Static separation method using non-porous cellulose beads

Abstract

An affinity separation method and system comprising an affinity separation media with low porosity and low non-specific binding, and a fluid containing a target compound to be isolated which is capable of binding onto the affinity separation media in a fluid mixing loop in a static filtration apparatus. The static filtration apparatus comprises an intermixing-chamber containing a filtration medium wherein a tangential flow is created for intermixing the affinity separation media and target compound in the fluid. The fluid is capable of passing through the filtration median while the affinity separation media are substantially incapable of passing through the filtration medium. The affinity separation media are separated from the fluid by opening the filtrate outlet so as to allow the fluid to pass through the filtration medium of the static filtration apparatus. The filtrate can be thereby rendering substantially free of the target compound.

We claim:  
     
         1 . A method of separation for isolating a target compound from a fluid comprising: 
 (a) forming a suspension comprising 
 i) a fluid containing a target compound to be isolated, and  
 ii) a plurality of low porosity affinity particles capable of binding the target compound,  
   (b) maintaining said low porosity affinity particles and said fluid in contact for a sufficient period of time for binding of said target compound onto said low porosity affinity particles to be effected,    (c) introducing said suspension into a static filtration apparatus having a filtration medium,    (d) creating a tangential flow in said static apparatus thereby aiding in separation between said fluid and said low porosity affinity particles with the target compound attached to the surface thereof,    (e) passing said fluid devoid of the target compound through said filtration medium of said static filtration apparatus,    (f) washing said low porosity affinity particles, and    (g) eluting said target compound from said low porosity affinity particles.    
     
     
         2 . The method of  claim 1 , wherein said low porosity affinity particles are nonporous.  
     
     
         3 . The method of  claim 2 , wherein said low porosity affinity particles are generally spherical.  
     
     
         4 . The method of  claim 3 , wherein said low porosity affinity particles are generally identical in diameter.  
     
     
         5 . The method of  claim 4 , wherein said low porosity affinity particles have an average diameter of between about 0.5 and about 25 microns.  
     
     
         6 . The method of  claim 4 , wherein said low porosity affinity particles have an average diameter between about 1 and about 3 microns.  
     
     
         7 . The method of  claim 4 , wherein said low porosity affinity particles have an average diameter at least about two-fold larger than the average pore size of said filtration medium.  
     
     
         8 . The method of  claim 7 , wherein said fluid contains particulate and said filtration medium has an average pore size at least about two-fold larger than the size of the largest particulate in said fluid.  
     
     
         9 . The method of  claim 4 , wherein said low porosity affinity particles have an average diameter at least about five-fold larger than the average pore size of said filtration medium.  
     
     
         10 . The method of  claim 7 , wherein said fluid contains particulate and said filtration medium has an average pore size at least about five-fold larger than the size of the largest particulate in said fluid.  
     
     
         11 . The method of  claim 1 , wherein said target compound is eluted from said low porosity affinity particles by transferring said affinity particles having said target compound thereto from said static filtration to a tank, passing an eluent through said tank to remove said target compound from said low porosity affinity particles, thereby providing eluded low porosity affinity particles from which said target compound has been removed, and collecting the resulting eluent containing said target compound.  
     
     
         12 . The method of  claim 10 , wherein said eluted low porosity affinity particles are then transferred back to said static filtration apparatus and the method is repeated.  
     
     
         13 . The method of  claim 1 , wherein said low porosity affinity particles have an average diameter between about 0.5 and about 120 um.  
     
     
         14 . The method of  claim 1 , wherein said low porosity affinity particles have an average diameter range of about 60 urn or less.  
     
     
         15 . The method of  claim 12 , wherein said low porosity affinity particles have an average diameter of about 20 um or less.  
     
     
         16 . The method of  claim 13 , wherein said filtration medium has an average pore rating of less than about 5 um.  
     
     
         17 . The method of  claim 1 , wherein said low porosity affinity particles have an average diameter of at least about 0.5 um.  
     
     
         18 . The method of  claim 15 , wherein said filtration medium is a microporous membrane having an average pore rating of less than about 1 um.  
     
     
         19 . A system for affinity separation of a target compound from a fluid comprising: 
 (a) a static filtration apparatus comprising a housing having a feed port, a concentrate port, a filtrate port, and a filtration medium disposed within the housing;    (b) the feed port and concentrate port being in fluid communication with the upstream side of the filtration medium, while the filtrate port is in fluid communication with the downstream side of the filtration medium; and    (c) a suspension comprising a plurality of low porosity affinity particles and a fluid containing a target compound to be isolated fed into the filtration apparatus via the feed port for inter-mixing.    
     
     
         20 . The system of  claim 19 , wherein said low porosity affinity particles are nonporous.  
     
     
         21 . The system of  claim 19 , wherein said low porosity affinity particles are generally spherical.  
     
     
         22 . The system of  claim 19 , wherein said low porosity affinity particles are generally identical in diameter.  
     
     
         23 . The system of  claim 20 , wherein said low porosity affinity particles have an average diameter at least about two-fold larger than the average pore size of said filtration medium.  
     
     
         24 . The system of  claim 21 , wherein said fluid contains particulates and said filtration medium has an average pore size at least about two-fold larger than the size of the largest particulate in said fluid.  
     
     
         25 . The system of  claim 20 , wherein said low porosity affinity particles have an average diameter at least about five-fold larger than the average pore size of said filtration medium.  
     
     
         26 . The system of  claim 23 , wherein said fluid contains particulates and said filtration medium has an average pore size at least about five-fold larger than the size of the largest particulate in said fluid.  
     
     
         27 . The system of  claim 19 , wherein said low porosity affinity particles have an average diameter of about 120 um or less.  
     
     
         28 . The system of  claim 19 , wherein said low porosity affinity particles have an average diameter of about 60 um or less.  
     
     
         29 . The system of  claim 19 , wherein said low porosity affinity particles have an average diameter or about 20 um or less.  
     
     
         30 . The system of  claim 27 , wherein said filtration medium has an average pore rating of less than about 5 um.  
     
     
         31 . The system of  claim 19 , wherein said low porosity affinity particles have an average diameter of between about 1 and about 3 um.  
     
     
         32 . The system of  claim 19 , wherein said filtration medium is a microporous membrane having an average pore rating of less than 1 um.  
     
     
         33 . The system of  claim 19  wherein said low porosity affinity particles are ungrafted for separation of a fusion protein comprising a cellulose binding protein segment fused with a target protein or peptide.  
     
     
         34 . The system of  claim 33  wherein said low porosity affinity particles comprise a bead having a surface, said surface of the bead as a whole representing an affinity site for attaching a cellulose binding fusion protein segment.  
     
     
         35 . The system of  claim 19  wherein said low porosity affinity particles have a plurality of chemical linkers attached thereto.  
     
     
         36 . The system of  claim 19  wherein said low porosity affinity particles are linker-coated polystyrene affinity particles having generally less than 40% porosity.  
     
     
         37 . The system of  claim 20  wherein said low porosity affinity particles are reconstituted cellulose affinity particles.  
     
     
         38 . The system of  claim 19  wherein said system is connected to other said systems forming a multi-system purification method wherein at least a product may be purified using said low porosity affinity particles capable of binding said products in each system for the purification of each product separate from the other using a single source of raw material having a mixture of each product.