Liquid purification or separation – Processes – Using magnetic force
Reexamination Certificate
1997-10-10
2001-06-05
Walker, W. L. (Department: 1723)
Liquid purification or separation
Processes
Using magnetic force
C210S222000
Reexamination Certificate
active
06241894
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to magnetic separation devices and methods of isolating magnetically labeled substances such as cells, organelles, subcellular components or fragments and the like from a non-magnetic medium by means of a high gradient magnetic field.
2. Background
The present invention uses a high gradient magnetic separation technique (HGMS) to remove magnetically charged or labeled substances distinguished from unlabeled substances from media. The present invention has particular utility in the purification of biological materials in the laboratory or in clinical applications. It can be used in either batch or continuous operation and the target substance to be removed may be either labeled substances or unlabeled substances.
HGMS refers to a procedure for selectively retaining magnetic substances or magnetically labeled substances in a channel or column disposed in a magnetic field. Usually, a biological material such as a cell is labeled with a very small magnetic particle. The magnetic particle is attached to a ligand. The ligand-magnetic particle complex then binds to the biological material making it susceptible to attraction by magnets or magnetic material in a HGMS separation device. The magnetically labeled biological substance is typically suspended in a liquid medium that is then placed in a HGMS device.
The labeled substance remains in the device while the liquid and ideally all other substances are expelled. Then the labeled substance can be removed.
HGMS is typically accomplished by using a device having a separation chamber with a mass of steel wool, steel wire or magnetically susceptible beads disposed between the poles of a conventional electro- or superconducting magnet and serves to generate large field gradients around the wire or beads which exert a strong attractive force on target substance-magnetic particle complexes.
Often such steel wool matrix HGMS devices give rise to disadvantages such as a tortuous path causing non-specific trapping of non-target substances. This occurs by virtue of the fact that the packing material has small dimensions to maximize induced field gradients but which trap non-target substances. These non-target substances are difficult to remove from the matrix; hence, these non-target substances are recovered along with the final product, thus decreasing product purity. This trapping also mandates that the internal matrix must be disposed of after each use. These types of HGMS devices also have the problem of direct contact of cells with the magnetic material which causes damage to cellular target substances.
Another type of HGMS device has unobstructed chambers to minimize non-specific entrapment, but require the generation of very high magnetic field gradients in order to capture the target substances. Such high fields and gradients are created by the appropriate design and placement of permanent or electromagnets. However, these open chamber HGMS devices suffer from a problem of zero field gradient in the center of the container and additionally, substantial regions of relatively low gradient where the velocity is greatest, and where more cells flow as described in U.S. Pat. No. 5,466,574.
From the foregoing, it is apparent that the prior HGMS devices and methods are useful but suffer from many problems. Therefore, there is a present need for a HGMS device and method which provides for separation of target substances with a high degree of purity and which will not damage the target substances during operation. The present invention solves the problems of the prior devices and methods by maximizing the magnetic force exerted on a magnetically labeled substance and minimizing the non-specific trapping of unlabeled substances. The presence of the novel segregating material which permits substantially unobstructed flow of medium through the channel and specifically shaped pole tips contribute to a high gradient magnetic field inside the container that minimizes the problem of a zero field gradient in the center of the container. The invention is also easily sterilized and does not trap unlabeled substances due to its flow through construction.
SUMMARY OF THE INVENTION
An object of the present invention is to avoid the problems of the prior devices. The primary consideration behind the design of the present HGMS device and the present method is to maximize the magnetic force exerted on a magnetically labeled substance, such as for example, cells, while minimizing the non-specific trapping of unlabeled substances.
The HGMS device of the present invention comprises a container having an interior surface defining a channel. The container further has an inlet and an outlet. On the interior surface of the channel are pole tips which may be in a sawtooth configuration or a configuration having sharp angles facing the interior of the channel that generate a high gradient magnetic field gradient in the channel. Within the channel may be incorporated separating material. The separating material eliminates the direct contact of cells with the magnetic pole material. The sawtooth pole tips also serve the purpose of assisting in creating a field gradient across the entire container to minimize the problem of zero field gradient in the center of the container where the velocity is greatest, and where more cells flow. The separating material is made of non-magnetic hollow fibers, flat tubes, sheets or other material which provides for a substantially unobstructed flow path of medium through the channel from the inlet to the outlet. Because there is a substantially unobstructed flow of medium through the channel, unlabeled substances are not trapped.
The specially shaped pole tips generate a large magnetic gradient across the entire interior of the container which is required to retain the labeled cells or substances in the device, while the unlabeled substances flow through.
In operation, magnetically labeled substances in a liquid medium are passed through the device and are subjected to a continuous high gradient magnetic field wherein no substantial volume of zero field gradient exists to remove the labeled substance from the medium. The inventive device can, by virtue of its superior magnetic attraction, retain the specifically labeled magnetic substances while unlabeled cells and liquid medium flow through. The labeled cells can then be released and collected by removing or decreasing the magnetic field.
From the foregoing summary, it will be appreciated that the present invention provides a separation device and methods of simple construction and operation which enable the efficient, safe separation with a high level of purification of labeled substances coupled with magnetic particles from a medium.
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Fan, Y. et al., “Selection and Purification of CD34+ Cells Using Monoclonal Antibody and Ferrofluids”,Advances in Bone Marrow Purging and Processing: Fourth Int'l
Briggs Donald David
Chary Srikanth Ranga
Jen Shang-Chih David
Schwartz Richard Merrill
Ocampo Marianne
Pillsbury & Winthrop LLP
Systemix
Walker W. L.
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