WO1990009230A1 - Composite membranes and method of making the same - Google Patents

Abstract

The invention provides composite membrane comprising a porous substrate carrying a synthetic hydrogel film of 0.5 to 50 microns in thickness with a hydration of at least 70 % by weight. The membranes are particularly useful as ultrafilters for the filtration of solutions containing proteins.

Description

COMPOSITE MEMBRANES AND METHOD OF MAKING THE SAME

Ultrafilters are filtration membranes which allow the permeation of small molecules but not macromolecules i.e. molecules of molecular weight

> 5000 daltons, though this limit may vary depending upon the nature of the ultrafi ltration film.

Solvent-cast cellulose acetate membranes are examples of highly selective films. CA-1 membrane from Celanese and Cuprophan PT-150 are formed fr_μom cellulose acetate and these membranes restrict the passage^*of most macromolecules though permeable to small molecular weight solutes. For this reason they may be .used in hae odialysis where small molecular weight materials such as urea are removed from the blood while the plasma proteins are conserved in the circulation. These materials to some degree have properties of hydrogels (see below) since they exhibit moderate swelling ratios in water. A different family of ultrafiltration membranes are the "anisotropic" or "assymetric" membranes. These may be cast using a range of techniques designed to i- produce a thin but compact array of pores of predetermined size on one surface of the membrane while the underlying material is of a much more open porous structure. These membranes may be constructed to allow the passage of molecules of sizes upto some stated limit e.g. <50000 or <200000 daltons. However the permeation barrier, a thin skin,, is sufficently thin to allow rapid water fluxes; the lower porous layer, offers little retardation to water movement. Such filters are produced by manufacturers such as Amicon, Sartorius, Biorad, Gelman and others.

A range of polymer materials is used in forming these membranes: polysulphones , cellulose derivatives, polyesters, polycarbonates, nylon 66, polypropylenes , polyvinylidene difluoride, polytetrafluoroethylene , polyvinylchloride , polyamides, and acrylic copolymers etc.

A third family of membranes may be produced by etching, where continuous polymer film is irradiated and the particle tracks then etched to produce pores. The time of etching defines the size of the pores allowing selectivity for molecular size to be determined. An example of this type of membrane is the Nucleopore membrane prepared from polycarbonate.

A problem encountered in using many, indeed all, of these membranes is that with prolonged use, the material being filtered binds to the membrane face, blocking channels; this reduces filter performance in terms of water fluxes and changed retention characteristics.

Hydrogels are three-dimensional crosslinked water-swollen interpenetrating networks of hydrophilic homopolymers or copolymers. These materials are readily wetted, show low protein adsorption and low thrombogenicity; their ion exchange capacity and surface chemical reactivity can be controlled. Their soft and rubbery consistency give them a strong, superficial resemblance to living tissue. The surface of hydrated hydrogel polymers provides a unique environment which differs from that of almost all other synthetic materials. Hydrogels obtained from acrylic and methacrylic acid are distinguished by an excellent resistance to chemical attack; they can be sterilised by boiling water or saline without deterioration though they suffer from mechanical weakness. In recent years, hydrogels have been used in applications such as haemodialysis membranes, artificial skin and coatings for prostheses. A common use is as contact lenses where polyhydroxyethylmethacrylate is the principal polymer used. Studies of the permeation propertied of true hydrogel membranes have been carried out by Lee et al¹, Chen^, κi_{m e}t _aι3_s Ratner and Miller^ mostly using polyhydroxethyl methacrylate (PHEMA). Earlier, Yasuda and associates'^ studied other methacrylate-bas'ed membranes including poly(glycerol m§thacrylate-co- methacrylic acid), poly(hydroxypropyl methacrylate-co- methacrylic acid) and also pure PHEMA. Limited studies of the use of poly(vinyl alcohol) and poly(N-vinyl- pyrollidone) have also been reported⁷. These studies have been restricted to small molecular weight solutes such as NaCl, urea, creatinine, glucose, tryptophan, sucrose, raffinose, vitamin B12, with some studies on insulin, heparin and dextran. There are relatively little data on the filtration of proteins ^"except for work by Colton et al.⁸ on myoglobin, <? -chymotrypsinogen , f? -lactoglobul in , ovalbumin and albumin using regenerated cellulose membranes.

The low fouling propensity of hydrogels has led to a recent patent^ describing hydrogel-coated microfi ltration membranes i.e. membranes which will retain particulates but not proteins. A porous polyvinyl idene fluoride membrane is directly coated with a crosslinked hydroxyalkyl acrylate polymer. The coating reduces fouling of the micro-filtration membrane, although filtration studies were not detailed in the application. The permeation of solutes is presumably through the pores of the composite membrane, a mode of operation fundamentally different from that described here.

It has been shown by the present inventors¹¹ that thin films of poly-2 ,3-dihydroxypropylmethacrylate behave in a similar manner to films of renal basement membrane in filtering a solution of myoglobin. The present invention stems from studies of filtration in the kidney where blood is continuously ultrafiltered across a connective tissue film termed basement membrane. This filtration barrier seems not to be subject to fouling. The renal basement membrane is an ultrathin (0.3 urn) connective tissue. Investigation of its filtration properties have indicated that it behaves as a randomly-arranged fibre matrix composed of collagen fibres and the filtration mechanism can be explained quantitatively by the fibre matrix theory¹⁰. It is assumed that the barrier is composed of a random matrix of fibres of defined radius packed at a given concentration, and that movement of solute through the barrier is hindered by collisions of solute molecules with flexing fibres; 90% of the space in basement membrane is occupied by water so that the material can be deemed a hydrogel. This theory offers new insights into filtration processes and the low fouling properties of the barrier can be explained, we believe, by there being no extensive solid surfaces to which proteins or other materials can become adsorbed.

There is no analogue of basement membrane currently in use as an ultrafilter and this invention proposes the use of hydrogels as ultrafilters; these mimic the properties of basement membrane. Hydrogels are formed into highly hydrated thin films with void volume of 0.7-0.9 or greater; these are carried by highly porous polymer matrices. This composite is a suitable ultrafiltration barrier for proteins and plasma exhibiting properties which resemble the natural renal basement membrane. The present invention provides a composite membrane comprising a porous polymer support carrying a synthetic hydrogel film wherein the hydrogel film is a homogeneous polymer film fonmed from one or more water soluble monomers with a t'hickness of from 0.5 to 50 microns and a hydration of at least 70% by weight.

The hydrogel film of the composite membranes according to the present invention is a homogeneous polymer film with a hydration of at least 70% by weight.

The hydrogel film is believed to be essentially a random fibre matrix throughout its structure. The film is thus not of the asymetric geometry seen for example with polyelectrolyte or cellulose acetate films wherein on the surface of the film forms a permselective barrier whilst the bulk of the film is an open-celled foam of gross porosity. The hydrogel film also contains essentially no regions of crystall inity although small crystallites may be present under certain conditions.^'

Suitable hydrogels include any formed from one or more water soluble monomers which are capable of forming a homogeneous polymer film with a hydration of at least 70%. Preferred hydrogels include those formed from acrylic and methacrylic monomers, including hydroxyalkyl acrylates and methacrylates, acrylamides and methacrylamides which may be copolymerised with other water soluble comonomers capable of forming homogeneous hydrogels thereof.

Hydrogels with a broad range of unusual physical and chemical properties may be obtained by selection of suitable monomers, and particularly by th use of appropriate concentrations when polymerising from aqueous solvent. The mechanical properties and th degree of hydration of homogeneous hydrogels are strongly dependent upon the proportions of monofunctional monomer and polyfunctional crosslinking agent included in the gel. The conventional method for preparing hydrophilic three-dimensional polymeric networks of hydrogels is simultaneously to polymerise a monomer of the esters of acrylic or methacrylic acid with alcohols possessing hydrophilic groups which impart hydrophilic properties to the polymer. Commonly the polymerisation is carried out at an elevated temperature, using a monoester of acrylic or methacrylic acid in conjunction with a bifunctional alcohol which has an esterifiable hydroxyl group and similar comonomers in an aqueous solution with a small amount of a diester of these acids and of an alcohol which has at least two esterifiable hydroxyl groups in the presence of an initiator.

By far the most widely used monomer is hydroxyethy methacrylate (containing about 0.1% ethylene glycol dimethacrylate and about 3% methacrylic acid), polymerisation gives a hard and brittle resin which swells in water to form a gel; an ethylene glycol-water mixture yields a porous sponge.

Thin hydrogel films may be formed by polymerising a monomer or comonomer solution between two spaced glass plates. The thickness of the film produced is dependant on the size of the spacer used to separate the plates and is generally at least 0.1mm thick. Typically polymerisation will be conducted at approximately 25°C for 24 hours. The hydrogel film is then removed from the glass plate by soaking. A problem with forming composite membranes is the porous polymer support must be bonded to the polymer film. This can be done by chemically modifying the surface of the polymer support. However such methods are not satisfactory since they can adversely affect the properties of either component of the formed composite membrane. A further method that can be employed is in situ polymerisation but adequate control of the polymer film can be difficult to achieve and can cause blockage of the pores of the porous polymer support.

In another aspect the present invention provides a method of making a composite membrane comprising a porous polymer support layer and a continuous synthetic hydrogel layer which method comprises polymerising a monomer or co ono er „ solution, capable of forming a hydrogel, on a hydrophobic surface for a period sufficient to allow^* gelling of the hydrogel film but less than that required for full equilibration of the film, applying a porous polymer matrix to the exposed hydrogel surface, leaving the hydrogel and porous polymer matrix for a time sufficient to allow them to become bonded together and removing the formed composite structure from the hydrophobic surface.

In a preferred method the hydrogel film is polymerised between two hydrophobic surfaces which method comprises polymerising a monomer or comonomer solution capable of forming a hydrogel between a first and a second surface at least one of which is hydrophobic, for a period sufficient to allow gelling of the hydrogel film but prior to full equilibration of the film, removing the first surface from the hydrogel film and applying to the exposed hydrogel surface a porous polymer matrix for a time sufficient to allow them to become bonded together and removing the formed composite structure from the second, hydrophobic, surface. According to the present invention a monomer or comonomer solution is polymerised on a hydrophobic surface, preferably between two surfaces, at least one of which, but preferably both, is hydrophobic to produce a hydrogel film. Shortly after gelling but before full equilibration of the film a porous polymer matrix support is applied to the exposed hydrogel surface. When the hydrogel film is polymerised between two surfaces one of the surfaces is first removed from the hydrogel film; in the case where only one surface is hydrophobic, it is the non- hydrophobic surface which is removed, a porous polymer matrix support is applied to the exposed hydrogel surface. The hydrogel surface should not be allowed to dry out. The porous polymer matrix is preferably applied to the hydrogel surface dry. The hydrogel film and porous polymer matrix are left for a time sufficient to allow complete equilibration of the hydrogel and bonding of the two layers which is achieved without any intermediate chemical binding layers. The composite structure so formed is then removed from the second, hydrophobic surface.

At least one of the surfaces on which polymerisation is carried out should be hydrophobic, if not the hydrogel films are difficult to handle and are unsatisfactory and the bond between the two components of the composite is inadequate so that the components of the composite will tend to separate. Preferably both polymerisation surfaces should be hydrophobic, since it is found that this results in an improved bond between the two components of the composite is obtained. Although no chemical bonding agents are used composite membranes produced according to the present invention show good stability and can be stored for several months without deterioration and are also stable to sterilisation by for example autoclaving.

The equi 1 ibration .of the hydrogel film must not be allowed to go to completion before application of the porous polymer matrix or the subsequent bonding of the two components of the composite membrane, will be inadequate and the two components will separate. Similarly if polymerisation is not allowed to proceed to a sufficient degree, hydrogel film formation will be irregular. For methacrylate monomer and comonomer solutions it has been found that a polymerisation time of between 30 and 60 minutes particularly 30-40 minutes was appropriate before applying the porous polymer matrix to the hydrogel. By observing these restraints gels may be first polymerised on a single hydrophobic surface and the porous polymer matrix applied to the exposed surface after an appropriate interval of time.

The present invention will be illustrated with particular reference to composite membrane ultrafilters comprising a hydrogel based on the monomer 2 ,3-dihydroxypropyl methacrylate (glyceryl methacrylate). It will be understood that hydrogels based on other known monomers are also included in the present invention. The ultrafilters produced according to the present invention preferably comprise a hydrogel film of from 0.5 to 50 microns, preferably between 1 and 20 microns. Hydrogel films of greater than 50 microns tend to reduce flow rate unduly whilst below about 0.5 microns the films do not provide adequate filtration. The hydrogel film will have a hydration value of 70%, and preferably at least 80%. The hydrogel film may optionally comprise no more than 20% crosslinking agent preferably no more than 10%. Above this degree of crosslinking the hydrogels become non uniform. Other particularly preferred monomers include hydroxyethoxyethyl methacrylate, hydroxydiethoxyethyl methacrylate and methoxydiethoxyethyl methacrylate or comono ers thereof and other acrylate or methacrylate monomers or comonomers or acrylamide or methacrylamide monomers or comonomers capable of forming hydrogels with a hydration of at least 70%.

Glyceryl methacrylate by virtue of its two hydroxyl groups, allows highly hydrated films to be formed with water contents of 70-90%; water contents between 80-85% are easy to achieve and the material is easy to handle when mechanically supported. The preparation of 2,3-dihydroxypropyl methacrylate (DHPM) can be achieved by mild acidic hydrolysis of 2 ,3-epoxypropyl methacrylate (glycidyl methacrylate). Polymerisation of glyceryl methacrylate in bulk or in reasonably concentrated aqueous solutions yielded water-insoluble polymers with variable water uptak capacities indicating some formation of crosslinks between the macromolecular chains. However, poly(glyceryl methacrylate) is water-soluble when prepared in very dilute aqueous solution where the formation of crosslinks between the growing macromolecules is less likely. It is possible however to adjust the composition of poly(glyceryl methacrylate) hydrogels so as to control the amount of water imbibed under equilibrium conditions. This is accomplished by varying the concentration of monomer prior to polymerisation, by varying the density of crosslinks, and by varying the concentration of chemical initiator used for polymerisation. Hydrogels with a higher water content have a more open structure which enhances water permeability and increases their permeability to solutes under some conditions. Thus the ultrafiltration characteristics of the gels may be modified though in a limited manner. The hydrophi 1 icity of the gel is also correlated to its structural composition which can be altered by using different monomers, by the addition of comonomers and by crosslinking agents. The crosslinking agents are not hydrophilic but influence hydration by their effect on polymer chain packing. Comonomers employed can be hydroxylalkyl acrylate and/or methacrylate, acrylamide and/or ethacrylamide , N-vinyl-pyroll idone, acrylic and/or methacrylic acid and/or glycidyl methacrylate. In essence, many derivatives of acrylic and methacrylic acid or other compounds used commonly in the preparation of hydrogels may be employed for this invention. Crosslinking agents are selected from diesters having no reactive hydrophilic groups. These are usually the water insoluble alkylene diesters containing acrylic or methacrylic groups e.g. ethylene glycol dimethacrylate. However, tetraethylene glycol dimethacrylate is routinely used in this invention because of its higher solubility in water. Hydration can also be controlled by monomers such as methacrylic acid which possess carboxyl groups which facilitate Van der Waal binding of water in the gel. The use of free carboxyls provides charged centres which undoubtedly influence ultrafiltration and indeed ultrafi ltration characteristics can be controlled to a degree by quantity of acid comonomer used. Similarly basic films can be produced by for example using allylamine as copolymer.

It is also possible to incorporate other components into the membranes of the present invention. These include enzymes, antibodies, chelators or ion exchange resins. These may be chemically linked or entrapped within the hydrogel layer. Uses of such modified membranes include enzymic assays cation or ^-,anion removal or concentration, metal separations or removal, peptide, amino acid or nucleotide separations and a variety of other such uses.

There are a number of initiators available for making hydrogels, the redox persulphate-metabisulphite system which generates free radicals for polymerisation is most common and is used extensively in this invention. No energy sources are needed except for the redox initiators and the polymerisation reaction is generally carried out at ambient temperature. However other radical generating initiators besides redox initiators may be used such as U.V. or gamma radiation.

A variety of porous, preferably polymer, supports to which the hydrogel is bonded may be used, most conveniently these are such as are used for microfiltration membranes. These may be prepared from the esters and mixed esters of cellulose, regenerated cellulose, polycarbonate, polyvinylidene difluoride, polyester, polythene, polytetrafluoroethylene, polyvinylchloride/acrylic, polysulphone, nylon 66 and a positively-charged membrane based on nylon 66. Most derivatives from the same class of the above matrices are also suitable as support. Thus, a wide range of matrices can be selected for their physical and chemical suitabilities for a particular application. In the present examples typically the pore size of the supporting mem^'branes ranged from 0.05 to 8.0 microns, but generally was between 0.2 to 1.0 microns. The size of the membrane disks ranged from 13 mm to 90 mm in diameter; but the 25 mm and 47 mm are more commonly used here. In general, the nature of the support is not critical to the functioning of the hydrogel films.

The composite membranes of the, .present invention are particularly useful for the filtration of solutions containing proteins since they show low fouling and low absorption of protein.. By appropriate selection of membrane composition and filtration conditions the membranes can be made permeable to proteins having a molecular weight up to approximately 100,000. Similarly the membranes can be made to reject proteins down to 20-30,000 Mwt.

It However, other factors such as the configuration of the protein molecules and the presence of charged groups on the protein or membrane will also influence rejection characteristics.

With reference to the figures:

Figure 1a shows profile of solvent flux (Jv) for the filtration of myoglobin using poly(glyceryl methacrylate films of different hydratio-ns. Figure 1b shows profile of solvent flux (Js).

Figure 1c shows profile of hydraulic pre eability coefficient (Lp).

Figure 1d shows profile of rejection.

Figure 2a shows profile of solvent flux (Jv) for filtration of myoglobin through poly(glyceryl methacrylate) hydrogel films of different hydrations prepared using tetraethylene glycol dimethacrylate as crossliker. Figure 2b shows profile of solute flux (Js). Figure 2c shows profile of hydraulic permeability coefficient (Lp).

Figure 2d shows profile of rejection. Figures 3a and 3b show profiles of solvent flux (Jv) for filtration of myglobin through films of copolymers of poly(glyceryl methacrylate)hydrogels.

Figures 3c and 3d show profiles of solute flux (Jv).

Figures 3e and 3f show profiles of hydraulic permeability coefficient (Lp).

Figures 3g and 3h show profiles of rejection. Figure 4 shows profile of rejection for commerically available filtration membranes.

Figure 5a shows profile of solvent flux (Jv) for the filtration of protein solutions using poly(glyceryl methacrylate) and poly(glyceryl methacrylate-co-methyacryl ic acid) films.

Figure 5b shows profiles of rejection of protein solutions using poly(glyceryl methacrylate), poly(glyceryl methacrylate-co-methacryl ic acid) and poly(glyceryl methacryla e-co-al lylamine) .

Figure 6 shows a section through a typical composite membrane of the present invention at 5000 X anification. The upper homogeneous hydrogel layer carried on a lower porous polymer layer.

Methods employed:

Glyceryl methacrylate was polymerised in aqueous solution using ammonium persulphate (12% w/v aqueous solution), and sodium metabisulphite (6% w/v aqueous solution), as redox initiators. Typically, 0.1 ml. of each of the persulphate and bisulphite solutions was added to 1 ml of glyceryl methacrylate appropriately diluted in water. The prepared monomer was used without further purification but it contains some difunctional, or even trifunctional impurities derived from the disproportionation of the monoester molecules. These impurities include glyceryl dimethacrylate, originating from ethylene methacrylate by esterification of the second alcohol function of DHPM, and also glycidyl methacrylate, reactive by virtue of its epoxy ring. The monomer was not purified further since the impurities provide sufficient crosslinking for gel formation. In cases where the monomers have been purified by solvent extraction in hexane/carbon tetrachloride , additional crosslinks can be introduced by adding a known amount of crosslinker to the polymer mixture in order to obtain suitable hydrogels. Polymerisation without crosslinkers yields hydrogels which are alcohol-soluble.

The main difficulty encountered is in preparin the hydrogels as thin/ultrathin films. When polymerised between glass plates using spacers, membranes of about 0.1 mm thickness can be separated from the glass plates'. The membranes prepare in this way were damaged easily because of the nature of the material which is very fragile and so must be supported before use in ultrafiltration cells. However these membranes proved to have unsatisfactory permeability characteristics. Samples of the polymeric membranes were produced between two soda glass plates without spacers in order to produce ultrathin films. The conditioning of the glass plates prior to, use to create a hydrophobic surface is particularly important The glass plates were cleaned- in chromic acid, washed thoroughly with water and finally rinsed with deionise water. A 4% solution of silicone fluid (Dow Corning 1107) in ethyl acetate was applied to the dried plates which were then baked in an oven at 110°C. After two hours, excess silicone was thoroughly wiped off leaving a permanent smooth water-proof surface. Without this treatment, the polymer films formed would adhere tenaciously to the walls of the glass plates. It would then be impossible to lift the polymer composite without damage. In addition the bond between the hydrogel and porous polymer components of the composite membrane is improved if the surfaces on which polymerisation of the hydrogel is done are hydrophobic. A polymer mixture with the desired composition was thoroughly mixed in an aqueous solution, redox initiators were then added and re-mixed thoroughly. An aliquot of 1 ml of the polymer mixture was applied to the centre of a siliconised glass plate. A second plate was then placed on top of the first plate with the siliconised side face down. The polymerising solution spread out by capillary action and covered the entire surface area. The plate sandwich was then held together by clips. The thickness of the membranes can be controlled to some extent by using different strength clips which provide variable applied pressures to the plates. Films of from 0.5 to 50 microns could be produced but typically the membrane thickness when measured under a light microscope varied between 2 to 15 microns, and usually between 6 to 9 microns. The polymer mixture was allowed to gel for 30-40 minutes and then separated; although the gel point corresponding to the incipient formation of an infinite network is reached within several minutes for glyceryl methacrylate. On separation, a ultra-thin layer of film was formed on one of the glass plates. The polymer was transparent, soft and gel-like to the touch, but becomes hard and brittle if allowed to dry out. A porous supporting polymer film (shiny "skin" side down where applicable) was laid over the slightly wet film quickly in order to prevent the film drying out, and this was left for a total of two hours from the start of the initial polymerisation time. At the end of two hours, the polymer composite can be peeled off using a razor blade. The hydrophilic methacrylate-based ^■hydrogel adhered very tightly to the underlying Support matrix giving a permanent laminate without the use of any adhesive or chemical treatment of the polymer or hydrogel surface. Hydrogen-bonding probably accounts for this adhesion. The membrane composites were soaked at room temperature in saline sodium chloride (0.15 mol/1) and Tris-hydrochloric acid (0.01 mol/1) buffer pH 7.4, containing 0.02% sodium azide as a preservative. This removed all traces of initiators, unpolymerised monomers, and other impurities and to allow the membrane to reach its equilibrium swelling. Films stored in this manner maintained normal filtration characteristics for several months without obvious signs of bacterial growth or degeneration. The two polymer matrices which made up the composite remained permanently bonded.

Ultrafilters according to the present invention show very low fouling. Proteins may be simply washed from the filter surface and recovered. The filter may be used and washed a number of times. It is envisaged that the ultrafilters of the present invention will be particularly useful in recovering proteins which are only present in very small quantities. Conventional filters tend to retain much of the filtered protein on their surface when small quantities of material are filtered. The ultrafilters of the present invention allow a higher degree of recovery. The ability to control the characteristics as described previously of the ultrafilters as described previously also means that a filter may be easily adapted for optimum performance in any given situation. Example 1

In this example, glyceryl methacrylate films of ^•different water contents were prepared according to the methods described above. The hydrogel thicknesses were generally between 2 to 15 microns, but 6 to 9 microns being more common. A hydrogel ultrafilter with a support of a mixed cellulose acetate/nitrate microporous membrane having a pore size of 0.45 micron, and a diameter of 47 mm is used for ultrafi ltration studies of protein solutions. The compositions of the polymer mixtures are summarised in Table 1 below. Volume quantity is in millilitres. Some filtration data are shown in Table 2 and in Figures 1a - 1d wherein •= membrane 1, =o membrane 2, ■ = membrane 3 and o = membrance 5.

Table 1. Polymer composition of hydrogels from l ceryl methacr late

GMA = glyceryl methacrylate monomer a = 12% w/v solution b = 6% w/v solution Table 2. Filtration data of hydrogel ultrafilters myoglobin (0.5 mg/ml )NaCl-Tris-HCl buffer pH 7.4

Membrane Pressures Rejection Jv Js (KPa) (%)

Jv = solvent flux (ml. min.^-1 cm^-2) for a 10 microns membrane. Js = solute flux (mg. min.^-1 cm^-2) for a 10 microns membrane. Lp = permeability coefficient (Cm.min^-1). % Hydration = Percentage solvent content at equilibrium a equilibrium with NaCl-Tris buffer. The permeability characteristics of these films resemble those of isolated renal basement membrane. It can be seen that, these films deviated from ideal behaviour, and under conditions where no concentration- polarisation occurred, Jv (Fig. 1a) increased in a non-linear fashion with increasing pressure so that Lp declined (Fig. 1c); Js (Fig. 1b) tended to remain constant so that rejection increased with increasing pressure (Fig. Id). For P-GMA-1:2 (membrane 1), P-GMA-1:3 (membrane 2), P-GMA-1:4 (membrane 3), with hydrations of 76.25%, 83.69% and ^•«84.25% respectively, these films behaved as expected, Lp increasing and rejection decreasing as void volume increased. When attempts were made to increase void volumes by reducing monomer ratio during polymerisation it was found that for P-GMA-1:5 (membrane 4) and P-GMA-1:6 (membrane 5) the hydrations contents were 91.0% and 90.9% respectively. It seems that these films had reached the limit of their solvent capacities; decreasing monomer/solvent ratio did not necessarily produce an increase in the overall water content within the gel structure. Highly hydrated films will have a very open structure and there may be limiting point where the polymer chains tend to collapse. Consequently the rejection profile of P-GMA-1:6 does not fall significantly below those of the denser films (Fig. 1c). Another aspect of behaviour that need be borne in mind^'is that films with higher hydrations are likely to be more compressible. However the water flux through the P-GMA-1:6 film was appreciably higher than for the more dense films (Fig. 1a) indicating that high hydration films may offer advantage in speed of operation.

Example 2

In these examples, increasing amount of tetraethylene glycol dimethacrylate is added as crosslinker to the monomer mixtures. The supporting matrix is a mixed cellulose acetate/nitrate microporous membrane having a pore size of 0.45 micron, and an t diameter of 47 mm. With increasing crosslinker concentration, there is a corresponding decrease in percentage of water within the hydrogels. The compositions of the polymer mixtures are summarised in Table 3 below. _r A

Table 3. Polymer composition of crosslinked glyceryl methacr late hydro els

TEGDMA = tetraethylene glycol dimethacrylate

Table 4 shows the composition of crosslinked poly(glyceryl methacrylate) films of different hydrations. The solvent flux, solute flux, permeability coefficients and rejection values for these membranes using a 0.5 mg/ml myoglobin solution are shown in Figures 2a- 2d where = membrane 10, Δ membrane 11, D = membrane 12.

Table 4. Polymer composition of crosslinked glyceryl methacrylate hydrogels.

Membrane GMA TEGDMA H₂0 (NH₄)₂S 0₈ Na₂S₂0₅ % hy tion

10 P-G A-1:2-X

11 P-GMA-1:3-X

12 P-GMA-1:4-X

TEGDMA = tetraethylene glycol dimethacrylate

Films prepared with 10% cross-linker (dimethacrylate) replacing monomer showed lower water fluxes for the P-GMA-1:2-X (membrane 10) and P-GMA-1:3-X (mMembrane 11) films but a higher flux for the P-GMA-A4-X film (cf. Figs. 2a and 1a); corresponding Js values are shown in Figs 1b and 2b; corresponding Lp values are shown in Figs. 1c and 2c. Rejection values were higher for the P-GMA-1:2-X (cross-linked) film compared with uncross-1 inked but lower for the P-GMA-1:3 and P-GMA-1:4 films (Fig.2d cf 1d). Void volumes were 70.0%, 76.9% and 77.5% respectively. Thus cross-linking allows film behaviour to be varied.

Example 3

In this example, hydrogels are formed from glyceryl methacrylate as the major monomer copolymerising with minor quantities of glycidyl methacrylate hydrophobic and N-vinyl-pyrrol idone hydrophylic. The appropriately mixed monomer mixtures are polymerised according to the method as described in the above examples. The support matrices used are listed below. The pore size ranged from 0.1 to 8.0 microns, but the more common ones being 0.2 and 0.45 microns. The diameter of the disks ranged from 13 mm to 90 mm, but the more common ones being 25 mm and 47 mm.

Table 5. Polymer composition of hydrogels from glyceryl methacrylate and glycidyl methacrylate and N-vinyl-pyrrol idone.

Membrane GMA Gly NVP H₂0 (NH₄)₂S₂θ8^{a N}a2S₂θ5^b % hydration 0

13^c 0.95 0.05 - 3.0 0.1 0.1 ' 86.77

14^d 0.90 0.10 - 3.0

15^c 0.95 - 0.05 3.0 5 16 0.90 - 0.10 3.0

Gly = Glycidyl methacrylate NVP = N-vinyl-pyrrol idone 0

Support matrices:

c = cellulose acetate, cellulose acetate/nitrate, pvc/acrylic, polycarbonate, nylon 66, polythene, 5 polytetrafluoroethylene, polysulphone.

d = cellulose nitrate, cellulose triacetate, charged nylon 66, polyvinylidene difluoride, polyester, regenerated cellulose. . 0

In exploring the influence of hydrophilicity on film behaviour, films were prepared to test the effect of the extend of hydration on filtration behaviour. These include a native poly(glyceryl methacrylate) (P-GMA)

35. membrane No. 2 (o ), film; and copolymers of glyperyl methacrylate with N-vinylpyrrolidone (P-GMA-NVP) membrane No. 16 (σ ), with glycidyl methacrylate (P-GMA-GLY membrane No. 13 (v ), with acrylamide/bisacrylamide membran No. 31 (P ) (P-GMA-A/BA) and two with hydroxyethyl methacrylate (P-GMA-HEMA-1 ) and (P-GMA-HEMA-2) membranes Nos. 26 (▼ ) and 27 ( • ). Flux values increase in the order of P-GMA-HEMA-1 (85.20%) < P-GMA-NVP (86.77%,) < P-GMA/BA (85.75%) which reflected increasing hydration and seemed to coincide with hydrophilicity of the monomers (Figs. 3a-3d). However, rejections were not very different (Figs. 3g and 3h) for P-GMA-GLY, P-GMA and P-GMA-NVP. P-GMA-NVP seems to have higher rejections than its hydration and water fluxes may have suggested. The rejections of P-GMA-A/BA and P-GMA-HEMA-1 are lower than others which seems to correlate with their degrees of hydrations. It is interesting that the copolymers of glyceryl methacrylate with hydroxyethyl methacrylate gave gels which have the lowest (P-GMA-HEMA-1) and highest (P-GMA-HEMA-2) rejection values (Fig. 3b). Hydroxyethyl methacrylate is relatively hydrophobic among the monomers used here so that the copolymer possesses a secondary structure hydrophobic in nature which may introduce heterogenity in the system. This heterogenity causes larger free volume voids in parts of the network (cf. crosslinking) which would explain the apparent higher fluxes but lower rejections. In P-GMA-HEMA-2, the greater proportion of the hydrophobic hydroxyethyl methacrylate used is reflected in the lowest water content of the copolymer which correlates with low fluxes and high rejections. When compared with P-GMA-1:2 which has almost the same water content, P-GMA-HEMA-2 while having slightly higher rejections, has much lower water fluxes. This is further evidence that the nature of the monomers is an important factor in governing filtration behaviour in hydrogel; since it would directly affect hydration which is important in determining water fluxes. Therefore, it seems.to be possible to manipulate film composition in order to produce a range of hydrogels with suitable fiftration characteristics. Lp values for these films are shown in (Figs 3e and 3f). The rejection values for^* two commercially available ultrafilters, A icon PM 10 ( ) and Amicon YM 10 (4 ) are shown for comparison (Fig. 4). It can be seen that the behaviour of the ultrafilter can be altered by the relative hydrophil icity of the membrane, It can be seen that the ultrafilters of the present invention have very different behaviour to the commercially available^" ultrafilters.

Example 4

In these examples, hydrogels are f rmed from glyceryl methacrylate as the major monomer copolymerising with minor amounts of acrylic or methacrylic acid. The appropriately mixed monomer mixtures are polymerised according to the method as described in the above examples.

Table 6. Polymer composition of hydrogels from glyceryl methacrylate and acrylic and methacrylic acid. Membrane GMA A-COOH M-COOH H 0 (NH )₂S 0₈ ^a Na₂S₂05^b % hydration

85.04

A-C00H = acrylic acid M-COOH = methacrylic acid

Support matrices:

e = cellulose acetate, pvc/acrylic, nylon 66, polythene, polytetrafluoroethylene, polysulphone, polycarbonate.

f = cellulose nitrate, cellulose triacetate, charged nylon 66, polyester.

g = cellulose acetate/nitrate, pvc/acrylic, polycarbonate, nylon 66, polythene, polysulphone.

h = cellulose acetate/nitrate, cellulose triacetate, charged nylon 66, polyvinylidene difluoride, polyester, regenerated cellulose, polycarbonate.

The solvent flux values and rejection values of protein solutions and plasma serum for membranes Nos. 2 (© ), 18 (• ) are shown in Figures 5a and 5b. Fig. 5b also includes rejection values for membrane 38 (■ ) P-GMA-ANH₂) a basic film. - 27 -

Water flux values were slightly higher for the more hydrated P-GMA-COOH film (Fig.5a), while rejection values for myoglobin (Fig. 5b) were unchanged. _Λ Rejection values for basic proteins, cytochrome c and lysozyme, were lower than for the uncharged film. The^' rejection of albumin, an acidic protein, were unchanged though rejection is high for this protein and it is, not a sensitive indicator of film behaviour.

Example 5

In this example, hydrogels are formed of glyceryl methacrylate as the major monomer copolymerising with minor amounts of hydroxyethyl methacrylate, hydroxypropyl methacrylate and tetraethylene glycol dimethacrylate. The appropriately mixed monomer mixtures are polymerised according to the method as described in the above examples.

Table 7. Polymer composition of hydrogels from glyceryl methacrylate and hydroxyethyl methacrylate, hydroxypropyl methacrylate and tetraethylene glycol dimethacrylate. HPMA TEGDMA H₂0 (NH )₂S₂0_ga Na₂S₂θ5^B % hydration

- 0.1

0.05 0.1

0.1

0.05 0.1 0.1 0.1 76.50

0.1 85.20

HEMA = hydroxyethyl methacrylate HPMA = hydroxypropyl methacrylate Exampl e 6

In these examples, hydrogels are formed of glyceryl methacrylate as the major monomer copolymerising with minor quantities of ^" acrylamide/bisacrylamide (29/1). The appropriately mixed monomer mixtures are polymerised according to the method as described in the above examples.

Table 8. Polymer composition of hydrogels from glyceryl methacrylate and acrylamide/ bisacrylamide.

Membrane GMA A/BA H₂0 (NH4) S₂0₈ ^a Na₂S 0s^b % hydrat

28^τ 0.1 29^f 0.1 30⁹ 0.1 319

0.1 85.75

A/BA = acrylamide/bisacrylamide (29/1, V,V)

Example 7

In this example, hydrogels are formed of hydroxyethyl methacrylate as the major monomer polymerising with tetraethylene glycol dimethacylate as crosslinker. The appropriately mixed monomer mixtures are polymerised according to the method as described in the above examples.

Table 9 .Polymer composition of hydrogels from hydroxyethyl methacrylate and tetraethylene glycol dimethacrylate. Membrane HEMA TEGDMA solvent* (NH₄)₂S2θ₈ ^a Na₂S₂0s^b

= water/ethylene glycol mixture (1/1; v/v)

cellulose acetate/nitrate, pvc/acrylic, regenerated cellulose, nylon 66, polysulphone, polycarbonate.

Example 8

In this example, glyceryl methacrylate ϊs polymer with latex beads as filler material. The appropriately mixed monomer mixtures are polymerised according to the method as described in the above examples.^" The support matrix is a mixed cellulose acetate/nitrate microporous membrane having a pore size of 0..45 micron, and an diameter of 47 mm.

Table 10. olymer composition of hydrogels from l cer l methacr late with latex beads

= latex beads incorporate into water expressed as percentage below. 1 = 0.1%, 2 = 0.2%, 3 = 0.5%, 4 '= 1.0%. Example 9

In this example, hydrogels are formed from glyceryl methacrylate as the major monomer copolymerising with a minor amount of allylamine. The appropriately mixed monomer mixture was polymerised according to the method as described in the above ^examples. The support matrices were a mixed cellulose acetate/nitrate microporous membrane and a PVC/acrylic 0 copolymer having pore size of 0.45 micron, and diameter of 47 mm. For these, the glass plates were separated after 35-40 minutes, and the composites were peeled off at a total of one hour after the initial polymerisation. 5

Table 11. Polymer composition of hydrogels from glyceryl methacrylate and allylamine.

Membrane GMA A-NH₂ H 0 (NH4) S₂08 Na₂S₂05 % hydrat

^• 38 0.99 0.01 3.0 0.1 0_ 85.75

Example 10 5

Poly(glyceryl methacrylate) films were used in filtration of a range of proteins over periods of over four months. Water flux measured in the absence of macrosolute did not change over this period. Studies with plasma, measuring buffer fluxes in the absence of

30 protein before and after plasma filtration showed no change in flux after 24 hours use. Using Amicon PM 10 j membranes there was a reduction in buffer flux of 20% after plasma filtration.

35 It will be apparent to those skilled In the art of making hydrogels that the composition of the polymerisation mixtures in terms of monomers and comonomers, crosslinking agents, initiators and coinitiators can be varied greatly in accordance to a particular need. Many derivatives , of the aforementioned materials may be copolymerised to form the polymer skeleton of the hydrogels of the invention so long as these are compatible with the process of production of the polymer composite. The same is true for the supporting matrices used as well as the conditions for polymerisation. The foregoing invention has been described in detail by way of illustration and example for purposes of clarity with the understanding that the foregoing disclosure relates to only preferred embodiments of the invention. It is understood that certain changes and modifications may be practised within the spirit of the invention but we intend to cover all changes and modifications of the examples of the invention herein chosen for the purpose of the disclosure. These do not constitute departures from the spirit and scope of the invention set forth in the appended claims, the invention can be practised otherwise than as specifically described.

References

1. Lee, .H., Jee, J.G., Thon, M.S. and Ree, T.,J.Bioeng., 2,269(1978).

2. Chen, R.Y.S., Polym. Prepr., 20(1), 1005(1979).

3. Kim, S.W.. Cardinal, J.R., Wisniewski, S. and Zenter, G.M., in Water in polymers, ACS Symp. Ser. 127, Rowland, S.P. Ed., Am. Chem. Soc, Washington, D.C., 347 (1980).

4. Ratner, B.D. and Miller, I.F., J.Biomed. Mater, Res., 7,353(1973).

5. Yasuda, H. and Lamaze, C.E., J. Macromol. Sci. Phys., 5,111(1971).

6. Ikenberry, L.D., Yasuda, H.K. and Clark, H.G., Chem. Eng. Progr. Symp. Ser., 64,69(1968).

7. Luttinger, M. and Cooper, C.W., J. Biomed. Mater. Res. 1,67(1967).

8. Colton, C.K., Smith, K.A., Merrill, E.W. and Farrell, P.C., J. Biomed. Mater. Res. 5,459(1971).

9. Millipore Corporation, European Patent, Publ. no. 0186758.

10. Robinson, G.B. and Walton, H.A. in Renal Basement Membranes in Health and Disease, Price, R.G. and Hudson, B.G. Ed., Acad. Press. 147 (1987).

11.Robinson, G.B. and Leung, B.K., Diffusion in Polymers - Plastics and Rubber Institute, 2nd International Conference University of Reading 22-24 March 1988.

Claims

1. The present invention provides a composite membrane comprising a porous substrate carrying a synthetic hydrogel film wherein the hydrogel film is a homogeneous polymer film formed from one or more water soluble monomers with a thickness of from 0.5 to 50 microns and a hydration of at least 70% by weight.

2. A composite membrane according to claim 1 wherein the hydrogel film is an acrylate, methacrylate, acrylamide or methacrylamide polymer which may be copolymerised with other water soluble comonomers capable of forming homogeneous hydrogels thereof.

3. A composite membrane according to claims 1 or

2 wherein the hydrogel film is from 1 to 20 microns * thick.

4. A composite membrane according to claims 1 to

3 wherein the hydration is at least 80%.

5. A composite membrane according to claims 1 to

4 wherein the hydrogel film is selected from polyhydroxyethoxyethyl methacrylate, polyhydroxydiethoxyethyl methacrylate, polymethoxydiethoxyethyl methacrylate or poly 2,3-dihydroxypropyl methacrylate or a copolymer thereof.

6. A composite membrane according to claims 1 to

5 wherein the hydrogel contains upto 20% crosslinking agent, preferably up to 10% crosslinking agent.

7. A composite membrane according to claims 1 to

6 wherein the hydrogel includes an acid or other charged species.

8. A composite membrane according to claims 1 to

7 wherein the hydrogel layer include immobilised enzymes, antibodies or liganding agents.

9. A composite ultrafiltration membrane according to any of claims 1 to 7 wherein the porous substrate has pores of from 0.05 to 8.0 microns.

10. A method of making a composite membrane comprising a porous support layer and a continuous synthetic hydrogel layer thereon which method comprises polymerising a monomer or comonomer solution capable of forming a hydrogel on a first hydrophobic surface for a period sufficient to allow gelling of the hydrogel film but less than that required for full equilibration of the film, applying a porous matrix to the exposed hydrogel surface, leaving the hydrogel and porous — matrix for a time sufficient to allow them to become bonded together and removing the formed composite structure from the hydrophobic surface.

11. A method according to claim 10 wherein the hydrogel film is polymerised between two surfaces, at least one of which is hydrophobic, one of the surfaces being removed from the hydrogel film prior to application of the porous matrix.

12. A method according to claims 10 or 11 wherein the hydrogel is polymerised for 30 to 60 minutes before applying the porous matrix.

13. A method according to claim 10 to 12 wherein the monomer is an acrylate, methacrylate, acrylamide, methacrylamide which may be copolymerised with other water soluble comonomers capable of forming homogeneous hydrogels thereof.

14. A method according to claim 10 to 13 wherein the monomer is 2,3 dihydroxypropyl methacrylate or a comonomer thereof.

15. A method according to claims 10 to 14 wherein , the polymerisation is initiated by a radical generating initiator.