Selective recognition of solutes in chromatographic media by artificially created affinity

• US 5,728,296 A
• Filed: 03/20/1996
• Issued: 03/17/1998
• Est. Priority Date: 03/20/1996
• Status: Expired due to Term

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Hjerten, Stellan, Liao, Jia-Li
• Primary Examiner(s): Therkorn, Ernest G.
• Application Number:
  US08/618,879
• Time in Patent Office: 727 Days
• Field of Search: 210/635, 210/656, 210/198.2, 210/502.1, 502/402, 502/404
• US Class Current: 210/198.2
• CPC Class Codes: B01D 15/3804 Affinity chromatography
• CPC Class Codes: B01D 15/3852 using imprinted phases or molecular recognition; using imprinted phases
• CPC Class Codes: B01J 20/26 Synthetic macromolecular compounds
• CPC Class Codes: B01J 20/268 Polymers created by use of a template, e.g. molecularly imprinted polymers
• CPC Class Codes: B01J 20/285 based on polymers
• CPC Class Codes: B01J 20/291 Gel sorbents
• CPC Class Codes: B01J 20/3057 Use of a templating or imprinting material molecularly imprinted polymers B01J20/268; filling pores of a substrate or matrix followed by the removal of the substrate or matrix
• CPC Class Codes: B01J 2220/54 Sorbents specially adapted for analytical or investigative chromatography
• CPC Class Codes: G01N 30/02 Column chromatography

Abstract

Chromatographic media that demonstrate an artificially created recognition of any preselected molecular species with only an affinity-type adsorption are formed by polymerization from solutions of non-ionizable monomers with a quantity of the species contained in the solution, followed by removal of the species by conventional washing. The media are of particular utility and interest when used to achieve affinity binding of proteins. Uses of the media include chromatography and the general isolation and removal of the adsorbed species.

Claims

1. An affinity chromatography medium with selective affinity for a preselected species, said medium formed by:

(a) forming an insoluble polymer matrix from a polymerizable substance in

the presence of said preselected species, said polymerizable substance being non-ionizable and one with respect to which said preselected species is inert; and

(b) extracting said preselected species from said insoluble polymer matrix.

2. An affinity chromatography medium in accordance with claim 1 in which said polymerizable substance is a member selected from the group consisting of acrylamide, agarose, methacrylate, substituted acrylamides and substituted methacrylates.

3. An affinity chromatography medium in accordance with claim 1 in which said polymerizable substance is a member selected from the group consisting of acrylamide, agarose, methacrylate, methyl methacrylate, and 2-hydroxyethyl methacrylate.

4. An affinity chromatography medium in accordance with claim 1 in which (a) is performed in aqueous solution at a polymerizable substance concentration of about 1% to about 60% by weight.

5. An affinity chromatography medium in accordance with claim 1 in which (a) is performed in the presence of a crosslinking agent.

6. An affinity chromatography medium in accordance with claim 5 in which said crosslinking agent is present in an amount ranging from about 0.2% to about 60% by weight relative to said polymerizable substance and said crosslinking agent combined.

7. An affinity chromatography medium in accordance with claim 1 in which said polymerizable substance is acrylamide and (a) is conducted in the further presence of a member selected from the group consisting of N,N'"'"'-methylenebisacrylamide, piperazinediacrylamide, N,N'"'"'-bisacrylylcystamine, and N,N'"'"'-diallyltartardiamide.

8. An affinity chromatography medium in accordance with claim 1 in which said polymerizable substance is agarose and (a) is conducted in the further presence of a member selected from the group consisting of divinyl sulfone and bis-epoxides.

9. An affinity chromatography medium in accordance with claim 1 in which said polymerizable substance is a mixture of 2-hydroxyethylmethacrylate and acrylamide and (a) is conducted in the further presence of N,N'"'"'-methylenebisacrylamide.

10. An affinity chromatography medium in accordance with claim 1 in which said preselected species is a molecule having a molecular weight of from about 100 to about 1,000,000.

11. An affinity chromatography medium in accordance with claim 1 in which said preselected species is a protein.

12. An affinity chromatography medium in accordance with claim 1 in which said preselected species is a protein having a molecular weight ranging from about 3,000 to about 300,000.

13. An affinity chromatography medium in accordance with claim 1 in which said preselected species is a protein having a molecular weight ranging from about 10,000 to about 100,000.

14. An affinity chromatography medium in accordance with claim 1 in which said preselected species is a virus.

15. An affinity chromatography medium in accordance with claim 1 in which said preselected species is a bacterium.

16. An affinity chromatography medium in accordance with claim 1 in which said preselected species is a nucleic acid.

17. An affinity chromatography medium in accordance with claim 1 in which said preselected species is a carbohydrate.

18. An affinity chromatography medium in accordance with claim 1 in (b) is performed by treating said insoluble polymer matrix with a member selected from the group consisting of salts, detergents, ethylene glycol and enzymes.