Diblock copolymer vesicles and separation membranes comprising aquaporin water channels and methods of making and using them

Abstract

A vesicle in a liquid composition including an amphiphilic diblock copolymer of the PMOXA.sub.a-b-PDMS.sub.c-d type as vesicle membrane forming material, further including as an additive from about 0.05% to about 1% v/v of reactive end group functionalised PDMS.sub.e-f, and a transmembrane protein. The vesicle optionally includes about 1 to about 12% v/v of triblock copolymer of the PMOXA.sub.a-b-PDMS.sub.c-d-PMOXA.sub.a-b type as membrane forming material.

Claims

1. A vesicle in a liquid composition, the vesicle comprising an amphiphilic diblock copolymer of the poly(2-methyloxazoline)-block-poly(dimethyl siloxane) (PMOXA-PDMS) type as vesicle membrane forming material, an additive from about 0.05% to about 1% (v/v) based on the liquid composition, of reactive end group functionalized poly(dimethyl siloxane) (PDMS), and a transmembrane protein, wherein the reactive end group is one, two, or more of amine, carboxylic, and/or hydroxy group(s).

2. The vesicle according to claim 1, wherein said PMOXA-PDMS is selected from the group consisting of PMOXA.sub.10-40-PDMS.sub.25-70 and mixtures thereof.

3. The vesicle according to claim 2, wherein the mixture comprises at least a first amphiphilic diblock copolymer of the general formula PMOXA.sub.10-28-PDMS.sub.25-70 and a second amphiphilic diblock copolymer of the general formula PMOXA.sub.28-40-PDMS.sub.25-70.

4. The vesicle according to claim 3, wherein the weight proportion between the first and the second amphiphilic diblock copolymer is in the range of 0.1:1 to 1:0.1.

5. The vesicle according to claim 1, wherein said reactive end group functionalized PDMS is PDMS.sub.30-50 functionalized with one or more of amine, carboxylic acid, and/or hydroxy group(s).

6. The vesicle according to claim 5, wherein the reactive end group functionalized PDMS is poly(dimethylsiloxane), bis(3-aminopropyl).

7. The vesicle according to claim 1, wherein the transmembrane protein is an aquaporin water channel.

8. The vesicle according to claim 1 further comprising from about 1% (v/v) to about 12% (v/v) based on the liquid composition, of a triblock copolymer of the PMOXA-PDMS-PMOXA type.

9. The vesicle according to claim 8, wherein said triblock copolymer of the PMOXA-PDMS-PMOXA type is selected from PMOXA.sub.10-20-PDMS.sub.25-70-PMOXA.sub.10-20.

10. The vesicle according to claim 1, wherein the liquid composition further comprises a flux improving agent.

11. The vesicle according to claim 10, wherein the flux improving agent is an alkylene glycol monoalkyl ether alkylate, beta cyclodextrin, or polyethylene glycol (15)-hydroxystearate.

12. The vesicle according to claim 10, wherein the flux improving agent is present in an amount of 0.1% to 10% by weight of the liquid composition.

13. A separation membrane comprising a vesicle according to claim 1.

14. The separation membrane according to claim 13, wherein the separation membrane comprises an active layer incorporating the vesicle and a porous support membrane.

15. The separation membrane according to anyone of claim 14, wherein the active layer comprises the vesicle incorporated in a thin film composite (TFC) layer formed on a porous substrate membrane.

16. A method of preparing vesicles in a liquid composition incorporating a transmembrane protein comprising the step of stirring a mixture of a solution of an amphiphilic diblock copolymer of the poly(2-methyloxazoline)-block-poly(dimethyl siloxane) (PMOXA-PDMS) type, 0.05% to about 1% (v/v) based on the of liquid composition, of reactive end group functionalized poly(dimethyl siloxane) (PDMS), and a transmembrane protein, wherein the reactive end group is one, two, or more of amine, carboxylic, and/or hydroxy group(s).

17. The method according to claim 16, further comprising a flux improving agent selected among the group comprising alkylene glycol monoalkyl ether alkylate, beta cyclodextrin, and polyethylene glycol (15)-hydroxystearate.

18. A method of preparing a thin film composite layer immobilizing vesicles incorporating a transmembrane protein on a porous substrate membrane, comprising the steps of a. providing a mixture of vesicles in a liquid composition prepared in accordance with claim 16 and a di-amine or tri-amine compound, b. covering the surface of a porous support membrane with the mixture of step a, c. applying a hydrophobic solution comprising an acyl halide compound, and d. allowing the aqueous solution and the hydrophobic solution to perform an interfacial polymerization reaction to form the thin film composite layer.

19. The method according to claim 18, wherein the porous support membrane is a flat sheet.

20. The method according to claim 19, comprising the further step of producing a spiral wound membrane module by winding the flat sheet membrane.

Description

DETAILED DESCRIPTION

(1) More specifically, the aspects of the disclosed embodiments relate to vesicles as disclosed herein, which vesicle comprises an amphiphilic diblock copolymer of the PMOXA.sub.a-b-PDMS.sub.c-d type optionally comprising from about 0.5% to less than about 8-10% v/v of a triblock copolymer of the PMOXA.sub.a-b-PDMS.sub.c-d-PMOXA.sub.a-b type as membrane forming material and further comprising as an additive from about 0.01% to about 0.2% v/v of a hydrophobic end-functionalized PDMS.sub.e-f, and a transmembrane protein.

(2) Examples of said end-functionalised PDMS are, e.g. bis(aminoalkyl) or bis(hydroxyalkyl) terminated PDMS.sub.e-f, where e-f represents the range of from 30 to 50, such as bis(aminopropyl) terminated poly(dimethyl siloxane) having the formula shown here below where (CAS Number 106214-84-0, Aldrich product No. 481246, average Mn ˜5,600 or CAS Number 106214-84-0, product No. 481696 Aldrich, average Mn ˜27,000:

(3) ##STR00001##

(4) and bis(hydroxyalkyl) terminated poly(dimethyl siloxane) having the formula shown here below where n is approximately 30 to 50 and m and p are both integers between 2 and 5, such as 3 or 4, (CAS Number 156327-07-0, Aldrich product No. 481246, average Mn ˜5,600):

(5) ##STR00002##

(6) Examples of transmembrane proteins are aquaporin water channels. i.e. aquaporins and aquaglyceroporins, such as those listed in the definitions below.

(7) In addition, the present disclosure relates to a method of making the liquid composition as disclosed, in which a solution of an amphiphilic diblock copolymer of the PMOXA.sub.a-b-PDMS.sub.c-d type optionally comprising about 2 to 10% of triblock copolymer of the PMOXA.sub.a-b-PDMS.sub.c-d-PMOXA.sub.a-b type as an additive, and from about 0.05% to about 1% of reactive end group functionalised PDMS.sub.e-f, as a cross-linking agent, is mixed with a transmembrane protein.

(8) As an example, the active layer may be a thin film composite layer formed on the support membrane. A TFC membrane may be formed using alternative reaction components, e.g. as described by Choumou Zhou et al. in Journal of Membrane Science, Volume 471, 1 Dec. 2014, Pages 381-391 “Thin-film composite membranes formed by interfacial polymerization with natural material sericin and trimesoyl chloride for nanofiltration”. A highly selective active layer may also be formed on the substrate by the layer-by-layer method (see Wang et al., Membranes, 5(3): 369-384, 2015).

(9) The filtration membrane according to the present disclosure may be prepared by adding a liquid composition comprising said diblock copolymer vesicles, e.g. with aquaporin water channel proteins as the transmembrane protein, during the membrane fabrication process, such as adding the liquid composition to an aqueous MPD solution when forming a TFC layer.

(10) In one aspect of the process of the present disclosure, the transmembrane protein may be an anion channel protein, such as voltage-dependent anion channels, which is useful in preparation of ion exchange membranes for reverse electrodialysis, cf. Dlugolecki et al. (Journal of Membrane Science, 319 214-222, 2008).

Definitions and Terms

(11) The term “transmembrane protein” (TP) as used herein is a type of membrane protein spanning the entirety of the biological membrane to which it is permanently attached in nature. That is, in nature, transmembrane proteins span from one side of a membrane through to the other side of the membrane. Examples of transmembrane proteins are ammonia transporters, urea transporters, chloride channels, and aquaporin water channels.

(12) The term “aquaporin water channel” as used herein includes a functional natural or synthetic aquaporin or aquaglyceroporin water channel, such as aquaporin Z (AqpZ), GIPf, SoPIP2; 1, aquaporin 1 and/or aquaporin 2. Aquaporin water channels include bacterial aquaporins and eukaryotic aquaporins, such as yeast aquaporins, plant aquaporins and mammalian aquaporins, as well as related channel proteins, such as aquaglyceroporins. Examples of aquaporins and aquaglyceroporins include: prokaryotic aquaporins such as AqpZ; mammalian aquaporins, such as Aqp1 and Aqp2; plant aquaporins, such as plasma intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), nodulin intrinsic proteins (NIP) and small intrinsic proteins (SIP), e.g. SoPIP2; 1, PttPIP2; 5 and PtPIP2; 2; yeast aquaporins, such as AQY1 and AQY2; and aquaglyceroporins, such as GlpF and Yfl054. Aquaporin water channel proteins may be prepared according to the methods described herein or as set out in Karlsson et al. (FEBS Letters 537: 68-72, 2003) or as described in Jensen et al. US 2012/0080377 A1 (e.g. see Example 6).

(13) The terms “separation membrane” as used herein includes membranes useful for separating water and, optionally, certain small size solutes including anions and cations, from other solutes, particles, colloids and macromolecules. Examples separation membranes are “filtration membranes” such as nanofiltration (NF) membranes, forward osmosis (FO) membranes and reverse osmosis (RO) membranes. One type of filtration membranes is a “thin film composite” (or TFC) membrane, often classified as nanofiltration and reverse osmosis membranes. TFC membranes are typically made by depositing a polyamide layer on top of a polyethersulfone or polysulfone porous layer on top of a non-woven or woven fabric support. The polyamide rejection layer is formed through interfacial polymerization of an aqueous solution of an amine with a solution of an acid chloride in an organic solvent. TFC membranes may be produced as described in WO 2013/043118 (Nanyang Technological University & Aquaporin A/S). Other types of filtration membranes are those formed by the layer-by-layer (LbL) deposition method, such as described in Gribova et al. (Chem. Mater., 24: 854-869, 2012) and Wang et al. (Membranes, 5(3): 369-384, 2015). For example, the self assembled nanostructure may be embedded or incorporated in the polyelectrolyte multilayer (PEM) films, as outlined in FIG. 4 of Gribova et al.

(14) “Thin-film-composite” or (TFC) membranes as used herein may be prepared using an amine reactant, preferably an aromatic amine, such as a diamine or triamine, e.g., 1,3-diaminobenzene (m-Phenylenediamine, >99%, e.g. as purchased from Sigma-Aldrich) in an aqueous solution, and an acyl halide reactant, such as a di- or triacid chloride, preferably an aromatic acyl halide, e.g. benzene-1,3,5-tricarbonyl chloride (CAS No. 84270-84-8, trimesoyl chloride (TMC), 98%, e.g. as purchased from Sigma-Aldrich) dissolved in an organic solvent where said reactants combine in an interfacial condensation polymerization reaction, cf. Khorshidi et al. (2016) Scientific Reports 6, Article number: 22069, and U.S. Pat. No. 4,277,344 which describes in detail the formation of a composite membrane comprising a polyamide laminated to a porous membrane support, at the surface of the support membrane, e.g. a polyethersulfone membrane. Benzene-1,3,5-tricarbonyl chloride (trimesoyl chloride) is dissolved in a solvent, such as a C.sub.6-C.sub.12 hydrocarbon including hexane (>99.9%, Fisher Chemicals), heptane, octane, nonane, decane etc. (straight chain or branched hydrocarbons) or other low aromatic hydrocarbon solvent, e.g. Isopar™ G Fluid which is produced from petroleum-based raw materials treated with hydrogen in the presence of a catalyst to produce a low odour fluid the major components of which include isoalkanes. Isopar™ G Fluid: Chemical Name: Hydrocarbons, C10-C12, isoalkanes, <2% aromatics; CAS No: 64742-48-9, chemical name: Naphtha (petroleum), hydrotreated heavy (from ExxonMobil Chemical). Alternatives to the reactant 1,3-diaminobenzene include diamines such as hexamethylenediamine etc., and alternatives to the reactant benzene-1,3,5-tricarbonyl chloride include a diacyl chloride, adipoyl chloride, cyanuric acid etc. as known in the art.

(15) The term “diblock copolymer” as used herein means a polymer consisting of two types of monomers, A and B. The monomers are arranged such that there is a chain of each monomer, and those two chains are grafted together to form a single copolymer chain.

(16) The abbreviation M.sub.n means number average molecular weight. It means the total weight of polymer divided by the number of polymer molecules. Thus, M.sub.n is the molecular weight weighted according to number fractions. The abbreviation M.sub.w means weight average molecular weight. The molecular weight weighted according to weight fractions. Molecular mass may be measured by gel permeation chromatography (GPC) in tetrahydrofuran. Polydispersity index defined as Mn/Mw will be determined from the elution curves obtained in GPC.

(17) Size of the vesicles: Preferably, the vesicles of the disclosed embodiments have a particle size of between about 10 nm diameter up to 200 nm diameter depending on the precise components of the vesicles and the conditions used for their formation. It will be clear to those skilled in the art that a particle size refers to a range of sizes and the number quoted herein refers to the average diameter, most commonly mean diameter of that range of particles. The vesicle compositions of the disclosed embodiments comprise vesicles having mean hydrodynamic diameters of 300 nm or less, in some cases mean diameters that are less than 400 nm such as less than 50 nm.

(18) Examples of molar ratios of transmembrane protein to block copolymer is dependent on the transmembrane protein used, the types of copolymers used, and the desired size of the vesicle. As an example, for vesicles of PDMS-PMOXA diblock based vesicles and aquaporin water channels, the molar ratio of transmembrane protein to block copolymer may be between 1:200 to 1:2000, such as 1:400 to 1:1500, such as 1:600 to 1:1000.

(19) The term “self-assembled” as used herein refers to the process by which vesicles are formed through hydrophilic and hydrophobic interaction of amphiphilic substances, such as the diblock copolymers described herein having a relatively hydrophilic PMOXA moiety and a relatively hydrophobic PDMS moiety.

(20) “Hydrodynamic diameter” as used herein represents the hydrodynamic size of nanoparticles in aqueous media measured by dynamic light scattering (DLS) defined as the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured.

(21) Forward osmosis (FO) is an osmotic process that uses a selectively-permeable membrane to effect separation of water from dissolved solutes. The driving force for this separation is an osmotic pressure gradient between a solution of high concentration, herein referred to as the draw and a solution of lower concentration, referred to as the feed. The osmotic pressure gradient induces a net flow of water through the membrane into the draw, thus effectively concentrating the feed. The draw solution can consist of a single or multiple simple salts or can be a substance specifically tailored for forward osmosis applications. The feed solution can be a dilute product stream, such as a beverage, a waste stream or seawater, cf. IFOA, http://forwardosmosis.biz/education/what-is-forward-osmosis/

(22) Most of the applications of FO, thus fall into three broad categories: product concentration, waste concentration or production of clean water as a bi-product of the concentration process. The term “PAFO” when used herein describes a pressure assisted forward osmosis process. The term “PRO” when used herein describes pressure retarded osmosis which is useful in the generation of osmotic power. Membranes of the disclosed embodiments are useful in all types of forward osmosis processes and may be specifically adapted for each FO type.

(23) The term “reverse osmosis” (RO) is used herein refers to when an applied feed water pressure on a selectively permeable membrane is used to overcome osmotic pressure. Reverse osmosis typically removes many types of dissolved and suspended substances from feed water, including bacteria, and is used in both industrial processes and in the production of potable water. During the RO process, the solute is retained on the pressurized side of the membrane and the pure solvent, the permeate, passes to the other side. Selectivity specifies that the membrane does not allow larger molecules or ions through its pores (holes), while allowing smaller components of the solution (such as solvent molecules) to pass freely. Low pressure reverse osmosis (LPRO) membranes typically operates at a feed water pressure of from about <5 bar and up to a maximum operating pressure of about 25 bar 15 specific flux LMH/bar. LPRO performed at the lower feed pressure ranges, e.g. 2 to 5 bar is sometimes designated ultra-low pressure reverse osmosis. LPRO membranes known in the art have typical operating limits for feed water temperature of about 45° C., feed water pH in the range of 2 to 11, and chemical cleaning in the range of pH 1 to 12.

(24) The present disclosure is further illustrated with reference to the following non-limiting examples

Experimental Section

(25) Equipment:

(26) Start FPLC connected with Laptop, using Unicorn operating system.

(27) Vacuum stream.

(28) Sterile 0.45 μM vacuum filter cup.

(29) 15 mL PP tubes.

(30) Abbreviations:

(31) CV: column volume.

(32) AQP: Aquaporin Z from E. coli.

(33) LDAO: N,N-Dimethyldodecylamine N-oxide (#40234, Sigma).

(34) PAGE: Polyacrylamide gel electrophoresis.

(35) Materials and Chemicals:

(36) HisTrap Gel filtration material (Ni Sepharose 6 Fast Flow #17-5318-03, GE Healthcare) packed into a XK16/20 column (GE Healthcare) at known volume or prepacked 1 ml, 5 ml HisTrap column.

(37) AQP Binding buffer: 20 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, 10% glycerol, 0.2% LDAO, pH8.0.

(38) LDAO-free AQP Binding buffer: 20 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH8.0.

(39) Imidazole-free AQP Binding buffer: 20 mM sodium phosphate, 300 mM NaCl, 10% glycerol, 0.2% LDAO pH8.0.

(40) AQP Elution buffer: 20 mM sodium phosphate, 300 mM NaCl, 200 mM imidazole, 10% glycerol, 0.2% LDAO, pH8.0, ddH.sub.2O.

(41) General Purification of Aquaporin and Preparation of Aquaporin Stock Solution Recombinant Production of Aquaporin Z

(42) All types and variants of aquaporin water channel proteins, including aquaglyceroporins, may be used in the manufacture of membranes and compositions according to the disclosed embodiments, cf. methods described in WO2010/146365. Representative examples include the spinach aquaporin SoPIP2; 1 protein and the bacterial aquaporin-Z from E. coli.

(43) Functional aquaporin-Z was overproduced in E. coli strain BL21(DE3) bacterial cultures as His-tagged protein with a tobacco etch virus cleavage site. The fusion protein has 264 amino acid and a Mw of 27234 Da. Genomic DNA from E. coli DH5 was used as a source for amplifying the AqpZ gene. The AqpZ gene was amplified using gene specific primers with the addition of a tobacco etch virus cleavage site (TEV); ENLYFQSN at the N-terminus of AqpZ. The amplified AqpZ was digested with the enzyme NdeI and BamHI and then ligated to the similarly digested 6-His tagged expression pET28b vector DNA. The positive clones were verified by PCR-screening. The authenticity of the constructs was then confirmed by DNA sequencing.

(44) The E. coli strain BL21(DE3) was used for expression of the protein. Luria Broth cultures containing 50 μg/ml kanamycin were incubated for 13-16 hours at 37 C, diluted 100-fold into fresh LB broth and propagated to a density of about 1.2-1.5 (OD at 600 nm). Expression of recombinant protein was induced by addition of 1 mM IPTG for 3 hour at 35° C. before centrifugation. Harvested cells were resuspended in ice-cold binding buffer (20 mM Tris pH 8.0, 50 mM NaCl, 2 mM β-mercaptoethanol, 10% glycerol) in the presence of 0.4 mg/ml lysozyme, 50 units Bensonase and 3% n-octyl β-D-Glucopyranoside. The sample was subjected to five times lysis cycles in a microfluidizer at 12,000 Pa. Insoluble material was pelleted by 30 minutes centrifugation at 40,000×g. The supernatant was passed through a Q-Sepharose fast flow column (Amersham Pharmacia), and the flow through was 10 collected. The flow though fraction was topped up with NaCl to 300 mM before loaded onto a pre-equilibrated Ni-NTA column. The column was washed with 100 column volumes of a wash buffer (20 mM Tris pH 8.0, 300 mM NaCl, 25 mM imidazole, 2 mM β-mercaptoethanol, 10% glycerol) to remove non-specifically bound material. Ni-NTA agarose bound material was eluted with five bed volumes of elution buffer (20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole, 2 mM β-mercaptoethanol, 10% 15 glycerol, containing 30 mM n-octyl β-D-Glucopyranoside). AqpZ was further purified with anion exchange chromatography; monoQ column (GE healthcare). The sample mixture was diluted and concentrated to bring the salt and imidazole concentration to approximately 10 mM with Amicon concentrator, membrane cut off 10,000 Da before loading to MonoQ column. The buffer used during anion exchange chromatography were (A) 20 mM Tris pH 8.0, 30 mM OG, 10% glycerol and (B) 20 mM 20 Tris pH 8.0, 1 M NaCl, 30 mM OG, 10% glycerol. The eluted peak fractions containing AqpZ from the ion exchange column was pooled. The purified AqpZ extract was kept frozen at −80° C.

(45) Procedure for Purification of Aquaporin Protein

(46) A batch of frozen extract of aquaporin protein, such as aquaporin Z, AQPZ, e.g. from an E. coli fermentation, was obtained and treated as follows for use in the experiments to produce and characterise membranes comprising protein-PAI nanostructures of the present disclosure.

(47) One day before the purification experiment, the AQP extract (stored at −80° C. freezer) was thawed on ice or in a 4° C. refrigerator. Portions of the buffers and ddH.sub.2O were readied at 4° C. The AQP extract was stirred in an adequate chilled beaker on ice bath by a magnetic stick to dissolve any precipitate. 1.5 volumes of pre-chilled LDAO-free AQP binding buffer was gradually added into 1 volume of the solubilized extract (using a further 0.5 volume buffer for rinsing the extract tubes and filtration cup), mixed well and filtered through a sterile 0.45 μM vacuum filter cup. Vacuum was applied to the filter cup to avoid excess foaming and the filtrate was placed on ice to use within 2 hours.

(48) A Histrap column was equilibrated with sterile water followed by AQP Binding buffer at RT. The flow rate was set at 1 ml/min (for 1 mL prepacked column) or 2.5 ml/min (for 5 ml prepacked column and self-packed column). The 3 times diluted extract (on ice water bath) was loaded onto the Histrap column using AKTA program. The flow rate was set at 1 ml/min (for 1 mL prepacked column) or 2.5 ml/min (for 5 mL prepacked column and self-packed column). The loading volume was less than 30 ml/ml resin. The extract flow-through on ice-water bath was collected and stored at 4° C. for further use. The column was washed with 10 CV (column volume) ice cold AQP binding buffer. The flow rate was set at 2.5 ml/min (for 5 ml prepacked column and self-packed column) or set at 1 ml/min for 1 ml prepacked column. The AQP protein was eluted with ice cold AQP elution buffer (10 column volume) at flow rate 2.5 ml/min using ÄKTA program. The fraction volume was set to 10 ml and collection started in 15 mL PP tubes after 0.5-1 CV.

(49) Eluted fractions were capped and stored on ice or 4° C. The AQP purity and conformation was examined by denaturing and native PAGE analysis respectively. Protein concentration was measured by Nanodrop. The extract flow-through may be processed a second and a third time as needed to produce an AQP composition of suitable quality.

(50) When AQP quality analyses are passed, the protein concentration may be adjusted to 5 mg/ml by adding ice cold imidazole-free AQP binding buffer containing 2% LDAO. Finally the AQP was sterilized by filtration through 0.45 μM sterilized cup and stored at 4° C. in refrigerator for use within a month or else stored at −80° C. in a freezer.

EXAMPLES

(51) Preparation of Handmade TFC FO Filtration Membranes

(52) These membranes were made according to the steps outlined below: a) Dissolve MPD in MilliQ water to get a 2.5% (W/W) concentration, see below b) Dissolve TMC in Isopar to a final concentration of 0.15% W/V c) Cover a rectangular shaped membrane, e.g. 5.5 cm×11 cm Membrana 1 FPH PES membrane with about 20 mL/m.sup.2 membrane of MPD solution and leave for 30 seconds under gentle agitation d) Dry the non-active side (back side) with lab drying paper (e.g. Kim-Wipe) for 5-10 seconds e) Put the membrane on a glass plate and dry gently with N.sub.2 until the surface turns from shiny to dim f) Apply tape around the edges of the membrane (≈1 mm) g) Put the glass plate with the taped membrane into a glass or metal container, add about 155 mL/m.sup.2 membrane TMC-Isopar to one end and rock gently back and forth for 30 seconds h) Remove glass plate from reservoir and dry with N.sub.2 for 10 to 15 seconds

(53) After removal of the tape the membrane can be transferred to MilliQ with the newly formed active side up and keep wet during handling in subsequent steps if necessary.

(54) MPD Solution Calculation:

(55) Weigh off 1.05 g of MPD and dissolve in 35 mL of MilliQ. Add 7 mL of liquid AQPZ composition prepared as described herein. Keep the solution topped with inert gas (Ar or N.sub.2) as much as possible. This MPD solution is used in Example 1 to 3. Weight off 1.25 g of MPD and dissolve in 46.25 mL of MiliQ. Add 2.5 mL of liquid AQPZ composition prepared as described herein. Keep the solution topped with inert gas (Ar or N.sub.2 as much as possible). This MPD solution is used in examples 4 to 6. TFC membranes with liquid AQPZ formulation of 5.5 cm×11 cm sizes was then mounted in a Sterlitech CF042 FO cell (www.sterlitech.com) and subjected to tests of 60 minutes (5 membranes) and tests of 900 minutes (4 membranes) duration in FO mode using 5 μM Calcein in deionised (MilliQ) water as feed and 1 M NaCl aqueous solution as draw and feed and draw speeds of 268 mL/min.

(56) Preparation of BWRO Handmade Membranes

(57) The membranes were made according to the steps outlined below: a) Provide a support membrane, e.g. a PES non-woven having fingerlike structure, size 5.5 cm×11 cm b) Mix 3 wt % MPD with 3 wt % ε-caprolactam, 0.5 wt % NMP, and 93.5 wt % DI water to obtain a solution c) Add 0.1 mg/mL of liquid AQPZ formulation to obtain a suspension d) Incubate the suspension from c) for 2 hours e) Prepare TMC solution from 0.09 wt % TMC, 0.9 wt % acetone, and 99.01 wt % Isopar E f) Dip coat the support membrane in the suspension d) for 30 seconds g) Apply drying with air knife h) Add the TMC solution from e) for interfacial polymerization i) Follow with 2 min drying in fume hood

(58) Optional post treatment of TFC membrane following the steps:

(59) 4 min 65° C. 10% Citric Acid

(60) 2 min DI water

(61) 1 min 5% IPA

(62) 2 min DI water

(63) 1 min 0.1% NaOCl

(64) 2 min DI water

(65) 1 min 0.2% NaHSO3

(66) Four membranes were made and mounted in a Sterlitech CF042 RO cell, www.sterlitech.com, operated at 5 bar using 500 ppm NaCl as feed for 60 minutes.

(67) Preparation of LPRO Handmade Membranes

(68) The membranes were made according to the steps outlined below: a) Provide a support membrane, e.g. a polysulfone membrane prepared on non-woven support b) Mix MPD to obtain 3 wt % and ε-caprolactam to obtain 3 wt % with DI water (3% are the final concentrations in the coating aqueous solution) c) Add liquid AQPZ formulation to obtain 3 wt % final concentration in the coating aqueous solution d) Incubate the coating aqueous solution obtained in c) for 15 minutes e) Prepare coating organic solution (TMC solution), by 0.09 wt % TMC and 99.1 wt % of Isopar E. f) Dip coat the support membrane in the coating aqueous solution from d) for 30 seconds g) Remove the excess of the solution from the surface of the support by the air knife set up to 1 bar h) Add the organic coating solution (TMC solution) from e) for interfacial polymerization i) Apply drying with air knife at 0.5 bar j) Post treatment of the TFC membrane: a. 4 min 70° C. 20% Citric Acid b. 2 min 70° C. DI water k) Optional post treatment of TFC membrane following the steps: a. 4 min 65° C. 10% Citric Acid b. 2 min DI water c. 1 min 5% IPA d. 2 min DI water e. 1 min 0.1% NaOCl f. 2 min DI water g. 1 min 0.2% NaHSO3

(69) Membranes were made and mounted in a Sterlitech CF042 RO cell, www.sterlitech.com, operated at a pressure of 5 bar and flow of 60 L/h using 500 ppm NaCl as feed for 60 minutes.

Example 1. Preparation of Vesicles from PMOXA.SUB.11.-PDMS.SUB.34 .Diblock Copolymer and Preparation of Water Membrane Using Said Vesicles

(70) Materials:

(71) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer PDMS.sub.34PMOXA.sub.11 was purchased from ChemPilots as a 36 mg/mL aqueous solution. Phosphate buffer 10 mM (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g of KH.sub.2PO.sub.4 in 800 mL MiliQ purified H.sub.2O, adjusting the pH to 7.2 with HCL and completing the volume to 1 L. N,N-Dimethyldodecylamine N-oxide BioXtra (Lauryldimethylamine N-oxide) (99% purity), LDAO was purchased from Sigma Aldrich.

(72) Poly(dimethylsiloxane), bis(3-aminopropyl) terminated with MW 2500 Da was purchased from Sigma Aldrich and was used as received.

(73) Preparation Method:

(74) 1. Prepare a fresh solution of PDMS.sub.34PMOXA.sub.11 by dissolving a 36 mg/mL PDMS.sub.34PMOXA.sub.11 stock solution existing in the stock MQ water to a final concentration of 3 mg/mL in a glass cylinder.

(75) 2. Add it in the flask used to prepare the Ex. 1 formulation. Let the solution stay without further stirring.

(76) 3. Add 1% poly(dimethylsiloxane), bis(3-aminopropyl) terminated with molecular weight 2500 Da. Stir in the presence of a magnetic stirrer at 170 rotations per min.

(77) 4. Stop the stirring and add AQPZ purified stock solution (purified as described above) to achieve a 1/400 AQPZ/PDMS34PMOXA11 molar protein ratio.

(78) 5. Stir the mixture overnight at 170 rotations per min (not more than 20 hours) at room temperature.

(79) 6. Next morning take the Ex. 1 formulation obtained in the sequence of steps 1 to 5, transfer it to the storage flask and keep it at room temperature (tested up to two months only).

(80) The vesicle formulation of Example 1 was tested from size, water permeability and zeta potential point of view by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl. The results are a mean of 5 different measurements corresponding to 5 different batches.

(81) TABLE-US-00001 TABLE 1 Ex. 1 vesicle formulation Dh/nm (DLS) 140 nm ± 20 (85% ± 10%) 30 nm ± 10 (15% ± 5%) Zeta potential/mV 27 ± 8 Ki/s.sup.−1 1890 ± 100

(82) Temperature stability and thermal behaviour were tested by warming up 5 mL of Ex. 1 vesicle formulation for 10 min at various temperatures ranging from 30° to 100° C. and their size and water permeability was further determined by DLS and stopped-flow measurements.

(83) Thermal treatment does not affect significantly the stability of the formulation where an increase of the larger size structures from around 120 nm at room temperature to 260 nm. From water permeability point of view no changes can be observed up to 100° C., Ki values from 1700 to 1900 s-1 were recorded.

(84) The formulation was immobilised in and tested on FO handmade membranes having a TFC active layer, e.g. produced such as is described above.

(85) For FO membranes tested the following results were obtained, and which showed a very high calcein rejection and a desired combination of water flux (Jw>5 L/m.sup.2 h) and high salt rejection (Js<1.5 g/,.sup.2 h) resulting in the ratio Js/Jw being well below 0.3.

(86) TABLE-US-00002 TABLE 2 No. of Jw Js Rejection Samples (L/m.sup.2h) (g/m.sup.2h) Js/Jw calcein (%) Ex. 1 form. 5 5.9 ± 1.29 ± 0.22 ± 99.93 ± 0.62 0.10 0.01 0.05

Example 2. Preparation of Vesicles from PMOXA.SUB.11.-PDMS.SUB.34 .Diblock Copolymer and Preparation of Water Membrane Using Said Vesicles

(87) Materials:

(88) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer PDMS34PMOXA11 was purchased from ChemPilots as a 36 mg/mL aqueous solution. Phosphate buffer 10 mM (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g of KH2PO4 in 800 mL MiliQ purified H2O, adjusting the pH to 7.2 with HCL and completing the volume to 1 L. N,N-Dimethyldodecylamine N-oxide BioXtra (Lauryldimethylamine N-oxide) (99% purity), LDAO was purchased from Sigma Aldrich.

(89) Poly(dimethylsiloxane), bis(3-aminopropyl) terminates with MW 2500 Da was purchased from Sigma Aldrich and was used as received.

(90) Preparation Method

(91) 1. Prepare a fresh solution of PDMS34PMOXA11 by dissolving a 36 mg/mL PDMS34PMOXA11 stock solution existing in the stock MQ water to a final concentration of 3 mg/mL in a glass cylinder.

(92) 2. Add it in the flask used to prepare the formulation 4 Amino. Let the solution stay without further stirring.

(93) 3. Add 0.1% poly(dimethylsiloxane), bis(3-aminopropyl) terminated with molecular weight 2500 Da. Stir in the presence of a magnetic stirrer at 170 rotation per min.

(94) 4. Stop the stirring and add AQPZ purified stock solution (purified as described above) to achieve a 1/400 molar protein:polymer ratio.

(95) 5. Stir the mixture overnight at 170 rotations per min (not more than 20 hours) at room temperature.

(96) 6. Next morning take the Ex. 2 vesicle formulation obtained in the sequence of steps 1 to 5, transfer it to the storage flask and keep it at room temperature (tested up to two months only).

(97) Ex. 2 vesicle formulation was tested from size, water permeability and zeta potential point of view by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl. The results are measured 5 times for 5 different batches.

(98) TABLE-US-00003 TABLE 3 Ex. 2 formulation Dh/nm (DLS) 140 nm ± 20 (90% ± 10%) 20 nm ± 10 (0% ± 10%) Zeta potential/mV 11 ± 4 Ki/s.sup.−1 1486 ± 200

(99) Temperature stability and thermal behaviour were tested by warming up 5 mL of Ex. 2 vesicle formulation for 10 min at various temperatures ranging from 30° to 100° C. and their size and water permeability was further determined by DLS and stopped-flow measurements.

(100) Thermal treatment does not affect significantly the stability of the formulation, however resulting in a hydrodynamic diameter increase of the larger size structures from around 140 nm at room temperature to around 290 nm. From water permeability point of view no changes can be observed up to 100° C., Ki values from 1400 to 1527 s-1 were recorded.

(101) The formulation was tested on RO, BW-RO low pressure handmade membranes and FO handmade membranes. Results are given in tables 4 and 5 below showing very good reproducibility (low std) of all performance parameters as well as parameters reaching desired values within both RO and FO commercial expectations.

(102) TABLE-US-00004 TABLE 4 Ex. 2 vesicle formulation tested on the BW-RO low pressure handmade membranes Jw/bar Applied No. of (L/m.sup.2h)/ Rejection pressure, Samples bar NaCl (%) bar Ex. 2 5 7 ± 0.5 98.9 ± 0.1 5 formulation

(103) TABLE-US-00005 TABLE 5 Ex. 2 vesicle formulation tested on the FO handmade membranes No. of Jw Js Samples (L/m.sup.2h) (gmh) Js/Jw Ex. 2 3 17.44 ± 1.3 2.21 ± 0.95 0.13 ± 0.07 formulation

Example 3. Preparation of Vesicles from PMOXA.SUB.24.-PDMS.SUB.65.+PMOXA.SUB.32.-PDMS.SUB.65 .Diblock Copolymer Blend and Preparation of Water Filtration Membrane Using Said Vesicles

(104) Main Vesicle Forming Materials:

(105) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer PDMS.sub.65PMOXA.sub.24 (DB1) purchased as a viscous white liquid used as received. Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer PDMS.sub.65PMOXA.sub.32 (DB2) purchased as a viscous white liquid used as received. As additives:

(106) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly-(2-methyloxazoline) triblock copolymer PMOXA.sub.12PDMS.sub.65PMOXA.sub.12 (TB) purchased as a viscous white liquid used as received as a hydrophobicity agent, and bis(3-aminopropyl) terminated poly(dimethylsiloxane) having a molecular weight of 2500 Da purchased as a liquid from Sigma Aldrich used as received as a cross-linking agent or functionalizing agent.

(107) Phosphate buffer 10 mM (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g of KH.sub.2PO.sub.4 in 800 mL MiliQ purified H.sub.2O, adjusting the pH to 7.2 with HCL and completing the volume to 1 L. Further detergent additives were N,N-Dimethyldodecylamine N-oxide BioXtra (Lauryldimethylamine N-oxide) (LDAO) was purchased from Carbosynth, and

(108) Poloxamer P123 purchased from Sigma Aldrich as a 30% solution in water. AqpZ 5 mg/mL in 0.2% LDAO in the stock (purified as described above).

(109) Preparation Method

(110) 1. Prepare P123 solution by dissolving 15 mL P123 in 1 L PBS.

(111) 2. Prepare a 0.05% LDAO solution in PBS by dissolving 0.05 g LDAO in 100 mL PBS.

(112) 3. In the preparation vessel weight DB1 to reach a concentration of 0.5 g DB1/L of prepared formulation.

(113) 4. In the same preparation vessel weight DB1 to reach a concentration on 0.5 g DB2/L of prepared formulation. (1:1 weight ratio DB1 and DB2)

(114) 5. In the same preparation vessel weight, add TB hydrophobicity additive to reach a concentration of 0.12 g TB/L of prepared formulation.

(115) 6. Add LDAO 5% prepared in step 2 in the proportion 100 mL/L of prepared formulation

(116) 7. Add the bis(3-aminopropyl) terminated poly(dimethylsiloxane) to reach a final concentration of 0.1%.

(117) 8. Add AqpZ stock solution to reach a concentration of 5 mg/L of prepared formulation and a 1/400 protein:polymer ratio.

(118) 9. Add poloxamer P123 solution prepared in step 1 to reach the desired volume of prepared formulation subtracting the volumes of LDAO, bis(3-aminopropyl) terminated poly(dimethylsiloxane) and AQPZ added in step 6 and 8.

(119) 10. Stir the mixture from step 10 overnight at 170 rotations per minute (not more than 20 hours) at room temperature to achieve the formulation.

(120) 11. Next morning take the prepared Ex. 3 formulation obtained in the sequence of steps 1 to 9, and filter it through 200 nm pore size filters to sterilize it, put it in a closed sealed bottle and keep it at room temperature for not more than 12 months.

(121) Ex. 3 vesicle formulation was tested from size, water permeability and zeta potential point of view by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl. The results are measured 5 times for 5 different batches.

(122) TABLE-US-00006 TABLE 6 Ex. 3 formulation Dh/nm (DLS) 317 nm ± 60 (60% ± 10%) 80 nm ± 20 (28% ± 5%) 11 nm ± 4 (5% ± 7%) Zeta potential/mV 13 ± 2 Ki/s.sup.−1 1286 ± 180

(123) Temperature stability and thermal behaviour were tested by warming up 5 mL of Ex. 3 formulation for 10 min at various temperatures ranging from 30° to 100° C. and their size and water permeability was further determined by DLS and stopped-flow measurements.

(124) Thermal treatment does not affect significantly the stability of the formulation where a decrease of the size of the formed structures from around 317 nm at room temperature to 290 nm at 40° C. and further to 185 nm at 80° C. was observed. From water permeability point of view no changes can be observed up to 100° C., Ki values from 1286 to 1321 s-1 up to 100° C. were recorded.

(125) The Ex. 3 vesicle formulation was incorporated in and tested on BW-RO low pressure handmade membranes and FO handmade membranes. Results are given in tables 7 and 8 below showing very good reproducibility (low std) of all performance parameters as well as parameters reaching desired values within both RO and FO commercial expectations.

(126) TABLE-US-00007 TABLE 7 Ex. 3 formulation tested on the BW-RO low pressure handmade membranes Jw/bar Applied No. of (L/m.sup.2h)/ Rejection pressure, Samples bar NaCl (%) bar Ex. 3 3 5.7 ± 0.2 99.3 ± 0.2 5 Formulation

(127) TABLE-US-00008 TABLE 8 Ex. 3 formulation tested on the FO handmade membranes No. of Jw Js Samples (L/m.sup.2h) (gmh) Js/Jw Ex. 3 3 16.36 ± 0.16 2.27 ± 0.58 0.14 ± 0.03 Formulation

Example 4

(128) Preparation of Vesicles from PMOXA.sub.24-PDMS.sub.65+PMOXA.sub.32-PDMS.sub.65 Diblock Copolymer Blend and Preparation of Water Filtration Membrane Using Said Vesicles

(129) Main Vesicle Forming Materials:

(130) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer (PDMS.sub.65PMOXA.sub.24-DB1) purchased as a viscous white liquid used as received. Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer (PDMS.sub.65PMOXA.sub.32-DB2) purchased as a viscous white liquid used as received.

(131) Additives:

(132) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly-(2-methyloxazoline) triblock copolymer PMOXA.sub.12PDMS.sub.65PMOXA.sub.12 (TB) purchased as a viscous white liquid used as received as a hydrophobicity agent, and bis(3-aminopropyl) terminated poly(dimethylsiloxane) having a molecular weight of 2500 Da purchased as a liquid from Sigma Aldrich used as received as a cross-linking agent or functionalizing agent.

(133) Phosphate buffer 10 mM (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g of KH.sub.2PO.sub.4 in 800 mL MiliQ purified H.sub.2O, adjusting the pH to 7.2 with HCl and completing the volume to 1 L. Further detergent additives were N,N-Dimethyldodecylamine N-oxide BioXtra (Lauryldimethylamine N-oxide—LDAO) was purchased from Carbosynth, and Propylene glycol monomethyl ether acetate (PGMEA, >99.5% purity) purchased from Sigma Aldrich.

(134) AqpZ 5 mg/mL in 0.2% LDAO in the stock (purified as described above).

(135) Preparation Method

(136) 1. Prepare a 5% by weight PGMEA solution by dissolving 50 g PGMEA in 11 PBS.

(137) 2. Prepare a 0.05% by weight LDAO solution in PBS by dissolving 0.05 g LDAO in 100 mL PBS.

(138) 3. In the preparation vessel, weigh DB1 to reach a concentration of 0.5 g DB1/L of prepared formulation.

(139) 4. In the same preparation vessel weigh DB2 to reach a concentration on 0.5 g DB2/L of prepared formulation. (1:1 weight ratio DB1 and DB2).

(140) 5. In the same preparation vessel, add TB hydrophobicity additive to reach a concentration of 0.12 g TB/L of prepared formulation.

(141) 6. Add LDAO 5% prepared in step 2 in the proportion 100 mL/L of prepared formulation

(142) 7. Add the bis(3-aminopropyl) terminated poly(dimethylsiloxane) to reach a final concentration of 0.1%.

(143) 8. Add AqpZ stock solution to reach a concentration of 5 mg/L of prepared formulation and a 1/400 protein:polymer ratio.

(144) 9. Add PGMEA 5% solution prepared in step 1 to reach the desired volume of prepared formulation subtracting the volumes of LDAO, bis(3-aminopropyl) terminated poly(dimethylsiloxane) and AQPZ added in step 6 and 8.

(145) 10. Stir the mixture from step 9 overnight at 170 rotations per minute (not more than 20 hours) at room temperature to achieve the formulation.

(146) 11. Next morning take the prepared Ex. 4 formulation obtained in the sequence of steps 1 to 10, and filter it through 200 nm pore size filters to sterilize it, put it in a closed sealed bottle and keep it at room temperature for not more than 12 months.

(147) Ex. 4 vesicle formulation was tested for size, water permeability and zeta potential by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl. The results are measured 5 times for 5 different batches.

(148) TABLE-US-00009 TABLE 8 Properties of ex. 4 vesicle formulation Ex. 4 formulation Dh/nm (DLS) 224 nm ± 30 (80% ± 10%) 58 nm ± 20 (28% ± 5%) Zeta potential/mV 4.8 ± 0.5 Ki/s.sup.−1  1194 ± 200 s.sup.−1
Preparation of Flat Sheet Membranes (AA Pilot)

(149) The membranes were made according to the steps outlined below: a. Prepare a support membrane by dissolving 17% of Polysulfone (PS)/Polyethersulfone (PES) in N-Methyl-2-pyrrolidone (NMP)/Dimethylformamide (DMF) and casting on non-woven polyester fabric support followed by phase inversion process in RO water to form support membrane, having and total thickness from 130 um to 180 um. Support membrane has a finger-like/sponge-like structure. b. Prepare an aqueous solution of 3 wt % MPD and 3 wt % ε-caprolactam using a stirrer. c. Add Ex. 4 vesicle formulation in an amount in accordance with table 9 below to the above solution to obtain a suspended aqueous solution. d. Incubate the aqueous solution from c) for 1 hours with stirrer mixing. e. Prepare organic solution from 0.09 wt % TMC and 99.91 wt % Isopar E f. Dispense support membrane from a roll and allow it to travel into a dip tank containing the above aqueous solution. Alternatively, a slot die is used to dispense the above mentioned aqueous solution on the support membrane. Aqueous solution contact time on the support membrane is controlled at 30-40 seconds. g. An air knife at vertical direction toward support membrane is used, pressure controlled at 0.2-2 bar, to remove excess aqueous solution. h. After removal of excess aqueous solution on membrane support, membrane is allowed to travel to a dip tank containing TMC solution prepared in step e). Alternatively, a slot die is used to dispense the TMC solution on the support membrane to allow interfacial polymerization reaction to occur. Organic solution contact time is controlled at 20-30 seconds. i. To remove excess organic solution, an air knife at vertical direction towards support membrane is used. The pressure is controlled at 0.2 bar to 1. j. The membrane is after polymerization and removal of excess organic solution directed into a tank containing 10% citric acid at 60-70° C. for around 4 minutes soaking. k. The membrane is after citric acid soaking allowed to travel into to a tank containing 15% aqueous IPA, at room temperature 22-25° C., for around 2 minutes soaking. l. Then the membrane is subjected to DI water soaking before hypochlorite post treatment. m. 2000 ppm aqueous hypochlorite solution is used to post treat the membrane for 1 minute soaking at room temperature, 22-25° C. followed by DI water rinsing. n. 1% sodium bisulfite is used to post treat the membrane for 1 minute soaking at room temperature, 22-25° C. followed by DI water soaking.

(150) The Ex. 4 formulation incorporated in TW-RO low pressure pilot line made membranes was tested. Results are given in table 9 below showing a flux increase when the amount of the PGMEA increases.

(151) TABLE-US-00010 TABLE 9 Ex. 4 formulation tested on the TW-RO low pressure pilot line made membranes Jw/bar Applied % PGMEA (L/m.sup.2h)/ Standard Rejection Standard pressure, solution bar deviation NaCl (%) deviation bar Ex. 4 0 7.40 1.09 95.44% 0.9% 5 Formulation 6% 8.79 0.53 95.06% 0.7% 5 8% 8.97 0.59 95.92% 0.4% 5 10%  9.91 0.41 94.42% 0.6% 5

(152) Test condition: 5 bar, 500 ppm NaCl, 25° C., 1 L/minute flowrate, coupon test.

(153) TABLE-US-00011 TABLE 10 Formulation tested on LPRO hand-made membranes Jw/bar Applied % PGMEA (L/m.sup.2h)/ Rejection pressure, solution bar NaCl (%) bar Ex. 4 0 4.19 ± 0.39 93.6 ± 2.5 5 Formulation 2% 4.76 ± 0.27 96.2 ± 2.6 5 4% 4.87 ± 0.51 97.0 ± 0.1 5

Example 5

(154) Preparation of Vesicles from PMOXA.sub.24-PDMS.sub.65+PMOXA.sub.32-PDMS.sub.65 Diblock Copolymer Blend and Preparation of Water Filtration Membrane Using Said Vesicles

(155) Main Vesicle Forming Materials:

(156) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer (PDMS.sub.65PMOXA.sub.24-DB1) purchased as a viscous white liquid used as received. Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer (PDMS.sub.65PMOXA.sub.32-DB2) purchased as a viscous white liquid used as received.

(157) Additives:

(158) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly-(2-methyloxazoline) triblock copolymer PMOXA.sub.12PDMS.sub.65PMOXA.sub.12 (TB) purchased as a viscous white liquid used as received as a hydrophobicity agent, and bis(3-aminopropyl) terminated poly(dimethylsiloxane) having a molecular weight of 2500 Da purchased as a liquid from Sigma Aldrich used as received as a cross-linking agent. Phosphate buffer 10 mM (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g of KH.sub.2PO.sub.4 in 800 mL MiliQ purified H.sub.2O, adjusting the pH to 7.2 with HCl and completing the volume to 1 L. Further detergent additives were N,N-Dimethyldodecylamine N-oxide BioXtra (Lauryldimethylamine N-oxide—LDAO) was purchased from Carbosynth, and Kolliphor® HS 15 or Polyethylene glycol (15)-hydroxystearate (KHS). AqpZ 5 mg/mL in 0.2% LDAO in the stock (purified as described above).

(159) Preparation Method

(160) 1. Prepare a 0.5% by weight KHS solution by dissolving 5 g KHS in 11 PBS.

(161) 2. Prepare a 0.05% by weight LDAO solution in PBS by dissolving 0.05 g LDAO in 100 mL PBS.

(162) 3. In the preparation vessel, weigh DB1 to reach a concentration of 0.5 g DB1/L of prepared formulation.

(163) 4. In the same preparation vessel weigh DB2 to reach a concentration on 0.5 g DB2/L of prepared formulation. (1:1 weight ratio DB1 and DB2).

(164) 5. In the same preparation vessel, add TB hydrophobicity additive to reach a concentration of 0.12 g TB/L of prepared formulation.

(165) 6. Add LDAO 0.05% prepared in step 2 in the proportion 100 mL/L of prepared formulation

(166) 7. Add the bis(3-aminopropyl) terminated poly(dimethylsiloxane) to reach a final concentration of 0.1%.

(167) 8. Add AqpZ stock solution to reach a concentration of 5 mg/L of prepared formulation and a 1/400 protein:polymer ratio.

(168) 9. Add KHS 0.5% solution prepared in step 1 in accordance with table 12 below, to reach the desired volume of prepared formulation subtracting the volumes of LDAO, bis(3-aminopropyl) terminated poly(dimethylsiloxane) and AQPZ added in step 6 and 8.

(169) 10. Stir the mixture from step 9 overnight at 170 rotations per minute (not more than 20 hours) at room temperature to achieve the formulation.

(170) 11. Next morning take the prepared Ex. 5 formulation obtained in the sequence of steps 1 to 10 and filter it through 200 nm pore size filters to sterilize it, put it in a closed sealed bottle and keep it at room temperature for not more than 12 months.

(171) Ex. 5 vesicle formulation was tested for size, water permeability and zeta potential by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl. The results are measured 5 times for 5 different batches.

(172) TABLE-US-00012 TABLE 11 Ex. 5 vesicle formulation properties Ex. 5 formulation Dh/nm (DLS) 306 nm ± 20 (40% ± 4%) 86 nm ± 10 (40% ± 4%) 14 nm ± 4 (20% ± 2%) Zeta potential/mV 1.73 ± 0.25 Ki/s.sup.−1  2561 ± 300 s.sup.−1

(173) TABLE-US-00013 TABLE 12 Formulation tested on LPRO hand-made membranes Jw/bar Applied % KHS (L/m.sup.2h)/ Rejection pressure, solution bar NaCl (%) bar Ex. 5 0 4.03 ± 0.08 97.9 ± 0.8 5 Formulation 3% 4.75 ± 0.08 98.2 ± 0.1 5 5% 5.23 ± 0.09 96.5 ± 1.7 5

(174) The results reported in table 12 indicates that the flux is improved by the addition of KHS to the coating aqueous solution in any of the tested concentrations. Furthermore, the salt rejection initially increases by the addition of 3% KHS, but it decreases when further amounts of KHS are added. Thus, a concentration of 3% say KHS appears to be the optimal concentration in which the water flux improves, without sacrificing the salt rejection.

(175) Flat sheet membranes were prepared using the pilot line method indicated in example 4 using the above example 5 formulation instead. The data is shown in Table 13 below.

(176) TABLE-US-00014 TABLE 13 Formulation tested on LPRO hand-made membranes Jw/bar Applied % KHS (L/m.sup.2h)/ Rejection pressure, solution bar NaCl (%) bar Ex. 5 0 7.96 98.46 5 Formulation 3% 10.33 98.34 5 5% 11.30 95.86 5 7% 11.49 94.18 5

(177) It is noted that the flux increases about 30% while the rejection remains at about the same level for 3% KHS in the coating aqueous solution. When the concentration of KHS is increased to a level of 5% or 7% the water flux increases, however sacrificing the salt rejection. Thus, the composition using 3% KHS appears to offer the optimal properties and is selected for further modification.

(178) The interfacial polymerisation of the TFC layer using the example 5 formulation comprising 3% KHS in the aqueous phase is further changed by modifying the organic phase with diethylketone (DEK) and mesitylene (Mes).

(179) TABLE-US-00015 TABLE 14 Formulation tested on LPRO hand-made membranes Jw/bar Applied % KHS % TFC (L/m.sup.2h)/ Rejection pressure, solution Modifier bar NaCl (%) bar Ex. 5 0 0 5.2 97.5 5 Formulation 3% 0 6.11 97.2 5 3% 3% DEK 7.47 96.3 5 3% 0.9% Mes 6.21 98.1 5 3% 1.8% Mes 6.42 98.3 5

(180) The results of the experiments reported in table 14 show that a flux increase of 22% can be obtained by the addition of 3% DEK to the organic phase. Thus, a total of 43% increase in the flux can be obtained by the addition of 3% KHS to the aqueous phase and 3% DEK to the organic phase without substantially sacrificing the salt rejection.

(181) The addition of Mes to the organic phase does not substantially increase the water flux further, however the salt rejection is increased. Thus, for applications where a high salt rejection is of importance Mes can be added to the organic phase and for applications where a high water flux is of importance DEK can be added to the organic phase.

Example 6

(182) Preparation of Vesicles from PMOXA.sub.24-PDMS.sub.65+PMOXA.sub.32-PDMS.sub.65 Diblock Copolymer Blend and Preparation of Water Filtration Membrane Using Said Vesicles.

(183) Main Vesicle Forming Materials:

(184) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer (PDMS.sub.65PMOXA.sub.24-DB1) purchased as a viscous white liquid used as received. Poly(2-methyloxazoline)-block-poly(dimethylsiloxane) diblock copolymer (PDMS.sub.65PMOXA.sub.32-DB2) purchased as a viscous white liquid used as received. Additives:

(185) Poly(2-methyloxazoline)-block-poly(dimethylsiloxane)-block-poly-(2-methyloxazoline) triblock copolymer PMOXA.sub.12PDMS.sub.65PMOXA.sub.12 (TB) purchased as a viscous white liquid used as received as a hydrophobicity agent, and bis(3-aminopropyl) terminated poly(dimethylsiloxane) having a molecular weight of 2500 Da purchased as a liquid from Sigma Aldrich used as received as a cross-linking agent. Phosphate buffer 10 mM (PBS) (pH 7.2, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g NaCl, 0.2 g KCl, 1.44 g Na.sub.2HPO.sub.4 and 0.24 g of KH.sub.2PO.sub.4 in 800 mL MiliQ purified H.sub.2O, adjusting the pH to 7.2 with HCl and completing the volume to 1 L. Further detergent additives were N,N-Dimethyldodecylamine N-oxide BioXtra (Lauryldimethylamine N-oxide—LDAO) was purchased from Carbosynth, and Beta Cyclodextrin (BCD-97% purity).

(186) AqpZ 5 mg/mL in 0.2% LDAO in the stock (purified as described above).

(187) Preparation Method

(188) 1. Prepare a 0.5% by weight BCD solution by dissolving 5 g BCD in 11 PBS.

(189) 2. Prepare a 0.05% by weight LDAO solution in PBS by dissolving 0.05 g LDAO in 100 mL PBS.

(190) 3. In the preparation vessel, weigh DB1 to reach a concentration of 0.5 g DB1/L of prepared formulation.

(191) 4. In the same preparation vessel weigh DB2 to reach a concentration on 0.5 g DB2/L of prepared formulation. (1:1 weight ratio DB1 and DB2).

(192) 5. In the same preparation vessel, add TB hydrophobicity additive to reach a concentration of 0.12 g TB/L of prepared formulation.

(193) 6. Add LDAO 0.05% prepared in step 2 in the proportion 100 mL/L of prepared formulation

(194) 7. Add the bis(3-aminopropyl) terminated poly(dimethylsiloxane) to reach a final concentration of 0.1%.

(195) 8. Add AqpZ stock solution to reach a concentration of 5 mg/L of prepared formulation and a 1/400 protein:polymer ratio.

(196) 9. Add BCD 0.5% solution prepared in step 1 in the mount indicated in table 14 below to reach the desired volume of prepared formulation subtracting the volumes of LDAO, bis(3-aminopropyl) terminated poly(dimethylsiloxane) and AQPZ added in step 6 and 8.

(197) 10. Stir the mixture from step 9 overnight at 170 rotations per minute (not more than 20 hours) at room temperature to achieve the formulation.

(198) 11. Next morning take the prepared Ex. 4 formulation obtained in the sequence of steps 1 to 10, and filter it through 200 nm pore size filters to sterilize it, put it in a closed sealed bottle and keep it at room temperature for not more than 12 months. Ex. 6 vesicle formulation was tested for size, water permeability and zeta potential by DLS, Zeta potential and stopped flow measurements in 0.5 M NaCl. The results are measured 5 times for 5 different batches.

(199) TABLE-US-00016 TABLE 13 Ex. 6 vesicle formulation properties Ex. 6 formulation Dh/nm (DLS) 198 nm ± 25 (100%) Zeta potential/mV −1.8 ± 0.15 Ki/s.sup.−1  1100 ± 100 s.sup.−1

(200) Flat sheet membranes were prepared using the pilot line method indicated in example 4 using the above example 6 solution instead. The data is shown in Table 14 below.

(201) TABLE-US-00017 TABLE 14 Formulation tested on LPRO hand-made membranes Jw/bar Applied (L/m.sup.2h)/ Rejection pressure, % BCD bar NaCl (%) bar Ex. 6 0 4.8 ± 0.13 95.7 ± 1.2 5 Formulation 3% 4.9 ± 0.21 97.2 ± 0.4 5

(202) The result of the tested formulations shows that the rejection of NaCl increases significantly, while the flux remains at the same level.