High gradient magnetic separation of biological material

Abstract

The invention relates to a high gradient magnet separation device for the separation or purification of magnetic or magnetically labeled biological material, comprising a magnet, a separating column, and a ferromagnetic matrix that can be arranged in an interior chamber of the separating column, and a storage container containing a buffer solution for the equilibration of the separating column and/or for the suspension of the biological material, wherein during operation a magnetic field created by the magnet creates a high gradient magnetic filed in the matrix and buffer solution can flow from the storage container through the separating column. For this purpose, the buffer solution comprises a base solution and macromolecules that can saturate nonspecific binding sites of the matrix.

1 - 14 . (canceled) 
     
     
         15 . High gradient magnetic separation apparatus for the separation or purification of magnetic or magnetically labeled biological material having a magnet, a separation column and a ferromagnetic matrix arrangeable in an inner space of the separation column, as well as a storage container containing a buffer solution for the equilibration of the separation column and/or for the suspension of the biological material, wherein, during operation, a magnetic field generated by the magnet can generate a high gradient magnetic field in the matrix and buffer solution can flow through the separation column from the liquid storage container, characterized in that the buffer solution encompasses a base solution and macromolecules which are able to saturate unspecific binding sites of the matrix. 
     
     
         16 . The apparatus in accordance with  claim 15 ,
 wherein the buffer solution has a density which matches the density of the particles of the biological material to be separated so far that a force due to gravity acting on the particles is largely compensated and therefore, the particles are almost suspended in the buffer solution.   
     
     
         17 . The apparatus in accordance with  claim 16 ,
 wherein the buffer solution has a high viscosity which facilitates a laminar flow through the separation column with a flow velocity suitable for the separation process.   
     
     
         18 . The apparatus in accordance with  claim 15 ,
 wherein the magnet is a permanent magnet or an electromagnet which is shaped so that the separation column can be arranged in the particularly homogenous magnetic field generated by it, wherein the separation column can selectively stand under the influence of the magnetic field or not by spatial separation and/or by switching it off.   
     
     
         19 . The apparatus in accordance with  claim 15 ,
 wherein the matrix is uncoated and/or has ordered or non-ordered filamentary, spherical or differently shaped material, in particular stainless, magnetic steel or steel wool.   
     
     
         20 . The apparatus in accordance with  claim 15 ,
 wherein the liquid storage container is connected to the separation column so that buffer solution can be introduced into the separation column via a flow limitation device with an adjustable flow velocity or not, and/or wherein the separation column has a flow limitation device which can influence the outflow out of, and the flow velocity inside the separation column.   
     
     
         21 . The apparatus in accordance with  claim 15 ,
 wherein the ionic strength of the base solution is adapted in dependence on the macromolecules so that aggregating effects of the macromolecules on the biological material are compensated, wherein the base solution has an isoosmolar concentration of the cations sodium, potassium, magnesium or calcium and of the anions chloride, phosphate, sulfate or carbonate, and is, in particular, a phosphate buffered saline or sucrose solution or a mixture thereof.   
     
     
         22 . The apparatus in accordance with  claim 15 ,
 wherein the macromolecules include natural or synthetic polyelectrolytes or polyampholytes, particularly synthetic polyelectrolyte or organic polyelectrolyte d-glucoronic acid, and/or wherein the macromolecules have an isoelectric point which leads, at the pH value of the base solution, to a charge which corresponds to the charge of the particles of the biological material to be separated, and/or wherein the macromolecules have a molecular weight of about 10,000 to about 100,000 kDa, particularly from about 30,000 to about 70,000 kDa.   
     
     
         23 . The apparatus in accordance with  claim 15 ,
 wherein the macromolecules include globular proteins, particularly albumins, bovine or human serumalbumin, ovoalbumin, lactoalbumin or plant albumins, β-lactoglobulin, κ-casein, histones, protamines, globulines, prolamines or glutelines with a concentration of about 3% to about 7%, particularly about 4% to about 5% in relation to the buffer solution.   
     
     
         24 . The apparatus in accordance with  claim 15 ,
 wherein the macromolecules include filamentary proteins, in particular hydrolysed collagens in a concentration of about 0.3 to about 20 wt-%, particularly of about 1 to about 10 wt-%, or gelatins, bovine gelatins, porcine gelatins or teleosteangelatins in a concentration of about 0.3% to about 1.5%, particularly from about 0.4% to about 0.8%, which have a low gel strength of about 150 Bloom or less, particularly of about 75 Bloom or less.   
     
     
         25 . A method for the separation or purification of intrinsically magnetic or preparatively magnetically labeled biological material by means of high gradient magnetic separation wherein the suspension with the biological material flows through a ferromagnetic matrix arranged in an external magnetic field so that the material adheres to the matrix, 
       characterized in that the matrix is uncoated and in that the biological material is suspended in a buffer solution with a base solution and macromolecules which saturate unspecific binding sites of the matrix during flow-through the matrix. 
     
     
         26 . The method in accordance with  claim 25 ,
 wherein the matrix and a separation column surrounding it is equilibrated before throughflow of the suspension by a pre-incubation with pure buffer solutions, i.e. buffer solution containing no biological material, and indeed over a sufficiently long period of time to saturate unspecific binding sites in the matrix, particularly over a duration of about 3 to about 20 or about 5 to about 10 minutes, and wherein the matrix is always kept covered by the buffer solution during equilibration.   
     
     
         27 . The method in accordance with  claim 26 ,
 wherein for the separation of unwanted biological material the eluate, i.e. a liquid leaving the matrix is captured after the throughflow, wherein after introduction of the suspension into the matrix additional pure buffer solution is flowed through the matrix, with the magnetic field still activated until it is ensured that the suspension has completely left the matrix, and wherein the matrix is always kept covered by the buffer solution during the throughflow.   
     
     
         28 . The method in accordance with  claim 26 ,
 wherein, for the separation of wanted biological material, after introduction of the suspension into the matrix with still activated magnetic field, additional pure buffer solution is flowed through the matrix until it is ensured that the suspension has completely left the matrix, and subsequently the matrix is washed with additional pure buffer solution with the external magnetic field deactivated by spatial separation or switch-off and wherein the eluate is captured, with the matrix being always kept covered by the buffer solution during the entire process.   
     
     
         29 . The method in accordance with  claim 26 ,
 wherein the eluate captured is centrifuged and the method is repeated with the centrifuged off biological material without the liquid phase of the eluate one or several times.