Perfusive chromatography
First Claim
1. A chromatography method comprising the steps of:
- (A) forming a chromatography matrix by packing a multiplicity of particles defining throughpores and solute interactive surface regions therewithin; and
(B) passing a fluid mixture of solutes comprising biological molecules through said matrix at a velocity sufficient to induce a convective fluid flow rate through said throughpores greater than the rate of solute diffusion through said throughpores and to produce a Peclet number in a said throughpore greater than 1.0.
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Abstract
Disclosed are chromatography methods and matrix geometries which permit high resolution, high productivity separation of mixtures of solutes, particularly biological materials. The method involves passing fluids through specially designed chromatography matrices at high flow rates. The matrices define first and second interconnected sets of pores and a high surface area for solute interaction in fluid communication with the members of the second set of pores. The first and second sets of pores are embodied, for example, as the interstices among particles and throughpores within the particles. The pores are dimensioned such that, at achievable high fluid flow rates, convective flow occurs in both pore sets, and the convective flow rate exceeds the rate of solute diffusion in the second pore set. This approach couples convective and diffusive mass transport to and from the active surface and permits increases in fluid velocity without the normally expected bandspreading.
109 Citations
34 Claims
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1. A chromatography method comprising the steps of:
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(A) forming a chromatography matrix by packing a multiplicity of particles defining throughpores and solute interactive surface regions therewithin; and (B) passing a fluid mixture of solutes comprising biological molecules through said matrix at a velocity sufficient to induce a convective fluid flow rate through said throughpores greater than the rate of solute diffusion through said throughpores and to produce a Peclet number in a said throughpore greater than 1.0. - View Dependent Claims (3, 4, 5, 16)
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2. A chromatography method comprising the steps of:
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(A) providing a chromatography matrix defining; interconnected first and second throughpore sets, the members of said first throughpore set having a greater means diameter than the members of said second throughpore set, and surface regions in fluid communication with the members of said second throughpore set which reversibly interact with a solute, and (B) passing a fluid mixture of solutes comprising biological molecules through said matrix at a rate sufficient to induce convective fluid flow through both said throughpore sets and to induce a convective flow rate within said second throughpore set greater than the rate of diffusion of said solute within said second throughpore set, thereby to produce a Peclet number in a said second throughpore greater than 1.0. - View Dependent Claims (14, 15)
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6. A chromatography method comprising the steps of
A. providing a chromatography matrix defining: -
interconnected first and second throughpore sets, each of which comprises a multiplicity of pores for channeling through said matrix a mixture of solutes disposed in a fluid, and surface regions in fluid communication with the members of the second throughpore set which sorb a solute in said mixture B. passing a fluid mixture of solutes comprising biological molecules through said matrix at a fluid flow rate sufficient to produce; convective fluid flow through both throughpore sets, a convective fluid flow velocity through said first throughpore set greater than the fluid flow velocity through the second throughpore set, and a convective fluid flow velocity through said second throughpores et greater than the diffusion rate of said solute within a member of said second throughpore set whereby the Peclet number therein is greater than 1.0, to load solutes from said fluid mixture onto said surface regions, and C. passing an eluant through said matrix to elute a fraction rich in a selected one of said solutes. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 17, 18, 19)
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20. A chromatography method comprising the steps of:
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(A) providing a chromatography matrix defining; interconnected first and second throughpore sets, each of which comprise a multiplicity of pores for channeling through said matrix a mixture of solutes disposed in a fluid and surface regions in fluid communication with the members of said second throughpores set which sorb a solute in said mixture (B) passing a fluid mixture of solutes through said matrix at a fluid flow rate greater than 1000 cm/hr to produce; convective fluid flow through both throughpore sets a convective flow velocity through said first throughpores set greater than the fluid flow velocity through said second throughpore set and a Peclet Number greater than 1.0, and a convective fluid flow velocity through a member of said second throughpore set greater than the diffusion rate of said solute within said member of said second throughpores set, and (C) passing an eluant through said matrix to elute a fraction rich in a selected one of said solutes. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28)
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29. A chromatography method comprising the steps of:
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(A) forming a chromatography matrix by packing a multiplicity of particles defining throughpores and solute interactive surface regions therewithin; and (B) passing a fluid mixture including a large biological molecule through said matrix at a velocity sufficient to induce a convective fluid flow rate through said throughpores greater than the rate of molecule diffusion through said throughpores and a Peclet Number greater than 1.0.
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30. A chromatography method comprising the steps of:
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(A) providing a chromatography matrix defining; interconnected first and second throughpore sets, the members of said first throughpore set having a greater mean diameter then the members of said second throughpore set, and surface regions in fluid communication with the members of said second throughpore set which reversibly interact with a large biological molecule, and (B) passing a fluid mixture of solutes including a said large biological molecule through said matrix at a rate sufficient to induce convective fluid flow through both said throughpore sets, to induce a convective flow rate within said second throughpore set greater than the rate of diffusion of said biological molecule within said second throughpore set and to produce in said second throughpore set a Peclet Number greater than 1.0.
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31. A chromatography method comprising the steps of
A. providing a chromatography matrix defining: -
interconnected first and second throughpore sets, each of which comprise a multiplicity of pores for channelling through said matrix a mixture of solutes including a large biological molecule disposed in a fluid, and surface regions in fluid communication with the members of the second throughpore set which sorb a said biological molecule in said mixture B. passing a fluid mixture of solutes including a said biological molecule through said matrix at a fluid flow rate sufficient to produce; convective fluid flow through both throughpore sets, a convective fluid flow velocity through said first throughpore set greater than the fluid flow velocity through the second throughpore set and a Peclet Number greater than 1.0, and a convective fluid flow velocity through a member of said second throughpore set greater than the diffusion rate of said biological molecule within said member of said second throughpore set, to load molecules from said fluid mixture onto said surface regions, and C. passing an eluant through said matrix to elute a fraction rich in a selected one of said biological molecules. - View Dependent Claims (32, 33, 34)
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Specification