Process for purifying biological material in a micro separation column

Abstract

A micro column system is provided for high gradient magnetic field separation of macromolecules and/or cells. The system provides fast kinetics and high efficiency as well as the purity and simplicity of a column separation. A yoke provides a magnetic field to a plurality of micro columns. A separation and release process for purifying biological material on the column includes release of the biological material from magnetic particles and elution from the column while the magnetic particles are still magnetically retained by the matrix inside the column.

1 . An HGMS micro separation column comprising: 
 first and second tubular portions, said first portion being integral with said second portion, said first portion having a first cross sectional area and said second portion having a second cross sectional area, said first cross sectional area being unequal to said second cross sectional area; and    a matrix adapted to selectively remove at least one component of a mixture as the mixture flows through said tube, wherein said matrix is contained in at least part of said first portion and at least part of said second portion, and wherein said matrix comprises ferromagnetic material.    
     
     
         2 . The micro separation column of  claim 1 , wherein said ferromagnetic material comprises ferromagnetic balls.  
     
     
         3 . The micro separation column of  claim 1 , wherein said ferromagnetic material comprises particles which are coated with a coating, said coating maintaining a relative positioning of said particles with respect to one another.  
     
     
         4 . The micro separation column of  claim 3 , wherein said coating comprises lacquer.  
     
     
         5 . The micro separation column of  claim 2 , wherein said ferromagnetic balls have a diameter of at least about 200 μm.  
     
     
         6 . The micro separation column of  claim 2 , wherein said ferromagnetic balls occupy at least 50 percent of an internal volume of said first and second portions.  
     
     
         7 . The micro separation column of  claim 1 , said tube further comprising a third portion, said third portion being integral with said second portion; said third portion having a third cross sectional area; said third cross sectional area being less than said second cross sectional area.  
     
     
         8 . The micro separation column of  claim 7 , said tube further comprising a fourth portion, said fourth portion being integral with said third portion; said fourth portion having an outside dimension which is less than a respective outside dimension of said third portion.  
     
     
         9 . The micro separation column of  claim 1 , said tube further comprising an upper portion, said upper portion being integral with said first portion; said upper portion having an upper cross sectional area; said upper cross sectional area being greater than said first cross sectional area.  
     
     
         10 . The micro separation column of  claim 1 , further comprising: 
 a retainer located in said second portion adjacent said matrix.    
     
     
         11 . The micro separation column of  claim 10 , wherein said retainer is substantially spherical.  
     
     
         12 . The micro separation column of  claim 10 , wherein said retainer comprises a porous mesh, frit or grid.  
     
     
         13 . The micro separation column of  claim 2 , further comprising 
 a retainer located in said second portion adjacent said matrix.    
     
     
         14 . The micro separation column of  claim 13 , wherein said retainer is substantially spherical and is substantially larger than each of said balls.  
     
     
         15 . The micro separation column of  claim 13 , wherein said retainer comprises a porous mesh, frit or grid.  
     
     
         16 . The micro separation column of  claim 1 , wherein said tube is formed from a material selected from the group consisting of PCTG, polyethylenes, polyamids, polypropylenes, acrylics and PET.  
     
     
         17 . The micro separation column of  claim 16 , wherein said tube is formed from PCTG.  
     
     
         18 . The micro separation column of  claim 11 , further comprising, at least one mount extending into said second portion, said retainer resting on said at least one mount.  
     
     
         19 . The micro separation column of  claim 18 , wherein said at least one mount comprises three mounts extending into said second portion.  
     
     
         20 . The micro separation column of  claim 1 , further comprising 
 an upper matrix retainer located in said first portion adjacent said matrix.    
     
     
         21 . The micro separation column of  claim 20 , wherein said upper matrix retainer comprises a grid, mesh or frit.  
     
     
         22 . The micro separation column of  claim 1 , wherein said matrix further comprises a nonmagnetic component.  
     
     
         23 . The micro separation column of  claim 22 , wherein said nonmagnetic component comprises glass.  
     
     
         24 . The micro separation column of  claim 22 , wherein said nonmagnetic component comprises plastic.  
     
     
         25 . The micro separation column of  claim 2 , wherein said matrix further comprises a nonmagnetic component.  
     
     
         26 . The micro separation column of  claim 25 , wherein said nonmagnetic component comprises glass balls or particles.  
     
     
         27 . The micro separation column of  claim 25 , wherein said nonmagnetic component comprises plastic balls or particles.  
     
     
         28 . The micro separation column of  claim 1 , wherein said column is gravity fed.  
     
     
         29 . The micro separation column of  claim 1 , wherein said column is pressure fed.  
     
     
         30 . A micro separation column comprising: 
 first and second tubular portions, said first portion being integral with said second portion;    a matrix adapted to selectively remove at least one component of a mixture as the mixture flows through said tubular portions, wherein said matrix is contained in at least part of said first portion and at least part of said second portion, and wherein an amount of said matrix contained in said first portion accomplishes a greater removal function than an amount of said matrix contained in said second portion.    
     
     
         31 . The micro separation column of  claim 30 , wherein said amount of said matrix contained in said second portion accomplishes a greater flow resistance function than said amount of said matrix contained in said first portion.  
     
     
         32 . A micro separation unit for use in performing micro separation, comprising: 
 a magnetic yoke having at least one notch formed along a length thereof, and    a pair of magnets placed within each of said at least one notch to form a gap therebetween, wherein each said gap is adapted to receive a micro separation column therein for performance of micro separation.    
     
     
         33 . The micro separation unit of  claim 32 , wherein said magnetic yoke is made of steel.  
     
     
         34 . The micro separation unit of  claim 32 , wherein said at least one notch comprises at least two notches.  
     
     
         35 . The micro separation unit of  claim 34 , wherein said at least two notches comprises four notches.  
     
     
         36 . The micro separation unit of  claim 32 , wherein each of said pairs of magnets forms a magnetic field in each respective gap of greater than about 0.2 Tesla.  
     
     
         37 . The micro separation unit of  claim 36 , wherein each of said pairs of magnets forms a magnetic field in each respective gap of greater than about 0.4 Tesla.  
     
     
         38 . The micro separation unit of  claim 37 , wherein each of said pairs of magnets forms a magnetic field in each respective gap of greater than about 0.5 Tesla.  
     
     
         39 . The micro separation unit of  claim 38 , wherein each of said pairs of magnets forms a magnetic field in each respective gap of greater than about 0.6 Tesla.  
     
     
         40 . The micro separation unit of  claim 32 , further comprising a non-fragile covering encasing said yoke and said at least one pair of magnets.  
     
     
         41 . The micro separation unit of  claim 40 , wherein said non-fragile covering comprises polyurethane rubber.  
     
     
         42 . The micro separation unit of  claim 40 , further comprising at least one mounting magnet within said covering for magnetically mounting said micro separation unit.  
     
     
         43 . A micro column system for high gradient magnetic field separation, comprising: 
 a micro separation unit including a magnetic yoke having at least one notch formed along a length thereof, and a pair of magnets placed within each of said at least one notch to form a gap therebetween; and    at least one micro separation column, each comprising: first and second tubular portions, said first portion being integral with said second portion, and a matrix adapted to selectively remove at least one component of a mixture as the mixture flows through said tubular portions, wherein said matrix is contained in at least part of said first portion and at least part of said second portion, and wherein an amount of said matrix contained in said first portion accomplishes a greater removal function than an amount of said matrix contained in said second portion.    wherein a number of said micro separation columns equals a number of said gaps.    
     
     
         44 . A micro separation column comprising: 
 a bipartite matrix contained within a portion of said column, said matrix having a height less than about 20 mm.    
     
     
         45 . The micro separation column of  claim 44 , wherein said matrix has a height less than about 15 mm.  
     
     
         46 . The micro separation column of  claim 45 , wherein said matrix has a height less than about 12 mm.  
     
     
         47 . A micro separation column comprising: 
 a porous matrix contained within a portion of said column; and    a void volume of less than about 85 μl defined in said column at a height of said matrix and below.    
     
     
         48 . The micro separation column of  claim 47 , wherein said void volume is less than about 50 μl.  
     
     
         49 . The micro separation column of  claim 48 , wherein said void volume is about 30 μl.  
     
     
         50 . The micro separation column of  claim 47 , wherein said matrix comprises a bipartite matrix contained within a portion of said column.  
     
     
         51 . A process for purifying biological material on a column, comprising: 
 retaining magnetic carriers bound to the biological material with ferromagnetic particles in a magnetic field; and    eluting the biological material by dissociating the biological material from the magnetic carriers while still in said magnetic field.    
     
     
         52 . The process of  claim 51 , wherein said eluting comprises a change of buffers.  
     
     
         53 . The process of  claim 51 , wherein said eluting comprises a change of temperature.  
     
     
         54 . The process of  claim 51 , wherein said eluting comprises a change of chemical or enzymatic reaction.