Immobilized artificial membranes

• US 4,931,498 A
• Filed: 02/25/1988
• Issued: 06/05/1990
• Est. Priority Date: 02/25/1988
• Status: Expired due to Term

• Current Assignee: Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Original Assignee: Purdue Research Foundation (The Trustees of Purdue University (d/b/a Purdue University))
• Inventors: Pidgeon, Charles
• Primary Examiner(s): Kight, III, John
• Assistant Examiner(s): Nutter, Nathan M.
• Application Number:
  US07/160,196
• Time in Patent Office: 831 Days
• Field of Search: 530/345, 530/811, 530/812, 530/813, 530/814, 530/815, 428/357, 428/402, 428/403, 428/406, 428/407, 210/656, 210/927, 525/54.1
• US Class Current: 525/54.1
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: B01J 20/3204 Inorganic carriers, supports or substrates
• CPC Class Codes: B01J 20/3206 Organic carriers, supports or substrates
• CPC Class Codes: B01J 20/3219 involving a particular spacer or linking group, e.g. for attaching an active group
• CPC Class Codes: B01J 20/3274 Proteins, nucleic acids, polysaccharides, antibodies or antigens
• CPC Class Codes: B01J 20/3282 Crosslinked polymers
• CPC Class Codes: B01J 20/3285 Coating or impregnation layers comprising different type of functional groups or interactions, e.g. different ligands in various parts of the sorbent, mixed mode, dual zone, bimodal, multimodal, ionic or hydrophobic, cationic or anionic, hydrophilic or hydrophobic
• CPC Class Codes: G01N 2030/527 sorbent material in form of a membrane
• CPC Class Codes: Y10S 530/811 Peptides or proteins is immobilized on, or in, an inorganic carrier
• CPC Class Codes: Y10S 530/812 Peptides or proteins is immobilized on, or in, an organic carrier
• CPC Class Codes: Y10S 530/813 Carrier is a saccharide
• CPC Class Codes: Y10S 530/814 Cellulose or derivatives thereof
• CPC Class Codes: Y10S 530/815 Carrier is a synthetic polymer
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]
• CPC Class Codes: Y10T 428/2991 Coated
• CPC Class Codes: Y10T 428/2996 Glass particles or spheres
• CPC Class Codes: Y10T 428/2998 including synthetic resin or polymer

Abstract

Methods and materials are described for the preparation of novel immobilized membrane compositions. The described compositions are useful for evaluating membrane association charcteristics of chemical compounds, and as a chromatographic support material for separation/purification of biomolecules and particularly those expressed by genetically transformed cells as novel hybrid proteins having covalently bound membrane-binding peptides. Novel phospholipid carboxylates are useful intermediates for the preparation of chromatography supports having surfaces formed as covalently bound artificial membranes which simulate natural cellular membranes. The immobilized membrane compositions are adapted for use in chromatographic systems to study interactions of biologically active substances with membranes in vitro. The immobilized membranes are expected to find use for vaccine production, protein purification, chiral separations/synthesis, as a combination reverse phase/normal phase HPLC support material, and for drug screening.

Claims

1. A composition of matter comprising

a particulate support material,

a synthetic membrane structure on the surface of said support material, said membrane structure comprising a hydrophilic outer portion and a hydrophobic inner portion and further comprising a layer of molecules of an amphiphilic compound, said molecules having hydrophilic and hydrophobic portions forming the outer and inner portions, respectively, of the membrane structure, said amphiphilic compounds selected from the group consisting of amphiphilic compound constituents of biological cell membranes and liposome-forming amphiphilic compounds, and

means for immobilizing the membrane structure on the surface of the support material.

3. The composition of claim 1 wherein the particulate support material is selected from the group consisting of silica, alumina, titania, and resin based chromatographic support material having a particle size from about 5 to about 100 microns in diameter.

4. The composition of claim 3 wherein the particulate support material has a substantially hydrophobic surface and the means for immobilizing the membrane structure comprises hydrophobic interaction of the hydrophobic surface and the hydrophobic portion of the amphiphilic molecules in said membrane structure.

5. The composition of claim 4 wherein the substantially hydrophobic surface of the particulate support structure is the hydrophobic portion of a second amphiphilic compound covalently linked to the particulate support structure through a functional group on the hydrophilic portion of said compound.

6. The composition of claim 4 wherein the hydrophobic portion of the amphiphilic molecules bears a functional group capable of forming covalent bonds with a second functional group on the substantially hydrophobic surface of the particulate support.

7. The composition of claim 4 wherein the amphiphilic compound is selected from the group consisting of lecithins, lysolecithins, cephalins, sphingomyelin, cardiolipin, glycolipids, gangliosides and cerebrosides.

8. The composition of claim 4 wherein the amphiphilic compound is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, N-methyl phosphatidylethanolamine, N,N-dimethylphosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol and the corresponding lysophospholipid derivatives.

9. The composition of claim 8 wherein the amphiphilic compound is a phospholipid and hydrophobic portion of the phospholipid molecules has at least one functional group for crosslinking the phospholipid molecules forming the synthetic membrane structure.

10. The composition of claim 9 wherein the amphiphilic compound is phosphatidylcholine, the hydrophobic portion of said compound comprising a diacetylenic C₈ -C₂₆ hydrocarbon chain having conjugated di-yne groups crosslinkable upon actinic irradiation.

11. The composition of claim 1 wherein the particulate support has a surface comprising a first functional group and the hydrophobic portion of the amphiphilic compound bears a second functional group capable of reacting with and forming covalent bonds with said first functional group, said means for immobilizing the membrane structure comprising covalent bonds between the first and second functional groups.

12. The composition of claim 11 wherein said first and second functional groups are selected so that said covalent bonds are selected from the group consisting of ester, ether and amide bonds.

13. The composition of claim 11 wherein the amphiphilic compound is a phospholipid.

14. The composition of claim 11 wherein the amphiphilic compound is selected from the group consisting of lecithins, lysolecithins, cephalins, sphingomyelin, cardiolipin, glycolipids, gangliosides and cerebrosides.

15. The composition of claim 14 wherein the amphiphilic compound is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, N-methylphosphatidylethanolamine, N,N-dimethylphosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol.

16. The composition of claim 11 wherein the amphiphilic compound is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, N-methyl phosphatidylethanolamine, N,N-dimethylphosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol.

17. The composition of claim 16 wherein the amphiphilic compound is covalently bound to the surface in an amount sufficient to cover the surface of the support structure at a concentration about 1 to about 2 molecules of amphiphilic compound per 100 square Angstroms of surface area of the support structure.

18. The composition of claim 17 wherein the particulate support structure is a silica, alumina, titania or resin based chromatographic support material having a particle size from about 5 to about 100 microns.

19. The composition of claim 11 wherein the immobilized membrane structure further comprises an absorbed component selected from the group consisting of lipids, peptides, saccharides, oligonucleotides, polynucleotides and membrane-binding analogues of peptides, saccharides, oligonucleotides and polynucleotides.

20. The composition of claim 19 wherein said adsorbed component is selected from the group consisting of phospolipids, cholesterol, cholesterol esters and peptides having a membrane binding amino acid sequence.

21. The composition of claim 11 wherein the amphiphilic compound is a phospholipid covalently bound in an amount sufficient to cover the surface of the support structure at a concentration of about 1.0 to about 2.0 molecules of phospholipid per 100 square Angstroms of surface area of the support structure.

22. The composition of claim 11 wherein the amphiphilic compound is phosphatidylcholine covalently bound in an amount sufficient to cover the surface of the support structure at a concentration of about 1.3 to about 1.6 molecules per 100 square Angstroms of surface area of the support structure.

23. The composition of claim 22 wherein cholesterol is adsorbed in the immobilized membrane structure formed by the covalently bound phosphatidylcholine.

2. The composition of claim 58 wherein the means for immobilizing the membrane structure on the surface of the support material comprises divalent functional groups covalently bonded to the surface of the support material and to the amphiphilic molecules forming the membrane structure.

24. A method for utilizing the membrane-association characteristics of chemical compounds to separate said compounds from compounds having dissimilar membrane-association characteristics, said method comprising the steps of contacting said compounds with the surface of a synthetic membrane-bearing chromatography support material in the presence of a mobile phase and

separating said mobile phase from said chromatography support material, said chromatography support material comprising

a support surface,

a synthetic membrane structure on the support surface, and

means for immobilizing the membrane structure on the support surface, said membrane structure comprising a hydrophilic outer portion and a hydrophobic inner portion and further comprising a layer of molecules of an amphiphilic compound, said molecules having hydrophilic and hydrophobic portions forming the outer an dinner portions, respectively, of the membrane structure, said amphiphilic compounds selected from the group consisting of amphiphilic compound constituents of biological cell membranes and liposome-forming amphiphilic compounds.

25. The method of claim 24 wherein the support surface is substantially hydrophobic and the means for immobilizing the membrane structure comprises hydrophobic interaction of the surface and the hydrophobic portion of the amphiphilic molecules forming said membrane structure.

26. The method of claim 25 wherein the amphiphilic compound is selected from the group consisting of phosphatidylcholine, phosphitidylethanolamine, N-methyl phosphatidylethanolamine, N,N-dimethylphosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol.

27. The method of claim 26 wherein the hydrophobic portion of said amphiphilic compound has at least one functional group for crosslinking the molecules of the amphiphilic compound forming the artificial membrane structure whereby said crosslinking functions to assist immobilization of said membrane structure on the support surface.

28. The method of claim 24 wherein the support surface comprises a first functional group and the hydrophobic portion of the amphiphilic compound has a second functional group capable of reacting with and forming covalent bonds with said first functional group, said means for immobilizing the membrane structure comprising covalent bonds between the first and second functional groups.

29. The method of claim 28 wherein said first and second functional groups are selected so that they react to form covalent bonds selected from the groups consisting of ether bonds, amide bonds, and ester bonds.

30. The method of claim 24 wherein the means for immobilizing the membrane structure on the support surface comprises divalent functional groups covalently bonded to the support surface and to the amphiphilic molecules forming the membrane structure.

31. The method of claim 30 wherein the amphiphilic compound is selected from the group consisting of lecithins, lysolecithins, cephalins, sphingomyelin, cardiolipin, glycolipids, gangliosides and cerebrosides.

32. The method of claim 31 wherein the amphiphilic compound is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, N-methyl phosphatidylethanolamine, N,N-dimethylphosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol.

33. The method of claim 24 wherein the chemical compounds are derived from a cell or a viral homogenate and wherein a membrane binding-fraction of said homogenate is separated for use in a multivalent vaccine formulation.

34. The method of claim 24 wherein the chemical compounds are proteins.

35. The method of claim 24 wherein the protein is one expressed in a host cell transformed with a DNA expression vector capable of expressing said protein.

36. The method of claim 35 wherein the protein is a hybrid polypeptide comprising a membrane-binding amino acid sequence coupled to a biologically active protein.

37. The method of claim 36 wherein the hybrid polypeptide further comprises a protease cleavable or chemically cleavable linking portion between the membrane-binding sequence and the protein.

38. A method for evaluating the membrane-association characteristics of a chemical compound, said method comprising the steps of contacting said compound with the surface of a synthetic membrane-bearing chromatography support material in the presence of a mobile phase, and separating said mobile phase from said chromatography support material, said synthetic membrane-bearing chromatography support material comprising

a support surface,

a synthetic membrane structure on the support surface, and means for immobilizing the membrane structure on the support surface, said membrane structure comprising a hydrophilic outer portion and a hydrophobic inner portion and further comprising a layer of molecules of an amphiphilic compound, said molecules having hydrophilic and hydrophobic portions forming the outer and inner portions, respectively, of the membrane structure, said amphiphilic compounds selected from the group consisting of amphiphilic compound constituents of biological cell membranes and liposome=forming amphiphilic compounds.

39. The method of claim 38 wherein the means for immobilizing the membrane structure on the support surface comprises divalent functional groups covalently bonded to the support surface and to the amphiphilic molecules forming the membrane structure.

40. The method of claim 38 wherein the support surface is substantially hydrophobic and the means for immobilizing the membrane structure comprises hydrophobic interaction of the surface and the hydrophobic portion of the amphiphilic molecules forming said membrane structure.

41. The method of claim 40 wherein the amphiphilic compound is selected from the group consisting of phosphatidylcholine, phosphitidylethanolamine, N-methyl phosphatidylethanolamine, N,N-dimethylphosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol.

42. The method of claim 41 wherein the hydrophobic portion of said amphiphilic compound has at least one functional group for crosslinking the molecules of the amphiphilic compound forming the artificial membrane structure whereby said crosslinking functions to assist immobilization of said membrane structure on the support surface.

43. The method of claim 38 wherein the support surface comprises a first functional group and the hydrophobic portion of the amphiphilic compound has a second functional group capable of reacting with and forming covalent bonds with said first functional group, said means for immobilizing the membrane structure comprising covalent bonds between the first and second functional groups.

44. The method of claim 43 wherein said first and second functional groups are selected so that they react to form covalent bonds selected from the group consisting of ether bonds, amide bonds, and ester bonds.

45. The method of claim 39 wherein the amphiphilic compound is selected from the group consisting of lecithins, lysolecithins, cephalins, sphingomyelin, cardiolipin, glycolipids, gangliosides and cerebrosides.

49. The method of claim 38 wherein the immobilized membrane structure further comprises a component selected from the group consisting of lipids, peptides, saccharides, oligonucleotides, polynucleotides and membrane-binding analogues of peptides, saccharides, oligonucleotides and polynucleotides.

46. The method of claim 46 wherein the amphiphilic compound is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, N-methyl phosphatidylethanolamine, N,N-dimethylphosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol.

47. The method of claim 46 wherein the immobilized membrane structure further comprises a component selected from the group consisting of lipids, peptides, saccharides, oligonucleotides, polynucleotides and membrane-binding analogues of peptides, saccharides, oligonucleotides and polynucleotides.

48. The method of claim 47 comprising the additional step of comparing the membrane association characteristics of said chemical compounds with those characteristics of known biologically active compounds to identify chemical compounds of probable biological activity.

50. The method of claim 47 wherein the component comprises a polypeptides segment of a transmembrane domain of a biologically active protein.

51. The method of claim 47 comprising the additional step of comparing the membrane association characteristics of said chemical compounds with those characteristics of known biologically active compounds to identify chemical compounds of probable biological activity.

52. A chromatography support material comprising a particulate support and a membrane mimetic structure comprising phosphate diester groups of the formula -CH₂ OPO₂ OR₁ covalently bonded to the surface of said support material, wherein R₁ is selected from the group consisting of 2-aminoethyl, 2-(N-methylamino)ethyl, 2-(N,N-dimethylamino)ethyl, 2-(trimethylammonium)ethyl, 2-carboxy-2-aminoethyl, inosityl, glyceryl, and galactopyranosyl.

53. The support material of claim 52 having covalently bound groups of the formula ##STR6## wherein R₄ is hydrogen or acyl group derived from a carboxylic acid.

54. The support material of claim 53 having covalently bound groups of the formula ##STR7## wherein n is an integer from 2 to 14 and m is an integer ≦

n.

55. The support material of claim 54 wherein the covalently bound groups are of the formula ##STR8## wherein n is an integer from 8 to 14.

56. In a chromatographic system comprising a mobile phase and a solid chromatography support material having a stationary phase on the surface of said support material, the improvement consisting essentially of forming said stationary phase as a synthetic membrane immobilized on the surface of the support material, said synthetic membrane comprising a hydrophilic outer portion and a hydrophobic inner portion and further comprising a layer of molecules of an amphiphilic compound, said molecules having hydrophilic and hydrophobic portions forming the outer and inner portions, respectively, of the synthetic membrane stationary phase, said amphiphilic compounds selected from the group consisting of amphiphilic compound constituents of biological cell membranes and liposome-forming amphiphilic compounds.

57. The improvement of claim 56 wherein the amphiphilic molecules forming the synthetic membrane stationary phase are covalently bonded to the surface of the support material.