Polymeric membranes and production methods thereof

Abstract

A method of producing a polymeric membrane for isolating a protein-of-interest from a protein mixture, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising a base polymer and an active polymer comprising an N-isopropylacrylamide (NIPAM)-itaconic acid co-polymer (NIA), and wherein the first immersion fluid comprises a solvent and / or a mixture of solvents and / or a mixture of a non-solvent and a solvent, and wherein the second immersion fluid comprises a non-solvent and / or a mixture of non-solvents and / or a mixture of a non-solvent and a solvent, wherein the second immersion fluid has a greater %v / v of non-solvent than the first immersion fluid.

Description

Polymeric membranes and production methods thereofTechnical Field

[0001] The present disclosure generally relates to polymeric membranes. The present disclosure also relates to methods for producing polymeric membranes. The present disclosure also relates to using the polymeric membranes for isolating a protein-of- interest from a protein mixture.Background

[0002] Protein purification is one of the most costly aspects of therapeutic protein production. Existing methods of protein purification include chromatography (e.g. affinity chromatography, anion exchange chromatography, hydrophobic interaction chromatography, SE-HPLC) and non-chromatography (e.g. precipitation and liquid extraction) purification methods. Major obstacles of existing methods are the high cost and time involved in purification and the need to ensure that the product is of a suitable quality (e.g. purity and stability) for therapeutic use. For example, affinity resins used in affinity chromatography can have relatively low binding capacity and chromatography purification from an average size batch can reach volumes of several hundred litres, being a huge capital investment in the amount of resin used, the infrastructure to handle and pack the chromatography columns, along with the running costs.

[0003] In some instances, co-purification of one or more proteins from the same protein mixture is required. For example, during plasma fractionation, blood plasma fractions containing one or more plasma proteins (e.g., immunoglobulin G (IgG), albumin and coagulation factors) are obtained from the same blood plasma sample. Each of these plasma fractions can then be processed for therapeutic use.

[0004] To date, several methods of plasma fractionation have been developed. Current methods require extremely high volumes of raw material and can often result in poor yield of some plasma protein fractions due to additional processing steps required to remove protein aggregates. At present, up to 70-75% of the IgG present in plasma may be recovered from plasma using existing technologies.

[0005] One method for protein purification is using polymeric membranes. The most widespread methods for preparation of porous polymeric films and membranes are based on phase separation of polymers from their solutions. Existing membrane substrates are predominantly fabricated using a single polymer via one-step non-solvent induced phase separation (NIPS).

[0006] Flat sheet membranes are formed by coating a porous mechanical support with a thin film of polymer solution. The polymer solution, or dope, is composed of at least one polymer, at least one good solvent, and may contain additives. The thin film and support are immersed into a coagulation bath, which consists of a poor solvent, i.e., the non- solvent, and may contain additives.

[0007] In the liquid NIPS process, the final pore morphology of the formed porous film / membrane strongly depends on the thermodynamic interactions between the involved components, the exchange rate between solvent and non-solvent (modulated by the compositions of the polymer solution and of the coagulation bath), the solvent volatility, and the temperature.

[0008] Although one-step NIPS is facile and has been commonly used in membrane fabrication, the membrane formation process in this method is very fast, completing within a few seconds, which makes it challenging to simultaneously tailor pore structure and internal surface morphology.

[0009] Additives, such as organic solvents, inorganic salts, surfactants, may be included into the into water bath has been employed in one- step NIPS for controlling membrane formation, it is still challenging to fabricate membranes with precisely tailorable structure due to the difficulty of controlling the complex membrane formation process in one- step NIPS.

[0010] Thus, there is a need for new methods of producing polymeric membranes which can provide increased control of the pore structure and internal surface morphology.

[0011] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledgein the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.Summary

[0012] The present disclosure is based on the inventors’ identification of a method of producing polymeric membranes that provides greater control of the properties of the produced membrane, for example the pore size, geometry and internal surface morphology and / or functionality of the membrane. The present disclosure is further based on the inventors’ identification of the inclusion of a hydrophobic backbone monomer in combination with a functional carboxylic acid monomer to form an active co-polymer with high selectivity for a compound (e.g. protein-of-interest) to be isolated and excellent dispersion in a base polymer.

[0013] According to one aspect of the present disclosure, there is provided a method of producing a polymeric membrane, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising a base polymer and an active polymer, the active polymer comprising a co-polymer of a hydrophobic backbone monomer and a carboxylic acid monomer, and wherein the first immersion fluid comprises a solvent and / or a mixture of solvents and / or a mixture of a non- solvent and a solvent, and wherein the second immersion fluid comprises a non-solvent and / or a mixture of non-solvents and / or a mixture of a non-solvent and a solvent, wherein the second immersion fluid has a greater %N / N of non-solvent than the first immersion fluid.

[0014] According to a further aspect of the present disclosure, there is provided a method of producing a polymeric membrane, the method comprising:immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising a base polymer and an active polymer, the active polymer comprising a co-polymer of a hydrophobic backbone monomer and a carboxylic acid monomer, and wherein the polymer composition has a higher solubility in the first immersion fluid than the second immersion fluid.

[0015] In some embodiments, the polymer membrane may be for isolation of a protein - of-interest from a protein mixture, whereby the functional carboxylic monomer has an affinity for the protein-of-interest.

[0016] In some embodiments, the active polymer is an N-isopropylacrylamide (NIPAM)-itaconic acid co-polymer (NIA), for example a block co-polymer (bNIA).

[0017] According to another aspect of the present disclosure, there is provided a method of producing a polymeric membrane, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising N- isopropylacrylamide (NIP AM), and wherein the first immersion fluid comprises a solvent and / or a mixture of solvents and / or a mixture of a non- solvent and a solvent, and wherein the second immersion fluid comprises a non-solvent and / or a mixture of non-solvents and / or amixture of a non-solvent and a solvent, wherein the second immersion fluid has a greater %N / N of non-solvent than the first immersion fluid.

[0018] According to another aspect of the present disclosure, there is provided a method of producing a polymeric membrane, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising N- isopropylacrylamide (NIP AM), and wherein the polymer composition has a higher solubility in the first immersion fluid than the second immersion fluid.

[0019] According to another aspect of the present disclosure, there is provided a polymeric membrane produced by a method in accordance with the present disclosure, wherein the polymeric membrane comprises a polymer matrix formed from the base polymer, the polymer matrix having one or more surfaces and a plurality of connected pores.

[0020] According to yet another aspect of the present disclosure, there is provided a method of isolating a protein-of-interest from a protein mixture, comprising contacting a polymeric membrane in accordance with the present disclosure with the protein mixture thereby to load the membrane with the protein-of-interest.

[0021] According to still another aspect of the present disclosure, there is provided a method of isolating a protein-of-interest from a protein mixture, comprising filtering the protein mixture using a polymeric membrane in accordance with the present disclosure.

[0022] The inventors of the present application have discovered that, according to various aspects and embodiments described herein, it is possible to produce a membrane capable of isolating a protein-of-interest from a protein mixture at relatively high flowrates of said mixture. Advantageously, this provides for increased productivity due to reduced processing time without impacting process efficiency and / or leading to a commercially appreciable reduction in the percentage of a desired protein recovered from a source protein mixture.Brief Description of Drawings

[0023] Embodiments of the present disclosure will now be described by way of example only with reference to the accompanying drawings in which:

[0024] Figure 1 - SEM images of commercial (A, C, E, F) and bNIA (B, D, F, G) membranes showing the top surface (A, B), bottom surface (C, D), cross section (E, F) , bottom surface (G, H);

[0025] Figure 2 - SEM images of a 36K bNIA membrane showing the top surface (A) and the bottom surface (B);

[0026] Figure 3 - Chromatographic profiles of a bNIA membrane after loading with: IgG and albumin feedstocks (results overlaid, A); IgG:HSA 1: 1 feedstock (B), IgG:HSA 1:2 feedstock (C), and IgG:HSA 1:4 feedstock (D);

[0027] Figure 4 - HPEC Peak Characterization of the feedstock, unbound and elution fractions for the IgG / HSA mixtures: IgG:HSA 1: 1 (A), IgG:HSA 1:2 (B), and IgG:HSA 1:4 (C);

[0028] Figure 5 - Chromatographic profiles comparing performance of bNIA and NIP AM membranes using pure IgG;

[0029] Figure 6 - Chromatographic profiles comparing performance of bNIA and NIP AM membranes using plasma (CRP);

[0030] Figure 7 - Chromatographic profiles for bNIA membrane for IgG feedstocks: purified IgG fraction (A); Paste (B); and plasma (CRP) (C).

[0031] Figure 8 - Chromatographic profile for 5-layer bNIA membranes for IgG feedstocks: purified IgG fraction (A); and plasma (CRP) (B).

[0032] Figure 9 - Chromatographic profile for bNIA membrane for purified IgG fraction shown before and after exposure to NaOH;

[0033] Figure 10 - Chromatographic profile for bNIA membrane for plasma (CRP) shown before and after exposure to NaOH;

[0034] Figure 11 - Chromatographic profile for purification (Figure I la) and recovery (Figure 1 lb) from purified IgG fraction feed;

[0035] Figure 12 - Chromatographic profile for purification (Figure 12a) and recovery (Figure 12b) from paste feed;

[0036] Figure 13 - Chromatographic profile for purification (Figure 13 a) and recovery (Figure 13b) from cryorich plasma (CRP) feed;

[0037] Figure 14 - Chromatographic profile for purification (Figure 14a) and recovery (Figure 14b) from purified mAb IgG2 feed;

[0038] Figure 15 - Shows the effect of pH on DBC for bNIA membranes for plasma (CRP);

[0039] Figure 16 - SEM images of a PVDF / bNIA membrane and a PVDF / bNIA / non- woven fabric membrane;

[0040] Figure 17 - Trans-membrane pressure drop data for a PVDF / bNIA membrane and a PVDF / bNIA / non-woven fabric membrane; and

[0041] Figure 18 - Mechanical strength data of a commercial membrane, PVDF / bNIA membrane and a PVDF / bNIA / non-woven fabric membrane.Description of Embodiments

[0042] In the following description, reference is made to the accompanying drawings which form a part hereof, and which is shown, by way of illustration, several embodiments. It is understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present disclosure.DefinitionsGeneral terms

[0043] With regards to the definitions provided herein, unless stated otherwise, or implicit from context, the defined terms and phrases include the provided meanings. Unless explicitly stated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired by a person skilled in the relevant art. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0044] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0045] Throughout this disclosure, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

[0046] Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the examples, steps, features, methods, hydrogels, processes, and compositions, referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0047] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0048] As used herein, the term “about”, unless stated to the contrary, typically refers to a range of up to + / - 10% of the designated value, and includes smaller ranges therein, for example + / - 5% or + / - 1% of the designated value.

[0049] It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

[0050] Throughout the present specification, various aspects and components of the invention can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 4.5, 4.75, and 5, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

[0051] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.Specific terms

[0052] As used herein "RAFT polymerization" or "RAFT" refers to reversible addition fragmentation-chain transfer polymerization.

[0053] As used herein, the solubility of a polymer in a fluid is used in relation to the ability of the polymer to dissolve in the solvent and form a solution. In particular, the relative solubilities of the immersion fluids refer to the amount of polymer able to be dissolved in the fluids in a fixed time period. These relative solubilities determine the rate of formation of the membrane from the polymer composition, which in turn provides the improved physical properties of the resulting membrane.

[0054] As used herein, the term “isolate”, “isolating” or “isolation” shall be taken to mean the separation of, whether completely or partially, a protein (e.g., a plasma protein such as IgG) present in a protein mixture (e.g., plasma or a plasma fraction).

[0055] As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

[0056] The term “immunoglobulin G (IgG)”, also known as “gamma globulin” or “immune globulin”, shall be taken to mean antibody of isotype G. There are several subclasses of IgG, for example, IgGl, IgG2, IgG3 and IgG4.

[0057] The term “plasma” shall refer to the straw-coloured / pale yellow component of blood obtained from one or more blood donor(s). Methods of obtaining plasma from a donor will be apparent to a skilled person and / or described herein. For example, plasma is obtained by removing red blood cells from donated blood. For example, plasma is obtained by plasmapheresis.

[0058] The term “plasma fraction” or “fraction thereof’ shall refer to plasma which has been fractionated to isolate one or more desirable protein components from the plasma. For example, plasma may be fractionated to isolate cryo-precipitates (proteins that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold) and cryo- supernatant (also known as cryo-poor plasma). For example, plasma may be fractionated by ethanol precipitation to produce IgG-containing Oncley fractions, Cohn fractions, ammonium sulphate precipitates, or Precipitates A (KN A), B (KN B), and the Precipitate of Supernatant B (KN B+l) from plasma as described in US patent 3,301,842. Plasma fractions include II+III precipitate produced according to Cohn methods such as Method 6, Cohn et. al. J. Am; Chem. Soc., 68 (3), 459-475 (1946), Method 9, Oncley et al. J. Am; Chem. Soc., 71, 541-550 (1946), or the I+II+III precipitate, Method 10, Cohn et.al. J. Am; Chem. Soc., 72, 465-474 (1950); as well as the method of Deutsch et.al. J. Biol. Chem. 164, 109-118 (1946) or the Precipitate-A, B and the Precipitate of Supernatant B of Nitschmann and Kistler Vox Sang. 7, 414-424 (1962); Helv. Chim. Acta 37, 866-873 (1954). For example, the plasma may be fractionated by octanoic acid fractionation as described in European application 893450. Typically, Cohn Fractions, and Kistler / Nitschmann Precipitate’s A (KN A), B (KN B) and the Precipitate of Supernatant B (KN B+l) exist as a suspended paste. In another example, the plasma sample or fraction thereof is obtained from a non-ethanolfractionation process. Other purification techniques including chromatography may be used.

[0059] As used herein, an “IgG intermediate product” refers to any material obtained between the manufacturing steps which comprises IgG. For example, supernatants or fractions of the starting material. In particular examples, the IgG intermediate product is used in the manufacture of an intramuscular immunoglobulin (IMIG), intravenous immunoglobulin (IVIG), or subcutaneous immunoglobulin (SCIG).

[0060] The term “cryo-precipitate” or “cryo-precipitates” refers to proteins in plasma that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold. Cryo-precipitates include factor VIII, fibrinogen, von Willebrand factor, factor XIII and platelet membrane microparticles.

[0061] The term “cryo-poor plasma” shall be taken to mean plasma removed of cryoprecipitates.

[0062] The term “cryo-rich plasma” shall be taken to mean plasma comprising components typically found in cryo-precipitates.

[0063] As used herein, the term “protein mixture” shall be understood to refer to a solution containing two or more proteins or a complex mix of proteins, including the protein-of-interest.

[0064] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0065] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds

[0066] The term “industrial or commercial scale” or “large scale” or “manufacturing scale” shall refer to the amount of product that would be produced in a batch that was designed for clinical testing, formulation, sale and / or distribution to the public. Forexample, industrial scale refers to large scale purification of IgG from the plasma or fraction thereof to produce the plasma protein product.

[0067] The term “dynamic binding capacity” or “DBC” of a membrane shall be taken to refer to the maximum amount of protein that the medium will bind under operating conditions before significant breakthrough of unbound protein occurs.Method of producing a polymeric membrane

[0068] In one aspect, there is provided a method of producing a polymer membrane, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising a base polymer and an active polymer, the active polymer comprising a co-polymer of a hydrophobic backbone monomer and a functional carboxylic acid monomer, and wherein the first immersion fluid comprises a solvent and / or a mixture of solvents and / or a mixture of a non- solvent and a solvent, and wherein the second immersion fluid comprises a non- solvent and / or a mixture of non- solvents and / or a mixture of a non- solvent and a solvent, wherein the second immersion fluid has a greater %N / N of non-solvent than the first immersion fluid.

[0069] In another aspect or embodiment, there is provided a method of producing a polymeric membrane, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting solvent, the polymer composition comprising a base polymer and an active polymer, the active polymer comprising a co-polymer of a hydrophobic backbonemonomer and a carboxylic acid monomer, and wherein the polymer composition has a higher solubility in the first immersion fluid than the second immersion fluid.

[0070] In some embodiments, the polymer membrane may be for isolation of a protein- of-interest from a protein mixture, whereby the functional carboxylic monomer has an affinity for the protein-of-interest.

[0071] The two-step method described herein has the advantage that it is possible to tailor the pore size, geometry and internal surface morphology and / or functionality of the membrane. The method described herein results in a two stage membrane formation mechanism wherein in the first immersion slow nucleation and crystallisation occur such that an intermediate polymeric phase is formed and in the second immersion fast solidification of the intermediate polymeric phase occurs to form the polymeric membrane. By controlling the conditions of first immersion and second immersion (e.g., composition, temperature, time), the pore geometry, internal surface morphology and / or functionality of the resulting membrane can be tailored. The membranes formed by the process described herein have excellent porosity and / or surface area making them especially suitable for protein binding. Advantageously, the method described herein is efficient and simple. In some embodiments, the method provides membranes with good ligand distribution and internal structure which can be utilised for direct capture target protein from plasma neat.

[0072] The hydrophobic backbone component of the active polymer of the present disclosure has been found to facilitate excellent blending with the base polymer.Membrane properties

[0073] Polymer membranes produced by aspects or embodiments of the method described herein may be characterised in terms of thickness as characterised by scanning electron microscopy. In some embodiments, the polymer membrane formed from the method disclosed herein will have a thickness (pm) about, or greater than about 10, 20, 40, 50, 60, 70, 80, 90, 100, 110 , 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, , 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000. In some embodiments, the polymer membrane formed from the method disclosed herein will have thickness (pm) of less than about 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500,450, 400, 350, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 80, 70, 60, or 50. The thickness of a membrane produced by aspects or embodiments of the method described herein may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 10 and 200, or about 150.

[0074] In some embodiments, the membrane casting mixture may be coated at a desired thickness onto an inert surface prior to immersion in the immersion fluids. In some examples, the membrane casting mixture may be spread on a glass plate prior to immersion. In some examples, the membrane casting mixture may be coated onto a thin sheet of non-woven fabric. The non-woven fabric may provide enhanced mechanical strength to the formed membrane. The non-woven fabric may facilitate the formation of a more porous network structure of the formed membrane. It will be appreciated that, where a non-woven fabric is used, the formed polymer membrane comprises the nonwoven fabric embedded therein. In such cases, the thickness of the polymer membrane comprising the non-woven fabric may be increased depending on the thickness of the non-woven fabric.

[0075] Polymer membranes produced by aspects or embodiments of the method described herein may have an excellent dynamic binding capacity. In some embodiments, the polymer membrane formed from the method disclosed herein will have a dynamic binding capacity for IgG (mg / mL) about, or greater than about 50, 60, 70, 80, 90, 100, 150, 200, 250, 275, 300, 325, 350, 400, 450, or 500. In some embodiments, the polymer membrane formed from the method disclosed herein will have a dynamic binding capacity (mg / ml) less than about 500, 450, 400, 350, 325, 300, 275, 250, 200, 190, 180, 170, 160, or 150. The dynamic binding capacity of a membrane produced by aspects or embodiments of the method described herein may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 50 to about 500, or between about 100 to about 400, or between about 100 to about 200, or between about 100 to about 160.

[0076] Polymer membranes produced by aspects or embodiments of the method described herein may have an excellent specific surface area. Large specific surface area is desirable as provides a membrane with a large adsorption capacity, Large specific surface area is desirable as it allows a higher degree of chemical functionalisationthrough the exposure of functional group on the membrane surface. In some embodiments, the polymer membrane formed from the method disclosed herein will have a specific surface area (m2 / g) about, or greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, the polymer membrane formed from the method disclosed herein will have a specific surface area (m2 / g) of less than about 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 or 20. The specific surface area of the membrane formed from the method disclosed may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 5 to about 50, or between about 10 to about 40.

[0077] Polymer membranes produced by aspects or embodiments of the method described herein may be characterised in terms of porosity. In some embodiments, the polymer membrane formed from the method disclosed herein will have a porosity (% void volume / total volume) of about, or greater than about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90. In some embodiments, the polymer membrane formed from the method disclosed herein will have a porosity of less than about 90, 85, 80, 70, 65, 60, 55, 50, or 45. The porosity of the membrane formed from the method disclosed may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 30 to about 90. In some examples, the porosity is greater than about 60.

[0078] The membrane described herein has unique core-shell structure. The active polymer serves as the shell while base polymer serves as the membrane matrix. In some embodiments, active polymer particles may be well distributed on the surface of base polymer membrane matrix. In some embodiments, the particles size distribution of active polymer may be tailored to specific ranges depending on the desired application of the membrane. In some embodiments, the polymer membrane formed from the method disclosed herein may have an active polymer particle size distribution (nm) defined by a lower bound and an upper bound. In some embodiments, the lower bound of the active polymer particle size distribution (nm) may be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150. In some embodiments, the upper bound of the active polymer particle size distribution (nm) may be about 500, 400, 300, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100. The active polymer particle size distribution (nm) of the membrane formed from the methoddisclosed may be in a range provided by any two of the lower and upper bounds, for example between about 1 to about 50, between about 10 to about 400, or between about 20 to about 300.

[0079] Polymer membranes produced by aspects or embodiments of the method described herein may have an excellent functional group density. High functional group density is desirable as it allows a higher degree of chemical functionalisation through the exposure of functional group on the membrane surface. In some embodiments, the polymer membrane formed from the method disclosed herein will have a functional group density (pmol / cm2) of about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the polymer membrane formed from the method disclosed herein will have a functional group density (pmol / cm2) of less than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5. The functional group density (pmol / cm2) of the membrane formed from the method disclosed may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 1 to about 15, or between about 5 to about 15.

[0080] Mechanical strength determines the durability and lifespan of a membrane in a particular industrial applications. The membranes described herein may demonstrate excellent mechanical strength due to their uniform structure. In some embodiments, the polymer membrane formed from the method disclosed herein may have a mechanical strength (MPa) of about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the mechanical strength (MPa) may be less than about 10, 19, 8, 7, 6, 5, 4, 3, 2, or 1. The mechanical strength (MPa) of the membrane formed from the method disclosed may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 1 to about 10, or between about 4 to about 6.

[0081] Pore size distribution and diameter influence the selectivity and retention capacity of a membrane. Polymer membranes produced by aspects or embodiments of the method described herein may have a narrow pore size distribution. In some embodiments, the polymer membrane formed from the method disclosed herein may have a pore size distribution (pm) defined by a lower bound and an upper bound. In some embodiments the lower bound (pm) may be about 0.05, 0.1, 0.15, 0.20, 0.25 0.30, 0.35, 0.40, 0.45, 0.50, 0.60, 0.70, 0.80, 0.90, 1, 2, 3, 4, or 5. In some embodiments, the upperbound (pm) may be about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. The pore size distribution (pm) of the membrane formed from the method disclosed may be in a range provided by any two of the lower and upper bounds, for example between about 0.05 to about 10, or between about 0.5 to about 3.

[0082] Permeability of a membrane may be defined as the flow rate for a defined pressure and frontal area. In some embodiments disclosed herein, the polymer membrane formed from the method disclosed herein may have a permeability (L / hr) at 1 bar and a frontal area of 3.14cm2of about, or greater than about 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000. In some embodiments, the permeability (L / hr) of the membrane may be less than about 20000, 19000, 18000, 17000, 16000, 15000, 14000, 13000, 12000, 11000, 10000, 9000, 8000, 7000, or 6000. The permeability (L / hr) of the membrane formed from the method disclosed may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 5000 to about 20000, or about 10000 to about 15000. The permeability (L / hr) of the membrane formed from the method disclosed may be about 13000.Polymer solubility

[0083] The inventors of the method disclosed herein have surprisingly discovered that by tailoring the composition of a casting solvent, a first immersion fluid and a second immersion fluids such that the relative solubilities of the base polymer and the copolymer in each composition membranes with the aforementioned properties can be obtained. Both the base polymer and the co-polymer have relatively high solubilities in the casting liquid, compared to their solubilities in at least one of the first and second immersion fluids such that the base polymer and the co-polymer are dissolved in the casting solvent to form a casting liquid. The co-polymer has a relatively higher solubility in the first immersion fluid relative to the base polymer such that upon immersion in the first immersion fluid the base polymer more readily precipitates (associates with itself rather than the first immersion fluid) to form a base polymer matrix. Upon immersion in the second immersion fluid, the co-polymer, more readily associates with the base polymer than the second immersion fluid and adsorbs, forming a shell, or a partial shell, on the surface of the base polymer matrix.Polymer composition

[0084] In the present disclosure, solvent casting is used to fabricate a membrane. The membranes fabricated are formed from a membrane casting mixture which comprises a polymer composition dissolved within a casting liquid. The polymer composition comprises a base polymer and an active polymer. The base polymer acts as the support skeleton or matrix of the membrane. In addition, the base polymer can contain one or more functional groups that promote adsorption of proteins on the membrane surface and / or allow for further functionalisation of the membrane. The active polymer provides additional functionality to the membrane. For example, the active polymer can contain one or more functional groups that promote adsorption of proteins on the membrane surface and / or allow for further functionalisation of the membrane.Active polymer

[0085] The active polymer according to the present disclosure comprises a co-polymer of a hydrophobic backbone monomer and a functional carboxylic acid monomer.

[0086] At least a first portion of the active polymer will have an affinity for the base polymer in the first immersion fluid and / or second immersion fluid. In accordance with the present disclosure, the hydrophobic backbone monomer facilitates blending of the active polymer with the base polymer. In a particularly advantageous embodiment, the hydrophobic backbone monomer comprises N-isopropylacrylamide (NIP AM).

[0087] At least a second portion of the active polymer will have a be capable of acting as a chemical ligand for binding a protein-of-interest. In accordance with the present disclosure, the second portion of the active polymer is the functional carboxylic acid monomer. In some embodiments, the functional carboxylic acid monomer comprises a dicarboxylic acid monomer. In a particularly advantageous embodiment, the functional carboxylic acid monomer comprises itaconic acid.

[0088] In some embodiments, the active polymer is a block co-polymer. In some embodiments, the block co-polymer is a RAFT polymerized block co-polymer. RAFT polymerisation is effective in providing highly reproducible polymers of predetermined molecular weight and monomer ratio. In some embodiments, the active polymer comprises a N-isopropylacrylamide / itaconic acid block co-polymer (bNIA). In someembodiments, the bNIA block co-polymer comprises a hydrophobic block and a hydrophilic block.

[0089] In some embodiments, the hydrophobic block comprises N- isopropylacrylamide. In some embodiments, the hydrophobic block comprises N- isopropylacrylamide only. In some embodiments, the hydrophilic block comprises N- isopropylacrylamide monomers. In some embodiments, the hydrophilic block comprises it itaconic acid monomers.

[0090] In some embodiments, the molar ratio of N-isopropylacrylamide monomers to itaconic acid monomers in the hydrophilic block of the active polymer is less than about 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.5: 1, 1: 1, 0.5: 1, or 0.1: 1. In some embodiments, the molar ratio of N-isopropylacrylamide monomers to itaconic acid monomers in the hydrophilic block of the active polymer is about, or more than about 0.1: 1, 0.5: 1, 1: 1, 1.5: 1, 2: 1, 2.5:1, 3: 1, 3.5: 1, 4: 1, 4.5: 1, or 5: 1. The molar ratio of N-isopropylacrylamide monomers to itaconic acid monomers in the hydrophilic block of the active polymer may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 0.1: 1 to 5: 1, or between about 0.5: 1 to 1.5: 1. In some embodiments, the molar ratio of N-isopropylacrylamide monomers to itaconic acid monomers in the hydrophilic block of the active polymer is about 1: 1.

[0091] In some embodiments, the molar ratio of N-isopropylacrylamide monomers to itaconic acid monomers in the active polymer is less than 15:3, 14:3, 13:3, 12:3, 11:3, 10:3, 9:3, 8:3, 7:3, 6:3, 5:3, 4:3, 3:3, 2:3, or 1:3. In some embodiments, the molar ratio of N-isopropylacrylamide monomers to itaconic acid monomers in the active polymer is about, or more than about 1:3, 2:3, 3:3, 4:3, 5:3, 6:3, 7:3, 8:3, 9:3, 10:3, 11:3, 12:3, 13:3, 14:3, or 15:3. The molar ratio of N-isopropylacrylamide monomers to itaconic acid monomers in the active polymer may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 1:3 to 15:3, or between about 5:3 to 10:3. In some embodiments, the molar ratio of N-isopropylacrylamide monomers to itaconic acid monomers in the active polymer is about 7:3.

[0092] In some embodiments, the molecular weight of the active polymer (Daltons) is about, or greater than about IK, 5K, 10K, 20K, 30K, 40K, 50K, 60K, 70K, 80K, 90K, or100K. In some embodiments, the molecular weight of the active polymer (Daltons) is less than about 100K, 90K, 80K, 70K, 60K, 50K, 40K, 30K, 20K, or 10K, or 5K. The molecular weight of the active polymer (Daltons) may be in a range provided by any two or more of the upper and / or lower amounts, for example between about IK to about 100K, or between about 10K to about 80K .In some embodiments, the molecular weight of the active polymer (Daltons) is about 12K, 36K, or 72K.Base polymer

[0093] When utilised in the process described herein the base polymer provides a robust support skeleton or matrix of the membrane. The base polymer may be selected from a polymer that possesses acceptable thermal and mechanical stability as well as filmforming properties In some embodiments, the base polymer is selected from cellulose, cellulose acetate (CA), polyacrylonitrile (PAN), polyimide, polysulfone (PSf), polyamide (PA), polycarbonate (PC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), and poly vinylidene fluoride (PVDF), and any combination thereof. In one example, the base polymer comprises polyvinylidene fluoride (PVDF).Ratio of active polymer to base polymer

[0094] The ratio of the active polymer to base polymer in the polymer composition may to selected to obtain various physical or performance characteristics of the resultant blended membrane. For instance, a higher ratio of active polymer can provide more reactive sites for pre and / or post membrane formation functionalisation.

[0095] In some embodiments, the weight ratio of base polymer to active polymer in the polymer composition is less than about 99: 1, 97.5:2.5, 95:5, 90: 10, 83: 17, 80:20, 77:23, 73:27, 72:28, 70:30, 6:40, 50:50. In some embodiments, the weight ratio of base polymer to active polymer in the polymer composition is about, or more than about 50:50, 60:40, 70:30, 72:28, 77:23, 80:20, 83: 17, 90: 10, 95:5, 97.5:2.5, or 99: 1. The ratio of base polymer to active polymer in the polymer composition may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 50:50 to 99: 1, or between about 70:20 to 97.5:2.5. In some embodiments, the weight ratio of base polymer to active polymer in the polymer composition is about 50:50, 77:23, or 99: 1. Inone example, the ratio of base polymer to active polymer in the polymer composition is about 73:27. In one example, the ratio of base polymer to active polymer in the polymer composition is about 83: 17.Casting liquid

[0096] The polymer composition is dissolved in a casting liquid. The casting liquid comprises a casting solvent. The composition of the casting solvent is selected to ensure the polymer composition is sufficiently soluble at the desired polymer concentration. In some embodiments, the casting solvent comprises a primary casting solvent. In some embodiments, the casting solvent further comprises one or more additional solvents and / or one or more non-solvents. It will be appreciated that the term non-solvent as used herein means a substance that is incapable of dissolving one or more components of the polymer composition.Primary casting solvent

[0097] In some embodiments, the primary casting solvent is an organic solvent. In some embodiments, the primary solvent is a polar solvent. In some embodiments, the primary casting solvent is a protic solvent. In some embodiments, the primary casting solvent is an organic protic solvent. In some embodiments, the primary casting solvent is selected from dimethylformamide (DMF), dimethylacetamide (DMAC), N-methyl-2-pyrrolidone (NMP), triethyl phosphate (TEP), and dimethyl sulfoxide (DMSO), and any combination thereof. In one example, the primary casting solvent is dimethylacetamide.Secondary casting solvent

[0098] In some embodiments, the secondary casting solvent is an organic solvent. In some embodiments, the secondary casting solvent is a polar solvent. In some embodiments, the secondary casting solvent is an alcohol. In some embodiments, the secondary casting solvent is selected from methanol, ethanol, n-propanol, and isopropanol, and any combination thereof. In one example, the secondary casting solvent is isopropanol.Ratio of primary casting solvent to secondary casting solvent

[0099] The amounts of primary casting solvent and secondary casting solvent in the casting liquid may be varied to achieve the desired solubility of the polymer composition within the membrane casting mixture. In some embodiments, the casting liquid does not include a secondary casting solvent. In some embodiments, the mass ratio of primary casting solvent to secondary casting solvent in the casting liquid is less than about 100:0, 90: 10, 80:20, or 70:30. In some embodiments, the mass ratio of primary casting solvent to secondary casting solvent in the casting liquid is about, or more than about 50:50, 60:40, 70:30, 80:20, 90: 10, or 100:0. The mass ratio of primary casting solvent to secondary casting solvent in the casting liquid may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 50:50 to 100:0, or between about 70:30 to 90:10. In some embodiments, the mass ratio of primary casting solvent to secondary casting solvent in the casting liquid is about 50:50, 80:20, or 100:0. In one example, the mass ratio of primary casting solvent to secondary casting solvent in the casting liquid is about 80:20.

[0100] In some embodiments, the primary casting solvent is dimethylacetamide (DMAc) and the secondary casting solvent is isopropanol. In one example, the primary casting solvent is dimethylacetamide (DMAc), the secondary casting solvent is isopropanol, and the mass ratio of the primary casting solvent to secondary casting solvent in the casting solvent is 80:20.Polymer amount in membrane casting mixture

[0101] The concentration of the polymer composition in the membrane casting mixture has a profound impact on the structure of the resulting membrane. Generally, a low concentration of polymer composition in the membrane casting mixture results in a membrane with a porous structure. Conversely, at higher concentrations of polymer composition in the membrane casting mixture, membranes with a more dense structure are produced. For example, a membrane solution mixture with a polymer concentration ranging from 15 to 20 wt.% can be used for the preparation of porous ultrafiltration membranes. Conversely, a membrane casting mixture containing higher concentrations of polymer, for example 25 wt.%, can be used for the preparation of reverse osmosis, gas separation, and pervaporation membranes. Thus, by varying the concentration of thepolymer composition in the membrane casting mixture, membrane with properties for different applications can be produced.

[0102] While any amount of polymer composition can be used in the membrane casting mixture in the process described herein, in some embodiments, the membrane casting mixture comprises less than about 30% w / w, 25% w / w, 20% w / w, or 15% w / w of the polymer composition based on the total weight of the membrane casting mixture. In some embodiments, the membrane casting mixture comprises about, or greater than about 1% w / w, 2% w / w, 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, or 30% w / w of the polymer composition based on the total weight of the membrane casting mixture. In some embodiments, the amount of polymer composition in the membrane casting mixture may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 1% w / w and 30% w / w, or between about 10% w / w and 20% w / w based on the total weight of the membrane casting mixture.Casting liquid amount in membrane casting mixture

[0103] While any amount of casting liquid can be used in the membrane casting mixture in the process described herein, in some embodiments, the membrane casting mixture comprises less than about 99% w / w, 98% w / w, 95% w / w, 90% w / w, or 85% w / w of the casting liquid based on the total weight of the membrane casting mixture. In some embodiments, the membrane casting mixture comprises about, or greater than about 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, 95% w / w, 98% w / w, or 99% w / w of the casting liquid based on the total weight of the membrane casting mixture. In some embodiments, the amount of casting liquid in the membrane casting mixture may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 70% w / w and 99% w / w, or between about 80% w / w and 90% w / w based on the total weight of the membrane casting mixture.Membrane casting mixture temperature

[0104] It will be appreciated that the temperature of the membrane casting mixture may influence the solubility of the polymer composition in the casting liquid. In some embodiments, the membrane casting mixture is heated to an initial temperature between about 20 °C and 100 °C, about 40 °C and 80 °C, or about 50 °C and 70 °C. In oneexample, the membrane casting mixture is heated to an initial temperature of about 59 °C.

[0105] In some embodiments, after the polymer composition has dissolved into the casting liquid and the membrane casting mixture is formed the membrane casting mixture is cooled from an initial temperature to a casting temperature. In some embodiments, the casting temperature is below the temperature at which the polymer composition was dissolved in the casting liquid. In some embodiments, the membrane casting mixture is cooled from an initial temperature to a casting temperature which is greater than the temperature of the first immersion fluid. In some embodiments, the membrane casting mixture is cooled from an initial temperature to a casting temperature between about 10 °C and 60 °C, about 20 °C and 50 °C, or about 40 °C and 60 °C. In one example, the membrane casting mixture is cooled from an initial temperature to a casting temperature of about 35 °C.Time in membrane casting mixture

[0106] It will be appreciated that depending on the composition of the casting liquid the time required to dissolve the polymer composition and form the membrane casting mixture may vary. In some embodiments, the time allowed for the polymer composition to dissolve in the casting liquid to form the membrane casting mixture (hours) is about, or greater than about 1, 2, 4, 6, 8, 12, 16, 18, or 24. In some embodiments, the time allowed for the polymer composition to dissolve in the casting liquid to form the membrane casting mixture (hours) is less than about 24, 18, 16, or 12. The time allowed for the polymer composition to dissolve in the casting liquid to form the membrane casting mixture (hours) may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 1 and 24, or between about 8 and 16. In some embodiments, the casting liquid and / or polymer composition are stirred for at least some of the time required for the formation of the membrane casting mixture.First immersionFirst immersion fluid

[0107] The composition of the first immersion fluid is selected to ensure that phase separation can be initiated and allow for slow nucleation and crystallisation of thepolymer composition. In some embodiments, the first immersion fluid comprises a primary first immersion solvent. In some embodiments, the first immersion fluid comprises a primary first immersion solvent and a secondary first immersion solvent. In some embodiments, the first immersion fluid comprises a primary first immersion solvent and a first immersion non- solvent. In some embodiments, the first immersion fluid comprises a primary first immersion solvent, a secondary first immersion solvent, and a first immersion non-solvent.

[0108] In some embodiments, the polymer composition has a higher solubility in the casting solvent than the first immersion fluid. In some embodiments, the base polymer has a relatively higher solubility in the casting solvent than the first immersion fluid. In some embodiments, the active polymer has a relatively higher solubility in the casting solvent than the first immersion fluid. In some embodiments, the active polymer has a relatively higher solubility in the first immersion fluid than the base polymer.Primary first immersion solvent

[0109] In some embodiments, the primary first immersion solvent is an organic solvent. In some embodiments, the primary first immersion solvent is a polar solvent. In some embodiments, the primary first immersion solvent is a protic solvent. In some embodiments, the primary first immersion solvent is an organic protic solvent. In some embodiments, the primary first immersion solvent is selected from dimethylformamide (DMF), dimethylacetamide (DMAC), N-methyl-2-pyrrolidone (NMP), triethyl phosphate (TEP), and dimethyl sulfoxide (DMSO), and any combination thereof. In one example, the primary first immersion solvent is dimethylacetamide.

[0110] In some embodiments, the solvent of the first immersion fluid and the casting solvent each comprise one or more solvent(s) in common. In some embodiments, the primary first immersion solvent is the same as the primary casting solvent. In some embodiments, the primary first immersion solvent is the same as the secondary casting solvent.Secondary first immersion solvent

[0111] In some embodiments, the secondary first immersion solvent is an organic solvent. In some embodiments, the secondary first immersion solvent is a polar solvent.In some embodiments, the secondary first immersion solvent is an alcohol. In some embodiments, the secondary first immersion solvent is selected from methanol, ethanol, n-propanol, and isopropanol, and any combination thereof. In one example, the secondary first immersion solvent is isopropanol.

[0112] In some embodiments, the primary first immersion solvent is dimethylacetamide (DMAc) and the secondary first immersion solvent is isopropanol

[0113] In some embodiments, the secondary first immersion solvent is the same as the primary casting solvent. In some embodiments, the secondary first immersion solvent is the same as the secondary casting solvent.First immersion non-solvent

[0114] In some embodiment, the first immersion non-solvent is water. In some embodiments, the first immersion non-solvent is deionised water.

[0115] In some embodiments, the primary first immersion solvent is dimethylacetamide (DMAc), the secondary first immersion solvent is isopropanol, and the non- solvent is water.

[0116] In some embodiments, the first immersion non-solvent is the same as the primary casting non- solvent.Composition of the first immersion fluid

[0117] The amounts of primary first immersion solvent, secondary first immersion solvent, and non-solvent in the first immersion fluid may be varied to achieve the desired phase separation in the of the membrane casting mixture in the first immersion.

[0118] In some embodiments, the first immersion fluid comprises less than about 80% w / w, 70% w / w, 60% w / w, or 50% w / w of the primary first immersion solvent based on the total weight of the first immersion fluid. In some embodiments, the first immersion fluid comprises about, or greater than about 30% w / w, 40% w / w, 50% w / w, 60% w / w, 70% w / w, or 80% w / w of the primary first immersion solvent based on the total weight of the first immersion fluid. In some embodiments, the amount of primary first immersion solvent in the first immersion fluid may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 30% w / w and 70% w / w, orbetween about 40% w / w and 60% w / w based on the total weight of the first immersion fluid.

[0119] In some embodiments, the first immersion fluid does not comprise a secondary first immersion solvent. In some embodiments, the first immersion fluid comprises less than about 20% w / w, 15% w / w, 10% w / w, or 5% w / w of the secondary first immersion solvent based on the total weight of the first immersion fluid. In some embodiments, the first immersion fluid comprises about, or greater than about 0% w / w, 5% w / w, 10% w / w, 15% w / w, or 20% w / w of the secondary first immersion solvent based on the total weight of the first immersion fluid. In some embodiments, the amount of secondary first immersion solvent in the first immersion fluid may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 0% w / w and 20% w / w, or between about 5% w / w and 10% w / w based on the total weight of the first immersion fluid.

[0120] In some embodiments, the first immersion fluid comprises less than about 80% w / w, 70% w / w, 60% w / w, or 50% w / w of the non-solvent based on the total weight of the first immersion fluid. In some embodiments, the first immersion fluid comprises about, or greater than about 20% w / w, 30% w / w, 40% w / w, 50% w / w, or 60% w / w , 70% w / w, or 80% w / w of the non-solvent based on the total weight of the first immersion fluid. In some embodiments, the amount of non-solvent in the first immersion fluid may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 20% w / w and 70% w / w, or between about 30% w / w and 50% w / w based on the total weight of the first immersion fluid.First immersion fluid temperature

[0121] It will be appreciated that the temperature of the first immersion fluid may influence the phase separation. In some embodiments, the temperature of the first immersion fluid is less than the temperature of the membrane casting mixture. In some embodiments, the temperature of the first immersion fluid is greater than the temperature of the second immersion fluid. In some embodiments, the temperature of the first immersion fluid is less than the temperature of the membrane casting mixture and greater than the temperature of the second immersion fluid. In some embodiments, thetemperature of the first immersion fluid is between about 0 °C and 50 °C, about 20 °C and 40 °C, about 25 °C and 35 °C. In one example, the temperature of the first immersion fluid is between about 25 °C and 35 °C. In one example, the temperature of the first immersion fluid is about 30 °C.First immersion time

[0122] It will be appreciated that depending on the composition of the membrane casting mixture the time required to initiate phase separation in the first immersion fluid will vary. In some embodiments, the first immersion period (mins) is about, or greater than about 0.01, 0.050.1, 0.5, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, or 60. In some embodiments, the first immersion period (mins) is less than about 60, 50, 40, 30, 20, 10, or 5. The first immersion period (mins) may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 0.01 and 60, or between about 0.1 and 5.Second immersionSecond immersion fluid

[0123] The composition of the second immersion fluid is selected to ensure that membrane solidification can occur. In some embodiments, the second immersion fluid comprises a second immersion solvent. In some embodiments, the second immersion fluid comprises a second immersion solvent and a second immersion non- solvent. In some embodiments, the second immersion fluid comprises a second immersion nonsolvent. In some embodiments, the second immersion fluid comprises a non-solvent and / or a mixture of non- solvents and / or a mixture of a non- solvent and a solvent. In some embodiments, the second immersion fluid has a greater %v / v of non-solvent than the first immersion fluid

[0124] In some embodiments, the polymer composition has a higher solubility in the first immersion fluid than the second immersion fluid. In some embodiments, the base polymer has a relatively higher solubility in the first immersion fluid than the second immersion fluid. In some embodiments, the active polymer has a relatively higher solubility in the first immersion fluid than the second immersion fluid. In someembodiment, the ratio of non-solvent to solvent in the second immersion fluid is higher than the ratio of non- solvent to solvent in the first immersion fluid.Second immersion solvent

[0125] In some embodiments, the second immersion solvent is an organic solvent. In some embodiments, the second immersion solvent is a polar solvent. In some embodiments, the second immersion solvent is a protic solvent. In some embodiments, the second solvent is an organic protic solvent. In some embodiments, the second immersion solvent is selected from dimethylformamide (DMF), dimethylacetamide (DMAC), N-methyl-2-pyrrolidone (NMP), triethyl phosphate (TEP), and dimethyl sulfoxide (DMSO), and any combination thereof. In one example, the second immersion solvent is dimethylacetamide. In some embodiments, the second immersion solvent is an alcohol. In some embodiments, the second immersion solvent is selected from methanol, ethanol, n-propanol, and isopropanol, and any combination thereof. In one example, the second immersion is isopropanol.Second immersion non-solvent

[0126] In some embodiment, the second immersion non- solvent is water. In some embodiments, the second immersion non-solvent is deionised water. In some embodiments, the first immersion non-solvent is the same as the second immersion nonsolvent.Composition of the second immersion fluid

[0127] The amounts of second immersion solvent and second immersion non-solvent in the second immersion fluid may be varied to achieve solidification in a desired time period.

[0128] In some embodiments, the first immersion fluid comprises less than about 40% w / w, 30% w / w, 20% w / w, or 10% w / w of the second immersion solvent based on the total weight of the second immersion fluid. In some embodiments, the second immersion fluid comprises about, or greater than about 0% w / w, 10% w / w, 20% w / w, or 30% w / w, or 40% w / w of the second immersion solvent based on the total weight of the second immersion fluid. In some embodiments, the amount of second immersion solvent in the firstimmersion fluid may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 0% w / w and 40% w / w, or between about 0% w / w and 20% w / w based on the total weight of the first immersion fluid.

[0129] In some embodiments, the second immersion fluid comprises less than about 100% w / w, 90% w / w, 80% w / w, or 70% w / w of the non-solvent based on the total weight of the second immersion fluid. In some embodiments, the second immersion fluid comprises about, or greater than about 60% w / w, 70% w / w, 80% w / w, 90% w / w, or 100% w / w of the non- solvent based on the total weight of the second immersion fluid. In some embodiments, the amount of non-solvent in the second immersion fluid may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 60% w / w and 100% w / w, or between about 90% w / w and 100% w / w based on the total weight of the first immersion fluid.Second immersion fluid temperature

[0130] It will be appreciated that the temperature of the second immersion fluid may influence the phase separation. In some embodiments, the temperature of the second immersion fluid is less than the temperature of the membrane casting mixture. In some embodiments, the temperature of the second immersion fluid is between about 0 °C and 30 °C, about 10 °C and 30 °C, about 15 °C and 25 °C. In one example, the temperature of the second immersion fluid is between about 15 °C and 25 °C. In one example, the temperature of the second immersion fluid is about 20 °C.Second immersion time

[0131] It will be appreciated that depending on the composition of the membrane casting mixture and the composition of the second immersion fluid the time required for membrane solidification will vary. In some embodiments, the second immersion period (mins) is about, or greater than about 1, 2, 3, 4, 5, 10, 20, 30, 60, 90, 120, or 240. In some embodiments, the second immersion period (mins) is less than about 240, 120, 90, 60, or 30. The second immersion period (mins) may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 1 and 240, between about 1 and 90, or between about 30 and 90.Membrane functionalisation

[0132] The base polymer and / or active polymer of the polymer composition can possess functional group which can be further reacted to covalently attach chemical linkers and / or additional functionalities including chemical or polypeptide ligands. The chemical linkers and / or additional functionalities can be selected for their affinity for a desired chemical and / or biological substance. Advantageously, the inclusion of chemical linkers and / or additional functionalities results in a membrane that can be used to preferentially adsorb a desired biological substance from a solution, for example a selected protein from a plasma or serum.

[0133] Alternatively, the inclusion of chemical linkers and / or additional functionalities results in a membrane that can be used to preferentially adsorb a desired compound from a fluid, for example a pollutant from an aqueous solution.

[0134] Functionalisation of the active polymer co-polymers can be carried out after polymer synthesis or post membrane fabrication. In some embodiments, the methods described herein further comprises incubating active polymer with ligands having an affinity for the biological substance such that the ligands are grafted onto the active polymer.

[0135] In some embodiments, the methods described herein further comprises incubating the polymer composition with ligands having an affinity for the biological substance such that the ligands are grafted onto the polymer composition. In some embodiments, the methods described herein further comprises incubating the polymer composition with ligands having an affinity for the biological substance such that the ligands are grafted onto the active polymer of the membrane.

[0136] In some embodiments, the methods described herein further comprises incubating the solidified polymeric membrane with ligands having an affinity for the biological substance such that the ligands are grafted onto the membrane. In some embodiments, the methods described herein further comprises incubating the solidified polymeric membrane with ligands having an affinity for the biological substance such that the ligands are grafted onto the co-polymer of active polymer of the membrane.

[0137] It will be appreciated that beyond possessing the required chemical reactivity to enable grafting onto a component of the polymer composition incorporated into themembrane no limitation placed is placed on the chemical linkers and / or additional functionalities. Ligands with the requisite functional groups to react with at least one monomer in the active co-polymer may be selected which have a particular affinity for a desired biological substance. In some embodiments, linkage is via thiol functional groups and / or amine functional groups. By way of non-limiting example, the ligands may be selected from Protein A, mercapto benzoic acid (thiol linkage), HS -polypeptide (thiol linkage), Anti A / Anti B (amine linkage), triazine (amine linkage) or AeAs (amine linkage).Applications

[0138] Membranes formed by the method disclosed herein may be used in a wide variety of applications for isolating a protein-of-interest from a protein mixture. The high degree of flexibility in the selection of functional monomers having an affinity for a target compound allows for further tailoring of membrane properties to make them more suitable to a selected application. Additionally or alternatively, the high degree of control of the produced membrane allow for tailoring the physical properties of the membrane, such as thickness and pore size / density, for filtration.

[0139] In some embodiments, the membranes of the present disclosure are for the isolation of a protein-of-interest from a protein mixture. Such a method may comprise contacting a polymeric membrane as disclosed herein with the protein mixture thereby to load the membrane with the protein-of-interest. The functional carboxylic monomer present in active polymer of the membrane may be selected for affinity with the protein- of-interest. In this way, the functional carboxylic acid monomer acts as a ligand for the protein-of-interest to be isolated from the protein mixture.

[0140] It will be appreciated that, although primarily described herein as recovery of a protein-of-interest that binds with the membrane, other applications may also include producing a protein mixture depleted in the protein-of-interest.

[0141] Once loading is complete, preferably to the dynamic binding capacity of the membrane, the protein-of-interest can be eluted and recovered from the membrane.

[0142] In some embodiments, such a method may comprise filtration of the protein mixture with the membrane. The filtration may be dead-end filtration or cross-flowfiltration. Such applications may include viral filtration, ultrafiltration / diafiltration and combinations thereof. The method of producing the polymeric membrane may be configured to provide a membrane having suitable properties for isolation of the protein- of-interest from the protein mixture. For filtration applications, the polymer composition may comprise base polymers only. In some embodiments, the base polymer may comprise N-isopropylacrylamide (NIP AM).

[0143] In one example, the protein mixture comprising the protein-of-interest is selected from the group consisting of a mammalian protein mixture and a human protein mixture. In one example, the protein mixture is a mammalian protein mixture. In one example, the protein mixture is a human protein mixture.

[0144] Exemplary proteins include plasma proteins, peptide hormones, growth factors, cytokines and polyclonal immunoglobulins proteins, plasma proteins selected from human and animal blood clotting factors including fibrinogen, prothrombin, thrombin, prothrombin complex, FX, FXa, FIX, FIXa, FVII, FVIIa, FXI, FXIa, FXII, FXIIa, FXIII and FXIIIa, von Willebrand factor, transport proteins including albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobulin and hemopexin, protease inhibitors including P-antithrombin, a- antithrombin, a-2-macroglobulin, Cl -inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), Protein C and Protein S, a-1 esterase inhibitor proteins, a-1 antitrypsin, antiangionetic proteins including latent- antithrombin, highly glycosylated proteins including a-1 -acid glycoprotein, antichymotrypsin, inter-a-trypsin inhibitor, a-2-HS glycoprotein and C-reactive protein and other proteins including histidine-rich glycoprotein, mannan binding lectin, C4- binding protein, fibronectin, GC-globulin, plasminogen, blood factors such as erythropoietin, interferon, tumor factors, tPA and / or yCSF. In one example, the feed suspension comprises a plasma protein.

[0145] In one example, the protein-of-interest is a plasma protein. For example, the protein-of-interest is a plasma protein selected from the group consisting of immunoglobulin G (IgG), an apolipoprotein Al, an albumin, a serine protease, a plasmin, plasminogen, a FXa, an alpha- 1- antitrypsin, an IgA, an IgM, a factor VIII, a fibrinogen, a von Willebrand factor, an activated clotting factor, factor XIII, a contact system factor, a prekallikrein activator (PKA) , a factor IX, a prothrombin complex, a Cl esteraseinhibitor, a protein C, an anti-thrombin III, a RhD immunoglobulin protein product, alpha acid glycoprotein, haptoglobin, hemopexin, transferrin, Factor H, coagulation factors such as Factor VII, Factor VIII and Factor IX and combinations thereof.

[0146] In one example, the protein-of-interest is an immunoglobulin. For example, the immunoglobulin is IgG. In another example, the immunoglobulin is IgA. In a further example, the immunoglobulin is IgM. In one example, the protein-of-interest is an apolipoprotein Al. In one example, the protein-of-interest is an albumin. For example, a-globulins and / or P-globulins. In one example, the protein-of-interest is a serine protease. In one example, the protein-of-interest is a plasmin. In one example, the protein-of-interest is plasminogen. In one example, the protein-of-interest is an alpha- 1- antitrypsin. In one example, the protein-of-interest is a fibrinogen. In one example, the protein-of-interest is a von Willebrand factor.

[0147] In one example, the protein-of-interest is an activated clotting factor. For example, the activated clotting factor is selected from a group consisting of FXa, FIXa, FVIIa and thrombin. For example, the activated clotting factor is FXa. For example, the activated clotting factor is FIXa. For example, the activated clotting factor is FVIIa. For example, the activated clotting factor is thrombin.

[0148] In one example, the protein-of-interest is a contact system factor. For example, the contact system factor protein is selected from a group consisting of FXIa, FXIIa and kallikrein. For example, the contact system factor protein is FXIa. For example, the contact system factor protein is FXII. For example, the contact system factor protein is kallikrein.

[0149] In one example, the protein-of-interest is a Prekallikrein activator (PKA). In one example, the protein-of-interest is a prothrombin complex. In one example, the protein- of-interest is a C 1 esterase inhibitor. In one example, the protein-of-interest is a protein C. In one example, the protein-of-interest is an anti-thrombin III. In one example, the protein-of-interest is a RhD immunoglobulin protein product. In one example, the protein-of-interest is alpha acid glycoprotein. In one example, the protein-of-interest is haptoglobin. In one example, the protein-of-interest is hemopexin. In one example, the protein-of-interest is transferrin. In one example, the protein-of-interest is Factor H.

[0150] In one example, the protein-of-interest is a coagulation factor. For example, the coagulation factor is selected from the group consisting of factor VII, factor VIII, factor IX, factor XIII and factor IX. In one example, the protein-of-interest is a factor VII (FVII). In one example, the protein-of-interest is a factor VIII (FVIII). In one example, the protein-of-interest is factor IX (FIX). In one example, the protein-of-interest is factor XIII (FXIII). In one example, the protein-of-interest is a factor IX (FIX).

[0151] In one example, the protein-of-interest is a serine protease inhibitor. For example, the serine protease inhibitor is selected from the group consisting of a Cl inhibitor, an alpha- 1- antitrypsin and an anti-thrombin.

[0152] In one example, the protein mixture is derived from plasma or a plasma fraction thereof. For example, the protein mixture is plasma or a plasma fraction thereof. In one example, the plasma fraction is selected from a group consisting of an IgG intermediate product, cryo-rich plasma, cryo-poor plasma, Supernatant I (SN I), Cohn Fraction II (Fr II), Cohn Fraction II+III (Fr II+III), Cohn Fraction I+II+III (FrI+II+III), Kistler / Nitschmann Precipitate A (KN A), Kistler / Nitschmann Precipitate B (KN B), Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), an organic acid salt precipitate, an organic acid salt precipitation supernatant and combinations thereof. In one example, the plasma fraction is an IgG intermediate product. In one example, the plasma fraction is cryo-rich plasma. For example, the plasma fraction is cryo-poor plasma. For example, the plasma fraction is Supernatant I (SN I). For example, the plasma fraction is Cohn Fraction II (Fr II). For example, the plasma faction is Cohn Fraction II+III (Fr II+III). For example, the plasma fraction is Cohn Fraction I+II+III (FrI+II+III). For example, the plasma fraction is Kistler / Nitschmann Precipitate A (KN A). For example, the plasma fraction is Kistler / Nitschmann Precipitate B (KN B). For example, the plasma fraction is Kistler / Nitschmann Precipitate of Supernatant B (KN B+l). In one example, the plasma fraction is an organic acid salt precipitate. In another example, the plasma fraction is an organic acid salt precipitation supernatant. In one example, the plasma sample or fraction thereof is obtained from a non-ethanol fractionation process.

[0153] In one example, the plasma fraction is a suspended paste. For example, the suspended paste is selected from a group consisting of Cohn Fraction II (Fr II), CohnFraction II+III (Fr II+III), Cohn Fraction I+II+III (FrI+II+III), Kistler / Nitschmann Precipitate A (KN A), Kistler / Nitschmann Precipitate B (KN B), Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), an organic acid salt precipitate and combinations thereof. For example, the suspended paste is a Cohn Fraction II (Fr II) paste. In one example, the suspended paste is a Cohn Fraction II+III (Fr II+III) paste. In another example, the suspended paste is a Cohn Fraction I+II+III (FrI+II+III) paste. In another example, the suspended paste is a Kistler / Nitschmann Precipitate A (KN A) paste. In another example, the suspended paste is a Kistler / Nitschmann Precipitate B (KN B) paste. In a further example, the suspended paste is a Kistler / Nitschmann Precipitate of Supernatant B (KN B+l) paste. In one example, the suspended paste is an organic acid salt precipitate paste.

[0154] In one example, the plasma or plasma fraction is clarified. Methods of clarification of the plasma or plasma fraction will be apparent to the skilled person and / or described herein. For example, the plasma or plasma fraction is clarified by passing the plasma or fraction thereof through a filter. For example, a depth or membrane filter can be used. For example, the plasma or plasma fraction is passed through a combination of filters. For example, the combination may be a 1.2 and 0.45 / 0.22 pm membrane filter combination. For example, the plasma or plasma fraction is clarified by passing the plasma or fraction thereof through a depth filter (e.g. BECO® depth filter). In one example, the plasma or plasma fraction is clarified by passing the plasma or plasma fraction through a filter press (e.g. BECO® integra plate or compact plate) comprising one or more depth filter(s). In one example, the filter press further comprises one or more filter aid(s) (e.g. cellulose-based filter aids such as Diacel® 150). In one example, the plasma or plasma fraction is clarified by passing the plasma or fraction thereof through a lipid- specific filter (e.g. Zeta Plus ™ DEL Series filter). For example, the plasma fraction is clarified Supernatant I (SN I). For example, the plasma fraction is clarified Cohn Fraction II (Fr II). For example, the plasma faction is clarified Cohn Fraction II+III (Fr II+III). For example, plasma fraction is clarified Cohn Fraction I+II+III (FrI+II+III). For example, the plasma fraction is clarified Kistler / Nitschmann Precipitate A (KN A). For example, the plasma fraction is clarified Kistler / Nitschmann Precipitate B (KN B).For example, the plasma fraction is clarified Kistler / Nitschmann Precipitate of Supernatant B (KN B+l).

[0155] In one example, the plasma is clarified cryo-rich plasma. In one example, the plasma fraction is clarified cryo-poor plasma.

[0156] In one example, the protein mixture is thawed at room temperature. For example, the protein mixture is thawed at a temperature of between 18°C to 26°C prior to filtration. In one example, the protein mixture is thawed at a temperature of between 18°C to 22°C prior to filtration.

[0157] The stability of the plasma or plasma fraction for feeding to the membrane described herein can be determined by assessing the pro-coagulant activity, proteolytic activity and particle size of the plasma or fraction thereof. Methods for assessing procoagulant activity, proteolytic activity and particle size will be apparent to a skilled person and include for example an in vitro coagulation assay, e.g., activated partial thromboplastin time (NaPTT) assay, microflow imaging (MFI) and / or using commercially available kits, such as thrombin activity assay kit (S-2238), general serine protease assay kit (S-2288), kallikrein activity assay kit (S-2302), plasmin activity assay kit (S-2251) and FXa activity kit (S-2765).

[0158] In one example, the protein mixture is at a temperature in the range of 2°C to 28°C before being fed to the membrane. In one example, the feed mixture thereof is at a temperature in the range of 2°C to 30°C during filtration using the filtration apparatus. For example, a temperature in the range of 2°C to 28°C, such as 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10°C, or 11°C, or 12°C, or 13°C, or 14°C, 15°C, or 16°C, or 17°C, or 18°C, or 19°C, or 20°C, or 21°C, or 22°C, or 23°C, or 24°C, or 25°C, or 26°C, or 27°C, or 28°C, or 29°C, or 30°C. In one example, the protein mixture is at a temperature in the range of 18°C to 26°C before being fed to the membrane and / or during loading of the membrane. For example, a temperature in the range of 18°C to 20°C. In one example, the plasma or fraction thereof is at a temperature in the range of from 20°C to 26°C.

[0159] The pH of a protein mixture being fed to the membrane may be controlled through the use of one or more buffers. Any appropriate buffer known to the person skilled in the art can be used. In some embodiments, the protein mixture being fed to themembrane has a pH of about, or greater than about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the protein mixture being fed to the membrane has a pH of less than about 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, or 6.0. The pH of the protein mixture being fed to the membrane may be in a range provided by any two or more of the upper and / or lower amounts. In some embodiments, the protein mixture being fed to the membrane has a pH of between about 6 to about 8. In some embodiments, the protein mixture being fed to the membrane has a pH of between about 6.5 to about7.5,

[0160] It will be appreciated by a person of skill in the art that the particular flow rate selected will be based upon the size of the membrane used and other, related, operational considerations. In some embodiments, when the membrane is as described in the Examples under “One step capture of IgG from resuspended paste (test 1 )”, the protein mixture is fed to the membrane at a flow rate (ml / min per ml of membrane) of about, or greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0. In some embodiments, when the membrane is as described in the Examples under “One step capture of IgG from resuspended paste (test 1)”, the protein mixture is fed to the membrane at a flow rate (ml / min per ml of membrane) of less than about 10.0, 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0,5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. The flow rate of the protein mixture fed to the membrane may be in a range provided by any two or more of the upper and / or lower amounts. In some embodiments, when the membrane is as described in the Examples under “One step capture of IgG from resuspended paste (test 1)”, the protein mixture is fed to the membrane at a flow rate (ml / min per ml of membrane) of between about 0.1 to about 10. In some embodiments, the protein mixture is fed to the membrane at a flow rate (ml / min per ml of membrane) of between about 1 to about 10. In some embodiments, when the membrane is as described in the Examples under “One step capture of IgG from resuspended paste (test 1 f the protein mixture is fed to the membrane at a flow rate (ml / min per ml of membrane) of between about 2.5 to about 10. In some embodiments, the protein mixtureis fed to the membrane at a flow rate (ml / min per ml of membrane) of between about 5 to about 10.

[0161] It will be appreciated by a person of skill in the art that the particular flow velocity selected will be based upon the size of the membrane used and other, related, operational considerations. In some embodiments, when the membrane is as described in the Examples under “One step capture of IgG from resuspended paste (test lf the flow velocity of the protein mixture through the membrane (cm / hr) is about, or greater than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000. In some embodiments, when the membrane is as described in the Examples under “One step capture of IgG from resuspended paste (test 1)”, the flow velocity of the protein mixture through the membrane (cm / hr) is less than about 5000, 4900, 4800, 4700, 4600, 4500, 4400, 4300,4200, 4100, 4000, 3900, 3800, 3700, 3600, 3500, 3400, 3300, 3200, 3100, 3000, 2900,2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900, 1800, 1700, 1600, 1500,1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100. The flow velocity of the protein mixture through the membrane may be in a range provided by any two or more of the upper and / or lower amounts. In some embodiments, when the membrane is as described in the Examples under “One step capture of IgG from resuspended paste (test 1)”, the flow velocity of the protein mixture through the membrane (cm / hr) is between about 100 to about 5000. In some embodiments, when the membrane is as described in the Examples under “One step capture of IgG from resuspended paste (test 1)”, the flow velocity of the protein mixture through the membrane (cm / hr) is between about 500 to about 2000.The recovery of the protein-of- interest may be determined after loading and eluting, or after filtration. That is, the recovery is defined as the mass of the eluted solution or filtrate as a percentage of the mass of the solution fed to the membrane. In embodiments, the recovery of the protein- of-interest may be at least 60%, for example at least 75%, at least 90%, at least 95%. In some examples, the recovery of the protein-of-interest may be at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55%, 60%, at least about 62.5%, at least about 65%, at least about 67.5%, at least about 70%, at least about 72.5%, at least about 75%, at least about 77.5%, at least about 80%, at least about 82.5%, at least about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, or at least about 95%.

[0162] In one example, at least 60% of the protein-of-interest is recovered from the protein mixture. In another example, at least 60% of the protein-of-interest is recovered from the protein mixture following separation using the membrane. For example, at least about 62.5%, at least about 65%, at least about 67.5%, at least about 70%, at least about 72.5%, at least about 75%, at least about 77.5%, at least about 80%, at least about 82.5%, at least about 85%, at least about 87.5%, at least about 90%, at least about 92.5%, or at least about 95% of the protein-of-interest is recovered from the protein mixture following the separation.

[0163] Methods of determining yield and purity of the protein-of-interest in the eluate or filtrate will be apparent to the skilled person and / or described herein. For example, purity is determined by SDS-PAGE and MALDI-TOF-MS peptide fingerprint analysis. Briefly, a purified or filtered protein product described herein is loaded onto a suitable SDS-PAGE gel (e.g. 8-16% TRIS-glycine), along with a protein size marker and a positive control for the protein-of-interest (e.g., IgG) under reduced and non-reduced conditions. Proteins are separated based on size and protein bands of interest are isolated, processed and analysed by MALDI-TOF-MS. In another example, impurities in the eluate described herein are measured in an Enzyme-Linked Immunosorbent Assay (ELISA) using impurity (e.g. IgA) specific antibodies. For example, the ELISA is performed using commercially available methods.

[0164] Additional purification steps may be performed after the membrane loading and eluting, or filtration. In one example, the method further comprises one or more steps selected from a group consisting of ion exchange chromatography, viral inactivation, viral filtration, ultrafiltration / diafiltration and combinations thereof. Additional purification steps will be apparent to the skilled person and / or described herein.

[0165] In one example, the method further comprises ion exchange chromatography. In one example, the ion exchange chromatography is anion exchange chromatography.For example, anion exchange chromatography may be used to remove IgA, remaining IgM and other plasma components (other than IgG).

[0166] The anion exchanger can be a resin -based anion exchanger, an anion exchange membrane adsorber, or any other format of anion exchanger with a positively charged substrate for capturing negatively charged particles. In one example, the anion exchanger is an anion exchange membrane adsorber. In another example, the anion exchanger is a resin-based anion exchanger. In a further example, the anion exchanger is a monolithic anion exchanger.

[0167] In one example, the method further comprises anion exchange chromatography using a resin-based anion exchanger. For example, the anion exchange chromatography resin is a strong anion exchanger. In one example, the strong anion exchange resin comprises a matrix consisting of a poly(styrene-divinylbenzene) matrix. In one example, the strong anion exchanger comprises a quaternized polyethyleneimine functional group. Suitable resin-based anion exchanges will be apparent to the skilled person and include, for example, POROSTM HQ 50.

[0168] In one example, the anion exchange chromatography step is performed in flow through mode. In another example, the anion exchange chromatography step is performed in bind-and-elute mode.

[0169] In one example, the anion exchange chromatography step comprises a buffer selected from the group consisting of sodium citrate, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium dihydrogen phosphate, Bis-Tris, phosphate, L-histidine and combinations thereof. In one example, the anion exchange chromatography step comprises a buffer comprising MES buffer. In another example, the anion exchange chromatography step comprises phosphate buffer.

[0170] In one example, the method further comprises viral inactivation. For example, viral inactivation may be effected by adjusting the solution to low pH. Low pH may be a pH of between 2 to 4. In one example, low pH viral inactivation is performed in the presence of caprylate. In another example, viral inactivation may be effected by contacting the plasma or fraction thereof, or an IgG-enriched preparation or IgG- containing pharmaceutical composition with n-Octyl-P-D-Glucopyranoside (OG),thereby forming an OG-IgG mixture. In a further example, low pH viral inactivation is performed in the presence of N,N-Dimethylmyristylamine N-oxide (TDAO).

[0171] In a further example, viral inactivation may be effected by exposing the protein mixture, plasma or plasma fraction, a protein depleted preparation or composition (e.g., a plasma protein depleted preparation e.g., an IgG-depleted preparation) to a solventdetergent inactivation step. Suitable solvent-detergent treatments would be apparent to the skilled person and include, for example environmentally friendly detergents. Exemplary environmentally friendly detergents suitable for use in the present disclosure and in particular for use in inactivating lipid enveloped viruses include N,N- Dimethylmyristylamine N-oxide (TDAO), polysorbate 80 (PS80), polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100-reduced), and a non-ionic surfactant prepared from glucose and alcohol (e.g., SimulsolTM formulations). In one example, the detergent is N,N-Dimethylmyristylamine N-oxide (TDAO). In one example, the detergent is polysorbate 80. In another example, the detergent is polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100-reduced). In a further example, the detergent is a non-ionic surfactant prepared from glucose and alcohol.

[0172] In one example, the method further comprises viral filtration. For example, viral filtration membranes of pore sizes from 15-20 nm may be used to remove microbes and viruses from a solution or eluate or pharmaceutical composition. Exemplary nanofilters include Planova S20N (Asahi), Virosart HC (Sartorius) and Planova 20N (Asahi).

[0173] In one example, the method further comprises ultrafiltration / diafiltration. An exemplary ultrafiltration / diafiltration membrane is Pellicon 2 Cassettes (Millipore) or Polyethersulfone or Hydrosart cassettes (Sartorius).

[0174] Although the present disclosure will be described with regard to the recovery of IgG, it will be appreciated that the membranes, systems and methods described herein are not limited to this use and may be applied to solid-liquid separation of any suitable slurry or suspension comprising a protein-of-interest, in particular derived from human blood plasma.

[0175] In one example, the method of the disclosure is performed at large scale. For example, the method is performed on an industrial or a commercial scale. Methods ofperforming on an industrial or a commercial scale will be apparent to a skilled person and / or described herein. For example, the method performed on an industrial scale comprises large scale isolation of plasma proteins from the plasma or plasma fraction.

[0176] In one example, large scale purification is performed using at least 500kg of the protein mixture. For example, large scale purification is performed using between 500kg to 1000kg, or 1000kg to 2500kg, or 2500kg to 5000kg, or 5000kg to 7500kg, or 7500kg, or 10000kg, or 10000kg to 12500kg, or 12500kg to 15000kg of the protein mixture. In one example, large scale purification is performed using at least 500kg of the plasma or fraction thereof. In one example, large scale purification is performed using at least 1000kg, or 2500kg, or 5000kg, or 7500kg, or 10000kg, or 12500kg, or 15000kg of the plasma or plasma fraction. In one example, large scale purification is performed using at least 1000kg of the plasma or plasma fraction. In one example, large scale purification is performed using at least 2500kg of the plasma or plasma fraction. In one example, large scale purification is performed using at least 5000kg of the plasma or plasma fraction. In one example, large scale purification performed using at least 7500kg of the plasma or plasma fraction. In one example, large scale purification is performed using at least 10000kg of the plasma or plasma fraction. In one example, large scale purification is performed using at least 12500kg of the plasma or plasma fraction. In one example, large scale is performed using at least 15000kg of the plasma or plasma fraction.

[0177] The present disclosure also provides a pharmaceutical composition comprising a protein-of-interest purified or produced by a method of the disclosure. The present disclosure also provides a pharmaceutical composition comprising IgG purified or produced as a protein-of-interest by a method of the disclosure. Proteins purified by the disclosure (syn. active ingredients) are useful for formulating into a pharmaceutical composition for parenteral, such as intravenous administration or subcutaneous administration, for therapeutic and prophylactic treatment.

[0178] In some embodiments, the protein-of-interest is an immunoglobulin, preferably human immunoglobulin G (IgG) such as immunoglobulin G from human plasma or a recombinantly produced immunoglobulin G.

[0179] In some embodiments, the active polymer of the polymer membrane comprises itaconic acid as the functional carboxylic monomer, and the protein-of-interest is IgG.

[0180] In some embodiments, a cartridge comprising a stack of membranes may be provided for separation of the protein mixture. The cartridge may comprise any suitable number of membrane layers, for example the cartridge may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 layers. In some embodiments, the cartridge comprises 5, 10, 15 or 20 layers.

[0181] The bed height of a stack of membranes may be defined as the total thickness of the stacked membranes. By way of example, a stack of 5 x 150pm membranes may have a bed height of 750pm. It will be appreciated that the individual membrane layers may have the same or different thicknesses. In addition, the individual membrane layers may have the same of different base polymers and / or active polymers.

[0182] Permeability of a stack of membranes may be defined as the flow rate for a defined pressure and frontal area. In some embodiments disclosed herein, for a stack of 5 x 150 pm polymer membranes formed from the method disclosed herein may have a permeability (mL / min) at 0.3 bar and a frontal area of 14.3cm2of about, or greater than about 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0. In some embodiments, the permeability (mL / min) of the membrane stack may be less than about 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5. The permeability (mL / min) of the membrane formed from the method disclosed may be in a range provided by any two or more of the upper and / or lower amounts, for example between about 0.1 to about 3.0, or about 1.5 to about 2.5. The permeability (mL / min) of the membrane formed from the method disclosed may be about 2.0.Example Embodiments

[0183] The present disclosure may be described by one or more of the following example embodiments.1. A method of producing a polymeric membrane for isolating a protein-of-interest from a protein mixture, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; andsequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising a base polymer and an active polymer comprising an N-isopropylacrylamide (NIPAM)-itaconic acid copolymer (NIA), and wherein the first immersion fluid comprises a solvent and / or a mixture of solvents and / or a mixture of a non- solvent and a solvent, and wherein the second immersion fluid comprises a non-solvent and / or a mixture of non-solvents and / or a mixture of a non-solvent and a solvent, wherein the second immersion fluid has a greater %N / N of non-solvent than the first immersion fluid.2. The method according to example embodiment 1, wherein the NIA co -polymer is a block co-polymer (bNIA).3. The method according to example embodiment 2, wherein the bNIA block copolymer comprises a first block comprising only NIP AM monomer and a second block comprising NIP AM and itaconic acid monomers.4. The method according to example embodiment 3, wherein the molar ratio of NIP AM monomer to itaconic acid monomer in the second block is 1: 1.5. The method according to any one of example embodiments 1 to 4, wherein the molar ratio of NIP AM monomer to itaconic acid monomer of the co-polymer of NIA is from 1: 1 to 3: 1.6. The method according to example embodiment 5, wherein the molar ratio of NIP AM monomer to itaconic acid monomer of the NIA co-polymer is approximately 7:3.7. The method according to any one of example embodiments 1 to 6, wherein the base polymer comprises polyvinylidene fluoride (PVDF).8. The method according to any one of example embodiments 1 to 7, wherein the weight ratio of base polymer to NIA co-polymer in the polymer composition is from 50:50 to 99: 1.9. The method according to example embodiment 8, wherein the weight ratio of base polymer to NIA co-polymer in the polymer composition is between about 70:30 to about 90: 10.10. The method according to example embodiment 9, wherein the weight ratio of base polymer to NIA co-polymer in the polymer composition is approximately 73:27.11. The method according to any one of example embodiments 1 to 10, wherein the NIA co-polymer has an average molecular weight of from 1,000 to 100,000.12. The method according to example embodiment 11 , wherein the NIA co-polymer has an average molecular weight of from 10,000 to 80,000.13. The method according to claim 12, wherein the NIA co-polymer has an average molecular weight of about 12,000, 32,000 or 72,000.14. The method according to any one of example embodiments 1 to 13, wherein, prior to immersing the membrane casting mixture into the first immersion fluid, the membrane casting mixture is coated onto a non-woven fabric membrane support.15. A method of producing a polymeric membrane for isolating a protein-of-interest from a protein mixture, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising a base polymer and an active polymer, an N-isopropylacrylamide (NIPAM)-itaconic acid co-polymer (bNIA), andwherein the polymer composition has a higher solubility in the first immersion fluid than the second immersion fluid.16. A polymeric membrane produced by a method according to any one of example embodiments 1 to 16, wherein the polymeric membrane comprises a polymer matrix formed from the base polymer, the polymer matrix having one or more surfaces and a plurality of connected pores.17. A polymeric membrane according to example embodiment 16, wherein the NIA co-polymer is distributed on the surface(s) of the polymer matrix.18. A polymeric membrane according to example embodiment 16 or example embodiment 17, wherein the NIA co-polymer is distributed in the pores of the polymer matrix.19. A polymeric membrane according to any one of example embodiments 16 to 18, wherein the NIA co-polymer is distributed substantially homogeneously throughout the polymer matrix.20. A method of isolating a protein-of-interest from a protein mixture, comprising: contacting a polymeric membrane according to any one of example embodiments 15 to 19 with the protein mixture thereby to load the membrane with the protein-of-interest.21. The method of example embodiment 20, wherein the protein-of-interest is selected from the group consisting of a plasma protein, peptide hormones, growth factors, cytokines and polyclonal immunoglobulins proteins, plasma proteins, transport proteins, protease inhibitors, a-1 esterase inhibitor proteins, a-1 antitrypsin, anti-angiogenic proteins and other proteins including histidine -rich glycoprotein, mannan binding lectin, C4-binding protein, fibronectin, GC-globulin, plasminogen, blood factors and combinations thereof.22. The method according to example embodiment 20 or example embodiment 21, wherein the protein-of-interest is a plasma protein selected from the group consisting of immunoglobulin G (IgG), an apolipoprotein Al, an albumin, a serine protease, a plasmin, plasminogen, a FXa, an alpha- 1- antitrypsin, an IgA, an IgM, a factor VIII, a fibrinogen,a von Willebrand factor, an activated clotting factor, factor XIII, a contact system factor, a prekallikrein activator (PKA) , a factor IX, a prothrombin complex, a Cl esterase inhibitor, a protein C, an anti-thrombin III, a RhD immunoglobulin protein product, alpha acid glycoprotein, haptoglobin, hemopexin, transferrin, Factor H, coagulation factors such as Factor VII, Factor VIII and Factor IX and combinations thereof.23. The method according to any one of example embodiments 20 to 22, wherein the protein mixture is or is derived from plasma or a plasma fraction thereof.24. The method of example embodiment 23, wherein the plasma fraction is selected from a group consisting of an IgG intermediate product, cryo-rich plasma, cryo-poor plasma, Supernatant I (SN I), Cohn Fraction II (Fr II), Cohn Fraction 11+111 (Fr II+III), Cohn Fraction I+II+III (FrI+II+III), Kistler / Nitschmann Precipitate A (KN A), Kistler / Nitschmann Precipitate B (KN B), Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), an organic acid salt precipitate, an organic acid salt precipitation supernatant and combinations thereof.25. The method according to any one of example embodiments 20 to 24, wherein the protein-of-interest is an immunoglobulin, preferably human immunoglobulin G (IgG) such as immunoglobulin G from human plasma or a recombinantly produced immunoglobulin G.26. The method according to any one of example embodiments 20 to 25, wherein the protein mixture is fed to the polymeric membrane at a flow rate (ml / min per ml of membrane) of between about 1 to about 10.ExamplesMembrane formation

[0184] A bNIA block co-polymer, featuring one block made of pure NIP AM (N- Isopropylacrylamide) and another block of NIP AM and itaconic acid at a molar ratio of 1: 1 was used. The overall molar ratio of NIP AM and itaconic acid within the bNIA copolymer is 7:3. The molecular weight of the bNIA block co-polymer was 12K.

[0185] Isopropanol and DMAc were mixed at a ratio of 20:80 to form a casting liquid. An amount of a polymer composition comprising a selected weight ratio of and the bNIAblock co-polymer to PVDF of 0.373: 1. The polymer composition was dissolved in the casting liquid to form a membrane casting mixture. Then, the membrane casting mixture was spread uniformly on to a glass plate to form a casted film with a thickness of 150 pm. The casted film together with the glass plate were immersed in a bath containing a first immersion fluid comprising isopropanol. The membrane was transferred a bath containing a second immersion fluid comprising deionized water to complete formation of the membrane.

[0186] A second membrane formed using a NIP AM polymer (i.e. with no itaconic acid) and PVDF was made using the same process for comparison.Membrane characterisation

[0187] The bNIA membrane and a commercially available membrane were both characterised by SEM as shown in Figure 1. SEM analysis of both the top and bottom surfaces of the commercial membrane (Figures 1A and 1C, respectively) revealed the existence of both relatively large and small pores within the membrane structure. In contrast, the bNIA membrane (Figures IB and ID) exhibited a uniform pore structure and layout, enabling a higher surface-to-volume ratio for immobilisation of ligands and more effective mass transfer of the protein-of-interest to the immobilized ligand on the membrane surface.

[0188] Referring to Figures 1E-1H, while both the commercial membrane (Figures IE and 1G) and the bNIA (Figures IF and 1H) membrane structure are formed on both sides of the supporting layer, the commercial membrane fails to achieve uniform coating, revealing exposed supporting structure. In contrast, the bNIA membrane achieves a uniform coating on both sides of the supporting structure, ensuring the stability and mechanical strength of the membrane.

[0189] A further bNIA membrane was prepared using a bNIA block co-polymer with a molecular weight of 36K. As shown in Figures 2A-2B showing SEM analysis of the top (Figure 2A) and bottom (Figure 2B) of the formed membrane, a similar uniformity in pore size was observed as for the 12K bNIA membrane.Separation oflgG from IgG / Albumin mixture

[0190] The ability of the 12K bNIA membrane to bind with IgG and albumin was investigated.

[0191] Referring initially to Figure 3A, a chromatographic profile of the membrane after loading with IgG and albumin are overlaid. As can be clearly seen, which binding was observed for IgG, no such binding occurred for albumin. The chromatographic profiles of the membrane after loading with various IgG / HSA mixtures are shown at Figures 3B (IgG:HSA 1: 1), 3C (IgG:HSA 1:2), and 3D (IgG:HSA 1:4).

[0192] Figures 4A-4C show HPLC Peak Characterization of the feedstock, unbound and elution fractions for the IgG / HSA mixtures as follows: Figure 4A (IgG:HSA 1: 1), 4B (IgG:HSA 1:2), and 4C (IgG:HSA 1:4), further demonstrating binding of the bNIA membrane to IgG but not albumin.Binding Capacity and Recovery with Pure IgG

[0193] To confirm the critical role of itaconic acid in protein binding performance the bNIA membrane and control membrane (without itaconic acid) were tested using purified IgG fraction as the feedstock. Two chromatographic runs were performed for the bNIA membrane and three were performed for the control membrane. The test conditions are shown in Table 1.Table 1: Operating condition for bNIA membranes exposed to Pure IgG

[0194] Results of the experiment are shown in Table 2 and Figure 5.Table 2: Dynamic binding capacity of IgG for bNIA membrane and NIP AM membranes when exposed to pure IgG.

[0195] It is clear that IgG binding was significantly reduced in the absence of itaconic acid, highlighting the critical role of this region of the active polymer.Capability to Purify IgG using. Plasma as Feedstock

[0196] To confirm the critical role of itaconic acid in protein binding performance the the bNIA membrane and control membrane were tested using Cryo-poor plasma (CRP) as the feedstock. Two chromatographic runs were performed for the bNIA membrane and three were performed for the control membrane. The test conditions are shown in Table 3.Table 3: Operating condition for bNIA membranes exposed to plasma

[0197] Results of the experiment are shown in Table 4 and Figure 6.Table 4: Dynamic binding capacity of IgG for bNIA membrane and NIP AM membranes when exposed to plasma.

[0198] It can be seen that when exposed to plasma containing IgG and other plasma proteins including IgG, IgM, fibrinogen, and other, the majority of IgG was bound to the membrane. The purity of IgG derived from plasma was about 75-80% highlighting that the membrane displays high selectivity for IgG under suitable / optimal buffer conditions.Further IgG binding studies

[0199] Additional IgG binding studies were conducted for various feedstocks using a 0.05 mL bNIA membrane. Results of the experiment are shown in Table 5 and Figures 7A (purified IgG fraction), 7B (Paste purification), and 7C (CRP purification).Table 5: Dynamic binding capacity of IgG from various feedstocks for bNIA membrane.

[0200] For the paste, an IgG purity of >95% was observed. For plasma, IgG purity was around 75%.

[0201] With reference to Figure 8, additional IgG binding studies were conducted for purified IgG fraction (Figure 8A) and for CRP (Figure 8B) using a cartridge comprising 5 stacked layers of bNIA membrane. A comparable performance between the cartridge and the single membrane of Table 5 was observed, with a DBC of 107.2 ± 3.7 for purified IgG fraction and 99.6 ± 6 for the CRP.Caustic exposure studies

[0202] The alkaline stability of the bNIA membrane was assessed by incubating the membrane in 0.5 M NaOH for 17 hours. With reference to Figure 9, the dynamic binding capacity of the membrane was maintained after exposure to the NaOH, with the DBC after caustic exposure (109 ± 3) substantially the same as the DBC pre-exposure (109 ± 4). The results demonstrate that the bNIA membrane can be cleaned with high concentration NaOH without compromising the DBC for the membrane.

[0203] Referring to Figure 10, similar experiments were conducted for a cartridge comprising 5 stacked layers of bNIA membrane, whereby the membranes were incubated with 0.5 NaOH for 24 hours and to 1.0M NaOH for 72 hours. Once again, the dynamic binding capacity of the membrane was not negatively impacted by the caustic exposure: pre-exposure (70 ± 1.4); post 0.5 M exposure (71.5 ± 6.4); and post 1.0 M exposure (80 + 1.4).Purification and recovery of IgG subclasses

[0204] The purification and recovery of IgG subclasses IgGl, IgG2, IgG3, and IgG4 from varying feedstocks using bNIA membranes was investigated. The feedstocks included: purified IgG fraction (Figures I la and 11b), re-suspended paste (Figures 12a and 12b), and cryorich plasma (Figures 13a and 13b). The IgG feedstock was loaded at 1 mL / min, with wash and elution at 4mL / min. The elution buffer was IM NaCl. The recovery of the IgG subclasses is summarised in Table 6 below.Table 6: Recovery of IgG subclasses from various feedstock using bNIA membranePurification and recovery from mAb IgG2 containing feed

[0205] The purification and recovery of IgG from a feed comprising 4 g / L mAb IgG2 using bNIA membranes was investigated. The mAb IgG2 feedstock was loaded at 2 mL / min, with wash and elution at 4 mL / min. The elution buffer was IM NaCl. With reference to Figures 14a and 14b, from 2 runs, the DBCbind / eiute was 103+4.0 mg IgG2 / mL MV and 90 mg IgG / mL MV and the recovery was 80+0.6 %.Plasma IgG binding to bNIA membrane at varying pH

[0206] The effect of pH on DBC for bNIA membranes for IgG was investigated. As shown in Figure 15, pH 6.5 was found to provide the highest DBC as well as the highest purity product.PVDF / bNIA / non-woven fabric membranes

[0207] PVDF / bNIA membranes with and without non-woven fabric were fabricated in accordance with the conditions summarized in Table 7 below.Table 7: Membrane fabrication conditionsMembrane No. PVDF bNIA DMAC IPA bNIA / PVDFPVDF / bNIA 12.5% 2.5% 68% 17% 20%PVDF / bNIA / non-woven 10.26% 3.83% 68.73% 17.18% 37%

[0208] With reference to Table 8 below, the presence of the non-woven fabric led to the formation of larger average pore size and greater flux for the formed membrane.Table 8: Pore size and flux of PVDF / bNIA membranes with and without non-woven support_ _ .TAverage pore size Water fluxMembrane No. ,x / T,91,(|jm) (L / m2hbar)PVDF / bNIA 0.16 2000PVDF / bNIA / non-woven 1.1 13000

[0209] The larger average pore size can also be seen in the SEM images of the two membranes shown in Figure 16.

[0210] As shown in Figure 17, the transmembrane pressure drop was assessed for each of the membranes at various flow rates, with the non-woven fabric supported membrane demonstrating a significantly lower pressure drop for each flow rate.

[0211] The benefit to mechanical strength of the provision of the non-woven fabric support was also demonstrated, as shown in Figure 18.One step capture of IgG from resuspended paste (test 1 )

[0212] Purification of all 4 IgG subclasses was performed from a resuspended precipitated IgG paste using 1 mL of the bNIA membrane was investigates. The 1 mL of the bNIA membrane comprised of five membranes stacked length ways,, with individual bNIA membranes with dimensions of length = 6.5 cm; width = 2.2 cm, and height(thickness) = 0.015 cm, wherein the protein mixture traversed the length of the stack. IgG paste was resuspended in 2 mM sodium acetate and 2 mM sodium citrate extraction buffer at pH 4.5 at a paste to buffer ratio of 1:5 and incubated for 2 hours at room temperature with stirring. The resulting mixture was then centrifuged and filtered to remove filter-aids. The pH was adjusted to 6.5 using 5 M NaOH before loading onto the bNIA membrane.

[0213] 90-95 mg of total protein was introduced to 1 ml of the bNIA membrane in 2.5 mM Na phosphate buffer solution at a flow rate of 5 ml / min flowrate for 2-3 minutes. The membrane wash then washed with 2.5 mM sodium phosphate pH 6.5 & conductivity 0.3 mS / cm at a flow rate of 5 ml / min flowrate for 7-8 minutes, and the IgG eluted using 0.5 M NaOH at a flow rate of 5 ml / min flowrate for 5-7 minutes. A similar procedure was used for chromatography performed at a pH of 7 with a 2.5 mM sodium phosphate buffer (pH 7.0) for equilibration and wash. The recovery of the IgG and subclass distribution is summarised in Tables 9 and 10 below.Table 9: Recovery of IgGFlowAvg IgG bound Recovery Flow rate . Residence® ®Jvelocity(mg) % (ml / min)(cm / hr)Time(s) pH 6.5 47 + 1.0 97 + 2.0 5 1818 13 pH 7.0 43 + 0.4 93 + 0.7 5 1818 13Table 10: Recovery of IgG subclassesOne step capture of IgG from resuspended paste (test 2)

[0214] Purification of all 4 IgG subclasses was performed from a resuspended paste comprising IgG using 1ml of the bNIA membrane. 1 mL of the bNIA membrane comprised five membranes stacked length ways, with individual bNIA membranes with dimensions of length = 6.5 cm; width = 2.2 cm, and height (thickness) = 0.015 cm, wherein the protein mixture traversed the length of the stack. IgG paste was resuspended in 2 mM sodium acetate and 2 mM sodium citrate extraction buffer at pH 4.5 at a paste to buffer ratio of 1:9 and incubated for 2 hours at room temperature with stirring. The resulting mixture was then centrifuged and the resulting supernatant filtered to remove filter-aids. The pH was adjusted to 6.5 using 5 M NaOH before loading onto the bNIA membrane.

[0215] 90-95 mg of total protein was introduced to 1 ml of the bNIA membrane in 2.5 mM Na phosphate buffer solution at a flow rate of 5 ml / min flowrate. The membrane was then washed with 2.5 mM sodium phosphate pH 6.5 & conductivity 0.3 mS / cm at a flow rate of 5 ml / min flowrate for 7-8 minutes. Elution was performed using 0.5 M NaOH at a flow rate of 5 ml / min flowrate for 5-7 minutes. A similar procedure was used for chromatography performed at a pH of 7 with a 2.5 mM sodium phosphate buffer (pH 7.0) for equilibration and wash. The recovery of the IgG subclasses is summarised in Tables 11 and 12 below.Table 11: Recovery of IgGTable 12: Recovery of IgG subclassesIgG recovery at different flow rates

[0216] The impact of flow rate on the purification of IgG from a precipitated plasma fraction (paste) comprising IgG was investigated. IgG was resuspended in 2 mM sodium acetate and 2 mM sodium citrate extraction buffer at pH 4.5 at a paste to buffer ratio of 1:5 and incubated for 2 hours at room temperature with stirring. The resulting mixture was then centrifuged and filtered to remove filter-aids. The clarified supernatant was split into two fractions: one adjusted to pH 6.5 and the other to pH 7.0. The impact of flow rate on the recovery of IgG was assessed by comparing chromatographic runs at flow rates of 1 mL / min per ml of membrane and 5 mL / min per ml of membrane and results are presented in Table 13.Table 13: Recovery of IgG at different flow rates

[0217] From the data in Table 13, it can be seen that increasing the flow rate did not impact the IgG binding nor influence the amount recovered through elution. Thus, membranes within the scope of the present disclosure can be operated at relatively high flowrates thereby providing the advantage of reduced the processing time for protein recovery without sacrificing process efficiency.

[0218] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The presentembodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A method of producing a polymeric membrane for isolating a protein-of-interest from a protein mixture, the method comprising: immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising a base polymer and an active polymer comprising an N-isopropylacrylamide (NIPAM)-itaconic acid copolymer (NIA), and wherein the first immersion fluid comprises a solvent and / or a mixture of solvents and / or a mixture of a non- solvent and a solvent, and wherein the second immersion fluid comprises a non-solvent and / or a mixture of non-solvents and / or a mixture of a non-solvent and a solvent, wherein the second immersion fluid has a greater %N / N of non-solvent than the first immersion fluid.

2. The method according to claim 1, wherein the NIA co-polymer is a block copolymer (bNIA).

3. The method according to claim 2, wherein the bNIA block co-polymer comprises a first block comprising only NIP AM monomer and a second block comprising NIP AM and itaconic acid monomers.

4. The method according to claim 3, wherein the molar ratio of NIP AM monomer to itaconic acid monomer in the second block is 1: 1.

5. The method according to any one of the preceding claims, wherein the molar ratio of NIP AM monomer to itaconic acid monomer of the co-polymer of NIA is from 1: 1 to 3: 1.

6. The method according to claim 5, wherein the molar ratio of NIP AM monomer to itaconic acid monomer of the NIA co-polymer is approximately 7:3.

7. The method according to any one of the preceding claims, wherein the base polymer comprises poly vinylidene fluoride (PVDF).

8. The method according to any one of the preceding claims, wherein the weight ratio of base polymer to NIA co-polymer in the polymer composition is from 50:50 to 99: 1.

9. The method according to claim 8, wherein the weight ratio of base polymer to NIA co-polymer in the polymer composition is between about 70:30 to about 90: 10.

10. The method according to claim 9, wherein the weight ratio of base polymer to NIA co-polymer in the polymer composition is approximately 73:27.

11. The method according to any one of the preceding claims, wherein the NIA copolymer has an average molecular weight of from 1,000 to 100,000.

12. The method according to claim 11, wherein the NIA co-polymer has an average molecular weight of from 10,000 to 80,000.

13. The method according to claim 12, wherein the NIA co-polymer has an average molecular weight of about 12,000, 32,000 or 72,000.

14. The method according to any one of the preceding claims, wherein, prior to immersing the membrane casting mixture into the first immersion fluid, the membrane casting mixture is coated onto a non-woven fabric membrane support.

15. A method of producing a polymeric membrane for isolating a protein-of-interest from a protein mixture, the method comprising:immersing a membrane casting mixture into a first immersion fluid at a first immersion temperature and for a first immersion period to form an intermediate polymeric phase; and sequentially, immersing the intermediate polymeric phase into a second immersion fluid at a second immersion temperature and for a second immersion period to form the polymeric membrane, wherein the membrane casting mixture comprises a polymer composition dissolved in a casting liquid, the polymer composition comprising a base polymer and an active polymer, an N-isopropylacrylamide (NIPAM)-itaconic acid co-polymer (bNIA), and wherein the polymer composition has a higher solubility in the first immersion fluid than the second immersion fluid.

16. A polymeric membrane produced by a method according to any one of the preceding claims, wherein the polymeric membrane comprises a polymer matrix formed from the base polymer, the polymer matrix having one or more surfaces and a plurality of connected pores.

17. A polymeric membrane according to claim 16, wherein the NIA co-polymer is distributed on the surface(s) of the polymer matrix.

18. A polymeric membrane according to claim 16 or claim 17, wherein the NIA copolymer is distributed in the pores of the polymer matrix.

19. A polymeric membrane according to any one of claims 16 to 18, wherein the NIA co-polymer is distributed substantially homogeneously throughout the polymer matrix.

20. A method of isolating a protein-of-interest from a protein mixture, comprising: contacting a polymeric membrane according to any one of claims 15 to 19 with the protein mixture thereby to load the membrane with the protein-of-interest.

21. The method of claim 20, wherein the protein-of-interest is selected from the group consisting of a plasma protein, peptide hormones, growth factors, cytokines and polyclonal immunoglobulins proteins, plasma proteins, transport proteins, protease inhibitors, a-1 esterase inhibitor proteins, a-1 antitrypsin, anti- angiogenic proteins and other proteins including histidine-rich glycoprotein, mannan binding lectin, C4-binding protein, fibronectin, GC-globulin, plasminogen, blood factors and combinations thereof.

22. The method according to claim 20 or claim 21, wherein the protein-of-interest is a plasma protein selected from the group consisting of immunoglobulin G (IgG), an apolipoprotein Al, an albumin, a serine protease, a plasmin, plasminogen, a FXa, an alpha- 1- antitrypsin, an IgA, an IgM, a factor VIII, a fibrinogen, a von Willebrand factor, an activated clotting factor, factor XIII, a contact system factor, a prekallikrein activator (PKA) , a factor IX, a prothrombin complex, a Cl esterase inhibitor, a protein C, an antithrombin III, a RhD immunoglobulin protein product, alpha acid glycoprotein, haptoglobin, hemopexin, transferrin, Factor H, coagulation factors such as Factor VII, Factor VIII and Factor IX and combinations thereof.

23. The method according to any one of claims 20 to 22, wherein the protein mixture is or is derived from plasma or a plasma fraction thereof.

24. The method of claim 23, wherein the plasma fraction is selected from a group consisting of an IgG intermediate product, cryo-rich plasma, cryo-poor plasma, Supernatant I (SN I), Cohn Fraction II (Fr II), Cohn Fraction II+III (Fr II+III), Cohn Fraction I+II+III (FrI+II+III), Kistler / Nitschmann Precipitate A (KN A), Kistler / Nitschmann Precipitate B (KN B), Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), an organic acid salt precipitate, an organic acid salt precipitation supernatant and combinations thereof.

25. The method according to any one of claims 20 to 24, wherein the protein-of- interest is an immunoglobulin, preferably human immunoglobulin G (IgG) such as immunoglobulin G from human plasma or a recombinantly produced immunoglobulin G.

26. The method according to any one of claims 20 to 25, wherein the protein mixture is fed to the polymeric membrane at a flow rate (ml / min per ml of membrane) of between about 1 to about 10.

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