GB2224668A - Filter - Google Patents

Abstract

A liquid permeable filter comprises a filter medium supporting a macromolecular-permeable gel which shows low non-specific binding, is easily derivatised and having high matrix loading capacity. The gel may comprise polyacrylamide or dextran and preferably occupies not more than 60% of the void space of the membrane.

Description

FILTER This invention relates to a filter for use in the separation of materials. The use of glass microfibre filters and of microporous membrane filters in material separation is now well known. Also known are so-called composite microporous membrane filters, which are used for filtration or as matrices for covalently immobilising active chemical species. Membranes with immobilised active components are commonly employed in clinical diagnostic systems or may be used in liquid chromatography systems.

The structure of known microporous membrane offers a very limited number of sites for immobilising active chemical species. At best, the structure could probably support a monolayer of molecules, that is to say the complete surface of the membrane structure is covered by a single molecular layer of active species. Furthermore, the majority of available membranes show a high level of nonspecific binding which is undesirable. The term nonspecific binding refers to the very strong and indiscriminate binding of protein species. Low nonspecific binding refers to binding of less than O.lmg of protein per cm3 of matrix. Yet still further, very few of the membranes in the prior art could be used to covalently bind the active chemicals to their structure.

Previous attempts in membrane modification did not address the mentioned areas of limitations. For example, US specification 4,066,512 describes a composite membrane structure where the coating is formed by adsorbing a layer of protein onto the surface of the membrane structure.

Such protein layer was shown in the same specification to bind other proteinaceous substances in a non-specific.

manner.

Accordingly, a need exists in the industry for membranes which can show combined high matrix loading capacity, very low non-specific binding and ease of derivatisation. The term matrix loading capacity refers to the membrane's ability to carry the immobilised groups and substances that may complex with them. A loading capacity is regarded as high if it exceeds lOmg per cm3 of matrix.

According to the invention a filter comprises a filter medium supporting a gel that is permeable to macromolecules, shows low non-specific binding, is easily derivatised and has a high matrix loading capacity, the gel content of the filter being such that liquid can flow through the filter medium.

Gels are classed as semi-solids, and their use as matrices in protein chromatography is known. Their compositions and characteristics are comprehensively described in the work of C R Lowe entitled "An Introduction to Affinity chromatography" published by Elsevier Biomedical Press in Amsterdam. It has been found, surprisingly, that a gel may be supported in a microporous membrane or other filter medium in such a way that severe impairment is not caused to the membrane, and in particular without causing an unacceptable reduction of flow of liquid through the filter medium.

The gel is preferably aqueous polymer gel containing from 2% to 10% by weight of polymeric material, the balance being water. The gel is desirably permeable to molecules in excess of 10,000 MW, and the polymeric material is conveniently selected from dextran and polyacrylamide.

Although the invention is applicable to a number of different filter media, for example to a glass microfibre filter, its preferred use is in the field where the filter medium is a microporous membrane.

The membrane is desirably impregnated with the gel, with the gel occupying no more than 60% of the void space in the unimpregnated membrane. This has been found to lead to very good gel retention, without materially reducing the flow rates of liquid through the membrane.

Many different membrane-forming polymeric materials are known. In the present invention, however, it is particularly preferred that the membrane is a cross-linked cellulose membrane. Such membranes are desirable as they do not exhibit non-specific binding, that is the indiscriminate binding of substances to the membrane material. The membranes are generally cast from cellulose esters such as cellulose acetate and/or cellulose nitrate which, after the phase inversion process, are converted to cellulose by hydrolysis of the nitrate or acetate groups.

With both the membrane and the gel materials exhibiting very low non-specific binding it will be appreciated that membranes according to the invention can readily be designed for specific binding applications. In preparing the membrane for a particular use, a specific functional group capable of covalent bonding to remove a particular substance from the material to be filtered will be immobilised onto the gel, which thus acts as a matrix for the functional groups.

The invention also extends to a method of forming a filter, comprising the steps of applying a gel-forming solution to a filter medium, precipitating the gel-forming material by use of a precipitating solution to give a gel that is permeable to macromolecules, shows low non-specific binding, is easily derivatised and has a high matrix loading capacity, the gel content of the filter being such that liquid can flow through the filter medium. A gel is considered to be easily derivatised if extreme conditions are not required, typically temperatures not greater than 800C and a pH range defined by the pH values of molar HC1 and molar NaOH.

Applicant believes, although not wishing to be bound by this belief, that while the structure of the membrane is surrounded by the impregnating solution which contains the gel forming materials, during precipitation the solid parts of the structure act as nuclei on which the materials will deposit as they become insoluble. Thus by ensuring that the membrane structure is impregnated uniformly, one should achieve a substantially uniform gel coating on all surfaces within the membrane structure.

Preferably the solution is capable of yielding a polymeric gel containing at least 90% water by weight, and a particularly preferred solution is one containing 2% to 10% dextran by weight.

The precipitated coat of gel may be quite stable and require no further fixing. However, in some cases fixing may be required and this can be achieved using a fixing solution that suitably contains a catalyst or cross-linking agent. A proportion of the cross-linking agent is incorporated into the gel, the remainder being washed off.

When the filter medium is a microporous membrane then preferably the gel-forming solution is applied by immersing the membrane in the solution so as to impregnate the membrane with the solution, excess solution is wiped off the membrane, and precipitation is effected by immersing the membrane in the precipitating solution. The removal of the excess solution from the membrane ensures that the surface of the membrane is not blocked or sealed off by the precipitated materials, and therefore that the membrane remains permeable.

When a glass microfibrous filter is used the thickness of the gel layer is preferably of the same order of magnitude as the fibres, for example in the range 0.1 to 1 micron.

In order that the invention may be better understood some examples of filters and formation methods in accordance with the invention will now be described in more detail, by way of example only.

EXAMPLES 1 - 8 In these examples, impregnating solutions were prepared by disolving dextran 50,000 - 300,00 MW in water.

The solutions were heated with stirring to 900C. Discs of filter material, 47 mm in diameter, were placed in the solutions for 30 minutes then they were removed. The excess impregnating liquid was scraped off the membranes with a knife. The membranes were then placed in a precipitating solution which contained a cross-linking agent for 20 hours. The precipitating solution was prepared by adding 1 part of 1M NaOH in water to 2 parts of acetone. The cross linking agent was either dibromopropanol (DBP) or epichlorohydrin (EPC) which was added to the precipitating solution to 10% on a volume to volume basis. After cross linking the membranes were washed first in acetone then in water.

EXAMPLE 9 In this example the impregnating solution was made up by adding the following components: 25 ml acrylamide; 25 ml of 1.5M Tris/HCl pH 8.8 containing 0.4% SDS; 48.9 ml distilled water and 0.1 ml NNN'N'-tetramethylethyldiamine (TEMED). The precipitating solution was 10% ammonium persulphate (AP) in water. The procedure comprised the steps of soaking the membrane in the impregnating solution for 10 minutes; scraping off excess liquid with a knife; then placing it in the precipitating solution for 10 minutes and finally washing the membrane in water.

Table 1 is a summary of the results of examples 1 - 9.

The dry weight was determined by placing the membranes in an oven at 1020C for 3 hours before weighing. The percentage weight increase is defined as the percentage weight difference between a sample that has been impregnated and an untreated sample. The water flowrate was determined by placing the samples in a holder and measuring the water flowrate at a pressure difference of 20 psi. The percentage water flowrate reduction is the percentage difference between the flowrate of an impregnated sample and an unimpregnated sample.

TABLE 1

Example I Filter Impregnating Precipitating % Dry % Water No. Medium Solution and Fixing Weight Flowrate Solution Increase Reduction 1 XLC 2% Dextran 10% EPC 23 ND 2 XLC 5% Dextran 10% EPC 35 12 3 XLC 7.5% Dextran 10% EPC 60 23 4 XLC 10% Dextran 10% EPC 88 50 5 XLC 2% Dextran 10% DBP 15 ND 6 XLC 5% Dextran 10% DBP 12 ND 7 NY 5% Dextran 10% EPC 22 ND 8 GMF 10% Dextran 10% EPC 91.4 7.5 9 9 CN See Text 10% AP ND ND XLC = 0.45 Micron Cross Linked Cellulose membrane NY = 0.45 Micron Nylon membrane GMF = Glass microfibre filter CN = 1.2 Micron Cellulose nitrate membrane ND = Not determined The above results clearly show that increasing the concentration of the impregnating solution increases the content of the coating but reduces the water flowrate progressively. The electron micrographs 1 - 5 show the micro-structure of various membrane samples. Micrograph 1 shows the structure of the sample from example 1 which is markedly different from the structure of an uncoated membrane (micrograph 2).Typically the width of the fibres increases with respect to its length in the impregnated samples as evidenced by example 3 (micrograph 3) and example 9 (micrograph 4). In all the impregnated samples the covering of the internal surfaces of the filter medium appeared extremely uniform except in the glass microfibre filter sample (micrograph 5). It is possible that in the latter case the pore size of the filter is already above the upper limit (greater than 10 micron), or it may be that the gel does not naturally bind to a glass surface in which case the surface could be modified by a suitable treatment prior to gel application.

EXAMPLE 10 Three membranes from example 3 above were dyed with Cibacron Blue (a trade mark of the Ciba Geigy Company, Switzerland) as follows. Three discs were placed in 50 ml of 0.6% w/v dye solution. After 15 minutes 10 ml of 1M NaOH was added. After 20 hours the discs were removed and washed thoroughly with water. The discs were then placed in a membrane holder and flushed with 50 ml of 0.05M sodium phosphate buffer. A solution containing 1 mg human serum albumin (HSA) per ml in the same buffer was recirculated through the membranes for 15 minutes and then flushed with 50 ml of the same buffer. The HSA bound to the membranes was eluted by flushing with 20 ml of 0.5M KSCN at a flowrate of lml/min. The amount of protein bound was determined by a UV absorption technique and was found to be 9.5 mg. The procedure was repeated for three untreated samples (XLC) as a control. The amount of protein bound to the control membranes was 3.7 mg.

It will be understood that these examples illustrate only some of the composite membranes of the invention and that by suitable selection of the membranes and gel materials and suitable control of the precipitation and gel fixing conditions, a wide range of gel membranes may be formed. The actual physical form of the finished composite membrane can be as required for the intended end use, and the membranes may generally take the form of any large surface area bodies, such as for example sheets, ribbons, tubes, hollow fibres or beaded particles.

Claims (19)

1. A filter comprising a filter medium supporting a gel that is permeable to macromolecules, shows low nonspecific binding, is easily derivatised and has a high matrix loading capacity, the gel content of the filter being such that liquid can flow through the filter medium.

2. A filter according to Claim 1 in which the gel is aqueous polymer gel containing from 2% to 10% by weight of polymeric material.

3. A filter according to Claim 2 in which the gel is permeable to molecules in excess of 10,000 MW.

4. A filter according to Claim 2 or Claim 3 in which the polymeric material is selected from dextran and polyacrylamide.

5. A filter according to any one of the preceding Claims, in which the filter medium is a microporous membrane.

6. A filter according to Claim 5 in which the membrane has a pore size of from 0.1 to 10 microns, preferably from 0.45 to 3 microns.

7. A filter according to Claim 5 or Claim 6, in which the membrane is impregnated with the gel, and the gel does not occupy more than 60% of the said space in the unimpregnated membrane.

8. A filter according to any one of Claims 5 to 7, in which the membrane is a cross-linked cellulose membrane.

9. A filter according to any one of Claims 1 to 4, in which the filter medium is a glass microfibre filter.

10. A method of forming a filter, comprising the steps of applying a gel-forming solution to a filter medium, precipitating the gel-forming material by use of a precipitating solution to give a gel that is permeable to macromolecules, shows low non-specific binding, is easily derivatised and has a high matrix loading capacity, the gel content of the filter being# such that liquid can flow through the filter medium.

11. A method according to Claim 10 in which the solution is capable of yielding a polymeric gel containing at least 90% water by weight.

12. A method according to Claim 11 in which the solution contains 2% to 10% dextran by weight.

13. A method according to any one of Claims 10 to 12, in which the precipitating gel is fixed by a fixing solution.

14. A method according to any one of Claims 10 to 13, in which the fixing solution contains a catalyst or crosslinking agent.

15. A method according to any one of Claims 10 to 14 in which the filter medium is a microporous membrane, the solution is applied by immersing the membrane in the solution so as to impregnate the membrane with the solution, excess solution is wiped off the membrane, and precipitation is effected by immersing the membrane in the precipitating solution.

16. A method according to Claim 15 in which the membrane has a pore size of from 0.1 to 10 micron, preferably from 0.45 to 3 micron.

17. A method according to Claim 15 or Claim 16, in which the membrane is a cross-linked cellulose membrane.

18. A filter substantially as hereinbefore described with reference to the Examples.

19. A method of forming a filter substantially as hereinbefore described with reference to the Examples.