EP4440739A1

EP4440739A1 - Process for preparation of functionalized fiber

Info

Publication number: EP4440739A1
Application number: EP22851441.0A
Authority: EP
Inventors: Ian Tomlinson; Simon Frisk; Martin Deetz
Original assignee: DDP Specialty Electronic Materials US LLC
Current assignee: DDP Specialty Electronic Materials US LLC
Priority date: 2021-12-02
Filing date: 2022-12-02
Publication date: 2024-10-09
Also published as: WO2023102465A1; WO2023102503A1; JP2024543596A; KR20240113786A; WO2023102505A1

Abstract

A method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 µm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb is described. The method comprises exposing the nanoweb to a vinyl monomer in an atmospheric plasma and then to a molecule which is capable of binding other molecules to an active site.

Description

PROCESS FOR PREPARATION OF FUNCTIONALIZED FIBER

Background

This invention relates generally to a method for producing a functionalized nanoweb useful in chromatography.

During the production of biopharmaceuticals, in particular antibody derived therapeutics, e.g., monoclonal antibodies, an affinity capture and elute purification step is frequently employed. This step most typically involves the binding of the desired therapeutic by a protein ligand conjugated to a chromatography support. Some support materials require surface functionalization prior to attachment of the protein ligand to the functionalized support surface. Fibers have been used as support materials, e.g., in Z. Ma et al., J. Chromatogr. B, 877 (2009) 3686-3694, fibers are exposed to an air plasma then immersed in a liquid-phase of methacrylic acid to react and produce a functionalized fiber. In a separate step, in a liquid-phase reaction, a protein is bound to the functionalized fiber via amino groups on the protein.

Statement of Invention

The present invention is directed to a method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb; said method comprising steps of: a) exposing said nanoweb and a vinyl monomer to an atmospheric plasma to produce a coated nanoweb; and b) exposing said coated nanoweb and an aerosol comprising a molecule capable of forming covalent bonds, preferably a protein, to an atmospheric plasma.

The present invention is further directed to a method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb comprising a protein; said method comprising steps of: a) exposing said nanoweb and a vinyl monomer to an atmospheric plasma to produce a coated nanoweb; and b) exposing said coated nanoweb to a protein which (i) is capable of binding other proteins to an active site, and (ii) comprises a free thiol group. Detailed Description

All percentages are weight percentages (wt%), and all temperatures are in °C, unless otherwise indicated. Averages are arithmetic averages unless indicated otherwise. All operations are performed at room temperature (from 18 to 25 °C) unless specified otherwise. The terms “(meth)acrylate”, “(meth)acrylic”, and “(meth)acrylamide” mean acrylate or methacrylate, acrylic or methacrylic, and acrylamide or methacrylamide, respectively (collectively “acrylic monomers”). An acrylic polymer is a polymer comprising at least 50 wt% polymerized units of (meth)acrylic acid, alkyl, glycidyl or hydroxyalkyl (meth)acrylates, alkyl, glycidyl and/or hydroxyalkyl (meth)acrylamides, or a combination thereof. A carbohydrate polymer is a polymer comprising polymerized units of sugar molecules, i.e., a polysaccharide; polysaccharides functionalized via ether or ester groups are considered to be within this definition.

A web of randomly distributed fibers is commonly referred to as a “nonwoven.” The fibers can be bonded to each other or unbonded, preferably unbonded. A “nanoweb” is a nonwoven web comprising at least one nanofiber. A nanoweb may also be referred to as a “nanofiber mat.” Preferably, the fibers in the nanoweb are “continuous,” i.e., having been laid down in one continuous stream to form the web. Fiber diameters may be determined by SEM picture examination. Preferably the fiber has a diameter from 0.1 to 1 pm. Preferably, at least 95% of the fiber has a diameter in the stated range, based on length of the fiber. Preferably, if less than 95% of the fiber has a diameter in the stated range, then fiber diameter is the arithmetic average of at least 50 measurements, preferably at least 100 measurements.

Preferably, the nanoweb has a mean flow pore size of at least 0.15 pm, preferably at least 0.2 pm, preferably at least 0.25 pm; preferably no more than 2 pm, preferably no more than 1 pm, preferably no more than 0.5 pm. Mean flow pore size is a calculated quantity from material porometry measurements, where the dry sample is subjected to airflow at various flow rates, then wetted with a fluid of known surface tension and air flows are returned at steadily increasing flow rate until the last wetted pore of the material is evacuated with air. A mean flow pore is determined from a /i slope of the dry air flow curve intersecting the wet flow curve. Relationships between fiber diameter and mean flow pore size have been determined. Simmonds et al proposed the following relationship to determine mean flow pore size, D_mean,pore from the mean fiber diameter of the nonwoven, Daber (see Simmonds GE, Bomberger JD, Bryner MA. Designing nonwovens to meet pore size specifications. J Eng Fibers Fabrics. 2007;2(l), 1 - 15.) Dmean,pore 0.39267 *Dfiber/(l -porosity)

Preferably, the nanoweb has a porosity from 40 to 90 volume %, preferably at least 50 volume%, preferably at least 60 volume %; preferably no more than 85 volume%, preferably no more than 80 volume%, preferably no more than 75 volume%. It is believed that fluid flow through the nanofiber mat is facilitated by high porosity, and binding capacity of a substrate is improved when the pore size is small. Preferably when pore size is 0.1 to 1 micron, porosity is 50 to 90 volume%; preferably when pore size is 0.1 to 0.5 micron, porosity is from 65 to 85 volume% Porosity is calculated from the following equation:

Porosity = 1- (mass of fiber in g/cm²/(Thickness of nanofiber mat in cm*Polymer Density in g/cm³))

Preferably, the fiber comprises a synthetic polymer; preferably poly(vinylidene fluoride) (PVDF), copolymers of PVDF such as poly(vinylidene fluoride-co-trifluoroethylene), polyamide, polyethersulfone (PES), polyethylene, polypropylene, polyester, polyimide or a combination thereof; preferably the fiber comprises PVDF, polyethersulfone, nylon or a combination thereof. Preferably, a PVDF polymer has a number average molecular weight is from 100,000 to 2,000,000 daltons, preferably from 200,000 to 500,000. Preferably, a polyamide has a number average molecular weight from 5,000 to 40,000, preferably from 10,000 to 20,000. Preferably, a polyethersulfone has a number average molecular weight from 20,000 to 80,000, preferably from 40,000 to 60,000. In a preferred embodiment of the invention the fiber comprises PVDF; preferably the fiber comprises at least 50 wt% PVDF, preferably at least 80 wt%, preferably at least 90 wt%, preferably at least 95 wt%.

Preferably, the vinyl monomer comprises at least one epoxy group; preferably from one to four, preferably from one to two, preferably one. Preferably, the vinyl monomer comprises an ester or ether of: (i) an epoxy alcohol, or (ii) an acetylenic alcohol. Preferably, the vinyl monomer is an ether of an epoxy alcohol, preferably glycidyl alcohol (e.g., vinyl glycidyl ether). Preferably, the vinyl monomer is an ester of an epoxy alcohol. Preferably, the ester is a (meth)acrylate ester. Preferably, the epoxy alcohol is glycidyl alcohol. Preferably, the vinyl monomer is a (meth)acrylate ester of glycidyl alcohol, preferably glycidyl (meth)acrylate. In a preferred embodiment, the nanoweb is exposed in plasma to a vinyl monomer comprising at least one hydrophilic group prior to introduction of a vinyl monomer comprising at least one epoxy group. Preferably the hydrophilic group is a hydroxyl, carboxylic acid, polyethylene glycol, methoxy polyethyleneglycol,; preferably hydroxyl. Preferably, the vinyl monomer is a (meth)acrylate ester having a hydrophilic group on the ester alkyl group, a PEG (meth)acrylate, (meth)acrylamide or (meth)acrylic acid; preferably a hydroxyalkyl (meth)acrylate; preferably 2-hydroxyethyl methacrylate (HEMA), 2-hydroxyethyl acrylate (HEA), 2-hydroxypropyl methacrylate, 2-hydroxypropyl acrylate, 4-hydroxybutyl methacrylate, 4-hydroxybutyl acrylate, or a mixture thereof; preferably HEMA or HEA. In another preferred embodiment, the vinyl monomer comprising at least one hydrophilic group is contacted with the vinyl monomer and the nanoweb concurrently.

The molecule capable of forming covalent bonds can include, but are not limited to, molecules that include nucleophilic groups. These nucleophilic groups can include, but are not limited to, hydroxyl, amine or thiol groups. These groups can be found in amino acids, peptides, proteins, nucleic acids, polynucleic acids, viruses and cells among others. Preferably the molecule capable of forming a covalent bond is a protein.

In one preferred embodiment of the invention, the fiber, vinyl monomer and protein are present in the plasma environment at the same time. In another preferred embodiment of the invention, the fiber is functionalized with the vinyl monomer in a plasma first and then the functionalized fiber is contacted with the protein in a plasma. In another preferred embodiment, the fiber is functionalized with the vinyl monomer in a plasma first and then the functionalized fiber is contacted with the protein in an aqueous medium.

Preferably, the protein comprises a free cysteine thiol group, i.e., a thiol group (-SH) which is not part of a disulfide linkage, and said protein is active with respect to binding other proteins to an active site. Preferably, the protein comprising a free cysteine thiol group is obtained by cleaving a disulfide bond in the protein which is not required for maintaining the integrity of the protein binding site, i.e., the protein’s activity for binding other proteins. Preferably, the disulfide is reduced using Tris(2-carboxyethylphosphine) hydrochloride (TCEP.HC1) which can be used at a pH between 1.5 and 9.0 depending upon the stability of the protein being reduced at that pH. A typical pH for the reduction is pH 3 to pH 8, using nonphosphate buffers e.g. TRIS, HEPES, Borate. One advantage of TCEP is that it does not contain a thiol group and therefore does not require removal before reacting the free sulfhydryl of the protein with the epoxide groups on the nanofiber. Additional reducing agents include dithiothreitol (DTT), mercaptoethanol (ME), 2-mercaptoethylamine hydrochloride (2-MEA.HC1) all of which are typically utilized around neutral pH. These three reagents all include free thiol groups and can be removed from the protein by filtration prior to reacting the protein with the nanofibers to reduce competitive reactions during the conjugation of the protein. Cysteine- Cysteine disulfide bonds are frequently found in proteins as a method of maintaining the protein structure such that cleavage of these disulfide bonds can result in denaturation of the protein. Other structural features such as P -sheets, helix bundles and hairpin structures can maintain a protein’s conformation without the use of disulfide linkages. Preferably, proteins useful for affinity binding of other proteins or useful biotherapeutic molecules are produced recombinantly or isolated from natural sources. Preferably, the protein is one which has been produced recombinantly so as to include a disulfide linkage that is not involved in the configurational stability of the resulting protein; preferred proteins of this type include Protein A, Protein A/G and Protein G. Other preferred proteins include antibodies, monoclonal and polyclonal, the recognition fragments F(ab’) and F(ab’)2 which can be produced recombinantly or following an enzymatic treatment to cleave the fragments from the intact antibody (Rosenstein et.al., Curr Protoc Mol Biol. 2020 Jun;131(l):el l9.doi: 10.1002/cpmb. l l9). Preferably, disulfide bonds which are reduced in the F(ab’)2 are distal to the binding site of the protein and can be cleaved to form the individual fragments without loss of the ability to bind to other proteins, most likely because disulfide linkages that help to maintain the protein configuration occur within a P- secondary structure such as a P-sheet which maintains the protein configuration and protects these disulfides from reduction. Preferably, the protein is in an aqueous solution at a concentration from 5 to 50 mM. Preferably the pH of the solution is from 8 to 9.5, preferably from 8.5 to 9.3, preferably from 8.7 to 9.1 and the molecular weight of the protein is no greater than 150,000 Daltons, preferably no greater than 100,000 Daltons and preferably no greater than 75,000 Daltons.

Preferably, a dielectric barrier discharge atmospheric pressure plasma process is used to attach the functional monomer and protein injected as liquid aerosol to the nanofiber substrate. The dielectric barrier discharge plasma process preferably is a homogenous glow discharge process. Homogeneous glow discharge plasma processes are known in the art to produce spatially uniform low temperature electrons from injected gases at atmospheric pressures. Ions collide with the injected monomers producing ionized species that may self-polymerize in aerosol prior to substrate deposition. Preferred gases suitable for plasma generation include carbon dioxide, nitrogen, argon, and/or helium. The flow, of gases in plasma form and the injected monomer aerosol, passes through a nozzle with a defined cross-sectional area. Preferably, for treating nonwoven roll goods, the nozzle is rectangular in geometry with an interelectrode gap and a width larger than the width of the nonwoven. The inter-electrode gap is preferably 0.5-10 mm, preferably, 0.8-2mm. The cross-sectional area of the nozzle used in the examples below is 5 cm² with a nozzle width of 40cm. Preferably, gas flow rates range from 2-150 slm/cm², where slm is in units of standard liters per minute with standard conditions of a gas volume are at temperature of 0°C and pressure of 1 atm (lOlkPa), preferably from 60 to 100 slm/cm². Gas flow rates through the plasma head will depend on plasma head size and substrate width to be coated with monomer as well as on environmental temperature and pressure of the gas. Injected monomer flow rates depend on the amount of monomer to be attached per area of substrate. High injection rates of monomer may lead to self-polymerization prior to deposition on the substrate. Preferably, injection rates of aerosolized monomer range from 0.2 slm/cm² to 5 slm/cm². Dilutions of monomers may be achieved by one or both means of liquid solution mixing, by dissolution or suspension of a solid monomer in water or an appropriate solvent, and by introduction of greater gas flow into the liquid monomer pure compound, suspension, or solvent mixture. Preferred dilution ratios (solvent or carrier to monomer) range from of 2: 1 to 50: 1, preferably from 3 : 1 to 20: 1. Preferably, alternating current powered covered electrodes generate the plasma through a narrow gap at atmospheric pressures from 0.9-1.1 atm (91-111 kPa). Preferably, plasma source voltage ranges from 1-100 kV with preferred range of 5-30 kV. Preferably supplied power for initial activation and reaction with monomer is in the range of 2 to 300W/cm², preferably 20 to 200 W/cm², preferably 30 to 160 W/cm². Preferably supplied power in the presence of protein is in the range of 1 to 100 W/cm², preferably 2 to 40 W/cm², preferably 3 to 20 W/cm². Preferably, the distance between the substrate and the plasma head is 1-10 mm, preferably 2-5 mm. Preferably, the substrate is pretreated using carbon dioxide, nitrogen, argon, and/or helium in an atmospheric plasma process without monomer. EXAMPLES

The nonwoven web from a non-carbohydrate based polymer, used as examples and shown in Table I, was prepared by an electrospinning process similarly to the one described in https://pubs.acs.org/doi/10.1021/acs.chemrev.8b00593. Similar non-carbohydrate webs may be produced by an electroblowing process as described in U.S. Patent number 7,618,579. These nonwoven webs were then optionally consolidated as described in U.S. Patent number US8697587B2. The spinning and consolidation processes were tuned such that porosity of the resulting webs could range between 35% and 95%, and the mean flow pore size between 0.1 pm and 5 pm.

Table I. Polymer Basis Weight Thickness Porosity MFP g/m2 pm % pm

1 PVDF 9.9 18 69 0.44

The example nonwoven substrate was pretreated through a plasma, and then processed through the plasma coating process (see, e.g., LIS 8,663,751, LIS 9,890,260 and LIS 9,938,388) using the functionalizing agent (“agent”) and process conditions resulting in various amounts of agent being injected into the plasma and applied to the substrates. Plasma process conditions for epoxide or methacrylic deposition on example nonwovens are shown in Table II as vinyl functionalized example numbers 1 and 2. Each nanofiber nonwoven web was subjected to an activation pass through the plasma treatment device, in which the top and bottom surfaces of the web are exposed to a plasma of CO2 and N2 at a flow rate of 80 slm/cm² and 8 slm/cm² power level of 300 W/m². The activated nanofiber web passes through a N2 plasma at flow rate of 80 slm/cm². The distance between the electrode and substrate was 2 mm.

Table II.

Plasma Process

Vinyl _T „ Plasma Vinyl . . .

» . . Nonwoven Gas » . . Aerosol Flow Dilution functionalized _c , . . . power, functional . , ,

. Substrate type _x rate, slm/cm ₂ 2 ratio example W/cnr agent

1 1 N₂ 200 GMA 1.1 7: 1

2 1 N₂ 200 HEMA 1.1 7: 1 Functionalized Example 1

Hydrolyzed recombinant protein A was thawed and solutioned into a nebulizer vial with deionized water at a concentration of 11 mg Protein A per mL of solution. No reduction reaction was carried out prior to aerosolizing the solution and co-inj ection with GMA into the plasma of Argon. The protein plasma was directed at a moving substrate of vinyl functionalized example 1 from Table II. Plasma process conditions used an atmospheric plasma process with a 3 : 1 blend of Ar to N2 gas at 60 slm/cm² of Ar, a distance from substrate to electrode of 2 mm, applied power to plasma of 40 W/cm², and 50:50 by volume co injection of protein A solution and GMA. Solution was diluted with additional Ar gas at 3 : 1 by volume with a total aerosol flow rate of 2 slm/cm². The resultant protein functionalized nanofiber substrate with Protein A was tested for saturated binding capacity as is.

Functionalized Example 2

Hydrolyzed recombinant protein A was thawed and solutioned into vial with deionized water at a concentration of 11 mg Protein A per mL of solution. A reduction reaction was carried out prior to aerosolizing the solution and co-inj ection with GMA into the plasma of Argon. Reduction to the disulfide bonds in the Protein A dimer was carried out using TCEP-HC1 (Tris(2- carboxyethyljphosphine hydrochloride) [See: Thermo Fisher Application Notes https://www.thermofisher.com/us/en/home/references/molecular-probes-the-handbook/thiol- reactive-probes/introduction-to-thiol-modification-and-detection.html] in aqueous solution at 5 - 50 mM concentration and pH 6-8. The excess TCEP was filtered from solution using a Sephadex column, followed by elution with deionized water. The filtered solution was adjusted for pH using 3M ammonium carbonate (AC), adding 0.5mL AC to each 1.5 mL of filtered reduced protein A solution until pH 9.5. The pH adjusted protein plasma was directed at a moving substrate of vinyl functionalized example 1 from Table II. Plasma process conditions used an atmospheric plasma process with a 3 : 1 blend of Ar to N2 gas at 60 slm/cm² of Ar, a distance from substrate to electrode of 2 mm, applied power to plasma of 40 W/cm², and 50:50 by volume co injection of protein A solution and GMA. Solution was diluted with additional Ar gas at 3 : 1 by volume with a total aerosol flow rate of 2 slm/cm². The resultant protein functionalized nanofiber substrate with Protein A was tested for saturated binding capacity as is. Functionalized Example 3

A 20mg/mL solution of recombinant Protein A in deionized water was prepared in a 50mL vial. TCEP at 0.5M was added to reduce the disulfide linkage and allowed to incubate for 10 minutes at room temperature. The reaction mixture was filtered through a Sephadex column, and reacted with GMA in 10³ equivalents, followed by treatment with IM ammonium formate. The resulting GMA modified recombinant protein A solution was filtered again, then injected as co-aerosol with HEMA into an Ar/N2 atmospheric plasma at 40 W/cm², same conditions as Functionalized Example 2 on to vinyl functionalized example 2. The resultant protein functionalized nanofiber substrate with Protein A was tested for saturated binding capacity as is.

Functionalized Example 4

The same GMA modified solution as prepared in Functionalized Example 3, is introduced in the same plasma process as Functionalized Example 2 onto vinyl functionalized example 2, except the dilution of the aerosol is increased 7: 1. The resultant protein functionalized nanofiber substrate with Protein A was tested for saturated binding capacity as is.

Functionalized Example 5

Vinyl functionalized example 1 from table II is conjugated in solution with a reactive cysteine on recombinant Protein A. This reaction involves first reducing disulfide bonds in the Protein A dimer using TCEP-HC1 (Tris(2-carboxyethyl)phosphine hydrochloride) [See: Thermo Fisher Application Notes https://www.thermofisher.com/us/en/home/references/molecular-probes-the- handbook/thiol-reactive-probes/introduction-to-thiol-modification-and-detection.html] in aqueous solution at 5 - 50 mM concentration and pH 6-8. The Protein A with a free reactive thiol is then allowed to react with the submerged epoxy-activated nanofiber mat in the presence of a salting out buffer e.g. sodium sulfate to increase the coating of the Protein A on the surface of the fibers and as a result increasing the local concentration of Protein A at the surface of the fiber which increases the amount of Protein A conjugated onto the nanofiber mat [See Thermo Fisher Application Notes: https://www.thermofisher.com1

Comparative Example 1.

Vinyl functionalized Example 2 is tested for saturated binding capacity as is, i.e. without plasma or solution-based conjugation with Protein A. The saturated binding capacity, SBC, can be measured by placing a 2.5 cm diameter disc of the membrane into a Swinnex filter holder and using an AKTA Pure™, elute a 10 mL solution of human IgG (0.2 mg/mL) in PBS buffer, pH 7.4 at 0.5 ml/min through the membrane. The filter is then washed with about ten bed volumes of the PBS buffer and then the human IgG is eluted from the membrane with 5 bed volumes of buffer (e.g. 0.1 M glycine at pH 3), quickly neutralized to about pH 7 to avoid denaturation and the concentration measured spectrophotometrically, using the formula below or by integration of the elution curve and comparing with a calibration curve (e.g. Hahn et. al. Journal of Chromatography A. 2005, 1093, 98):

Protein concentration

Protein concentration (mg/ml): Absorbance xdilution x (Molecular weight / Molar extinction coefficient)

Results of saturated binding capacity measurements on functionalized nanofiber surfaces are shown in Table III indicating the performance increase of capability of the total structure to bind when exposed to protein A in an atmospheric plasma or solution-based process.

Table III.

Functionalized Coated Vinyl Functional SBC / membrane volume

Example Example Agent mg/cm³

Comparative 1 1 HEMA 1.4

1 1 GMA 4.0

2 1 GMA 3.7

3 2 HEMA 4.5

4 2 HEMA 4.4

5 1 GMA 32.4 Functionalized Example 6

A vinyl -functionalized nanoweb of Example 1 was conjugated with Protein A supplied by the Repligen Corporation of Waltham, MA. This type of Protein A binds to the vinyl-functionalized nanoweb via an amine group. The successful conjugation was demonstrated by the binding of IgG to the Protein A-functionalized nanoweb, as demonstrated by the results in Table IV. The capacity testing was performed as described above in the paragraph after comparative Example 1.

Table IV

Claims

1. A method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb; said method comprising steps of: a) exposing said nanoweb and a vinyl monomer to an atmospheric plasma to produce a coated nanoweb; and b) exposing said coated nanoweb and an aerosol comprising a molecule capable of forming covalent bonds to an atmospheric plasma.

2. The method of claim 1 in which the nanoweb is not a carbohydrate polymer.

3. The method of claim 1 or claim 2 in which the nanoweb comprises PVDF, polyamide, polyethersulfone, polyethylene, polypropylene, polyester or polyimide.

4. The method of any preceding claim in which the vinyl monomer comprises at least one epoxy group.

5. The method of any preceding claim in which the molecule capable of forming covalent bonds comprises molecules that include at least one of hydroxyl, amine or thiol groups.

6. The method of any preceding claim in which the aerosol comprises a non-volatile buffer.

7. The method of any preceding claim in which the vinyl monomer comprises an ester or ether of: (i) an epoxy alcohol, or (ii) an acetylenic alcohol.

8. The method of any preceding claim in which the molecule is capable of binding proteins to an active site.

9. The method of any preceding claim in which said molecule comprises at least one protein. The method of claim 9 in which the protein is capable of binding other proteins to an active site. A method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb; said method comprising steps of: a) exposing said nanoweb and a vinyl monomer to an atmospheric plasma to produce a coated nanoweb; and b) exposing said coated nanoweb to a molecule capable of forming covalent bonds. he method of claim 11 in which the nanoweb is not a carbohydrate polymer. he method of claim 11 or 12 in which the nanoweb comprises PVDF, polyamide, polyethersulfone, polyethylene, polypropylene, polyester or polyimide. The method of any of claims 11 to 13 in which the vinyl monomer comprises at least one epoxy group. The method of any of claims 11 to 14 in which the molecule capable of forming covalent bonds comprises molecules that include at least one of hydroxyl, amine or thiol groups. The method of any of claims 11 to 15 in which the vinyl monomer comprises an ester or ether of: (i) an epoxy alcohol, or (ii) an acetylenic alcohol. A method for functionalizing a nanoweb having mean flow pore size from 0.1 and 5 pm and a porosity from 40 to 90 volume % to produce a functionalized nanoweb comprising a protein; said method comprising steps of: a) exposing said nanoweb, a first vinyl monomer and a second vinyl monomer to an atmospheric plasma to produce a coated nanoweb; and b) exposing said coated nanoweb to a molecule capable of forming covalent bonds. The method of claim 17 in which the nanoweb is not a carbohydrate polymer.

19. The method of claim 17 or 18 in which the nanoweb comprises PVDF, polyamide, polyethersulfone, polyethylene, polypropylene, polyester or polyimide.

20. The method of any of claims 17 to 19 in which the first vinyl monomer comprises at least one epoxy group.

21. The method of any of claims 17 to 20 further comprising a second vinyl monomer which comprises at least one hydrophilic group

22. The method of any of claims 17 to 21 in which the molecule capable of forming covalent bonds comprises molecules that include at least one of hydroxyl, amine or thiol groups.

23. The method of any of claims 17 to 22 in which the aerosol comprises a non-volatile buffer.

24. The method of any of claims 17 to 23 in which the first vinyl monomer comprises an ester or ether of: (i) an epoxy alcohol, or (ii) an acetylenic alcohol.

25. The method of claim 17 to 24 in which said molecule comprises at least one protein.

26. The method of claim 25 in which the protein is capable of binding other proteins to an active site.

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