This invention relates generally to the field of the immobilisation of enzymes on solid supports for carrying out enzyme-catalyzed reactions.

In one aspect the invention relates to methods and apparatus for retarding the initial action of an enzyme on its substrate, whilst not retarding, or at least retarding to a lesser extent, the subsequent reaction. Such a retardation pattern can be useful for example when the enzyme substrate is a disinfectant, such as hydrogen peroxide, and it is desired to produce a disinfection system in which articles to be disinfected can be introduced into the disinfectant solution in the presence of an enzyme, such that the substrate disinfects the article, and is subsequently decomposed by the enzyme, without the need for user intervention. An example of this kind of system is a container for the disinfection of a contact lens using hydrogen peroxide.

In a second aspect, the invention is concerned with methods for the immobilisation of enzymes on polymeric materials such as nylon. The immobilisation of enzymes is useful in a number of fields, but is particularly useful, as indicated above, in the production of systems for the automatic disinfection of contact lenses, using hydrogen peroxide. An example of a system for contact lens disinfection using hydrogen peroxide and an immobilised catalase catalyst is described in EP-A-0229807. In this reference, a catalase is deposited on an internal surface of a container used for the contact lens disinfection. The aim is to produce a rate of peroxide decomposition by the catalase which is such that lens disinfection has been effectively completed, -by the time that all the peroxide is

decomposed by the catalase.

Although it is possible to make contact lens disinfection systems as described in EP-A-0229807 which are effective, in practice it is difficult to deposit an amount of enzyme which will decompose all the peroxide within a reasonable time span (say, 4 to 6 hours) whilst not acting unduly quickly initially and thereby reducing the peroxide level below that needed for effective disinfection, before the desired disinfection has been achieved.

The reaction of the peroxide with the immobilized enzyme generally follows pseudo-first order reaction kinetics, such that the initial rate of peroxide decomposition is very high, falling to a much lower level as the reaction progresses.

Various attempts have been made therefore to control the rate of the enzyme catalyzed peroxide decomposition so as to retard the reaction. Methods for retarding enzyme catalyzed decomposition include film or matrix techniques.

Film techniques involve providing the enzyme in solid form with an external barrier, for example a coating. This coating is sparingly soluble in the solution of the enzyme substrate, and so the coating dissolves after a period, thereby bringing the substrate into contact with the enzyme, and initiating the enzyme-catalyzed decomposition of the substrate. The time for which the coating prevents contact between the enzyme and the substrate depends upon the thickness and nature of the coating. This parameter can therefore be adjusted to control the length of time that the substrate is present at its initial concentration.

Matrix techniques typically involve the entrapment of the enzyme within the interstitial regions of a matrix.

For example, catalase can be entrapped by using polyacrylamide or polyHema. This process alters the kinetic profile of an enzyme-catalyzed reaction by virtue of altering the environment of the enzyme.

Film techniques usually result in standard reaction kinetics. In order to achieve a reasonable delay of the enzyme-catalyzed decomposition, it may be necessary to provide a relatively thick coating on the enzyme. This inevitably leads to long substrate neutralization times, for example 24 hours, since the enzyme takes longer to come into contact with the substrate (Figure 1, for example, shows that even with relatively thin coatings, a small decrease in initial rate of decomposition leads to a large increase in the final rate).

Long substrate neutralization times present problems for certain applications of the technique, for example, in the disinfection of a contact lens. In this case, hydrogen peroxide is used as the enzyme substrate that disinfects the lens, and catalase is present as the enzyme which catalyses the decomposition of the hydrogen peroxide, once the disinfection of the lens is complete. Since the human eye can tolerate only very small amounts of H2O2 (typically <50ppm), it is important that the H2O2 is almost completely decomposed before the lens is inserted into the eye. The H2O neutralization times given by film techniques are therefore unsatisfactory for practical use in contact lens disinfection. Similar problems occur when using matrix techniques.

It would be advantageous to provide a method of controlling the enzyme-catalyzed decomposition of an enzyme substrate so as to retard the initial rate of substrate decomposition whilst not delaying the overall rate of neutralization to an unacceptable degree.

Accordingly, in a first aspect of the invention, there is provided apparatus for carrying out the enzyme catalyzed decomposition of a solution of an enzyme substrate, which apparatus comprises a container for containing the said solution, and an enzyme suitable for catalysing the decomposition of the said substrate, the enzyme being immobilized on a surface with which, in use, the said solution comes into contact, wherein the container is separated into a primary chamber and a secondary chamber in communication with the primary chamber, wherein the surface to which the enzyme is immobilized is provided within the said secondary chamber, and wherein the communication between the primary and secondary chambers is such as to permit limited circulation of the solution so as to decrease the initial rate of decomposition of the enzyme substrate in the primary chamber as compared with the initial rate of decomposition which would occur if the said enzyme were immobilized on a surface in the primary chamber.

The primary chamber may be a reaction chamber for the reaction of the enzyme substrate with a reactant. The enzyme substrate may be hydrogen peroxide, and the primary chamber may be a chamber for the disinfection of an article such as a contact lens. The preferred enzyme is catalase.

In a preferred embodiment of the invention, the primary chamber is separated from the secondary chamber by means of a divider. This divider can be made of nylon or any other activatable polymer surface. In a particular embodiment, the divider can be removably mounted in the container. Communication between the two chambers can be provided by a hole in the divider.

In a preferred embodiment, the communication between the chambers may be generally at the edge of the divider,

for example by providing a series of holes around the edge of the divider, or by means of a shaft passing through the divider and connecting the two chambers. The communication between the chambers may also be at the edge of the divider.

In a particularly preferred embodiment of the invention, the enzyme is immobilized on that surface of the divider which faces the secondary chamber.

The ratio of the volume of the primary chamber to that of the secondary chamber is preferably from 3:1 to 20:1, more preferably about 10:1.

The invention also provides a method for carrying out the enzyme-catalyzed decomposition of a solution of an enzyme substrate, which method comprises introducing the solution into the primary chamber of a container as described above.

A second aspect o'f the invention is concerned, as indicated above, with methods for the immobilisation of enzymes to polymeric materials such as nylon. In EP-A- 0229807, enzymes are adsorbed on the internal surfaces of a contact lens container. From an ease of manufacturing standpoint however, it is difficult to coat the interior of a container of this kind with an enzyme in a manner which is reproducible, and which is satisfactory from the point of view of quality control. It is far more straight forward from a manufacturing standpoint to coat a sheet of material with an enzyme, and then to incorporate the sheet of material into a container, typically either as a part of the exterior wall of the container, or as a divider as discussed above. A sheet of material is much more susceptible to coating methods providing a uniform coating, air drying and the like; than is the interior of a

container. Large sheets of material can be coated with enzyme, and subsequently used to produce discs or inserts, for incorporation in the contact lens case. We have discovered however, that sheets of polymeric material, in particular nylon sheets, do not perform in a satisfactory manner, in that only relatively low amounts of enzyme can be bound to the surface. In practice, the amounts of enzyme which it is possible to bind to nylon sheets are found to be insufficient to make a workable contact lens disinfection system.

We have now discovered that the amount of catalyst which can be caused to adhere to a polymeric material, in particular a polyamide material, can be substantially increased if the polymer has been orientated by the application of shear during the production of the sheet.

Accordingly, in a second aspect of the invention, there is provided a method of producing a sheet of a polymeric material having a layer of an enzyme bound thereto, which method comprises providing a polymer sheet which has been formed by a method which includes the step of applying shear to a shear orientatable polymer, to orientate the polymer in the sheet, and binding the enzyme to the orientated sheet.

In one embodiment of the second aspect of the invention, a preformed shear orientated sheet is taken and coated with an enzyme layer. In an alternative embodiment, the method includes within its scope the preliminary step of forming the polymer sheet by applying a shear to a shear-orientatable polymer, as well as the subsequent step of coating the sheet with the enzyme. The enzyme may be a catalase, as disclosed in EP-A-0229807.

The shear may be applied to the polymeric material by

melting the polymer, applying a shear force to the molten polymer, and cooling the melt to crystallise the polymer thereby fixing the orientation of the polymer in the sheet.

The shear force may be applied by causing a rapid change of direction of the melt, for example by injection moulding the polymer melt into a mould which is shaped such that the melt flows in a moulding path which causes it to experience a change in direction during moulding, sufficient to introduce the desired shear.

In accordance with the further aspect of the invention, there is provided a polymer sheet having an enzyme bound thereto, produced by a method as described above.

A number of preferred embodiments of the invention will now be illustrated, by reference to the accompanying drawings, in which :-

Figure 1 shows the variation of rate of the enzyme- catalyzed decomposition of H2O2 for different enzyme coat numbers;

Figure 2 is a schematic drawing of a container in accordance with the invention for disinfecting a contact lens;

Figure 2a is a schematic drawing of a further embodiment of a container in accordance with the invention;

Figure 3 is a graph showing the variation in rate of the enzyme-catalyzed decomposition of H2O2 solution in the reaction chamber (2) of the apparatus: of Figure 2, when the height of

the secondary chamber (3) is altered;

Figure 4 is a graph showing the variation in neutralization time of H2O2 solution in the reaction chamber (2) of the apparatus of Figure 2, when the surface area of the hole (5) and peripheral holes (6) is varied;

Figure 5 is a graph showing the change in concentration of H2O2 solution in the reaction chamber (2) of the apparatus of Figure 2, over an expanded time range.

Figure 6 shows a schematic cross-section through a mould used for injection moulding an orientated polymer sheet; and

Figure 7 is a schematic perspective drawing of a polymer sheet produced in the mould of Figure 6.

Figure 1 is a graph showing the normal first-order reaction curve resulting from the decomposition of H ₂ θ2 catalyzed by catalase, in which the initial rate of decomposition of H2O2 is delayed using a film technique. The film technique involves coating a surface (in this case, a nylon surface) with a layer of catalase, and subsequently applying a layer of a sparingly soluble coating, in order to minimise the initial rate of H2O2 decomposition, and therefore maximise the contact lens disinfection time. The coating comprises polymethylmethacrylate copolymer and is of such a thickness as is produced when the surface is immersed in a solution of the coating, removed and allowed to dry. Thus, a coating of double thickness is produced as a result of a second immersion, after- the first has dried. The catalase

does not begin to catalyse the decomposition of the H2O2 until the catalase comes into contact with the , that is until a part of the. coating has dissolved. Consequently, the initial rate of decomposition of the H2O2 depends upon the thickness of the coating: the thicker the coating, the slower the initial rate. However, the nature of first order reaction kinetics is that the rate is much faster at the beginning of the reaction than it is at the end. This means that a coating that leads to a slow initial rate will also lead to a very slow final rate, and therefore to a long H2O2 neutralization time. Figure 1 illustrates this point. It shows that with one coat, catalase effects neutralization in under 50 minutes, whereas when two coats are used, the initial rate of H ₂ θ2 decomposition is only slightly slower and neutralization takes 120 minutes. Thus film techniques are not particularly suitable for use in the disinfection of contact lenses, since they result in impractically long H2O2 neutralization times, and only provide a marginal decrease in the initial rate of H2O2 decomposition.

Figure 2 illustrates apparatus in accordance with the invention for disinfection of a contact lens using hydrogen peroxide, in which the peroxide is decomposed by catalase immobilized on a surface in the apparatus.

Referring to Figure 2, the apparatus comprises a contact lens container (1) with a reaction chamber (2) and a secondary chamber (3) . A nylon divider (4) separates reaction chamber (2) and secondary chamber (3), divider (4) being 3mm above the base of container (1) . Catalase (7) is immobilized on that surface of divider (4) which faces secondary chamber (3) <thickness of catalase (7) is exaggerated in Figure 2).

In the embodiment shown, divider (4) is removably

mounted in container (1), by means of retention ring (11). This enables replacement dividers to be provided (not shown), with replacement catalase immobilized thereon, for use when catalase (7) is contaminated and no longer catalyses the decomposition of the H2O2. In a preferred embodiment however, divider (4) is non-replaceable, the entire container being disposable after use.

Communication is provided between reaction chamber (2) and secondary chamber (3) by means of a central hole (5), and peripheral holes (6) in divider (4). Central hole (5) has a diameter of 1.7mm. Lid (8) of container (1) has contact lens support (9) attached thereon, so that lens support (9) is suspended in reaction chamber (2) . Fill line (10) is marked on container (1), and the dimensions of the apparatus are such that, when the container is filled with H2O2 solution to the level of fill line (10) and lens support (9) is immersed therein, the solution rises to the same height as the top edge of lens support (9) .

Lens support (9) may be provided separately from lid (8). In a further embodiment, support (9) may be adapted to support an article other than a contact lens in reaction chamber (2), in order to disinfect the article.

Divider (4) may be permanently joined to container (1), in which case container (1) has no retention ring (11).

For the apparatus of Figure 2, the ratio of the volume of reaction chamber (2) to secondary chamber (3) is 10:1. Generally, this ratio may be between 3:1 and 20:1.

Figure 2a illustrates an alternative embodiment of apparatus in accordance with the invention. Container (1) is substantially the same as that illustrated in Figure 2,

except that it has a vent shaft (6) instead of the peripheral holes (6) of Figure 2. Vent shaft (6) passes through divider (4) and provides communication between reaction chamber ( 2 ) and secondary chamber (3). In use, shaft (6) allows escape from secondary chamber (3) of liberated gases, thereby reducing the likelihood of the system becoming air-locked.

The apparatus of Figures 2 and 2a are used to disinfect a contact lens in the following manner. Lid (8) (together with attached lens support (9) ) is removed from container (1), and a contact lens is then supported on lens support (9) . H2O2 solution with a concentration of 3 percent (w/v) is introduced into container (1) to the level of fill line (11), where the solution is distributed between reaction chamber (2) and secondary chamber (3). Hole (5) and peripheral holes (6) enable limited circulation of the H2O2 between these two chambers. Lid (8) is replaced on container (1), thereby lowering lens support (9) and the contact lens into reaction chamber (2).

Typically, the concentration of H2O2 solution is from 0.3% (w/v) to 3.0% (w/v).

Catalyzed decomposition of the H2O2 takes place in secondary chamber (3) due to contact between the H2O2 and catalase (7). Limited circulation of the H2O2 solution results in a decreased initial rate of decomposition in the reaction chamber (2) as compared with the initial rate of decomposition which would occur if the catalase (7) had been immobilized on a surface in the reaction chamber (2). After a period of time, a sudden increase in H2O2 decomposition rate occurs, resulting in the complete decomposition of the H2O2 solution (see Figure 5 below). The H2O2 concentration in reaction chamber (2) is therefore initially maintained at- a value close to 3 percent (w/v),

enabling the H2O2 to disinfect the contact lens before the H2O2 is neutralized.

Thus the contact lens is left immersed in reaction chamber (2) until the concentration of the H2O2 is reduced by decomposition to a level that the human eye can tolerate (typically <50 ppm). Lid (8) is then removed from container (1) together with lens support (9); the contact lens is removed and can be worn.

It has been found that both the volume of the secondary chamber (3) icharacterized by the height of the divider (4) above the base of container (1) for a particular container), and the combined surface area of hole (5) and peripheral holes (6), determine the reaction profile of the H2O2 decomposition. Figure 3 is a plot of concentration of H2O2 against time for apparatus in which the hole (5) has a diameter of 1.7mm and the height of the divider (4) above the base of container (1) is a) 0.5 mm and b) 2.0mm. It can be seen from Figure 3 that when the height is 0.5mm the concentration of the H2O2 solution is maintained at a level close to the initial concentration (i.e. 3 percent w/v), whereas when secondary chamber (3) is larger, the initial rate of H2O2 decomposition is greater, leading to a shorter H2O2 neutralization time. It can be seen, therefore, that the delaying effect of catalase action upon the H2O2 increases as the ratio of the size of reaction chamber (2) to that of secondary chamber (3) increases.

Figure 4 is a plot of the combined surface area of the hole (5) and peripheral holes (6) in divider (4) against the time that it takes for the H2O2 solution to be fully neutralized. It can be seen that the greater the extent of the communication between secondary chamber (3) and reaction chamber (2), the faster is the H2O2 neutralization

time. This demonstrates that the longest delay in catalase action (and consequently, the longest H2O2 neutralization time) is given by apparatus in which the circulation of the H2O2 solution between reaction chamber (2) and secondary chamber (3) is limited because the extent of the communication between the two chambers is limited.

Figure 5 is a plot of the concentration of H2O2 against time for three reactions in which the diameter of the hole (5) is 1.7mm and the height of divider (4) above the base of the container (1) is 2.0mm. The expanded time axis (as compared to that of Figure 3) shows that the catalytic effect of the catalase is delayed for about 60 minutes. This means that the concentration of H2O2 is maintained at about its initial value (i.e. 3 percent w/v) for about 60 minutes, at which point the decomposition of the H2O2 takes place so that its concentration is reduced to below 2ppm between 75 minutes and 100 minutes after the start of the experiment. Thus, in application to the disinfection of a contact lens, the H2O2 would typically have about 60 minutes in which to disinfect the lens, and then the contact lens may be ready to wear after only about another 20 minutes.

Figure 7 illustrates a nylon sheet produced by injection moulding, suitable for producing a coated sheet in accordance with the second aspect of the invention.

The sheet of Figure 7 is injection moulded in a mould of the kind as illustrated in Figure 6.

Steel mould (21) has a first cavity (22) with an inlet port (25) , and second cavity (23) . First cavity (22) and second cavity (23) connect at a right angle at edge (24). Mould (21) can be opened to gain access to the injection moulded sheet having sides of 112mm, formed in second

cavity ( 23 ) .

In use, molten nylon-6 is injected into first cavity (22) at inlet port (25). The melt flows through first cavity (22) and around edge (24), where it undergoes a shear force due to the forced change of direction.

The temperature of mould (21) is carefully controlled, so that the melt is just below its crystallisation temperature as it approaches edge (24) . The temperature is such that the melt then rapidly crystallises to form a nylon sheet, such as nylon sheet (30) shown in Figure 7. Sheet (30) has sides of approximately 112mm, and a lip (32) at right-angles to square section (31).

After nylon sheet (30) was removed from mould (21), it was coated with a catalase by covalent immobilisation. The immobilisation was achieved by binding a molecule to nylon sheet (30), reacting gluteraldehyde with the bound molecule to provide an enzyme binding site, coating the activated surface of sheet (30) with an aqueous solution of the catalase in order to bind the catalase to the surface, and stabilising the bound catalase by coating sheet (30) with a suitable stabilising composition. Discs approximately 3cm in diameter were then pressed from the coated sheet.

The amount of catalase bound to sheet (30) was measured by immersing a disc pressed from sheet (30) in 3% (w/v) hydrogen peroxide solution. The time taken for the catalase to decompose the hydrogen peroxide was measured, and a rate constant was calculated. This rate constant is a measure of the amount of catalase bound to the disc. The same process was carried out for a disc pressed from a commercially available nylon sheet coated with catalase by the same method, and the rate constants for each disc were compared. The following rate constants were found:

Conventional nylon sheet 0.025 min ^-1

Injection shear moulded nylon sheet 0.53 min ^-1

It can be seen that the application of a shear force to a nylon melt immediately before crystallisation of the melt produces a nylon sheet to which 20-fold higher levels of catalase can be bound compared to a conventional nylon sheet.

Without wishing to be bound by any theory of operation, it is thought that the application of shear force to the melt as it is forced around edge (24) affects the orientation of the polymer molecules within the melt. The shear force is thought to result in a melt in which the polymer molecules are linearly orientated. It is thought that rapid crystallisation of the melt after the application of a shear force "fixes" the orientation of the polymer molecules. The higher levels of catalase which can be bound to a nylon sheet produced in this way are thought to be due to the linear orientation of the nylon molecules within the sheet.

It will be appreciated that a shear force can be applied to shear orientatable molecules within a polymer sheet by methods other than that described above. For instance, cold drawing of the polymer sheet after crystallisation of a polymer melt may be used to produce an orientated sheet to which high levels of a catalase can be bound.

It will be appreciated that apparatus in accordance with the first aspect of invention can be used in the disinfection of other objects, for example dentures, and in other applications in which it is desired to retard the initial rate of an enzyme-catalyzed reaction.