Immobilized artificial membranes - Patent Review 4931498

I claim:

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.

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.

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 C.sub.8 -C.sub.26 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.

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.

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.

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.

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 -CH.sub.2 OPO.sub.2 OR.sub.1 covalently bonded to the surface of said support material, wherein R.sub.1 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 R.sub.4 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 .ltoreq. 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.

Description Submit all comments and votes

This invention relates to novel chromatography supports uniquely adapted for the study and purification of biologically active compounds. More particularly, this invention relates to the use of amphiphilic constituents of biological membranes as the stationary phase in chromatographic systems. Membrane constituents are immobilized on the surface of commercially available chromatography support materials to form artificial membranes designed to mimic the associative characteristics of natural biological membranes. Used in chromatography systems, particularly high pressure liquid chromatography (HPLC), the support materials of this invention provide a powerful tool for isolation of chemical substances and identification of new drug leads.

Background and Summary of the Invention

With the rapid growth of research and development in the field of biotechnology there has been an ever increasing demand for new methods and materials critical to the efficient performance of biotechnology research. Indeed, whole new industries have evolved to support the biotechnology revolution. One technology area that has received much attention in service of the needs of the biotech industry is that of chromatographic systems. The separation and purification of biomolecules is of paramount importance, not only to the molecular biologist performing preliminary cloning experiments, but also to the biochemical engineer responsible for the commercial production of high purity products. Much emphasis has been placed on the adaptation of traditional chromatography techniques and systems to meet the many special purification problems of the biotechnology industry. The literature is replete with disclosures of chromatographic theories, techniques and materials for separation of purification of biomolecules.

Types of chromatography which have been applied to the purification of biomolecules, that is, molecules derived from biological sources, include size exclusion chromatography, ion exchange chromatography, bioaffinity chromatography, reversed-phase chromatography and hydrophobic interaction chromatography, among others. The application and efficiency of each of those types of chromatography procedures relies on the selectivity of surface-surface interactions between the solute molecules and the stationary phase of the chromatography system, each interacting with the mobile liquid phase. A wide variety of stationary phase chromatography support materials are commercially available.

The present invention is directed to a new stationary phase chromatographic support material, the surface of which is designed to mimic the structure and surface-surface interactivity of biological cell membranes. Consequently, separation of biomolecules in chromatographic systems utilizing the immobilized artificial membrane supports of the present invention are the result of molecular interactions similar to the interactions of said biomolecules and biological membranes in vivo. More specifically, use of the present supports allow separation of a wide variety of peptides/proteins using an aqueous mobile phase without (or with minimum use of) the added protein-denaturing solvents commonly used in the now popular reversed-phase chromatographic systems. Many peptides can be separated in the same undenatured form they have when they interact with cell membranes in vivo.

The present compositions having covalently bound artificial membrane structure can be employed in chromatographic systems using a highly polar or non-polar mobile phase.

The immobilized artificial membrane-bearing chromatographic supports of this invention also find application to a novel generic protein purification procedure for hybrid proteins expressed by genetically engineered microorganisms. A biologically active protein is expressed as a hybrid protein covalently linked to a membrane-binding peptide through a selectively cleavable peptide linkage. The hybrid peptide is first purified using an immobilized membrane chromatographic support in accordance with this invention, and thereafter, the hybrid peptide is subjected to predetermined conditions for selective cleavage at a cleavage site between the active protein and the membrane binding peptide.

Possibly more significant than use of the present chromatographic supports as a tool for separation and purification of biomolecules is the potential offered by use of the present supports in a high performance chromatographic system for studying the interaction between solute molecules in the mobile phase and an immobilized membrane-excipient stationary phase which can include receptors, enzymes, antibodies and the like. Thus, the present chromatographic supports can serve as a powerful tool for the evaluation and study of drug-membrane/membrane excipient interactions, and they can find use as an in vitro indicator of potential or probable drug activity. Moreover, the artificial membrane bearing supports of the present invention can be used for catalysis reactions and chiral syntheses known to take place in biological or artificial membrane (liposome) environments. The present supports will also find use for vaccine preparation in that it will allow isolation and purification of membrane-binding fractions of viral homogenates.

In accordance with the present invention a composition of matter is provided which comprises a mechanically stable particulate support structure dimensioned for use in a chromatographic system, an artificial membrane structure on the surface of said support material and a means for immobilizing the membrane structure on said surface. The membrane structure comprises an amphiphilic compound having a hydrophilic headgroup portion and a hydrophobic portion. The molecules of the amphiphilic compound collectively define the membrane structure on the surface of the support material so that the membrane structure, in the preferred embodiment, has a hydrophobic inner portion and a hydrophilic outer headgroup portion.

"Immobilized" as used to describe the membrane structure on the surface of the present chromatographic supports is to be regarded as relative to the mobile phase. Thus while the molecules of the amphiphilic compound can be covalently bonded for "immobilization", they can also be "immobilized" relative to a hydrophilic polar (aqueous) liquid phase by hydrophobic interaction between the hydrophobic portion of said amphiphilic compounds and a hydrophobic surface on the particulate support structure. The preferred amphiphilic compounds forming the immobilized membrane structure are those occurring in artificial membranes (liposomes) and biological cell membranes. Phospholipids are most preferred. The immobilized membrane structures can be modified by adsorption of other biological membrane constituents, such as lipids, including phospholipids other than that used as the principal membrane-forming phospholipid, peptides/proteins, saccharides and the like.

Preparation of preferred covalently immobilized membrane chromatographic supports in accordance with this invention can be accomplished utilizing a novel phospholipid carboxylates derived by reaction of C.sub.10 -C.sub.16 cyclic dicarboxylic acid anhydrides with glycero-phosphatides and lysophospholipids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of biological membranes.

FIGS. 2(a) and 2(b) are cross-sectional views of chromatography support particles of the invention.

FIGS. 3(a) and 3(b) are partial cross-sectional views of chromatography support particles of the invention.

FIG. 4 and FIG. 5 are partial cross-sectional views of chromatography support particles of this invention having covalently bound phospholipids and lysophospholipids respectively.

FIG. 6 illustrates infrared absorbance spectra for several intermediate compounds and a mixture thereof.

FIG. 7 shows .sup.13 C solid state magnetic resonance spectra for a lecithin carboxylic acid and a chromatographic support of the invention.

FIG. 8 is a graphic illustration of the relationship between micromoles of bonded lecithin to the surface of Nucleosil-300(7NH.sub.2) and the area in square Angstroms per molecule of lecithin on the support surface.

FIG. 9 is a reflectance IR spectrum of an immobilized membrane bearing support of the invention.

FIGS. 10, 11 and 12 are chromatograms showing separation of compounds on an HPLC column utilizing a support material of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention a chromatography support material is prepared to have a surface designed to mimic the structural and physico-chemical characteristics of natural biological membranes.

Biological membranes elicit affinity for virtually every type of biomolecule. All solutes in biological fluids/systems, including drugs, sugars, lipids, nucleic acids, amino acids, peptides and proteins interact with biological cell membranes. Indeed, such interactions between biomolecules and cell membranes are fundamental to cell function and viability. The chromatography supports in accordance with this invention take advantage of the selective interaction of biomolecules with biological membranes; they are designed to have an immobilized amphiphilic surface structure which resembles that of biological membranes.

There are many illustrations in the literature of membrane structure and the arrangement of membrane constituents. One such illustration is shown in FIG. 1. It is the so-called fluid mosaic model for membrane structure originally proposed by Singer and Nicholson in Science, 175, 720, 1972, and later adapted by Hancock and Sparrow in High Performance Liquid Chromatography, Vo. 3, Academic Press 1983 at page 51. The biological membrane structure 10 is shown as a bi-layer 12(a), 12(b) of amphiphilic phospholipid molecules 13, each having a headgroup portion 14 and a hydrophobic portion 15. The headgroup portions 14 of the respective bi-layer phospholipids collectively define outer hydrophilic membrane surfaces separated by a non-polar (hydrophobic) environment defined by the hydrophobic portions 15 of the predominant phospholipid constituents of the membrane structure. In addition to the amphiphilic phospholipid molecules 13, biological membranes have other constituents 16 present, including, for example, membrane-associated proteins, such as receptor molecules, enzymes, and the like critical for regulation of cell function and other constituents such as lipids (cholesterol, for example) and saccharides.

In accordance with this invention there is provided a novel composition of matter comprising a mechanically stable particulate structure dimensioned for use in a chromatographic system, an artificial membrane structure on the surface of the support material and means for immobilizing said membrane structure on the surface of the support material. The membrane structure comprises an amphiphilic compound having a hydrophilic headgroup portion and a hydrophobic portion, said amphiphilic compound selected from phospholipids and other biological membrane constituents. The immobilized membrane structure is formed to have, like biological membranes, an outer hydrophilic headgroup portion and an inner non-polar (hydrophobic) portion.

Immobilization of the membrane structure can be accomplished either by covalent bonding of the component amphiphilic molecules, by hydrophobic interaction of the amphiphilic molecules with a hydrophobic surface on the particulate support structure or by a combination of hydrophobic interaction and covalent bonding. Each method of membrane immobilization offers some advantage and the preferred method can be determined after consideration of the intended application of the immobilized membrane support material. Thus, while covalent bonding of the constituent amphiphilic compounds forming the membrane structure will likely provide a more stable (i.e, more resistance to "mobilization" of component molecules) immobilized membrane structure and one usable with a wide range of mobile phases, amphiphilic molecules covalently bound to the support surface to collectively define the immobilized membrane structure do not have the lateral mobility of amphiphilic molecules in biological membranes. On the other hand, artificial membrane structures immobilized solely by the, hydrophobic interaction of the membrane forming amphiphilic with a hydrophobic surface on a particulate support will have the lateral mobility such molecules inherently have in biological membranes. Use of hydrophobic interaction as a means for immobilizing the artificial membrane structure suffers from the disadvantage of reduced chromatography support life and mobile phase restrictions. Thus, an immobilized membrane support prepared by contacting a membrane-forming solution of a phospholipid such as phosphatidylcholine with, for example, a commercially available reversed-phase chromatographic support material will be limited to use with polar mobile phases which do not interfere with the hydrophobic interaction of the immobilization of the artificial membrane structure. Further, even where the mobile phase is limited to water, one can expect that the phospholipids, due to their amphiphilic character and the character of solute molecules in the mobile phase, will be leached slowly from the "hydrophobic interaction immobilized" membrane structure. Regeneration of such hydrophobic interaction immobilized membrane supports can be accomplished, for example, by perfusing a fresh solution of the phospholipid through a chromatographic column containing the membrane-bearing support material.

Immobilized membrane supports can also be prepared making use of both hydrophobic interaction and covalent bonding. For example, a hydrophobic reversed-phase particulate support material can be used as a base for forming an immobilized membrane from phospholipids having cross-linkable functional groups on the hydrophobic portion of the phospholipid molecules. Exemplary of such phospholipids are those described by Chapman in U.S. Pat. No. 4,348,329, issued Sept. 7, 1982, the disclosure of which is expressly incorporated herein by reference. The phospholipids disclosed by Chapman have in the hydrophobic portion a C.sub.8 -C.sub.26 fatty acid ester having a conjugated di-yne functionality that can be crosslinked by actinic radiation with like groups to give intermolecular and intramolecular crosslinking. Thus such conjugated di-yne phospholipids can be brought into contact in solution with hydrOphobiC reversed-phase particles or a resinous (polymeric) particulate support material having a hydrophobic surface, to form a membrane-like phospholipid monolayer on the particle surface which thereafter can be exposed to radiation and thereby crosslinked to form a membrane-like pellicular coating effectively immobilized by covalent bonding on the particle surface.

Alternatively, phospholipids having reactive functional groups on their hydrophobic portion can be used to form the artificial membrane structure and thereafter, or in conjunction with membrane formation, they can be reacted with functional groups bound to the surface of the particulate support to covalently bond all or a substantial portion of the amphiphilic molecules constituting the immobilized membrane structure to the surface of the support.

The nature of such covalent bonding is not critical to function of the immobilized artificial membrane supports in accordance with this invention. Thus, covalent bonding of amphiphilic molecules bearing reactive functional groups to support surfaces through, or by reaction with, surface functional groups can be accomplished by a radiation-induced crosslinking reaction or, for example, by nucleophilic or condensation-type reactions resulting in formation of ester, ether, or amide bonds, for example.

It will be recognized that the immobilized artificial membrane-bearing supports prepared in accordance with this invention can be formed from single amphiphilic compounds or from mixtures of different amphiphilic compounds, if necessary, to modify the surface characteristics of the immobilized membrane-bearing chromatography support materials.

The mechanically stable particulate support structures used as a foundation for the present immobilized membrane structures are well known in the art. They can be porous or non-porous and formed of silica, alumina, titania or of resins having sufficient structural integrity to withstand the pressures found in high performance chromatography systems. Particle size can range from about 5 to about 100 microns, more preferably from about 5 to about 50 microns in diameter. Commercially available C.sub.8 and C.sub.18 reversed-phase particulate supports are conveniently used to form immobilized membrane supports in accordance with this invention wherein the membranes are immobilized through hydrophobic interaction of the hydrophobic surface of said supports and the hydrophobic portion of applied amphiphilic compounds. Other commercially available particulate supports can be used advantageously to form covalently immobilized membrane supports. Thus, for example in a preferred embodiment of this invention, Nucleosil-300(7NH.sub.2), a silica based support having a particle size of about 7 microns and bearing covalently bound propylamine groups can be used as described in more detail hereinbelow to provide an immobilized membrane bearing chromatography support wherein the amphiphilic phospholipid molecules constituting the membrane structure are each covalently bound to the particle surface. Again, the nature of the covalent linkage is not critical to the function of the immobilized membrane supports in accordance with this invention.

Other commercially available particulate support structures having functional groups capable of reacting with and forming covalent bonds with functional groups on the hydrophobic portions of amphiphilic phospholipid compounds can be predictably employed. Thus, for example, commercially available pellicular coated chromatography support materials having available functional groups for forming covalent bonds with amphiphilic compounds are acceptable for use as mechanically stable support structures. More particularly, supports having a pellicular coating formed form a polyamine, such as polyethyleneimine, crosslinked with an epoxy resin or other amine crosslinking agents are suitable support structures for the present compositions. Such supports are described by Regnier et al. in U.S. Pat. No. 4,245,005, issued Jan. 13, 1981.

FIGS. 2 and 3 illustrate immobilized membrane-bearing particulate chromatography supports in accordance with this invention. With reference to FIG. 2(a) the immobilized membrane structure comprises covalently bound amphiphilic molecules 18 having a hydrophilic headgroup portion 14 and a hydrophobic portion 15. The molecules of amphiphilic compounds are covalently bonded through a linkage 22 to the surface of a mechanically stable particulate support 19. The immobilized membrane structure can be viewed practically as one-half of the bi-layer 12(a), 12(b) of the biological membrane 10 illustrated in FIG. 1, although supports having a bi-layer-like surface are also completed by this invention. The membrane structure presents an outer hydrophilic surface, i.e., the hydrophilic headgroups 14, and a hydrophobic inner portion collectively defined by the hydrophobic portions 15 of the covalently bound amphiphilic molecules 18.

FIGS. 3(a) and (b) illustrate an immobilized membrane structure 24 comprised of a multiplicity of amphiphilic molecules 18, most preferably phospholipid molecules, having a headgroup portion 14 and a hydrophobic portion 15 on a particle 19 having a hydrophobic surface structure 21 and a locus of hydrophobic interaction 26 between the hydrophobic surface structure 21 and the hydrophobic portion 15 of the amphiphilic molecules 18 FIG. 3(b) likewise illustrates a hydrophilic interaction immobilized membrane structure 24 on the surface of a particle 19 having a hydrophobic surface structure 21. In FIG. 3(b), however, the membrane structure 24 is shown such that the hydrophobic portion 15 of the amphiphilic molecules 18 are interdigitated with hydrophobic substituents collectively defining the hydrophobic surface structure 21, resulting in an enhanced locus of hydrophobic interaction 26 and therefore an increased stability (resistance to mobilization) of the immobilized artificial membrane structure.

Preferred amphiphilic molecules used for forming the immobilized membranes in accordance with this invention are amphiphilic molecules found in natural biological membranes. Exemplary of such are lecithins, lysolecithins, cephalins, sphingomyelin, cardiolipin, glycolipids, gangliosides and cerebrosides. A preferred group of amphiphilic compounds adapted for use in accordance with this invention are phospholipids. Of those phospholipids, lecithins and lysolecithins are particularly preferred. Thus immobilized membranes in accordance with this invention can be formed on support materials utilizing phosphatidylcholine, phosphatidylethanolamine, N-methylphosphatidylethanolamine, N,N-dimethylphosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Phosphatidylcholine, the most predominant phospholipid in biological membranes, is particularly preferred. Such amphiphilic compounds can be used to form immobilized membranes directly on hydrophobic reversed-phase particulate supports to provide hydrophobic interaction immobilized membrane surfaces. Alternatively, those compositions can be modified chemically to have reactive functional groups at or near the terminus of the hydrophobic portion of those molecules, said functional groups being capable of forming covalent bonds with reactive functional groups on the surface of the support material.

The am