Patent Description:
Porous membranes have many biological applications, for example: bioprocessing; biopharmaceutical processes; and cell and gene therapeutics. Constant advancements in the understanding of biological mechanisms and cellular activity mean that there is a need for more controlled membrane filtering so that retainates and filtrates that result from filtering can be better defined. Typically these retainates or filtrates are target molecules or materials, such as cells, proteins, peptides, DNA, RNA, antibodies, viruses, bacteria or phages or other microorganisms or macromolecules. Commercially available polymer membranes consist of a generally random array of pores formed by: casting a liquid web of polymer dissolved in a solvent spread across a support substrate; drying the web until the solvent evaporates; and leaving a residual solid polymer structure which has a multiplicity of pores. One manufacturing method is described in more detail in publication <CIT>. Other manufacturing methods are also known. See, for example, <CIT>, <CIT>, and <CIT>.

The pore size, pore size consistency, pore shape, pore size deviation from one side of the membrane to the other, and number of pores per unit area are all very much dependent on the composition of the initial casting liquid, the thickness of the web and the web drying conditions. To some extent, to generate a membrane with a new thickness or pore size, or in a new material requires considerable trial and error, even for experienced producers of membranes. Membranes of a thickness ranging from <NUM> to <NUM> are commonly used, having pores of an average size of <NUM> to <NUM>.

Another technique to produce membrane with very small pore sizes for example <NUM> and <NUM> is by means of exposing a polymer such as polycarbonate to an electron beam pulse, analogous to a shotgun blast. The technique is known as 'track etching' and one example is described in <CIT>. The result of the technique is a largely random pattern of fine pores which have inconsistent properties, even when consistent production parameters are used.

Three-dimensional (<NUM>-D) microfabrication methods are known which are based on so called two-photon polymerization of a volume of photosensitive material using ultrashort laser pulses, for making solid objects. When focused into a volume of a photosensitive material, the pulses initiate polymerization via two-photon absorption and subsequent polymerization. After laser illumination of the photosensitive material in a pattern resembling the desired <NUM>-D form in a dimensionally controlled environment and subsequent washing away of the nonilluminated external regions, the polymerized material remains in the prescribed <NUM>-D form. This allows fabrication of any computer-generated <NUM>-D structure by direct laser "recording" into the volume of a photosensitive material. Under such conditions, artefacts having elements of a dimension of about <NUM> or more are possible.

<NUM>-D fabrication methods for membranes are discussed but largely dismissed in "<NPL>". However, the <NUM>-D fabrication of fine pores in membranes is largely dismissed because the time taken to produce such pores by conventional techniques described above is said to be impracticable. For example, the above paper quotes a manufacturing time of several months for a <NUM> by <NUM> sample membrane with a pore size of <NUM>, presumably with a practically useful pore density, although a pore density is not mentioned. In addition, a practical technique for producing such membranes is not disclosed.

Conventional membranes rely on various filtration mechanisms comprising both surface filtration- fundamental a sieving action, and depth filtration, where filtration relies at least partial on physical retention of an obstructing pore structure, surface interactions of specific or non-specific type or surface modification with affinity type ligands. Known depth filtration structures may comprise multilayer membrane sandwich structures where pore size, pore structure, porosity, tortuosity and other physical pore characteristics in the layers may vary to provide asymmetric features along the path of liquid and particles passing through the membrane.

Filtering mechanisms conventional include retention of waste (clarification or flow through chromatography), retention and subsequent release of a target e.g. proteins, cells etc (also called purification) in depth filtration, or retention of a product in surface filtration (commonly called ultra- and diafiltration incl. cell washing) upstream of the filter by size exclusion.

The conventional filtration mechanisms mentioned in the above two paragraphs are part of conventional processes, and so any improved membranes need to be compatible with such mechanisms, and therefore need to have broadly equivalent physical properties.

The inventor has addressed the problems mentioned above in a novel way by taking a completely novel approach to manufacturing polymer membranes. The inventor has realised that, in one approach, low energy laser photo-polymerisation of a monomer or oligomer composition can be used for producing a polymer structure, or part thereof, which has solid photopolymerised areas analogous to the polymer structure of a conventional polymer membrane, and can leave unpolymerized regions analogous to the pores left by solvent evaporation in a conventional polymer membrane, which can be removed from the remaining polymerised regions by solvent washing. In a refinement of that approach, the photopolymerised areas may each envelope unpolymerised area, such that once the unpolymerized region (pores) have been washed out, the enveloped unpolymerised regions may be wide-area photopolymerised to reduce processing time. In another approach, low energy laser photo polymerisation of the composition can be used for producing a male polymer structure which has solid polymerised areas analogous to the pores of a conventional polymer membrane, and can leave unpolymerized regions of the composition analogous to the polymer structure in a conventional polymer membrane, followed by a washing of the unpolymerized composition to leave the male regions only, and followed by casting of a further solid material at least around the male polymer structure and then removal of that male polymer structure to leave just the further solid material, now with voids left by the removed male polymer structure analogous to said pores of conventional polymer membrane. In this paragraph the term 'analogous to' means 'having the same effect as', because in practice the pores and surrounding material of a conventional polymer membrane are the result of solvent evaporation, or electron beam exposure, and so they are generally random in form, whereas photo-polymerisation can be undertaken in a dimensionally controlled way, so that there is no need, unless it is desired, to have randomness. In turn, this means that virtually any pore size(s), pore shape, pore geometry (e.g. spiral/circuitous pores), pore density, and consistency of pore sizes, can be achieved, within manufacturing tolerances. Further in turn, this means that the inadequacies of conventional polymer membranes do not have to be tolerated, because the manufacturing process described herein will allow complete control over the key geometric form of the pores produced, without the randomness of conventional membranes. For example, tortuous paths formed by evaporation of solvents during manufacture of nitrocellulose membranes can be eliminated or emulated or at will.

Hence, various aspects and embodiments of the present invention, as defined by the appended claims, are provided.

In this way a polymer membrane having a predictable pore size and shape can be manufactured, and the unpredictable nature of the conventional evaporation process is largely eliminated, the only limitation being the manufacturing tolerances of the process mentioned immediately above.

In various embodiments, the focal polymerisation position(s) of the laser light can be moved relative to the composition by optical means and/or the position of already polymerised polymer composition can be moved by mechanical means relative to the position of the laser light. Since optical movements are relatively quick and precise, but relatively restricted in dimension, one small first region of membrane, or one layer of membrane can be produced by optical repositioning of the light, before moving the new part formed region of the membrane itself mechanically and by a relatively larger distance on to a second region adjacent the first region such that the optical repositioning movements can begin again in the second region. Such a process can be repeated again and again at subsequent regions until the whole membrane is produced, formed from multiple regions of the membrane after being formed or part formed by optical repositioning of the polymerisation position. However, for small areas, solely optical repositioning of the laser light can be undertaken with good results. In order to realign the polymerised membrane with the laser focal point(s) once the membrane is moved to the second or subsequent regions, it is possible to finely position the membrane relative to laser focal point(s) by recognising a pore (or part pore) pattern using a microscope and image recognition software, and by then moving the membrane further, or adjusting the focal point position such that the pore pattern to be produced at the second or subsequent region is in register with the pore pattern produced previously. The same technique can be used to provide a datum for subsequent layers after producing layers, for example, so that asymmetric pores can be produced by deliberately stepping one layer away from an underlying layer, e.g. to form a zig-zag pore.

In various embodiments, only pore walls and outer surfaces of the membrane need to be photopolymerised in dimensionally controlled conditions, in essence forming a shell, with the internal areas of the final membrane structure remaining liquid. Wide area photopolymerisation of that remaining interspace liquid composition can be effected once the unpolymerized composition of the pores has been washed out, for example by using a wide area light source such as a high energy laser or lasers. Polymerisation by heat or UV light are also possible. This technique reduces production times significantly because much of the polymerisation can be done without dimensional control.

The invention can be put into effect in numerous ways, illustrative embodiments of which are described below with reference to the drawings, wherein:.

The invention, together with its objects and the advantages thereof, may be understood better by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals, or reference numerals with the same last two digits, identify like elements.

Referring to <FIG> there is shown a micrograph of a printed polymer membrane <NUM> with a pore <NUM> in plan view, manufactured according to the protocol described below. In this example the generally circular pore <NUM> is shown as a black area having a diameter of about <NUM>, with a white 'halo' due to the effect of charge concentration on sharp edges where the dynamic range of the optical system is not wide enough to show the edge and it saturates the pixels of the micrograph.

Referring to <FIG> there is shown a micrograph of a membrane <NUM> formed form deliberately delineated printed regions A, B and C each having different sized printed test pores, and all shown less magnified than the membrane <NUM> shown in <FIG>. The pore sizes are approximately, <NUM>, <NUM> and <NUM>, <NUM> respectively. The multiple pores shown are suitable for use as a membrane of the type described above. Using the protocol described below, these pores were manufactured by solidifying liquid polymer layer by layer, leaving a pore-size area unsolidified, and the unsolidified polymer was then washed out of the pore-size hole, to reveal the pore. Each region A, B and C etc, was printed in turn using optical manipulation, before moving on to the next region by mechanical movement of the partial membrane relative to the optical elements used to form each pore.

<FIG> shows a further membrane <NUM> having different regions D and E again having multiple pores <NUM> made in the same way as mentioned above, but much smaller in diameter, in this case pore of <NUM> and <NUM> respectively, have been formed. It will be apparent that the spacing between the pores <NUM>, <NUM> and <NUM> shown could be increased or decreased to some extent, and the repeating grid pattern could be irregular or a different pattern, for example a tessellating pattern of regular shaped pores. In addition the shape of the pore could be other than circular, for example hexagonal pores could be used to reduce the amount of material that has be polymerised, where the hexagonal pores tessellate like a honey comb. In this way, membranes having the desired characteristics similar to conventionally manufacture membranes can be achieved. Pore density can be adjusted to suit the membrane's use, but up to <NUM>% pore to solid material density is possible for example with regularly spaced pore rows space about <NUM>. 018D apart where D is the mean pore diameter and where the rows are staggered.

Referring to <FIG>, there is shown schematically, an example of a production technique. Shown in section is: a portion of a bath of liquid composition <NUM>, including a silicon rubber substrate floor <NUM>. The composition includes a mixture of at least: photo-activatable monomer molecules, photo activation initiator molecules and photo-activation quencher molecules as described below. In this illustration a linear series <NUM> of bits of the polymerised composition <NUM>, have been formed using a laser beam L, focused at the a constant bit height in the Z axis, having a femtosecond laser pulse triggered by a controller (<NUM> <FIG>) when the focal point is aligned in a desired X and Y axis coordinate, where Z is up and down the page of the illustration, X is left and right of the illustration, and Y is into and out of the illustration. Such a pulse forms overlapping polymerised bits <NUM> but control of the laser is used to leave unpolymerized 'pores' <NUM>, and the laser is then optically repositioned at an adjacent position in order to repeat the solidification of the next series <NUM> of bits. In the illustration, the laser focal point P has been scanned in X (from left to right) and periodically energised, to produce the series <NUM> of bits, but was not energised at the positions where pores <NUM> are intended to be located.

In <FIG> a second layer of polymerised bits has been produced, slightly overlapping the first layer shown in <FIG>, in this case by rescanning the focal point P in the same pattern as for <FIG> but raised in Z by a height of less than one bit size to overlap the first series of bits, but again allowing un-energised scanning of the laser over the pore areas <NUM>. That layer by layer polymerisation can be repeated over and over to produce a membrane of a desired thickness, and with almost any pore size and pore pattern.

<FIG> shows a final membrane <NUM>, formed by yet more layers of bits formed onto previous layers having unpolymerized pore areas <NUM>. In practice those pore areas <NUM> are emptied of unpolymerized composition, by washing the membrane <NUM> with solvent. For clarity, the bits are shown far larger than they are when formed in practice. Interstitial areas <NUM> formed between the edges <NUM> of the pore areas <NUM> are shown polymerised. However, it is within the ambit of this invention to leave at least a portion of those interstitial areas <NUM> unpolymerized, and once the pore areas <NUM> are washed out, a wide area final polymerisation can take place which polymerises the area <NUM> also.

For convenience the pore areas <NUM> have been shown as a simple vertical pore path, but it is just as simple to produce other shaped pore paths, for example a spiralling pore, a zig-zag pore, a pseudo random path, or the like, which in practice may be a better shape to retain or capture analytes of interest for example large molecules such as proteins, or cells, whilst allowing other matter to pass through the membrane. Such a circuitous path provided for depth filtering as described above.

It should be understood that <FIG> show schematically a membrane in cross section, with pores112 shown oversize in relation to the solidified areas <NUM>, for ease of understand.

The pore diameter may be less than <NUM> or larger, with typical pore path lengths (not necessarily membrane thickness) of <NUM>, <NUM>, <NUM>, <NUM> or <NUM> times the pore diameter. If the pore edge <NUM> only is to be polymerised, leaving the area <NUM>' still liquid, then for tall pores which have relatively small diameter, that then tall hollow structure will be weak once the liquid in the pore area <NUM> has been washed out. It is possible to strengthen those solidified edges <NUM> by polymerising a brace <NUM>, joining, say, adjacent edges <NUM> of adjacent pore areas <NUM>. Multiple braces between pores edges <NUM> could be employed for increased strength and rigidity, for example cross braces <NUM> shown in <FIG> and c.

<FIG> shows one example of equipment <NUM> used to manufacture the above mentioned membrane. In this example a laser <NUM> is oriented to propagate a beam of coherent light L toward two galvanic reflective mirrors <NUM> and <NUM> each under the influence of a controller <NUM>. An adjustable objective lens or lenses <NUM>, again under the influence of the controller <NUM> is used to focus the beam L to an exact polymerisation point P in the bath <NUM>, for polymerisation as described above. Controlled movements of the mirrors 204and <NUM>, provide rapid repositioning of the focal point P, and with rapid firing of the laser with a femtosecond laser pulse, then the series of polymerised bits <NUM> mentioned above are achieved in the region A shown. Layers of bits can be made by adjustments in the Z height of the lens <NUM> using a linear actuator <NUM> under the control of the controller <NUM>. The bath <NUM> is repositioned in the X axis using a further linear actuator <NUM>, and in the Y axis by a similar arrangement not shown, in each case the actuators are under the influence of the controller <NUM> so that the next regions B and C etc can be polymerised in the same manner by the rapid mirror movements mentioned, before the next region is selected. For better accuracy, the space between the objective <NUM> and a glass layer <NUM> above the bath <NUM> is filled with light transmissive oil <NUM>, so that the light path of the laser light pulse L is largely through materials with about the same refractive index.

Whilst the above equipment will provide adequately fast production, faster production is desirable, and so the use of multiple laser beams L as shown in <FIG> is preferred, which in this case come from multiple lasers <NUM>, of equipment <NUM>' but could be produced by splitting a more powerful laser beam into numerous beams using plural beam splitters. The latter approach is less costly, but requires more optical alignment, and so multiple lasers are preferred. The equipment <NUM>' shown in <FIG> operates in a similar manner to that described above with reference to <FIG>, except that plural lasers <NUM> are grouped together with their output beams formed into a convergent pattern, in this case nine lasers are arranged in a three by three, two-dimensional pattern. The beams propagate toward the bath of polymer composition <NUM> as explained above and can be moved stepwise to form the polymerised bits as detailed above.

In order to mitigate heat build-up, the laser beam L (<FIG>) or laser beams L' (<FIG>) can be moved by more than the width between adjacent pores (i.e. the pore pitch), and/or the beams L' can be more than one pore pitch apart. Thus, for example if the beams L' are space by X pitches and moved by X-<NUM> pitches stepwise then, excluding edge pores, the remaining pores can be formed with less heat build-up than by moving just one pitch at a time. Whilst <FIG> shows multiple lasers, it would be possible to replace those lasers with an array of LED lasers.

Irrespective of which method of polymerisation is used, the successive layering of polymerised material can lead to reduced transparency and diffraction issues. To mitigate those issues, the laser light system may take advantage of modelling the exact underlying 3D structure by evaluating and optimizing the dose, angle etc of the localized light and energy application to account for non-uniformity in the structure, diffraction patterns etc..

Another membrane manufacturing technique is shown in <FIG>, where a male mould having artefacts <NUM> is formed by photo polymerisation using the same techniques as described above. The unpolymerized remainder <NUM> of the polymer is washed away, to leave a male mould, where the now voids <NUM> can be filled with a membrane forming material such as an in-filling thermosetting polymer. Once the in-filling polymer is set the moulds can be removed mechanically or by washing away with a suitable solvent, or by means of heat, to leave pores equivalent to the pores <NUM>, <NUM>, <NUM><NUM> etc as described above.

An example of the composition <NUM> and manufacturing protocol follow:
The flowable composition <NUM> in one example comprises a transparent photo-activatable acrylate monomer resin, with the addition of up to <NUM>% of a photo activation initiator, such as an acylphosphine oxide such as <NUM>,<NUM>,<NUM>-trimethylbenzoyldiphenylphosphine oxide molecules, or a benzophenone, a xanthone, or a quinone, or a combination of these molecules, and a photo-activation quencher such as tertiary amine molecules. The Laser can be an exciplex laser (also known as a excimer laser) having at output wavelength of about <NUM> with a pulse length of <NUM> to <NUM> femtoseconds (fs), although about <NUM> to <NUM> fs, for example <NUM> fs is preferred, and a repetition rate of about <NUM> is possible.

Where it is the pores that are of principal dimensional interest, the interspace between the pores could be filed-in with lower resolution, for example by using higher energy laser light where possible to photopolymerize a larger area more quickly, and thereby speeding up the manufacturing process, or by the introduction of material by jetting, i.e. liquid thermoplastic introduction, which need not be the same material that surrounds the pore.

The energy required to induce local polymerisation is provide by a focused laser pulse at at least one focal point in the composition <NUM> by means of two-photon absorption polymerisations, i.e. two or more photons are simultaneously absorbed by the above photo activation initiator (photoinitiators) to create the active species that start polymerisation of the monomer resin. Under these conditions, multiphoton absorption occurs only in the region where light intensity is at a maximum. That confines polymerization within the volume of the focused laser beam (known as a voxel). Slightly overlapping, for example <NUM>% overlapping bits of polymerised material are thus produced. The quenching molecules provide fluorescence quenching to inhibit or halt the dendritic spread of polymer branches, which in turn provides a more consolidated and defined polymerisation volume. The membrane is revealed by washing away the unsolidified part of the resin using an organic solvent.

The results of the above-mentioned techniques and materials used, provide a suitable equivalent to conventional laminar nitrocellulose membranes and to Trak Etch membranes, including their thickness and pore density. However, it is envisaged that variants could be produced to enhance the properties of the membranes made according to the invention. For example, as shown in <FIG>, a membrane <NUM>' is shown having fibres <NUM>, for example nano-fibres introduced into the composition bath <NUM> during manufacture, which do not unduly interfere with the formation of the pores described above. The finished membrane <NUM>' has improved mechanical strength. Additionally, or alternatively, a mesh or web of material (not shown) having a weave which is relatively course compared with the size and positioning of the membrane pores could be immersed in the bath prior to polymerisation, to achieve the same effect. Once the pores have been formed, the mesh etc becomes incorporated into the membrane, again adding significant strength, and thereby allowing greater trans-membrane pressure differential.

The substrate support <NUM> mentioned, is intended in the examples above to be a removable surface on which to form the membrane. In addition, the substrate's <NUM> surface may be employed to mount one or more micro-sensors <NUM> (<FIG>) thereon, and if needed, electrically conductive paths <NUM> for communication and/or power to/from such sensors, for example formed on the surface of the support <NUM> prior to the polymer bath being present, by 3D printing. Peeling off the support <NUM> from the finished membrane or vice versa, will leave the sensors and any conductive tracks in place ready to be used on or in the finished membrane. In this case the micro-sensor <NUM> is a capacitive gap sensor which measures transmembrane pressure differential, which can give an indication of the filtering performance of the membrane. Other sensors could be used, for example, other pressure sensors, flow sensors, conductivity sensors, pH sensors, osmolality sensors, chemical composition or concentration sensors etc, which can provide data in real time as filtration takes place, for example membrane performance. In another example (not illustrated) the membrane can be formed on a substrate, which substrate includes a light absorbance sensor and the membrane includes an inlet and outlet to the sensor, such that the photo-adsorbent properties of the fluid passing through the membrane can be monitored remotely. Thereby, the concentration of proteins or the like can be monitored. In yet another variant, the membrane can be formed over microfluidic valves and over a pressure sensor, which can produce a signal for opening the valves, for example if the side to side membrane pressure differential exceeds a predetermined amount. Other sensors could be used, for example an electrical conductivity sensor where an interruption in an electrical path, for example if a membrane were to rupture would signal membrane failure, or temperature sensing, could be used. The use of more conventional, lower resolution material additive manufacturing to produce the addition sensors or other ancillary parts of a membrane, e.g. a frame or other physical support, can be combined with the membrane manufacturing methods described herein with good results.

The embodiments shown, provide a flat membrane, but membranes can be useful in other shapes, for example tubular membranes which act as hollow fibres, in hollow fibre filtration. Thus, the term membrane used herein is intended to cover sheet like materials and other thin materials which are not necessarily flat.

Although numerous embodiments have been described and illustrated, it will be apparent to the skilled addressee that additions, omissions and modifications are possible to those embodiments without departing from the scope of the invention claimed.

The techniques described above can be used to achieve said asymmetric features in a single membrane layer, may even have multiple asymmetries in physical properties (for example wider, then narrower, then wider pores) and/or contoured surface characteristics to promote surface or depth filtering, such as a funnel pore opening or narrowed pore opening.

Chemical ligands or anchors for subsequent ligand attachment may be printed, allowing controlled placement and subsequent modifications of non-isotropic, asymmetric character for improved function and/or more efficiency use of (expensive) ligands.

The techniques described above require a relatively small optical head scanning distance for producing polymerised areas of a small dimension, then moving the optical head on to another region to be polymerised, or moving the material to be polymerised relative to the head. In that case it is desirable to match the pore pattern at each region, but it is not essential. It is also possible to have discontinuities in pores, folds or pleats at boundaries between regions. Then folded parts of the membrane can be adhered to relatively rigid parts to form filter cassettes or cartridges.

Above in relation to <FIG> is described the technique of printing over known sensors or other hardware, and thereby incorporating such hardware into the then printed membrane. In other embodiments it is possible to form the sensor, or part of the sensor from the polymerised material. For example for the pressure senor mention above, it is possible to form a flexible compressible cavity this wall formed from polymerised material, where a sensing surface is precisely formed as a flexible, pressure sensitive membrane and the rest of the cavity would act as a fluid conduit to a pressuring sensitive sensor device, thereby allowing measurement of fluid pressure at some internal part of the membrane.

Further, in an embodiment it is possible to light conduits or light guides. Such light guides may provide for a secondary polymerization step, for example inside a structure with poor transparency. Where the lights have terminal light diffusers or lenses, then light guided into the part-polymerised membrane into the structure can be used to fully polymerise the membrane.

Discrete sheet membrane production has been described and illustrated, but it will be apparent that other techniques could be employed, for example a continuous manufacturing technique could be used, for example the finished membrane could be peeled off its support <NUM>, washed to produce the pores and then rolled onto a roll.

Claim 1:
A method for the production of a porous polymer membrane (<NUM>, <NUM>') suitable for liquid filtration or analyte capture, comprising the following steps:
providing a flowable liquid composition (<NUM>) on a substrate (<NUM>) the composition including at least: photo-activatable monomer molecules, photo activation initiator molecules and photo-activation quencher molecules;
i) providing one or more pulses (L) of laser light at least one focal point (P) in the liquid composition (<NUM>) of sufficient energy to locally polymerise the liquid composition (<NUM>);
ii) moving the or each focal point (P) relative to the previously polymerised liquid composition (<NUM>) in a continuous or stepwise predetermined manner to a multiplicity of further positions; and
iii) repeating the pulse(s) at those further positions such that a three dimensional matrix of the liquid composition (<NUM>) is polymerised leaving unpolymerized areas of a size equivalent to conventional polymer membrane pores; and
iv) repeating steps i) to iii) to solidify the liquid composition (<NUM>) layer by layer.