Liquid purification or separation – Magnetic
Reexamination Certificate
2000-04-20
2002-10-29
Savage, Matthew O. (Department: 1723)
Liquid purification or separation
Magnetic
C210S223000, C210S695000, C435S173400, C435S173700
Reexamination Certificate
active
06471860
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the application of high gradient magnetic separation (HGMS) to the separation of biological materials, including cells, organelles and other biological materials. Specifically, this invention relates to micro columns and micro column systems for high gradient magnetic field separation of macromolecules and cells.
BACKGROUND ART
High gradient magnetic separation (HGMS) refers to a process for selectively retaining magnetic materials in a chamber or column disposed in a magnetic field. This technique can also be applied to non-magnetic targets labeled with magnetic particles. This technique is thoroughly discussed in U.S. Pat. Nos. 5,411,863 and 5,385,707, which are hereby incorporated by reference in their entireties.
The material of interest, being either magnetic or coupled to a magnetic particle, is suspended in a fluid and applied to the chamber. In the presence of a magnetic field supplied across the chamber, the material of interest, being magnetic, is retained in the chamber. Materials which are non-magnetic and do not have magnetic labels pass through the chamber. The retained materials can then be eluted by changing the strength of, or by eliminating the magnetic field.
U.S. Pat. No. 4,508,625 to Graham (Graham '625), discloses a process of contacting chelated paramagnetic ions with particles having a negative surface charge and contained in a carrier liquid to increase the magnetic susceptibility of the particles. A magnetic field is then applied to the carrier liquid and particles to separate at least a portion of the particles from the carrier liquid.
U.S. Pat. No. 4,666,595 to Graham (Graham '595), discloses an apparatus for dislodging intact biological cells from a fluid medium by HGMS. The fluid containing the cells is passed through a flow chamber containing a separation matrix having interstices through which the fluid passes. The matrix is subjected to a strong magnetic field during the time that the fluid passes therethrough. At least some of the cells are thereby magnetically retained by the matrix while the rest of the fluid passes therethrough.
Graham '595 further discloses a piezoelectric transducer in fluid communication with the matrix by means of the carrier fluid. When the matrix reaches its loading capacity for cells, the carrier fluid is replaced by an elutriation fluid. The piezoelectric transducer is then excited, to generate high frequency acoustic waves through the fluid in the chamber. The acoustic waves dislodge the cells (particles) from the matrix and are carried out by the elutriation fluid.
U.S. Pat. No. 4,664,796 to Graham et al. (Graham et al. '796) discloses an HGMS system for separating intact biological cells from a fluid medium. The system includes a flow chamber containing a separation matrix having interstices through which the fluid passes, and an associated magnetizing apparatus for coupling magnetic flux with the matrix. The magnetizing apparatus includes a permanent magnet having opposing North and South poles, and field guiding pole pieces. The flux coupler is positioned to pass a strong magnetic field through the matrix during the time that the carrier fluid passes therethrough to permit capture of the cells or particles by the matrix.
The flux coupler is positioned so that the magnetic flux is diverted away from the matrix during the elutriation phase, when the carrier fluid is replaced by an elutriation fluid, so that the viscous forces of the elutriation fluid exceed the weakened magnetic attractive forces between the matrix and the cells or particles, thereby permitting the elutriation fluid to carry away the cells or particles. Additionally, a piezoelectric transducer may be provided to be used in conjunction with the diversion of the magnetic flux by the flux coupler during the elutriation phase, to allow for a slower flow of elutriation fluid.
The matrix is positioned within the flow chamber so as to be subjected to the full magnetic flux of the magnet when the flow chamber is in a first position, during separation of the cells from the carrier fluid. When the flow chamber is rotated approximately 90° from the first position, during the elutriation phase, the matrix is positioned such that the magnetic flux substantially bypasses the matrix.
Graham et al. '795 further discloses the option of using a piezoelectric transducer in fluid communication with the matrix for use in conjunction with the positioning of the flux coupler to bypass the strong magnetic field around the matrix, to allow lower flow rates of the elutriation fluid.
The prior art addresses various methods of HGMS and methods of recapturing the cells/particles once they have been separated by HGMS. For very small samples, however, such as those encounter in molecular biology applications, the prior art is far from ideal for performing HGMS. Very small elution volumes are needed to efficiently elute very small samples, such as, for example, in the separation of messenger RNA from total RNA or cell lysates. Larger elution volumes require larger volumes of enzymes for downstream applications, which become prohibitively expensive and render the procedure inefficient and unusable. Additionally, small void volumes are important in situations where chemical reactions are intended to be performed within the column itself. The present invention is directed to more efficient and effective use of the HGMS technique for separation of very small samples, especially for use in clinical and commercial settings.
DISCLOSURE OF THE INVENTION
The present invention provides improvements in high gradient magnetic separation of materials contained within very small volumes. The present invention combines the advantages of a binding reaction in suspension (e.g., fast kinetics, high efficiency) with those of a separation on a column (e.g., purity, simplicity), while at the same time keeping the elution volume requirements low. Also, a small void volume is provided for performance of chemical reactions within the column.
The separation techniques may be employed in a continuous process or sequential processes, with the different steps of the separation being performed by simply adding different buffers, chemicals, etc., also with potentially different temperatures, e.g., hot water, etc., into a column. Thus, the complete procedure is very fast.
The present invention provides a micro separation column having first and second tubular portions, where the first portion is integral with the second portion. The first portion has a first cross sectional area which is unequal to the cross sectional area of the second portion. A matrix which is adapted to selectively remove at least one component of a mixture as the mixture flows through the tube is contained in at least part of the first portion and at least part of the second portion.
The matrix contains ferromagnetic material, preferably ferromagnetic balls or other ferromagnetic particles. The ferromagnetic material may be coated with a coating which maintains the relative position of the particles with respect to one another. Preferably, the coating comprises lacquer, and more preferably, a lacquer as described in at least one of U.S. Pat. Nos. 5,691,208; 5,693,539; 5,705,059; and 5,711,871, each of which are hereby incorporated by reference in their entireties. The ferromagnetic balls or particles preferably have a diameter or size of at least 100 &mgr;m, more preferably greater than about 200 &mgr;m and less than about 2000 &mgr;m, still more preferably greater than about 200 &mgr;m and less than about 1000 &mgr;m, and most preferably about 280 &mgr;m. The matrix (i.e., ferromagnetic particles and coating) preferably occupies at least about 50 percent of the internal volume of the first and second portions. The void volume of the column, that is the interstitial volume which is not occupied by the matrix (i.e., the matrix void volume) and the volume of the portion of the column that is below the matrix is preferably less than about 85 &mgr;l, more preferably
Koester Mathias
Miltenyi Stefan
Siebenkotten Gregor
Bozicevic Field & Francis LLP
Miltenyi Biotech GmbH
Ocampo Marianne
Savage Matthew O.
LandOfFree
Magnetic micro separation column and method of using it does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Magnetic micro separation column and method of using it, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Magnetic micro separation column and method of using it will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2957803