Preparation and use of cellulose nanofiber membrane

Abstract

A filtration membrane comprising cellulose fibres, the membrane having a pore size distribution such that the modal pore diameter is between 10 nm and 25 nm and/or wherein less than 5% of the pore volume comprises pores of greater than 40 nm and having a total porosity greater than 30%.

Claims

1. A filtration membrane comprising a paper formed from cellulose fibres, the cellulose fibres comprising elementary fibrils of diameter greater than 10 nm, the membrane having a pore size distribution such that the modal pore diameter is between 10 nm and 25 nm and/or wherein less than 5% of the pore volume comprises pores of greater than 40 nm; wherein the pore size distribution is determined according to BJH desorption analysis.

2. A filtration membrane according to claim 1 wherein the cellulose fibres are derived from green filamentous algae.

3. A filtration membrane comprising cellulose fibres derived from green filamentous algae, the membrane having a pore size distribution such that the modal pore diameter is between 10 nm and 25 nm and/or wherein less than 5% of the pore volume comprises pores of greater than 40 nm.

4. A filtration membrane according to claim 3, wherein the cellulose fibres have elementary fibrils of diameter greater than 10 nm.

5. A filtration membrane according to claim 1, wherein at least half of the cellulose fibres have a degree of crystallinity greater than 90%.

6. A filtration membrane comprising cellulose fibres, at least half of the cellulose fibres having a degree of crystallinity greater than 90%, the membrane having a pore size distribution such that the modal pore diameter is between 10 nm and 25 nm and/or wherein less than 5% of the pore volume comprises pores of greater than 40 nm.

7. A filtration membrane according to claim 6 wherein the cellulose fibres are derived from green filamentous algae.

8. A filtration membrane according to claim 6, wherein the cellulose fibres have elementary fibrils of diameter greater than 10 nm.

9. A filtration membrane according to claim 1, wherein at least half of the cellulose fibres are derived from algae Cladophorales and/or algae Siphonocladales.

10. A filtration membrane according to claim 1, wherein at least half of the cellulose comprises elementary fibrils having a diameter greater than 15 nm.

11. A filtration membrane according to claim 1, wherein the modal pore diameter is between 10 nm and 25 nm.

12. A filtration membrane according to claim 1 wherein the membrane has a porosity of greater than 10%.

13. A filtration membrane according to claim 1, wherein the cellulose fibres are modified by a cross-linking additive.

14. A method of removing particles from a feed fluid, the method comprising passing the feed fluid through a filtration membrane, said filtration member comprising a paper formed from cellulose fibres, the cellulose fibres comprising elementary fibrils of diameter greater than 10 nm, the membrane having a pore size distribution such that the modal pore diameter is between 10 mn and 25nm and/or wherein less than 5% of the pore volume comprises pores of greater than 40 nm; wherein the pore size distribution is determined according to BJH desorption analysis; and retaining at least a portion of the particles on the membrane and produce a filtrate containing a lower concentration of the particles than the feed fluid.

15. A method according to claims 14, wherein the particles have a diameter greater than 10nm.

16. A method according to claims 14, wherein the particles comprise microorganisms such as viruses.

17. A method according to claims 14, wherein the method provides a particle removal probability log reduction value (LRV) of greater than 1.

18. A method according to claim 14 wherein the particles comprise proteins such as protein aggregates and/or protein prion particles.

19. A method according to claim 16 wherein the fluid is passed through the membrane under a pressure differential of approximately 10 to 600 kPa.

20. A method of manufacturing a filtration membrane according to claim 1, the method comprising: (i) dispersing cellulose in a fluid, (ii) removing fluid from the cellulose to lay a green membrane, (iii) at least partially drying the cake, (iv) subjecting the cake to heat under pressure to form the membrane.

21. A method according to claim 20 wherein step (iv) comprises subjecting the cake to a temperature above 90° C.

22. A method according to claim 20, wherein step (i) is performed by defibrillating the cellulose.

23. A method according to claim 20 comprising a further step of applying a cross-linking agent to the membrane.

24. A filtration membrane according to claim 1, wherein at least half of the cellulose fibres are derived from algae Cladophora species.

25. A filtration membrane according to claim 1, wherein at least half of the cellulose comprises elementary fibrils having a diameter between 20 nm and 30 nm.

26. A filtration membrane according to claim 1, wherein the modal pore diameter is between 10 nm and 20 nm.

27. A filtration membrane according to claim 1 wherein the membrane has a porosity of greater than 20%.

Description

DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the present invention will now be described with reference to the following drawings:

(2) FIG. 1 shows a plot of BJH N.sub.2 desorption profile of membrane according to the invention.

(3) FIG. 2 shows fluorospectrophotometric profiles of the starting fluorophore tagged polystyrene (PS) latex bead dispersions and the filtrate produced according to the method of the invention: (a) 500 nm beads; (b) 100 nm beads; and (c) 30 nm beads. The results are the average of three measurements. The inserts represent photographs of the Cladophora membranes following the filtration.

(4) FIG. 3 shows SEM images of PS latex beads following filtration on Cladophora cellulose paper filter according to the invention: (a) 500 nm beads; (b) 100 nm beads; (c) 30 nm beads.

(5) FIG. 4 shows SEM images of Au nanoparticles (NPs) following filtration on Cladophora cellulose paper filter according to the invention: (a) 50 nm beads; (b) 30 nm beads; (c) 20 nm.

(6) FIG. 5 shows photographs of Au NPs following filtration on Cladophora cellulose paper filter according to the invention: (a) 50 nm beads; (b) 30 nm beads; (c) 20 nm.

DETAILED DESCRIPTION

(7) Embodiments of the present invention provide a cellulose based filtration membrane which presents a narrow pore size distribution such that it acts a barrier to the passage of viruses while allowing smaller species, such as proteins to pass through. It is preferred that the filtration membrane is formed from a material comprising cellulose extracted from algae such as a green filamentous algae including Cladophorales and Siphonocladales orders.

(8) The characteristic features of cellulose nanofibers derived from these green filamentous algae is their high degree of crystallinity, viz.≥90% (as measured with XRD). The crystallinity index measured with XRD is calculated by using the following formula:

(9) $\mathrm{CrI}=\frac{I_{22}-I_{18}}{I_{22}}⨯100$

(10) Where I.sub.22 is the intensity of the peak at 22° and I.sub.18 is the intensity of the background at 18°. Another characteristic property of highly crystalline cellulose from filamentous green algae are two well-resolved peaks at 14 and 16° as opposed to wood-type cellulose which shows a broad halo in this region. The high degree of crystallinity of cellulose from green filamentous algae is related to thick and stiff elementary fibrils, which are rectangular in shape and have a thickness of around 20-30 nm. For comparison, elementary fibrils from wood-type cellulose are only 4-5 nm in thickness. The peculiarities of solid-state structure of nanocellulose extracted from filamentous green algae are believed to be advantageous to produce porous type structures in the dry state—both as powder and sheets.

(11) The filtration membranes of the present invention may have a modal pore diameter of less than 40 nm and preferably in the region of 10 to 25 nm. Furthermore, the filtration membrane preferably has pores whose diameter is smaller than 40 nm. In some embodiments, the maximum pore diameter is, for example, 35 nm or 30 nm. The pore size distribution of the filtration membranes is derived from Barett-Joiner-Halenda (BJH) N.sub.2 gas desorption analysis.

(12) The preferred cellulose has a high degree of crystallinity, for example in the region of 85% or greater crystallinity, e.g. around 90% or 95% or greater as measured by X-ray diffraction. The preferred cellulose may have elementary fibrils with a mean diameter greater than 10 nm, more preferably greater than 15 nm and most preferably greater than 20 nm, for example 20 to 30 nm.

(13) The filtration membranes of the present invention allow, for example, protein solutions and other biological samples to pass therethrough with little or no effect on the component parts of that solution, while providing a barrier to the passage of viruses. Furthermore, the filtration membranes of the present invention provide sufficient wet strength to withstand pressure gradients required for fine filtration techniques.

(14) The filtration membranes of the present invention may be prepared by a method whereby cellulose is obtained (for example from green filamentous algae including Cladophorales and Siphonocladales, as described above) and dispersed in water, using high-shear homogenisation or optionally with the aid of sonication. The dispersed sample may be collected on a porous support and the collected cellulose wet cake is allowed to dry at least partially. The cellulose product may then be removed from the support and dried by use of a hot press to form the filtration membrane.

(15) It is particularly surprising that the use of a hot press provides a membrane having the characteristics of the present invention, i.e. of narrow pore size distribution. Hot presses are commonly used in papermaking with highly defibrillated cellulose from land-based plants to produce non-porous cellulose films with excellent gas barrier properties. It would be clear to the person skilled in the art that a non-porous film would not provide an effective filtration membrane. Alternatively, highly defibrillated cellulose from land-based plants may be rendered porous in the dry state by employing sophisticated, tedious and difficult to scale-up drying techniques, such as solvent-exchange/critical point drying or freeze-drying. The latter processes are energy intensive, thus costly, and may optionally involve flammable and/or hazardous organic solvents, which greatly limits their industrial utility.

(16) The filtration membranes, as described above, may be used to remove viruses in the production of therapeutic proteins, for example, coagulation factors (such as for example Factor VIII, Factor IX, Factor XI, prothrombin complex or von Willebrand factor), immunoglobins, protease inhibitors, transferrin, interferons or haemoglobins.

(17) Moreover, the filtration membrane of the invention may be used, for example, to remove viruses in the production of therapeutic proteins from transgenic plants (tobacco, tomato, potato, arabidopsis, rice, turnip, canola), such as for example protein C, hirudin, granulocyte-macrophage colony-stimulating factor, somathropin, erythropoietin, enkephalins, epidermal growth factors, interferons, serum albumin, hemoglobins, homotrimeric collagen, lactoferin, angiotensin-converting enzyme, α-tricosanthin, glucocerbrosidase.

(18) Moreover, the filtration membranes of the invention may be used to remove viruses, for example, in the production of monoclonal antibodies in cell lines such as Chinese hamster ovary cell lines. In particular, the filtration membranes of the invention may be used for example to remove viruses in the production of recombinant proteins, such as for example anti-EGRF mAb, α-glucosidase, laronidase, Ig-CTLA4 fusion protein, N-acetylgalactosamine-4-sulfatase, luteinizing hormone, anti-VEGF mAb, Factor VIII (engineered), anti-IgE mAb, anti-CD11a mAb, α-galactosidase, interferons, erythropoietin (engineered), anti-CD52 mAb, tissue plasminogen activator (engineered), anti-HER2 mAb, TNFα fusion, factor IX, follicle stimulating hormone, antiCD20 mAb, glucocerbrosidase, dexyribonuclease I, tissue plasminogen activator.

(19) Additionally or alternatively, the filtration membranes of the present invention may be used, for example to remove viruses, in the production of proteins derived from milk of transgenic mammal, for example human antithrombin III. The filtration membranes may also be used to remove virus related particles from process streams from manufacturing processes, which employ viruses as biological expression systems, for example vaccine manufacturing processes.

(20) It is also envisaged that the filtration membranes may be used to remove from a fluid PrPs, which may induce pathologies such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, Kuru disease, ovine scrapie, Alpers syndrome, fatal familial insomnia or Gerstmann-Strassler-Scheinker syndrome. For example, such PrPs could be removed in the uses described above.

(21) It is preferred that membranes for use in the removal of PrPs have a maximum pore diameter of 35 nm or less. It is more preferred that membranes for use in the removal of PrPs have a maximum pore diameter of 30 nm or less, for example 25 nm or less, 20 nm or less or 15 nm or less.

(22) In a further embodiment, the filtration membranes of the present invention may be used to isolate viral particles such that they may be used, for example, in biopharmaceutical applications and/or research such as in relation to therapeutic gene delivery.

(23) The present invention will be further described by reference to the following non limiting examples.

Example 1

(24) About 300 mg of Cladophora cellulose was dispersed in deionized water using high-shear ultra sonic treatment (750 W; 20 kHz; 13 mm probe; Vibracell, Sonics, USA) for 10 minutes at 71% amplitude. The dispersed sample was then drained on a nylon filter having an average pore size distribution of 100 nm (R01SP09025; 90 mm; GE Water and Process Technologies).

(25) The collected cellulose mass was allowed to dry until slightly damp—just enough to allow the hydrogen bonds to form a coherent layer. Subsequently, the nylon support was then easily delaminated using tweezers without affecting the integrity of the cellulose layer.

(26) The sample was then dried completely under load using a heat-press (Rheinstern, Germany) at 105° C. to produce a flat paper sheet. The produced paper sheet was 70 μm thick, 26 mm in diameter and had total porosity of 37%. The porosity was measured by BJH N.sub.2 desorption analysis. The results are shown in the graph in FIG. 1.

(27) A suspension (5 μl) of uniform polystyrene latex beads (2.5% solids) was diluted in 10 ml of water. The particle size of latex beads was 500, 100, and 30 nm, respectively. The diluted dispersion was filtered through the Cladophora cellulose membrane in a Büchner funnel at a suction pressure of 10-15 kPa.

(28) The filtrate was collected and the fluorescence intensity was measured using a fluorospectrophotometer (Tecan Infinite M200, Austria) at the specified excitation and emission wavelengths. The intensity of the filtrate was similar to that of pure water (as shown in FIG. 2). SEM analysis verified the presence of screened latex particles on the surface of nanocellulose-based filter paper, as shown in FIG. 3.

Example 2

(29) Filtration membranes were prepared according to the method in Example 1. The membranes were then sterilised in an autoclave at 121° C. for 20 min. Swine influenza viruses A (SIV A, 80-120 nm) were propagated as described in Virus Genes 2011, 42, 236 Matreveli et al to produce a stock solution.

(30) A feed solution was produced by diluting the stock solution 10.sup.−1 with phosphate buffered saline (PBS). Twenty (20) ml of the produced SIV feed solution was filtered through Cladophora cellulose membrane (26 mm in diameter) in a Büchner funnel. The suction pressure was adjusted to 10-15 kPa, and the filtrate was subsequently collected. Another 10 ml of the diluted SIV feed solution was frozen in −70° C. to be used as the hold-control to measure the factual virus litre in the feed.

(31) The SIV titre was analysed by the end-point titration through cytopathogenic effect (cpe). Standard 96-well plates containing MDCK cells were used in tenfold dilutions by assaying eight replicates of 50 μl per dilution. Negative controls were EMEM-trypsin and PBS-trypsin. The virus titres after 8 days were calculated according to Karber and expressed as log.sub.10 tissue culture infectious dose TCID.sub.50 ml.sup.−1. The results of the titres of the stock, feed and filtrate solutions are shown in Table 1, below. No infectious SIV particles were found in each of the triplicate 96-well plates in the filtrate. The corresponding virus removal probabilities are then LRV≥5.2 or LRV≥6.3 depending on the log.sub.10 titre value of the feed solution. For large viruses, i.e. viruses, with particle size≥50 nm, the state of the art industrial filters exhibit LRV≥6-7.

(32) TABLE-US-00001 TABLE 1 TCID.sub.50 titres in log.sub.10 ml.sup.−1 with calculated LRV for the SIV retention test. Titre log.sub.10 ml.sup.−1 LRV ml.sup.−3 Stock 7.2 ± 0.4 (n = 3) na.sup.b Feed (diluted) 6.0 ± 0.3 (n = 3) na.sup.b Filtrate ≤0.8.sup.a ≥5.2 ± 0.3 Stock 7.2 (n = 1) na.sup.b Feed (undiluted) 7.1 (n = 1) na.sup.b Filtrate ≤0.8.sup.a ≥6.3 .sup.anon-detectable; .sup.bna = non-applicable

Example 3

(33) Filtration membranes according to Example 1 were prepared. Prior to the virus retention test, the membranes were sterilised in an autoclave at 121° C. for 20 min. A solution spiked with Murine leukemia virus (MuLV), particle size 80-110 nm, was used as a model. A volume of 7.5 ml having log 10 total virus load of 5.43±0.27 was filtered. No cytopathogenic effect was detected in the filtrate fraction. LRV≥5.25 was calculated according to the Kärber's method.

Example 4

(34) Filtration membranes according to Example 1 were prepared. The pore size mode of the functional nanocellulose layer was centered around 11 nm as obtained by BJH N.sub.2 gas desorption analysis. The functional nanocellulose filtration layer was then laminated on an ordinary filter paper acting as support. A suspension (500 μl) of standard Au nanoparticles (50, 30, 20 nm stabilised with 0.1 mM PBS) was diluted in 10 ml of water and then filtered through the 2-layer nanocellulose filter paper in a Büchner funnel with the suction pressure gradient of 37 kPa.

(35) The filtrate solution was collected, and its UV intensity was measured using a spectrophotometer (Tecan infinite M200, Austria) at in the region between 450 and 650 nm. The intensity of the filtrate solution was insignificantly different from that of pure water. The efficiency of filtration was verified by visual inspection of the 2-layer filter paper: dark red-blue precipitate was clearly seen. The scanning electron microscopy analysis also confirmed the interception of Au nanoparticles by the paper filter (see FIG. 4).

Example 5

(36) Filtration membranes according to Example 1 were prepared. The sample was then cross-linked with polycarboxylic acid to enhance the wet strength properties. The prepared paper filter was soaked in 16% wt citric acid solution in presence of 1% sodium hypophosphate overnight before being cured at 160° C. under load using a heat-press (Rheinstern, Germany) for 10 min. The cured filter paper was then dialysed in water to remove unreacted chemicals and then dried again in a heat-press at 100° C. to produce the final flat paper sheet.

(37) The wet (tensile) strength was improved 10-fold (from about 2 to 20 MPa) and the strain to failure value was improved by 60% (from 1.6 to 2.2%). Pore size mode of the cross-linked nanocellulose membrane was centered around 15 nm as obtained by BJH N.sub.2 gas desorption analysis.

(38) The filtering properties were tested using a feed solution spiked with 20 nm Au nanoparticles. A suspension (500 μl) of standard Au nanoparticles (20 nm stabilised with 0.1 mM PBS) was diluted in 10 ml of water and then filtered through the nanocellulose membrane in a Büchner funnel with the corresponding suction pressure gradient of 37 kPa. The filtrate solution was collected, and its UV intensity was measured using a spectrophotometer (Tecan Infinite M200, Austria) at in the region between 450 and 650 nm. The intensity of the filtrate solution was insignificantly different from that of pure water. The retention of 20 nm Au nanoparticles was clearly detectable by visual observation of the paper filter (see FIG. 5) following the filtration by characteristic dark red-blue precipitate.

Example 6

(39) A filtration membrane according to Example 4 was prepared. A suspension (1000 μl) of standard Au nanoparticles (nominal size 5 nm stabilised with 0.1 mM PBS; Sigma Aldrich, product number 752568-25ML) was diluted in 50 ml of water and then filtered through the pressure gradient of 300 kPa. The filtrate solution was collected, and its UV intensity was measured using a spectrophotometer (Tecan Infinite M200, Austria) at in the region between 450 and 650 nm. The intensity of the filtrate solution was insignificantly different from that of pure water. The efficiency of filtration was verified by visual inspection of the 2-layer filter paper: dark red-blue precipitate was clearly seen. As such, it appears that the membrane is suitable for removing very small particles. As such, the membrane could be used to remove, say, PrPs from a fluid.

(40) Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.