Protein A-silica immunoadsorbent and process for its production

• US 4,681,870 A
• Filed: 01/11/1985
• Issued: 07/21/1987
• Est. Priority Date: 01/11/1985
• Status: Expired due to Term

• Current Assignee: Cypress Systems Incorporated (Logisteed Limited)
• Original Assignee: Cypress Bioscience, Inc.
• Inventors: Hargreaves, Richard E., Balint, Joseph P. Jr.
• Primary Examiner(s): Garvin, Patrick P.
• Application Number:
  US06/690,781
• Time in Patent Office: 921 Days
• Field of Search: 502/403, 128/DIG. 3
• US Class Current: 502/403
• CPC Class Codes: A61M 1/3679 by absorption A61M1/3675 takes precedence
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: B01J 20/103 comprising silica
• CPC Class Codes: B01J 20/28004 Sorbent size or size distribution, e.g. particle size
• CPC Class Codes: B01J 20/3078 Thermal treatment, e.g. calcining or pyrolizing
• CPC Class Codes: B01J 20/3085 Chemical treatments not covered by groups B01J20/3007 - B01J20/3078
• CPC Class Codes: B01J 20/3204 Inorganic carriers, supports or substrates
• CPC Class Codes: B01J 20/3217 Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
• CPC Class Codes: B01J 20/3219 involving a particular spacer or linking group, e.g. for attaching an active group
• CPC Class Codes: B01J 20/3257 the functional group or the linking, spacer or anchoring group as a whole comprising at least one of the heteroatoms nitrogen, oxygen or sulfur together with at least one silicon atom, these atoms not being part of the carrier as such
• CPC Class Codes: B01J 20/3263 comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. an heterocyclic or heteroaromatic structure
• CPC Class Codes: B01J 20/3274 Proteins, nucleic acids, polysaccharides, antibodies or antigens
• CPC Class Codes: B01J 2220/58 Use in a single column
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: Y10S 128/03 Heart-lung

Abstract

An immunoadsorbent material for removing IgG and IgG-complexes from biological fluids is prepared by covalently binding protein A to a solid-phase silica matrix. It has been found that particularly stable, high-capacity immunoadsorbents are obtained by derivatizing the silica with amino and/or carboxyl groups, and reacting the protein A with a carbodiimide at a pH in the range from 3.5 to 4.5. Binding through free hydroxyl groups may be achieved with cyanogen halides at a pH in the range from 11.0 to 11.5. After acid washing (pH 2.0-2.5) to remove non-covalently bound protein A, the immunoadsorbent may be employed in a column for therapeutic treatment of various cancers and autoimmune disorders where IgG-complexes are implicated as suppressing factors in inhibiting a normal immune response.

Claims

1. A method for preparing an immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said method comprising:

introducing free amino or carboxyl groups onto a silica matrix;

reacting the silica matrix with purified protein A in the presence of a carbodiimide at a pH in the range from 3.5 to 4.5 to covalently link the protein A to the matrix through the amino or carboxyl groups; and

washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.

2. A method as in claim 1, wherein amino groups are introduced onto the silica matrix by reaction with a silane.

3. A method as in claim 2, wherein the amino silane is γ

-aminoproplytriethoxysilane.

4. A method as in claim 1, wherein carboxyl groups are introduced by first reacting the silica matrix with an amino silane to introduce amino groups, followed by reacting the silica matrix with succinic anhydride to replace the amino groups with carboxyl groups.

5. A method as in claim 1, wherein the silica matrix is amorphous silica.

6. A method as in claim 5, wherein the amorphous silica is diatomite.

7. A method as in claim 6, wherein the diatomite is sized from 45 to 60 mesh.

8. A method as in claim 1, wherein the protein A is present at at least 0.05 weight percent of the silica matrix.

9. An immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said material comprising purified protein A covalently coupled to a silica matrix prepared by the method comprising:

introducing free amino or carboxyl groups onto a silica matrix;

reacting the silica matrix with purified protein A in the presence of a carbodiimide at a pH in the range from 3.3 to 3.7 to covalently link the protein A to the matrix through the amino or carboxyl groups; and

washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.

10. An immunoadsorbent material as in claim 9, wherein amino groups are introduced onto the silica matrix by reaction with a silane.

11. An immunoadsorbent material as in claim 10, wherein the amino silane is γ

-aminoproplytriethoxysilane.

12. An immunoadsorbent material as in claim 9, wherein carboxyl groups are introduced by first reacting the silica matrix with an amino silane to introduce amino groups, followed by reacting the silica matrix with succinic anhydride to replace the amino groups with carboxyl groups.

13. An immunoadsorbent material as in claim 9, wherein the silica matrix is amorphous silica.

14. An immunoadsorbent material as in claim 13, wherein the amorphous silica is diatomite.

15. An immunoadsorbent material as in claim 9, wherein the protein A is present at at least 0.05 weight percent of the silica matrix.

16. A method for preparing an immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said method comprising:

introducing free hydroxyl groups onto a silica matrix;

activating the silica matrix in the presence of cyanogen bromide at a pH in the range from 11.0 to 11.5, and covalently linking the protein A to the matrix at a pH in the range from 8.5 to 9.0; and

washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.

17. A method as in claim 16, wherein hydroxyl groups are introduced onto the silica matrix by reaction with a silane.

18. A method as in claim 17, wherein the silane is γ

-glycidoxypropyltrimethoxysilane.

19. A method as in claim 16, wherein the silica matrix is amorphous silica.

20. A method as in claim 19, wherein the amorphous silica is diatomite.

21. An immunoadsorbent material useful for removing IgG and IgG-complexes from biological fluids, said material comprising purified protein A covalently coupled to a silica matrix prepared by the method comprising:

introducing free hydroxyl groups onto a silica matrix;

activating the silica matrix in the presence of cyanogen bromide at a pH in the range from 11.0 to 11.5, and covalently linking the protein A at a pH in the range from 8.5 to 9.0; and

washing the silica matrix at a pH in the range from 2.0 to 2.5 to remove loosely bound protein A from the matrix.

22. An immunoadsorbent material as in claim 21, wherein hydroxyl groups are introduced onto the silica matrix by reaction with a silane.

23. An immunoadsorbent material as in claim 22, wherein the silane is γ

-glycidoxypropyltrimethoxysilane.

24. An immunoadsorbent material as in claim 21, wherein the silica matrix is amorphous silica.

25. An immunoadsorbent material as in claim 24, wherein the amorphous silica is diatomite.

26. An immunoadsorbent material as in claim 25, wherein the diatomite is sized from 45 to 60 mesh.