Lignocellulosic and cellulosic beads for use in affinity and immunoaffinity chromatography of high molecular weight proteins

• US 5,328,603 A
• Filed: 08/19/1992
• Issued: 07/12/1994
• Est. Priority Date: 03/20/1990
• Status: Expired due to Fees

• Current Assignee: Virginia Tech Intellectual Properties, Inc. (Virginia Polytechnic Institute and State University)
• Original Assignee: The Center For Innovative Technology
• Inventors: Kaster, Jeffrey A., Velander, William H., Glasser, Wolfgang G.
• Primary Examiner(s): Therkorn, Ernest G.
• Application Number:
  US07/932,710
• Time in Patent Office: 692 Days
• Field of Search: 536/56, 536/57, 536/124, 210/635, 210/656, 210/198.2, 210/502.1, 502/401, 502/404, 96/101
• US Class Current: 210/198.2
• CPC Class Codes: B01J 20/291 Gel sorbents
• CPC Class Codes: B01J 20/3212 consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
• CPC Class Codes: B01J 20/3219 involving a particular spacer or linking group, e.g. for attaching an active group
• CPC Class Codes: B01J 20/3227 by end-capping, i.e. with or after the introduction of functional or ligand groups
• CPC Class Codes: B01J 20/3274 Proteins, nucleic acids, polysaccharides, antibodies or antigens
• CPC Class Codes: B01J 20/3287 Layers in the form of a liquid
• CPC Class Codes: B01J 20/3293 Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
• CPC Class Codes: B01J 2220/54 Sorbents specially adapted for analytical or investigative chromatography

Abstract

Improved cellulosic beads for use as supports in bioaffinity chromatography are produced by dissolution of cellulose in a chaotropic cellulose solvent, formation of the dissolved cellulose into droplets, and immersion of the droplets into a non-solvent capable of solvent interchange with the cellulose solvent to form generally spherical porous cellulose beads of narrow particle size distribution. The beads formed are preferably made with cellulose having a degree of polymerization between 100 and 200, and the resulting beads when saturated with water without drying contain between 1% and 7% cellulose by weight and have a particle size of at least about 0.3 mm. The beads can be activated by a suitable activation method, and specific bioaffinity ligands are bound to the active sites in the beads. The beads reacted ligands, the beads then used in bioaffinity chromatography to isolate specific bioaffinity molecules having molecular weights between 5,000 and 500,000 from complex solutions. The beads are particularly useful in bioaffinity chromatography of antibodies, therapeutic proteins, enzymes, and other high molecular weight proteins. The cellulose beads have similar properties to agarose beads used in the prior art for bioaffinity chromatography of high molecular weight proteins, but the cellulosic beads of the present invention have much greater mechanical strength and resist crushing under higher column flow rates without chemical cross linking are much cheaper to produce than prior art agarose and dextran beads.

Claims

1. Beads for use in affinity chromatography, comprising:

from about 1% to about 7% of said beads being cellulose, said beads being sufficiently porous to allow molecules with molecular weights ranging from 5,000 to 5,000,000 to have access to their interiors, having a particle size greater than about 0.3 mm, and having sufficient strength to avoid crushing when used in chromatographic separation at linear flow rates above 20 cm/min in a 15 cm column bed;

active sites on said cellulose;

bioaffinity ligands attached to said active sites, said ligands having molecular weights up to about 500,000; and

specific molecular recognition sites on said ligands, said molecular recognition sites capable of selectively attracting and binding specific bioaffinants, said bioaffinants having molecular weights ranging up to about 500,000;

wherein, said pores are of sufficient size to allow the attachment of said ligands to said active sites on said cellulose without altering the capability of said specific molecular recognition sites to selectively attract and bind specific bioaffinants.

2. Beads according to claim 1, wherein:

said ligands and said bioaffinants are proteins selected from the group consisting of antibodies, therapeutic proteins, and enzymes.

3. Beads according to claim 2, wherein:

said beads are of substantially spherical shape, and

said cellulose has a degree of polymerization ranging less than about 200.

4. Beads according to claim 1, wherein:

said beads are never dried, and are combined with an aqueous medium to form a gel.

5. Beads according to claim 1, wherein:

said beads having a particle size of at least about 0.5 mm.

6. A chromatography support, comprising:

highly porous cellulose beads which contain when saturated with water a content of cellulose between about 1% to 7% by weight, and a particle size greater than about 0.3 mm;

said cellulose imparting to said beads sufficient strength to avoid crushing when used in chromatographic separation at linear flow rates above 20 cm/min in a 15 cm high bed, wherein said support is a bioaffinity support, active sites on said cellulose;

bioaffinity ligands attached to said active sites, said bioaffinity ligands having molecular weights ranging up to about 500,000 said ligands having specific molecular recognition sites thereon to selectively attract and bind specific bioaffinants having molecular weights ranging up to about 500,000; and

the pores of said porous beads being of sufficient size to allow the attachment of said ligands to said active sites on said cellulose without altering the capability of said molecular recognition sites in said ligands to selectively attract and bind specific bioaffinants and being sufficiently porous to allow molecules with molecular weights ranging from 5,000 to 5,000,000 to have access to their interiors.

7. The chromatography support, of claim 6, wherein

said beads have a particle size greater than about 0.5 mm.

8. The chromatorgraphy support of claim 6, wherein said bioaffiants are proteins selected from the group consisting of antibodies, therapeutic proteins, and enzymes.