Use of a double-network hydrogel resin comprising agarose and a sugar-alcohol for purification of molecules

Abstract

The present invention relates to a double-network (DN) hydrogel resin comprising a physico-chemically cross-linked agarose as the first network, the cross-linked agarose being non-covalently linked to at least one physico-chemically cross-linked sugar-alcohol of the general formula (CHOH)nH2, where n = 4–12, as the second network, the two networks interpenetrate to form a double network structure hydrogel. The DN resin can be further cross-linked chemically, i.e. via covalent bonds created by a bifunctional cross-linker. A method of preparing the DN hydrogel resin, use thereof as an adsorbent material and preferably as chromatography media, a packed or fluidised bed comprising beads of the DN hydrogel resin, a housing comprising same, as well as processes for purifying a sample comprising a macromolecule or for purifying a protein using beads of the DN hydrogel resin are also disclosed.

Description

Use of a double-network hydrogel resin comprising agarose and a sugar-alcohol for purification of moleculesTECHNICAL FIELD

[0001] The present invention generally relates to a doublenetwork hydrogel suitable for the purification of molecules via a chromatographic or adsorptive method. More particularly, the present invention relates to a double-network hydrogel made of agarose and a sugar-alcohol suitable for different types of chromatography and adsorptive systems used for purifying molecules, including macromolecules such as proteins.BACKGROUND OF THE INVENTION

[0002] Agarose (AG) is considered the best material for manufacturing adsorbents for biomolecule purification. This is due to the benefits associated with AG-based adsorbents, such as high internal porosity, their hydrophilic nature and their easy chemical modification and functionalisation. A major drawback for using AG as a chromatography support material is the relatively low mechanical strength of the beaded / cross-linked material; this, in turn, has a negative effect on adsorbent lifetime and stability under operating pressures. A second major drawback of AG-based adsorbents is their fragility when exposed to mechanical stress (e.g., stirring, pumping, conveying) which prevents the transport of such particles without damage.

[0003] Alternatively, polymeric adsorbents - that are made of synthetic polymers - have high mechanical strength but, due to limited internal porosity, they are better suited for smaller molecules. These adsorbents may also suffer from lower binding capacity. Additionally, continuous processing schemes require the transport of such adsorbent beads further limiting their use due to mechanical instability.

[0004] Several hydrogel technologies have been reported in the art providing products suitable for applications in di f ferent technical fields , e . g . biomedical , personal care as well as in nano-sensor applications . Hydrogels fabricated from single polymers have been extensively investigated . However, in many cases a single polymer alone cannot meet divergent demands in terms of both properties and performance .

[0005] Double-network ( DN) hydrogels have been reported as promising sof t-and-tough materials that intrinsically possess extraordinary mechanical strength and toughness due to their unique contrasting network structures , strong interpenetrating network entanglement , and ef ficient energy dissipation .

[0006] DN hydrogels are widely used materials due to their biocompatibility, their ability to mimic the hydrated and porous extracellular microenvironment , as well as their ability to tune both mechanical and biochemical properties , so their use in the medical field is very promising . Tissue engineering and repair of cartilage tissue is considered one of their most prominent applications . DN hydrogels have also attracted much attention as environmental adsorption materials because their large particle morphology and pore structure , excel lent water absorption . The large number of functional groups on the polymer chains , good anti-swelling performance and the doping of nanoparticles lead to excellent adsorption capacity . However, the interconnection between the two networks is found to be unclear and uncontrolled .

[0007] Lyons et al . , 2009 reported hydrogels prepared by physically blending the natural polymer agar with the high molecular weight synthetic polymer polyvinyl alcohol ( PVA) in varying ratios to produce a new biosynthetic polymer applicable for a variety of purposes . Hydrogen bonding was observed to take place between the polyvinyl alcohol and the agar molecules in the composite materials leading to changes in the thermal ,mechanical and swelling characteristics of the composite hydrogels .

[0008] Shao et al., 2019 report a DN hydrogel consisting of a fully physically linked poly (vinyl alcohol ) / agarose (PVA / AG) dual-network, which is of high toughness and self-healing properties .

[0009] However, constructing a double-network hydrogel as an adsorbent for chromatography or adsorptive recovery purposes has not been reported in the art.

[0010] There is a need in the art for the development of agarose- based adsorbent material with increased mechanical strength / stability and adsorption capacity, particularly suitable for purification of biomolecules. There is also a need in the art for the development of agarose-based beads that can be transported without damage.SUMMARY

[0011] The object of the invention is to improve the agarose- based material for chromatography and adsorption applications disclosed in the art. Of special relevance are adsorptive methods that require a true moving bed, where particle transport is required .

[0012] The object is achieved by the use of a double-network hydrogel resin as an adsorbent material (also called adsorption media) as defined in claim 1. Advantageous embodiments are recited in the dependent claims. The double-network hydrogel resin is preferably used as a chromatography support material (also called chromatography media) , and more preferably as an ion-exchange chromatography media, and even more preferably as a cation-exchange chromatography media.

[0013] Furthermore, the object is achieved by a process as defined in claim 7 for purifying a sample comprising amacromolecule, said process implementing the double-network hydrogel resin; a process as defined in claim 9 for purifying proteins, said process implementing the double-network hydrogel resin; a double-network hydrogel resin as defined in claim 1 and more particularly as defined in claim 10; a method as defined in claim 11 of preparing the double-network hydrogel resin; a packed bed or fluidised bed as defined in claim 14 comprising the double-network hydrogel resin; and a housing as defined in claim 15 comprising the packed bed or fluidised bed.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1. Microscopic optical images for a) control (also called SN-SP beads) after 12 pumping cycles, b) DN1-SP beads after 12 pumping cycles, c) DN2-SP beads after 12 pumping cycles, d) control (also called SN-SP beads) after 48 pumping cycles, e) DN1-SP beads after 48 pumping cycles and f) DN2-SP beads after 48 pumping cycles. All adsorbents were functionalized with SP ligand .Figure 2: Bar graphic on the measurement of the tensile strength of the DN casted hydrogels (DN1-CH and DN2-CH) , and control (also called SN-SP beads) .Figure 3: Bar graphic on the measurement of the compression strength of the DN casted hydrogels (DN1-CH and DN2-CH) , and control (also called SN-SP beads) .Figure 4: Measurement of the maximum binding capacity of the single network (SN-SP beads) hydrogel and the DN hydrogels (DN1- SP beads and DN2-SP beads) , all adsorbents being functionalized with SP groups, with a particle size of 250-350 pm (■) and 350- 500 pm (■) .Figure 5: Measurement of viscoelastic properties of DN2-SP beads (A) , comparative DN2-SP beads (■) , and control SN-SP beads (•) .Figure 6: Comparison of maximum binding capacities of DNhydrogels (DN1-SP beads and DN2-SP beads) and control (SN-SP beads hydrogel) , all adsorbents being functionalized with SP groups, with particle size range of 100-500 pm.Figure 7: Measurement of the maximum binding capacity of control (SN-SP beads hydrogel) , DN1-SP beads and DN2-SP beads, all adsorbents being functionalized with SP groups, under static conditions with a particle size of 250-350 pm (^) and 350-500 pm (^) .Figure 8: Measurement of the adsorption capacity under operational conditions at 10% breakthrough value for adsorbents with a particle size range of 100-500 pm for control (■) (SN-SP beads hydrogel) , DN1-SP beads (A) and DN2-SP beads (•) , all adsorbents being functionalized with SP groups.Figure 9: Measurement of the total ionic strength of SN-SP beads, Comparative DN1-SP beads, Comparative DN2-SP beads, DN1-SP beads and DN2-SP beads with a particle size of 250-350pm.Figure 10: Recorded values on first moment as a function of theoretically calculated superficial residence time for pulse injection of BSA (bovine serum albumin) under non-binding conditions into a column packed with SN-SP beads, Comparative DN1-SP beads and DN-SP beads adsorbents of the present invention (DN1-SP beads and DN2-SP beads) with a particle size of 250- 350pm. / z-t is calculated according to Equation 1 / 0°°C(t)tdt l ~ Jo C(t)dt 1 and plotted against theoretically calculated superficial residence time where L is the length of the column (m) and Uis the linear flow velocity (m / s) . L / U is thus in seconds and can be converted into minutes.DETAILED DESCRIPTION

[0014] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

[0015] "Double-network (DN) hydrogels" are generally formed by crosslinking two different polymers chemically and / or physically and are considered as a special class of interpenetrating polymer network (IPN) hydrogels that are characterized by: (i) two networks that are interpenetrating yet independent, with asymmetric and contrasting properties; and (ii) the molar concentration of the first network being 20-300 times higher than that of the secondary network. The primary network with high stiffness (i.e., brittle) acts as a sacrificial network, whereas the secondary network is ductile and can sustain large deformations, resulting in synergistically high strength and toughness. In the present invention, the double network (DN) hydrogel comprises two intertwined cross-linked networks, a first network represented by agarose and a second network represented by a specific sugar-alcohol . The two networks are interpenetrating thanks to at least physical crosslinking between both networks, and the molar concentration of the first network is higher than that of the secondary network, preferably the molar concentration of the first network (agarose) is 1.05 to 5 times, and more preferably around 1.5 (e.g. 1.44) times higher than that of the secondary network (specific sugaralcohol) .

[0016] The terms "microspheres", "beads", "adsorbent beads" and "adsorbents" refer to spherical or substantially spherical particles, and are understood as having the same meaning. These terms may therefore be used herein interchangeably.

[0017] The term "sugar-alcohol" as utilized herein, also called polyhydric alcohols, polyalcohols, alditols or glycitols are a class of low molecular weight polyols, commonly obtained by hydrogenation of sugars. In the present invention, they have the general formula (CHOH)nH2, where n = 4-12. The general formula(CHOH)nH2can also be written CnOnH2n+2or

[0018] The term "adsorptive functionality" and "adsorptive functionality groups" are used herein interchangeably.

[0019] The term "bifunctional cross-linkers" refers to chemical compounds with two reactive ends that can form covalent bonds with the available -OH groups on the hydrogel forming substances, creating a stable (chemically) cross-linked network (also known as chemical crosslinking) . Examples of bifunctional crosslinkers are bis-oxiranes or bis-epoxides (e.g. diethylene glycol diglycidyl ether, diglycidyl ether) , diamines (e.g. ethylenediamine, hexamethylenediamine) , dicarboxylic acids (succinic acid, adipic acid) , diisocyanates (e.g. hexamethylene diisocyanate) , and dialdehydes (e.g. glutaraldehyde) .

[0020] The term "macromolecule" generally refers to a molecule having a high molecular weight, for example having an average molecular weight of 1,000 Daltons or greater. For the purposes of the invention, the macromolecules have preferably an average molecular weight of 10,000 Daltons or greater, 60,000 Daltons or greater, or even 140,000 Daltons or greater. Of particular interest of the invention, the macromolecules are biomolecules, particularly proteins.

[0021] "Protein mixture" (respectively "nucleic acid mixture") comprises a protein (respectively a nucleic acid) of interest (for which purification is desired) and one or more contaminant, i.e., impurities. In one embodiment, the mixture is produced from a host cell or organism that expresses the protein of interest (either naturally or recombinantly ) . Such mixtures include, for example, cell cultures, cell fermentation broth, cell lysates, and clarified bulk (e.g., clarified cell culture supernatant) .

[0022] In a first aspect, the present invention relates to a double-network hydrogel resin, comprising a physico-chemically cross-linked agarose as the first network, the cross-linked agarose being non-covalently linked to at least one physico- chemically cross-linked sugar-alcohol of the general formula (CHOH)nH2, where n = 4-12, as the second network, the two networks interpenetrating to form a double-network structure hydrogel. In the present invention, the term "physico-chemically crosslinked" refers to at least physical crosslinking and optionally chemical crosslinking. It is well-known that physical crosslinking is obtained through physical interactions (e.g. hydrogen bonds, electrostatic interactions, hydrophobic interaction, etc.) whereas chemical crosslinking is obtained for example through the use of a chemical cross-linker. Physical crosslinking produces non-covalent bonds whereas chemical crosslinking produces covalent bonds. In the present invention, the agarose and the sugar-alcohol are at least cross-linked through physical interactions, and preferably through hydrogen bonds or hydrogen bonding.

[0023] The present invention in particular relates to a non- covalently cross-linked network formed by mixtures of agarose and sugar-alcohols of the general formula (CHOH)nH2, where n = 4-12.

[0024] The sugar-alcohol may be selected from the group consisting of mannitol (n=6) , sorbitol (n=6) , galactitol (n=6) , erythritol (n=4) , xylitol (n=5) , threitol (n=4) , arabitol (n=5) , ribitol (also called adonitol) (n=5) , iditol (n=6) , and volemitol (n=7) . Preferably, the sugar-alcohol is mannitol or sorbitol .

[0025] Advantageously, the mass ratio of the first network or network-forming substance of agarose to the second network or network-forming substance of sugar-alcohol is between 6:0.1 and 0.1:6, preferably between 5:1 and 1:5, and more preferably between 5:1 and 1:1.1. Advantageously, the agarose and sugaralcohols of the general formula (CHOH)nH2, where n = 4-12 represent from 0.5 to 15% by weight, and more preferably from 1 to 10% by weight, with respect to the total weight of the DN hydrogel resin. Preferably, water molecules represent at least 90% by weight, and more preferably at least 95% by weight, with respect to the total weight of the DN hydrogel resin.

[0026] The two networks of the DN hydrogel resin of the present invention are preferentially non-covalently linked via hydrogen bonding (which is a specific physical crosslinking) . Hydrogen bonding accounts for a significant increase of the mechanical strength and confers self-healing properties to the material. In particular, agarose as the first network is linked to at least one sugar-alcohol of the general formula (CHOH)nH2, where n = 4- 12, as the second network thanks to hydrogen bonds, which are well-known to be non-covalent bonds. By contrast, the traditional crosslinking step (chemical crosslinking) , which provides increased mechanical strength for the agarose resin alone is not used in the present invention, as this procedure does not improve mechanical properties of the DN hydrogel resin.

[0027] In a particular embodiment, the DN hydrogel resin (which is preferably at least physically cross-linked thanks to hydrogen bonds between agarose and at least one sugar-alcohol asdefined in the present invention) can be further chemically cross-linked, i.e. via covalent bonds created by a bifunctional cross-linker. Suitable bifunctional cross-linkers for use in the present invention are e.g. bis-oxiranes or bis-epoxides (e.g., diethylene glycol diglycidyl ether, diglycidyl ether) , diamines (e.g. ethylenediamine, hexamethylenediamine) , dicarboxylic acids (succinic acid, adipic acid) , diisocyanates (e.g. hexamethylene diisocyanate) , or dialdehydes (e.g. glutaraldehyde) , and preferably bis-oxiranes or bis-epoxides. The bifunctional cross-linkers can react at both extremes with available -OH groups on the hydrogel forming substances. However, such chemical crosslinking is only optional (i.e. not mandatory) in the DN hydrogel resin of the present invention. Indeed, chemical crosslinking can decrease the adsorption capacity of the DN hydrogel resin of the present invention.

[0028] The DN hydrogel resin may be conveniently adapted for use as an adsorbent material (also called adsorption media) . Advantageously the double-network hydrogel resin is used as a chromatography support material (also called chromatography media) . In particular, the DN hydrogel resin can be used as the stationary phase for different types of chromatography and other adsorptive methods, depending on the target macromolecule to be purified and the type of envisioned process. Accordingly, the DN hydrogel resin may be modified with a functionality enabling reversible bioproduct binding by a method selected from the group consisting of ion-exchange, hydrophobic interaction, mixed-mode, and affinity chromatography. The double-network hydrogel resin is more preferably used as an ion-exchange chromatography media. In that embodiment, the DN hydrogel resin may be modified with a functionality enabling reversible bioproduct binding by ionexchange chromatography method. Likewise, the use of a densifier would allow for the use in fluidised bed systems or moving bed systems .

[0029] Reversible product binding refers to the ability of the adsorbent to capture the product of interest while it is possible to release such product from the solid phase upon changing the conditions of the liquid phase (e.g., pH, salt concentration, addition of organic modifiers, addition of competing species, etc . ) .

[0030] In certain embodiments, the adsorptive functionality consists of pendant sulfopropyl (SP) groups (enabling a cationexchange chromatography) , diethylaminoethyl (DEAE) weak anion exchange groups, or trimethylammonium strong anion- exchange groups, or other pendant groups that enable other types of chromatography modes, e.g., hydrophobic interaction chromatography, mixed-mode and affinity chromatography or mixedmode chromatography. Such functionalization follows classical routes to chemical ligand immobilisation onto polysaccharide matrices .

[0031] In one particular embodiment, the functionality is an ion-exchange adsorptive functionality, preferably a cationexchange adsorptive functionality. In a preferred embodiment, the adsorptive functionality consists of pendant SP groups, most preferably pendant SP groups.

[0032] The DN hydrogel resin of the present invention may be provided as spherical or substantially spherical particles, or "beads". It is known that in certain contexts, irregularly shaped particles create the uneven flow resulting from inhomogeneous packing of the column, the so-called channelling, besides other problems. Spherical material tends to reduce or eliminate these problems. Generation of the DN hydrogels-based spherical particles reduces or eliminates channelling and flow defects.

[0033] The DN adsorbents can be manufactured with any particle size depending on the application (packed / f luidized bed) , but for chromatographic beads the typical size range is between 50-500 pm. Likewise, population particle size distribution can be adjusted, depending on the degree of uniformity required.

[0034] The size of the spherical particles of the DN hydrogel resin of the present invention, i.e. in the form of beads, varies between about 10 pm and about 500 pm in diameter, preferably between 50 pm and about 500 pm in diameter, more preferably between about 250 pm and about 350 in diameter or between about 350 pm and about 500 pm in diameter.

[0035] In another aspect, the invention relates to a method of preparing the double network hydrogel resin of the invention, comprising the step of contacting agarose and at least one physico-chemically cross-linked sugar-alcohol of general formula (CHOH)nH2, where n = 4-12.

[0036] According to one embodiment, the sugar-alcohol is selected from the group consisting of mannitol (n=6) , sorbitol (n=6) , galactitol (n=6) , erythritol (n=4) , xylitol (n=5) , threitol (n=4) , arabitol (n=5) , ribitol (n=5) , iditol (n=6) , and volemitol (n=7) .

[0037] Advantageously, the agarose and the at least one sugaralcohol are contacted in a mass ratio between 6:0.1 and 0.1:6, preferably 5:1 and 1:5, more preferably between 5:1 and 1:1.1 (i.e. first network of agarose to second network of sugar alcohol) .

[0038] The agarose and at least one sugar-alcohol can be contacted by using a solvent, e.g., demineralized water, and preferably subjected to heat (e.g. microwave) until dissolved (so as to form a resulting solution) . A densifier can then be added to the resulting solution.

[0039] The DN hydrogels can be activated and / or functionalized using methods known in the art generally applied for agarose and any chemistry available for hydroxyl group can be applied.

[0040] The densifier used in the process for preparation of the DN hydrogel beads of the present invention may be a metal oxide, e.g. those metal dioxides which are members of the group of dioxides comprising titanium dioxide, aluminum dioxide, silicon dioxide and mixtures thereof. Barium sulfate, stainless steel powder and glass are also suitable densifiers. In a preferred embodiment, the densifier is titanium dioxide.

[0041] Optionally, spherical particles ("beads") of the DN hydrogel of the present invention may be provided, following the step of addition of a densifier. The beads of the DN hydrogel may be formed by using any emulsification technology known in the art, such as the one described by Palsson, Gustavsson, & Larsson, 2000. The densifier can represent from 1 to 5% by weight, with respect to the total weight of the DN hydrogel of the present invention.

[0042] In a preferred embodiment, the size of the beads varies between about 50 pm and about 500 pm in diameter, preferably between about 250 pm and about 350 or preferably between about 350 pm and about 500 pm in diameter.

[0043] In a particular embodiment, the method of preparing the double-network hydrogel resin of the invention further comprises the (optional) step of subjecting the DN hydrogel resin to a f reeze-and-thaw (FT) method. This method enables physical crosslinking between agarose and the at least one sugar-alcohol as defined in the present invention.

[0044] The f reeze-and-thaw method preferably comprises or consists of drying the DN hydrogel resin, which may be in form of beads, by vacuum and placing it into a freezer at very low temperature, e.g. -15 to -25°C, for several hours (e.g. 10-14 hours) followed by thawing for several hours (e.g. 3-7 hours) at room temperature, e.g. from 20 to 25°C. Of note, the freeze-and- thaw method cannot be applied for the agarose-single network (SN) hydrogel. To a certain degree, hydroxyl groups in agarosecould potentially form hydrogen bonds among each other, but this would reduce the available functional groups, thus resulting in decreased adsorption capacity. On the other hand, in DN hydrogels, two polymers are involved, the agarose and the at least one sugar-alcohol , increasing the amount of hydroxyl groups and consequently the amount of hydrogen bonds formed during (physical) crosslinking.

[0045] The DN hydrogel resin of the present invention exhibits increased protein binding capacity, mechanical strength and density compared to the single network (SN) agarose resin alone. One or more of these properties may be further improved by subjecting the DN hydrogel resin, or beads thereof, to the f reeze-and-thaw method. Different number of cycles of freezethaw may be needed to achieve an optimal biding capacity, depending on the composition of the DN hydrogel resin. For instance, subjecting the DN hydrogel resin to 2 to 6 freeze-thaw cycles may increase the maximum binding capacity of the DN hydrogel resin of the present invention.

[0046] The DN hydrogel resin, or beads thereof, which may have or may have not been subjected to the optional step of the f reeze-and-thaw (FT) method, may be then functionalized by applying conventional conjugation methods for use as the stationary phase in different chromatography methods.

[0047] Accordingly, the DN hydrogel resin, or beads thereof, may be modified with a functionality enabling reversible bioproduct binding by a method selected from the group consisting of ionexchange, hydrophobic interaction, mixed-mode, and affinity chromatography, preferably wherein the functionality is an ionexchange adsorptive functionality, more preferably a cationexchange adsorptive functionality.

[0048] The surface functionalization of the double-network hydrogel resin of the invention may be achieved by attaching pendant allyl groups to the DN hydrogel surface with hydroxylgroups by treatment thereof with an allylation agent, such as allyl bromide, allyl chloride or any other suitable allylation agent known in the art. The attachment of a pendant allyl group may be performed as described by Shi and co-workers (2010) . The pendant allyl groups then serve as anchoring sites on the DN hydrogel as attachment points for the pendant functionality groups .

[0049] In a preferred embodiment, the adsorptive functionality consists of pendant SP groups.

[0050] In yet another aspect, the present invention relates to the use of the double-network hydrogel resin, or beads thereof, as an ion-exchange chromatography media, optionally a cationexchange chromatography media.

[0051] As a preferred embodiment, the DN hydrogel resin, or beads thereof, of the present disclosure is suitable for protein or nucleic acid purification.

[0052] In yet another aspect, the present invention relates to a packed or fluidised bed comprising the DN hydrogel resin, or beads thereof, according to the invention as the chromatography media .

[0053] The fluidised or moving beds comprising the beads of the DN network hydrogel resin described herein in the appropriate densities and particle sizes achieves good separation performance inter alia higher loading capacities, higher throughput, higher binding capacity or the less complexity of scale-up .

[0054] In a further aspect, a housing comprising the packed bed or fluidised bed as disclosed herein is provided.

[0055] Moreover, in yet another aspect, it is provided a process for purifying a sample comprising a macromolecule, comprising contacting said sample with a packed bed or a fluidised bed asdisclosed herein. In one embodiment, the purification is carried out at a pH ranging from 5 to 8.

[0056] The DN network hydrogel resin, or beads thereof, of the present invention as a chromatography media enables purification in a continuous flow-through mode or a bind / elute mode. Advantageously, a continuous flow-through mode is used, especially due to the increased resistance of the DN network hydrogel resin, or beads thereof, to mechanical treatment.

[0057] In addition, as the DN hydrogel resin, or beads thereof, of the present invention has increased density compared to standard agarose-based resin beads, they are particularly suitable for use in fluidised beds (DBA) or expanded beds (EBA) or true moving beds (TMB) . The present DN hydrogel resins beads may further be less prone to biomass fouling, if the DN hydrogel resin beads are prepared by using a densifier, preferably titanium oxide as densifier.

[0058] In still another aspect, the DN hydrogel resin as described herein is particularly suitable for purification of biomacromolecules or macromolecules, including, but not limited to, proteins (inter alia immunoglobulins including human recombinant immunoglobulins such as the monoclonal antibody adalimumab (Humira®) ) , nucleic acids (DNA and RNA) , carbohydrates and lipids.

[0059] It is therefore another embodiment of the present invention a process for purifying a protein or a nucleic acid, comprising; providing a protein or nucleic acid mixture, contacting the protein or nucleic acid mixture with beads of the double network hydrogel as disclosed herein, washing the DN hydrogel resin beads to remove unbound species, compressing the DN hydrogel resin beads, and washing the compressed DN hydrogel resin beads to extract bound protein or bound nucleic acid.

[0060] In one aspect, the protein is an antibody, such as a monoclonal antibody (including, but not limited to a human, humanized and chimeric antibody) .

[0061] One will appreciate that the selection of chromatographic conditions, elutes and buffers suitable for performing the processes for purification of macromolecules or proteins as disclosed herein is within the ability of those skilled in the art .ExamplesExample 1: Preparation of functionalized adsorbent DN hydrogen resin beads la) Preparation of the DN hydrogen resin

[0062] The double-network hydrogen resin of the present invention is prepared according to the following steps: i. Agarose and selected sugar-alcohols (mannitol / sorbitol ) are weighed and dissolved into demineralized water in an optimal mass ratio (5:1 agarose to sugar-alcohol ) and heated in a microwave until completely dissolved; ii. Titanium dioxide (TiCy) powder as a densifier is added to the resulting solution and stirred for 10 min in a water bath kept at 90°C. lb) Preparation of the DN hydrogen resin spherical beads through emulsification technology according to Palsson, Gustavsson, & Larsson, 2000 iii. The solution is poured into an oil phase containing paraffin oil and Span 80 (1g span 80 / 100 mL of paraffin oil) heated at 55°C with a magnetic stirrer at 750 rpm. iv. Formed microspheres are collected by centrifugation and washed with 20% by volume acetone and demineralized water three times.lc) Optimization of the DN hydrogen resin using the freeze-thaw (FT) method v. The DN hydrogel beads are then vacuum dried to remove excess water and kept at -20 °C for 12 hours followed by thawing at 20°C for 5 hours. vi . Microspheres (beads) are kept at -20°C for 12h and thawed at 20°C for 5h (not applied for SN-SP control) . ld) After (optional) step 1c) , the DN hydrogel beads were functionalized by applying conventional conjugation methods for use in different chromatography methods. For example, The DN hydrogel beads prepared in Example la) -c) were then functionalized with pendant SP groups as follows:

[0063] The DN hydrogel beads were coupled with SP ligand groups leading to cation-exchange properties (Shi, Jia, & Sun, 2010) . The hydroxyl groups were ally-activated by adding 0.6mL of dimethyl sulfoxide (DMSO) , ImL of 0.4M NaOH and 0.6mL of allyl bromide (AlBr) per 1g of microspheres to an Erlenmeyer flask kept with stirring at 25°C for 24h. Afterwards, the residual reactants were washed off with distilled water and the pendant SP groups were then coupled to the beads by adding 0.3 g of sodium metabisulfite (Na2S20s) per 1g of adsorbent beads into the Erlenmeyer flask with 10 ml of demineralized water per 1g of beads followed by adjusting pH to 6.0 and placing flask shaking for 18h at 25°C. DN1-SP ( sugar-alcohol = mannitol) and DN2-SP ( sugar-alcohol = sorbitol) were obtained from this protocol. DN1-SP and DN2-SP are in the form of beads in the following examples, except indicated otherwise.

[0064] Comparative functionalized DN hydrogels 1 and 2 (Comparative DN1-SP and Comparative DN2-SP) made from PVA (instead of selected sugar-alcohols ) were also prepared according to the method of preparation la) -d) described above. Comparative DN1-SP and Comparative DN2-SP are in the form of beads in the following examples, except indicated otherwise.

[0065] The control material (agarose adsorbent coupled with SP groups - "SN-SP") was manufactured according to established protocols, with 5% by weight agarose in water followed by epoxy crosslinking and further SP-ligand coupling. SN-SP is in the form of beads in the following examples except indicated otherwise .

[0066] The composition of the exemplary DN hydrogels of the invention, the comparative DN hydrogels and the control (agarose alone) are shown in Table 1.Table 1: Compositions of Control, Comparative DN hydrogels and the DN hydrogels of the invention (weight! with respect to the total weight of the composition) .FTN:number of freeze-thaw (FT) cycles.

[0067] Table 1 includes optimal compositions and the results for all the other DN compositions evaluated are not shown.Example 2: Results under mechanical stress

[0068] The mechanical strength of the functionalized SP-DN1 and SP-DN2 hydrogel adsorbent beads prepared in Example 1 and control adsorbent beads have been evaluated, after subjecting the beads to the shear stress produced by continuous processing with a peristaltic pump, 1 pumping cycle corresponding to the time needed to circulate all the particles through the pump one time.Afterwards, the samples were examined under optical microscope for comparison.

[0069] As shown in Figure 1, the single network-based control SN-SP (a) are extensively damaged after 12 cycles of pumping. On the other hand, the DN1-SP (b) and DN2-SP (c) hydrogel beads produced according to the invention were resistant to such mechanical action under same conditions. The DN1-SP and DN2-SP hydrogel beads actually withstood at least 48 cycles until any sign of deterioration was noticed (see Figure 1 (e) and (f) respectively, compared to (d) ) .Example 3: Quantification of mechanical strength by compression and tensile strength for casted hydrogels.

[0070] Figure 2 and 3 show the results for tensile and compression strength, respectively, for hydrogels manufactured according to the following protocol:Casted hydrogels DN1-CH and DN2-CH were manufactured in the following manner:Agarose and selected sugar-alcohols (mannitol / sorbitol ) are weighed and dissolved into demineralized water in an optimal mass ratio (5:1 agarose to sugar-alcohol ) and heated in a water bath sonicator to remove any excess air. The compositions are the same as the ones described in Table 1 for DN1-SP and DN2- SP.The hydrogels obtained were then poured into petridishes to cast a round gel with a thickness around 2 mm.After the gels had settled at room temperature, they were stored in the fridge at 4 degrees Celsius (DN1-CH and DN2-CH) . According to the results, the casted gels have similar or better mechanical properties in terms of tensile strenght and compression strength) than the control.Example 4 : Results for adsorption capacity

[0071] Figure 4 shows the adsorption capacity ( lysozyme ) of spherical particles manufactured according to Example 1 . According to the results , the DN hydrogel adsorbent beads of the present invention ( DN1-SP and DN2-SP ) have around twice the adsorption capacity of the control ( SN-SP ) . The DN adsorbents of the invention underwent through two FT cycles as shown in Table 1 .

[0072] Under equilibrium adsorption conditions , the pendant SP groups- functionali zed DN hydrogel resin beads of the present invention, all prepared by FT method, can achieve maximum capacity between 178-214 mg of lysozyme per g of adsorbent which is almost twice the capacity compared to agarose alone functionali zed with SP groups with capacity of 130 mg of lysozyme per g of adsorbent under identical conditions and similar beads si zes .Example 5 : Mechanical properties of DN2-SP beads compared to comparative DN2-SP beads

[0073] The viscoelastic properties were measured using a rheometer to perform an amplitude sweep in the linear viscoelastic range using adsorbent particles with a si ze range of 354-500 pm and plate-plate geometry with a gap si ze of 0 . 5mm . The results are shown in Figure 5 ( figures 5A and 5B ) .

[0074] As can be observed in Figure 5A, the crossover for storage and loss modulus which indicates the point of failure for a given system is increased for the DN adsorbent particles in comparison to the SN-SP . As it can be observed in Figure 5B, the values for both shear stress and strain are increased for the comparative DN2-SP and more signi ficantly for the DN2-SP in comparison to the SN-SP . .Example 6: Comparison of maximum binding capacities between the optimized DN1-SP beads and DN2-SP beads, Comparative DN1-SP beads and Comparative DN2-SP beads and Control beads, under static conditions

[0075] The protein binding capacity of the DN hydrogel resin prepared according to the invention was demonstrated by binding lysozyme, which is used as a model protein in all experiments.

[0076] The same amount of a known concentration of glucose isomerase was measured into six Erlenmeyer flasks. Different amounts of carrier material made according to the methods of the invention were added to each flask and stirred for five hours. The contents of the flask were filtered and a residual enzyme activity was measured from the filtrates. The binding capacity was obtained by dividing the initial activity in the flask to the interpreted minimum weight of the carrier that would leave zero activity in the filtrate. The desirable capacity is about 2,000 glucose isomerase units per gram of carrier.Methods

[0077] Tensile strength was measured according to DIN 53504.

[0078] Compression strength was measured by exposing a sample with a 45mm diameter and 4mm to a downward compression force (no standard method available) .Results

[0079] The maximum binding capacities of the optimized DN1-SP and DN2-SP hydrogel adsorbent beads of the present invention , and the control, with particle size range of 50-500 pm, are compared, as shown in Figure 6. The optimization was done for both network ratio and number of FT cycles: The optimal conditions for DN1-SP and DN2-SP were obtained with two cycles of freeze-thaw method.

[0080] The samples were then divided based on the particle diameter into groups of 250-350 pm and 350-500 pm to exclude effects caused by differences in particle size. The results, comparing the performance of the adsorbents with similar particle size range, are shown in Figure 7.

[0081] Therefore, it is shown that the developed adsorbents with a double network structure (DN1-SP and DN2-SP) are able to achieve under both static conditions approximately twice the binding capacity in comparison to a single network adsorbent (control) , regardless of particle size.

[0082] By comparing the static binding capacities for lysozyme of the DN1-SP and DN2-SP hydrogel adsorbents of the invention with those of the Comparative DN1-SP and Comparative DN2-SP (Table 2) , it is clear that the particle size does not play a crucial role in this property when a sugar alcohol is used instead of a high molecular weight polyol (PVA) .Table 2: Static binding capacity for lysozyme (mg / mL)Size SN-SP Comp DN1- Comp DN2- DN1-SP DN2-SP(pm) SP SP100-500 131±5 226±8 282±0.03 178±7 214±2250-350 81±12 216±18 182±20 165±4 176±7350-500 75±6 163±1 165±13 165±6 147±3Example 7: Comparison of maximum binding capacities between the optimized DN1-SP and DN2-SP hydrogel adsorbents and control, under dynamic conditions.

[0083] Samples were packed in 1 ml columns and measurements were performed with AKTA FPLC (fast protein liquid chromatography) system. The results were then expressed as dynamic binding capacity, DBC, determined as bound protein in mg per ml of adsorbent and measured at 10% sample breakthrough concentration. 3 mg / ml lysozyme was used as a sample.

[0084] The results of adsorption capacity under operational conditions are given in Figure 8 for control (■) (SN-SP beads hydrogel) , DN1-SP beads (A) and DN2-SP beads (•) , as well in first row of Table 3, showing that DCB of the optimized DN1-SP and DN2-SP hydrogel adsorbents is also nearly doubled compared to the control (SN-SP) .

[0085] Under operational conditions, the advantages of DN1-SP and DN2-SP are most apparent with a wide size distribution of 50-500 pm, resulting in higher DBG in comparison to SN-SP and comparative DN1-SP. With a size range of 350-500 pm, DN1-SP has a better performance over the Comparative DN1-SP. Additionally, DN1-SP had a comparable DBC to Comparative DN2-SP that is manufactured with significantly higher ratio of second network polymer .

[0086] Under dynamic conditions measured at 10% of breakthrough the pendant SP groups-functionalized DN hydrogel resin adsorbent (size of the particles 100-500 pm) , the DN1-SP achieved a capacity of 88 mg of lysozyme per ml of adsorbent which is 87% higher than the binding capacity of agarose alone functionalized with SP groups which achieved capacity of 44 mg of lysozyme per ml of adsorbent. A similar result was obtained with the DN2-SP. On the other hand, the Comparative DN1-SP hydrogel exhibited inconsistent results at all different particle sizes measured.

[0087] The results were obtained at residence time of 1.3 minutes, corresponding to linear flow velocity of 150 cm / h under the experimental conditions used.Table 3: Dynamic binding capacity at 150cm / h for lysozyme (mg / mL)Size SN-SP Comp DN1-SP Comp DN2-SP DN1-SP DN2-SP(pm)100-500 4410.4 5410.1 8011.5 8811.2 7813.6250-350 3812.7 8217.0 5514.2 4910.6 4311.9350-500 3110.9 29.512.6 4716.3 4311.7 2711.6

[0088] Pendant SP groups-functionalized DN hydrogel resin beads of the present invention, with a particle size distribution of smaller than 500 pm, can actually achieve comparable results of 200 mg of lysozyme per g of adsorbent to commercial SP- functionalized AG resin with a particle size distribution of 45- 165 pm (SP Sepharose® Fast Flow, Cytiva) , hence mitigating effect of particle size on capacity. Nevertheless, manufacturing the DN hydrogel resins in smaller particle size may achieve even higher binding capacity.

[0089] Particle size is the size of an isolated particle or sometimes a way to referring to the average particle size in a population of particles. Particle size distribution refers to the dispersion in sizes observed within a population. A population of uniform particles of exactly the same size would have a distribution of 1. The D90 / 10 method for reporting distribution is commonly used. D90 / 10 is defined as the particle diameter (dp) at 90% of the distribution (a larger number) divided by dp at 10% of the distribution (a smaller number) . Particles used for preparing HPLC columns can range from values less than 1.1 to 1.5 or more.

[0090] Under equilibrium adsorption conditions, the pendant SP groups-functionalized DN hydrogel resins of the present invention (e.g., as prepared by the FT method) can achieve maximum capacity of 225 mg of lysozyme per g of adsorbent which is more than twice the capacity compared to agarose alone functionalized with SP group with capacity of 130 mg of lysozyme per g of adsorbent under identical conditions and similar beads sizes.Example 8: Effect of Ionic StrengthMethods

[0091] To determine the ion-exchange capacity of porous media, transient pH phenomenon was employed using two buffer solutions with the same pH value but different ionic strength with anestablished method by Nika. For cationic porous media, the column is equilibrated with a buffer of low ionic strength followed by a step change to a buffer with added salt (NaCl) to start ion exchange between Na+and other two cations in the resin resulting in a drop of pH of the solution. The magnitude and duration of the pH transient are dependent on the type as well as on the amount of ion exchange groups in the column. The ionic capacity was calculated from the relative elution volumes, normalized by column volume, as described with the following expression:where K is relative elution volume, At (pH) is the time interval calculated from the pH transition profile by subtracting the time at the beginning of the pH transition, to, from the time at which pH has returned 50% of the lowest pH value and C2is the concentration of buffer B. The constants a and b for cationic systems have been determined to be 1.57 and 0.5, respectively. The pH transient experiments were performed by mounting a column into AKTA FPLC system and equilibrating the adsorbent with 20mM Tris-HCl at pH 7.4 (buffer A) followed by equilibration with the same buffer containing 1.0M of NaCl (buffer B) . The buffer solutions were prepared by adjusting the pH with HC1 water solution (1:1) .

[0092] To further understand the mechanism effecting improved adsorption capacity of the DN hydrogel adsorbent, the ionic strength was measured using a pH gradient technique and the results were calculated according to Equation 1. The total ionic strength of the adsorbent was measured with a particle size distribution of 250-350pm to minimize any differences caused by particle size.Results

[0093] The results are shown in Figure 9. It was expected that the improved adsorption capacity of the DN adsorbents was due to increased amount of hydroxyl groups leading to higher total ionic strength. However, Comparative DN1-SP and Comparative DN2-SP showed similar values between 1.25-1.28 mmol / mL in comparison to SN-SP that had an average value of 1.33 mmol / mL. For the DN1-SP and DN2-SP inventive beads the total ionic strength had a significant reduction with values in the range of 0.75-0.82 mmol / mL indicating that the amount of hydroxyl groups was not the contributing factor for enhanced adsorption capacity.Example 9: Effect of PorosityMethods

[0094] The total bed porosity, £,, consists of void fraction, a, specified as the ratio of liquid between the particles and particle porosity, ap, calculated according to Equation 2.Eb= E + (1 - a)ap(2)Ebcan be measured using moment analysis method by injecting a pulse of acetone under non-binding conditions. The first moment, .lfis calculated according to Equation 3, and plotted againsttheoretically calculated superficial residence time, andobtaining the total porosity from slope of the fitted data by linear regression from Equation 3.

[0095] The protein sample for measurement of spwas prepared from Bovine serum albumin at 10 g / L in 20 mM phosphate buffer containing 250 mM sodium chloride and 5% (v / v) ethanol to help mitigate non-specific interactions, namely electrostatic and hydrophobic. 10 pL of protein solution corresponding to 1% of total column volume was injected in the column at flow velocities ranging between 0.75-3mL / min corresponding to linear velocitiesbetween 150-600cm / h. The pulse method was repeated without column to determine the external volume from tubes etc. s was measured with a solution containing Ig / L of blue dextran (2000kDa) .

[0096] Adsorption in a stirred batch vessel at beginning of an experiment is predominantly governed by external mass transfer, and the film mass transfer coefficient kfis determined from the measured data between 0-20 minutes according towhere C is the measured concentration at time t, Co is the initial protein concentration, Vais the adsorbent volume, V is the solute volume, and Rpis the radius of the adsorbent particle.Results

[0097] The particle porosity was measured for SN-SP and DN adsorbents beads with a particle size of 250-350pm to minimize differences resulting from the adsorbent size distribution. The recorded values for first moment as a function of theoretically calculated superficial residence time are plotted in Figure 10. The measured values show a linear correlation with a coefficient of determination of 0.99 for all the adsorbents.

[0098] The value for particle porosity of Comparative DN1-SP (0.24) is lower in comparison to the one of the control (0.42) . The highest particle porosity wis measured for DN1-SP (0.54) which is higher than the one of the control (0.42) as well as the one of the Comparative DN1-SP (0.24) . DN2-SP has at least an increased porosity compared to comparative DN1-SP. All the calculated values for porosity are listed in Table 4 calculated from results presented in Figure 10..Table 4: Particle porosity of control, Comparative adsorbents and the DN adsorbents of the invention.Particle PorosityControl (SN-SP) 0.42Comparative DN1-SP 0.24DN1-SP 0.54DN2-SP 0.31

[0099] The differences in particle porosities between the manufactured adsorbents indicate that the enhanced adsorption capacity of DN1-SP adsorbent is due to increased porosity providing more space for the protein inside the support material. Comparative DN1-SP has the lowest particle porosity which could explain that the performance under dynamic conditions is hindered due to restrictions at pore level. Therefore, increased binding capacity is at least partially related to particle internal pore size and film mass transfer coefficients were determined from batch adsorption experiments listed in Table 5.Table 5: Film mass transfer coefficients of control, Comparative adsorbents and the DN adsorbents beads of the invention.Kf(m / s)Control (SN-SP) 3.44 E-09Comparative DN1-SP 4.00 E-07DN1-SP 4.89 E-08DN2-SP 5.00 E-08

[0100] The results demonstrate that the DN structure DN1-SP and DN2-SP significantly enhances the mass transfer from the solute to the network regardless of particle porosity. Therefore, the DN formation offers a novel method for construction of novel adsorbents by tuning the mass transfer properties according to the target molecule. Mannitol was identified to provide both enhanced porosity and film mass transfer, out of all the hydroxyl containing polymers cross-linked with agarose.BIBLIOGRAPHYJohn G. Lyons, Luke M. Geever, Michael J.D. Nugent, James E. Kennedy, Clement L. Higginbotham, Development and characterisation of an agar-polyvinyl alcohol blend hydrogel, Journal of the Mechanical Behavior of Biomedical Materials, Volume 2, Issue 5, 2009, Pages 485-493, ISSN 1751-6161, https: / / doi.Org / 10.1016 / j . mbbm.2008.12.003.Shao, J., Zhang, Z . , Zhao, S., Wang, S., Guo, Z . , Xie, H. and Hu, Y. (2019) , Self-Healing Hydrogel of Poly (Vinyl Alcohol ) / Agarose with Robust Mechanical Property. StarchStarke, 71: 1800281. https: / / doi.org / 10.1002 / star.201800281Palsson, E., Gustavsson, P., & Larsson, P. (2000) . Pellicular expanded bed matrix suitable for high flow rates. Journal of Chromatography A, 878 (1) , 17-25. doi : 10.1016 / S0021- 9673 (00) 00223-5Shi, Q. , Jia, G., & Sun, Y. (2010) . Dextran-grafted cation exchanger based on superporous agarose gel: Adsorption isotherms, uptake kinetics and dynamic protein adsorption performance. Journal of Chromatography A, 1217 (31) , 5084-5091. doi:10.1016 / j . chroma .2010.05.065

Claims

CLAIMS1. Use of a double-network hydrogel resin comprising:- a physico-chemically cross-linked agarose as a first network,- at least one physico-chemically cross-linked sugar-alcohol of the general formula (CHOH)nH2, where n = 4-12, as a second network, said physico-chemically cross-linked agarose being non- covalently linked to said at least one physico-chemically crosslinked sugar-alcohol via hydrogen bonding,- said first and second networks interpenetrating to form a double-network structure, and- said double-network hydrogel resin being in the form of beads, as an adsorbent material.

2. Use according to claim 1, wherein the sugar-alcohol is selected from the group consisting of mannitol, sorbitol, galactitol, erythritol, xylitol, threitol, arabitol, ribitol, iditol, and volemitol, and preferably mannitol and sorbitol.

3. Use according to claim 1 or 2, wherein the mass ratio of the first network of agarose to the second network of sugar alcohol is between 6:0.1 and 0.1:6, preferably between 5:1 and 1:5, and more preferably between 5:1 and 1:1.1.

4. Use according to any of claims 1 to 3, wherein the doublenetwork hydrogel resin is used as a chromatography media, and in particular as a stationary phase in different chromatography methods, wherein the chromatography is preferably selected from the group consisting of ion-exchange chromatography, hydrophobic interaction chromatography and affinity chromatography.

5. Use according to any of claims 1 to 4, wherein the double-network hydrogel resin is modi f ied with a functionality enabling reversible bioproduct binding by a method selected from the group consisting of ion-exchange , hydrophobic interaction, mixed-mode interaction, and af finity chromatography, preferably wherein the functionality is an ion-exchange adsorptive functionality, more preferably a cation-exchange adsorptive functionality .6 . Use according to any of claims 1 to 5 , wherein the beads have a particle si ze of between 10 pm and 500 pm in diameter, preferably between 50 pm and 500 pm in diameter, and more preferably between 250 pm and 350 or between 350 pm and 500 pm in diameter .7 . A process for puri fying a sample comprising a macromolecule , comprising contacting said sample with a packed or fluidised bed comprising beads of a double-network hydrogel resin as defined in any of claims 1 to 6 as a chromatography media .8 . The process of claim 7 , wherein it is implemented in a continuous flow-through mode or a bind / elute mode , and preferably a in a continuous flow-through mode .9 . A process for puri fying a protein or a nucleic acid, comprising; providing a protein or a nucleic acid mixture , contacting the protein or a nucleic acid mixture with beads of the double-network hydrogel resin as defined in any of claims 1 to 6 , washing the double-network hydrogel resin beads to remove unbound species , compressing the double-network hydrogel resin beads , andwashing the compressed double-network hydrogel resin beads to extract bound protein or bound nucleic acid.

10. A double-network hydrogel resin comprising:- a physico-chemically cross-linked agarose as a first network,- at least one physico-chemically cross-linked sugar-alcohol of the general formula (CHOH)nH2, where n = 4-12, as a second network, said physico-chemically cross-linked agarose being non- covalently linked to said at least one physico-chemically crosslinked sugar-alcohol via hydrogen bonding,- said first and second networks interpenetrating to form a double-network structure,- said double-network hydrogel resin being in the form of beads, and- wherein agarose and the sugar-alcohol are in a mass ratio between 5:1 to 1:5.

11. A method of preparing a double network hydrogel resin according to claim 10, comprising a step of contacting agarose (AG) and at least one physico-chemically cross-linked sugar- alcohol of general formula (CHOH)nH2, where n = 4-12, wherein agarose and the sugar-alcohol are contacted in a mass ratio between 5:1 and 1:5; and further comprising a step of subjecting the double-network hydrogel to a f reeze-and-thaw method.

12. The method of claim 11, wherein agarose and the sugar-alcohol are contacted by using a solvent, subjected to heat until dissolved to form a resulting solution, and a densifier is added to the resulting solution.

13. The method of claim 11 or 12, further comprising a step of forming the beads of the double network hydrogel by an emulsification technology.14 . A packed or fluidised bed comprising beads of the doublenetwork hydrogel resin of claim 10 as a chromatography media . 15 . A housing comprising the packed or fluidised bed of claim14 .

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