresis equipment. A centrifuge separates cells and other materials by inertial force. Heavier material is forced outward while lighter material remains on the top of the solution. This process may be beneficial when the cells separated can handle that kind of force and are able to separate based solely on size and/or density. This technique can be especially damaging to a cell, due to the high forces imposed when the unit propels cells into a container wall.
In U.S. Pat. No. 5,974,901, Zborowski et al. teach a method in which a nearly constant force field, e.g. magnetic, is applied in a region that contains cells that are caused to migrate in the force field. By capturing a series of microscope images in the force field, particle (cell) velocities can be measured and, through software, a histogram of velocities that indicate the degree of magnetization of the particles can be produced when the force field is a magnetic force field. One application of this method is the measurement of magnetophoretic mobility, the ratio of particle velocity to the applied force field, from which additional physical and chemical information about the particle can be derived. The present invention is distinguished from the Zborowski et al reference in that while Zborowski analyzes particles on the basis of a distribution of magnetic properties, the instant invention provides a means to capture them on the basis of said properties, collecting and separating particles on the basis of their magnetophoretic mobility and is not limited to the collection of merely analytical data as taught by the Zborowski reference.
In U.S. Pat. No. 5,968,820, Zborowski et al. teach a method in which a mixture of biological cells upon whose surface is affixed a number of magnetic particles in proportion to the number of receptors of interest to the researcher can be separated on that basis in a flowing stream in which they are suspended. The flowing stream flows between two magnet pole pieces, and cells within said stream are deflected toward the pole pieces at a velocity that depends on their magnetophoretic mobility and hence magnetic susceptibility and hence receptor density. The separated cells or particles are finally collected utilizing multiple outlets in fractions with each fraction containing cells having a specified range of receptor densities. Contrary to the teachings of Zborowski et al., the instant invention uses a static feed sample in a cuvette and, through the application of magnetic force, causes cells or particles to emerge from said feed cuvette with a velocity that is proportional to magnetophoretic mobility and hence magnetic susceptibility and hence receptor density.
In U.S. Pat. No. 5,053,344, Zborowski et al. teaches a system consisting of a gap between two magnetic pole pieces in which a suspension of particles is caused to flow through a thin chamber with parallel walls by gravity or some other driving means,.The chamber is positioned so as to allow the particles suspended in the flowing stream to experience a spatially graded magnetic force. The spatially graded magnetic force causes the capture of particles spatially distributed on a plane according to their magnetic susceptibility in a process traditionally termed “ferrography”. Subsequent to capture, some particles, especially biological cells, can be examined according to the position at which they were captured and classified, but not collected in suspension according to magnetic susceptibility and hence, if labeled with liganded magnetic particles, receptor density. This system does not separate particles collectible in suspension and therein differs from the instant invention, which is designed to accomplish such separation and collection.
Improved techniques for separating living cells and proteins are increasingly important to biotechnology because separation is frequently the limiting factor for many biological processes. In response to that need, the present invention was developed to provide a method for quantitatively separating cells, particles, ligands, proteins, and other chemcial species using a magnetic and/or an electromagnetically-assisted separat
Barton Ken
Deuser Mark S.
Dunn Scott
Todd Paul W.
Vellinger John
Carrithers David W.
Carrithers Law Office
Ketter James
SHOT, Inc.
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