Immobilization of biologically active protein on a support with a 7-18 carbon spacer and a bifunctional phospholipid

• US 5,405,766 A
• Filed: 03/25/1993
• Issued: 04/11/1995
• Est. Priority Date: 03/26/1992
• Status: Expired due to Fees

• Current Assignee: Her Majesty The Queen In Right of Canada As Represented By The Minister of National Defence
• Original Assignee: Her Majesty The Queen In Right of Canada As Represented By The Minister of National Defence
• Inventors: Thompson, Michael, Lee, William E., Kallury, Krishna M. R.
• Primary Examiner(s): Naff, David M.
• Application Number:
  US08/036,867
• Time in Patent Office: 747 Days
• Field of Search: 435/176, 435/177, 435/180, 435/181, 435/182, 436/518, 436/524, 436/532, 530/810, 530/811, 530/816
• US Class Current: 435/174
• CPC Class Codes: C07K 17/06 attached to the carrier via a bridging agent
• CPC Class Codes: C12N 11/06 attached to the carrier via a bridging agent
• CPC Class Codes: C12N 11/14 Enzymes or microbial cells immobilised on or in an inorganic carrier
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: Y10S 530/81 Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
• CPC Class Codes: Y10S 530/811 Peptides or proteins is immobilized on, or in, an inorganic carrier
• CPC Class Codes: Y10S 530/816 Attached to the carrier via a bridging agent

Abstract

Enzymes and certain other bioactive substances are immobilized on solid substrates which have sufficient functional groups such as hydroxyl or carboxyl. The bioactive substances are linked to the substrates through spacer compounds having a long open alkyl chain with 7-18 carbon atoms and also through phospholipid intermediates. The spacer compound is chemically linked to the substrate. The phospholipid is covalently linked to the spacer compound. Immobilized bioactive substances of the invention exhibit a marked increase in activity and stability. In a preferred embodiment, immobilized enzymes having a high degree of resistance to thermal inactivation are prepared.

Claims

1. A preparation comprising

a solid support

a monolayer forming spacer compound having an alkyl chain containing 7-18 carbon atoms, said spacer compound covalently bound to said support,

a monolayer forming bifunctional phospholipid with two dissimilar functional groups, said phospholipid covalently bound to said spacer compound with one of said functional groups, and

a biologically active protein covalently bound to said phospholipid through the other functional group of the latter.

2. A preparation of claim 1 wherein the spacer compound is a non-polymerizable alkoxyalkylsilane with an alkyl chain containing 7 to 18 carbon atoms, including a terminal carbon atom, free of substituents on the alkyl chain, said alkoxyalkylsilane carrying an added function on the terminal carbon atom of said chain.

3. The preparation of claim 2 wherein the added function on the alkoxyalkylsilane is a carboxylic moiety.

4. The preparation of claim 1 wherein the spacer compound is an aliphatic dicarboxylic acid.

5. The preparation of claim 2 where the alkoxysilane is an omega-carboxyacylaminopropylalkoxysilane.

6. The preparation of claim 1 wherein the spacer compound is a diamine.

7. The preparation according to claim 1 wherein the biologically active protein is an enzyme.

8. A preparation of claim 1 wherein the solid support is selected from the group consisting of metals, metal oxide composites, silicon-based semiconductors, ceramics, and natural or synthetic polymers having, prior to the linkage with the spacer compound, hydroxyl groups or functional groups that can be converted into hydroxyls.

9. The preparation according to claim 1 wherein said biologically active substance is an antibody.

10. The preparation according to claim 1 wherein said biologically active substance is an antigen.

11. The preparation according to claim 1 wherein said phospholipid has two constituent sn chains and each of said functional groups is located at terminal carbon atoms of the two constituent sn chains.

12. A method of making a preparation comprising a biologically active protein immobilized on a solid support, the method comprising the steps of:

a) providing a solid support with hydroxyl groups

b) covalently linking to said support a monolayer-forming spacer compound having an alkyl chain containing 7-18 carbon atoms,

c) covalently bonding to said spacer compound a monolayer-forming bifunctional phospholipid having two dissimilar functional groups through one of said functional groups, and

d) covalently bonding to said bifunctional phospholipid a biologically active protein through the other functional group of said phospholipid.

13. The method of claim 12 wherein the biologically active protein is an enzyme.

14. The method of claim 12 wherein the spacer compound of step b) is an alkoxysilane having a straight alkyl chain with 7-18 carbon atoms and carrying a masked terminal carboxylic function that is capable of liberating the free carboxyl upon hydrolysis.

15. The method of claim 12 wherein the bifunctional phospholipid is a modified bifunctional phosphatidylcholine.

16. The method of claim 12 wherein the bifunctional phospholipid is a modified monofunctional phosphatidylethanolamine.

17. The method according to claim 12 wherein the spacer compound is linked to said support through a siloxane bond.

18. The method according to claim 12 comprising the step of forming in step b) a carboxyalkylsilyl structure of the spacer compound on said support.

19. The method according to claim 15 wherein the bifunctional phosphatidylcholine prior to its bonding to the spacer compound is prepared by the steps of:

a) providing a glycerophosphorylcholine having an sn-1 hydroxyl group

b) protecting the sn-1 hydroxyl group of the choline of step a),

c) treating the choline of step b) with omega-protected aminocarboxylic acid,

d) deprotecting the sn-1 protection of the choline of step c), and

e) introducing an omega-protected hydroxyacyl chain onto the sn-1 carbon by reacting the choline of step d) with omega-protected hydroxycarboxylic acid.

20. The method of claim 12 wherein the phospholipid before bonding to said spacer compound has protective moieties on said two functionalities, the method comprising the steps of selectively removing one of said protective moieties of the phospholipid for bonding with the spacer compound and subsequently removing the other protective moiety for bonding with the biologically active protein.