WO2024062258A1 - Functionalised polysaccharide bead preparation - Google Patents

Functionalised polysaccharide bead preparation Download PDF

Info

Publication number
WO2024062258A1
WO2024062258A1 PCT/GB2023/052461 GB2023052461W WO2024062258A1 WO 2024062258 A1 WO2024062258 A1 WO 2024062258A1 GB 2023052461 W GB2023052461 W GB 2023052461W WO 2024062258 A1 WO2024062258 A1 WO 2024062258A1
Authority
WO
WIPO (PCT)
Prior art keywords
beads
solvent
various embodiments
polysaccharide
alkyl
Prior art date
Application number
PCT/GB2023/052461
Other languages
French (fr)
Inventor
Davide CALIFANO
Ferdinando RADICE
Original Assignee
Naturbeads Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Naturbeads Ltd filed Critical Naturbeads Ltd
Publication of WO2024062258A1 publication Critical patent/WO2024062258A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
    • C08L1/08—Cellulose derivatives
    • C08L1/26—Cellulose ethers
    • C08L1/28—Alkyl ethers
    • C08L1/288—Alkyl ethers substituted with nitrogen-containing radicals
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B11/00—Preparation of cellulose ethers
    • C08B11/02—Alkyl or cycloalkyl ethers
    • C08B11/04—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals
    • C08B11/14—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals with nitrogen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/78—Cellulose

Definitions

  • the present disclosure relates generally to methods for preparing polysaccharide beads, and more particularly, to methods for preparing functionalised polysaccharide beads, for example functionalised cellulose beads.
  • the functionalised polysaccharide beads are suitable for, but not limited to, use in cell culture.
  • the functionalised polysaccharide beads are well suited for use in the culture of cells for purposes such as the production of biopharmaceuticals and food products while having improved environmental characteristics.
  • the present disclosure also provides functionalised beads obtained by the inventive methods, uses thereof, and methods for attaching cells thereto.
  • Biopolymers such as polysaccharides are an important development in the reduction of the environmental footprint of consumer and industrial products.
  • Biopolymers and biopolymer particles such as beads are often biodegradable as well as being derived from renewable and sustainable raw materials.
  • Cellulose for example, is the most abundant biopolymer on Earth and can be obtained from many sources, including waste. Products made from biopolymers can thus benefit from closed-loop production processes as part of a circular economic model.
  • Cell culture is a critical and ubiquitous method in research and industry for the production of proteins, antibodies, vaccines and the like.
  • Mammalian cell culture is particularly favoured in the ‘biotech’ industry due to its ability to propagate human viruses, express monoclonal antibodies, incorporate necessary post-translational modifications and achieve correct protein folding.
  • mammalian cell cultures are typically slow growing and low yielding, particularly compared to bacterial cultures.
  • industrial-scale cell culture requires the use of extremely large culture volumes to produce the necessary quantities of the desired product, for instance to produce kilogram quantities of a therapeutic monoclonal antibody.
  • Cultured meat which may also be referred to variously as “healthy meat”, “slaughter-free meat”, “in vitro meat”, “vat-grown meat”, “lab-grown meat”, “cell-based meat”, “clean meat”, “cultivated meat” or “synthetic meat”, is a meat-like product produced by in vitro cultures of animal cells using cell culture and tissue engineering techniques that were originally developed for biotechnology and medicine. Cultured meat has been proposed as an alternative to meat obtained by conventional farming and slaughter as a way to address the environmental impact of meat production, animal welfare, food security, and human health.
  • Microcarrier culture is one way in which the volume of cell culture per unit mass of cells produced can be reduced.
  • Microcarriers are typically spherical particles that provide a substrate within the cell culture upon and/or in which mono- and/or multi-layers of cells can grow. Each microcarrier particle can carry several hundred cells such that expansion capacity of the culture can be multiplied at least several times.
  • Microcarriers may be produced from synthetic or natural materials. In most cases, the microcarrier material requires chemical modification to provide favourable interactions (e.g. electrostatic interactions) that facilitate binding of the cells to the microcarrier. Microcarriers must withstand the rigours of the culture environment, for example in a stirred bioreactor. In particular, it is desirable that microcarriers do not contaminate the culture with debris such as fragments of degraded microcarrier material. It is also desirable that microcarriers do not comprise small molecules that could leach into the culture and which may be prohibited, regulated, and/or toxic. The production of food products is strictly regulated and so any production method in this field must be controllable to an extremely high standard.
  • microcarriers produced from sustainable, renewable materials without the use of toxic, regulated, and/or prohibited reagents that are suitable for use broadly in cell culture applications, particularly the production of biopharmaceuticals and food products such as cultured meat.
  • the present disclosure meets this need with the various aspects and embodiments defined herein.
  • the present disclosure provides a method for preparing functionalised cellulose beads, said method comprising the steps of:
  • A is an optionally substituted epoxide group
  • L is a linker group
  • B is a quaternary ammonium group
  • X is a counterion.
  • the present disclosure provides a method for preparing functionalised polysaccharide beads, said method comprising the steps of:
  • polysaccharide beads are prepared by extruding a dispersed phase into an anti-solvent to form beads of the polysaccharide, wherein the dispersed phase comprises the polysaccharide in a solvent, and wherein each of the solvent and anti-solvent comprises water; optionally wherein extruding the dispersed phase into an anti-solvent to form beads of the polysaccharide comprises extruding the dispersed phase through a fluid medium into a mould and then contacting the extruded dispersed phase with the anti-solvent.
  • the present disclosure provides a method for preparing functionalised polysaccharide beads, said method comprising the steps of:
  • step (ii) reacting the product of step (i) with a salt represented by Formula (I): A-L-B .X
  • polysaccharide beads are prepared by: a. a membrane emulsification of a dispersed phase into a continuous phase wherein the dispersed phase comprises the polysaccharide in a solvent, and wherein passing the dispersed phase through the membrane forms an emulsion of the polysaccharide in the continuous phase; and b. a phase inversion with an anti-solvent to form beads of the polysaccharide; wherein each of the solvent and anti-solvent comprises water.
  • the present disclosure provides a method for attaching cells to functionalised beads, wherein the method comprises preparing functionalised beads by any of the methods described herein and contacting the functionalised beads with one or more cells, preferably wherein the functionalised beads are contacted with one or more cells during cell culture.
  • the present disclosure provides functionalised beads prepared by the methods described herein.
  • Features described herein in the context of the methods are also therefore applicable to the functionalised beads obtained by the methods.
  • the present disclosure provides for the use of the functionalised beads obtained by the methods described herein in cell culture.
  • the functionalised beads obtained by the methods described herein in cell culture.
  • Features described herein in the context of the methods are also therefore applicable to the use.
  • Figure 1 is a schematic representation of an embodiment wherein the polysaccharide beads are prepared by extruding a dispersed phase into an anti-solvent.
  • Figure 2 contains a schematic representation of an embodiment wherein the polysaccharide beads are prepared by membrane emulsification ( Figure 2(a)), and a representation of a further embodiment wherein the emulsion is cooled as described herein ( Figure 2(b)).
  • Figure 3 shows the change in charge density (expressed as milliequivalents per gram (meq/g) as defined herein) as well as the dry weight (expressed as the percentage of dry cellulose per unit of hydrated cellulose mass) with reaction time (expressed in hours) as determined for the functionalisation of cellulose beads with GTMAC according to Examples 2 and 3 of the present disclosure.
  • Figure 4 shows z-stack projections of images of stained C2C12 cells attached to functionalised cellulose beads prepared according to the Examples described herein, as well as unmodified cellulose beads and a commercial microcarrier. Bright white spots represent live cells stained with fluorescein diacetate. The cells were stained and images taken after four days of culture.
  • Figure 5 shows the degree of cell attachment determined by cell counting for the functionalised cellulose beads according to the Examples, unmodified cellulose beads and a commercial microcarrier after 1 , 4, and 7 days of culture according to the Examples herein.
  • Figure 5(a) compares the number of cells per bead
  • Figure 5(b) compares the number of cells per unit surface area (cells/mm 2 ). Error bars represent means of standard deviations determined from three biological replicates (cell cultures). For each biological replicate cells were counted on 100 beads, with the exception of the large CCBs where the cells on 25 beads were counted.
  • the term "about” modifying the quantity of a component refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making concentrates, mixtures or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the materials employed, or to carry out the methods; and the like.
  • the term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about”, the claims include equivalents to the quantities.
  • the term “at least” includes the end value of the range that is specified. For example, “at least 10 cm” includes the value 10 cm.
  • wt% means “weight percentage” as the basis for calculating a percentage. Unless indicated otherwise, all % values are calculated on a weight basis, and are provided with reference to the total weight of the product in which the substance is present.
  • % water in the solvent of the dispersed phase refers to the wt% water based on the total weight of the solvent.
  • % polysaccharide in the dispersed phase refers to wt% polysaccharide based on the total weight of the dispersed phase.
  • w/v means “weight by volume” as the basis for calculating a percentage.
  • v/v means “volume by volume” as the basis for calculating a percentage.
  • substantially free means no more than trace amounts, i.e. the amount of the substance(s) concerned is negligible. In various embodiments, “substantially free” means no more than 1000 ppm, preferably no more than 100 ppm, more preferably no more than 10 ppm, even more preferably no more than 1 ppm of the substance(s) concerned.
  • the disclosure includes, where appropriate, all enantiomers and tautomers of the compounds disclosed herein.
  • a person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics.
  • the corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
  • Some of the compounds disclosed herein may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms.
  • the present disclosure contemplates the use of all the individual stereoisomers and geometric isomers of those compounds, and mixtures thereof.
  • the terms used in the claims encompass these forms.
  • hydrocarbyl refers to a group comprising at least C and H. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon.
  • Non-limiting examples of such hydrocarbyls are alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, and isomeric forms thereof; cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloocytyl, 2-methylcyclopentyl, 2,3-dimethyl- cyclobutyl, 4-methylcyclobutyl, 3-cyclopentylpropyl, and the like; cyclo
  • the hydrocarbyl group may be an aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group.
  • the term “hydrocarbyl” refers to a group having carbon atoms directly attached to the remainder of the molecule, wherein the group consists of carbon and hydrogen atoms.
  • the hydrocarbyl group may be an aliphatic or aromatic group.
  • non-limiting examples of such hydrocarbyls are alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, and isomeric forms thereof; cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloocytyl, 2-methylcyclopentyl, 2,3-dimethyl-cyclobutyl, 4-methylcyclobutyl, 3- cyclopentyl propyl, and the like
  • alkyl includes both saturated straight chain and branched alkyl groups which may be substituted (mono- or poly-) or unsubstituted.
  • the alkyl group is a C1.20 alkyl group, more preferably a C1.15, more preferably still a C1.12 alkyl group, more preferably still, a Ci-e alkyl group, more preferably a C1.3 alkyl group.
  • Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl.
  • Suitable substituents include, for example, one or more groups selected from OH, O-alkyl, halogen, NH 2 , NH-alkyl, N-(alkyl) 2 , CF 3 , NO 2 , CN, COO-alkyl, COOH, CONH 2 , CO-NH-alkyl, CO-N(alkyl)2, SO2-alkyl, SO2NH2 and SO2-NH-alkyl.
  • Preferable substituents include one or more groups selected from OH, O-alkyl, halogen other than fluorine, NH2, NH- alkyl, N-(alkyl) 2 , NO 2 , CN, COO-alkyl, COOH, CONH 2 , CO-NH-alkyl, CO-N(alkyl) 2 , SO 2 -alkyl, SO2NH2 and SO2-NH-alkyl.
  • substituents include one or more groups selected from OH, O-alkyl, chlorine, NH 2 , NH-alkyl, N-(alkyl) 2 , NO 2 , CN, COO-alkyl, COOH, CONH 2 , CO-NH-alkyl, CO-N(alkyl)2, SO2-alkyl, SO2NH2 and SO2-NH-alkyl.
  • substituents include one or more groups selected from OH, O-alkyl, NH2, NH-alkyl, N-(alkyl)2, NO 2 , CN, COO-alkyl, COOH, CONH 2 , CO-NH-alkyl, CO-N(alkyl) 2 .
  • aryl refers to a Ce-12 aromatic group which may be substituted (mono- or poly-) or unsubstituted. Typical examples include phenyl and naphthyl etc. Suitable substituents include, for example, one or more groups selected from OH, O-alkyl, halogen, NH 2 , NH-alkyl, N-(alkyl) 2 , CF 3 , NO 2 , CN, COO-alkyl, COOH, CONH 2 , CO-NH-alkyl, CO- N(alkyl) 2 , SO 2 -alkyl, SO 2 NH 2 and SO 2 -NH-alkyl.
  • aralkyl is used as a conjunction of the terms alkyl and aryl as given above.
  • Cyclic alkyl groups may be referred to as “cycloalkyl” and include those with 3 to 10 carbon atoms having single or multiple fused rings.
  • Non-limiting examples of cycloalkyl groups include adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
  • alkenyl refers to both straight and branched carbon chains which have at least one carbon-carbon double bond.
  • alkenyl groups may include C2- C12 alkenyl groups.
  • alkenyl includes C2-C10, C2-C8, C2-C6 or C2-C4 alkenyl groups.
  • the number of double bonds is 1-3; in another embodiment of alkenyl, the number of double bonds is one.
  • Other ranges of carbon-carbon double bonds and carbon numbers are also contemplated depending on the location of the alkenyl moiety on the molecule.
  • “C2-C -alkenyl” groups may include more than one double bond in the chain.
  • Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1- methyl-ethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1 -propenyl, 2-methyl-1-propenyl, 1- methyl-2-propenyl, 2-methyl-2-propenyl; 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1- methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2- butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1 ,1- dimethyl-2-propenyl, 1 ,2-dimethyl-1-propenyl, 1 ,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl,
  • Heteroaryl refers to a monovalent aromatic group of from 1 to 15 carbon atoms, preferably from 1 to 10 carbon atoms, having one or more oxygen, nitrogen, and sulfur heteroatoms within the ring, preferably 1 to 4 heteroatoms, or 1 to 3 heteroatoms.
  • the nitrogen and sulfur heteroatoms may optionally be oxidized.
  • Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple fused rings provided that the point of attachment is through a heteroaryl ring atom.
  • heteroaryls examples include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, indolyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinnyl, furanyl, thiophenyl, furyl, pyrrolyl, imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyrazolyl benzofuranyl, benzothiophenyl, imidazopyridyl, imidazopyrimidyl, or pyrrolopyrimidyl.
  • Heteroaryl rings may be unsubstituted or substituted by one or more moieties as described for aryl above.
  • Alkoxy refers to alkyl-O-, wherein alkyl is as defined above.
  • Examples of Ci-Ce-alkoxy include, but are not limited to, methoxy, ethoxy, OCH2-C2H5, OCH(CHs)2, n-butoxy, OCH(CHs)- C2H5, OCH2-CH(CHS)2, OC(CHS)3, n-pentoxy, 1 -methylbutoxy, 2-methylbutoxy, 3- methylbutoxy, 1 ,1 -dimethylpropoxy, 1 ,2-dimethylpropoxy, 2,2-dimethyl-propoxy, 1- ethylpropoxy, n-hexoxy, 1 -methylpentoxy, 2-methylpentoxy, 3-methylpentoxy, 4- methyl pentoxy, 1 ,1-dimethylbutoxy, 1 ,2-dimethylbutoxy, 1 ,3-dimethylbutoxy, 2,2- dimethylbutoxy, 2,3-dimethylbutoxy, 3,3-d
  • the general inventive concept is centred on providing a method for preparing functionalised polysaccharide beads, for example functionalised cellulose beads, where the functionalised polysaccharide beads are suitable for use in applications in, but not limited to, cell culture, biomedicine, biomanufacture, pharmaceuticals, as well as food production such as the production of cultured meat and have improved environmental benefits.
  • the polysaccharide beads of the present disclosure are functionalised by reaction with compounds comprising quaternary ammonium groups, which alters the properties of the polysaccharide beads in order to enable or improve their suitability for use in one or more of the above-mentioned applications.
  • polysaccharide/cellulose beads are (i) contacted with a base; and in step (ii) the product of step (i) is reacted with a salt represented by Formula (I):
  • polysaccharide beads with quaternary ammonium moieties ensures cell attachment under a range of culture and process conditions such as pH, temperature, CO2 concentration, and stirring.
  • the quaternary ammonium moieties remain ionised over a broad pH range.
  • use of the beads as microcarriers in cell culture can protect the cells from shear forces in the culture (e.g. from stirring) and thus reduce stress on the cells and improve proliferation.
  • the functionalised beads of the present disclosure have notably been found to be mechanically robust even in the absence of cross-linking. This is in contrast to many commercially available microcarriers which are cross-linked materials.
  • the functionalised beads of the present disclosure can be produced from sustainable, renewable, and cost- effective materials such as cellulose.
  • functionalisation of the polysaccharide beads by the methods described herein can be performed in innocuous solvent systems (e.g. aqueous solvents) and with non-toxic reagents that do not lead to the production of harmful side-products.
  • the size of the beads may also be tuned over a wide range to be suitable for specific applications.
  • the functionalised beads described herein are particularly suitable for use as microcarriers in cell culture.
  • beads made of polysaccharides such as cellulose may be particularly suitable for food applications of cell culture such as cultured meat because cellulose is a non-toxic, naturally occurring polysaccharide that is safe to eat.
  • Cellulose from plant material constitutes insoluble dietary fibre in natural food products, for example.
  • the methods for preparing the functionalised beads described herein allow the use of mild, aqueous processes and nonharmful reagents such that the functionalised beads may be used in downstream applications such as food culture without the need for extensive and costly purification before use.
  • polysaccharide beads for example cellulose beads.
  • Polysaccharides are biopolymers (polymers produced by living organisms, i.e. polymeric biomolecules). Polysaccharides are typically polymeric carbohydrate structures.
  • the polysaccharide may be, for example, starch, cellulose, chitin, and chitosan. Even more preferably, the polysaccharide is starch or cellulose. Most preferably, the polysaccharide is cellulose.
  • Cellulose is a linear polymer made up of p-D-glucopyranose units covalently linked with 1 ⁇ 4 glycosidic bonds.
  • Cellulose may be obtained from many different sources and the present disclosure is not necessarily limited as to the origin, form, or other characteristics of the cellulose.
  • Cellulose is typically obtained from plant sources, for example from virgin or recycled wood pulp.
  • Pulp is a lignocellulosic fibrous material prepared by chemically or mechanically separating cellulose fibers from wood, fiber crops, waste paper, or rags. Cellulose may be obtained from virgin sources or advantageously from recycled sources.
  • the cellulose is selected from the group consisting of virgin, recycled, pulp, and microcrystalline cellulose, and combinations thereof.
  • the cellulose is virgin cellulose.
  • the cellulose is recycled cellulose.
  • the cellulose is pulp cellulose.
  • the cellulose is microcrystalline cellulose.
  • Microcrystalline cellulose is typically made from high-grade, purified wood or cotton cellulose. Hydrolysis is used to remove amorphous cellulose until the microcrystalline form remains. With its amorphous cellulose portions removed, it becomes an inert, white, free- flowing powder. It can be processed in a number of ways, for example through reactive extrusion, steam explosion, and acid hydrolysis.
  • An example of a commercially available MCC is Avicel® produced by DuPont.
  • bead refers to a discrete entity with defined size and shape that does not have any dimension or dimensions substantially greater than any other dimension and which may be described as “spherical” or “substantially spherical”.
  • a person skilled in the art is readily able to identify a “bead” as opposed to e.g. an elongate particle.
  • the term “bead” as used herein does not include elongate particles such as (nano/micro)fibres, (nano/micro)fibrils and the like, nor does said term include flat/sheet-like structures such as membranes and the like.
  • embodiments referring to “beads” as such apply equally to each of the aspects of the present disclosure unless specifically indicated and include any polysaccharide bead as encompassed by the present disclosure.
  • the beads are approximately spherical. Approximately spherical beads may be advantageous in certain applications, for example in biomanufacture and food production, where such a shape can facilitate industrial processing and recovery processes such as filtration.
  • the size of the beads of the present disclosure is not limited, and the skilled person will be able to select sizes according to a desired application.
  • the size of the beads may be readily identified by a person skilled in the art, for example, using an optical microscope image and image analysis software with a suitable detection algorithm (e.g. Imaged using an edge detection algorithm), laser diffraction with commercially available equipment such as Mastersizer from Malvern Panalytical (e.g. Mastersizer 3000), with an appropriately sized sieve, or by using a caliper.
  • the beads may have a diameter of at least about 1 pm. In some embodiments, the beads may have a diameter of at least about 10 pm. In some embodiments, the beads may have a diameter of at least about 25 pm. In some embodiments, the beads may have a diameter of at least about 50 pm. In some embodiments, the beads may have a diameter of at least about 80 pm. In some embodiments, the beads may have a diameter of at least about 100 pm.
  • the beads may have a diameter of less than about 5 mm. In some embodiments, the beads may have a diameter of less than about 4 mm. In some embodiments, the beads may have a diameter of less than about 3 mm. In some embodiments, the beads may have a diameter of less than about 2 mm. In some embodiments, the beads may have a diameter of less than about 1 mm.
  • the beads have a diameter of from about 1 pm to about 3 mm. In various embodiments, the beads have a diameter of from about 10 pm to about 3 mm. In various embodiments, the beads have a diameter of from about 25 pm to about 3 mm. In various embodiments, the beads have a diameter of from about 50 pm to about 3 mm. In various embodiments, the beads have a diameter of from about 80 pm to about 3 mm. [0057] In various embodiments, the beads have a diameter of from about 0.1 mm to about 3 mm. In various embodiments, the beads have a diameter of from about 0.15 mm to about 3 mm. In various embodiments, the beads have a diameter of about 0.2 mm to about 3 mm.
  • the beads may have a diameter greater than or equal to about 0.2 mm. In various embodiments, the beads may have a diameter of from about 0.2 mm to about 3 mm, from about 1 to about 3 mm or from about 1 to about 2 mm.
  • Polysaccharide beads may be obtained from various methods of production in a form wherein said beads are wetted or immersed in a solvent such as water. Such beads may be referred to as “wet” beads and may be provided in this form for further use. Alternatively, beads may be subsequently dried to provide “dry beads”.
  • the unmodified beads i.e. the starting material in the methods of the present disclosure have not previously been dried.
  • the functionalised beads obtained by the methods of the present disclosure can be dried and then re-suspended, e.g. in water, such that the dried functionalised beads fully regain their prior hydration/solvation and the size, shape, and structure of the beads are unaffected by the drying and rehydration/resolvation steps.
  • the beads are in the form of a hydrogel for step (i) of the methods of the present disclosure.
  • a hydrogel is defined by IIIPAC as a gel in which the swelling agent is water.
  • the term “hydrogel” means a non-fluid polymer network (i.e. formed by the polysaccharide) that is expanded throughout its whole volume by a fluid, namely water.
  • Polysaccharide beads in the form of a hydrogel may be preferred as they can provide a higher effective surface area for reaction with base according to step (i) of the functionalisation methods described herein.
  • an increased effective surface area leads to an increased density of functional groups for subsequent functionalisation with quaternary ammonium groups in step (ii) of the methods described herein.
  • the enhanced effective surface area of the functionalised beads may lead to an enhanced degree of cell attachment and thus higher cell culture densities.
  • good cell growth, increased cell densities and/or increased yields in culture may be achieved with beads in the form of a hydrogel compared to non-hydrogel beads such as solid beads.
  • the polysaccharide beads of the present disclosure are mechanically robust, even in the absence of additional cross-linking.
  • cross-linking refers to the formation of additional bonds other than the bonds linking monomer units in the polysaccharide by reaction with a cross-linking agent, where said additional bonds may be formed between functional groups in the same polysaccharide chain or between functional groups in neighbouring (i.e. different) polysaccharide chains.
  • the beads of the present disclosure are not cross-linked.
  • the present disclosure is not limited in this regard; for some applications, it may be desired to cross-link the polysaccharide in the beads of the present disclosure.
  • the beads of the present disclosure are cross-linked.
  • the polysaccharide beads are cross-linked by reaction with a cross-linking agent.
  • Cross-linking agents are typically multi-functional molecules that comprise more than one reactive group capable of reacting with functional groups on the polysaccharide.
  • the functional groups on the polysaccharide that react with the cross-linking agent are hydroxyl groups present in the saccharide monomers of the polysaccharide, for example secondary hydroxyl groups.
  • the cross-linking agent is selected from the group consisting of formaldehyde; methylolated nitrogen compounds such as dimethylolurea, dimethylolethyleneurea, and dimethylolimidazolidone; dicarboxylic acids such as maleic acid; dialdehydes such as glyoxal and glutaraldehyde; diepoxides; diioscyanates; divinyl compounds such as divinyl sulfone, dihalogen containing compounds such as dichloroacetone and 1 ,3-dichloropropan-2-ol, and halohydrins such as epichlorohydrin.
  • formaldehyde methylolated nitrogen compounds such as dimethylolurea, dimethylolethyleneurea, and dimethylolimidazolidone
  • dicarboxylic acids such as maleic acid
  • dialdehydes such as glyoxal and glutaraldehyde
  • diepoxides diioscyanates
  • the cross-linking agent is selected from the group consisting of methylolated nitrogen compounds such as dimethylolurea, dimethylolethyleneurea, and dimethylolimidazolidone; dicarboxylic acids such as maleic acid; dialdehydes such as glyoxal and glutaraldehyde; diepoxides; diioscyanates; divinyl compounds such as divinyl sulfone, dihalogen containing compounds such as dichloroacetone and 1 ,3-dichloropropan-2-ol, and halohydrins such as epichlorohydrin.
  • dicarboxylic acids such as maleic acid
  • dialdehydes such as glyoxal and glutaraldehyde
  • diepoxides diioscyanates
  • divinyl compounds such as divinyl sulfone, dihalogen containing compounds such as dichloroacetone and 1 ,3-dichloropropan-2-ol, and hal
  • the cross-linking agent is selected from the group consisting of methylolated nitrogen compounds such as dimethylolurea, dimethylolethyleneurea, and dimethylolimidazolidone; dicarboxylic acids such as maleic acid; dialdehydes such as glyoxal and glutaraldehyde; diepoxides; diioscyanates; divinyl compounds such as divinyl sulfone. of beads with base
  • step (i) of the methods of the present disclosure the beads are contacted with a base.
  • base takes its usual meaning in the chemical arts, for instance a chemical species or molecular entity having an available pair of electrons capable of forming a covalent bond with a proton or with a vacant orbital of some other species.
  • the base is not limited, and the skilled person will be able to select bases appropriate to the polysaccharide beads being contacted with said base.
  • the propensity of a hydroxyl group in a polysaccharide to be removed by a base can be measured and/or predicted in terms of its pK a .
  • the definition of pK a , the calculation/measurement thereof, and the use of said parameter in relation to acid/base reactions is specifically part of the common general knowledge of a person skilled in the art of the present disclosure.
  • a lower pK a corresponds to a stronger acid, i.e. where the proton in question is more weakly held and thus more easily removed by a base.
  • a person skilled in the chemical arts will understand that hydroxyl groups are typically considered weak acids.
  • secondary hydroxyl groups will typically be weaker acids (have a higher pK a ) than primary hydroxyl groups.
  • the primary hydroxyl groups in cellulose are believed to have a pK a of approximately 12.5 in water; however, this may vary, for example depending on the degree of deprotonation of the cellulose polymer and/or the source of the cellulose.
  • Basicity may be measured/predicted in terms of the pKa of the corresponding conjugate acid (pK a H).
  • the base of the present disclosure may have a pK a H of at least about 12, at least about 13, at least about 14, or at least about 15.
  • the base is a strong base. Strong bases are generally considered to be those that are fully dissociated in aqueous solution.
  • a strong base may be a base having a pK a n greater than the pKa of at least one hydroxyl group on the saccharide repeat unit of the polysaccharide.
  • a strong base as used in the present disclosure has a pK a n of at least 12.5.
  • the base is a hydroxide salt.
  • the base is preferably a hydroxide salt that is soluble in said aqueous solution under the conditions of the reaction as described herein.
  • the base may be a Group I hydroxide.
  • the base may be potassium hydroxide or sodium hydroxide.
  • the base is sodium hydroxide.
  • hydroxide salts may also be advantageous for certain applications because they form non-toxic water upon deprotonation of the polysaccharide hydroxyl groups.
  • alkali metal ions such as sodium from sodium hydroxide will typically contribute non-toxic dissolved salts (e.g. NaCI) in combination with counterions such as chloride, which salts are either allowable in the final product or which can be readily removed from the product such as by washing and sieving as discussed in more detail below.
  • the base will dissolved in an appropriate solvent prior to contacting the beads with the basic solution.
  • the method is carried out in an aqueous solvent, and thus in further embodiments the base is dissolved in said aqueous solvent prior to contacting the beads with the solution of the base in the aqueous solvent.
  • the beads may be (re-)suspended in said solution.
  • the beads are in the form of a hydrogel and contacted with the basic solution.
  • the concentration of the base in solution prior to contacting the beads is not limited and the skilled person will be able to select an appropriate concentration based on their common general knowledge.
  • the base is present in solution at a concentration of at least about 0.02 M, at least 0.05 M, or at least 0.1 M prior to contacting said solution with the beads according to step (i).
  • the method is carried out in aqueous solvent and the base is present in solution in said aqueous solvent at a concentration of at least about 0.02 M, at least 0.05 M, or at least 0.1 M prior to contacting said solution with the beads according to step (i).
  • the method is carried out in aqueous solvent and the base is a hydroxide salt that is present in solution in said aqueous solvent at a concentration of at least about 0.02 M, at least 0.05 M, or at least 0.1 M prior to contacting said solution with the beads according to step (i).
  • the base is present in solution at a concentration of up to 0.2 M prior to contacting said solution with the beads according to step (i).
  • the base is present in solution at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i).
  • the method is carried out in aqueous solvent and the base is present in solution in said aqueous solvent at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i).
  • the base has a pK a n of at least 12.5 and is present in solution at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i).
  • the method is carried out in aqueous solvent, the base has a pK aH of at least 12.5 and said base is present in solution in said aqueous solvent at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i).
  • the method is carried out in aqueous solvent and the base is a hydroxide salt that is present in solution in said aqueous solvent at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i).
  • step (ii) of the methods of the present disclosure the product of step (i) is reacted with a salt represented by Formula (I):
  • A is an optionally substituted epoxide group
  • L is a linker group
  • B is a quaternary ammonium group
  • X is a counterion.
  • linker group refers to any moiety covalently attached to both A and B.
  • the linker group may be an optionally substituted hydrocarbyl group as defined hereinabove.
  • the linker group is an unsubstituted hydrocarbyl group as defined hereinabove.
  • the linker group is an optionally substituted aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H and optionally substituted alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H and methyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1 .
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H and optionally substituted aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H and optionally substituted phenyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is H. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, m is 1 and each R 4 and R 5 is H.
  • suitable substituents include one or more groups selected from OH, O-alkyl, NH2, NH-alkyl, N-(alkyl)2, NO2, CN, COO-alkyl, COOH, CONH2, CO-NH-alkyl, CO-N(alkyl) 2 .
  • the linker group may be a polyether group such as those derived from ethylene glycol or a polyethylene glycol.
  • the linker group may have the formula [O-(CH 2 ) n ]o wherein n is an integer from 1 to 5 and o is an integer from 1 to 10.
  • n is an integer from 1 to 4, 1 to 3, or 1 to 2
  • o is an integer from 1 to 10.
  • n is 2 and o is an integer from 1 to 10.
  • n is an integer from 1 to 5 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • n is an integer from 1 to 5 and o is 1.
  • n is an integer from 1 to 4, 1 to 3, or 1 to 2
  • o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • n is 2 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • Quaternary ammonium cations are positively charged polyatomic ions with the structure N + R 1 R 2 R 3 R 4 wherein each R 1 , R 3 , R 3 , and R 4 is independently alkyl, aryl, and aralkyl.
  • the quaternary ammonium group B has the formula -N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aralkyl, and aryl.
  • each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl. In further embodiments, each R 1 , R 2 , and R 3 is independently alkyl. In yet further embodiments, each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, preferably C1-C3 alkyl. For instance, each R 1 , R 2 , and R 3 may be independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In some embodiments, each R 1 , R 2 , and R 3 is methyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted phenyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted phenyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted phenyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci- Ce alkyl and aryl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1 .
  • m is 1 and each R 4 and R 5 is H.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted phenyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently alkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently alkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted phenyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, methyl, ethyl, n- propyl, and isopropyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted phenyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, methyl, ethyl, n- propyl, and isopropyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n- propyl and isopropyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted phenyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is methyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is methyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is methyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted Ci-e alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is methyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted phenyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is methyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H and optionally substituted C1.3 alkyl
  • the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is methyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is methyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10, each R 4 and R 5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N + R 1 R 2 R 3 wherein each R 1 , R 2 , and R 3 is methyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the epoxide group A is substituted with 1 , 2 or 3 alkyl groups, preferably wherein each alkyl group is independently selected from Ci-Ce alkyl, more preferably wherein each alkyl group is independently selected from C1-C3 alkyl. More preferably, the epoxide group A is unsubstituted.
  • counterion means an ion that accompanies another ionic species in order to maintain electric neutrality.
  • the counterion X is a monovalent anion.
  • X is a halide ion.
  • X is a halide ion other than fluoride. More preferably, X is a chloride ion.
  • the salt represented by Formula (I) is a salt represented by Formula (la):
  • each of R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl, and X' is halide.
  • each of R 1 , R 2 , and R 3 is independently selected from Ci- Ce alkyl, aryl, and aralkyl.
  • L is the linker group as defined above.
  • each R 1 , R 2 , and R 3 is independently selected from alkyl and aryl. In various embodiments of the salt represented by Formula (la), each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, each R 1 , R 2 , and R 3 is independently alkyl. In yet further embodiments, each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, preferably C1-C3 alkyl.
  • each R 1 , R 2 , and R 3 may be independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In some embodiments, each R 1 , R 2 , and R 3 is methyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl.
  • the salt represented by Formula (I) is a salt represented by Formula (lb): wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl, each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and X' is halide.
  • each R 1 , R 2 , and R 3 is independently selected from alkyl and aryl.
  • each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R 1 , R 2 , and R 3 is independently alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl
  • each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl
  • each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl
  • each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and m optionally substituted aryl, and each R 1 , R 2 , and R 3 is methyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the linker group may be a polyether group such as those derived from ethylene glycol or a polyethylene glycol.
  • the linker group may have the formula [O-(CH2)n]o wherein n is an integer from 1 to 5 and o is an integer from 1 to 10.
  • n is an integer from 1 to 4, 1 to 3, or 1 to 2
  • o is an integer from 1 to 10.
  • n is 2 and o is an integer from 1 to 10.
  • n is an integer from 1 to 5 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • n is an integer from 1 to 5 and o is 1.
  • n is an integer from 1 to 4, 1 to 3, or 1 to 2
  • o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • n is 2 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • the salt represented by Formula (I) is a salt represented by Formula (Ic): wherein each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl, and X’ is halide.
  • each R 1 , R 2 , and R 3 is independently selected from alkyl and aryl.
  • each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl.
  • each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl.
  • each R 1 , R 2 , and R 3 is independently alkyl. In further embodiments, each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl. In yet further embodiments, each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl. In yet further embodiments, each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In any of the foregoing, at least two of R 1 , R 2 , and R 3 may be different. In other embodiments, each of R 1 , R 2 , and R 3 are different. In yet further embodiments, each R 1 , R 2 , and R 3 is methyl.
  • X- is preferably chloride (Cl’).
  • the salt represented by Formula (I) is glycidyltrimethylammonium chloride (GTMAC; also known as 2,3- epoxypropyl)trimethylammonium chloride).
  • GTMAC glycidyltrimethylammonium chloride
  • Said salt is commercially available (CAS Number: 3033-77-0), for example from Sigma-Aldrich (Merck KGaA).
  • the optionally substituted epoxide group A reacts in step (ii) with the deprotonated hydroxyl groups of the beads formed by step (i).
  • This type of reaction is commonly referred to as a ring-opening etherification and results in in the quaternary ammonium group being covalently attached to the polysaccharide beads via the linker group L and a residue derived from reaction of the epoxide group with the deprotonated hydroxyl group of the polysaccharide.
  • An exemplary reaction where the salt is glycidyltrimethylammonium chloride and the polysaccharide is cellulose is illustrated in Scheme 2:
  • the ring-opening etherification mechanism means that the beads are functionalised with quaternary ammonium groups in a direct coupling reaction without condensation, i.e. without the release of leaving groups, for example, that would contribute additional species to the reaction mixture.
  • Functionalisation of beads without forming condensation products may be advantageous for various applications where such condensation products may be prohibited or regulated, e.g. in food products, and would thus need to be removed in additional purification steps that would reduce efficiency and increase costs of the process.
  • the salt of Formula (I), for example a salt of Formula (la), (lb), (Ic), or glycidyltrimethylammonium chloride is produced in situ.
  • a precursor compound is added to the product of step (i) such that the precursor reacts to form a salt of Formula (I) or embodiments thereof as discussed herein and then said salt thus formed proceeds to react with the deprotonated beads formed in step (i) to yield the functionalised beads of the present disclosure.
  • the salt represented by Formula (la) may be produced in situ by reacting the product of step (i) with a compound of Formula (Ila):
  • Y is chlorine.
  • X- is chloride.
  • X and Y are the same.
  • Y is chlorine and X' is chloride.
  • each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, aralkyl, and aryl. In various embodiments, each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl. [0122] In various embodiments of the compound of Formula (Ila), each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, each R 1 , R 2 , and R 3 is independently alkyl. In yet further embodiments, each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl, preferably C1-C3 alkyl.
  • each R 1 , R 2 , and R 3 may be independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In some embodiments, each R 1 , R 2 , and R 3 is methyl.
  • the linker group L is (CR 4 R 5 ) m wherein m is an integer from 1 to 10 and each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl
  • each R 1 , R 2 , and R 3 is independently selected from alkyl, aryl, and aralkyl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl
  • each R 1 , R 2 , and R 3 is independently selected from alkyl and aryl.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R 1 , R 2 , and R 3 is independently alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl
  • each R 1 , R 2 , and R 3 is independently selected from Ci-Ce alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl
  • each R 1 , R 2 , and R 3 is independently selected from C1-C3 alkyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl
  • each R 1 , R 2 , and R 3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • m is an integer from 1 to 10
  • each R 4 and R 5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R 1 , R 2 , and R 3 is methyl.
  • m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1.
  • m is 1 and each R 4 and R 5 is H.
  • the linker group may be a polyether group such as those derived from ethylene glycol or a polyethylene glycol.
  • the linker group may have the formula [O-(CH2)n]o wherein n is an integer from 1 to 5 and o is an integer from 1 to 10.
  • n is an integer from 1 to 4, 1 to 3, or 1 to 2; and o is an integer from 1 to 10.
  • n is 2 and o is an integer from 1 to 10.
  • n is an integer from 1 to 5 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • n is an integer from 1 to 5 and o is 1.
  • n is an integer from 1 to 4, 1 to 3, or 1 to 2; and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In various embodiments, n is 2 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
  • m is 1
  • each R 4 and R 5 is H
  • each R 1 , R 2 and R 3 is methyl
  • Y is chlorine
  • X' is chloride.
  • the compound of Formula (Ila) is (3-chloro- 2-hydroxypropyl)trimethylammonium chloride (‘CHTAC’), which is commercially available (CAS Number: 3327-22-8).
  • the base of step (i) may be a hydroxide salt, for example sodium hydroxide.
  • a hydroxide salt for example sodium hydroxide.
  • Scheme 3 shows the in situ production of the salt represented by Formula (I) from a compound of Formula (Ila) for the case wherein said salt is GTMAC, the compound of Formula (Ila) is CHTAC, and the base of step (i) is a hydroxide salt.
  • aqueous solvents are advantageous as they are generally considered to be environmentally friendly.
  • environmentally friendly is meant not harmful to the environment such that the solvent can be disposed of without the need for specialist equipment or process(es), i.e. non-toxic.
  • use of aqueous solvents may be preferable in applications where the presence of potentially toxic organic solvents is prohibited or strictly regulated, and which would thus necessitate additional purification steps.
  • the aqueous solvent is substantially free of organic solvents.
  • substantially free is defined above.
  • the beads are suspended in the aqueous solvent.
  • the base may be added to the aqueous solvent prior to suspension of the beads in the solution of the base in the aqueous solvent. In this way, good solvent accessibility to the beads is achieved and thus efficient deprotonation and functionalisation of said beads.
  • the reaction mixture may be mixed during reaction, for example in a stirred vessel.
  • the relative amounts of beads and each respective reactant are not limited.
  • the ratio of the mass of beads to the reaction solvent e.g. aqueous solvent
  • the ratio of the mass of beads to reaction solvent is not limited, although preferably the ratio of the mass of beads to reaction solvent is such that the beads can be homogenously distributed in the reaction mixture.
  • Epoxides may be susceptible to hydrolysis in aqueous conditions and in the presence of base.
  • the salt of the amount of the salt represented by Formula (I), (la), (lb), or (Ic) e.g.
  • GTMAC w/v
  • a 25 mL volume of aqueous 1 M NaOH in which 10 g of cellulose beads are suspended may be contacted with 31.3 g of GTMAC, i.e. about 125% w/v GTMAC based on the volume of the aqueous solvent and base.
  • the molar ratio of the salt of Formula (I), Formula (la), Formula (lb), or Formula (Ic) (e.g. GTMAC) to anhydrous glucose units of the beads is at least about 1 :1 , at least about 2:1 , at least about 3:1 , or at least about 4:1. In various embodiments, the molar ratio of the salt of Formula (I), Formula (la), Formula (lb), or Formula (Ic) (e.g. GTMAC) to anhydrous glucose units of the beads is up to 5:1. Thus, in various embodiments, the molar ratio of the salt of Formula (I), Formula (la), Formula (lb), or Formula (Ic) (e.g.
  • GTMAC to anhydrous glucose units of the beads is from about 1 :1 , from about 2:1 , from about 3:1 , or from about 4:1 to about 5:1.
  • the foregoing ratios are calculated based on the number of moles of the salt and the anhydrous glucose units prior to the reaction of said salt and anhydrous glucose units, i.e. before the reactants are consumed/transformed.
  • anhydrous glucose unit takes its normal meaning in the art and refers to a single sugar molecule (i.e. monomer) in the polysaccharide of the beads of the present disclosure.
  • step (i) and/or step (ii) are performed at a temperature of from about 10°C to about 30°C.
  • step (i) and/or step (ii) are performed at room temperature (about 20°C).
  • both steps (i) and (ii) are performed at room temperature.
  • all steps of the methods disclosed herein are performed at room temperature. Conducting the various steps at room temperature contributes to the minimal environmental impact of the methods disclosed herein.
  • reaction time is not limited, and a person skilled in the art will be able to determine a suitable duration for the reaction, for example to ensure that the reaction reaches completion.
  • the functionalisation of the beads according to the methods of the present disclosure leads to the introduction of quaternary ammonium groups on said beads.
  • the beads thus functionalised bear multiple positive charges, i.e. are polycationic, in contrast to the unfunctionalised beads that are typically electrically neutral prior to performing steps (i) and (ii) of the methods described herein.
  • the progress of the functionalisation reaction of step (ii) can thus be monitored via the charge density of the beads.
  • a person skilled in the art of the present disclosure will be able to select suitable analytical methods for determining charge density of the beads. For example, charge density may be measured by the conductometric titration of chloride ions against silver nitrate. In such a method, the beads after reaction are thoroughly washed, e.g.
  • the functionalised beads of the present disclosure have a charge density of at least about 0.5 meq/g, at least about 0.75 meq/g, at least about 1 meq/g, or at least about 2 meq/g as defined hereinabove. In various embodiments, the functionalised beads of the present disclosure have a charge density of up to about 3 meq/g as defined hereinabove. Thus, in various embodiments, the functionalised beads of the present disclosure have a charge density of from about 0.5 meq/g, from about 0.75 meq/g, from about 1 meq/g, or from about 2 meq/g to about 3 meq/g as defined hereinabove. In various embodiments, the functionalised beads of the present disclosure have a charge density of from about 0.5 meq/g, from about 0.75 meq/g, or from about 1 meq/g to about 2 meq/g as defined hereinabove.
  • the functionalised beads of the present disclosure have a dry weight of less than about 10%, less than about 8%, less than about 6%, or less than about 4%. In various embodiments, the functionalised beads of the present disclosure have a dry weight of at least about 1 %. Thus, in various embodiments, the functionalised beads of the present disclosure have a dry weight of from about 1% to about 10%, to about 8%, to about 6%, or to about 4%.
  • the functionalised beads of the present disclosure have a charge density of from about 0.5 meq/g, from about 0.75 meq/g, from about 1 meq/g, or from about 2 meq/g to about 3 meq/g and a dry weight of from about 1 % to about 10%, to about 8%, to about 6%, or to about 4%.
  • the functionalised beads of the present disclosure have a charge density of from about 0.5 meq/g, from about 0.75 meq/g, or from about 1 meq/g to about 2 meq/g as defined hereinabove, and a dry weight of from about 1% to about 10%, to about 8%, to about 6%, or to about 4%.
  • the functionalised beads of the present disclosure have a charge density of from about 0.75 meq/g to about 2 meq/g and a dry weight of from about 2% to about 4%.
  • said methods may further comprise the step of (iii) neutralising the product of step (ii) with an acid.
  • neutralising means contacting the product of step (ii) comprising a base as described herein with an acid as described herein such that the resulting pH is about 7.
  • the acid includes a counterion which is the same as X in Formula (I).
  • the anions contributed by said salt and said acid may be advantageous to avoid the need for additional purification steps.
  • the acid includes a counterion which is the same as X in Formula (I) and Y in Formula (Ila). Again, in this way, the presence of multiple different anionic species in the reaction can be avoided, and thus the need for additional purification steps may also be avoided.
  • the acid is hydrochloric acid.
  • X' is chloride and the acid of step (iii) is hydrochloric acid.
  • the salt of Formula (I), (la), (lb) or (Ic) is prepared from a compound of Formula (Ila)
  • X' is chloride
  • Y is chlorine
  • the acid of step (iii) is hydrochloric acid.
  • the base of step (i) is a hydroxide salt and the acid includes a counterion which is the same as X in Formula (I).
  • the base of step (i) is a hydroxide salt, the acid is hydrochloric acid, and X- is chloride.
  • the acid includes a counterion which is the same as X in Formula (I) and the leaving group corresponding to Y in the compound of Formula (lla)ln further embodiments wherein the salt of Formula (I), (la), (lb) or (Ic) is prepared from a compound of Formula (Ila), X' is chloride, Y is chlorine, the base of step (i) is a hydroxide salt and the acid of step (iii) is hydrochloric acid.
  • the methods disclosed herein further comprise the step of (iv) separating the functionalised beads by filtration and optionally (v) washing the filtrate from step (iv) with water.
  • the filtration process is not limited. Techniques suitable for the separation of step (iv) will be known to a person skilled in the art and include but are not limited to those described herein.
  • the functionalised beads may be separated from the reactants by vacuum filtration or simply by sieving.
  • the filtration process may involve mechanical or any other type of filtration (e.g. using equipment known in the art such as a hydrocyclone).
  • a filtration medium e.g. a filter
  • the functionalised beads may be allowed to settle in a vessel and the reaction solution comprising reactants (e.g. the base, unreacted salt of Formula (I), and/or compound of Formula (Ila), and/or acid etc.) removed or decanted to leave beads wetted in residual reaction solution.
  • reactants e.g. the base, unreacted salt of Formula (I), and/or compound of Formula (Ila), and/or acid etc.
  • the beads may be separated by a centrifugal separator or a disk stack separator.
  • the functionalised beads may be washed one or more times, for example with an aqueous solvent including water.
  • the aqueous solvent is substantially free of organic solvents. The term “substantially free” is defined above. By avoiding such organic solvents, functionalised beads produced by the methods disclosed herein may be immediately suitable for use in downstream applications, such as in the food industry, without further costly purification steps.
  • the functionalised beads are washed one or more times with water.
  • the functionalised beads may be separated as described above and then washed with water.
  • One or more cycles of separation and washing steps may be performed, preferably until the remaining amounts of reactants and/or ionic products (e.g. salts formed by neutralisation of the base with acid) are negligible.
  • the progress of washing steps may be monitored by measuring the electrical conductivity of water in which the functionalised beads are suspended. Such techniques are expressly within the common general knowledge of a person skilled in the art of the present disclosure.
  • the functionalised beads are stored in an aqueous solvent, preferably wherein said aqueous solvent is substantially free of organic solvents.
  • the term “substantially free” is defined above.
  • the functionalised beads may be stored in water, preferably deionised water.
  • the functionalised beads may preferably be stored at temperatures lower than room temperature, for example less than about 20 °C.
  • functionalised beads may be stored in deionised water at less than about 10 °C.
  • the functionalised beads are stored in deionised water at about 4 °C.
  • the functionalised beads are stored in the manner according to any of the foregoing embodiments prior to use as described herein.
  • the functionalised beads may be sterilised prior to storage as described herein.
  • the functionalised beads may be sterilised prior to their subsequent use as described herein.
  • the present disclosure is not limited in terms of suitable sterilisation methods and the skilled person will be able to select suitable sterilisation methods.
  • the functionalised beads may be sterilised by irradiation (e.g. UV- irradiation or gamma-ray irradiation), heat treatment (autoclavation), or chemical disinfection (e.g. 70% v/v ethanol in water).
  • the functionalised beads may be dried (for example in a vacuum oven, by air drying, in a rotovaporator or by freeze-drying) and stored in a dry state. Such dried beads may be rehydrated by suspension in an aqueous solvent prior to use in a desired application as described herein.
  • the aqueous solvent is substantially free of organic solvents. The term “substantially free” is defined above. As already discussed above, avoiding organic solvents may be advantageous in that the functionalised beads produced by the methods disclosed herein may be immediately suitable for use in downstream applications, such as in the food industry, without further costly purification steps.
  • the aqueous solvent is water.
  • cell culture refers to methods wherein eukaryotic or prokaryotic cells taken out of their natural environment (e.g. from the tissue of an originating organism) are grown under controlled artificial conditions, for example in a reactor in a laboratory.
  • the types of cells cultured are not limited and include bacteria, archaea and eukaryotes.
  • Non-limiting examples of eukaryotes contemplated by the present disclosure include plant, fungi, and animal cells.
  • Animal cells may include non-mammalian and mammalian cells.
  • Microcarriers provide within the cell culture a substrate upon and/or in which mono- and/or multi-layers of cells can grow.
  • the microcarriers are typically suspended in the cell culture medium, for example by stirring.
  • adherent anchorage-dependent cells
  • adherent animal cells such as adherent mammalian cells.
  • Adherent cells are cells that require fixation to a surface for them to grow in vitro. Consequently, an equivalent mass of cultured cells can be obtained with a smaller culture volume when using microcarriers, thus leading to process and cost efficiencies particularly for large-scale industrial mammalian cell cultures.
  • the functionalised beads of the present disclosure are further advantageous in that they are produced in a highly cost- effective manner due to the simplicity of the methods described herein. Further, the functionalisation methods described herein are environmentally friendly, for example because the use of organic solvents and/or the production of toxic, regulated, and/or prohibited side products can be avoided.
  • each of the functionalisation methods and methods for producing the non-functionalised bead starting material disclosed herein can be performed in aqueous solution where the aqueous solution is substantially free or completely free of organic solvents.
  • cellulose beads cellulose is both cost-effective and sustainable as a starting material. For instance, cellulose is the most abundant biopolymer on Earth and can be obtained from many sources, including waste, enabling closed-loop production and recycling.
  • the present disclosure provides the use of functionalised beads prepared by any of the methods disclosed herein in cell culture. Also provided is a method for attaching cells to functionalised polysaccharide beads, wherein the method comprises preparing functionalised beads by any of the methods described herein and contacting the functionalised beads with one or more cells, preferably wherein the functionalised beads are contacted with one or more cells during cell culture.
  • bacterial, yeast, insect and mammalian cells include bacterial, yeast, insect and mammalian cells.
  • bacterial, yeast and mammalian cells may be used for the overexpression of recombinant proteins.
  • Mammalian cells are most commonly used for the production of e.g. biologies because of their ability to propagate human viruses, express monoclonal antibodies, and incorporate necessary post-translational modifications.
  • cultured meat to reduce environmental impacts from farming and animal husbandry as well as on ethical and animal welfare grounds.
  • the cells of the use and method for attaching cells to functionalised polysaccharide beads as described herein are animal cells, preferably mammalian cells, and more preferably adherent mammalian cells.
  • animal cells preferably mammalian cells, and more preferably adherent mammalian cells.
  • Non-limiting examples of commonly cultured mammalian cells for industrial purposes include Chinese hamster ovary cells (CHO), lymphoma cells (e.g. NS0, SP2/0), baby hamster kidney (BHK) cells, hybridoma cells, Vero cells (e.g. ATCC CCL-81), and human embryonic kidney (HEK) cells.
  • Cell lines and information relating thereto can typically obtained from a public repository such as that maintained by the American Type Culture Collection (ATCC) as well as commercial vendors.
  • ATCC American Type Culture Collection
  • Meat tissue is made up of various cell types such as myofibers, adipocytes, fibroblasts, chondrocytes, and endothelial cells.
  • Cultured meat approaches typically obtain the starting cells for culture in one of two ways: (i) by taking a tissue biopsy or using post-slaughter tissue from a livestock species of interest, i.e. primary cell culture, or (ii) differentiating pluripotent stem cells.
  • stem cells are preferred as these have the capacity to proliferate and then differentiate into the mature cell types present in meat.
  • Two such stem cell types are adult stem cells and pluripotent stem cells.
  • Adult stem cells are undifferentiated progenitor cells that can be obtained from specific organs and tissues in animals.
  • Adult stem cells are multipotent (can differentiate into a number of cell types). Examples useful for cultured meat production include muscle satellite cells, mesenchymal stem/stromal cells, and fibro/adipogenic progenitors.
  • Pluripotent stem cells are also attractive for cultured meat production because they are highly proliferative in culture and can be differentiated into a wide variety of mature cell types.
  • Pluripotent stem cells may be embryonic stem cells, which are derived from blastocysts, or induced pluripotent stem cells obtained by cell reprogramming of somatic cells by the induction of genes associated with pluripotency.
  • stem cells contemplated herein will be exclusively from non-human animals. Further, the collection of stem cells as well as any processes and uses of embryos are expressly not encompassed by the present disclosure.
  • the functionalised beads prepared by the methods described herein are used in cell culture for food production, preferably for the production of cultured meat.
  • the functionalised beads prepared by the methods described herein may be used in animal cell culture for food production.
  • the conditions of the cell culture are not limited. The skilled person will be able to select appropriate cell culture conditions for the cell line of interest as part of their common general knowledge.
  • a standard textbook in the art regarding all aspects of animal cell culture include techniques and methods is Freshney’s Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 5 th Edition, John Wiley & Sons, Inc. 2005, ISBN: 978-0-471- 74759-8.
  • Guidance on the culture of specific cell lines is also provided by suppliers of commercial cell lines and from repositories such as the ATCC.
  • Another reference covering microbial, mammalian and plant cell culture is Advanced Fermentation and Cell Technology, Wiley-Blackwell, 2021 , ISBN: 978-1-119-04276-1.
  • Cells are typically cultured in a liquid culture medium.
  • DMEM Dulbecco’s Modified Eagle Medium
  • the culture medium may contain one or more supplements, such as serum, antibiotics, amino acids, or growth factors.
  • the culture medium may be supplemented with fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • FBS may be supplemented into culture media, e.g. DMEM, in an amount of about 10%.
  • serum-free culture media may be preferred in some embodiments.
  • Antibiotics and/or fungicides may be used to avoid contamination of cell cultures, however for food production purposes such as cultured meat it may be preferred to omit these in favour of production under strictly controlled sterile conditions.
  • the cell culture will typically be warmed to a physiologically relevant temperature, for example 37 °C, to support the growth of the cells. Further, cell culture is typically performed under high humidity, for example to limit evaporation of culture medium, such as 85-95% relative humidity. Many culture media use sodium bicarbonate buffering systems to maintain the pH of the culture at around 7.4. In such embodiments, the culture is performed in a mixture of air and carbon dioxide, preferably 4-10% CO2, and more preferably 5% CO2. The skilled person in the art will be able to modify such environmental conditions as appropriate to the cell line of interest as part of their common general knowledge.
  • Cell culture is typically performed for research purposes at small scale in dishes for stationary culture, in roller bottles for mixed cultures and glass spinner flasks for stirred cultures.
  • Large-scale cell culture may be performed in culture vessels such as large stirred tanks having capacities of around 15,000 litres.
  • microcarriers may be used in such large-scale vessels, but may also be used in other configurations such as packed beds, and fluidised beds.
  • Microcarriers may be retained from the cell culture medium by various methods and the present disclosure is not limited in this regard.
  • microcarriers can be retained by filtration or centrifugation.
  • the cells may be detached from the microcarriers enzymatically, for example by treatment with trypsin, and/or a chelating agent such as EDTA.
  • the detached cells may subsequently be separated from the microcarriers by various methods that the skilled person will be able to select from their common general knowledge.
  • Non-limiting examples include differential sedimentation, filtration, density gradient centrifugation, fluidised bed separation, or use of a vibromixer.
  • the present disclosure provides for the use of functionalised beads prepared by any of the methods described herein in the culture of animal cells, preferably mammalian cells.
  • the animal/mammalian cells are adherent cells.
  • the animal cells are adherent mammalian cells.
  • the functionalised beads described herein are used as microcarriers in said cell culture.
  • the cell culture comprises animal cells and a liquid culture medium.
  • the cell culture may comprise mammalian cells, preferably adherent mammalian cells, and a liquid culture medium.
  • the liquid culture medium comprises at least one supplement selected from the list consisting of foetal bovine serum, amino acids, growth factors, antibiotics and antifungals.
  • the cell culture is carried out in a bioreactor, preferably a stirred bioreactor.
  • the cell culture is carried out at a temperature of around 37°C in the presence of air containing from about 4% to about 10% CO2 and with a relative humidity of from about 85% to about 95%. Any of the foregoing characteristics of the described use are also applicable to the method for attaching cells to functionalised beads as described herein.
  • the cell culture comprises animal cells, a liquid culture medium, and functionalised beads prepared according to any of the methods described herein; wherein the liquid culture medium comprises at least one supplement selected from the list consisting of foetal bovine serum, amino acids, growth factors, antibiotics and antifungals; preferably wherein the cell culture is carried out in a bioreactor at a temperature of around 37°C in the presence of air containing from about 4% to about 10% CO2 and with a relative humidity of from about 85% to about 95%.
  • the present disclosure provides a generally applicable methodology for the functionalisation of polysaccharide beads. Accordingly, the present disclosure is not limited in terms of the means by which the polysaccharide beads are prepared.
  • polysaccharide beads may be prepared by extrusion, and (ii) membrane emulsification followed by phase inversion.
  • a dispersed phase is extruded into an anti-solvent to form beads of the polysaccharide.
  • the dispersed phase comprises the polysaccharide in a solvent as discussed further below, and the extrusion of such a dispersed phase is known in the art. It is a process wherein the dispersed phase is forced, pressed, or pushed out, for example through an aperture or opening.
  • the opening may be in a syringe as shown in Figure 1 or any other suitable extrusion device as known in the art.
  • FIG. 1 A schematic representation of an exemplary embodiment of the extrusion process is shown in Figure 1.
  • the dispersed phase (1) comprising the polysaccharide in a solvent is extruded through a needle (2) of a syringe (3). Extrusion is specifically into the anti-solvent (4) to form polysaccharide beads (5).
  • the extruded dispersed phase is dropped from a height, d, above the surface of the anti-solvent.
  • the latter exemplary method of preparing polysaccharide beads comprises a membrane emulsification step and a phase inversion step.
  • Membrane emulsification is known in the art; it is a technique in which a dispersed phase is forced through the pores of a microporous membrane directly into a continuous phase, where emulsified droplets are formed and detached at the end of the pores with a drop- by-drop mechanism.
  • a schematic representation of a membrane emulsification process is shown in Figure 2, where the arrow indicates the direction of flow.
  • the dispersed phase generally includes a first liquid containing the polysaccharide dissolved in a solvent, and the continuous phase includes a second liquid which is immiscible with the first liquid.
  • the interaction of the two liquids when the dispersed phase is pushed or otherwise transported through the membrane is called a dispersion process, and their inhomogeneous mixture is termed an emulsion, i.e. droplets of the dispersed phase surrounded by the continuous phase.
  • phase inversion is a chemical phenomenon exploited in the fabrication of artificial membranes, and is performed by removing solvent from a liquid-polymer solution.
  • phase inversion includes immersing the polymer solution into a third liquid called the anti-solvent.
  • anti-solvent based phase inversion has proven to be particularly effective in precipitating droplets of polysaccharide into beads from an emulsion of dispersed/continuous phase.
  • Ionic liquids are salts that are in liquid form at a temperature between ambient temperature and 100°C, for example imidazolium based ionic liquids such as 1-ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc) or the like.
  • imidazolium based ionic liquids such as 1-ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc) or the like.
  • ionic liquids are essentially non-volatile (avoiding fugitive emissions) and are considered to have environmental benefits over other solvents. Ionic liquids can, for example, be readily recycled by distillation to remove the anti-solvent.
  • Ionic liquids are typically not used in pure form, however.
  • An amount of a co-solvent is often added to the ionic liquid when dissolving polysaccharides such as cellulose.
  • the use of a co-solvent may assist in dissolution of the polysaccharide, and may reduce the amount of costly ionic liquid required.
  • the inclusion of a cosolvent may also improve the efficiency and yield of the process by modifying the viscosity of the dispersed phase, which may in turn reduce the amount of deformation exhibited by the beads.
  • Typical co-solvents employed in combination with ionic liquids are dipolar aprotic solvents, such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and the like.
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • Typical co-solvents employed in combination with ionic liquids are dipolar aprotic solvents, such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and the like.
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • co-solvents therefore cannot be used in processes for the preparation of polysaccharide beads for use in food production as well as other applications.
  • the use of dipolar aprotic solvents may also complicate the recycling of the ionic liquid and increase costs.
  • organic solvents such as ethanol are typical. Again, however, these substances may reduce the overall environmental benefits of the process, may be associated with safety concerns, and may complicate and increase the cost of recycling of ionic liquids.
  • an aqueous solvent and an aqueous anti-solvent may be used.
  • the use of an aqueous solvent and anti-solvent may obviate the use of reagents associated with environmental and safety concerns and may also simplify and reduce the cost of solvent recycling.
  • use of such solvents and anti-solvents may increase the stability of ionic liquids against temperature-based degradation [Williams et al., Thermochimica Acta (2016), 669: 126-139], which may allow an increased number of recycling cycles to be performed, for example.
  • the inclusion of water in the dispersed phase may increase the likelihood of bead sphericity.
  • the dispersed phase from which the polysaccharide beads may be prepared comprises a solvent in which the polysaccharide is dispersed or dissolved, which solvent comprises water.
  • solvent is therefore meant any substance (e.g. liquid) which disperses or dissolves the polysaccharide.
  • solvent also includes solvent mixtures.
  • the solvent of the dispersed phase may comprise water and may comprise an ionic liquid, an organic solvent, an inorganic nonaqueous solvent, or a combination thereof.
  • the solvent for the dispersed phase comprises water and at least one of an ionic liquid, an organic solvent, an inorganic nonaqueous solvent, or a combination thereof.
  • the solvent for the dispersed phase comprises water and one or more ionic liquid(s).
  • Non-limiting examples of solvents for the dispersed phase other than water include methanol, ethanol, ammonia, acetone, acetic acid, n-propanol, n-butanol, isopropyl alcohol, ethyl acetate, dimethyl sulfoxide, sulfuryl chloride, phosphoryl chloride, carbon disulfide, morpholine, N-methylmorpholine, NaOH without and with association of urea and thiourea, bromine pentafluoride, hydrogen fluoride, sulfuryl chloride fluoride, acetonitrile, dimethylformamide, hydrocarbon oils and blends thereof, toluene, chloroform, carbon tetrachloride, benzene, hexane, pentane, cyclopentane, cyclohexane, 1 ,4-dioxane, dichloromethane, nitromethane, propylene carbonate, formic acid,
  • the dispersed phase will depend on the polysaccharide being used.
  • suitable solvents for the dispersed phase of the present disclosure is specifically within the common general knowledge of the skilled person.
  • the solvent for the dispersed phase comprises water and an ionic liquid.
  • the ionic liquid may be selected from 1-ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc), and a combination thereof.
  • the solvent for the dispersed phase comprises water and one or more organic solvents. In other embodiments, the solvent for the dispersed phase is substantially free of organic solvents.
  • the term “substantially free” is defined above.
  • the solvent of the dispersed phase consists of water and an ionic liquid
  • the total wt% of water and ionic liquid in the dispersed phase solvent will total 100 wt%.
  • water is present, for example, in an amount of at least 0.5 wt%
  • an ionic liquid may be present in an amount of at least 99.5 wt%, with the proviso that the total of water and ionic liquid is 100 wt%.
  • the ionic liquid may be present as the remainder of the solvent.
  • the solvent used for the dispersed phase is environmentally friendly.
  • environmentally friendly is meant not harmful to the environment such that the solvent can be disposed of without the need for specialist equipment or process(es), i.e. non-toxic.
  • polysaccharides have limited dissolution in most of the common solvents.
  • those solvents which do dissolve polysaccharides are often toxic and/or highly selective.
  • the solvent for the dispersed phase may comprise an ionic liquid in addition to water.
  • the concentration of polysaccharide in the dispersed phase is not limited and may be any concentration suitable for the methods discussed herein.
  • the polysaccharide is present in the dispersed phase in an amount from about 0.1 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.5 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1.5 wt% to about 15 wt%.
  • the polysaccharide is present in the dispersed phase in an amount from about 2 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2.5 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3.5 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 4 wt% to about 15 wt%.
  • the polysaccharide is present in the dispersed phase in an amount from about 0.1 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.5 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 .5 wt% to about 12 wt %.
  • the polysaccharide is present in the dispersed phase in an amount from about 2 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2.5 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3.5 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 4 wt% to about 12 wt %.
  • the polysaccharide is present in the dispersed phase in an amount from about 0.1 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.5 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 .5 wt% to about 10 wt %.
  • the polysaccharide is present in the dispersed phase in an amount from about 2 wt% to about 10 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2.5 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3.5 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 4 wt% to about 10 wt %.
  • the dispersed phase may further include optional components. These optional components include, but are not limited to, surfactants, porogens, active ingredients, pockets of air, double emulsions, pigments, and dyes. The level of any of the optional components is not significant in the present disclosure.
  • the dispersed phase includes a co-solvent.
  • the surfactant may be any suitable surfactant known in the art, for example, any ionic or non-ionic surfactant.
  • Ionic surfactants may include sulfates, sulfonates, phosphates and carboxylates such as alkyl sulfates, ammonium lauryl sulfates, sodium lauryl sulfates, alkyl ether sulfates, sodium laureth sulfate and sodium myreth sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorobutanesulfonate, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, and alkyl carboxylates.
  • Non-ionic surfactants may include polyethers, polyoxyalkylene derivatives of hexitol, partial long-chain fatty acid esters such as sorbitan oleates, ethylene oxide derivatives of long-chain alcohols, ethoxylated vegetable oil, polydimethylsilxoxanes, and ethylene oxide/propylene oxide copolymers.
  • the temperature of the dispersed phase is not limited.
  • temperature of the dispersed phase or “the dispersed phase is at a temperature of”, or the like, is meant the temperature of the dispersed phase prior to extrusion or membrane emulsification (e.g. when it is placed in the apparatus for such extrusion or emulsification), and/or the temperature of the apparatus during extrusion or emulsification of the dispersed phase.
  • the extrusion or emulsification means may be heated so that the dispersed phase remains at an elevated temperature in situ.
  • the extrusion means is heated directly by one or more heating means. This is discussed further below.
  • the dispersed phase is at ambient or room temperature, namely between about 20 and about 25°C. In various embodiments, the dispersed phase is heated above ambient temperature.
  • the dispersed phase may be heated using any suitable means.
  • the dispersed phase is preferably heated in situ such that there is no temperature loss prior to extrusion or membrane emulsification, for example by heating a vessel containing the dispersed phase and/or the extrusion or emulsification means. In the extrusion process, a heated syringe and/or needle may, for example, be used.
  • Suitable heating apparatus may comprise a heating element, for example a Peltier element, as well as a means of regulating the temperature, such as a thermocouple and controller.
  • the temperature of the dispersed phase is from about 5°C to less than about 100°C, from about 10°C to less than about 100°C, from about 15°C to less than about 100°C, from about 20°C to less than about 100°C, from about 25°C to less than about 100°C, or from about 30°C to less than about 100°C.
  • the maximum temperature will be set by the point at which the evaporation of water from the dispersed phase becomes prohibitive and/or decomposition of the ionic liquid begins to occur. This will readily be determined by the person skilled in the art.
  • the temperature of the dispersed phase is from about 5°C to about 90°C, from about 10°C to about 90°C, from about 15°C to about 90°C, from about 20°C to about 90°C, from about 25°C to about 90°C, or from about 30°C to about 90°C.
  • the temperature of the dispersed phase is from about 5°C to about 80°C, from about 10°C to about 80°C, from about 15°C to about 80°C, from about 20°C to about 80°C, from about 25°C to about 80°C, from about 30°C to about 80°C, or from about 40°C to about 80°C.
  • the anti-solvent may comprise water, i.e. it may be aqueous.
  • the anti-solvent may comprise water and an organic solvent such as an alcohol or acetone, or any other organic solvent known in the art. Suitable alcohols include ethanol and/or methanol.
  • the anti-solvent is environmentally friendly. More preferably, the solvent and antisolvent are both environmentally friendly. Thus, in various embodiments, the anti-solvent is substantially free of organic solvents. In various embodiments, the anti-solvent is or consists of water.
  • the anti-solvent further comprises an ionic liquid.
  • the anti-solvent may comprise water and an ionic liquid before phase inversion or extrusion of the dispersed phase.
  • the ionic liquid may be introduced into the anti-solvent during the phase inversion or extrusion.
  • the dispersed phase comprises an ionic liquid
  • the ionic liquid may be introduced into the antisolvent from the dispersed phase during the phase inversion or extrusion process.
  • the concentration of ionic liquid in the anti-solvent is up to about 50 wt% - the term “up to” being understood to mean greater than zero. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 40 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 30 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 20 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 10 wt%.
  • the anti-solvent comprises water and an ionic liquid
  • the ionic liquid may be 1- ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc), or mixtures thereof.
  • the ionic liquid is 1-ethyl-3- methylimidazolium acetate (EmimOAc).
  • the temperature of the anti-solvent is not limited. In various embodiments, the temperature of the anti-solvent is from about 5°C to about 80°C. In various embodiments, the temperature of the anti-solvent is from about 10°C to about 70°C. In various embodiments the temperature of the anti-solvent is from about 15°C to about 60°C.
  • the temperature of the antisolvent may be ambient such that phase inversion is carried out at ambient temperature, namely between about 20 and about 25°C.
  • the anti-solvent has a temperature between about 20 and about 25°C.
  • the anti-solvent is cooled to a temperature below ambient temperature, namely below about 20°C.
  • the antisolvent may be cooled to a temperature T 2 , for the phase inversion (b), T 2 being less than T d is P .
  • T 2 is substantially equal to Ti, more preferably T 2 is equal to Ti, where Ti is defined above.
  • the advantage of controlling the temperature of the anti-solvent (T 2 ) in such embodiments is to prevent pre-mature thawing of the frozen droplets.
  • T 2 temperature of the anti-solvent
  • the anti-solvent is able to contact the surface of the droplets, causing precipitation of the polysaccharide and hardening of the precipitate surface.
  • the anti-solvent will convert the droplet of dissolved polysaccharide to a bead thereof, whilst leaching the solvent system into the anti-solvent.
  • the dispersed phase is extruded through a fluid medium by capillary extrusion.
  • the fluid medium may, for example, be air.
  • capillaries through which the dispersed phase may be extruded are glass capillaries, microfluidic channels, and (hypodermic) needles.
  • the material from which such capillaries are prepared is not limited and the skilled person will be able to select suitable capillaries compatible with the dispersed phase.
  • the surface of the capillary may also be modified.
  • the capillary may, for example, be treated, coated, or lined, in order to alter its wetting properties.
  • Such modifications of the capillary material may, for example, alter the hydrophilicity/hydrophobicity of the capillary material, thereby altering the wettability of the capillary surface.
  • Capillaries may, for example, be treated with reactive hydrophobic compounds such as silanes to form a hydrophobic surface layer, or hydrophobic compounds may be deposited onto a capillary surface by methods such as chemical vapour deposition.
  • metal needles may be lined with PTFE (polytetrafluoroethylene). The identification of suitable surface modifications is specifically within the common general knowledge of the skilled person.
  • the size of the aperture or opening e.g. the diameter of the capillary or the gauge of the needle, is not limited. It will be immediately apparently to a person skilled in the art that the size of the aperture or opening will, however, influence the size of the droplets of the dispersed phase extruded therefrom. Generally, a larger aperture or opening would be expected to produce larger droplets of the dispersed phase, and conversely a smaller aperture or opening would be expected to produce smaller droplets of the dispersed phase. The skilled person will be able to select appropriately sized openings/apertures.
  • the diameter of the aperture or opening through which the dispersed phase is extruded may be less than about 3 mm, less than about 2.5 mm, less than about 2 mm, less than about 1.5 mm, less than about 1 mm, less than about 0.75 mm, less than about 0.5 mm, less than about 0.4 mm, less than about 0.3 mm, or less than about 0.2 mm. In various embodiments, the diameter of the aperture or opening through which the dispersed phase is extruded may be greater than about 0.1 mm.
  • the diameter of the aperture or opening through which the dispersed phase is extruded may be greater than about 0.1 mm and less than about 3 mm, greater than about 0.1 mm and less than about 2.5 mm, greater than about 0.1 mm and less than about 2 mm, greater than about 0.1 mm and less than about 1.5 mm, greater than about 0.1 mm and less than about 1 mm, greater than about 0.1 mm and less than about 0.75 mm, greater than about 0.1 mm and less than about 0.5 mm, greater than about 0.1 mm and less than about 0.4 mm, or greater than about 0.1 mm and less than about 0.3 mm.
  • the diameter of the aperture or opening through which the dispersed phase is extruded may be from about 0.1 mm to about 1 mm, from about 1 mm to about 2 mm, or from about 2 mm to about 3 mm.
  • the dispersed phase is extruded through a needle.
  • the needle may be blunt-tipped, although the present disclosure is not limited in this respect.
  • the needle gauge size is 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , or 10 gauge.
  • the rate of extrusion is not limited and may be controlled using standard laboratory equipment, for example a syringe pump. In various embodiments, the rate of extrusion is less than about 1 mL/min, less than about 100 pL/min, less than about 10 pL/min, less than about 1 pL/min, or less than about 100 nL/min. In other embodiments, the rate of extrusion is from about 1 pL/min to about 1 mL/min, or from about 10 pL/min to about 100 pL/min.
  • the dispersed phase is first extruded through a fluid medium into a mould and then the extruded dispersed phase is contacted with the anti-solvent.
  • the mould may impart a shape to the polysaccharide beads formed upon contacting the extruded dispersed phase with the anti-solvent.
  • the shape of the polysaccharide beads is not limited, and will be determined by the shape of the mould in this instance.
  • the mould may be formed of any suitable material that is compatible with the dispersed phase and anti-solvent, and may, for example, be a silicone polymer such as polydimethylsiloxane (PDMS).
  • PDMS polydimethylsiloxane
  • the mould may be prepared by casting the mould material, or may be prepared by 3D printing the mould material.
  • the extruded dispersed phase may be contacted with the anti-solvent by submerging the mould containing the extruded dispersed phase in the anti-solvent.
  • the mould may be removed after the polysaccharide beads have formed, or may be retained during further processing steps, such as washing and filtration/extraction of the polysaccharide beads.
  • extrusion may occur within the anti-solvent; that is to say, the dispersed phase may be exposed to the anti-solvent immediately upon extrusion (for example where the aperture or opening is submerged in the anti-solvent).
  • the extruded dispersed phase is dropped from a height above the surface of the anti-solvent. This can be seen in Figure 1 , wherein the extruded dispersed phase is dropped from a height, d, above the surface of the anti-solvent.
  • the dropping height may influence the sphericity of the beads obtained by the extrusion process. Without wishing to be bound by any one theory, it is believed that a greater dropping height may minimize tailing (i.e. improve sphericity) by allowing more time for cohesive forces to act on the falling droplet.
  • the extruded phase is dropped from a height of at least 10 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 20 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 30 cm above the surface of the anti-solvent.
  • the extruded phase is dropped from a height of at least 40 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 50 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 70 cm above the surface of the anti-solvent, or at least 80 cm above the surface of the anti-solvent.
  • the maximum dropping height will typically be determined by the distance at which non-spherical beads are formed. This is known in the art and readily understood by the skilled person. It may, for instance, be determined by eye. In various embodiments, however, the extruded phase is dropped from a height of less than 80 cm above the surface of the antisolvent. In various embodiments, the extruded phase is dropped from a height of less than 70 cm above the surface of the anti-solvent. In various embodiments, the extruded phase is dropped from a height of less than 60 cm above the surface of the anti-solvent. In various embodiments, the extruded phase is dropped from a height of less than 50 cm above the surface of the anti-solvent.
  • the extruded phase is dropped from a height of about 1 cm to about 80 cm above the surface of the anti-solvent, preferably from a height of about 5 cm to about 70 cm, more preferably from a height of about 10 cm to about 60 cm.
  • the extruded phase is dropped from a height of about 10 cm to about 80 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 70 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 50 cm above the surface of the antisolvent.
  • the extruded phase is dropped from a height of about 20 cm to about 80 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 70 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 50 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 40 cm above the surface of the anti-solvent.
  • the extrusion process may further comprise the step of separating the polysaccharide beads from the anti-solvent.
  • the means by which the polysaccharide beads may be separated from the anti-solvent are not limited and will be known to a person skilled in the art.
  • the polysaccharide beads may be separated from the antisolvent by a filtration process.
  • the filtration process is not limited and may involve mechanical or any other type of filtration (e.g. using equipment known in the art such as a hydrocyclone).
  • a filtration medium e.g. a filter
  • the polysaccharide beads may be allowed to settle in a vessel and anti-solvent removed or decanted to leave polysaccharide beads wetted in residual antisolvent.
  • the polysaccharide beads may be separated by a centrifugal separator or a disk stack separator.
  • the polysaccharide beads may be washed one or more times, for example with an aqueous solvent including water. Such washing steps may be performed to remove residual ionic liquid that may be present.
  • the solvent in which the polysaccharide beads are immersed may be exchanged for an alternative solvent.
  • membrane emulsification involves passing a dispersed phase through a membrane into a continuous phase so as to form an emulsion.
  • the membrane is not limited; it can be any porous structure suitable for a membrane emulsification process.
  • the membrane may be a plate with holes acting as pores (e.g. micron-sized holes), a perforated metal tube, or sintered porous glass.
  • emulsion is meant the class of two-phase systems of matter where both phases are liquid. Emulsions are a type of colloid, and generally consist of two immiscible liquids. In various embodiments, the emulsion may be a macro-emulsion; this is an emulsion in which the particles of the dispersed phase have diameters of approximately 1 to 1000 microns.
  • sol refers to a general class of two-phase systems of matter where the continuous phase is liquid and the dispersed phase is solid.
  • the membrane emulsification is also not limited and may be any membrane emulsification process known in the art.
  • the membrane emulsification process may be a cross-flow membrane emulsification, a rotational membrane emulsification, a vibrational membrane emulsification, or a combination thereof.
  • cross-flow As is understood in the art, the terms “cross-flow”, “rotational” and “vibrational” refer to the method used to generate shear on the membrane surface.
  • a continuous phase could, for example, move relative to a stationary membrane to create shear, or the membrane could move relative to stationary phases.
  • the dispersed phase could be injected into a stationary continuous phase.
  • the membrane emulsification may involve a cross flow system, a stirred-cell tube membrane, a stirred cell-flat membrane, a rotating flat membrane, a vibrating/rotating tube membrane and/or a premixed membrane emulsification.
  • the membrane emulsification is a cross-flow membrane emulsification.
  • an emulsification process in which the continuous phase moves relative to a stationary membrane is preferred.
  • the dispersed phase and continuous phase will depend on the polysaccharide being used.
  • Various features of the solvent for the dispersed phase have already been discussed above, and said features individually or in any combination thereof are combinable with the embodiments disclosed herein.
  • the continuous phase will comprise a solvent which is immiscible with the dispersed phase such that an emulsion is formed when the dispersed phase is forced through the porous membrane.
  • solvent has the meaning as already defined hereinabove.
  • the solvent of the continuous phase is not limited other than it must be immiscible with the dispersed phase.
  • the solvent of the continuous phase may be a non-polar solvent.
  • the solvent of the continuous phase may be selected from hydrocarbon oils and blends thereof. Such hydrocarbon oils may be mineral oils, vegetable oils, or synthetic oils.
  • the solvent of the continuous phase may further comprise water and/or one or more ionic liquids that may be present in residual amounts. Such residues of water and/or ionic liquid may arise as a result of solvent recycling processes.
  • the solvent used for the continuous phase is environmentally friendly. More preferably the solvent used for both the dispersed phase and continuous phase is environmentally friendly.
  • the term “environmentally friendly” has the meaning as already defined hereinabove.
  • the continuous phase may further include optional components.
  • optional components include, but are not limited to, co-solvents, surfactants, pigments, and dyes. The level of any of the optional components is not significant in the present disclosure.
  • the continuous phase includes a co-solvent.
  • the co-solvent is not limited and may be any solvent known in the art.
  • the co-solvent may be selected from hydrocarbon oils and blends thereof.
  • Such hydrocarbon oils may be mineral oils, vegetable oils, or synthetic oils.
  • the co-solvent may further be a co-solvent mixture.
  • the surfactant is as defined above.
  • the emulsion is cooled to a temperature Ti, Ti being greater than the pour point of the continuous phase (T CO nt), and equal to or less than a transition temperature selected from the group consisting of the freezing point, glass transition temperature and pour point, of the dispersed phase (Tdisp): wherein Tdisp > T CO nt.
  • Ti is not, however, critical to the present disclosure; rather it is the relationship of Ti to the respective temperatures of the dispersed phase and continuous phase that is important.
  • pour point refers to the temperature below which a substance (e.g. liquid) loses its flow characteristics. It is typically defined as the minimum temperature at which the liquid (e.g. oil) has the ability to pour down from a beaker.
  • the pour point can be measured with standard methods known in the art. ASTM D7346, Standard Test Method for No Flow Point and Pour Point of Petroleum Products and Liquid Fuels may, for example be used. For commercially available materials, the pour point is often provided by the supplier or manufacturer.
  • freeze point refers to the temperature at which a substance changes state from liquid to solid at standard atmospheric pressure (1 atmosphere).
  • the freezing point can be measured with standard methods known in the art. ASTM E794, Standard Test Method for Melting and Crystallization Temperatures by Thermal Analysis may, for example, be used. For commercially available materials, the freezing point may be provided by the supplier or manufacturer.
  • glass transition point or “glass transition temperature” refers to the temperature at which a polymer structure transitions from a hard or glassy material to a soft, rubbery material. This temperature can be measured by differential scanning calorimetry according to the standard test method: ASTM E1356, Standard Test Method for Assignment of the Glass Transition Temperature by Differential Scanning Calorimetry. For commercially available materials, the glass transition temperature may be provided by the supplier or manufacturer.
  • the dispersed phase having a transition temperature - the transition temperature being selected from the group consisting of freezing point, glass transition temperature and pour point - which is higher than the continuous phase pour point means that the continuous phase surrounding the solidified dispersed phase is still able to function as a transport medium.
  • a diagrammatic representation of an emulsion undergoing cooling and temporary conversion to a sol within a cooling coil heat exchanger is shown in Figure 2(b).
  • the method of cooling is not also limited.
  • the emulsion may be cooled by any means known in the art for removing heat (energy) from a system.
  • the emulsion may further be cooled at any point prior to phase inversion. In various embodiments, this means the emulsion is cooled simultaneously with or separately from the membrane emulsification process.
  • the emulsion may, for example, be cooled as it is formed (e.g. by a cooling means located at the outlet of the membrane).
  • the emulsion may be cooled in a step following membrane emulsification, e.g. in a cooling apparatus separate from the membrane emulsification apparatus.
  • the cooling should take place as soon as possible after the emulsification takes place in order to reduce the possibility of liquid state dispersed phase droplets coalescing and/or aggregating.
  • the emulsion may be cooled by a cooling medium (e.g. water, ice etc.) at least partially surrounding the vessel where the emulsion is formed.
  • a cooling medium e.g. water, ice etc.
  • the vessel (e.g. pipe) where the emulsion is formed may have a cooling jacket containing a cooling medium.
  • the cooling medium is not limited, and includes any medium having a lower temperature than the emulsion.
  • the emulsion may be cooled by a cooling apparatus connected to the membrane emulsification unit.
  • the cooling apparatus may be a heat exchanger, such as an immersion heat exchanger.
  • a coil heat exchanger is immersed in a cooling medium (e.g. a cold water bath) but the disclosure is not limited in this respect.
  • Any type of heat exchanger could, for instance, be used such as a tube-and-shell heat exchanger, a plate-and-frame heat exchanger, or a jacketed tube.
  • an immersion heat exchanger could be used with another cooling medium such as anti-freeze, dry ice or the like, in order to cool the emulsion to Ti.
  • phase inversion is carried out under shear; the skilled person will be aware of suitable shear conditions for phase inversion.
  • Shear may, for example, be achieved through the use of a stirred vessel (e.g. a mechanically stirred vessel) or a settling vessel (e.g. a gravity settling vessel).
  • the term “shear” is used herein to refer to an external force acting on an object or surface parallel to the slope or plane in which it lies, the stress tending to produce strain.
  • phase inversion comprises a filtration process.
  • the filtration process is not limited and may involve mechanical or any other type of filtration (e.g. using equipment known in the art such as a hydrocyclone).
  • a filtration process may also be encompassed by the phase inversion being carried out under shear as described above.
  • a filtration medium e.g. filter
  • the emulsion may gravity settle (shear) through the anti-solvent and into the filter, whilst the continuous phase passes through the filter (the filtrate). The frozen droplets may then be collected in the filter as the filter cake.
  • the polysaccharide beads may be separated from the anti-solvent/continuous phase mixture or the anti-solvent/continuous phase mixture may be removed from the beads.
  • the method of removal is not limited. In various embodiments, however, the method of removal depends on whether the method is being operated in batch or continuous mode.
  • the phase inversion step may first be performed in a closed vessel and the resulting mixture then transferred into a decanter vessel and allowed to reach a settled stage. Once settled, layers may be removed sequentially from the bottom of the vessel.
  • the order of the layers can be (1) continuous phase, (2) an interfacial layer comprising wetted polysaccharide beads and (3) the remaining anti-solvent.
  • the disclosure is not, however, limited in this respect and the skilled person will appreciate that the order of the layers will depend on their respective densities.
  • the method is continuous and to operate in continuous mode
  • the phase inversion step may be performed under continuous input of emulsion and antisolvent and continuous output of the multi-phase mixture to a decanter.
  • a steady-state partition of the mixture may exist and there can be a continuous and preferably simultaneous removal from each of the phases.
  • the order of these layers will of course vary and the method is not limited to any particular order.
  • the multi-phase (e.g. three phase) mixture may be separated using techniques known in the art, such as a disc stack separator (e.g. a centrifugal separator such as the one manufactured by Andritz).
  • the cooling medium e.g. a medium surrounding the vessel containing the emulsion or used with a heat exchanger connected to the membrane emulsification unit
  • a device such as a recirculating chiller (ThermoFlex available from ThermoFisher Scientific) may, for example, be used to keep the cooling medium at the desired temperature.
  • phase inversion is followed by or involves removal of the polysaccharide beads as described above.
  • Phase inversion may be followed by decanting and then polysaccharide bead removal from the mixture and/or phase inversion may involve mechanical filtration of the wetted beads from the anti-solvent/continuous phase/bead mixture.
  • the polysaccharide beads may be removed from the continuous phase before phase inversion.
  • wetted frozen droplets may be removed from the sol (e.g. using filtration) and then phase inversion carried out to precipitate the polysaccharide and form beads thereof.
  • Microcrystalline cellulose (MCC, from Sigma-Aldrich®) and EmimOAc were dried in a vacuum oven at 80°C for 1 h to remove traces of water.
  • Cellulose solutions were prepared at a concentration of 4-10 wt% MCC in the EmimOAc with 8 wt% deionized water content.
  • the water was first added to the EmimOAc under stirring, followed by the MCC.
  • the mixture was shaken by hand for a minute, then transferred to rollers for 24 h.
  • the samples were placed in a 70°C oven for 24 h, stirred with a spatula, left in the oven for a further 24 h, and then finally transferred to the rollers once again for 24 h.
  • a dispersed phase comprising 6 wt% microcrystalline cellulose and 8 wt% water in 1- ethyl-3-methylimidazolium acetate was prepared according to routine methods known in the art.
  • An oily continuous phase was also prepared according to routine methods known in the art.
  • the dispersed phase and continuous phase were fed into a membrane emulsification unit and an emulsion thereby formed.
  • the emulsion was then transferred into a phase inversion unit with an aqueous anti-solvent to form cellulose beads.
  • Example 1 Cellulose epoxide ring-opening etherification to form Cationic Cellulose Beads (CCBs)
  • the size of the beads of the present disclosure can be readily varied, including to obtain beads of similar size to commercial microcarriers.
  • beads can be prepared by the methods disclosed herein to meet the needs of different applications, for example by varying the cell-carrying capacity of each bead, as well as to be compatible with process and equipment requirements (e.g. separation).
  • Example 3 Cell culture of mammalian adherent cells on CCBs
  • CCBs Cell culture was performed to assess the suitability and adhesion efficiency of CCBs as microcarriers.
  • the cell line used was the murine myoblast C2C12 cell line (ECACC 91031101).
  • the cells were cultured in proliferation media composed of high glucose Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich D5796) supplemented with 10% (v/v) foetal bovine serum (FBS; GibcoTM, Thermo Fisher Scientific) and 1 % (v/v) penicillin/streptomycin (P/S; Sigma-Aldrich).
  • DMEM high glucose Dulbecco's Modified Eagle's Medium
  • FBS foetal bovine serum
  • P/S penicillin/streptomycin
  • Commercial microcarriers were used as a positive control, while unmodified cellulose microbeads were used as negative control.
  • CCBs and controls were equilibrated in culture medium at 37°C, high humidity, and 5% CO2 for at least 15 minutes. Seeding was performed by transferring aliquots of cell suspension (at a known concentration) in a 24-well plate to reach 66,000 cells per well. Cells were grown in the cell culture incubator for one day while kept in suspension on a plate shaker at 100 rpm.
  • Figure 4 and Figure 5 both show that unmodified beads do not exhibit affinity for cell attachment, demonstrating that modification of the cellulose according to the methods disclosed herein is necessary to permit cell binding and proliferation.
  • the beads of the present disclosure enable high densities of cells to be achieved in microcarrier culture while also possessing the advantageous physicochemical and environmental/sustainability characteristics discussed hereinabove.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Polymers & Plastics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Materials Engineering (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

The present disclosure provides methods for preparing functionalised polysaccharide beads. One aspect provides a method for preparing functionalised cellulose beads. Also provided are a method for attaching cells to said functionalised polysaccharide beads, functionalised polysaccharide beads obtained from the methods, and uses thereof.

Description

Functionalised polysaccharide bead preparation
FIELD
[0001] The present disclosure relates generally to methods for preparing polysaccharide beads, and more particularly, to methods for preparing functionalised polysaccharide beads, for example functionalised cellulose beads. The functionalised polysaccharide beads are suitable for, but not limited to, use in cell culture. In particular, the functionalised polysaccharide beads are well suited for use in the culture of cells for purposes such as the production of biopharmaceuticals and food products while having improved environmental characteristics. The present disclosure also provides functionalised beads obtained by the inventive methods, uses thereof, and methods for attaching cells thereto.
BACKGROUND
[0002] Biopolymers such as polysaccharides are an important development in the reduction of the environmental footprint of consumer and industrial products. Biopolymers and biopolymer particles such as beads are often biodegradable as well as being derived from renewable and sustainable raw materials. Cellulose, for example, is the most abundant biopolymer on Earth and can be obtained from many sources, including waste. Products made from biopolymers can thus benefit from closed-loop production processes as part of a circular economic model.
[0003] Cell culture is a critical and ubiquitous method in research and industry for the production of proteins, antibodies, vaccines and the like. Mammalian cell culture is particularly favoured in the ‘biotech’ industry due to its ability to propagate human viruses, express monoclonal antibodies, incorporate necessary post-translational modifications and achieve correct protein folding. However, mammalian cell cultures are typically slow growing and low yielding, particularly compared to bacterial cultures. Thus, industrial-scale cell culture requires the use of extremely large culture volumes to produce the necessary quantities of the desired product, for instance to produce kilogram quantities of a therapeutic monoclonal antibody.
[0004] There is also a rapidly growing interest in the use of cell culture for the production of food products, in particular ‘cultured meat’. Cultured meat, which may also be referred to variously as “healthy meat”, “slaughter-free meat”, “in vitro meat”, “vat-grown meat”, “lab-grown meat”, “cell-based meat”, “clean meat”, “cultivated meat” or “synthetic meat”, is a meat-like product produced by in vitro cultures of animal cells using cell culture and tissue engineering techniques that were originally developed for biotechnology and medicine. Cultured meat has been proposed as an alternative to meat obtained by conventional farming and slaughter as a way to address the environmental impact of meat production, animal welfare, food security, and human health. However, for cultured meat to become commercially viable, significant economies will need to be achieved. In particular, the mass of cells that would need to be produced to meet demand is extremely large, and this would need to be achieved while reaching an acceptable price-point to compete with conventionally produced meat. Given the motivations for pursuing cultured meat discussed above, it must also be ensured that the process for producing cultured meat has a smaller environmental impact than conventional meat production.
[0005] Accordingly, in both biotechnology and the nascent cultured meat industry there is a significant need for products and processes that increase the yield of cell culture, particularly animal cell culture, to increase unit productivity and decrease costs and environmental impacts.
[0006] Microcarrier culture is one way in which the volume of cell culture per unit mass of cells produced can be reduced. Microcarriers are typically spherical particles that provide a substrate within the cell culture upon and/or in which mono- and/or multi-layers of cells can grow. Each microcarrier particle can carry several hundred cells such that expansion capacity of the culture can be multiplied at least several times.
[0007] Microcarriers may be produced from synthetic or natural materials. In most cases, the microcarrier material requires chemical modification to provide favourable interactions (e.g. electrostatic interactions) that facilitate binding of the cells to the microcarrier. Microcarriers must withstand the rigours of the culture environment, for example in a stirred bioreactor. In particular, it is desirable that microcarriers do not contaminate the culture with debris such as fragments of degraded microcarrier material. It is also desirable that microcarriers do not comprise small molecules that could leach into the culture and which may be prohibited, regulated, and/or toxic. The production of food products is strictly regulated and so any production method in this field must be controllable to an extremely high standard.
[0008] In summary, there is a need for microcarriers produced from sustainable, renewable materials without the use of toxic, regulated, and/or prohibited reagents that are suitable for use broadly in cell culture applications, particularly the production of biopharmaceuticals and food products such as cultured meat. The present disclosure meets this need with the various aspects and embodiments defined herein. SUMMARY
[0009] In a first aspect, the present disclosure provides a method for preparing functionalised cellulose beads, said method comprising the steps of:
(i) contacting cellulose beads with a base; and
(ii) reacting the product of step (i) with a salt represented by Formula (I):
A-L-B .X
(I) wherein A is an optionally substituted epoxide group, L is a linker group, B is a quaternary ammonium group, and X is a counterion.
[0010] In a second aspect, the present disclosure provides a method for preparing functionalised polysaccharide beads, said method comprising the steps of:
(i) contacting polysaccharide beads with a base; and
(ii) reacting the product of step (i) with a salt represented by Formula (I):
A-L-B .X
(I) wherein A is an optionally substituted epoxide group, L is a linker group, B is a quaternary ammonium group, and X is a counterion; wherein the polysaccharide beads are prepared by extruding a dispersed phase into an anti-solvent to form beads of the polysaccharide, wherein the dispersed phase comprises the polysaccharide in a solvent, and wherein each of the solvent and anti-solvent comprises water; optionally wherein extruding the dispersed phase into an anti-solvent to form beads of the polysaccharide comprises extruding the dispersed phase through a fluid medium into a mould and then contacting the extruded dispersed phase with the anti-solvent.
[0011] In a third aspect, the present disclosure provides a method for preparing functionalised polysaccharide beads, said method comprising the steps of:
(i) contacting polysaccharide beads with a base; and
(ii) reacting the product of step (i) with a salt represented by Formula (I): A-L-B .X
(I) wherein A is an optionally substituted epoxide group, L is a linker group, B is a quaternary ammonium group, and X is a counterion; wherein the polysaccharide beads are prepared by: a. a membrane emulsification of a dispersed phase into a continuous phase wherein the dispersed phase comprises the polysaccharide in a solvent, and wherein passing the dispersed phase through the membrane forms an emulsion of the polysaccharide in the continuous phase; and b. a phase inversion with an anti-solvent to form beads of the polysaccharide; wherein each of the solvent and anti-solvent comprises water.
[0012] In another aspect, the present disclosure provides a method for attaching cells to functionalised beads, wherein the method comprises preparing functionalised beads by any of the methods described herein and contacting the functionalised beads with one or more cells, preferably wherein the functionalised beads are contacted with one or more cells during cell culture.
[0013] In a further aspect, the present disclosure provides functionalised beads prepared by the methods described herein. Features described herein in the context of the methods are also therefore applicable to the functionalised beads obtained by the methods.
[0014] In a yet further aspect, the present disclosure provides for the use of the functionalised beads obtained by the methods described herein in cell culture. Features described herein in the context of the methods are also therefore applicable to the use.
[0015] These aspects and embodiments are set out in the appended independent and dependent claims. It will be appreciated that features of the dependent claims may be combined with each other and with features of the independent claims in combinations other than those explicitly set out in the claims. Furthermore, the approaches described herein are not restricted to specific embodiments such as those set out below, but include and contemplate any combinations of features presented herein.
[0016] The foregoing and other objects, features, and advantages of the present disclosure will appear more fully hereinafter from a consideration of the detailed description that follows along with the accompanying drawings. It is to be expressly understood, however, that the drawings are for illustrative purposes and are not to be construed as defining the limits of the disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 is a schematic representation of an embodiment wherein the polysaccharide beads are prepared by extruding a dispersed phase into an anti-solvent.
[0018] Figure 2 contains a schematic representation of an embodiment wherein the polysaccharide beads are prepared by membrane emulsification (Figure 2(a)), and a representation of a further embodiment wherein the emulsion is cooled as described herein (Figure 2(b)).
[0019] Figure 3 shows the change in charge density (expressed as milliequivalents per gram (meq/g) as defined herein) as well as the dry weight (expressed as the percentage of dry cellulose per unit of hydrated cellulose mass) with reaction time (expressed in hours) as determined for the functionalisation of cellulose beads with GTMAC according to Examples 2 and 3 of the present disclosure. Charge density values are indicated with diamonds, and dry weight values as squares. Error bars represent standard deviations (n = 3).
[0020] Figure 4 shows z-stack projections of images of stained C2C12 cells attached to functionalised cellulose beads prepared according to the Examples described herein, as well as unmodified cellulose beads and a commercial microcarrier. Bright white spots represent live cells stained with fluorescein diacetate. The cells were stained and images taken after four days of culture.
[0021] Figure 5 shows the degree of cell attachment determined by cell counting for the functionalised cellulose beads according to the Examples, unmodified cellulose beads and a commercial microcarrier after 1 , 4, and 7 days of culture according to the Examples herein. Figure 5(a) compares the number of cells per bead, and Figure 5(b) compares the number of cells per unit surface area (cells/mm2). Error bars represent means of standard deviations determined from three biological replicates (cell cultures). For each biological replicate cells were counted on 100 beads, with the exception of the large CCBs where the cells on 25 beads were counted. DETAILED DESCRIPTION
[0022] While various exemplary embodiments are described or suggested herein, other exemplary embodiments utilizing a variety of methods and materials similar or equivalent to those described or suggested herein are encompassed by the general inventive concepts. Those aspects and features of embodiments which are implemented conventionally may not be discussed or described in detail in the interests of brevity. It will thus be appreciated that aspects and features of apparatus and methods described herein which are not described in detail may be implemented in accordance with any conventional techniques for implementing such aspects and features.
[0023] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
[0024] In this specification, unless otherwise stated, the term "about" modifying the quantity of a component refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making concentrates, mixtures or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the materials employed, or to carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents to the quantities.
[0025] The ranges provided herein provide exemplary amounts of each of the components. Each of these ranges may be taken alone or combined with one or more other component ranges.
[0026] As used herein, the term “at least” includes the end value of the range that is specified. For example, “at least 10 cm” includes the value 10 cm.
[0027] As used herein, wt% means “weight percentage” as the basis for calculating a percentage. Unless indicated otherwise, all % values are calculated on a weight basis, and are provided with reference to the total weight of the product in which the substance is present. For example, % water in the solvent of the dispersed phase refers to the wt% water based on the total weight of the solvent. Similarly, % polysaccharide in the dispersed phase refers to wt% polysaccharide based on the total weight of the dispersed phase. [0028] As used herein, w/v means “weight by volume” as the basis for calculating a percentage. Similarly, v/v means “volume by volume” as the basis for calculating a percentage.
[0029] As used herein, “substantially free” means no more than trace amounts, i.e. the amount of the substance(s) concerned is negligible. In various embodiments, “substantially free” means no more than 1000 ppm, preferably no more than 100 ppm, more preferably no more than 10 ppm, even more preferably no more than 1 ppm of the substance(s) concerned.
[0030] In all aspects of the present disclosure, the disclosure includes, where appropriate, all enantiomers and tautomers of the compounds disclosed herein. A person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
[0031] Some of the compounds disclosed herein may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present disclosure contemplates the use of all the individual stereoisomers and geometric isomers of those compounds, and mixtures thereof. The terms used in the claims encompass these forms.
[0032] As used herein, the term “hydrocarbyl” refers to a group comprising at least C and H. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon. Non-limiting examples of such hydrocarbyls are alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, and isomeric forms thereof; cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloocytyl, 2-methylcyclopentyl, 2,3-dimethyl- cyclobutyl, 4-methylcyclobutyl, 3-cyclopentylpropyl, and the like; cycloalkenyl groups, such as cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like, and isomeric forms thereof; cycloalkadienyl groups, such as cyclopentadientyl, cyclohexadienyl, cycloheptadienyl, and the like; aryl groups, such as phenyl, tolyl, xylyl, naphthyl, biphenylyl, and the like; aralkyl groups, such as benzyl, phenethyl, phenpropyl, naphthmethyl, and the like. The hydrocarbyl group may be an aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group. [0033] In other embodiments, the term “hydrocarbyl” refers to a group having carbon atoms directly attached to the remainder of the molecule, wherein the group consists of carbon and hydrogen atoms. For example, the hydrocarbyl group may be an aliphatic or aromatic group. In such embodiments, non-limiting examples of such hydrocarbyls are alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, and isomeric forms thereof; cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloocytyl, 2-methylcyclopentyl, 2,3-dimethyl-cyclobutyl, 4-methylcyclobutyl, 3- cyclopentyl propyl, and the like; cycloalkenyl groups, such as cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like, and isomeric forms thereof; cycloalkadienyl groups, such as cyclopentadientyl, cyclohexadienyl, cycloheptadienyl, and the like; aryl groups, such as phenyl, tolyl, xylyl, naphthyl, biphenylyl, and the like; aralkyl groups, such as benzyl, phenethyl, phenpropyl, naphthmethyl, and the like. The hydrocarbyl group may be an aryl, alkyl, cycloalkyl, aralkyl or alkenyl group.
[0034] As used herein, the term “alkyl” includes both saturated straight chain and branched alkyl groups which may be substituted (mono- or poly-) or unsubstituted. Preferably, the alkyl group is a C1.20 alkyl group, more preferably a C1.15, more preferably still a C1.12 alkyl group, more preferably still, a Ci-e alkyl group, more preferably a C1.3 alkyl group. Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. Suitable substituents include, for example, one or more groups selected from OH, O-alkyl, halogen, NH2, NH-alkyl, N-(alkyl)2, CF3, NO2, CN, COO-alkyl, COOH, CONH2, CO-NH-alkyl, CO-N(alkyl)2, SO2-alkyl, SO2NH2 and SO2-NH-alkyl. Preferable substituents include one or more groups selected from OH, O-alkyl, halogen other than fluorine, NH2, NH- alkyl, N-(alkyl)2, NO2, CN, COO-alkyl, COOH, CONH2, CO-NH-alkyl, CO-N(alkyl)2, SO2-alkyl, SO2NH2 and SO2-NH-alkyl. More preferable substituents include one or more groups selected from OH, O-alkyl, chlorine, NH2, NH-alkyl, N-(alkyl)2, NO2, CN, COO-alkyl, COOH, CONH2, CO-NH-alkyl, CO-N(alkyl)2, SO2-alkyl, SO2NH2 and SO2-NH-alkyl. Even more preferable substituents include one or more groups selected from OH, O-alkyl, NH2, NH-alkyl, N-(alkyl)2, NO2, CN, COO-alkyl, COOH, CONH2, CO-NH-alkyl, CO-N(alkyl)2.
[0035] As used herein, the term “aryl” refers to a Ce-12 aromatic group which may be substituted (mono- or poly-) or unsubstituted. Typical examples include phenyl and naphthyl etc. Suitable substituents include, for example, one or more groups selected from OH, O-alkyl, halogen, NH2, NH-alkyl, N-(alkyl)2, CF3, NO2, CN, COO-alkyl, COOH, CONH2, CO-NH-alkyl, CO- N(alkyl)2, SO2-alkyl, SO2NH2 and SO2-NH-alkyl. [0036] The term “aralkyl” is used as a conjunction of the terms alkyl and aryl as given above.
[0037] Cyclic alkyl groups, may be referred to as “cycloalkyl” and include those with 3 to 10 carbon atoms having single or multiple fused rings. Non-limiting examples of cycloalkyl groups include adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
[0038] The term “alkenyl” refers to both straight and branched carbon chains which have at least one carbon-carbon double bond. In some embodiments, alkenyl groups may include C2- C12 alkenyl groups. In other embodiments, alkenyl includes C2-C10, C2-C8, C2-C6 or C2-C4 alkenyl groups. In one embodiment of alkenyl, the number of double bonds is 1-3; in another embodiment of alkenyl, the number of double bonds is one. Other ranges of carbon-carbon double bonds and carbon numbers are also contemplated depending on the location of the alkenyl moiety on the molecule. “C2-C -alkenyl” groups may include more than one double bond in the chain. Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1- methyl-ethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1 -propenyl, 2-methyl-1-propenyl, 1- methyl-2-propenyl, 2-methyl-2-propenyl; 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1- methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2- butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1 ,1- dimethyl-2-propenyl, 1 ,2-dimethyl-1-propenyl, 1 ,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1- ethyl-2-propenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1 -pentenyl,
2-methyl- 1-pentenyl, 3-methyl-1 -pentenyl, 4-methyl- 1-pentenyl, 1-methyl-2-pentenyl, 2- methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl-3-pentenyl, 2-methyl-
3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4- pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1 ,1-dimethyl-2-butenyl, 1 ,1-dimethyl-3- butenyl, 1 ,2-dimethyl-1-butenyl, 1 ,2-dimethyl-2-butenyl, 1 ,2-dimethyl-3-butenyl, 1 ,3-dimethyl- 1-butenyl, 1 ,3-dimethyl-2-butenyl, 1 ,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3- dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1- butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1 ,1 ,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2- propenyl, 1-ethyl-2-methyl-1-propenyl and 1-ethyl-2-methyl-2-propenyl.
[0039] “Heteroaryl” refers to a monovalent aromatic group of from 1 to 15 carbon atoms, preferably from 1 to 10 carbon atoms, having one or more oxygen, nitrogen, and sulfur heteroatoms within the ring, preferably 1 to 4 heteroatoms, or 1 to 3 heteroatoms. The nitrogen and sulfur heteroatoms may optionally be oxidized. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple fused rings provided that the point of attachment is through a heteroaryl ring atom. Examples of heteroaryls include pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, indolyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinnyl, furanyl, thiophenyl, furyl, pyrrolyl, imidazolyl, oxazolyl, isoxazolyl, isothiazolyl, pyrazolyl benzofuranyl, benzothiophenyl, imidazopyridyl, imidazopyrimidyl, or pyrrolopyrimidyl. Heteroaryl rings may be unsubstituted or substituted by one or more moieties as described for aryl above.
[0040] “Alkoxy” refers to alkyl-O-, wherein alkyl is as defined above. Examples of Ci-Ce-alkoxy include, but are not limited to, methoxy, ethoxy, OCH2-C2H5, OCH(CHs)2, n-butoxy, OCH(CHs)- C2H5, OCH2-CH(CHS)2, OC(CHS)3, n-pentoxy, 1 -methylbutoxy, 2-methylbutoxy, 3- methylbutoxy, 1 ,1 -dimethylpropoxy, 1 ,2-dimethylpropoxy, 2,2-dimethyl-propoxy, 1- ethylpropoxy, n-hexoxy, 1 -methylpentoxy, 2-methylpentoxy, 3-methylpentoxy, 4- methyl pentoxy, 1 ,1-dimethylbutoxy, 1 ,2-dimethylbutoxy, 1 ,3-dimethylbutoxy, 2,2- dimethylbutoxy, 2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1 -ethylbutoxy, 2-ethylbutoxy, 1 ,1 ,2-trimethylpropoxy, 1 ,2,2-trimethylpropoxy, 1-ethyl-1-methylpropoxy, 1-ethyl-2- methylpropoxy and the like.
[0041] The general inventive concept is centred on providing a method for preparing functionalised polysaccharide beads, for example functionalised cellulose beads, where the functionalised polysaccharide beads are suitable for use in applications in, but not limited to, cell culture, biomedicine, biomanufacture, pharmaceuticals, as well as food production such as the production of cultured meat and have improved environmental benefits. In particular, the polysaccharide beads of the present disclosure are functionalised by reaction with compounds comprising quaternary ammonium groups, which alters the properties of the polysaccharide beads in order to enable or improve their suitability for use in one or more of the above-mentioned applications.
[0042] Thus, in each of the methods of the first, second, and third aspects described herein, polysaccharide/cellulose beads are (i) contacted with a base; and in step (ii) the product of step (i) is reacted with a salt represented by Formula (I):
A-L-B .X
(I) wherein A is an optionally substituted epoxide group, L is a linker group, B is a quaternary ammonium group, and X is a counterion. [0043] In the present disclosure, it has been found that the functionalisation of the polysaccharide beads described herein confers advantageous physicochemical properties over unmodified polysaccharide beads. In particular, the cationic functionalisation of polysaccharide beads according to the methods disclosed herein has been found to facilitate the binding of cells to said beads and thereby facilitate the proliferation of said cells. Moreover, functionalisation of the polysaccharide beads with quaternary ammonium moieties ensures cell attachment under a range of culture and process conditions such as pH, temperature, CO2 concentration, and stirring. In particular, the quaternary ammonium moieties remain ionised over a broad pH range. Further, use of the beads as microcarriers in cell culture can protect the cells from shear forces in the culture (e.g. from stirring) and thus reduce stress on the cells and improve proliferation.
[0044] The functionalised beads of the present disclosure have notably been found to be mechanically robust even in the absence of cross-linking. This is in contrast to many commercially available microcarriers which are cross-linked materials. The functionalised beads of the present disclosure can be produced from sustainable, renewable, and cost- effective materials such as cellulose. Moreover, functionalisation of the polysaccharide beads by the methods described herein can be performed in innocuous solvent systems (e.g. aqueous solvents) and with non-toxic reagents that do not lead to the production of harmful side-products. The size of the beads may also be tuned over a wide range to be suitable for specific applications.
[0045] In view of the advantageous characteristics described above, the functionalised beads described herein are particularly suitable for use as microcarriers in cell culture. In particular, beads made of polysaccharides such as cellulose may be particularly suitable for food applications of cell culture such as cultured meat because cellulose is a non-toxic, naturally occurring polysaccharide that is safe to eat. Cellulose from plant material constitutes insoluble dietary fibre in natural food products, for example. Further, the methods for preparing the functionalised beads described herein allow the use of mild, aqueous processes and nonharmful reagents such that the functionalised beads may be used in downstream applications such as food culture without the need for extensive and costly purification before use.
[0046] For ease of reference, these and further features of the present disclosure are now discussed under appropriate section headings. However, the teachings under each section are not limited to the section in which they are found. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Polysaccharide beads
[0047] All aspects of the present disclosure concern polysaccharide beads, for example cellulose beads. Polysaccharides are biopolymers (polymers produced by living organisms, i.e. polymeric biomolecules). Polysaccharides are typically polymeric carbohydrate structures. In various embodiments of the present disclosure, the polysaccharide may be, for example, starch, cellulose, chitin, and chitosan. Even more preferably, the polysaccharide is starch or cellulose. Most preferably, the polysaccharide is cellulose.
[0048] Cellulose is a linear polymer made up of p-D-glucopyranose units covalently linked with 1^4 glycosidic bonds. Cellulose may be obtained from many different sources and the present disclosure is not necessarily limited as to the origin, form, or other characteristics of the cellulose. Cellulose is typically obtained from plant sources, for example from virgin or recycled wood pulp. Pulp is a lignocellulosic fibrous material prepared by chemically or mechanically separating cellulose fibers from wood, fiber crops, waste paper, or rags. Cellulose may be obtained from virgin sources or advantageously from recycled sources.
[0049] In some embodiments, the cellulose is selected from the group consisting of virgin, recycled, pulp, and microcrystalline cellulose, and combinations thereof. In some embodiments, the cellulose is virgin cellulose. In some embodiments, the cellulose is recycled cellulose. In some embodiments, the cellulose is pulp cellulose. In some embodiments, the cellulose is microcrystalline cellulose.
[0050] Microcrystalline cellulose (MCC) is typically made from high-grade, purified wood or cotton cellulose. Hydrolysis is used to remove amorphous cellulose until the microcrystalline form remains. With its amorphous cellulose portions removed, it becomes an inert, white, free- flowing powder. It can be processed in a number of ways, for example through reactive extrusion, steam explosion, and acid hydrolysis. An example of a commercially available MCC is Avicel® produced by DuPont.
[0051] The term “bead” as used herein refers to a discrete entity with defined size and shape that does not have any dimension or dimensions substantially greater than any other dimension and which may be described as “spherical” or “substantially spherical”. A person skilled in the art is readily able to identify a “bead” as opposed to e.g. an elongate particle. In other words, the term “bead” as used herein does not include elongate particles such as (nano/micro)fibres, (nano/micro)fibrils and the like, nor does said term include flat/sheet-like structures such as membranes and the like. As used herein, embodiments referring to “beads” as such apply equally to each of the aspects of the present disclosure unless specifically indicated and include any polysaccharide bead as encompassed by the present disclosure.
[0052] In various embodiments of the present disclosure, the beads are approximately spherical. Approximately spherical beads may be advantageous in certain applications, for example in biomanufacture and food production, where such a shape can facilitate industrial processing and recovery processes such as filtration.
[0053] The size of the beads of the present disclosure is not limited, and the skilled person will be able to select sizes according to a desired application. The size of the beads may be readily identified by a person skilled in the art, for example, using an optical microscope image and image analysis software with a suitable detection algorithm (e.g. Imaged using an edge detection algorithm), laser diffraction with commercially available equipment such as Mastersizer from Malvern Panalytical (e.g. Mastersizer 3000), with an appropriately sized sieve, or by using a caliper.
[0054] As recited herein, “diameter” takes its usual meaning and is used in relation to approximately spherical beads. Thus, the skilled person will understand that the diameter of an approximately spherical bead as recited herein will be approximately the same when measured in any direction through the centre of said bead. In some embodiments, the beads may have a diameter of at least about 1 pm. In some embodiments, the beads may have a diameter of at least about 10 pm. In some embodiments, the beads may have a diameter of at least about 25 pm. In some embodiments, the beads may have a diameter of at least about 50 pm. In some embodiments, the beads may have a diameter of at least about 80 pm. In some embodiments, the beads may have a diameter of at least about 100 pm.
[0055] In some embodiments, the beads may have a diameter of less than about 5 mm. In some embodiments, the beads may have a diameter of less than about 4 mm. In some embodiments, the beads may have a diameter of less than about 3 mm. In some embodiments, the beads may have a diameter of less than about 2 mm. In some embodiments, the beads may have a diameter of less than about 1 mm.
[0056] In various embodiments, the beads have a diameter of from about 1 pm to about 3 mm. In various embodiments, the beads have a diameter of from about 10 pm to about 3 mm. In various embodiments, the beads have a diameter of from about 25 pm to about 3 mm. In various embodiments, the beads have a diameter of from about 50 pm to about 3 mm. In various embodiments, the beads have a diameter of from about 80 pm to about 3 mm. [0057] In various embodiments, the beads have a diameter of from about 0.1 mm to about 3 mm. In various embodiments, the beads have a diameter of from about 0.15 mm to about 3 mm. In various embodiments, the beads have a diameter of about 0.2 mm to about 3 mm.
[0058] In various embodiments, larger beads may be preferred to facilitate their use in industrial processes, for example for ease of separation. Thus, in various embodiments, the beads may have a diameter greater than or equal to about 0.2 mm. In various embodiments, the beads may have a diameter of from about 0.2 mm to about 3 mm, from about 1 to about 3 mm or from about 1 to about 2 mm.
[0059] Polysaccharide beads may be obtained from various methods of production in a form wherein said beads are wetted or immersed in a solvent such as water. Such beads may be referred to as “wet” beads and may be provided in this form for further use. Alternatively, beads may be subsequently dried to provide “dry beads”. In the present disclosure, it is preferable that the unmodified beads, i.e. the starting material in the methods of the present disclosure have not previously been dried. On the other hand, it has been advantageously found that the functionalised beads obtained by the methods of the present disclosure can be dried and then re-suspended, e.g. in water, such that the dried functionalised beads fully regain their prior hydration/solvation and the size, shape, and structure of the beads are unaffected by the drying and rehydration/resolvation steps.
[0060] In various embodiments of the disclosure, the beads are in the form of a hydrogel for step (i) of the methods of the present disclosure. A hydrogel is defined by IIIPAC as a gel in which the swelling agent is water. As used herein, the term “hydrogel” means a non-fluid polymer network (i.e. formed by the polysaccharide) that is expanded throughout its whole volume by a fluid, namely water.
[0061] Polysaccharide beads in the form of a hydrogel may be preferred as they can provide a higher effective surface area for reaction with base according to step (i) of the functionalisation methods described herein. In turn, an increased effective surface area leads to an increased density of functional groups for subsequent functionalisation with quaternary ammonium groups in step (ii) of the methods described herein. In particular, the enhanced effective surface area of the functionalised beads may lead to an enhanced degree of cell attachment and thus higher cell culture densities. Thus, good cell growth, increased cell densities and/or increased yields in culture may be achieved with beads in the form of a hydrogel compared to non-hydrogel beads such as solid beads. [0062] Advantageously, the polysaccharide beads of the present disclosure are mechanically robust, even in the absence of additional cross-linking. As used herein, “cross-linking” refers to the formation of additional bonds other than the bonds linking monomer units in the polysaccharide by reaction with a cross-linking agent, where said additional bonds may be formed between functional groups in the same polysaccharide chain or between functional groups in neighbouring (i.e. different) polysaccharide chains.
[0063] Mechanical robustness may be important in industrial applications, for example use in stirred reactors or other process steps where it is important that the beads maintain their integrity and avoid degradation that might contribute debris and thus require additional removal/purification steps, as well as prolonging the useful life of the beads. Thus, in various embodiments, the beads of the present disclosure are not cross-linked.
[0064] Nonetheless, the present disclosure is not limited in this regard; for some applications, it may be desired to cross-link the polysaccharide in the beads of the present disclosure. Thus, in alternative embodiments, the beads of the present disclosure are cross-linked. In various embodiments, the polysaccharide beads are cross-linked by reaction with a cross-linking agent. Cross-linking agents are typically multi-functional molecules that comprise more than one reactive group capable of reacting with functional groups on the polysaccharide. In various embodiments, the functional groups on the polysaccharide that react with the cross-linking agent are hydroxyl groups present in the saccharide monomers of the polysaccharide, for example secondary hydroxyl groups. Examples of reactive functional groups capable of reacting with such groups on the polysaccharide include but are not limited to aldehyde groups, acid groups, acyl halide (e.g. chloride) groups, halide groups, and epoxide groups. In various embodiments the cross-linking agent is selected from the group consisting of formaldehyde; methylolated nitrogen compounds such as dimethylolurea, dimethylolethyleneurea, and dimethylolimidazolidone; dicarboxylic acids such as maleic acid; dialdehydes such as glyoxal and glutaraldehyde; diepoxides; diioscyanates; divinyl compounds such as divinyl sulfone, dihalogen containing compounds such as dichloroacetone and 1 ,3-dichloropropan-2-ol, and halohydrins such as epichlorohydrin. In various embodiments the cross-linking agent is selected from the group consisting of methylolated nitrogen compounds such as dimethylolurea, dimethylolethyleneurea, and dimethylolimidazolidone; dicarboxylic acids such as maleic acid; dialdehydes such as glyoxal and glutaraldehyde; diepoxides; diioscyanates; divinyl compounds such as divinyl sulfone, dihalogen containing compounds such as dichloroacetone and 1 ,3-dichloropropan-2-ol, and halohydrins such as epichlorohydrin. In further embodiments, the cross-linking agent is selected from the group consisting of methylolated nitrogen compounds such as dimethylolurea, dimethylolethyleneurea, and dimethylolimidazolidone; dicarboxylic acids such as maleic acid; dialdehydes such as glyoxal and glutaraldehyde; diepoxides; diioscyanates; divinyl compounds such as divinyl sulfone.
Figure imgf000017_0001
of beads with base
[0065] In step (i) of the methods of the present disclosure, the beads are contacted with a base. The term “base” as used herein takes its usual meaning in the chemical arts, for instance a chemical species or molecular entity having an available pair of electrons capable of forming a covalent bond with a proton or with a vacant orbital of some other species. The base is not limited, and the skilled person will be able to select bases appropriate to the polysaccharide beads being contacted with said base.
[0066] Treatment of polysaccharide beads with base results in the deprotonation of hydroxyl groups present in the saccharide subunits of the polysaccharide of the beads as illustrated in Scheme 1 for cellulose (“Cell”).
Cell — o .H + OH <3 _ ► Cell — o <9
Scheme 1
[0067] The propensity of a hydroxyl group in a polysaccharide to be removed by a base can be measured and/or predicted in terms of its pKa. The definition of pKa, the calculation/measurement thereof, and the use of said parameter in relation to acid/base reactions is specifically part of the common general knowledge of a person skilled in the art of the present disclosure. A lower pKa corresponds to a stronger acid, i.e. where the proton in question is more weakly held and thus more easily removed by a base. A person skilled in the chemical arts will understand that hydroxyl groups are typically considered weak acids. Moreover, secondary hydroxyl groups will typically be weaker acids (have a higher pKa) than primary hydroxyl groups. The primary hydroxyl groups in cellulose, for example, are believed to have a pKa of approximately 12.5 in water; however, this may vary, for example depending on the degree of deprotonation of the cellulose polymer and/or the source of the cellulose.
[0068] Basicity may be measured/predicted in terms of the pKa of the corresponding conjugate acid (pKaH). For example, the base of the present disclosure may have a pKaH of at least about 12, at least about 13, at least about 14, or at least about 15. In various embodiments of the present disclosure, it is desirable to maximise the degree of deprotonation of hydroxyl groups in the beads, e.g. to maximise in turn the degree of functionalisation in step (ii) of the methods disclosed herein. Accordingly, in some embodiments the base is a strong base. Strong bases are generally considered to be those that are fully dissociated in aqueous solution. For the purposes of the present disclosure, a strong base may be a base having a pKan greater than the pKa of at least one hydroxyl group on the saccharide repeat unit of the polysaccharide. For instance, in various embodiments a strong base as used in the present disclosure has a pKan of at least 12.5.
[0069] In various embodiments, the base is a hydroxide salt. In embodiments where the methods of the present disclosure are performed in aqueous solution, the base is preferably a hydroxide salt that is soluble in said aqueous solution under the conditions of the reaction as described herein. Thus, in various embodiments the base may be a Group I hydroxide. For example, the base may be potassium hydroxide or sodium hydroxide. Preferably, the base is sodium hydroxide.
[0070] As well as being strong bases, hydroxide salts may also be advantageous for certain applications because they form non-toxic water upon deprotonation of the polysaccharide hydroxyl groups. Further, alkali metal ions such as sodium from sodium hydroxide will typically contribute non-toxic dissolved salts (e.g. NaCI) in combination with counterions such as chloride, which salts are either allowable in the final product or which can be readily removed from the product such as by washing and sieving as discussed in more detail below.
[0071] In various embodiments, the base will dissolved in an appropriate solvent prior to contacting the beads with the basic solution. In various embodiments, the method is carried out in an aqueous solvent, and thus in further embodiments the base is dissolved in said aqueous solvent prior to contacting the beads with the solution of the base in the aqueous solvent. For example, the beads may be (re-)suspended in said solution. Alternatively the beads are in the form of a hydrogel and contacted with the basic solution.
[0072] The concentration of the base in solution prior to contacting the beads is not limited and the skilled person will be able to select an appropriate concentration based on their common general knowledge. In various embodiments, the base is present in solution at a concentration of at least about 0.02 M, at least 0.05 M, or at least 0.1 M prior to contacting said solution with the beads according to step (i). In various embodiments, the method is carried out in aqueous solvent and the base is present in solution in said aqueous solvent at a concentration of at least about 0.02 M, at least 0.05 M, or at least 0.1 M prior to contacting said solution with the beads according to step (i). In various embodiments, the method is carried out in aqueous solvent and the base is a hydroxide salt that is present in solution in said aqueous solvent at a concentration of at least about 0.02 M, at least 0.05 M, or at least 0.1 M prior to contacting said solution with the beads according to step (i).
[0073] In various embodiments, the base is present in solution at a concentration of up to 0.2 M prior to contacting said solution with the beads according to step (i). Thus, in various embodiments the base is present in solution at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i). In various embodiments, the method is carried out in aqueous solvent and the base is present in solution in said aqueous solvent at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i).
[0074] In various embodiments, the base has a pKan of at least 12.5 and is present in solution at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i). In various embodiments, the method is carried out in aqueous solvent, the base has a pKaH of at least 12.5 and said base is present in solution in said aqueous solvent at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i).
[0075] In various embodiments, the method is carried out in aqueous solvent and the base is a hydroxide salt that is present in solution in said aqueous solvent at a concentration of from about 0.02 M, from about 0.05 M, or from about 0.1 M to about 0.2 M prior to contacting said solution with the beads according to step (i).
Functionalisation reactions
[0076] In step (ii) of the methods of the present disclosure, the product of step (i) is reacted with a salt represented by Formula (I):
A-L-B .X
(I) wherein A is an optionally substituted epoxide group, L is a linker group, B is a quaternary ammonium group, and X is a counterion.
[0077] As used herein, the term “linker group” refers to any moiety covalently attached to both A and B. For example, the linker group may be an optionally substituted hydrocarbyl group as defined hereinabove. In some embodiments, the linker group is an unsubstituted hydrocarbyl group as defined hereinabove. In some embodiments, the linker group is an optionally substituted aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group.
[0078] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
[0079] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
[0080] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H and optionally substituted alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
[0081] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
[0082] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
[0083] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
[0084] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H and methyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1 .
[0085] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H and optionally substituted aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
[0086] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H and optionally substituted phenyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1.
[0087] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is H. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, m is 1 and each R4 and R5 is H.
[0088] In any of the above embodiments of the linker group L, suitable substituents include one or more groups selected from OH, O-alkyl, NH2, NH-alkyl, N-(alkyl)2, NO2, CN, COO-alkyl, COOH, CONH2, CO-NH-alkyl, CO-N(alkyl)2.
[0089] In other embodiments, the linker group may be a polyether group such as those derived from ethylene glycol or a polyethylene glycol. For example, the linker group may have the formula [O-(CH2)n]o wherein n is an integer from 1 to 5 and o is an integer from 1 to 10. In various embodiments, n is an integer from 1 to 4, 1 to 3, or 1 to 2, and o is an integer from 1 to 10. In various embodiments, n is 2 and o is an integer from 1 to 10. In various embodiments, n is an integer from 1 to 5 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In various embodiments, n is an integer from 1 to 5 and o is 1. In various embodiments, n is an integer from 1 to 4, 1 to 3, or 1 to 2, and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In various embodiments, n is 2 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
[0090] Quaternary ammonium cations are positively charged polyatomic ions with the structure N+R1R2R3R4 wherein each R1, R3, R3, and R4 is independently alkyl, aryl, and aralkyl. In various embodiments of the present disclosure, the quaternary ammonium group B has the formula -N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aralkyl, and aryl. In various embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In various embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In further embodiments, each R1, R2, and R3 is independently alkyl. In yet further embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl, preferably C1-C3 alkyl. For instance, each R1, R2, and R3 may be independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In some embodiments, each R1, R2, and R3 is methyl. [0091] In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted phenyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0092] In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted phenyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0093] In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted phenyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci- Ce alkyl and aryl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1 . In some embodiments, m is 1 and each R4 and R5 is H.
[0094] In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted phenyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently alkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently alkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0095] In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted phenyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, methyl, ethyl, n- propyl, and isopropyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0096] In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from C1-C3 alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from C1-C3 alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from C1-C3 alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from C1-C3 alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted phenyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from C1-C3 alkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from C1-C3 alkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, methyl, ethyl, n- propyl, and isopropyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from C1-C3 alkyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from C1-C3 alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0097] In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n- propyl and isopropyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted phenyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0098] In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is methyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted Ci-e alkyl and optionally substituted aryl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is methyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is methyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted Ci-e alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is methyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted phenyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is methyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H and optionally substituted C1.3 alkyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is methyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H, methyl, ethyl, n-propyl, and isopropyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is methyl. In various embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10, each R4 and R5 is independently selected from H and methyl, and the quaternary ammonium group B has the formula N+R1R2R3 wherein each R1, R2, and R3 is methyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0099] In various embodiments, the epoxide group A is substituted with 1 , 2 or 3 alkyl groups, preferably wherein each alkyl group is independently selected from Ci-Ce alkyl, more preferably wherein each alkyl group is independently selected from C1-C3 alkyl. More preferably, the epoxide group A is unsubstituted.
[0100] As used herein, “counterion” means an ion that accompanies another ionic species in order to maintain electric neutrality. Thus, as the quaternary ammonium group bears a single positive charge under the conditions described herein, in various embodiments the counterion X is a monovalent anion. For example, in various embodiments X is a halide ion. Preferably, X is a halide ion other than fluoride. More preferably, X is a chloride ion.
[0101] In further embodiments, the salt represented by Formula (I) is a salt represented by Formula (la):
.X
Figure imgf000028_0001
wherein each of R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl, and X' is halide. In various embodiments, each of R1, R2, and R3 is independently selected from Ci- Ce alkyl, aryl, and aralkyl. L is the linker group as defined above.
[0102] In various embodiments of the salt represented by Formula (la), each R1, R2, and R3 is independently selected from alkyl and aryl. In various embodiments of the salt represented by Formula (la), each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, each R1, R2, and R3 is independently alkyl. In yet further embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl, preferably C1-C3 alkyl. For instance, each R1, R2, and R3 may be independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In some embodiments, each R1, R2, and R3 is methyl.
[0103] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl. Accordingly, in various embodiments the salt represented by Formula (I) is a salt represented by Formula (lb):
Figure imgf000029_0001
wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and X' is halide. In various embodiments, each R1, R2, and R3 is independently selected from alkyl and aryl. In various embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl.
[0104] In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0105] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0106] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0107] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently selected from C1-C3 alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0108] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0109] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and m optionally substituted aryl, and each R1, R2, and R3 is methyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0110] In other embodiments, the linker group may be a polyether group such as those derived from ethylene glycol or a polyethylene glycol. For example, the linker group may have the formula [O-(CH2)n]o wherein n is an integer from 1 to 5 and o is an integer from 1 to 10. In various embodiments, n is an integer from 1 to 4, 1 to 3, or 1 to 2, and o is an integer from 1 to 10. In various embodiments, n is 2 and o is an integer from 1 to 10. In various embodiments, n is an integer from 1 to 5 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In various embodiments, n is an integer from 1 to 5 and o is 1. In various embodiments, n is an integer from 1 to 4, 1 to 3, or 1 to 2, and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In various embodiments, n is 2 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
[0111] In various embodiments, the salt represented by Formula (I) is a salt represented by Formula (Ic):
Figure imgf000030_0001
wherein each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl, and X’ is halide. In various embodiments, each R1, R2, and R3 is independently selected from alkyl and aryl. In various embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aryl, and aralkyl. In various embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl.
[0112] In various embodiments, each R1, R2, and R3 is independently alkyl. In further embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In yet further embodiments, each R1, R2, and R3 is independently selected from C1-C3 alkyl. In yet further embodiments, each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In any of the foregoing, at least two of R1, R2, and R3 may be different. In other embodiments, each of R1, R2, and R3 are different. In yet further embodiments, each R1, R2, and R3 is methyl.
[0113] In any of the embodiments described herein, X- is preferably chloride (Cl’).
[0114] Thus, in an exemplary embodiment, the salt represented by Formula (I) is glycidyltrimethylammonium chloride (GTMAC; also known as 2,3- epoxypropyl)trimethylammonium chloride). Said salt is commercially available (CAS Number: 3033-77-0), for example from Sigma-Aldrich (Merck KGaA).
[0115] In the methods of the present disclosure, the optionally substituted epoxide group A reacts in step (ii) with the deprotonated hydroxyl groups of the beads formed by step (i). This type of reaction is commonly referred to as a ring-opening etherification and results in in the quaternary ammonium group being covalently attached to the polysaccharide beads via the linker group L and a residue derived from reaction of the epoxide group with the deprotonated hydroxyl group of the polysaccharide. An exemplary reaction where the salt is glycidyltrimethylammonium chloride and the polysaccharide is cellulose is illustrated in Scheme 2:
Figure imgf000031_0001
Scheme 2
[0116] As can be seen from Scheme 2, the ring-opening etherification mechanism means that the beads are functionalised with quaternary ammonium groups in a direct coupling reaction without condensation, i.e. without the release of leaving groups, for example, that would contribute additional species to the reaction mixture. Functionalisation of beads without forming condensation products may be advantageous for various applications where such condensation products may be prohibited or regulated, e.g. in food products, and would thus need to be removed in additional purification steps that would reduce efficiency and increase costs of the process.
[0117] In some embodiments of the present disclosure, the salt of Formula (I), for example a salt of Formula (la), (lb), (Ic), or glycidyltrimethylammonium chloride is produced in situ. In such embodiments, a precursor compound is added to the product of step (i) such that the precursor reacts to form a salt of Formula (I) or embodiments thereof as discussed herein and then said salt thus formed proceeds to react with the deprotonated beads formed in step (i) to yield the functionalised beads of the present disclosure.
[0118] For example, the salt represented by Formula (la) may be produced in situ by reacting the product of step (i) with a compound of Formula (Ila):
OH
Y\ ,NR1R2R3 y-
(Ha) wherein X; R1, R2, and R3 are as defined herein, L is the linker group as defined herein and Y is a halogen.
[0119] The reaction of the compound of Formula (Ila) to form the salt represented by Formula (la) is promoted by the presence of a base, which base is conveniently provided by (and is therefore the same as) the base of step (i) as present in the product of step (i) of the methods of the present disclosure.
[0120] In various embodiments, Y is chlorine. In various embodiments, X- is chloride. In various embodiments, X and Y are the same. Thus, in various embodiments, Y is chlorine and X' is chloride.
[0121] In various embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl, aralkyl, and aryl. In various embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. [0122] In various embodiments of the compound of Formula (Ila), each R1, R2, and R3 is independently selected from Ci-Ce alkyl and aryl. In various embodiments, each R1, R2, and R3 is independently alkyl. In yet further embodiments, each R1, R2, and R3 is independently selected from Ci-Ce alkyl, preferably C1-C3 alkyl. For instance, each R1, R2, and R3 may be independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In some embodiments, each R1, R2, and R3 is methyl.
[0123] In some embodiments, the linker group L is (CR4R5)m wherein m is an integer from 1 to 10 and each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0124] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently selected from alkyl, aryl, and aralkyl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently selected from alkyl and aryl. In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0125] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently selected from Ci-Ce alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0126] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently selected from C1-C3 alkyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H. [0127] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is independently selected from the group consisting of methyl, ethyl, n-propyl and isopropyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0128] In various embodiments, m is an integer from 1 to 10, each R4 and R5 is independently selected from H, optionally substituted alkyl and optionally substituted aryl, and each R1, R2, and R3 is methyl. In further embodiments, m is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In other embodiments, m is 1. In some embodiments, m is 1 and each R4 and R5 is H.
[0129] In other embodiments, the linker group may be a polyether group such as those derived from ethylene glycol or a polyethylene glycol. For example, the linker group may have the formula [O-(CH2)n]o wherein n is an integer from 1 to 5 and o is an integer from 1 to 10. In various embodiments, n is an integer from 1 to 4, 1 to 3, or 1 to 2; and o is an integer from 1 to 10. In various embodiments, n is 2 and o is an integer from 1 to 10. In various embodiments, n is an integer from 1 to 5 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In various embodiments, n is an integer from 1 to 5 and o is 1. In various embodiments, n is an integer from 1 to 4, 1 to 3, or 1 to 2; and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In various embodiments, n is 2 and o is an integer from 1 to 5, 1 to 4, 1 to 3, or 1 to 2.
[0130] In some embodiments, m is 1 , each R4 and R5 is H, each R1, R2 and R3 is methyl, Y is chlorine and X' is chloride. In such embodiments, the compound of Formula (Ila) is (3-chloro- 2-hydroxypropyl)trimethylammonium chloride (‘CHTAC’), which is commercially available (CAS Number: 3327-22-8).
[0131] In any of the above-described embodiments, the base of step (i) may be a hydroxide salt, for example sodium hydroxide. The above description of the base would be understood by the person skilled in the art to apply in this respect.
[0132] By way of example, Scheme 3 shows the in situ production of the salt represented by Formula (I) from a compound of Formula (Ila) for the case wherein said salt is GTMAC, the compound of Formula (Ila) is CHTAC, and the base of step (i) is a hydroxide salt.
Figure imgf000035_0001
Scheme 3
Reaction conditions
[0133] The skilled person will be able to select suitable reaction conditions for each of steps (i) and (ii) of the methods of the present disclosure as well as optional steps (iii), (iv) and (v). In various advantageous embodiments, the methods of the present disclosure are carried out in aqueous solvent. Such aqueous solvents are advantageous as they are generally considered to be environmentally friendly. By the term “environmentally friendly” is meant not harmful to the environment such that the solvent can be disposed of without the need for specialist equipment or process(es), i.e. non-toxic. Further, the use of aqueous solvents may be preferable in applications where the presence of potentially toxic organic solvents is prohibited or strictly regulated, and which would thus necessitate additional purification steps. Thus, in various embodiments, the aqueous solvent is substantially free of organic solvents. The term “substantially free” is defined above. By avoiding such organic solvents, functionalised beads produced by the methods disclosed herein may be immediately suitable for use in downstream applications such as in the food industry without further costly purification steps.
[0134] In further embodiments, the beads are suspended in the aqueous solvent. As already discussed above, the base may be added to the aqueous solvent prior to suspension of the beads in the solution of the base in the aqueous solvent. In this way, good solvent accessibility to the beads is achieved and thus efficient deprotonation and functionalisation of said beads. The reaction mixture may be mixed during reaction, for example in a stirred vessel.
[0135] The relative amounts of beads and each respective reactant are not limited. For example, the ratio of the mass of beads to the reaction solvent (e.g. aqueous solvent) is not limited, although preferably the ratio of the mass of beads to reaction solvent is such that the beads can be homogenously distributed in the reaction mixture.
[0136] It will be generally be preferable to ensure as many deprotonated hydroxyl groups on the polysaccharide of the beads are functionalised as possible. Epoxides may be susceptible to hydrolysis in aqueous conditions and in the presence of base. Thus, in various embodiments wherein the reaction solvent is an aqueous solvent, the salt of the amount of the salt represented by Formula (I), (la), (lb), or (Ic) (e.g. GTMAC) contacted with the beads in step (ii) is at least about 50% w/v, preferably at least about 60% w/v, preferably at least about 70% w/v, preferably at least about 80% w/v, preferably at least about 90% w/v, preferably at least about 100% w/v, based on the volume of the aqueous solvent and base. For example, a 25 mL volume of aqueous 1 M NaOH in which 10 g of cellulose beads are suspended may be contacted with 31.3 g of GTMAC, i.e. about 125% w/v GTMAC based on the volume of the aqueous solvent and base.
[0137] In various embodiments, the molar ratio of the salt of Formula (I), Formula (la), Formula (lb), or Formula (Ic) (e.g. GTMAC) to anhydrous glucose units of the beads is at least about 1 :1 , at least about 2:1 , at least about 3:1 , or at least about 4:1. In various embodiments, the molar ratio of the salt of Formula (I), Formula (la), Formula (lb), or Formula (Ic) (e.g. GTMAC) to anhydrous glucose units of the beads is up to 5:1. Thus, in various embodiments, the molar ratio of the salt of Formula (I), Formula (la), Formula (lb), or Formula (Ic) (e.g. GTMAC) to anhydrous glucose units of the beads is from about 1 :1 , from about 2:1 , from about 3:1 , or from about 4:1 to about 5:1. The foregoing ratios are calculated based on the number of moles of the salt and the anhydrous glucose units prior to the reaction of said salt and anhydrous glucose units, i.e. before the reactants are consumed/transformed. As used herein, “anhydrous glucose unit” takes its normal meaning in the art and refers to a single sugar molecule (i.e. monomer) in the polysaccharide of the beads of the present disclosure.
[0138] In various embodiments, step (i) and/or step (ii) are performed at a temperature of from about 10°C to about 30°C. In various embodiments, step (i) and/or step (ii) are performed at room temperature (about 20°C). Preferably, both steps (i) and (ii) are performed at room temperature. More preferably, all steps of the methods disclosed herein are performed at room temperature. Conducting the various steps at room temperature contributes to the minimal environmental impact of the methods disclosed herein.
[0139] The reaction time is not limited, and a person skilled in the art will be able to determine a suitable duration for the reaction, for example to ensure that the reaction reaches completion.
[0140] The functionalisation of the beads according to the methods of the present disclosure leads to the introduction of quaternary ammonium groups on said beads. The beads thus functionalised bear multiple positive charges, i.e. are polycationic, in contrast to the unfunctionalised beads that are typically electrically neutral prior to performing steps (i) and (ii) of the methods described herein. The progress of the functionalisation reaction of step (ii) can thus be monitored via the charge density of the beads. A person skilled in the art of the present disclosure will be able to select suitable analytical methods for determining charge density of the beads. For example, charge density may be measured by the conductometric titration of chloride ions against silver nitrate. In such a method, the beads after reaction are thoroughly washed, e.g. with distilled water. Washing may be performed until the conductivity of the water has stabilised. Titration with silver nitrate then determines the amount of chloride ions coordinated to the functionalised beads, i.e. via the complementary charges of the quaternary ammonium groups. Charge density may be expressed as milliequivalents (corresponding to millimoles of quaternary ammonium groups) per gram of dry polysaccharide (meq/g), e.g. cellulose.
[0141] In various embodiments, the functionalised beads of the present disclosure have a charge density of at least about 0.5 meq/g, at least about 0.75 meq/g, at least about 1 meq/g, or at least about 2 meq/g as defined hereinabove. In various embodiments, the functionalised beads of the present disclosure have a charge density of up to about 3 meq/g as defined hereinabove. Thus, in various embodiments, the functionalised beads of the present disclosure have a charge density of from about 0.5 meq/g, from about 0.75 meq/g, from about 1 meq/g, or from about 2 meq/g to about 3 meq/g as defined hereinabove. In various embodiments, the functionalised beads of the present disclosure have a charge density of from about 0.5 meq/g, from about 0.75 meq/g, or from about 1 meq/g to about 2 meq/g as defined hereinabove.
[0142] As can be seen from Figure 3 discussed in more detail below, a longer reaction time can lead to a higher degree of functionalisation (and thus a higher charge density) and a lower dry weight of the beads of the present disclosure. Dry weight as used herein is expressed as the percentage of dry polysaccharide per unit of hydrated polysaccharide mass. Thus, in various embodiments the functionalised beads of the present disclosure have a dry weight of less than about 10%, less than about 8%, less than about 6%, or less than about 4%. In various embodiments, the functionalised beads of the present disclosure have a dry weight of at least about 1 %. Thus, in various embodiments, the functionalised beads of the present disclosure have a dry weight of from about 1% to about 10%, to about 8%, to about 6%, or to about 4%.
[0143] In various embodiments, the functionalised beads of the present disclosure have a charge density of from about 0.5 meq/g, from about 0.75 meq/g, from about 1 meq/g, or from about 2 meq/g to about 3 meq/g and a dry weight of from about 1 % to about 10%, to about 8%, to about 6%, or to about 4%. In various embodiments, the functionalised beads of the present disclosure have a charge density of from about 0.5 meq/g, from about 0.75 meq/g, or from about 1 meq/g to about 2 meq/g as defined hereinabove, and a dry weight of from about 1% to about 10%, to about 8%, to about 6%, or to about 4%. [0144] In various embodiments, the functionalised beads of the present disclosure have a charge density of from about 0.75 meq/g to about 2 meq/g and a dry weight of from about 2% to about 4%.
Further processing and storage of functionalised beads
[0145] In any of the embodiments of the methods described herein, said methods may further comprise the step of (iii) neutralising the product of step (ii) with an acid. As used herein, the term “neutralising” means contacting the product of step (ii) comprising a base as described herein with an acid as described herein such that the resulting pH is about 7.
[0146] Such neutralisation reactions are a matter of routine practice in the chemical arts and a person skilled in the art of the present disclosure is able to perform such a reaction based on their common general knowledge. The skilled person will be able to select suitable acids for said purpose and the appropriate amounts to achieve neutralisation, and the present disclosure is not limited in this regard.
[0147] In various embodiments, the acid includes a counterion which is the same as X in Formula (I). Such embodiments where the anions contributed by said salt and said acid are the same may be advantageous to avoid the need for additional purification steps. Similarly, in various embodiments wherein the salt of Formula (I), (la), (lb) or (Ic) is prepared from a compound of Formula (Ila), the acid includes a counterion which is the same as X in Formula (I) and Y in Formula (Ila). Again, in this way, the presence of multiple different anionic species in the reaction can be avoided, and thus the need for additional purification steps may also be avoided.
[0148] In various embodiments, the acid is hydrochloric acid. In some embodiments, X' is chloride and the acid of step (iii) is hydrochloric acid. In further embodiments wherein the salt of Formula (I), (la), (lb) or (Ic) is prepared from a compound of Formula (Ila), X' is chloride, Y is chlorine, and the acid of step (iii) is hydrochloric acid.
[0149] In various embodiments, the base of step (i) is a hydroxide salt and the acid includes a counterion which is the same as X in Formula (I). In various embodiments, the base of step (i) is a hydroxide salt, the acid is hydrochloric acid, and X- is chloride. In further embodiments wherein the salt of Formula (I), (la), (lb) or (Ic) is prepared from a compound of Formula (Ila), the acid includes a counterion which is the same as X in Formula (I) and the leaving group corresponding to Y in the compound of Formula (lla)ln further embodiments wherein the salt of Formula (I), (la), (lb) or (Ic) is prepared from a compound of Formula (Ila), X' is chloride, Y is chlorine, the base of step (i) is a hydroxide salt and the acid of step (iii) is hydrochloric acid.
[0150] In various embodiments of the present disclosure, the methods disclosed herein further comprise the step of (iv) separating the functionalised beads by filtration and optionally (v) washing the filtrate from step (iv) with water. The filtration process is not limited. Techniques suitable for the separation of step (iv) will be known to a person skilled in the art and include but are not limited to those described herein. For example, the functionalised beads may be separated from the reactants by vacuum filtration or simply by sieving. The filtration process may involve mechanical or any other type of filtration (e.g. using equipment known in the art such as a hydrocyclone). In various embodiments, a filtration medium (e.g. a filter) may be used to filter the beads from the reactants and thereby collect the beads.
[0151] In various embodiments, the functionalised beads may be allowed to settle in a vessel and the reaction solution comprising reactants (e.g. the base, unreacted salt of Formula (I), and/or compound of Formula (Ila), and/or acid etc.) removed or decanted to leave beads wetted in residual reaction solution. Alternatively, the beads may be separated by a centrifugal separator or a disk stack separator.
[0152] In various embodiments, the functionalised beads may be washed one or more times, for example with an aqueous solvent including water. In various embodiments, the aqueous solvent is substantially free of organic solvents. The term “substantially free” is defined above. By avoiding such organic solvents, functionalised beads produced by the methods disclosed herein may be immediately suitable for use in downstream applications, such as in the food industry, without further costly purification steps.
[0153] In various embodiments, the functionalised beads are washed one or more times with water. For example, the functionalised beads may be separated as described above and then washed with water. One or more cycles of separation and washing steps may be performed, preferably until the remaining amounts of reactants and/or ionic products (e.g. salts formed by neutralisation of the base with acid) are negligible. In various embodiments, the progress of washing steps may be monitored by measuring the electrical conductivity of water in which the functionalised beads are suspended. Such techniques are expressly within the common general knowledge of a person skilled in the art of the present disclosure.
[0154] In various embodiments, the functionalised beads are stored in an aqueous solvent, preferably wherein said aqueous solvent is substantially free of organic solvents. The term “substantially free” is defined above. For example, the functionalised beads may be stored in water, preferably deionised water. In such embodiments, the functionalised beads may preferably be stored at temperatures lower than room temperature, for example less than about 20 °C. For example, functionalised beads may be stored in deionised water at less than about 10 °C. In various embodiments, the functionalised beads are stored in deionised water at about 4 °C. In various embodiments, the functionalised beads are stored in the manner according to any of the foregoing embodiments prior to use as described herein. In such embodiments, this may be advantageous to remove the need for additional drying and swelling steps as discussed below. In various embodiments where the avoidance of microbial contamination is required such as cell culture, the functionalised beads may be sterilised prior to storage as described herein. Alternatively or additionally, the functionalised beads may be sterilised prior to their subsequent use as described herein. The present disclosure is not limited in terms of suitable sterilisation methods and the skilled person will be able to select suitable sterilisation methods. For example, the functionalised beads may be sterilised by irradiation (e.g. UV- irradiation or gamma-ray irradiation), heat treatment (autoclavation), or chemical disinfection (e.g. 70% v/v ethanol in water).
[0155] In other embodiments, the functionalised beads may be dried (for example in a vacuum oven, by air drying, in a rotovaporator or by freeze-drying) and stored in a dry state. Such dried beads may be rehydrated by suspension in an aqueous solvent prior to use in a desired application as described herein. In various embodiments, the aqueous solvent is substantially free of organic solvents. The term “substantially free” is defined above. As already discussed above, avoiding organic solvents may be advantageous in that the functionalised beads produced by the methods disclosed herein may be immediately suitable for use in downstream applications, such as in the food industry, without further costly purification steps. Thus, in various embodiments, the aqueous solvent is water. As discussed herein, it has been found that functionalised beads obtained by the methods of the present disclosure can be dried and then resuspended, e.g. in water, such that the dried functionalised beads fully regain their prior hydration/solvation and the size, shape, and structure of the beads are unaffected by the drying and rehydration/resolvation steps.
Applications in cell culture
[0156] As discussed above, the functionalised beads prepared by the methods of the present disclosure have been found to be particularly useful as microcarriers in cell culture. As used herein “cell culture” refers to methods wherein eukaryotic or prokaryotic cells taken out of their natural environment (e.g. from the tissue of an originating organism) are grown under controlled artificial conditions, for example in a reactor in a laboratory. The types of cells cultured are not limited and include bacteria, archaea and eukaryotes. Non-limiting examples of eukaryotes contemplated by the present disclosure include plant, fungi, and animal cells. Animal cells may include non-mammalian and mammalian cells.
[0157] All forms of cell culture including primary culture and the culture of cell lines derived from primary culture (including cell lines that have been selected or genetically or otherwise modified) are contemplated by the present disclosure.
[0158] Microcarriers provide within the cell culture a substrate upon and/or in which mono- and/or multi-layers of cells can grow. The microcarriers are typically suspended in the cell culture medium, for example by stirring. By providing a high surface/volume ratio, microcarriers in cell culture can enable the achievement of higher yields than in suspension cultures without microcarriers, and in particular for adherent (anchorage-dependent) cells such as adherent animal cells such as adherent mammalian cells. Adherent cells are cells that require fixation to a surface for them to grow in vitro. Consequently, an equivalent mass of cultured cells can be obtained with a smaller culture volume when using microcarriers, thus leading to process and cost efficiencies particularly for large-scale industrial mammalian cell cultures.
[0159] As well as being beneficial as microcarriers in cell culture, the functionalised beads of the present disclosure are further advantageous in that they are produced in a highly cost- effective manner due to the simplicity of the methods described herein. Further, the functionalisation methods described herein are environmentally friendly, for example because the use of organic solvents and/or the production of toxic, regulated, and/or prohibited side products can be avoided. In particular, each of the functionalisation methods and methods for producing the non-functionalised bead starting material disclosed herein can be performed in aqueous solution where the aqueous solution is substantially free or completely free of organic solvents. In the case of cellulose beads, cellulose is both cost-effective and sustainable as a starting material. For instance, cellulose is the most abundant biopolymer on Earth and can be obtained from many sources, including waste, enabling closed-loop production and recycling.
[0160] As discussed above, beads produced by the methods of the present disclosure are mechanically robust, even in the absence of cross-linking, and the size of the beads can be readily tuned to particular applications through the methods of production of the starting material disclosed herein. Accordingly, in a further aspect, the present disclosure provides the use of functionalised beads prepared by any of the methods disclosed herein in cell culture. Also provided is a method for attaching cells to functionalised polysaccharide beads, wherein the method comprises preparing functionalised beads by any of the methods described herein and contacting the functionalised beads with one or more cells, preferably wherein the functionalised beads are contacted with one or more cells during cell culture.
[0161] Commonly cultured cell types in industry include bacterial, yeast, insect and mammalian cells. For instance, bacterial, yeast and mammalian cells may be used for the overexpression of recombinant proteins. Mammalian cells are most commonly used for the production of e.g. biologies because of their ability to propagate human viruses, express monoclonal antibodies, and incorporate necessary post-translational modifications. As discussed herein above, there has also been significant recent interest in the use of cell culture for the production of food, e.g. so-called cultured meat to reduce environmental impacts from farming and animal husbandry as well as on ethical and animal welfare grounds.
[0162] Thus, in various embodiments, the cells of the use and method for attaching cells to functionalised polysaccharide beads as described herein are animal cells, preferably mammalian cells, and more preferably adherent mammalian cells. Non-limiting examples of commonly cultured mammalian cells for industrial purposes include Chinese hamster ovary cells (CHO), lymphoma cells (e.g. NS0, SP2/0), baby hamster kidney (BHK) cells, hybridoma cells, Vero cells (e.g. ATCC CCL-81), and human embryonic kidney (HEK) cells. Cell lines and information relating thereto can typically obtained from a public repository such as that maintained by the American Type Culture Collection (ATCC) as well as commercial vendors.
[0163] In cultured meat applications, the aim is typically to culture high yields of cells that mimic naturally farmed and slaughtered meat. Meat tissue is made up of various cell types such as myofibers, adipocytes, fibroblasts, chondrocytes, and endothelial cells. Cultured meat approaches typically obtain the starting cells for culture in one of two ways: (i) by taking a tissue biopsy or using post-slaughter tissue from a livestock species of interest, i.e. primary cell culture, or (ii) differentiating pluripotent stem cells. Generally, to achieve quantities sufficient for the production of cultured meat, stem cells are preferred as these have the capacity to proliferate and then differentiate into the mature cell types present in meat. Two such stem cell types are adult stem cells and pluripotent stem cells. Adult stem cells are undifferentiated progenitor cells that can be obtained from specific organs and tissues in animals. Adult stem cells are multipotent (can differentiate into a number of cell types). Examples useful for cultured meat production include muscle satellite cells, mesenchymal stem/stromal cells, and fibro/adipogenic progenitors. Pluripotent stem cells are also attractive for cultured meat production because they are highly proliferative in culture and can be differentiated into a wide variety of mature cell types. Pluripotent stem cells may be embryonic stem cells, which are derived from blastocysts, or induced pluripotent stem cells obtained by cell reprogramming of somatic cells by the induction of genes associated with pluripotency.
[0164] As is evident from the applications described herein, the stem cells contemplated herein will be exclusively from non-human animals. Further, the collection of stem cells as well as any processes and uses of embryos are expressly not encompassed by the present disclosure.
[0165] As cultured meat applications require substantial scale-up of the chosen cell source, it is particularly desirable to use cell culture methods that maximise yield while minimising culture volumes for reasons of cost and environmental impact, for example reducing the amount of cell culture medium required during production. Microcarriers such as the functionalised beads of the present disclosure are thus particularly useful in such applications. Accordingly, in some embodiments the functionalised beads prepared by the methods described herein are used in cell culture for food production, preferably for the production of cultured meat. For instance, the functionalised beads prepared by the methods described herein may be used in animal cell culture for food production.
[0166] The conditions of the cell culture are not limited. The skilled person will be able to select appropriate cell culture conditions for the cell line of interest as part of their common general knowledge. A standard textbook in the art regarding all aspects of animal cell culture include techniques and methods is Freshney’s Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 5th Edition, John Wiley & Sons, Inc. 2005, ISBN: 978-0-471- 74759-8. Guidance on the culture of specific cell lines is also provided by suppliers of commercial cell lines and from repositories such as the ATCC. Another reference covering microbial, mammalian and plant cell culture is Advanced Fermentation and Cell Technology, Wiley-Blackwell, 2021 , ISBN: 978-1-119-04276-1.
[0167] Cells are typically cultured in a liquid culture medium. A non-limiting example commonly used in animal cell culture applications such as those described herein is DMEM (Dulbecco’s Modified Eagle Medium). The culture medium may contain one or more supplements, such as serum, antibiotics, amino acids, or growth factors. For example, the culture medium may be supplemented with fetal bovine serum (FBS). Typically, FBS may be supplemented into culture media, e.g. DMEM, in an amount of about 10%. However, for cultured meat applications that are at least in part motivated by ethical concerns, the use of serum-free culture media may be preferred in some embodiments. Antibiotics and/or fungicides may be used to avoid contamination of cell cultures, however for food production purposes such as cultured meat it may be preferred to omit these in favour of production under strictly controlled sterile conditions.
[0168] The cell culture will typically be warmed to a physiologically relevant temperature, for example 37 °C, to support the growth of the cells. Further, cell culture is typically performed under high humidity, for example to limit evaporation of culture medium, such as 85-95% relative humidity. Many culture media use sodium bicarbonate buffering systems to maintain the pH of the culture at around 7.4. In such embodiments, the culture is performed in a mixture of air and carbon dioxide, preferably 4-10% CO2, and more preferably 5% CO2. The skilled person in the art will be able to modify such environmental conditions as appropriate to the cell line of interest as part of their common general knowledge.
[0169] Cell culture is typically performed for research purposes at small scale in dishes for stationary culture, in roller bottles for mixed cultures and glass spinner flasks for stirred cultures. Large-scale cell culture may be performed in culture vessels such as large stirred tanks having capacities of around 15,000 litres. Similarly, microcarriers may be used in such large-scale vessels, but may also be used in other configurations such as packed beds, and fluidised beds.
[0170] Microcarriers may be retained from the cell culture medium by various methods and the present disclosure is not limited in this regard. For example, microcarriers can be retained by filtration or centrifugation.
[0171] In various embodiments, it will be necessary to harvest the cultured cells from the microcarriers, and preferably while minimising damage to said cells. The present disclosure is not limited in this regard. For example, the cells may be detached from the microcarriers enzymatically, for example by treatment with trypsin, and/or a chelating agent such as EDTA. The detached cells may subsequently be separated from the microcarriers by various methods that the skilled person will be able to select from their common general knowledge. Non-limiting examples include differential sedimentation, filtration, density gradient centrifugation, fluidised bed separation, or use of a vibromixer.
[0172] Accordingly, in various embodiments the present disclosure provides for the use of functionalised beads prepared by any of the methods described herein in the culture of animal cells, preferably mammalian cells. In various embodiments, the animal/mammalian cells are adherent cells. In various embodiments, the animal cells are adherent mammalian cells. In further embodiments of said use, the functionalised beads described herein are used as microcarriers in said cell culture. In various embodiments, the cell culture comprises animal cells and a liquid culture medium. For instance, the cell culture may comprise mammalian cells, preferably adherent mammalian cells, and a liquid culture medium. In further embodiments, the liquid culture medium comprises at least one supplement selected from the list consisting of foetal bovine serum, amino acids, growth factors, antibiotics and antifungals. In various embodiments, the cell culture is carried out in a bioreactor, preferably a stirred bioreactor. In further embodiments, the cell culture is carried out at a temperature of around 37°C in the presence of air containing from about 4% to about 10% CO2 and with a relative humidity of from about 85% to about 95%. Any of the foregoing characteristics of the described use are also applicable to the method for attaching cells to functionalised beads as described herein.
[0173] Thus, in an exemplary embodiment of the present disclosure, the cell culture comprises animal cells, a liquid culture medium, and functionalised beads prepared according to any of the methods described herein; wherein the liquid culture medium comprises at least one supplement selected from the list consisting of foetal bovine serum, amino acids, growth factors, antibiotics and antifungals; preferably wherein the cell culture is carried out in a bioreactor at a temperature of around 37°C in the presence of air containing from about 4% to about 10% CO2 and with a relative humidity of from about 85% to about 95%.
Preparation of polysaccharide beads
[0174] The present disclosure provides a generally applicable methodology for the functionalisation of polysaccharide beads. Accordingly, the present disclosure is not limited in terms of the means by which the polysaccharide beads are prepared.
[0175] Two non-limiting examples of methods by which polysaccharide beads may be prepared are (i) extrusion, and (ii) membrane emulsification followed by phase inversion. To prepare polysaccharide beads by extrusion, a dispersed phase is extruded into an anti-solvent to form beads of the polysaccharide. The dispersed phase comprises the polysaccharide in a solvent as discussed further below, and the extrusion of such a dispersed phase is known in the art. It is a process wherein the dispersed phase is forced, pressed, or pushed out, for example through an aperture or opening. The opening may be in a syringe as shown in Figure 1 or any other suitable extrusion device as known in the art.
[0176] A schematic representation of an exemplary embodiment of the extrusion process is shown in Figure 1. In the exemplary embodiment of Figure 1 , the dispersed phase (1) comprising the polysaccharide in a solvent is extruded through a needle (2) of a syringe (3). Extrusion is specifically into the anti-solvent (4) to form polysaccharide beads (5). In the exemplary embodiment of Figure 1 , the extruded dispersed phase is dropped from a height, d, above the surface of the anti-solvent.
[0177] The latter exemplary method of preparing polysaccharide beads comprises a membrane emulsification step and a phase inversion step.
[0178] Membrane emulsification is known in the art; it is a technique in which a dispersed phase is forced through the pores of a microporous membrane directly into a continuous phase, where emulsified droplets are formed and detached at the end of the pores with a drop- by-drop mechanism. A schematic representation of a membrane emulsification process is shown in Figure 2, where the arrow indicates the direction of flow.
[0179] The dispersed phase generally includes a first liquid containing the polysaccharide dissolved in a solvent, and the continuous phase includes a second liquid which is immiscible with the first liquid. The interaction of the two liquids when the dispersed phase is pushed or otherwise transported through the membrane is called a dispersion process, and their inhomogeneous mixture is termed an emulsion, i.e. droplets of the dispersed phase surrounded by the continuous phase.
[0180] In the context of producing polysaccharide beads, the droplets of dispersed phase in continuous phase have been successfully isolated by phase inversion. In the context of cellulose, this is described in ACS Sustainable Chem. Eng. 2017, 5, 7, 5931-5939, which is incorporated herein by reference. Phase inversion is a chemical phenomenon exploited in the fabrication of artificial membranes, and is performed by removing solvent from a liquid-polymer solution. There are various methods of phase inversion including immersing the polymer solution into a third liquid called the anti-solvent. The use of anti-solvent based phase inversion has proven to be particularly effective in precipitating droplets of polysaccharide into beads from an emulsion of dispersed/continuous phase.
[0181] Common to both exemplary processes for preparing polysaccharide beads is the use of a solvent into which the polysaccharide is dissolved to form the dispersed phase, and the use of an anti-solvent to form the polysaccharide beads.
[0182] Solvents for use in the preparation of polysaccharide beads, particularly by membrane emulsification or extrusion, are known and ionic liquids are commonly favoured as they are able to solubilise recalcitrant polysaccharides. Ionic liquids are salts that are in liquid form at a temperature between ambient temperature and 100°C, for example imidazolium based ionic liquids such as 1-ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc) or the like. Moreover, ionic liquids are essentially non-volatile (avoiding fugitive emissions) and are considered to have environmental benefits over other solvents. Ionic liquids can, for example, be readily recycled by distillation to remove the anti-solvent.
[0183] Ionic liquids are typically not used in pure form, however. An amount of a co-solvent is often added to the ionic liquid when dissolving polysaccharides such as cellulose. The use of a co-solvent may assist in dissolution of the polysaccharide, and may reduce the amount of costly ionic liquid required. In methods for forming polysaccharide beads, the inclusion of a cosolvent may also improve the efficiency and yield of the process by modifying the viscosity of the dispersed phase, which may in turn reduce the amount of deformation exhibited by the beads.
[0184] Typical co-solvents employed in combination with ionic liquids are dipolar aprotic solvents, such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and the like. However, such solvents are not generally considered to be environmentally friendly, and the use thereof may therefore have a negative impact on the overall environmental benefits of ‘green’ processes that use ionic liquids. Moreover, many solvents are prohibited from food products, for example DMF is associated with toxic effects. Such co-solvents therefore cannot be used in processes for the preparation of polysaccharide beads for use in food production as well as other applications. The use of dipolar aprotic solvents may also complicate the recycling of the ionic liquid and increase costs. For example, some degree of distillation of DMSO is to be expected during recycling and the presence of aprotic solvent has been reported to reduce the thermal stability of 1-ethyl-3-methylimidazolium acetate (EmimOAc) [see Williams et al., Thermochimica Acta (2018), 669 126-139, for example],
[0185] Turning now to the anti-solvents that may be used when preparing polysaccharide beads, organic solvents such as ethanol are typical. Again, however, these substances may reduce the overall environmental benefits of the process, may be associated with safety concerns, and may complicate and increase the cost of recycling of ionic liquids.
[0186] Thus, in various embodiments, an aqueous solvent and an aqueous anti-solvent may be used. The use of an aqueous solvent and anti-solvent may obviate the use of reagents associated with environmental and safety concerns and may also simplify and reduce the cost of solvent recycling. In particular, use of such solvents and anti-solvents may increase the stability of ionic liquids against temperature-based degradation [Williams et al., Thermochimica Acta (2018), 669: 126-139], which may allow an increased number of recycling cycles to be performed, for example. Finally, the inclusion of water in the dispersed phase may increase the likelihood of bead sphericity.
[0187] Accordingly, in various embodiments, the dispersed phase from which the polysaccharide beads may be prepared comprises a solvent in which the polysaccharide is dispersed or dissolved, which solvent comprises water. By the term “solvent” is therefore meant any substance (e.g. liquid) which disperses or dissolves the polysaccharide. The term “solvent” also includes solvent mixtures.
[0188] The solvent of the dispersed phase may comprise water and may comprise an ionic liquid, an organic solvent, an inorganic nonaqueous solvent, or a combination thereof. In various embodiments of the present disclosure, the solvent for the dispersed phase comprises water and at least one of an ionic liquid, an organic solvent, an inorganic nonaqueous solvent, or a combination thereof. In various embodiments of the present disclosure, the solvent for the dispersed phase comprises water and one or more ionic liquid(s).
[0189] Non-limiting examples of solvents for the dispersed phase other than water include methanol, ethanol, ammonia, acetone, acetic acid, n-propanol, n-butanol, isopropyl alcohol, ethyl acetate, dimethyl sulfoxide, sulfuryl chloride, phosphoryl chloride, carbon disulfide, morpholine, N-methylmorpholine, NaOH without and with association of urea and thiourea, bromine pentafluoride, hydrogen fluoride, sulfuryl chloride fluoride, acetonitrile, dimethylformamide, hydrocarbon oils and blends thereof, toluene, chloroform, carbon tetrachloride, benzene, hexane, pentane, cyclopentane, cyclohexane, 1 ,4-dioxane, dichloromethane, nitromethane, propylene carbonate, formic acid, tetrahydrofuran, diethyl ether, phosphoric acid, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methylimidazolium chloride, 1-methoxymethyl-3-methylimidazolium bromide, N-ethylpyridinium chloride, N- methylmorpholine-N-oxide, 1 -methylimidazole, N,N-dimethylformamide, N,N'- dimethylimidazolidin-2-one, N,N-dimethylacetamide, sulfolane, y-valerolactone, y- butyrolactone, N,N,N',N'-tetramethylurea, N-methylpyrrolidinone, and methylene chloride. The skilled person will readily recognise which of the exemplary solvents are ionic liquids, organic solvents, and/or inorganic non-aqueous solvents.
[0190] As will be understood by the skilled person in the art, the dispersed phase will depend on the polysaccharide being used. The identification of suitable solvents for the dispersed phase of the present disclosure is specifically within the common general knowledge of the skilled person. [0191] In various embodiments, the solvent for the dispersed phase comprises water and an ionic liquid. The ionic liquid may be selected from 1-ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc), and a combination thereof. In some embodiments, the solvent for the dispersed phase comprises water and one or more organic solvents. In other embodiments, the solvent for the dispersed phase is substantially free of organic solvents. The term “substantially free” is defined above. The skilled person will understand that when the solvent of the dispersed phase consists of water and an ionic liquid, the total wt% of water and ionic liquid in the dispersed phase solvent will total 100 wt%. If water is present, for example, in an amount of at least 0.5 wt%, an ionic liquid may be present in an amount of at least 99.5 wt%, with the proviso that the total of water and ionic liquid is 100 wt%. In other words, the ionic liquid may be present as the remainder of the solvent.
[0192] Preferably, the solvent used for the dispersed phase is environmentally friendly. By the term “environmentally friendly” is meant not harmful to the environment such that the solvent can be disposed of without the need for specialist equipment or process(es), i.e. non-toxic. It is known in the art that polysaccharides have limited dissolution in most of the common solvents. It is also known in the art that those solvents which do dissolve polysaccharides are often toxic and/or highly selective. Thus, for polysaccharides such as cellulose, starch, chitin, glycogen, and/or chitosan, the solvent for the dispersed phase may comprise an ionic liquid in addition to water. The dissolution of cellulose with the ionic liquid 1-butyl-3-methylimidazolium chloride is, for example, discussed in Richard et al., J. Am. Chem. Soc. 2002, 124, 4974-4975. Verma et al., Sustainable Chemistry and Pharmacy 13 (2019), 100162 similarly discusses the solubility of cellulose in ionic liquids and ionic liquids with co-solvents. Each of these disclosures is incorporated herein by reference.
[0193] The concentration of polysaccharide in the dispersed phase is not limited and may be any concentration suitable for the methods discussed herein. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.1 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.5 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1.5 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2.5 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3.5 wt% to about 15 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 4 wt% to about 15 wt%.
[0194] In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.1 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.5 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 .5 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2.5 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3.5 wt% to about 12 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 4 wt% to about 12 wt %.
[0195] In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.1 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 0.5 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 1 .5 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2 wt% to about 10 wt%. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 2.5 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 3.5 wt% to about 10 wt %. In various embodiments, the polysaccharide is present in the dispersed phase in an amount from about 4 wt% to about 10 wt %.
[0196] The dispersed phase may further include optional components. These optional components include, but are not limited to, surfactants, porogens, active ingredients, pockets of air, double emulsions, pigments, and dyes. The level of any of the optional components is not significant in the present disclosure. In various embodiments, the dispersed phase includes a co-solvent.
[0197] The surfactant may be any suitable surfactant known in the art, for example, any ionic or non-ionic surfactant. Ionic surfactants may include sulfates, sulfonates, phosphates and carboxylates such as alkyl sulfates, ammonium lauryl sulfates, sodium lauryl sulfates, alkyl ether sulfates, sodium laureth sulfate and sodium myreth sulfate, dioctyl sodium sulfosuccinate, perfluorooctanesulfonate, perfluorobutanesulfonate, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, and alkyl carboxylates. Non-ionic surfactants may include polyethers, polyoxyalkylene derivatives of hexitol, partial long-chain fatty acid esters such as sorbitan oleates, ethylene oxide derivatives of long-chain alcohols, ethoxylated vegetable oil, polydimethylsilxoxanes, and ethylene oxide/propylene oxide copolymers.
[0198] The temperature of the dispersed phase is not limited. By the expression “temperature of the dispersed phase” or “the dispersed phase is at a temperature of”, or the like, is meant the temperature of the dispersed phase prior to extrusion or membrane emulsification (e.g. when it is placed in the apparatus for such extrusion or emulsification), and/or the temperature of the apparatus during extrusion or emulsification of the dispersed phase. As discussed in more detail below, the extrusion or emulsification means may be heated so that the dispersed phase remains at an elevated temperature in situ. Preferably the extrusion means is heated directly by one or more heating means. This is discussed further below.
[0199] In some embodiments, the dispersed phase is at ambient or room temperature, namely between about 20 and about 25°C. In various embodiments, the dispersed phase is heated above ambient temperature. The dispersed phase may be heated using any suitable means. The dispersed phase is preferably heated in situ such that there is no temperature loss prior to extrusion or membrane emulsification, for example by heating a vessel containing the dispersed phase and/or the extrusion or emulsification means. In the extrusion process, a heated syringe and/or needle may, for example, be used. Suitable heating apparatus may comprise a heating element, for example a Peltier element, as well as a means of regulating the temperature, such as a thermocouple and controller.
[0200] Thus, in various embodiments, the temperature of the dispersed phase is from about 5°C to less than about 100°C, from about 10°C to less than about 100°C, from about 15°C to less than about 100°C, from about 20°C to less than about 100°C, from about 25°C to less than about 100°C, or from about 30°C to less than about 100°C. The maximum temperature will be set by the point at which the evaporation of water from the dispersed phase becomes prohibitive and/or decomposition of the ionic liquid begins to occur. This will readily be determined by the person skilled in the art.
[0201] In various embodiments, the temperature of the dispersed phase is from about 5°C to about 90°C, from about 10°C to about 90°C, from about 15°C to about 90°C, from about 20°C to about 90°C, from about 25°C to about 90°C, or from about 30°C to about 90°C. In various embodiments, the temperature of the dispersed phase is from about 5°C to about 80°C, from about 10°C to about 80°C, from about 15°C to about 80°C, from about 20°C to about 80°C, from about 25°C to about 80°C, from about 30°C to about 80°C, or from about 40°C to about 80°C.
Anti-solvent
[0202] The anti-solvent may comprise water, i.e. it may be aqueous. In various embodiments, the anti-solvent may comprise water and an organic solvent such as an alcohol or acetone, or any other organic solvent known in the art. Suitable alcohols include ethanol and/or methanol. Preferably, the anti-solvent is environmentally friendly. More preferably, the solvent and antisolvent are both environmentally friendly. Thus, in various embodiments, the anti-solvent is substantially free of organic solvents. In various embodiments, the anti-solvent is or consists of water.
[0203] In various embodiments, the anti-solvent further comprises an ionic liquid. In some embodiments, the anti-solvent may comprise water and an ionic liquid before phase inversion or extrusion of the dispersed phase. In other embodiments, the ionic liquid may be introduced into the anti-solvent during the phase inversion or extrusion. In some embodiments where the dispersed phase comprises an ionic liquid, the ionic liquid may be introduced into the antisolvent from the dispersed phase during the phase inversion or extrusion process.
[0204] In various embodiments, the concentration of ionic liquid in the anti-solvent is up to about 50 wt% - the term “up to” being understood to mean greater than zero. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 40 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 30 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 20 wt%. In various embodiments the concentration of ionic liquid in the anti-solvent is up to about 10 wt%. [0205] Where the anti-solvent comprises water and an ionic liquid, the ionic liquid may be 1- ethyl-3-methylimidazolium acetate (EmimOAc), 1-butyl-3-methylimidazolium chloride (BmimOAc), or mixtures thereof. In various embodiments, the ionic liquid is 1-ethyl-3- methylimidazolium acetate (EmimOAc).
[0206] The temperature of the anti-solvent is not limited. In various embodiments, the temperature of the anti-solvent is from about 5°C to about 80°C. In various embodiments, the temperature of the anti-solvent is from about 10°C to about 70°C. In various embodiments the temperature of the anti-solvent is from about 15°C to about 60°C.
[0207] In the membrane emulsification process discussed herein, the temperature of the antisolvent may be ambient such that phase inversion is carried out at ambient temperature, namely between about 20 and about 25°C. In such embodiments, the anti-solvent has a temperature between about 20 and about 25°C. Alternatively, the anti-solvent is cooled to a temperature below ambient temperature, namely below about 20°C. For example, the antisolvent may be cooled to a temperature T2, for the phase inversion (b), T2 being less than TdisP. Preferably T2 is substantially equal to Ti, more preferably T2 is equal to Ti, where Ti is defined above.
[0208] The advantage of controlling the temperature of the anti-solvent (T2) in such embodiments is to prevent pre-mature thawing of the frozen droplets. Without wishing to be bound by any one theory, it is believed that by cooling the anti-solvent to T 2, the droplets remain in a frozen state (and hence spherical and non-aggregated) whilst the continuous phase surrounding them is stripped away by the phase inversion. The anti-solvent is able to contact the surface of the droplets, causing precipitation of the polysaccharide and hardening of the precipitate surface. Additionally, as the frozen dispersed phase droplet thaws, the anti-solvent will convert the droplet of dissolved polysaccharide to a bead thereof, whilst leaching the solvent system into the anti-solvent.
(i) Extrusion
[0209] As discussed above, while the present disclosure is not limited by the means by which the polysaccharide beads are prepared, a non-limiting example of a suitable method is wherein the dispersed phase is extruded into the anti-solvent to form beads of the polysaccharide.
[0210] In various embodiments, the dispersed phase is extruded through a fluid medium by capillary extrusion. The fluid medium may, for example, be air. Examples of capillaries through which the dispersed phase may be extruded are glass capillaries, microfluidic channels, and (hypodermic) needles. The material from which such capillaries are prepared is not limited and the skilled person will be able to select suitable capillaries compatible with the dispersed phase.
[0211] The surface of the capillary may also be modified. The capillary may, for example, be treated, coated, or lined, in order to alter its wetting properties. Such modifications of the capillary material may, for example, alter the hydrophilicity/hydrophobicity of the capillary material, thereby altering the wettability of the capillary surface. Capillaries may, for example, be treated with reactive hydrophobic compounds such as silanes to form a hydrophobic surface layer, or hydrophobic compounds may be deposited onto a capillary surface by methods such as chemical vapour deposition. In another example, metal needles may be lined with PTFE (polytetrafluoroethylene). The identification of suitable surface modifications is specifically within the common general knowledge of the skilled person.
[0212] The size of the aperture or opening, e.g. the diameter of the capillary or the gauge of the needle, is not limited. It will be immediately apparently to a person skilled in the art that the size of the aperture or opening will, however, influence the size of the droplets of the dispersed phase extruded therefrom. Generally, a larger aperture or opening would be expected to produce larger droplets of the dispersed phase, and conversely a smaller aperture or opening would be expected to produce smaller droplets of the dispersed phase. The skilled person will be able to select appropriately sized openings/apertures.
[0213] The diameter of the aperture or opening through which the dispersed phase is extruded may be less than about 3 mm, less than about 2.5 mm, less than about 2 mm, less than about 1.5 mm, less than about 1 mm, less than about 0.75 mm, less than about 0.5 mm, less than about 0.4 mm, less than about 0.3 mm, or less than about 0.2 mm. In various embodiments, the diameter of the aperture or opening through which the dispersed phase is extruded may be greater than about 0.1 mm. In various embodiments, the diameter of the aperture or opening through which the dispersed phase is extruded may be greater than about 0.1 mm and less than about 3 mm, greater than about 0.1 mm and less than about 2.5 mm, greater than about 0.1 mm and less than about 2 mm, greater than about 0.1 mm and less than about 1.5 mm, greater than about 0.1 mm and less than about 1 mm, greater than about 0.1 mm and less than about 0.75 mm, greater than about 0.1 mm and less than about 0.5 mm, greater than about 0.1 mm and less than about 0.4 mm, or greater than about 0.1 mm and less than about 0.3 mm. In other embodiments, the diameter of the aperture or opening through which the dispersed phase is extruded may be from about 0.1 mm to about 1 mm, from about 1 mm to about 2 mm, or from about 2 mm to about 3 mm. [0214] In various embodiments, the dispersed phase is extruded through a needle. The needle may be blunt-tipped, although the present disclosure is not limited in this respect. In various embodiments, the needle gauge size is 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , or 10 gauge.
[0215] The rate of extrusion is not limited and may be controlled using standard laboratory equipment, for example a syringe pump. In various embodiments, the rate of extrusion is less than about 1 mL/min, less than about 100 pL/min, less than about 10 pL/min, less than about 1 pL/min, or less than about 100 nL/min. In other embodiments, the rate of extrusion is from about 1 pL/min to about 1 mL/min, or from about 10 pL/min to about 100 pL/min.
[0216] In some embodiments, the dispersed phase is first extruded through a fluid medium into a mould and then the extruded dispersed phase is contacted with the anti-solvent. In various embodiments, the mould may impart a shape to the polysaccharide beads formed upon contacting the extruded dispersed phase with the anti-solvent. The shape of the polysaccharide beads is not limited, and will be determined by the shape of the mould in this instance. The mould may be formed of any suitable material that is compatible with the dispersed phase and anti-solvent, and may, for example, be a silicone polymer such as polydimethylsiloxane (PDMS). The mould may be prepared by casting the mould material, or may be prepared by 3D printing the mould material. The extruded dispersed phase may be contacted with the anti-solvent by submerging the mould containing the extruded dispersed phase in the anti-solvent. The mould may be removed after the polysaccharide beads have formed, or may be retained during further processing steps, such as washing and filtration/extraction of the polysaccharide beads.
[0217] When a mould is not used, extrusion may occur within the anti-solvent; that is to say, the dispersed phase may be exposed to the anti-solvent immediately upon extrusion (for example where the aperture or opening is submerged in the anti-solvent). Alternatively, and preferably, in various embodiments the extruded dispersed phase is dropped from a height above the surface of the anti-solvent. This can be seen in Figure 1 , wherein the extruded dispersed phase is dropped from a height, d, above the surface of the anti-solvent.
[0218] The dropping height may influence the sphericity of the beads obtained by the extrusion process. Without wishing to be bound by any one theory, it is believed that a greater dropping height may minimize tailing (i.e. improve sphericity) by allowing more time for cohesive forces to act on the falling droplet. Thus, in various embodiments, the extruded phase is dropped from a height of at least 10 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 20 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 30 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 40 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 50 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of at least 70 cm above the surface of the anti-solvent, or at least 80 cm above the surface of the anti-solvent.
[0219] The maximum dropping height will typically be determined by the distance at which non-spherical beads are formed. This is known in the art and readily understood by the skilled person. It may, for instance, be determined by eye. In various embodiments, however, the extruded phase is dropped from a height of less than 80 cm above the surface of the antisolvent. In various embodiments, the extruded phase is dropped from a height of less than 70 cm above the surface of the anti-solvent. In various embodiments, the extruded phase is dropped from a height of less than 60 cm above the surface of the anti-solvent. In various embodiments, the extruded phase is dropped from a height of less than 50 cm above the surface of the anti-solvent.
[0220] In various embodiments the extruded phase is dropped from a height of about 1 cm to about 80 cm above the surface of the anti-solvent, preferably from a height of about 5 cm to about 70 cm, more preferably from a height of about 10 cm to about 60 cm.
[0221] In various embodiments the extruded phase is dropped from a height of about 10 cm to about 80 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 70 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 10 cm to about 50 cm above the surface of the antisolvent.
[0222] In various embodiments the extruded phase is dropped from a height of about 20 cm to about 80 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 70 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 60 cm above the surface of the anti-solvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 50 cm above the surface of the antisolvent. In various embodiments the extruded phase is dropped from a height of about 20 cm to about 40 cm above the surface of the anti-solvent.
[0223] The extrusion process may further comprise the step of separating the polysaccharide beads from the anti-solvent. The means by which the polysaccharide beads may be separated from the anti-solvent are not limited and will be known to a person skilled in the art. For example, in various embodiments, the polysaccharide beads may be separated from the antisolvent by a filtration process. The filtration process is not limited and may involve mechanical or any other type of filtration (e.g. using equipment known in the art such as a hydrocyclone). In various embodiments, a filtration medium (e.g. a filter) may be used to filter the polysaccharide beads from the anti-solvent and thereby collect the polysaccharide beads.
[0224] In various embodiments, the polysaccharide beads may be allowed to settle in a vessel and anti-solvent removed or decanted to leave polysaccharide beads wetted in residual antisolvent. Alternatively, the polysaccharide beads may be separated by a centrifugal separator or a disk stack separator.
[0225] In various embodiments, the polysaccharide beads may be washed one or more times, for example with an aqueous solvent including water. Such washing steps may be performed to remove residual ionic liquid that may be present. In various embodiments, the solvent in which the polysaccharide beads are immersed may be exchanged for an alternative solvent.
(ii) Membrane emulsification
[0226] As discussed above, another non-limiting example of a suitable method for preparing polysaccharide beads is membrane emulsification followed by phase inversion. Membrane emulsification involves passing a dispersed phase through a membrane into a continuous phase so as to form an emulsion. The membrane is not limited; it can be any porous structure suitable for a membrane emulsification process. For example, the membrane may be a plate with holes acting as pores (e.g. micron-sized holes), a perforated metal tube, or sintered porous glass.
[0227] By the term “emulsion” is meant the class of two-phase systems of matter where both phases are liquid. Emulsions are a type of colloid, and generally consist of two immiscible liquids. In various embodiments, the emulsion may be a macro-emulsion; this is an emulsion in which the particles of the dispersed phase have diameters of approximately 1 to 1000 microns. The term “sol” refers to a general class of two-phase systems of matter where the continuous phase is liquid and the dispersed phase is solid.
[0228] The membrane emulsification is also not limited and may be any membrane emulsification process known in the art. For example, the membrane emulsification process may be a cross-flow membrane emulsification, a rotational membrane emulsification, a vibrational membrane emulsification, or a combination thereof. As is understood in the art, the terms “cross-flow”, “rotational” and “vibrational” refer to the method used to generate shear on the membrane surface. A continuous phase could, for example, move relative to a stationary membrane to create shear, or the membrane could move relative to stationary phases. Alternatively, the dispersed phase could be injected into a stationary continuous phase. Known process parameters such as membrane type, average pore size and porosity, crossflow velocity, transmembrane pressure and emulsifier may also be used. In various embodiments, the membrane emulsification may involve a cross flow system, a stirred-cell tube membrane, a stirred cell-flat membrane, a rotating flat membrane, a vibrating/rotating tube membrane and/or a premixed membrane emulsification.
[0229] International Patent Application No. WO 01/45830 describes an example of a rotational membrane emulsification. International Patent Application No. WO 2012/094595 describes an example of a cross-flow membrane emulsification. Pedro S. Silva et al., “Azimuthally Oscillating Membrane Emulsification for Controlled Droplet Production", AIChE Journal 2015 Vol. 00, No. 00, describes a vibrational membrane emulsification: specifically a membrane emulsification system comprising a tubular metal membrane which is periodically azimuthally oscillated in a gently cross flowing continuous phase. WO 2019/092461 describes a cross-flow membrane emulsification. Each of these method descriptions is incorporated herein by reference.
[0230] In various embodiments, the membrane emulsification is a cross-flow membrane emulsification. Preferably an emulsification process in which the continuous phase moves relative to a stationary membrane.
[0231] As will be understood by the skilled person in the art, the dispersed phase and continuous phase will depend on the polysaccharide being used. Various features of the solvent for the dispersed phase have already been discussed above, and said features individually or in any combination thereof are combinable with the embodiments disclosed herein. The continuous phase will comprise a solvent which is immiscible with the dispersed phase such that an emulsion is formed when the dispersed phase is forced through the porous membrane. The term “solvent” has the meaning as already defined hereinabove.
[0232] The two phases - namely the dispersed phase and the continuous phase - must be immiscible with one another. It therefore follows that the solvents for each of the phases must be immiscible with one another. The identification of suitable solvents for the dispersed phase and continuous phase of the second aspect is specifically within the common general knowledge of the skilled person.
[0233] The solvent of the continuous phase is not limited other than it must be immiscible with the dispersed phase. The solvent of the continuous phase may be a non-polar solvent. In various embodiments, the solvent of the continuous phase may be selected from hydrocarbon oils and blends thereof. Such hydrocarbon oils may be mineral oils, vegetable oils, or synthetic oils. The solvent of the continuous phase may further comprise water and/or one or more ionic liquids that may be present in residual amounts. Such residues of water and/or ionic liquid may arise as a result of solvent recycling processes.
[0234] Preferably the solvent used for the continuous phase is environmentally friendly. More preferably the solvent used for both the dispersed phase and continuous phase is environmentally friendly. The term “environmentally friendly” has the meaning as already defined hereinabove.
[0235] The continuous phase may further include optional components. These optional components include, but are not limited to, co-solvents, surfactants, pigments, and dyes. The level of any of the optional components is not significant in the present disclosure. In various embodiments, the continuous phase includes a co-solvent.
[0236] The co-solvent is not limited and may be any solvent known in the art. In various embodiments, the co-solvent may be selected from hydrocarbon oils and blends thereof. Such hydrocarbon oils may be mineral oils, vegetable oils, or synthetic oils. The co-solvent may further be a co-solvent mixture.
[0237] The surfactant is as defined above.
[0238] In various embodiments, the emulsion is cooled to a temperature Ti, Ti being greater than the pour point of the continuous phase (TCOnt), and equal to or less than a transition temperature selected from the group consisting of the freezing point, glass transition temperature and pour point, of the dispersed phase (Tdisp): wherein Tdisp > TCOnt. The absolute value of Ti is not, however, critical to the present disclosure; rather it is the relationship of Ti to the respective temperatures of the dispersed phase and continuous phase that is important.
[0239] The term “pour point” refers to the temperature below which a substance (e.g. liquid) loses its flow characteristics. It is typically defined as the minimum temperature at which the liquid (e.g. oil) has the ability to pour down from a beaker. The pour point can be measured with standard methods known in the art. ASTM D7346, Standard Test Method for No Flow Point and Pour Point of Petroleum Products and Liquid Fuels may, for example be used. For commercially available materials, the pour point is often provided by the supplier or manufacturer.
[0240] The term “freezing point” refers to the temperature at which a substance changes state from liquid to solid at standard atmospheric pressure (1 atmosphere). The freezing point can be measured with standard methods known in the art. ASTM E794, Standard Test Method for Melting and Crystallization Temperatures by Thermal Analysis may, for example, be used. For commercially available materials, the freezing point may be provided by the supplier or manufacturer.
[0241] The term “glass transition point” or “glass transition temperature” refers to the temperature at which a polymer structure transitions from a hard or glassy material to a soft, rubbery material. This temperature can be measured by differential scanning calorimetry according to the standard test method: ASTM E1356, Standard Test Method for Assignment of the Glass Transition Temperature by Differential Scanning Calorimetry. For commercially available materials, the glass transition temperature may be provided by the supplier or manufacturer.
[0242] Since deformation and aggregation are believed to take place when dispersed phase droplets are in a liquid state, the cooling of the emulsion to or below the pour point of the dispersed phase is believed to temporarily change - at least partially - the emulsion’s “colloid class” from an emulsion - i.e. liquid-in-liquid - to a sol - solid-in-liquid - and thereby result in the dispersed phase being easier to work with in downstream processes.
[0243] In addition, the dispersed phase having a transition temperature - the transition temperature being selected from the group consisting of freezing point, glass transition temperature and pour point - which is higher than the continuous phase pour point, means that the continuous phase surrounding the solidified dispersed phase is still able to function as a transport medium. A diagrammatic representation of an emulsion undergoing cooling and temporary conversion to a sol within a cooling coil heat exchanger is shown in Figure 2(b).
[0244] The method of cooling is not also limited. The emulsion may be cooled by any means known in the art for removing heat (energy) from a system. The emulsion may further be cooled at any point prior to phase inversion. In various embodiments, this means the emulsion is cooled simultaneously with or separately from the membrane emulsification process. The emulsion may, for example, be cooled as it is formed (e.g. by a cooling means located at the outlet of the membrane). Alternatively, the emulsion may be cooled in a step following membrane emulsification, e.g. in a cooling apparatus separate from the membrane emulsification apparatus. Advantageously, the cooling should take place as soon as possible after the emulsification takes place in order to reduce the possibility of liquid state dispersed phase droplets coalescing and/or aggregating.
[0245] In various embodiments, the emulsion may be cooled by a cooling medium (e.g. water, ice etc.) at least partially surrounding the vessel where the emulsion is formed. In a preferred embodiment, the vessel (e.g. pipe) where the emulsion is formed may have a cooling jacket containing a cooling medium. The cooling medium is not limited, and includes any medium having a lower temperature than the emulsion.
[0246] In various embodiments the emulsion may be cooled by a cooling apparatus connected to the membrane emulsification unit. The cooling apparatus may be a heat exchanger, such as an immersion heat exchanger. In an exemplary embodiment, a coil heat exchanger is immersed in a cooling medium (e.g. a cold water bath) but the disclosure is not limited in this respect. Any type of heat exchanger could, for instance, be used such as a tube-and-shell heat exchanger, a plate-and-frame heat exchanger, or a jacketed tube. Additionally, an immersion heat exchanger could be used with another cooling medium such as anti-freeze, dry ice or the like, in order to cool the emulsion to Ti.
[0247] The temperature of the anti-solvent during phase inversion is discussed above.
[0248] In various embodiments of the present disclosure, phase inversion is carried out under shear; the skilled person will be aware of suitable shear conditions for phase inversion. Shear may, for example, be achieved through the use of a stirred vessel (e.g. a mechanically stirred vessel) or a settling vessel (e.g. a gravity settling vessel). The term “shear” is used herein to refer to an external force acting on an object or surface parallel to the slope or plane in which it lies, the stress tending to produce strain. [0249] In various embodiments, phase inversion comprises a filtration process. The filtration process is not limited and may involve mechanical or any other type of filtration (e.g. using equipment known in the art such as a hydrocyclone). A filtration process may also be encompassed by the phase inversion being carried out under shear as described above. In various embodiments, a filtration medium (e.g. filter) may be used to filter the emulsion through the anti-solvent and thereby collect the polysaccharide beads. In such embodiments, the emulsion may gravity settle (shear) through the anti-solvent and into the filter, whilst the continuous phase passes through the filter (the filtrate). The frozen droplets may then be collected in the filter as the filter cake.
[0250] If not collected as part of phase inversion (e.g. via filtration or otherwise), the polysaccharide beads may be separated from the anti-solvent/continuous phase mixture or the anti-solvent/continuous phase mixture may be removed from the beads. The method of removal is not limited. In various embodiments, however, the method of removal depends on whether the method is being operated in batch or continuous mode.
[0251] When the method of the second aspect is being operated in batch mode, the phase inversion step may first be performed in a closed vessel and the resulting mixture then transferred into a decanter vessel and allowed to reach a settled stage. Once settled, layers may be removed sequentially from the bottom of the vessel. Typically the order of the layers can be (1) continuous phase, (2) an interfacial layer comprising wetted polysaccharide beads and (3) the remaining anti-solvent. The disclosure is not, however, limited in this respect and the skilled person will appreciate that the order of the layers will depend on their respective densities.
[0252] In various embodiments, the method is continuous and to operate in continuous mode, the phase inversion step may be performed under continuous input of emulsion and antisolvent and continuous output of the multi-phase mixture to a decanter. Within the decanter, a steady-state partition of the mixture may exist and there can be a continuous and preferably simultaneous removal from each of the phases. For example, there may be continuous and preferably simultaneous removal from: (1) the continuous phase, (2) anti-solvent and (3) wetted polysaccharide beads. The order of these layers will of course vary and the method is not limited to any particular order.
[0253] Alternatively, the multi-phase (e.g. three phase) mixture may be separated using techniques known in the art, such as a disc stack separator (e.g. a centrifugal separator such as the one manufactured by Andritz). [0254] To provide continuous cooling alongside a continuous phase inversion, the cooling medium (e.g. a medium surrounding the vessel containing the emulsion or used with a heat exchanger connected to the membrane emulsification unit) may need to be recycled or recirculated with a suitable device. A device such as a recirculating chiller (ThermoFlex available from ThermoFisher Scientific) may, for example, be used to keep the cooling medium at the desired temperature.
[0255] In various embodiments, phase inversion is followed by or involves removal of the polysaccharide beads as described above. Phase inversion may be followed by decanting and then polysaccharide bead removal from the mixture and/or phase inversion may involve mechanical filtration of the wetted beads from the anti-solvent/continuous phase/bead mixture.
[0256] Alternatively, the polysaccharide beads may be removed from the continuous phase before phase inversion. In such embodiments, wetted frozen droplets may be removed from the sol (e.g. using filtration) and then phase inversion carried out to precipitate the polysaccharide and form beads thereof.
[0257] Having generally described this disclosure, a further understanding can be obtained by reference to certain specific examples illustrated below which are provided for purposes of illustration only and are not intended to be all inclusive or limiting unless otherwise specified.
Examples
Materials and methods
Preparation of cellulose solutions
[0258] Microcrystalline cellulose (MCC, from Sigma-Aldrich®) and EmimOAc were dried in a vacuum oven at 80°C for 1 h to remove traces of water. Cellulose solutions were prepared at a concentration of 4-10 wt% MCC in the EmimOAc with 8 wt% deionized water content. The water was first added to the EmimOAc under stirring, followed by the MCC. The mixture was shaken by hand for a minute, then transferred to rollers for 24 h. The samples were placed in a 70°C oven for 24 h, stirred with a spatula, left in the oven for a further 24 h, and then finally transferred to the rollers once again for 24 h.
Preparation of cellulose beads by extrusion
[0259] The cellulose solution prepared as detailed above was loaded into a syringe fitted with a blunt-tipped needle. The solution was immediately extruded from the needle dropwise at 0.1 mL/min via a syringe pump (KdScientific-210) into an anti-solvent that was water or ethanol. An appropriate dropping height for optimal sphericity was selected by eye for each sample. The beads were washed with deionised water.
Preparation of cellulose beads by membrane emulsification
[0260] A dispersed phase comprising 6 wt% microcrystalline cellulose and 8 wt% water in 1- ethyl-3-methylimidazolium acetate was prepared according to routine methods known in the art. An oily continuous phase was also prepared according to routine methods known in the art.
[0261] The dispersed phase and continuous phase were fed into a membrane emulsification unit and an emulsion thereby formed. The emulsion was then transferred into a phase inversion unit with an aqueous anti-solvent to form cellulose beads.
Example 1 : Cellulose epoxide ring-opening etherification to form Cationic Cellulose Beads (CCBs)
[0262] 10 g of cellulose beads were prepared according to the methods detailed above, suspended in 25 mL of 0.1 M sodium hydroxide solution and then transferred to a round bottom flask. The bead suspension was heated to 65 °C and then 31.3 g of Glycidyl trimethylammonium chloride (GTMAC) was added in a dropwise manner. The reaction was then allowed to proceed for 1 , 2, 4, 6 or 8 hours under mild agitation. After reaction, the suspension was neutralised with hydrochloric acid (HCI) and the CCBs separated from any remaining reagents by vacuum filtration. The separated CCBs were thoroughly washed with distilled water until conductivity values were stable. Purified CCBs were then stored at 4°C. Charge density of CCBs was measured by conductometric titration of chloride (Ch) ions against silver nitrate (AgNOs).
Example 2: Physicochemical characterisation of CCBs
Charge density and dry weight
[0263] The CCBs taken from the reaction of Example 1 after reaction for 1 , 2, 4, 6, or 8 hours were analysed to determine their dry weight and charge density. Charge density is expressed in terms of milliequivalents as defined hereinabove. Accordingly, in the present Example, meq refers to the number of moles of trimethylammonium chloride moieties per gram of dry cellulose. The dry weight is expressed herein as the percentage of dry cellulose per unit of hydrated cellulose mass. The determined dry weights and charge densities at each time point are shown in Figure 3.
[0264] As can be seen from Figure 3, the charge density increased with time, while the dry weight correspondingly decreased with time.
[0265] The reduction in dry weight with the addition of charged moieties on the cellulose indicates that the cellulose network is influenced by charge-charge repulsive forces impeding formation of crystalline and/or highly packed polymer regions.
CCB morphology
[0266] Brightfield images of wet CCBs and wet unmodified cellulose beads were obtained with a microscope (EVOS M5000 Imaging System). The average diameter was evaluated using the software Imaged, measuring at least 25 beads of each type. Measurements for CCBs of varying size according to the present disclosure as well as unmodified cellulose beads and a commercial microcarrier not encompassed by the present disclosure are summarised in Table 1.
Table 1 : Average diameter [ m] of unmodified cellulose beads, ‘large’ CCBs, ‘medium’ CCBs, ‘small’ CCBs and a commercial microcarrier (n = 25 in each case). Values were obtained by Imaged analysis of swollen beads.
Figure imgf000065_0001
★Manufacturer information.
[0267] As can be seen from Table 1 , the size of the beads of the present disclosure can be readily varied, including to obtain beads of similar size to commercial microcarriers. Thus, beads can be prepared by the methods disclosed herein to meet the needs of different applications, for example by varying the cell-carrying capacity of each bead, as well as to be compatible with process and equipment requirements (e.g. separation). Example 3: Cell culture of mammalian adherent cells on CCBs
[0268] Cell culture was performed to assess the suitability and adhesion efficiency of CCBs as microcarriers. The cell line used was the murine myoblast C2C12 cell line (ECACC 91031101). The cells were cultured in proliferation media composed of high glucose Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich D5796) supplemented with 10% (v/v) foetal bovine serum (FBS; Gibco™, Thermo Fisher Scientific) and 1 % (v/v) penicillin/streptomycin (P/S; Sigma-Aldrich). Commercial microcarriers were used as a positive control, while unmodified cellulose microbeads were used as negative control. Before cell seeding, CCBs and controls were equilibrated in culture medium at 37°C, high humidity, and 5% CO2 for at least 15 minutes. Seeding was performed by transferring aliquots of cell suspension (at a known concentration) in a 24-well plate to reach 66,000 cells per well. Cells were grown in the cell culture incubator for one day while kept in suspension on a plate shaker at 100 rpm.
[0269] Prior to observation and cell counting, cells bound on microcarriers were stained with fluorescent dyes and imaged with a fluorescent microscope (EVOS M5000 Imaging System, ThermoFisher). Staining dyes used were: Hoechst 33342 (ThermoFisher Scientific, H21492), fluorescein diacetate (“FDA”; Acros Organic, 191660050), propidium iodide (“PI”; ThermoFisher Scientific, 11425392). Hoechst 33342 stains the nuclei of both live and dead cells; FDA stains the cytoplasm of live cells; PI stains the nuclei of dead cells.
[0270] For staining, spent media was discarded and the Hoechst 33342 solution was then added to the wells and incubated at room temperature in the dark for 10 minutes. The solution was then removed and replaced with the PI and FDA solution. Following an incubation time of 5 minutes in the dark, the solution was removed, and the wells washed once with PBS (phosphate buffered saline) prior to imaging. Images of cells were obtained as Z-stacks on the microcarriers to allow a sharper visualisation of cells attached on the microcarriers. Cells were analysed on day 1 , day 4 and day 7 after seeding. Projections of the Z-stacks are shown in Figure 4. As can be seen from Figure 4, cell attachment to unmodified beads was negligible whereas cell attachment was seen for both the CCBs according to the invention and the commercial microcarrier.
[0271] To quantitate the degree of cell attachment, cell counting using Imaged based on the images obtained as discussed above was performed. Stained cells were identified using the command “Find Maxima” which allows the automatic counting of higher intensity regions on the image, corresponding to the living cells. The average number of cells per mm2 was obtained by dividing the number of cells by the surface area of each type of microcarrier. Surface area was calculated based on the diameter of the microcarriers, assuming them to be perfectly spherical. The results are shown in Figure 5.
[0272] As can be seen from Figure 5, the absolute number of cells attached to the beads correlated with the bead size, while the number of cells per unit surface area varied depending on the type of beads used. ‘Medium’ CCBs showed a similar value compared to the commercial microcarriers. ‘Big’ and ‘small’ CCBs exhibited a higher average value. Cells were found to attach and proliferate irrespective of the size of the CCBs.
[0273] Figure 4 and Figure 5 both show that unmodified beads do not exhibit affinity for cell attachment, demonstrating that modification of the cellulose according to the methods disclosed herein is necessary to permit cell binding and proliferation. Moreover, the beads of the present disclosure enable high densities of cells to be achieved in microcarrier culture while also possessing the advantageous physicochemical and environmental/sustainability characteristics discussed hereinabove.
[0274] The various embodiments described herein are presented only to assist in understanding and teaching the claimed features. These embodiments are provided as a representative sample of embodiments only, and are not exhaustive and/or exclusive. It is to be understood that advantages, embodiments, examples, functions, features, structures, and/or other aspects described herein are not to be considered limitations on the scope of the invention as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope of the claimed invention. Various embodiments of the invention may suitably comprise, consist of, or consist essentially of, appropriate combinations of the disclosed elements, components, features, parts, steps, means, etc., other than those specifically described herein. In addition, this disclosure may include other inventions not presently claimed, but which may be claimed in future.

Claims

1. A method for preparing functionalised cellulose beads, said method comprising the steps of:
(iii) contacting cellulose beads with a base; and
(iv) reacting the product of step (i) with a salt represented by Formula (I):
A-L-B .X
(I) wherein A is an optionally substituted epoxide group, L is a linker group, B is a quaternary ammonium group, and X is a counterion.
2. The method of claim 1 , wherein the salt is represented by Formula (la): .X
Figure imgf000068_0001
wherein each of R1, R2, and R3 is independently selected from Ci-Ce alkyl, aralkyl and aryl, and X- is a halide ion; preferably wherein X- is a chloride ion.
3. The method of claim 2, wherein each of R1, R2, and R3 is independently selected from
Ci-Ce alkyl, preferably C1-C3 alkyl, more preferably wherein each R1, R2, and R3 is methyl.
4. The method of claim 2 or claim 3, wherein the salt of Formula (la) is produced in situ by reacting the product of step (i) with a compound of Formula (Ila):
Figure imgf000068_0002
wherein X; R1, R2, and R3 are as defined in claim 2, L is the linker group and Y is a halogen, preferably wherein Y is chlorine. 5. The method of any preceding claim, wherein L has the formula (CR4R5)m wherein m is an integer from 1 to 10 and each of R4 and R5 is independently selected from H, alkyl and aryl; preferably wherein m is 1 and each R4 and R5 is H.
6. The method of any preceding claim, wherein the base is a hydroxide salt.
7. The method of any preceding claim, wherein the method is carried out in aqueous solvent, preferably wherein the cellulose beads are suspended in the aqueous solvent.
8. The method of claim 7, wherein the aqueous solvent is substantially free of organic solvents.
9. The method of claim 7 or claim 8, wherein the amount of the salt represented by Formula (I) or (la) in step (ii) is at least about 50% w/v, preferably at least about 100% w/v, based on the volume of the aqueous solvent and base.
10. The method of any preceding claim, wherein the molar ratio of the salt of Formula (I) or Formula (la) to anhydrous glucose units of the cellulose beads is at least about 1:1, preferably at least about 2: 1 , more preferably at least about 3: 1.
11. The method of any preceding claim, wherein the method further comprises the step of
(iii) neutralising the product of step (ii) with an acid, preferably wherein the acid includes a counterion which is the same as X in Formula (I).
12. The method of any preceding claim, wherein the method further comprises the step of
(iv) separating the functionalised cellulose beads by filtration and optionally (v) washing the filtrate from step (iv) with water.
13. The method of any preceding claim, wherein the cellulose is selected from the group consisting of virgin, recycled, pulp, and microcrystalline cellulose, and combinations thereof.
14. The method of any preceding claim, wherein the cellulose beads are in the form of a hydrogel for step (i).
15. The method of any preceding claim, wherein the cellulose beads are not cross-linked.
16. A method for preparing functionalised polysaccharide beads, said method comprising the steps of: (ii) contacting polysaccharide beads with a base; and
(ii) reacting the product of step (i) with a salt represented by Formula (I):
A-L-B .X
(I) wherein A is an optionally substituted epoxide group, L is a linker group, B is a quaternary ammonium group, and X is a counterion; wherein the polysaccharide beads are prepared by extruding a dispersed phase into an anti-solvent to form beads of the polysaccharide, wherein the dispersed phase comprises the polysaccharide in a solvent, and wherein each of the solvent and anti-solvent comprises water; optionally wherein extruding the dispersed phase into an anti-solvent to form beads of the polysaccharide comprises extruding the dispersed phase through a fluid medium into a mould and then contacting the extruded dispersed phase with the anti-solvent. A method for preparing functionalised polysaccharide beads, said method comprising the steps of:
(i) contacting polysaccharide beads with a base; and
(ii) reacting the product of step (i) with a salt represented by Formula (I):
A-L-B .X
(I) wherein A is an optionally substituted epoxide group, L is a linker group, B is a quaternary ammonium group, and X is a counterion; wherein the polysaccharide beads are prepared by: a. a membrane emulsification of a dispersed phase into a continuous phase wherein the dispersed phase comprises the polysaccharide in a solvent, and wherein passing the dispersed phase through the membrane forms an emulsion of the polysaccharide in the continuous phase; and b. a phase inversion with an anti-solvent to form beads of the polysaccharide; wherein each of the solvent and anti-solvent comprises water. The method of claim 16 or 17, wherein the solvent of the dispersed phase further comprises an ionic liquid.
19. The method of any one of claims 16 to 18, wherein the anti-solvent is substantially free of organic solvents.
20. The method of any one of claims 16 to 19, wherein the anti-solvent further comprises an ionic liquid, or wherein the anti-solvent consists of water. 21. A method for attaching cells to functionalised beads, wherein the method comprises preparing functionalised beads by the method of any one of claims 1 to 20 and contacting the functionalised beads with one or more cells, preferably wherein the functionalised beads are contacted with one or more cells during cell culture.
22. The method of claim 21 wherein the one or more cells are animal cells, preferably mammalian cells, more preferably adherent mammalian cells.
23. Functionalised beads prepared by the method according to any preceding claim, preferably wherein said beads are approximately spherical and/or have a diameter from about 80 pm to about 3 mm.
24. Use of functionalised beads prepared by the method of any one of claims 1 to 22 in cell culture.
PCT/GB2023/052461 2022-09-23 2023-09-22 Functionalised polysaccharide bead preparation WO2024062258A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB2213888.7 2022-09-23
GBGB2213888.7A GB202213888D0 (en) 2022-09-23 2022-09-23 Functionalised polysaccharide bead preparation

Publications (1)

Publication Number Publication Date
WO2024062258A1 true WO2024062258A1 (en) 2024-03-28

Family

ID=83978828

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2023/052461 WO2024062258A1 (en) 2022-09-23 2023-09-22 Functionalised polysaccharide bead preparation

Country Status (2)

Country Link
GB (1) GB202213888D0 (en)
WO (1) WO2024062258A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1081717A1 (en) * 1998-04-28 2001-03-07 Chisso Corporation Method for treating radioactive waste
WO2001045830A1 (en) 1999-12-22 2001-06-28 University Of Leeds Rotating membrane
WO2012094595A2 (en) 2011-01-07 2012-07-12 Brotech Corp., D/B/A Purolite Method of producing uniform polymer beads of various sizes
CN107522877A (en) * 2017-07-18 2017-12-29 武汉工程大学 Quaternized cellulose bead, preparation method and purposes
WO2019092461A1 (en) 2017-11-13 2019-05-16 Micropore Technologies Ltd Cross-flow assembly and method for membrane emulsification controlled droplet production

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1081717A1 (en) * 1998-04-28 2001-03-07 Chisso Corporation Method for treating radioactive waste
WO2001045830A1 (en) 1999-12-22 2001-06-28 University Of Leeds Rotating membrane
WO2012094595A2 (en) 2011-01-07 2012-07-12 Brotech Corp., D/B/A Purolite Method of producing uniform polymer beads of various sizes
CN107522877A (en) * 2017-07-18 2017-12-29 武汉工程大学 Quaternized cellulose bead, preparation method and purposes
WO2019092461A1 (en) 2017-11-13 2019-05-16 Micropore Technologies Ltd Cross-flow assembly and method for membrane emulsification controlled droplet production

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
"A Manual of Basic Technique and Specialized Applications", 2005, JOHN WILEY & SONS, INC, article "Freshney's Culture of Animal Cells"
"Advanced Fermentation and Cell Technology", 2021, WILEY-BLACKWELL
ACS SUSTAINABLE CHEM. ENG., vol. 5, no. 7, 2017, pages 5931 - 5939
DATABASE WPI Week 201810, Derwent World Patents Index; AN 2018-01836G, XP002810745 *
MARTIN GERICKE ET AL: "Functional Cellulose Beads: Preparation, Characterization, and Applications", vol. 113, no. 7, 29 March 2013 (2013-03-29), pages 4812 - 4836, XP002739481, ISSN: 0009-2665, Retrieved from the Internet <URL:http://pubs.acs.org/doi/ipdf/10.1021/cr300242j> [retrieved on 20150512], DOI: 10.1021/CR300242J *
OBRIEN JAMES COOMBS ET AL: "Continuous Production of Cellulose Microbeads via Membrane Emulsification", ACS SUSTAINABLE CHEM. ENG., vol. 5, 31 May 2017 (2017-05-31), pages 5931 - 5939, XP055979096 *
PEDRO S. SILVA ET AL.: "Azimuthally Oscillating Membrane Emulsification for Controlled Droplet Production", AICHE JOURNAL, vol. 00, no. 00, 2015
RICHARD ET AL., J. AM. CHEM. SOC., vol. 124, 2002, pages 4974 - 4975
VERMA ET AL., SUSTAINABLE CHEMISTRY AND PHARMACY, vol. 13, 2019, pages 100162
WILLIAMS ET AL., THERMOCHIMICA ACTA, vol. 669, 2018, pages 126 - 139

Also Published As

Publication number Publication date
GB202213888D0 (en) 2022-11-09

Similar Documents

Publication Publication Date Title
US8337901B2 (en) Process for the manufacture of cellulose sulfate with improved characteristics
Tavassoli et al. Large-scale production of stem cells utilizing microcarriers: a biomaterials engineering perspective from academic research to commercialized products
Gericke et al. Functional cellulose beads: preparation, characterization, and applications
JP6581650B2 (en) Culture container for spheroid formation using temperature-sensitive glycol chitosan derivative and method for forming spheroid using the same
CN112409553B (en) Method for preparing injectable porous hydrogel microspheres by micro-fluidic ice crystal method and application thereof
Campiglio et al. Cross-linking optimization for electrospun gelatin: Challenge of preserving fiber topography
Khan et al. Optimization of a 3D bioprinting process using ultrashort peptide bioinks
Kim et al. Multiscale modulation of nanocrystalline cellulose hydrogel via nanocarbon hybridization for 3D neuronal bilayer formation
WO2024062258A1 (en) Functionalised polysaccharide bead preparation
CN109135226A (en) A kind of polylactic acid/polyaniline/nano micro crystal cellulose composite conductive thin film and preparation method thereof
Chow Electrospinning functionalized polymers for use as tissue engineering scaffolds
Li et al. Preparation of polycarbonate/gelatine microspheres using a high-voltage electrostatic technique for enhancing the adhesion and proliferation of mesenchymal stem cells
Godoy-Gallardo et al. Advanced Binary Guanosine and Guanosine 5'-Monophosphate Cell-Laden Hydrogels for Soft Tissue Reconstruction by 3D Bioprinting
Marapureddy et al. Carbamoylated chitosan hydrogels with improved viscoelastic properties and stability for potential 3D cell culture applications
Jang et al. Sulfur (VI) fluoride exchange (SuFEx)-mediated synthesis of the chitosan-PEG conjugate and its supramolecular hydrogels for protein delivery
CN105106967A (en) Chitosan microparticles with shapes and sizes identical to lurid cells and preparation method of chitosan microparticles
Kim et al. Fabrication of biomimetic bundled gel fibres using dynamic microfluidic gelation of phase-separated polymer solutions
CN110511405A (en) A kind of antimicrobial form keratin based aquagel and preparation method thereof
EP3992277A1 (en) Cell culturing vessel and cell culturing apparatus
WO2017109755A1 (en) Gellan gum-based hydrogels, methods and uses thereof
Mojares et al. Strong Elastic Protein Nanosheets Enable the Culture and Differentiation of Induced Pluripotent Stem Cells on Microdroplets
Vassilev et al. Manufacturing human pluripotent stem cells and differentiated progenitors
Kamperman et al. Smart Building Blocks for Modular Tissue Engineering Based on Sequential Desthiobiotin/Biotin Coupling
Hayakawa et al. Suspension Culture System for Isolating Cancer Spheroids using Enzymatically Synthesized Cellulose Oligomers
Zhang et al. Engineering Cell Microenvironment Using Nanopattern-Derived Multicellular Spheroids and Photo-Crosslinked Gelatin/Hyaluronan Hydrogels

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23783498

Country of ref document: EP

Kind code of ref document: A1