Functionalized, polymer-conjugated polyamide-based chromatography media

Abstract

The present invention relates to a polyamide-based chromatography media comprising high surface area polyamide fibers having a cross-section comprising a region having a main body defining a substantially longitudinal axis, and five projections extending radially outwardly from said main body, wherein the polyamide fibers are modified with a immobilization polymer and further modified with an adsorptive functionality enabling a chromatography selected from the group consisting of ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography, wherein the immobilization polymer is selected from the group consisting of dextran, dextrin, amylose, pullulan, agarose or derivatives thereof, cellulose, glucomannan, and derivatives thereof, and polymethacrylate A packed bed comprising the polyamide-based chromatography media, a housing comprising same, processes for purifying a sample comprising a macromolecule or nanoparticles or for purifying a protein are also disclosed.

Description

[0001] Functionalized, polymer-conjugated polyamide-based chromatography media TECHNICAL FIELD

[0001] The present invention generally relates to fibrous chromatography media suitable for the purification of macromolecules and supramolecular complexes via a chromatographic or adsorptive method. More particularly, the present invention relates to a polymer-conjugated polyamide- based chromatography media which is suitable for different types of chromatography columns or adsorptive systems usable for purifying macromolecules. BACKGROUND OF THE INVENTION

[0002] Purification of various therapeutic biomolecules, e.g. monoclonal antibodies, is currently achieved by using bead-based chromatography resins, which are prepared with different ligand structures that enable the beads to function in affinity, cation- exchange, or anion-exchange modes, and hydrophobic interaction and / or mixed-mode.

[0003] Functionalized resin-based chromatography media, such as Sepharose FF, Sepharose XL, is the most widely used chromatographic column media in downstream processing of biopharmaceuticals so far, which is often accompanied by a high price accounting for over 50% of the total manufacturing costs. However, resin-based ion exchange columns typically require sophisticated equipment and long set-up times, exhibit low flow rates, and suffer from limited yields. The small pore sizes and large bed volumes of conventional columns contribute to size exclusion and dilution effects, which limit the ability to purify larger macromolecules and other biological entities, demand further process steps to concentrate dilute eluates and increase reagent consumption. Large scale production columns usually require significant amounts of packing material, so that the manufacturers of biopharmaceuticals usually recycle chromatography resins several times, which ends up consuming substantial quantities of media, and additional costs associated with the validation of each cleaning, sterilization, and column packing operation.

[0004] Several technologies are described in the literature and marketed commercially for biopharmaceutical purifications based on functionalized fibrous media and / or composites. Different methods have been reported in the literature for the modification of polyamide materials used in non-chromatography applications, and some attempts of modifying polyamide fibers to be used as adsorbent material for purification of biomacromolecules, e.g. proteins, via chromatography have also been disclosed.

[0005] Polyamide-based supports (e.g. nylon 6) have been studied for ion exchange-based protein separations based on the acid / base character of the natural carboxylic acid and primary amine end groups of the polyamides. However, due to the high surface hydrophobicity of native nylon 6 fibers, poor wettability and nonspecific protein adsorption easily take place. Additional surface modifications on the native nylon fibers could improve the chromatographic properties thereof as the stationary phase.

[0006] WO2017223048A1 reports the modification of nylon 6 capillary-channelled polymer fibers with 2-acrylamido-2- methylpropanesulfonic acid (AMPS) via microwave-assisted grafting polymerization to attain a strong cation exchange stationary phase. The cation exchange ligand densities on the resultant nylon-SO3H fibers were greater in comparison to the cation (COOH) density of native nylon 6 fibers and the modified fiber stationary phase showed increased lysozyme dynamic loading capacities compared to native nylon 6.

[0007] WO2012015908A2 discloses a chromatography media combining a high surface area fiber, including fibers made of a thermoplastic polymer, e.g. polyamides, with pendant adsorptive functionality directly introduced on the fiber surface for biomolecule chromatography applications. SUMMARY

[0008] The object of the invention is to improve the fibrous material for chromatography applications disclosed in the art.

[0009] The object is achieved by a polyamide-based chromatography media according to claim 1. Advantageous embodiments are recited in the dependent claims.

[0010] Furthermore, the object is achieved by a packed bed comprising the polyamide-based chromatography media according to claim 5, a housing comprising the packed bed according to claim 6, a process for purifying a sample comprising a macromolecule or nanoparticles according to claim 9 and a process for purifying a protein according to claim 12. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1: Reaction scheme for obtaining a polyamide-based chromatography media according to a preferred embodiment of the invention, further including SEM images of native nylon-6 fibers before and after pre-treatment.

[0012] Figure 2: Influence of conjugated dextran density in the Sakura nylon 6-dextran-SP chromatographic media. Static binding capacity (SBC), dynamic binding capacity (DBC), and ratio between SBC and DBC are plotted as functions of conjugated dextran quality. Conditions: 20 mM pH 7.4 phosphate buffer as equilibration buffer and model protein (2 mg / ml lysozyme for DBC at 60 cm / h and 1 mg / ml lysozyme for SBC for 3 hours contact with mild shaking) was dissolved in the same buffer.

[0013] Figure 3: Representative scanning electron microscope images in different scales of (a) pristine (unmodified backbone with exposed amino groups) and (b) modified Sakura nylon 6 fabrics according to a preferred embodiment of the invention.

[0014] Figure 4: Zeta potential values, as a function of pH, for modified and unmodified Sakura nylon 6 fabrics.

[0015] Figure 5: DBC of adsorbent Sakura nylon 6-dextran-SP according to a preferred embodiment of the invention at different flow rates from 60 to 480 cm / h. Conditions: 20 mM phosphate buffer pH 7.4 was used as equilibration buffer and model protein (2 mg / ml lysozyme) was dissolved in the same buffer.

[0016] Figure 6: (a) Chromatographic separation profiles between lysozyme and bovine serum albumin (BSA) using Sakura nylon 6 fabric-dextran-sulphopropyl fabric packed column (column volume = 2.5 ml); UV as solid line and conductivity as dashed line. (b) Reducing SDS-polyacrylamide gel electrophoresis of pooling from (a). M is a molecular weight marker as indicated. Conditions: 20 mM phosphate buffer pH 7.4 was used as equilibration buffer and 5 ml 2 mg / ml lysozyme + 2 mg / ml BSA in the same buffer was loaded as sample, 60 cm / h.

[0017] Figure 7: (a) Chromatographic purification of CHO_K1 cell line fermentation broth producing recombinant human IgG1 mAb using Sakura nylon 6-dextran-sulfopropyl fabric packed column (column volume = 0.33 ml); UV as black solid line, conductivity as black dashed line, and pressure in grey. (b) Reducing SDS- polyacrylamide gel electrophoresis of pooling from (a). M is a molecular weight marker as indicated. Conditions: 20 mM phosphate buffer pH 5.1 was used as equilibration buffer and 5 ml crude fermentation broth was loaded as sample, at 60 cm / h.

[0018] Figure 8: Moving belt prototype for direct protein capture.

[0019] Figure 9: Reducing SDS-PAGE image of CHO_K1 cell line fermentation broth (F), protein left in sampling chamber at the end of process (S), and eluted protein from elution chamber at the end of process (E). M is a molecular weight marker as indicated. DETAILED DESCRIPTION

[0020] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.

[0021] Polyamide is a polymer with repeating units linked by amide bonds, which can occur both naturally and artificially. Examples of naturally occurring polyamides are proteins, such as wool and silk. Artificially made polyamides can be made through step-growth polymerization or solid-phase synthesis yielding materials such as nylons, aramids, and sodium polyaspartate. The term “nylon” is used as a generic designation for a family of synthetic polymers composed of polyamides, so that for the purpose of the present invention, polyamide and nylon can be used interchangeably. In the present invention, polyamide include homopolymers and copolymers.

[0022] The term “polysaccharide” refers to natural or synthetic polymeric carbohydrates. The selection criteria for the polymeric carbohydrates to be suitable for the present invention are: 1) easy to react, offering sufficient hydroxyl groups for the following ligand introduction and hydrophilization, 2) flexible conformation, and 3) inexpensive. Examples of natural polysaccharides meeting such criteria for the purpose of the present invention are polysaccharides soluble in water such as dextran (α (1-6)-linked glucan), dextrin, amylose, pullulan, agarose, cellulose, glucomannan, and derivatives thereof, and preferably dextran.

[0023] The term “immobilization polymer” or “immobilized polymer” according to the invention includes the polymeric carbohydrates according to the invention as well as synthetic polymers that also meet the three selection criteria defined above, such as polymethacrylates. This immobilization polymer is covalently immobilized on the polyamide fiber surface to yield (immobilization / immobilized) polymer-layer coated polyamide fibers. As utilized herein, “immobilization” is also called surface conjugation, which may be performed, e.g. by using standard methods of grafting polymerization or known chemical approaches to covalent bonding.

[0024] The term “adsorptive functionality” and “adsorptive functionality groups” are used herein interchangeably. This term refers to the presence of chemical (or biochemical) pendant groups (or molecules) that allow for (selective) product binding and de-binding.

[0025] Sakura refers to fibers of nylon 6 commercially available. Sakura is a fibre type characterised by a five-petal cross-section shape (see for example the website https: / / www.kuraray.co.jp / , and the fabric sold under the reference no. 56T24 [AE-13691-B]).

[0026] Pristine: unmodified Sakura type fiber backbone with exposed amino groups, after having been treated with a water- soluble finish (40 wt.%) and subsequently contacted with hot water (about 80°C) for about half an hour for removal / dissolution of said finish / sheath.

[0027] 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. The molecular weight is measured thanks to well-known methods by a person skilled in the art such as by mass spectrometry, size exclusion chromatography, or denaturing gel electrophoresis, etc...The term “nanoparticles" generally refers to extracellular vesicles and virus-like particles, etc, for example having a diameter size ranging from 30 to 5000 nm.

[0028] "Protein mixture" comprises a protein 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 protein mixtures include, for example, cell cultures, cell fermentation broth, cell lysates, and clarified bulk (e.g., clarified cell culture supernatant).

[0029] According to one embodiment of the invention, a polyamide-based chromatography media is provided, comprising high surface area polyamide fibers having a cross-section comprising a region having a main body region defining a substantially longitudinal axis, and five projections extending radially outwardly from said main body region, wherein the polyamide fibers are immobilized with a immobilization polymer and the surface of said polyamide fibers is further modified with an adsorptive functionality enabling a chromatography selected from the group consisting of ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography, wherein the immobilization polymer is selected from the group consisting of dextran (α (1-6)-linked glucan), dextrin, amylose, pullulan, agarose, cellulose, glucomannan, and derivatives thereof, and polymethacrylate. Preferably, the immobilization polymer used for producing the polyamide-based adsorbent fibrous material of the present disclosure is a polysaccharide selected from the group consisting of dextran (α (1-6)-linked glucan), dextrin, amylose, pullulan, agarose, cellulose, glucomannan, and derivatives thereof, and more preferably the polysaccharide dextran. The specific surface area of the polyamide fibers is measured according to Brunauer-Emmett-Teller (BET) surface area method, preferably with nitrogen gas. A suitable apparatus can be a Gemini 2360 analyzer. In the present invention, a high specific surface area means a specific surface area ranging from 0.01 to 100 m2 / g, preferably ranging from 0.05 to 10 m2 / g, and more preferably ranging from 0.1 to 5 m2 / g.

[0030] Polyamide fibers may be of any length and diameter and are preferably cut or staple fibers or a non-woven fabric. They need not be bonded together as an integrated structure but can serve effectively as individual discrete entities. They may be in the form of a continuous length such as thread or monofilament of indeterminate length or they may be formed into shorter individual fibers such as by chopping fibrous materials (e.g. staple fibers) such as non-woven or woven fabrics, cutting the continuous length fiber into individual pieces, formed by a crystalline growth method and the like. The polyamide-based fiber according to the invention have a diameter preferably ranging from 1 to 100 µm, and preferably from 1 to 50 µm.

[0031] According to the present invention, the polyamide is selected from the group consisting of nylon-6, nylon-6,6, nylon- 4,6, nylon-12, nylon-4,12, nylon-6,12 and nylon-8, etc. Preferably, the polyamide used is nylon-6 or nylon-6,6, most preferably nylon-6.

[0032] The polyamide-based fiber according to the invention has a five-petal cross-sectional shape, with a main body region defining a substantially longitudinal axis, and five projections extending radially outwardly from the main body region, acquiring an increased surface area compared to the native fibrous material (e.g. a cylindrical fibre with comparable diameter).

[0033] The immobilization polymer(s) is (are) (covalently) immobilized on the polyamide fiber surface to yield immobilized polymer-layer coated polyamide fibers. As utilized herein, such immobilization, also called surface conjugation, may be performed by using grafting polymerization. Molecules containing two or more epoxy groups can be used as a crosslinking agent between amine-containing surface of the polyamide fibers, preferably pre-treated to remove a water-soluble finish / sheath to expose the amino groups, and the immobilization polymer (hydroxyl rich immobilization polymer). Indeed, one epoxy group can react with the amino groups of the polyamide fibers and the remaining epoxy groups then serve as anchoring sites on the fiber surface as attachment points for the immobilization polymer(s). Others bifunctional compounds can be used as crosslinking agents such as bisepoxyrane.

[0034] Before immobilization, polyamide fibers (e.g. commercially available polyamide fibers such as sakura polyamides fibers) are preferably pre-treated to remove a water- soluble finish / sheath (40 wt.%), commercially available under the trade name MINT. This enables to expose the amino groups of the polyamide fibers. As an example, the pre-treatment can be performed by contacting Sakura type fiber / fabric at about 80 °C with water, for approximately 30 min, before any further application (e.g., immobilization polymer layer conjugation such as dextran layer conjugation and adsorptive functionality groups introduction such as sulfopropyl (SP) groups introduction) to expose surface amino groups of polyamide fibers, and preferably nylon fibers.

[0035] For the purposes of the invention, the approach for the epoxide-opening reaction was based on the procedures described by Tang et al, 2012 with modification, but different methods available in the art may be used for the epoxide-opening reaction.

[0036] The crosslinking agent may be an epoxy compound, and preferably a polyepoxy compound, more preferably selected from the group consisting of ethylene glycol diglycidyl ether (EGDE), triglycidyl ether, trimethylolpropane triglycidyl ether, N,N,N′,N′-tetraglycidylethylene diamine, glycerol diglycidyl ether, etc. Preferably, the crosslinking agent is ethylene glycol diglycidyl ether (EGDE).

[0037] There is actually an optimal density of immobilization polymer onto the polyamide fiber surface, as more immobilization polymer could represent a higher amount of hydroxyl groups, thus leading to a higher degree of crosslinking, which, in turn, could block the path of the transport of solutes to their binding sites. In a certain embodiment, an amount of immobilization polymer of 0.005 to 0.140 g, preferably 0.007 to 0.138 g, per gram of polyamide fibre is used for obtaining a suitable density of the polymer-immobilized polyamide fiber.

[0038] The surface functionalization of the high surface area polyamide-based fibers can be accomplished by a two-step process. A suitable functionalization process is grafting polymerization. In one embodiment, the adsorptive functionality consists of pendant groups grafted to said fibers. It can begin with the attachment of pendant allyl groups to the polymer- immobilized polyamide fiber surface by treatment of the fibers with 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 fiber surface as attachment points for the adsorptive functionality groups. The adsorptive functionality groups are thus preferably pendant adsorptive functionality groups.

[0039] In certain embodiments, the adsorptive functionality consists of pendant sulfopropyl (SP) groups or pendant acrylic polymer groups (both enabling a cation-exchange chromatography) or trimethylammonium groups (enabling anion-exchange chromatography), or even other pendant groups that would enable other types of chromatographic methods, e.g. hydrophobic interaction chromatography and affinity chromatography. Such functionalization follows classical routes to chemical ligand immobilisation onto polymeric matrices.

[0040] In one embodiment, the adsorptive functionality is an ion-exchange adsorptive functionality, preferably a cation- exchange adsorptive functionality. In a preferred embodiment, the adsorptive functionality consists of pendant SP groups or pendant acrylic polymer groups, most preferably pendant SP groups.

[0041] Importantly, due to a very low amount or density of -NH2 groups, polyamide fibers are unlikely to directly react with functional moieties, and thus to specifically bind macromolecules or nanoparticles. However, the presence of the immobilized polymer layer according to the invention (e.g. dextran) brings notable advantages to the polyamide-based chromatography media of the invention: 1. Further increase of the polyamide fiber (reactive) surface area is achieved, from an already high baseline resulting from the five-petal cross-sectional shape present in the native polyamide material; 2. No main structural changes compared to the native polyamide fiber, but only a degree of roughness is introduced upon functionalization; and 3. The hydroxyl groups of immobilized polymers, a hydroxyl rich donor, results in outstanding biocompatibility, hydrophilicity, and chemical reactivity e.g. functionalization, serving as a modification point for the addition of a pendant adsorptive functionality.

[0042] In another embodiment, a packed bed comprising the polyamide-based chromatography media according to the invention is provided. The bed may be packed with the isolated polyamide- based fibers or pre-woven polyamide-based fibers.

[0043] The bed comprising the polyamide-based chromatography media as described herein in the appropriate densities and thickness achieves good chromatographic properties inter alia higher loading capacities, higher throughput, higher binding capacity or the less complexity of scale-up. It is estimated to cost as low as EUR 200-230 per gram constructed polyamide-based chromatography media, considering all materials, solvents, plastic embodiments / systems / cartridges, and reagents used, so that it can be used a “disposable” technology, which may be discarded after single or multiple use(s). Recycling the bed packed with polyamide-based chromatography media for further uses.

[0044] In another embodiment, a housing comprising the packed bed is provided. The polyamide-based chromatography media of the packed bed can be in the form of woven polyamide-based fibers material, non woven polyamide-based fibers material, or random polyamide-based fibers material.

[0045] In one aspect, the packed bed may be delivered to the user in a dry, prepacked (“ready-to-use”) format, unlike bead- based media. The fibers can be fused either by thermal or chemical means to form a semi-rigid structure that can be housed in a pressure vessel. Chromatographic bead-based media is generally delivered as loose material (wet) wherein the user is required is load a pressure vessel (column) and by various means create a well-packed bed without voids or channels. In contrast, in accordance with certain embodiments of the housing disclosed herein, no packing is required by the user as the product is delivered as ready for service.

[0046] Moreover, in another embodiment, it is provided a process for purifying a sample comprising a macromolecule or nanoparticles, comprising contacting said sample with a bed of the polyamide-based chromatography media as disclosed herein.

[0047] In one aspect, the purification is carried out at a pH ranging from 3 to 9, and more preferably ranging from 5 to 8.

[0048] In another aspect, the polyamide-based chromatography media enables purification in a continuous (flow-through) mode or a bind / elute mode. Preferably, a continuous (flow-through) mode is used, which may be achieved by using a continuous moving belt interface. In other terms, the process can be implemented in a continuous mode or a batch mode, and preferably, in a continuous mode, for example in a SMB (simulated moving bed) or PCC (periodic counter-current) mode.

[0049] Accordingly, in the present invention, the fibres can be packed in a column or cartridge for classical chromatography as mentioned above or, due to the elasticity of the woven material, used in a “belt” allowing continuous recovery (as shown in figure 8). In one aspect of the present invention, the polyamide-based chromatography media of the present invention is thus used as an adsorbent belt (e.g. based on mechanism of belt conveyors), preferably in a continuous purification system. The continuous purification system can comprises said adsorbent belt, and a four-chamber tank, wherein each chamber is responsible for association, unbound washing, elution, and re-equilibrium, respectively. Said system is able to possess its own buffer volume and liquid speed. The developed polyamide-based chromatography media of the present invention passes through each chamber in sequence via rollers readily for continuous bioproduct recovery from a feedstock.

[0050] For example, in mAb purification, cation exchange chromatography is typically conducted wherein, operating at a pH below the isoelectric point of the antibody protein and at a modestly depressed solution conductivity, the antibody protein will ionically bind to the support via the ion exchange ligand while unbound contaminants (host cell proteins, nucleic acids, etc.) pass freely through the chromatography bed. These contaminants are further eliminated by flushing the packed bead bed with appropriate buffer solution before releasing the bound mAb product with a buffer of high conductivity sufficient to shield the ionic interaction between bead resin and protein. In contrast, anion exchange chromatography is often used downstream in monoclonal antibody production to further remove residual cell culture contaminants wherein the operation is conducted at solution conditions of pH and conductivity such that the mAb protein will not bind to the cationic surface of the bead resin but instead passes freely through the chromatography column. Proteins and nucleic acids on the other hand that bear a net negative charge will effectively bind to the anion exchange resin and thereby are eliminated from the product.

[0051] In still another aspect, the polyamide-based chromatography media as described herein is particularly suitable for purification of biomacromolecules, including, but not limited to, proteins (inter alia immunoglobulins including human recombinant immunoglobulins such as the recombinant human IgG1 monoclonal antibody specific for human tumor necrosis factor (TNF), i.e. adalimumab (Humira®)), nucleic acids (DNA and RNA), carbohydrates and lipids.

[0052] It is therefore another embodiment of the present invention a process for purifying a protein, comprising: providing a protein mixture, contacting the protein mixture with the polyamine-based chromatography media as disclosed herein, washing the polyamine-based chromatography media to remove unbound species, compressing the polyamine-based chromatography media, and washing the compressed polyamine-based chromatography media to extract bound protein.

[0053] In one aspect, the protein is an antibody, such as a monoclonal antibody (including, but not limited to a human, humanized and chimeric antibody), preferably adalimumab.

[0054] One will appreciate that the selection of chromatographic conditions, elutes and buffers suitable for performing the processes for purification of macromolecules or proteins, or nanoparticles as disclosed herein is within the ability of those skilled in the art. Examples: Example 1: Preparation of the polyamide-based adsorbent fiber from commercial Sakura nylon 6 fibers

[0055] The surface functionalization of the polyamide fibers can be accomplished by a three-step process, as shown in the scheme in Figure 1, having the polysaccharide dextran as the exemplary immobilization polymer and pendant sulfopropyl (SP) groups as the exemplary adsorptive functionality. (a) Pre-treatment of Sakura nylon 6 fibers (1) to remove water-soluble sheath:

[0056] A pre-treatment of the commercial Sakura nylon 6 fibers (1) is carried out according to a step (a): the round cross- sectional shape commercial Sakura nylon 6 fibers (1) as shown in figure 1 is conjugated with a water-soluble finish (40 wt.%) when delivered. Indeed, it has a water-soluble sheath (made of polymer). This water-soluble polymer was removed according to a step (a) by dissolution in hot water at 80 °C for half an hour. Solid concentration (fiber to water ratio) in step (a) was 4 mg / ml for the dissolution. Afterwards, the obtained fibers (2) were washed thoroughly with demineralized water and dried in oven overnight at 60°C, resulting in pre-treated Sakura nylon 6 fibers (2) with exposed amino groups, having a five-petal cross- sectional shape (also called sakura cross-section shape). The obtained pre-treated Sakura nylon 6 fibers (2) are also called pristine or unmodified Sakura type fiber backbone with exposed amino groups. (b) Conjugation with the immobilization polymer:

[0057] 900 mg dextran was completely dissolved in 25 ml dimethyl sulfoxide (DMSO) so as to form a dextran DMSO solution. After adding pre-treated 250 mg Sakura nylon 6 fibers (dry weight) (2), 14.3 ml EGDE (ethylene glycol diglycidyl ether) and 5.75 ml 2.0 M sodium hydroxide (NaOH) solution were added into the dextran DMSO solution. The reaction according to a step (b) was proceeded for 18 hours under stirring (250 rpm), at 40°C. The obtained dextran-grafted fibers (3) (also called dextran-grafted Sakura nylon 6 fibers) was washed thoroughly with demineralized water and dry in oven overnight at 60°C. (c) Functionalization step: c.1) Prior to the introduction of the adsorptive functionality, the dextran-grafted Sakura nylon 6 fibers (3) were activated with allyl bromide according to a step (c.1) as described by Shi and co-workers [Shi, Q.H., Jia, G.D., & 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), pp. 5084–5091]. 2 ml demineralized water, 0.5 ml 2 M NaOH, 1.5 ml DMSO, and 1.5 ml allyl bromide was added to 250 mg dextran- grafted Sakura nylon 6 fibers (3) in a 25 ml round-bottom flask. The mixture was allowed to react for 24 hours at 25°C under stirring (200 rpm). Afterwards, the obtained allylated fibers (4) (also called allylated dextran-grafted Sakura nylon 6 fibers) were washed 3 times with demineralized water, 0.1 M NaOH, 25% ethanol (EtOH), 0.5 M sodium chloride (NaCl), and demineralized water and dried by tissue. c.2) SP groups were introduced according to a step (c.2) by submerging the allylated dextran-grafted Sakura nylon-6 fibers (4) in 10 ml demineralized water and then sulfonated by reacting the vinyl groups with sodium sulphite (Na2SO3), generated by titrating a suspension of 0.3 g sodium metabisulfite (Na2S2O5) to pH 6 with 2 M NaOH. The reaction was allowed to proceed for 18 h at 25°C in a 50 ml round-bottom flask under stirring (170 rpm). The obtained functionalized products (5) (also called sulfopropyl (SP) functionalized dextran-grafted Sakura nylon 6 fibers) were washed for 3 times with demineralized water and dried in oven at 60°C overnight. Example 2: Comparison of the static binding capacity (SBC) and dynamic binding capacity (DBC) of naked Sakura nylon 6 (2), Sakura nylon 6-dextran (3), Sakura nylon 6-dextran-SP (5), and SP Sepharose fast flow.

[0058] As shown in Table 1 below, the immobilization of dextran to naked Sakura nylon 6 fibers (with exposed amino groups) (2), carried out according to Example 1, led to more than threefold increase in Brunauer-Emmett-Teller (BET) surface area and almost seven times decreases in nonspecific protein adsorption when lysozyme was used as model protein, under the following conditions: 20 mM pH 7.4 phosphate buffer was used as equilibration buffer and lysozyme (2 mg / ml lysozyme for DBC at 60 cm / h and 1 mg / ml lysozyme for SBC for 3 hours contact with mild shaking) was dissolved in the same buffer.

[0059] Together with BET surface area, SBC and DBC were compared among naked Sakura nylon 6 (2), Sakura nylon 6-dextran (3), Sakura nylon 6-dextran-SP (5), and bead-based chromatographic adsorbent (SP Sepharose fast flow). DBC of Sakura nylon 6-dextran (3) was too small to be detected and BET surface area of SP Sepharose fast flow was too large (~ 50 m2 / g).

[0060] In addition, the data in Table 1 show that, after introducing the pendant SP group, SBC and DBC of Sakura nylon 6- dextran-SP (5) enhanced 4.5 and 27 times, respectively, compared to naked Sakura nylon 6 (2). The difference between two increase amplitudes should be related to the utilization rate of added specific surface area. For SBC, sufficient contact time partially compensates the advantages of the increased surface area. For DBC, the influence of the increased specific surface area becomes dominant under dynamic conditions. Table 1: Comparison of the SBC, DBC, and BET surface area of naked Sakura nylon 6 (2), Sakura nylon 6-dextran (3), Sakura nylon 6-dextran-SP (5), and a bead-based chromatographic adsorbent Sakura Sakura nylon 6 Naked nylon 6 with dextran Bead-based sakura with and chromatographic 6 dextran sulfopropyl adsorbent (2) (3) (5) Static binding capacity (mg / g) Dynamic binding 2.0 ± 0.1 n / a 54.5 ± 2.0 94.2 ± 3.2 capacity (mg / g) Specific surface 0.23 ± 0.0 0.76 ± 0.0 0.95 ± 0.0 ~ 50 area (m2 / g) not part of the invention Example 3: Influence of mass of immobilized polymer in the SBC of the polyamide-based chromatography media according to the invention.

[0061] The influence of the density of immobilization polymer in the SBC is shown in Figure 2, using dextran as the exemplary immobilization polymer and pendant sulfopropyl (SP) groups as the exemplary adsorptive functionality. The peak SBC of the constructed polyamide-based adsorbent media (curve with squares) is superior to the commercial SP Sepharose fast flow. However, DBC (curve wiht triangles) reaches its peak value earlier than its SBC counterpart (with less conjugated dextran on the surface), mainly because under the limited contact time, advantages of higher SP group density are not obvious. The dextran density value selected, achieved in Example 1 above, corresponds to the maximum attainable DBC and an increased dextran density would instead reduce the DBC. More dextran could represent a higher amount of hydroxyl groups leading to a higher degree of crosslinking which could block the path of the transport of solutes to their binding sites. Curve with spheres represents the ratio SBC / DBC.

[0062] The data point with zero abscissa from SBC graph refers to the direct introduction of SP functional groups to the pristine Sakura nylon 6 fibers (2) (with exposed amino groups, without conjugated dextran layer), which evidences the need of the immobilization polymer, in this case dextran layers.

[0063] The resulting dextran coverage (i.e., density) was actually far higher than it would be expected due to the presence of scarce -NH2groups in the native Sakura nylon 6 fibers. Without being bound by theory, it is believed that one or more unknown polymer(s) exist(s) on the surface of the Sakura type fiber / fabric which may assist the dextran introduction. Example 4: Evaluation of the effect of the modification process on the mechanical properties of the polyamide fibers

[0064] Figure 3 depicts scanning electron microscope (SEM) images for comparison of the pristine (unmodified) material (2) (pre- treated Sakura nylon 6 fibers) (Figure 3a) with the modified Sakura nylon 6 fibers (5) obtained in Example 1 (sulfopropyl (SP) functionalized dextran-grafted Sakura nylon 6 fibers) (Figure 3b). As shown in Figure 3b, the modified Sakura nylon 6 fibers (5) exhibit a specific evident five-petal Sakura cross-sectional shape, for a given denier per filament, providing relatively larger surface areas, higher thermal and acoustical insulation, luster, and better crackbridging performance than round native fiber. The pristine fabric can be observed as characteristically smooth while some degree of roughness and diameter increase were introduced upon functionalization, which evidence the presence of a grafted dextran layer. The spatial structure of fabrics persists essentially unchanged, which is also a qualitative indication of appropriate convective flow and physical robustness.

[0065] In addition, although not expected, a homogeneous chain scission within the backbone polymer during the chemical reaction was obtained. Without being bound by theory, it is believed that modifying the polyamide fibers with an immobilization polymer (the polysaccharide dextran as the exemplary polymeric carbohydrate) prevents the de-polymerization of backbone and preserve backbone mechanical properties, via the formation of a coating layer between and around the fabrics, while introducing hydroxyl groups.

[0066] Zeta potential is a parameter employed to assess not only the surface charge of a solid immersed in liquid media of defined solution chemistry but also the hydrophobicity of the solid. As shown in Figure 4, there was a significant decrease of the zeta potential in the acid range for the sulfopropyl (SP) functionalized dextran-grafted Sakura nylon 6 fibers (5) obtained in Example 1 (curve with triangles) compared to the zeta potential of the unmodified pristine one (2) (curve with spheres), proving the introduction of the SP groups and increase of the surface hydrophilic group. The sulfopropyl (SP) functionalized dextran-grafted Sakura nylon 6 fibers (5) had negative zeta potential values over the whole observed pH range from 2.3 to 9.3 because of its strong cation nature. Example 5: Mass transfer resistance and column performance

[0067] The effects of mass transfer resistance and dispersion on the DBC, as well as the performance of a column packed with Sakura nylon 6-dextran-SP fabric (5) obtained in Example 1, were studied at increasing flow rate. Although adsorbent Sakura nylon 6-dextran-SP (5) showed a slight decrease in DBC from 60 to 120 cm / h which could be explained by a small increase in axial dispersion, the values were independent of flow rate after 120 cm / h until 480 cm / h, indicating convective flow (see Figure 5). Additionally, the peclet number of the adsorbent remained constant (> 63.6), further confirming plug flow characteristics with minimal axial mixing. In addition, the bed was porous in nature (30%), which prevented any backpressure to develop. Example 6: Chromatographic separation profiles between lysozyme and bovine serum albumin

[0068] A study of the chromatographic separation profiles between lysozyme and bovine serum albumin (BSA) using a column packed with Sakura nylon 6-dextran-SP fabric (5) obtained in Example 1 was conducted aiming at proving the capture abilities of lysozyme from lysozyme / BSA mixture (see Figure 6). BSA breakthrough before the end of loading (figure 6a) and, according to the information from polyacrylamide gel electrophoresis (PAGE) (figure 6b), only lysozyme was detected from second peak in spite of a small tail. Example 7: Purification CHO_Kl cell line fermentation broth producing adalimumab

[0069] A CHO_Kl cell line fermentation broth producing adalimumab was purified using a column packed with Sakura nylon 6-dextran-SP fabric (5) obtained in Example 1. 5 ml crude fermentation broth was injected to a disposable fixed-adaptor SepFast™ 6.2 mm ID / 0.33 ml column (Biotoolomics, UK) with a more than 15 times difference in injection volume and column volume. This high throughput was mainly due to the high DBC. The stuck cell debris or other solid impurities from fermentation broth occupied almost 60% of the column volume without clogging and back pressure development, demonstrating the potential of a much more throughput. Combinations of convective flow of the Sakura nylon 6-dextran-SP fabric adsorbent (5) and high DBC, the purification performance was amplified.

[0070] According to the information from PAGE, the first peak after breakthrough contains purified adalimumab (see Figure 7a and 7b). Thanks to its open structure and mechanical stability, low back pressure was preserved through the whole process in spite of a sharp spike during breakthrough. Combinations of convective flow of fabric adsorbent and high DBC, the purification performance was amplified.

[0071] Cell debris or other solid impurities from fermentation broth were stuck inside the column and could not be washed out. However, as purification can be accomplished in one step, this is of no great importance due to the very low cost involved in producing the polyamide-based chromatography media according to the invention, which may be discarded after a single or multiple use(s). Example 8: Direct protein purification using a continuous moving belt prototype

[0072] A continuous benchtop protein recovery and purification system (10) based on the true moving bed concept was designed, constructed, and tested. The continuous moving belt prototype (10a, 10b) (Figure 8) has the form of a belt conveyor and a tank that is divided into four chambers (11) (e.g. volume of at most 45 ml each) containing either feedstock or buffer solutions, each chamber being responsible for association (product binding), unbound washing (removal of unbound compounds), elution (product recovery), and re-equilibrium respectively. Adsorbent belts (12) such as a monolayer fabric belt (712 x 20 mm) made of the polyamide-based chromatography media of the present invention passed through each chamber in sequence via rollers (13) readily for continuous biomolecule recovery from a crude feedstock.The developed polyamide-based chromatography media of the present invention can thus be used in a continuous purification system based on mechanism of belt conveyors. The system comprises a four-chamber tank, wherein each chamber is responsible for association, unbound washing, elution, and re-equilibrium, respectively. It is able to possess its own buffer volume and liquid speed. The developed polyamide- based chromatography media of the present invention passes through each chamber in sequence via rollers readily for continuous bioproduct recovery from a feedstock.

[0073] The tensile strength and the elasticity of the belt constructed serves the purpose of belt construction and enables the “moving bed” based on the belt system. This is not possible with the use of resin-based ion exchange chromatographic media or functional belts constructed from inelastic materials.

[0074] Results observed from the prototype operation showed that the developed prototype was capable of protein (lysozyme) separations with productivity up to 0.5 mg / cm2 / h. Recombinant human IgG1 mAb adalimumab was directly recovered from unclarified CHO_K1 cell line fermentation broth with high purity, as shown in Figure 9 (image of SDS-PAGE - sodium dodecyl sulfate–polyacrylamide gel electrophoresis – under reducing conditions), and high purification factor (5.8) by only one step, confirming the selectivity of the purification procedure. In particular, the continuous moving belt prototype was challenged in continuous mode by a CHO_K1 cell line fermentation broth (13.2 mS / cm, pH 6.84) producing Humira mAb, donated by the University of Natural Resources and Life Sciences (Vienna, Austria). The purification was attempted under the following conditions: belt rate at 7.00 cm / min, 10-times diluted fermentation broth as sample, 0.75M NaCl (~52.7 mS / cm) as disassociation buffer, pH 5.1 20 mM phosphate buffer (~3.5 mS / cm) as unbound washing and re-equilibrium buffer, 30 mL liquid per chamber initially, and fresh buffer flowing into the washing and re-equilibrium chambers continuously with a speed of 1.95 mL / min. In addition, 15 mL eluted Humira mAb was taken out from the elution chamber, 2.5 mL undiluted fermentation broth as sample was added to the sampling chamber, and 20 mL 1M NaCl was added to the elution chamber, once the conductivity started out less than the critical point in the elution chamber. Normally, mAb purification may involve several steps to attain a desired level of purity. In this example, the targeted Humira mAb was directly recovered from the fermentation broth via only one step with high purity, as ascertained by SDS-PAGE (see Figure 9). This data confirms the selectivity of the purification procedure and Humira mAb’s identity as well, since unique bands near 28 kDa (light chain) and 49 kDa (heavy chain) were clearly observed.

Claims

Claims 1. Polyamide-based chromatography media comprising high surface area polyamide fibers having a cross-section comprising a region having a main body region defining a substantially longitudinal axis, and five projections extending radially outwardly from said main body region, wherein the polyamide fibers are immobilized with a immobilization polymer and the surface of said polyamide fibers is further modified with an adsorptive functionality enabling a chromatography selected from the group consisting of ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography, wherein the immobilization polymer is selected from the group consisting of dextran, dextrin, amylose, pullulan, agarose, cellulose, glucomannan, and derivatives thereof, and polymethacrylate.

2. The media of claim 1, wherein the adsorptive functionality is an ion-exchange adsorptive functionality, preferably a cation-exchange adsorptive functionality.

3. The media of claim 1 or 2, wherein the adsorptive functionality consists of pendant groups grafted to said fibers.

4. The media of any of claims 1 to 3, wherein the adsorptive functionality consists of pendant sulfopropyl groups or pendant acrylic polymer groups, preferably pendant sulfopropyl groups.

5. A packed bed comprising the polyamide-based chromatography media of any of claims 1 to 4.

6. A housing comprising a packed bed of claim 5.

7. A housing according to claim 6, wherein the polyamide- based chromatography media is in the form of woven polyamide- based fibers material, non woven polyamide-based fibers material, or random polyamide-based fibers material.

8. Use of the polyamide-based chromatography media of any of claims 1 to 4, as an adsorbent belt, preferably in a continuous purification system.

9. A process for purifying a sample comprising a macromolecule or nanoparticles, comprising contacting said sample with a bed of the polyamide-based chromatography media of any of claims 1 to 4.

10. The process of claim 9, wherein the process is implemented in a continuous mode.

11. The process of claim 9 or 10, wherein said purification is carried out at a pH ranging from 3 to 9.

12. A process for purifying a protein, comprising: providing a protein mixture, contacting the protein mixture with the polyamine-based chromatography media of any of claims 1 to 4, washing the polyamine-based chromatography media to remove unbound species, compressing the polyamine-based chromatography media, and washing the compressed polyamine-based chromatography media to extract bound protein.

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