Functionalized chromatography media comprising polymer nanofibers and methods for preparing the same

Abstract

The present invention provides a functionalized polymer chromatography medium prepared by the following method, the method comprising: (i) providing a substrate formed of one or more polymer nanofibers, (ii) grafting one or more neutral polymer chains from the substrate, and (iii) contacting the grafted product with a reagent, the reagent functionalizing the product of step (ii) into a chromatography medium, wherein step (ii) comprises reacting a plurality of compounds of the formula and / or their enantiomers and / or their derivatives of formula (I) and / or their enantiomers and / or diastereomers: with one or more functional groups present on the nanofiber substrate, wherein R 1 , R 2 , R 3 , R 4 and R 5 may be the same or different and are selected from H, halogen, C 1 ‑C 4 Alkyl or C 1 ‑C 4 Alkoxy, provided that R 1 , R 2 , R 3 , R 4 or R 5 At least one of is not hydrogen.

Description

Field of the Invention

[0001] The present invention relates to functionalized chromatographic media suitable for separating biomolecules from a mobile phase. Background of the Invention

[0002] The biotechnology market is the fastest growing segment of the world’s pharmaceutical market, accounting for 20% ($153 billion) of all market sales in 2012. Market share has grown 41% from 10% in 2002, from $153 billion to $215 billion between 2012 and 2018. With approximately 200 monoclonal antibody (MAb) products currently on the market and more than 1,000 in clinical trials, the need for technological advancement in this area is clear. Over the past few decades, typical fermentation titers of biomolecules in industrial settings have increased from 0.5 g / L to ~3 g / L, and based on advances in activation in molecular biology, levels as high as 10 g / L are believed to be achievable in the near future. However, while downstream purification processes have also been studied and developed to some extent, improvements in this area have not matched those in the upstream.

[0003] The manufacture of therapeutic proteins needs to achieve high purity during processing so that the protein to be administered is substantially free of harmful contaminants. At present, on an industrial scale, chromatography is the main method used to obtain high-purity proteins. From an economic point of view, the chromatographic unit operation that is heavily relied on is the key to the progress of downstream processing of biomolecules such as MAbs. Chromatography accounts for up to 60% (Re-use of Protein A Resin: Fouling and Economics, March 1, 2015, BioPharm International, Vol. 28, No. 3, Anurag S. Rathore, Mili Pathak, Guijun Ma, Daniel G. Bracewell) of biotherapeutic drug processing.

[0004] When a liquid phase containing a target molecule and impurities is contacted with a solid phase, this chromatographic separation involves the binding of i) the target molecule and / or ii) one or more impurities to the solid phase. The interaction between the target molecule / impurity and the solid phase can be based on charge, hydrophobicity, affinity or a combination thereof.

[0005] Historically, conventional packed bed chromatography using porous beads has been a very powerful separation tool. The porosity of these beads creates a high surface area to bind the target or impurities. This results in a high capacity material, meaning that a smaller amount of adsorbent material can be used. High capacity also increases the concentration effect achieved during the separation process, since more target can be bound per unit volume of adsorbent compared to the relative concentration of the loaded suspension. These aspects are critical for industrial scale processing, where each batch may require purification of several kilograms of material from a liquid volume of up to 20,000 L. Typical binding capacities for porous beads are in the range of 35-120 mg / mL, depending on the functionality of the solid phase and the substance being bound.

[0006] In porous bead-based systems, the binding events between the target molecule / impurity and the solid phase depend on diffusion into the porous beads. Therefore, there is a strong correlation between the residence time and the flow rate in porous bead-based systems. Therefore, the binding capacity decreases with a decrease in residence time. This in turn is accompanied by a rapid decrease in capacity, where less than 2 minutes are used in porous bead-based systems. The high flow rates required for short residence times may also be incompatible with porous beads, especially at production scales where many liters of bead suspension are packed into columns. Here, the mechanical instability of the porous beads may lead to compression or collapse events, which in turn lead to an uneven column bed.

[0007] Since flow rate affects residence time, it is critical to maximize the amount of target that can be bound to the solid phase per unit time. This allows for the use of less adsorbent amounts and / or separations to be performed in a shorter time. This metric can be defined as grams bound per unit volume per unit time (mg / mL / min). The typical binding capacity and residence time of the porous beads discussed above result in an overall productivity of about 10-120 mg / mL / min for a single column porous bead system.

[0008] As an alternative to porous bead-based systems, monoliths or membranes can be used. Flow through such materials is convective rather than diffusive, so their binding capacity is much less sensitive to flow than porous bead-based systems. These materials can be run at much higher flow rates than porous bead-based materials, with typical residence times of about 0.2-0.5 minutes.

[0009] However, under dynamic flow, the typical binding capacities of monoliths (10-20 mg / mL) and membranes (7.5-29 mg / mL) at 10% breakthrough of the target are lower than those of porous beads (Gottschalk, U. (2008). Biotechnol Prog, 24(3), 496-503. doi: 10.1021 / bp070452g). The poorer binding capacities of monoliths and membrane materials (compared to porous bead-based materials) can be compensated to some extent by using higher flow rates.

[0010] The typical binding capacities and residence times for the monoliths and membranes discussed above result in a total productivity of binding events for the monolith and membrane system of about 10-145 mg / mL / min.

[0011] There is a need for chromatography materials that combine the high binding capacity associated with porous bead-based materials with the higher flow rates achievable with monolith / membrane materials. Such materials would provide high capacity at high flow rates to achieve maximum productivity (mg / mL / min).

[0012] The inventor has surprisingly found that the nanofiber material produced by the two-step method presents such favorable properties, in which the functionalization step is carried out independently of the grafting step. The material produced by the method has a significantly increased binding capacity compared to non-grafted materials. The capacity shown by the product of the present invention is comparable to or exceeds the capacity that can only be realized with porous beads usually, and is several times higher than the capacity that can be realized by current commercial membranes and monolithic technology. In addition, these capacities can be realized under sub-second residence time, resulting in productivity values ​​10-100 times or even thousands of times higher than conventional commercially available materials.

[0013] The materials produced according to the present invention rely on several surprising innovations. First, the inventors have discovered that increasing the amount of grafting can increase the binding capacity of the chromatographic material for a set density of ligand groups, i.e., the groups that bind to the chromatographic medium to which the target biomolecule selectively binds. It has previously been known in the context of functionalizing chromatographic media with charged groups that increasing the amount of those charged groups increases the charge density of the material and the binding capacity of the material. Therefore, previous methods of maximizing the binding capacity of a material have focused on maximizing the charge density. However, the inventors have demonstrated for the first time that by increasing grafting, the binding capacity can be increased independently of the charge density of the material.

[0014] In other cases, it has been found that varying the functionalization step has little effect on charge density, but when combined with a grafting step, significant increases in binding capacity can be achieved for the same degree of functionalization.

[0015] It has therefore been found that the charge density and binding capacity of chromatographic materials can be controlled by varying the degree of functionalization and the degree of grafting to provide highly optimized materials. The inventors have studied and exploited this relationship between the grafting step and the subsequent functionalization step to produce materials with very high binding capacities at high flow rates. It has been found that the relationship between charge density and dynamic binding capacity is far more subtle than previously recognized. Thus, for materials with the same charge density, it has been found that the dynamic binding capacity can be varied by controlling the degree of grafting. This is an unexpected and highly advantageous result.

[0016] This is very important because previous attempts to increase the capacity of chromatography materials have focused solely on increasing the density of ligand groups on the material. They have therefore failed to address how to control and increase the capacity of the material by independently varying the conditions of grafting and functionalization as isolated steps. In many known modification methods, this degree of control is simply not possible because the chromatography material is modified with a charged polymer, i.e. the grafting and functionalization steps are performed simultaneously.

[0017] Importantly, in addition to increasing the binding capacity of the material, grafting and functionalization also have the effect of modulating the material's resistance to flow, which can have an adverse effect on the productivity of the chromatographic material. This is discussed in Menkhaus et al. (Menkhaus, TJ, Varadaraju, H., Zhang, L., Schneiderman, S., Bjustrom, S., Liu, L., & Fong, H. (2010). Chemical Communications, 46(21), 3720-3722. doi: 10.1039 / C001802C), who report that increased grafting may be associated with increased resistance to flow and increased mass transfer diffusion in convective media. This may be because known methods for modifying chromatographic materials generally involve grafting charged polymer chains from the surface of the material. Therefore, increasing the degree of grafting will also increase the charge density of the material, and these cannot be independently controlled to optimize the binding capacity and flow characteristics of the material.

[0018] These disadvantages are overcome in the present invention by separating the grafting step from the step of functionalizing the material. It has also been found that increasing the degree of grafting does not itself increase the resistance to flow through the material. Only when the grafted material is functionalized does it begin to affect the resistance to flow.

[0019] Furthermore, the present inventors have found that glycidol and its derivatives are particularly preferred monomer units for use in the grafting step. Surprisingly, when the grafting step employs glycidol polymerization, the resulting chromatography media exhibit improved properties (e.g., they benefit from higher capacity and higher productivity) over chromatography media produced by other grafting methods such as atom transfer radical polymerization (ATRP). SUMMARY OF THE INVENTION

[0020] Therefore, the present invention provides a method for preparing a functionalized polymer chromatography medium. The method comprises:

[0021] (i) providing a substrate formed of one or more polymer nanofibers,

[0022] (ii) grafting one or more neutral polymer chains from the substrate, and

[0023] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) as a chromatographic medium,

[0024] wherein step (ii) comprises making a plurality of formula Compounds and / or enantiomers thereof and / or derivatives thereof of formula (I) and / or enantiomers and / or diastereomers thereof:

[0025]

[0026] reacting with one or more functional groups present on the nanofiber substrate,

[0027] wherein R1, R2, R3, R4 and R5 may be the same or different and are selected from H, halogen, C1-C4 alkyl or C1-C4 alkoxy, provided that at least one of R1, R2, R3, R4 or R5 is not hydrogen.

[0028] The present invention also provides:

[0029] - functionalized chromatographic media obtainable by the process of the invention;

[0030] - a method for preparing a chromatographic column, said method comprising carrying out the method of the invention and incorporating into the column the product thus obtained;

[0031] - a chromatography column which (a) is obtainable by the process described, or (b) comprises one or more functionalized chromatography media according to the invention;

[0032] - Use of the functionalized chromatographic medium according to the invention or the chromatographic column according to the invention in chromatography;

[0033] - A method for separating one or more biomolecules from a mobile phase, the method comprising contacting the one or more biomolecules in the mobile phase with a functionalized chromatographic medium of the invention or a chromatographic column of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown are the dynamic binding capacity (DBC) of several commercially available porous bead-based systems as a function of residence time.

[0035] Figure 2 The effect of increasing the amount of functionalizing agent on the charge density of trimethylammonium functionalized materials without a grafting step is shown.

[0036] Figure 3 The effect of increasing the charge density on the dynamic binding capacity (DBC) of trimethylammonium functionalized materials without a grafting step is shown.

[0037] Figure 4 The effect of increasing the degree of polymer grafting and the amount of trimethylammonium functionalizing agent on the charge density of several materials prepared according to the present invention is shown.

[0038] Figure 5 Shows Figure 4 The average of the values ​​shown in .

[0039] Figure 6 The effect of increasing polymer grafting on the dynamic binding capacity (DBC) of several trimethylammonium functionalized materials prepared according to the present invention at a set charge density is shown.

[0040] Figure 7 Shown is the effect of increasing the charge density at a set polymer grafting on the dynamic binding capacity (DBC) of several materials prepared according to the invention compared to ungrafted material.

[0041] Figure 8 The effect of increasing the degree of polymer grafting and functionalization reaction time on the charge density of the glycidol / sulfonic acid functionalized materials is shown.

[0042] Fig. 9 The effect of the individual grafting steps on the dynamic binding capacity of the glycidol / sulfonic acid functionalized materials according to the invention is shown.

[0043] Fig.10 The effect of grafting on the dynamic binding capacity (DBC) of glycidol / carboxymethyl functionalized materials according to the invention is shown.

[0044] Fig.11 The effect of increasing charge density and degree of polymer grafting on the flow resistance of several materials prepared according to the present invention is shown.

[0045] Fig.12 The effect of increasing the degree of polymer grafting on the flow resistance of several uncharged materials is shown.

[0046] Fig.13 The effect of increasing the amount of glycidol reagent on the density of -OH groups in the grafted product is shown. DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention relates to a two-step method for preparing a functionalized polymer chromatography medium from a polymer nanofiber substrate. The two steps are a polymer grafting step and a functionalization step.

[0048] Polymer Nanofibers

[0049] The functionalized polymer chromatography medium of the present invention is formed by a polymer nanofiber substrate. Each substrate is formed by one or more polymer nanofibers.

[0050] Polymer nanofiber is generally electrospun polymer nanofiber.Such electrospun polymer nanofiber is well known to those skilled in the art, and the optimized conditions for its preparation are found in, for example, O.Hardick, et al., J.Mater.Sci.46 (2011) 3890, the entire contents of which are incorporated herein by reference.Method of the present invention generally includes electrospun polymer to produce the initial step of one or more polymer nanofibers.This may include electrospun polymer to produce one or more nonwoven sheets, each nonwoven sheet comprising one or more polymer nanofibers.

[0051] The polymer nanofibers used in the present invention generally have an average diameter of 10 nm to 1000 nm. For some applications, polymer nanofibers with an average diameter of 200 nm to 800 nm are suitable. For some applications, polymer nanofibers with an average diameter of 200 nm to 400 nm may be suitable.

[0052] The length of the polymer nanofibers used in the present invention is not particularly limited. Therefore, conventional electrospinning processes can produce polymer nanofibers hundreds of meters or even thousands of meters long. However, the length of the one or more polymer nanofibers can generally be up to 10 km, preferably 10 m to 10 km.

[0053] Generally, the one or more polymer nanofibers are provided in the form of one or more nonwoven sheets, and each nonwoven sheet comprises one or more polymer nanofibers.Therefore, substrate is usually formed by one or more nonwoven sheets, and each nonwoven sheet comprises one or more polymer nanofibers.The nonwoven sheet comprising one or more polymer nanofibers is the pad of the one or more polymer nanofibers, wherein each nanofiber is substantially randomly oriented, that is, it is not yet manufactured so that the one or more nanofibers present specific pattern.The nonwoven sheet comprising polymer nanofibers is generally provided by known methods, such as O.Hardick, et al., those disclosed in J.Mater.Sci.46 (2011) 3890.In some cases, nonwoven sheet can be composed of single polymer nanofiber.Or, nonwoven sheet can comprise two or more polymer nanofibers, for example 2,3,4,5,6,7,8,9 or 10 polymer nanofibers.

[0054] Nonwoven sheets usually have a weight of 1 to 40 g / m 2 , preferably 5 to 25 g / m 2 , in some cases 1 to 20 or 5 to 15 g / m 2 Surface density.

[0055] The nonwoven sheet generally has a thickness of 5 to 120 μm, preferably 10 to 100 μm, in some cases 50 to 90 μm, in other cases 5 to 40, 10 to 30 or 15 to 25 μm.

[0056] The polymer for preparing the nanofiber used in the method of the present invention is not particularly limited, provided that the polymer is suitable for use in chromatographic applications. Therefore, usually, the polymer is a polymer suitable for use as a chromatographic medium, i.e., an adsorbent, in chromatography. Suitable polymers include polyamides such as nylon, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polysulfones such as polyethersulfone (PES), polycaprolactone, collagen, chitosan, polyethylene oxide, agarose, acetate agarose, cellulose, cellulose acetate and combinations thereof. Preferred polyethersulfone (PES), cellulose, cellulose acetate and combinations thereof. In some cases, preferred cellulose, cellulose acetate and combinations thereof.

[0057] In some embodiments, substrate comprises one or more nanofibers, which are formed by one or more polymer nanofibers, and the one or more polymer nanofibers are formed by different polymers. Therefore, in this embodiment, substrate comprises one or more different polymers. Typical polymers are as defined above.

[0058] Typically, the method of the present invention is used to prepare a functionalized cellulose chromatographic medium, and the method includes providing a substrate formed by one or more cellulose acetate nanofibers. Preferably, the method includes providing a substrate formed by one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers. Cellulose acetate is easy to electrospin and can be easily converted into cellulose after electrospinning. Therefore, preferably the method includes providing a substrate formed by one or more nonwoven sheets, each nonwoven sheet comprising one or more electrospun cellulose acetate nanofibers.

[0059] The substrate formed by one or more polymer nanofibers comprises the one or more polymer nanofibers, usually in the form of one or more nonwoven sheets as discussed above. In certain embodiments, the one or more polymer nanofibers or one or more nonwoven sheets do not undergo any physical processing steps before the grafting step.

[0060] Physical modification of nanofibers

[0061] However, in certain preferred embodiments of the present invention, the provision of substrate includes optionally physically modifying the polymer nanofibers in the nonwoven sheet before the grafting step. Specifically, the physical modification may include heating and / or pressing the polymer nanofibers / nonwoven sheet, preferably heating and pressing the polymer nanofibers / nonwoven sheet. These steps will improve the structural stability of the material. The pressing and heating conditions may also be changed to change the thickness and / or porosity of the resulting material.

[0062] The use of a plurality of nonwoven sheets of polymer nanofibers enables the preparation of thicker materials having greater adsorption capacity (once grafted and functionalized). Thus, the provision of a substrate typically comprises providing two or more nonwoven sheets stacked one on top of another, each of said sheets comprising one or more polymer nanofibers, and simultaneously heating and pressing the stack of sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0063] In the case of cellulose chromatography media, providing the substrate typically includes providing two or more nonwoven sheets stacked one upon another, each of the sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse contact points between the nanofibers of adjacent sheets.

[0064] Preferred processing conditions for pressing and heating the polymer nanofiber / nonwoven sheet can be found in WO-A-2015 / 052460 and WO-A-2015 / 052465, the entire contents of which are incorporated herein by reference.

[0065] Grafted nanofiber substrate

[0066] The method of the present invention comprises a grafting step (step (ii)) which comprises grafting one or more neutral polymer chains from the substrate provided in step (i).

[0067] Grafting one or more neutral polymer chains from a substrate generally comprises growing one or more polymer chains from one or more functional groups present on the substrate, optionally in the presence of one or more catalysts. Thus, generally, the substrate comprises one or more functional groups, preferably one or more functional groups from which polymer chains can be grown. Growing polymer chains from the one or more functional groups means building up a polymer from corresponding monomer building blocks at the one or more functional groups.

[0068] Thus, the grafting step generally involves growing polymer chains directly from the substrate, rather than bonding preformed polymer chains to the substrate. Thus, as polymerization proceeds, the corresponding monomers are added to the ends of the growing polymer chains, which are anchored to the substrate at the distal end.

[0069] Growing polymer chains directly from the substrate enables control over the structure of the polymer coating, particularly using a polymerization strategy that allows the polymers to all grow simultaneously at a uniform rate. This enables the formation of dense and well-defined polymer coatings.

[0070] In the present invention, the grafting step comprises making a plurality of Compounds and / or enantiomers thereof and / or derivatives thereof of formula (I) and / or enantiomers and / or diastereomers thereof:

[0071]

[0072] react with one or more functional groups present on the nanofiber substrate,

[0073] Wherein R1, R2, R3, R4 and R5 can be the same or different and are selected from H, halogen, C1-C4 alkyl or C1-C4 alkoxy, provided that at least one of R1, R2, R3, R4 or R5 is not hydrogen. Therefore, usually only one type of polymer is grafted to the substrate. However, in other embodiments, more than one type of polymer can be grafted to the substrate.

[0074] Typically, R1, R2, R3, R4 and R5 may be the same or different and are selected from fluorine, chlorine, bromine, methyl or ethyl.

[0075] Thus, in the compound of formula (I):

[0076] R1 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl;

[0077] R2 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl;

[0078] R3 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl;

[0079] R4 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl; and

[0080] R5 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl.

[0081] Typically, when the grafting step (ii) comprises reacting a substrate having one or more functional groups from which a polymer can grow with a plurality of compounds of formula (I) and / or enantiomers thereof, at least one of R1, R2, R3, R4 and R5 is hydrogen. In this case, preferably, at least two of R1, R2, R3, R4 and R5 are hydrogen. More preferably, at least three of R1, R2, R3, R4 and R5 are hydrogen. Even more preferably, four of R1, R2, R3, R4 and R5 are hydrogen. Preferably, R1 is hydrogen and at least one of R2 and R3 is hydrogen. Alternatively, R2 and R3 are hydrogen. Yet more preferably, R1, R2 and R3 are hydrogen.

[0082] More preferably, the grafting step (ii) comprises reacting a substrate having one or more functional groups from which a polymer can grow with a plurality of compounds of the formula and / or enantiomers thereof.

[0083] In embodiments where the substrate is formed from nanofibers formed from different polymers, each different type of polymer nanofiber may be grafted with a different polymer during the grafting step. This may, for example, be due to the presence of different functional groups on the different polymer nanofibers. Alternatively, the same polymer may be grafted to each different type of polymer nanofiber in the substrate.

[0084] Typical functional groups include hydroxyl, amino and carboxyl. In the case where the substrate is formed from one or more cellulose or cellulose acetate nanofibers, the functional groups are typically hydroxyl groups.

[0085] Typically, between step (i) and (ii), substrate is treated to introduce the one or more functional groups, or between step (i) and (ii), substrate is treated to deprotect or activate any functional group on substrate, or between step (i) and (ii), substrate is treated to increase the number / density of functional groups on substrate. Preferably, between step (i) and (ii), substrate is treated to introduce the one or more functional groups, or any functional group on substrate is deprotected or activated. More preferably, between step (i) and (ii), substrate is treated to deprotect or activate any functional group on substrate. Even more preferably, between step (i) and (ii), substrate is treated to deprotect any functional group on substrate.

[0086] However, in alternative embodiments, there is no additional processing step between steps (i) and (ii). In these embodiments, typically, the grafting step (ii) is carried out under conditions such that the one or more functional groups are also introduced in the same step, or any functional group on the substrate is deprotected or activated, or the number / density of functional groups on the substrate is increased. Preferably, the grafting step (ii) is carried out under conditions such that the one or more functional groups are also introduced in the same step, or any functional group on the substrate is deprotected or activated. More preferably, the grafting step (ii) is carried out under conditions such that any functional group on the substrate is also deprotected or activated in the same step. Even more preferably, the grafting step (ii) is carried out under conditions such that any functional group on the substrate is also deprotected in the same step.

[0087] In a particularly preferred embodiment, the functional groups are hydroxyl groups. In this particularly preferred embodiment, the grafting step (ii) is generally carried out under conditions such that the hydroxyl groups on the substrate are also deprotected in the same step.

[0088] Deprotection of the functional group is generally performed so that the functional group can have one or more polymer chains growing therefrom.

[0089] For example, when the chromatography medium is a cellulose chromatography medium, a cellulose acetate substrate is typically provided and, prior to the grafting step, the cellulose acetate is treated to convert it to cellulose. This involves deprotection of the acetylated hydroxyl groups to produce hydroxyl groups. The conversion of cellulose acetate to cellulose is typically achieved using an alkaline aqueous solution, preferably a solution of NaOH in water:ethanol, more preferably 2:1 water:ethanol, for greater than 12 hours, such as 12 to 36 hours.

[0090] Alternatively, when the chromatographic medium is a cellulose chromatographic medium, a cellulose acetate substrate is provided and treated in a grafting step (ii) under conditions wherein the cellulose acetate is converted to cellulose and the cellulose is subsequently grafted with a plurality of The grafting step (ii) is carried out in the presence of an alkaline aqueous solution for a period of 4-6 hours, preferably a solution of NaOH or KOH, more preferably KOH in water or water: ethanol, preferably water.

[0091] When the chromatographic medium is a cellulose chromatographic medium, the present invention generally provides a method for preparing a functionalized cellulose chromatographic medium, the method comprising:

[0092] (i) providing a substrate formed of one or more cellulose acetate nanofibers, treating the cellulose acetate to convert it into cellulose,

[0093] (ii) grafting one or more neutral polymer chains from the resulting cellulose substrate, and

[0094] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) as a chromatographic medium.

[0095] When the chromatographic medium is a cellulose chromatographic medium, as an alternative, the present invention provides a method for preparing a functionalized cellulose chromatographic medium, the method comprising:

[0096] (i) providing a substrate formed of one or more cellulose acetate nanofibers,

[0097] (ii) subjecting the substrate to conditions whereby the cellulose acetate is converted to cellulose and subsequently one or more neutral polymer chains are grafted onto the resulting cellulose substrate, and

[0098] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) as a chromatographic medium.

[0099] The activation of functional groups is discussed below in the context of step (iii) of the claimed method. This discussion also applies to the activation of functional groups in step (ii) of the claimed method.

[0100] Methods of increasing the number and / or density of functional groups on a substrate are known to the skilled person.

[0101] When the one or more functional groups are introduced into the substrate, the substrate is treated in a further step (ia) of modifying the functional groups present on the substrate between steps (i) and (ii) to introduce functional groups from which one or more polymer chains can grow, followed by a step (ii) of growing polymer chains from the substrate thus modified. Step (ia) may comprise a single step or a plurality of steps which together modify the functional groups present on the substrate into functional groups from which one or more polymer chains can grow.

[0102] In embodiments involving polymerization of glycidol, the substrate is typically treated between steps (i) and (ii) to deprotect any functional groups on the substrate.

[0103] Comparative methods involving ATRP and RAFT polymerization in the grafting step are also described herein. In these methods, the substrate is typically treated between steps (i) and (ii) to modify the functional groups present on the substrate to functional groups from which one or more polymer chains can grow. Prior to this step, the substrate may also be treated to deprotect any functional groups on the substrate.

[0104] In methods involving ATRP polymerization, the hydroxyl groups on the substrate are typically modified to introduce alkyl halide or aryl halide groups. Typically, the alkyl halide is an alkyl fluoride, alkyl chloride, alkyl bromide or alkyl iodide. Alkyl bromides are preferred. Tertiary alkyl halides are more preferred. Tertiary alkyl bromides are even more preferred.

[0105] The one or more polymer chains grafted to the substrate are neutral. The polymer chains do not contain any group that is considered by those skilled in the art to be a charged group, for example, the charged groups discussed below. Typically, the polymer chains grafted to the substrate in step (ii) do not contain any charged groups as defined herein.

[0106] The neutrality of a polymer can be assessed by whether the polymer contains any groups that are ionizable, i.e., protonated or deprotonated at a substantially neutral pH, e.g., pH 6-8, typically pH 6.5-7.5, often pH 6.75-7.25, or about pH 7. Typically, a neutral polymer is substantially free of acidic or basic centers, i.e., substantially free of functional groups that are protonated or deprotonated at pH 6-8, typically pH 6.5-7.5, often pH 6.75-7.25, or about pH 7. This can be determined by a skilled artisan by assays typical in the art. Typical procedures for assessing acidity and basicity and their theoretical aspects are discussed in "Acidity and basicity of solids: Theory, assessment and utility", J. Fraisard and L. Petrakis, eds., NATO ASI Series C, Vol. 444, Kluwer Academic Publishers, Dordrecht, Boston and London, 1994, particularly on page 513, the entire contents of which are incorporated herein by reference. As used herein, substantially means less than 1 mol%, preferably less than 0.1 mol%, even more preferably less than 0.01 mol%, or even less than 0.001 mol%.

[0107] As mentioned above, the grafting step (ii) comprises bonding a substrate having one or more functional groups from which a polymer can grow to a plurality of The compound and / or its enantiomer and / or its derivative of formula (I) and / or its enantiomer and / or diastereomer is reacted:

[0108]

[0109] wherein R1, R2, R3, R4 and R5 may be the same or different and are selected from H, halogen, C1-C4 alkyl or C1-C4 alkoxy, provided that at least one of R1, R2, R3, R4 or R5 is not hydrogen;

[0110] A polymerization process called glycidol polymerization is used. Thus, the growth of polymer chains is achieved using glycidol polymerization.

[0111] Typically, R1, R2, R3, R4 and R5 may be the same or different and are selected from fluorine, chlorine, bromine, methyl or ethyl.

[0112] Thus, in the compound of formula (I):

[0113] R1 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl;

[0114] R2 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl;

[0115] R3 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl;

[0116] R4 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl; and

[0117] R5 is H, halogen, C1-C4 alkyl or C1-C4 alkoxy, preferably H, fluorine, chlorine, bromine, methyl or ethyl.

[0118] Typically, when the grafting step (ii) comprises reacting a substrate having one or more functional groups from which a polymer can grow with a plurality of compounds of formula (I) and / or enantiomers thereof, at least one of R1, R2, R3, R4 and R5 is hydrogen. In this case, at least two of R1, R2, R3, R4 and R5 are hydrogen. More preferably, at least three of R1, R2, R3, R4 and R5 are hydrogen. Even more preferably, four of R1, R2, R3, R4 and R5 are hydrogen. Preferably, R1 is hydrogen and at least one of R2 and R3 is hydrogen. Alternatively, R2 and R3 are hydrogen. Yet more preferably, R1, R2 and R3 are hydrogen.

[0119] More preferably, the grafting step (ii) comprises bonding a substrate having one or more functional groups from which a polymer can grow to a plurality of reacting compounds and / or their enantiomers.

[0120] Glycidol polymerization is a technique known to those skilled in the art. Glycidol polymerization does not generally require the presence of a catalyst. However, the polymerization may optionally be carried out in the presence of one or more suitable catalysts. In such embodiments, chemical or biological catalysts are generally used. Glycidol polymerization is generally carried out in an aqueous environment. Generally, glycidol polymerization is carried out at room temperature. Generally, glycidol polymerization is carried out under mild alkaline conditions. Generally, glycidol polymerization is carried out for greater than about 5 hours, preferably greater than about 10 hours, more preferably greater than about 15 hours, for example about 16 hours. After the glycidol polymerization, the grafted product is generally washed in water and then washed with a weak acid.

[0121] The polymerization of glycidol comprises polymerizing glycidol and / or a glycidol derivative of formula (I) from one or more functional groups as defined herein present on the substrate. Typically, these functional groups are hydroxyl groups. Thus, typically, step (ii) of the claimed method comprises polymerizing a plurality of glycidol derivatives of formula (I) from one or more functional groups as defined herein present on the substrate. Typically, these functional groups are hydroxyl groups. and / or its derivatives of formula (I) and / or its enantiomers and / or diastereomers are reacted with one or more hydroxyl groups present on the nanofiber substrate. Preferably, step (ii) of the claimed method comprises reacting a plurality of formula The compound and its enantiomers react with one or more hydroxyl groups present on the nanofiber substrate.

[0122] Polymerization of glycidol typically produces one or more polyglycerol chains.

[0123] Thus, in one embodiment, the present invention provides a method for preparing a functionalized polymer chromatography medium, the method comprising:

[0124] (i) providing a substrate formed of one or more polymer nanofibers,

[0125] (ii) grafting one or more neutral polymer chains from the substrate, and

[0126] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) as a chromatographic medium,

[0127] The growing polymer chain comprises polymerizing glycidol and / or a glycidol derivative of formula (I), preferably glycidol, from one or more functional groups present on the substrate.

[0128] Therefore, in one embodiment, the present invention also provides a method for preparing a functionalized polymer chromatography medium, the method comprising:

[0129] (i) providing a substrate formed of one or more polymer nanofibers,

[0130] (ii) grafting one or more neutral polymer chains from the substrate, and

[0131] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) as a chromatographic medium,

[0132] Wherein the one or more polymer chains are one or more polyglycerol chains.

[0133] Glycidol polymerization inevitably leads to branching of the polymer chains, producing a "bushes" structure. Thus, typically, one or more polymer chains are branched.

[0134] Another polymerization method well known to those skilled in the art is controlled radical polymerization (CRP). Controlled radical polymerization is a term well known to those skilled in the art and generally refers to a free radical addition polymerization of monomers, wherein the molecular weight of the growing polymer can be controlled to a certain extent with respect to time. Examples of CRP include atom transfer radical polymerization (ATRP), Atom Generation by AGET (Atoms Generated by Electron Transfer)-ATRP and Reversible Addition-Fragmentation Chain Transfer (RAFT). Polymerization methods are not completely limited to CRP, but may include free radical polymerization (FRP) and ring-opening metathesis polymerization (ROMP).

[0135] ATRP can be carried out in an aqueous environment or optionally in a mixture of water and another solvent, which solvent can be, for example, methanol, DMSO, THF, DMF or NMP. ATRP polymerization is preferably carried out under substantially oxygen-free conditions. Typically, ATRP is carried out at room temperature. Typically, after carrying out ATRP, the resulting material is washed with water.

[0136] The catalyst for ATRP polymerization is generally a transition metal or transition metal salt complexed with one or more ligands. As used herein, a ligand is a compound coordinated with a metal ion (usually a transition metal ion). Suitable transition metals include copper, cobalt, molybdenum, rhodium, osmium, ruthenium, palladium, nickel and rhenium. Copper is preferred. Examples of suitable ligands for copper coordination catalysts include 2,2'-bipyridine (bpy), 4,4'-di(5-nonyl)-2,2-bipyridine (dNbpy), N,N,N',N'-tetramethylethylenediamine (TMEDA), N-propyl(2-pyridyl)methylimine (NPrPMI), 2,2':6',2''-terpyridine (tpy), 4,4',4''-tri(5-nonyl)- 2,2':6',2''-terpyridine (tNtpy), N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA), N,N-bis(2-pyridylmethyl)octylamine (BPMOA), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), tris[2-(dimethylamino)ethyl]amine (Me6TREN), tris[(2-pyridyl)methyl]amine (TPMA) and 1,4,8,11-tetraaza-1,4,8,11-tetramethylcyclotetradecane (Me4CYCLAM).

[0137] The use of appropriate monomers and, if appropriate, chain transfer agents can induce branching of the polymer at the growing ends of the polymer chains in the CRP process, thereby providing the possibility of a bush-type structure. In other cases, branching is less preferred and a substantially unbranched "brush-like" polymer structure can be formed.

[0138] ATRP polymerisation can be controlled to produce substantially linear polymer chains or to produce a degree of branching. Substantially unbranched polymers will produce polymer "brush" structures.

[0139] ATRP polymerization can use one or more different types of monomers, wherein a monomer is any individual unit used to form a polymer. Each monomer includes one or more polymerizable groups and is typically selected from monofunctional monomers and difunctional monomers. The polymerizable group typically contains a C=C double bond.

[0140] Examples of suitable monofunctional monomers are methacrylates and methacrylamides, such as N-[3-(dimethylamino)propyl]methacrylamide. Other typical methacrylates and methacrylamides include 2-hydroxypropyl methacrylamide (HPMA) and hydroxyethyl methacrylate (HEMA).

[0141] Difunctional monomers, such as dimethacrylate or divinyl sulfone, can be used to introduce branching and / or crosslinking in ATRP polymerizations.

[0142] Functionalization of grafted products

[0143] The method of the present invention comprises a step of functionalizing the grafted product (step (iii)), which functionalizes the product into a chromatographic medium, for example by introducing one or more ligand groups onto the grafted product. This step comprises contacting the grafted product with a reagent, which functionalizes the grafted product into a chromatographic medium by introducing one or more ligand groups onto the grafted product.

[0144] For the avoidance of doubt, step (iii) may comprise a single step or a plurality of steps which together functionalise the product of step (ii) as a chromatographic medium.

[0145] The reagents are usually used to functionalize the chromatographic medium by introducing one or more ligand groups, which makes the functionalized product containing the one or more ligand groups suitable for use as a chromatographic medium. The one or more ligand groups introduced will depend on the specific chromatographic technique using the medium. The ligand group is a group introduced into the grafted product, which makes the grafted product suitable for use as a chromatographic medium. Suitable ligand groups and reagents will be discussed further below.

[0146] In some embodiments, the grafted product is functionalized with only one type of ligand group. In other embodiments, the grafted product is functionalized with two or more types of ligand groups. Thus, step (iii) of the claimed method involves contacting the grafted product with a reagent that functionalizes the product of step (ii) into a chromatographic medium by introducing one or more ligand groups, which may be the same or different, onto the grafted product.

[0147] In embodiments where the substrate comprises one or more polymer nanofibers formed from different polymers, each different species of polymer nanofiber may be functionalized (after grafting) with one or more ligand groups, which may be the same or different.

[0148] In embodiments where more than one type of polymer may be grafted to the substrate, each polymer graft may be functionalized with one or more ligand groups, which may be the same or different.

[0149] Although the present invention contemplates methods involving only a single treatment with a reagent, methods involving multiple functionalization steps may also be used. Such embodiments include functionalization by contacting the grafted product with the reagent two or more times in a batch manner. Batch functionalization refers to reacting the grafted product with a reagent to functionalize it, then stopping the reaction and reacting the resulting (partially) functionalized material with a separate batch of reagents. Reacting in a batch manner does not simply mean, for example, adding more portions of reagent to a reaction vessel.

[0150] The batch functionalization is usually carried out between two and ten times, i.e. 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0151] Preferred processing conditions for batch functionalization may be found in WO-A-2015 / 052460 and WO-A-2015 / 052465, the entire contents of which are incorporated herein by reference.

[0152] In some cases, contacting with a reagent may include placing the grafted product in a reservoir and flowing the reagent through the reservoir so that the reagent contacts the grafted product as it flows, which functionalizes the grafted product into a chromatographic medium. In some cases, functionalizing the material in this manner may be more effective than simply contacting the grafted product with a reagent in, for example, a flask or beaker.

[0153] Preferred processing conditions for flow functionalization may be found in WO-A-2015 / 052460 and WO-A-2015 / 052465, the entire contents of which are incorporated herein by reference.

[0154] The reagent functionalizes the grafted product to produce a chromatographic medium, in particular a functionalized chromatographic medium. Typically, the reagent functionalizes the grafted product so that it is suitable for use in ion exchange, affinity capture or hydrophobic chromatography methods. Thus, contact with the reagent will produce a chromatographic medium that is functionalized with one or more ligand groups (i.e., one or more negatively charged moieties, one or more positively charged moieties, one or more proteins, simulated or synthetic ligands that mimic the effects of protein ligands, peptides, antibodies or fragments thereof, dyes, histidine, groups containing metal cations or hydrophobic groups). In particular, when the functionalized chromatographic medium is used in an anion exchange chromatography method, 2-chloro-N,N-diethylamine hydrochloride (DEACH) and glycidyl trimethylammonium are preferably used as reagents. In particular, when the functionalized chromatographic medium is used in a cation exchange chromatography method, other preferred reagents are 1,4-butane sultone; sodium chloroacetate; TEMPO, followed by sodium perchlorate; or allyl glycidyl ether, followed by sodium bisulfite. Particularly when the functionalized chromatography medium is used in an affinity chromatography method, another preferred reagent is NaIO4; divinyl sulfone, then protein A; allyl glycidyl ether, then first a halohydrin forming reagent (e.g., N-bromosuccinamide), then protein A; or allyl glycidyl ether, then first an epoxide forming reagent, then protein A. Particularly when the functionalized chromatography medium is used in a hydrophobic chromatography method, another preferred reagent is styrene oxide.

[0155] Typically, the reagent is glycidyltrimethylammonium; 1,4-butanesultone; sodium chloroacetate; NaIO4, followed by protein A; divinyl sulfone, followed by protein A; allyl glycidyl ether, followed by a halohydrin forming reagent (e.g., N-bromosuccinamide) first, followed by protein A; or allyl glycidyl ether, followed by an epoxide forming reagent first, followed by protein A. Preferably, the reagent is divinyl sulfone, followed by protein A; or allyl glycidyl ether, followed by a halohydrin forming reagent (e.g., N-bromosuccinamide) first, followed by protein A.

[0156] Thus, in a preferred embodiment, the present invention provides a method for preparing a functionalized polymeric chromatography medium, wherein the functionalization step (iii) comprises a plurality of steps which together functionalize the product of step (ii) into a chromatography medium.

[0157] In a particularly preferred embodiment, the present invention provides a method for preparing a functionalized polymeric chromatography medium, wherein in the functionalization step (iii):

[0158] (a) first contacting the grafted product with a reagent selected from divinyl sulfone, allyl glycidyl ether and a combination thereof;

[0159] (b) optionally treating the product of step (a) with a halohydrin forming reagent or an epoxide forming reagent, preferably a halohydrin forming reagent; and

[0160] (c) contacting the product of step (b) with Protein A.

[0161] In this particularly preferred embodiment, if the grafted product is first contacted with divinyl sulfone in step (a), the product of step (a) is generally not treated with a halohydrin-forming agent or an epoxide-forming agent. Thus, in some embodiments, in the functionalization step (iii), the grafted product is first contacted with divinyl sulfone and the product of this step is then contacted with protein A. In this particularly preferred embodiment, if the grafted product is first contacted with allyl glycidyl ether in step (a), the product of step (a) is then treated with a halohydrin-forming agent or an epoxide-forming agent, preferably a halohydrin-forming agent, in step (b). Thus, in some embodiments, in the functionalization step (iii), the grafted product is first contacted with allyl glycidyl ether and the product of this step is treated with a halohydrin-forming agent and the product of this step is then contacted with protein A.

[0162] In another particularly preferred embodiment, the present invention provides a method for preparing a functionalized polymeric chromatography medium, wherein in the functionalization step (iii):

[0163] (a) contacting the grafted product with a reagent selected from the group consisting of divinyl sulfone, allyl glycidyl ether, and combinations thereof;

[0164] (b) optionally treating the product of step (a) with a halohydrin forming reagent or an epoxide forming reagent, preferably a halohydrin forming reagent; and

[0165] (c) contacting the product of step (b) with Protein A.

[0166] In this particularly preferred embodiment, if the grafted product is first contacted with divinyl sulfone in step (a), the product of step (a) is generally not treated with a halohydrin-forming agent or an epoxide-forming agent. Thus, in some embodiments, in the functionalization step (iii), the grafted product is first contacted with divinyl sulfone and the product of this step is then contacted with protein A. In this particularly preferred embodiment, if the grafted product is first contacted with allyl glycidyl ether in step (a), the product of step (a) is then treated with a halohydrin-forming agent or an epoxide-forming agent, preferably a halohydrin-forming agent, in step (b). Thus, in some embodiments, in the functionalization step (iii), the grafted product is contacted with allyl glycidyl ether and the product of this step is treated with a halohydrin-forming agent and the product of this step is then contacted with protein A.

[0167] Chromatographic media and methods

[0168] The product of the process of the invention is a functionalized chromatography medium, ie a chromatography medium from which a polymer has been grafted and which has then been functionalized by introducing one or more ligand groups onto the grafted product to render them suitable for use in one or more chromatography methods.

[0169] Specific chemical functionalization is discussed in more detail below. Generally speaking, such chemical functionalization changes the chemical and / or physical properties of the functionalized chromatographic medium by introducing one or more charged groups. This in turn affects the behavior of the functionalized chromatographic medium when used in a chromatographic method. For example, compared to its unfunctionalized form, the modification can change the polarity, hydrophobicity or biobinding properties of the functionalized chromatographic medium. In some cases, compared to its unfunctionalized form, the modification can change more than one of the polarity, hydrophobicity or biobinding properties of the functionalized chromatographic medium. In one embodiment, compared to its unfunctionalized form, the modification will change the polarity and hydrophobicity of the functionalized chromatographic medium.

[0170] Functionalized chromatographic media are typically in the form of membranes. Such membranes are suitable for use in membrane chromatography methods. Membrane chromatography methods are well known to those skilled in the art and are discussed in "Membrane Processes in Biotechnologies and Pharmaceutics" edited by Catherine Charcosset, Elsevier, 2012, the entire contents of which are incorporated herein by reference.

[0171] Typically, the functionalized polymer chromatography medium is suitable for use in a chromatography method selected from ion exchange chromatography, affinity capture chromatography, hydrophobic chromatography and mixed mode chromatography. In some cases, the chromatography method is operated in a "mixed mode", i.e., utilizing more than one form of interaction, i.e., ion exchange, affinity capture and hydrophobic interaction. Typically, such a "mixed mode" chromatography involves ion exchange (ions) and hydrophobic interaction. Preferably, the functionalized polymer chromatography medium is suitable for use in a chromatography method selected from ion exchange chromatography, affinity capture chromatography and hydrophobic chromatography, preferably ion exchange chromatography and affinity capture chromatography. In operation, such chromatography methods involve passing a mobile phase containing the desired molecule through an adsorbent phase (here, a functionalized chromatography medium). The adsorbent phase is typically selected so that the desired molecule is preferentially retained thereon relative to other components also present in the mobile phase.

[0172] Typically, the polymeric chromatography medium is functionalized with DEAE, Q, SP, CM, Protein A, phenyl or MEP groups, for example with DEAE, Q, SP, CM or Protein A groups. Typically, the polymer is cellulose and the chromatography medium is functionalized with DEAE, Q, SP, CM, Protein A, phenyl or MEP groups, for example with DEAE, Q, SP, CM or Protein A groups. Thus, the functionalized chromatography medium may be a cellulose derivatized with DEAE, Q, SP, CM, Protein A, phenyl or MEP groups, for example with DEAE, Q, SP, CM or Protein A groups.

[0173] In a preferred embodiment, the polymeric chromatography medium is functionalized with Protein A.

[0174] Ion exchange chromatography is a technique for separating molecules (usually ions or polar molecules) based on their ionic charge. Therefore, the functionalized chromatographic media used in such methods contain one or more positively or negatively charged moieties. The positive and / or negative charges in the functionalized chromatographic media are usually balanced with one or more counterions. Ion exchange chromatography includes one or more of cation exchange chromatography and anion exchange chromatography.

[0175] Functionalized chromatographic media for use in cation exchange chromatography contain one or more negatively charged moieties. Typical negatively charged moieties include one or more carboxylate, sulfonate or phosphonate groups or mixtures thereof, i.e., the moieties typically contain one or more -COO - 、-SO3 - or -P(OH)2O - Typical functionalized chromatographic media used in cation exchange chromatography contain one or more -O-CH2COO - 、-CH2COO - 、-SO3 - 、-CH2CH2CH2SO3 - 、-CH2CH2SO3 - or -P(OH)2O - part.

[0176] Functionalized chromatographic media for use in anion exchange chromatography contain one or more positively charged moieties. Typical positively charged moieties include one or more quaternary amine groups. Typical functionalized chromatographic media for use in anion exchange chromatography contain one or more -N + (CH3)3, -N + (C2H5)H, -CH2CH2N + (C2H5)H, -CH2CH2N + (C2H5)2(CH2CH(OH)CH3)、-O-CH2CH2-N + (CH3)3, -CH2CH2N + (CH3)3 or -CH2CH2N + (CH3)2H moiety.

[0177] For the avoidance of doubt, a "charged group" refers to a group that contains an ionized moiety such that it carries a positive or negative charge, ie a "charged group" includes an anionic or cationic moiety. A charged group is a specific example of a ligand group.

[0178] Typically, the one or more charged groups include one or more carboxylates (-COO - ), sulfonate (-SO3 - ) or phosphonate (-P(OH)2O - ) group, or a quaternary amine group, or a mixture thereof. Typically, the one or more charged groups include all anionic groups or all cationic groups, but in some cases, mixtures of anionic and cationic groups are contemplated. Typically, only one type of anionic group is used, but mixtures may also be used. Typically, only one type of cationic group is used, but mixtures may also be used.

[0179] Representative charged groups include -O-CH2COO - 、-CH2COO - 、-SO3 - 、-CH2CH2CH2SO3 - 、-CH2CH2SO3 - 、-P(OH)2O - 、-N + (CH3)3, -N + (C2H5)H, -CH2CH2N + (C2H5)H, -CH2CH2N + (C2H5)2(CH2CH(OH)CH3)、-O-CH2CH2-N + (CH3)3, -CH2CH2N + (CH3)3 and -CH2CH2N + (CH3)2H moiety.

[0180] Typical charged groups include DEAE, Q, SP and CM groups. Typically, the functionalized polymeric chromatographic medium is functionalized with DEAE, Q, SP or CM groups. Thus, the functionalized chromatographic medium can be cellulose derivatized with DEAE, Q, SP or CM groups.

[0181] Affinity capture chromatography is a technique for separating molecules based on the affinity of a molecule to a specific ligand (usually but not always a biological ligand). This method can, for example, rely on the attraction between an antibody and an antigen or an enzyme and a substrate. The functionalized chromatographic medium used in affinity capture chromatography usually contains one or more parts selected from one or more proteins, peptides, antibodies or fragments thereof, dyes, histidine or groups containing metal cations. Therefore, the one or more ligand groups may comprise one or more such parts. Alternatively, the functionalized chromatographic medium used in affinity capture chromatography may contain a simulation or synthetic ligand that simulates the effect of a protein ligand.

[0182] Typical proteins used in affinity capture chromatography are well known to those skilled in the art and include Protein A, Protein G and Protein L. Protein A is preferred.

[0183] Protein A is a protein well known to the skilled person. As used herein, reference to "protein A" includes recombinant protein A (which may have an altered sequence compared to the protein A found in Staphylococcus aureus) and tagged protein A (as described in EP-B-0873353 and US6399750, the entire contents of which are incorporated herein by reference). Protein A may be a modified variant of protein A, such as a cysteine ​​modified variant of protein A.

[0184] Typical antibodies and fragments thereof for use in affinity capture chromatography are well known to those skilled in the art and include IgG.

[0185] Typical dyes used in affinity capture chromatography are well known to those skilled in the art and include Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G.

[0186] Typical groups containing metal cations for use in affinity capture chromatography are well known to those skilled in the art. Such groups typically contain a chelating agent to immobilize the metal cation. The metal cation is typically selected from copper, nickel, zinc and cobalt cations, preferably Cu 2+ 、Ni 2+ 、Zn 2+ and Co 2+ .

[0187] Hydrophobic interaction chromatography is a technique for separating molecules based on their hydrophobicity. Therefore, the functionalized chromatographic media used in such methods contain one or more moieties containing one or more hydrophobic groups. Typical hydrophobic groups include propyl, butyl, phenyl and octyl.

[0188] Mixed mode (or multi-mode) chromatography is a technique for separating molecules based on two or more characteristics (usually hydrophobicity and ionic charge). This may involve a combination of hydrophobicity and anionicity, or a combination of hydrophobicity and cationicity. Therefore, the functionalized chromatographic medium used in such methods generally contains one or more positively charged or negatively charged parts generally as defined above, and it contains one or more hydrophobic groups generally as defined above. The positive charge and / or negative charge in the functionalized chromatographic medium is generally balanced with one or more counterions. The functionalized chromatographic medium used in such methods may also contain one or more ionizable hydrophobic groups to be used in so-called hydrophobic charge induction chromatography (HCIC). Therefore, in one embodiment, mixed mode chromatography is hydrophobic charge induction chromatography. The suitable groups used in such methods have 4-mercapto-ethyl-pyridine (MEP) groups and octylamine groups.

[0189] Functionalized chromatographic media for use in mixed mode chromatographic methods involving a combination of hydrophobic and anionic interactions contain one or more generally positively charged moieties as defined above and one or more generally hydrophobic groups as defined above. Suitable groups for use in such methods include N-benzylmethylethanolamine groups and N-benzoyl-homocysteine ​​groups. Functionalized chromatographic media for use in mixed mode chromatographic methods involving a combination of hydrophobic and cationic interactions contain one or more generally negatively charged moieties as defined above and one or more generally hydrophobic groups as defined above. Suitable groups for use in such methods include N-benzoyl-homocysteine ​​groups.

[0190] The method for preparing functionalized chromatographic media claimed in the present invention generally involves, in step (iii), introducing one or more ligand groups into the grafted product so that the resulting functionalized product containing the one or more ligand groups is suitable for use as a chromatographic medium in a chromatographic method. Typical parts, media, reagents and methods are as defined above. The one or more ligand groups are introduced by reacting a suitable reagent with one or more functional groups contained on the grafted product. Typical functional groups include hydroxyl, amino, halogen and carboxyl. Since step (ii) of the method involves polymerization of glycidol, the functional group is generally hydroxyl. Suitable reagents for introducing the one or more ligand groups will be discussed elsewhere.

[0191] The one or more functional groups can be activated before reacting with the reagent. Conventional activation methods known in the art can be used. Therefore, in the case where the functional group is a hydroxyl group, such a group can be activated by treating with carbonyl diimidazole (CDI), dioxirane, cyanuric acid, N-hydroxysuccinimide ester (NHS), 2-fluoro-1-methylpyridinium toluene-4 sulfonate (FMP), NaIO4, divinyl sulfone or allyl glycidyl ether. In the case where the functional group is amino, such a group can be activated by treating with epichlorohydrin, glutaraldehyde or epoxide. In the case where the functional group is carboxyl, such a group can be activated by treating with CDI or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). In the case where the functional group is a halogen atom, such a group can be activated by treating with divinyl sulfone.

[0192] The skilled artisan can select suitable reagents to introduce specific groups and moieties into specific polymers, for example based on the desired ligand groups and moieties and the functional groups contained in those polymers. Typical reagents include 2-chloro-N,N-diethylamine hydrochloride (DEACH); glycidyltrimethylammonium chloride (GMAC); 1,4-butanesultone; sodium chloroacetate; NaIO4, followed by protein A; divinyl sulfone, followed by protein A; allyl glycidyl ether, followed by a halohydrin forming reagent first, followed by protein A; or allyl glycidyl ether, followed by an epoxide forming reagent first, followed by protein A.

[0193] Typical halohydrin formation reagent comprises the electrophilic source of halogen and the nucleophilic source of hydroxyl.Therefore, typical halohydrin formation reagent comprises diatomic halogen (being generally Cl2, Br2 or I2) in water, N-chlorosuccinamide, N-bromosuccinamide or N-iodosuccinamide.Usually, halohydrin formation reagent produces halohydrin material from alkene.Halohydrin formation reaction can be regioselective to mainly produce the halohydrin product of the terminal carbon atom bonding of halogen atom and polymer chain thereon, or it can be regioselective to mainly produce the halohydrin product of the terminal carbon atom bonding of hydroxyl and polymer chain thereon, or it can have low regioselectivity.Preferably, halohydrin formation reaction can be regioselective to mainly produce the halohydrin product of the terminal carbon atom bonding of halogen atom and polymer chain thereon.

[0194] Typical epoxide-forming agents include peroxyacids (e.g. between -chloroperoxybenzoic acid (mcpba)), hydrogen peroxide in the presence of NaOH or H2O, tert-butyl hydroperoxide in the presence of vanadyl acetylacetonate, i Pr)4 and diethyl tartrate in the presence of tert-butyl hydroperoxide, in the presence of Ti(O i tert-butyl hydroperoxide in the presence of Pr)4 and diisopropyl tartrate, or potassium persulfate in the presence of a fructose-derived catalyst and a base.

[0195] generally,

[0196] - the chromatography method is a cation exchange method and the reagent functionalizes the chromatography medium with one or more charged groups including one or more carboxylate, sulfonate or phosphonate moieties; or

[0197] - the chromatography method is an anion exchange method and the reagent functionalizes the chromatography medium with one or more charged groups including one or more quaternary amino or diethylamine moieties;

[0198] - the chromatographic method is affinity capture chromatography, the reagent being a chromatographic medium functionalized with one or more proteins, peptides, antibodies or fragments thereof, dyes, histidine groups or groups containing metal cations;

[0199] - the chromatography method is hydrophobic interaction chromatography and the reagents are chromatographic media functionalized with one or more propyl, butyl, phenyl or octyl groups; or

[0200] - The chromatography method is mixed mode chromatography and the reagents are chromatographic media functionalized with one or more MEP, octylamine, N-benzylmethylethanolamine or N-benzoyl-homocysteine ​​groups.

[0201] Specific embodiments of the invention

[0202] As described above, the present invention provides a two-step method for preparing a functionalized polymer chromatography medium from a polymer nanofiber substrate, the two steps being (i) a polymer grafting step and (ii) a functionalization step.

[0203] The polymer grafting step generally introduces 500-60,000 μmol / g, preferably 1000-60,000 μmol / g of polymer into the substrate. In some embodiments, the grafting step introduces 1000-2000 μmol / g. In other embodiments, the grafting step introduces 5000-10,000 μmol / g. In still other embodiments, the grafting step introduces 30,000-60,000 μmol / g. This amount can generally be determined by measuring the amount of a specific functional group (e.g., hydroxyl) added to the substrate by the grafting method. The technician should be aware of the appropriate method for determining the amount of a specific functional group present in a given grafted material sample.

[0204] Increased polymer density may be obtained by using increased amounts or higher concentrations of the polymerizing agent, or by carrying out the reaction at higher temperatures or for longer times.

[0205] In the case of glycidol polymerization, the grafting step typically introduces 500-60,000 μmol / g of glycidol into the grafted product as determined by the -OH density of the grafted product. The -OH density of the grafted product can be determined by titration, typically with tetrabutylammonium hydroxide.

[0206] An increased -OH density can be obtained by using increased amounts of glycidol or a glycidol derivative having formula (I).

[0207] Preferably, the -OH content of a particular polymer sample is determined by the following assay:

[0208] 1) placing a sample of the grafted material on a wetted filter paper in a Buchner filter funnel and washing with ultrapure water while applying a vacuum to ensure that the water washes through the sample;

[0209] 2) Dry the sample in an oven at 60°C to constant weight and measure the mass of the sample.

[0210] 3) mince the sample and suspend it in a 50 mL centrifuge tube containing 10 mL of p-toluenesulfonyl isocyanate solution (20 mL p-toluenesulfonyl isocyanate / 500 mL acetonitrile);

[0211] 4) The tube was sealed and the mixture was stirred under heating (60° C.) in a water bath for 1 hour;

[0212] 5) Allow the tube and its contents to cool to room temperature, carefully quench with 10 mL of ultrapure H2O, then transfer the mixture to a titration cup and make up to 80 mL with isopropanol;

[0213] 6) The mixture was stirred at room temperature for 30 minutes and then titrated with tetrabutylammonium hydroxide (0.481 M solution in methanol) to determine the absolute concentration of hydroxyl groups present.

[0214] The -OH concentration (mol / g) of a particular polymer sample can be determined by measuring the absolute concentration of hydroxyl groups in the sample using the above-described determination method and dividing by the total mass of the dried sample obtained in step (2) of the determination method.

[0215] After steps (ii) and (iii), the density of the ligand groups introduced in step (iii) of the method of the present invention is typically 100-2,000 μmol / g of functionalized chromatography medium. The density is preferably 300-1,500 μmol / g, more preferably 500-1200 μmol / g. In some embodiments, the density is 100-500 μmol / g. In other embodiments, the density is 1200-2000 μmol / g. Densities of functionalized chromatography media much higher than 2,000 μmol / g may render the material difficult to use as a chromatography medium.

[0216] The number of moieties in the functionalized material is typically determined by titrating the density. The skilled person will be aware of suitable methods for determining the amount of a particular moiety present in a given sample of functionalized material.

[0217] In the case of functionalization with trimethylammonium chloride, the density can be determined as the trimethylammonium chloride density, which can be determined by the following determination method:

[0218] 1) Wash 50 mg of material on a Buchner filter funnel with 100 mL of 0.1 M HCl solution, and then wash with 100 mL of 0.01 M HCl solution;

[0219] 2) The material was dried in a drying oven at 75°C to constant weight, then torn into small pieces and placed in a 50 mL centrifuge tube equipped with a small magnetic stirring bar;

[0220] 3) Add 15 mL of deionized water and approximately 1 mL (added via a rubber-tipped pipette) of potassium chromate solution, which turns the mixture yellow;

[0221] 4) The mixture was stirred vigorously for 20 minutes and then titrated with 0.1 M silver nitrate. The endpoint of the titration was identified by the color change from clear yellow to cloudy brown.

[0222] 5) Calculate the trimethylammonium chloride content (μmol / g) as micromoles of silver nitrate added to reach the endpoint per gram of nanofiber material used in the titration.

[0223] In the case of functionalization with sulfonic acid (S) groups, the density can be determined as the sulfonic acid density, which can be determined by the following determination method:

[0224] 1) Wash the dried functionalized material sample with 0.1 M HCL and 0.01 M HCl;

[0225] 2) Dry the material in an oven and weigh it;

[0226] 3) Determine the molarity of the material using the amount of NaOH that must be added to reach pH 7;

[0227] 4) The sulfonic acid (S) content (μmol / g) was calculated as micromoles of NaOH added to reach pH 7 per gram of nanofiber material used in the titration.

[0228] The two-step method of the present invention allows a high degree of control of the pressure drop on the material sample. This is advantageous because the absolute pressure drop on the chromatographic material is a limiting factor on an industrial scale. Specifically, the normal operating parameters of most commercially available pumps make a pressure drop of 2MPa the maximum allowable pressure drop. The inventors have found that surprisingly high flow rates of materials can be passed through the material produced according to the present invention at a pressure drop of 2Mpa. Therefore, when the liquid phase passes through a functionalized polymer chromatographic medium with a thickness of 0.05 to 10mm at a flow rate of 1 to 640 times the volume of film per minute, the pressure drop on the medium is usually less than 2MPa. This can be determined using standard means, such as the AKTA protein purification system. This is a measure of the flow resistance of the material.

[0229] Typically, the pressure drop is less than 1 MPa, preferably less than 0.5 MPa.

[0230] Typically, the flow rate through the medium is between 1 and 60 membrane volumes per minute, preferably between 5 and 40.

[0231] The liquid phase of the medium is not particularly important. Typical liquid phases include standard buffers, such as Tris buffer, preferably 10 mM Tris.

[0232] Typically, the liquid phase is passed through a material having a thickness of 0.1 to 5 mm.

[0233] Preferably, when Tris buffer passes through a functionalized polymer chromatography medium having a thickness of 0.1 to 5 mm at a flow rate of 1 to 60 times the membrane volume per minute, the pressure drop across the medium is less than 1 MPa.

[0234] More preferably, when 10 mM Tris passes through a functionalized polymer chromatography medium having a thickness of 0.1 to 5 mm at a flow rate of 5 to 40 membrane volumes per minute, the pressure drop across the medium is less than 0.5 MPa.

[0235] The combination of grafting and functionalization steps allows to achieve materials with high dynamic binding capacity (DBC). Thus, typically, the functionalized chromatographic material has a DBC of 10 to 210 mg / mL (10% penetration), preferably 20 to 195 mg / mL (10% penetration), 30 to 180 mg / mL (10% penetration), 40 to 165 mg / mL (10% penetration), or 50 to 150 mg / mL (10% penetration). For materials wherein step (ii) involves polymerization of glycidol, typically the functionalized chromatographic material has a DBC of 50-150 mg / mL (10% penetration).

[0236] The DBC at 10% breakthrough can be determined by standard means, for example using an AKTA Pure system.

[0237] DBC at 10% penetration is usually determined by the following method:

[0238] 1) Passing a loading material (for anion exchange material, the loading material was 1 mg / mL BSA / 10 mM Tris (pH 8). For cation exchange material, the loading material was 1 mg / mL lysozyme / sodium acetate pH 4.7 10 mM) through the functionalized material contained in the reservoir of an AKTAPure system (GE Healthcare);

[0239] 2) loading the material at a determined membrane volume flow rate per minute (mV / min) until the concentration after the reservoir outlet exceeds 10% of the loading amount, as measured by a UV flow cell;

[0240] 3) The total amount of protein loaded onto the disc at 10% breakthrough was determined by analyzing the chromatograms in Unicorn software (GE Healthcare), taking into account the dead volume in the system and reservoir equipment.

[0241] The high dynamic binding capacity of the functionalized material of the present invention and the high possible flow rate of the functionalized material of the present invention can advantageously achieve high productivity. Therefore, typically, the productivity of the functionalized polymer chromatographic medium is 50 mg / mL / min to 75,000 mg / mL / min. Preferably, the productivity is 600 mg / ml / min or higher. More preferably, the productivity is 1200 mg / ml / min or higher. Even more preferably, the productivity is 2400 mg / ml / min or higher. The productivity can be 10,000 mg / ml / min or higher or even 15,000 mg / ml / min or higher, or 20,000 mg / ml / min or higher. Using glycidol polymerization, productivity of 10,000 mg / ml / min, 15,000 mg / ml / min or 20,000 mg / ml / min or higher can be achieved.

[0242] As used herein, the productivity of a material is determined by how much material can be loaded onto an adsorbent material per unit volume of material per unit time. In practice, this is determined as DBC (10% breakthrough) divided by the residence time of the loaded material in the reservoir. The residence time can in turn be determined by the flow rate of the material through the reservoir.

[0243] As discussed above, the present inventors have thoroughly investigated the relationship between the grafting step (ii) and the functionalisation step (iii) and have found that by separating these steps from each other a highly optimised material can be produced.

[0244] Thus, in certain embodiments, the grafting step (ii) has been found to have the effect of increasing the dynamic binding capacity (DBC) of the functionalized polymeric chromatography medium. Thus, for two materials of a given ligand group density, it has been found that the material with the higher degree of grafting generally has the higher DBC. This is particularly the case for materials functionalized with positively charged groups - i.e. when the functionalized chromatography material is suitable for use in anion exchange chromatography.

[0245] Thus, in one embodiment, the present invention provides a method for preparing a functionalized polymer chromatography medium, the method comprising:

[0246] (i) providing a substrate formed of one or more polymer nanofibers as defined herein,

[0247] (ii) grafting one or more neutral polymer chains from a substrate as defined herein, and

[0248] (iii) contacting the grafted product with an agent as defined herein which functionalizes the product of step (ii) into a chromatographic medium as defined herein, wherein the grafting step (ii) comprises contacting a plurality of compounds of the formula Compounds and / or enantiomers thereof and / or derivatives thereof of formula (I) and / or enantiomers and / or diastereomers thereof:

[0249] ,

[0250] reacting with one or more functional groups present on the nanofiber substrate, wherein R1, R2, R3, R4 and R5 may be the same or different and are selected from H, halogen, C1-C4 alkyl or C1-C4 alkoxy, with the proviso that at least one of R1, R2, R3, R4 or R5 is not hydrogen, and having the effect of increasing the DBC of the functionalized polymeric chromatographic material.

[0251] In other embodiments, typically materials functionalized with negatively charged groups - i.e. when the functionalized chromatography material is suitable for use in cation exchange chromatography, it has been found that varying the amount of functionalizing agent used in step (iii) of the claimed method has only a small effect on the charge density, but when combined with the grafting step, a significant increase in binding capacity can be achieved for the same degree of functionalization. Again, it is therefore clear that the combination of the grafting and functionalization steps has a positive impact on DBC.

[0252] Typically, the functionalized chromatography medium is a functionalized cellulose chromatography medium and the substrate is formed from one or more cellulose acetate nanofibers, between steps (i) and (ii), the cellulose acetate is treated to convert it into cellulose, the step of grafting one or more neutral polymer chains from the substrate involves glycidol polymerization, and the grafted product is contacted with a reagent selected from the following: glycidyl trimethylammonium chloride (GMAC); 1,4-butane sultone; sodium chloroacetate; NaIO4, followed by protein A; divinyl sulfone, followed by protein A; allyl glycidyl ether, followed by a halohydrin forming reagent (e.g., N-bromosuccinamide), followed by protein A; or allyl glycidyl ether, followed by an epoxide forming reagent, followed by protein A.

[0253] Alternatively, the functionalized chromatography medium is a functionalized cellulose chromatography medium and the substrate is formed from one or more cellulose acetate nanofibers, the grafting step comprises both conversion of cellulose acetate to cellulose and grafting of one or more neutral polymer chains onto the resulting cellulose substrate via glycidol polymerization, the grafted product being contacted with a reagent selected from the group consisting of: glycidyltrimethylammonium chloride (GMAC); 1,4-butanesultone; sodium chloroacetate; NaIO4, followed by protein A; divinyl sulfone, followed by protein A; allyl glycidyl ether, followed by a halohydrin forming reagent (e.g., N-bromosuccinamide), followed by protein A; or allyl glycidyl ether, followed by an epoxide forming reagent, followed by protein A.

[0254] In a preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatographic medium, the method comprising:

[0255] (i) providing a substrate formed of one or more cellulose acetate nanofibers, treating the cellulose acetate to convert it into cellulose,

[0256] (ii) grafting one or more neutral polymer chains, as defined herein, from the resulting cellulose substrate, and

[0257] (iii) contacting the grafted product with a reagent as defined herein which functionalizes the product of step (ii) into a chromatographic medium as defined herein,

[0258] wherein the grafting step (ii) comprises grafting a plurality of formula The compound and / or its enantiomer reacts with one or more functional groups present on the nanofiber substrate.

[0259] Typically, in this preferred embodiment, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0260] Preferably, in this preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse contact points between the nanofibers of adjacent sheets.

[0261] In an alternative preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0262] (i) providing a substrate formed of one or more cellulose acetate nanofibers,

[0263] (ii) subjecting the substrate to conditions under which cellulose acetate is converted to cellulose and subsequently one or more neutral polymer chains are grafted onto the resulting cellulose substrate, and

[0264] (iii) contacting the grafted product with a reagent as defined herein which functionalizes the product of step (ii) into a chromatographic medium as defined herein,

[0265] wherein the grafting step (ii) comprises grafting a plurality of formula The compound and / or its enantiomer reacts with one or more functional groups present on the nanofiber substrate.

[0266] Typically, in this alternative preferred embodiment, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0267] Preferably, in this alternative preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse contact points between the nanofibers of adjacent sheets.

[0268] In a more preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0269] (i) providing a substrate formed of one or more cellulose acetate nanofibers, treating the cellulose acetate to convert it into cellulose,

[0270] (ii) By making multiple The compound and / or its enantiomer reacts with one or more hydroxyl groups present on the substrate to graft one or more polymer chains from the resulting cellulose substrate, and

[0271] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) as a chromatographic medium as defined herein.

[0272] Typically, in this more preferred embodiment, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0273] Preferably, in this more preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0274] In an alternative more preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0275] (i) providing a substrate formed of one or more cellulose acetate nanofibers,

[0276] (ii) subjecting the substrate to a reaction wherein the cellulose acetate is converted to cellulose and subsequently one or more neutral polymer chains are reacted through a plurality of formulas The compound and / or its enantiomers are grafted onto the resulting cellulose substrate by reacting with one or more hydroxyl groups present on the substrate under alkaline aqueous conditions, and

[0277] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) as a chromatographic medium as defined herein.

[0278] Typically, in this alternative more preferred embodiment, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0279] Preferably, in this alternative more preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse contact points between the nanofibers of adjacent sheets.

[0280] In an even more preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0281] (i) providing a substrate formed of one or more cellulose acetate nanofibers, treating the cellulose acetate to convert it into cellulose,

[0282] (ii) By making multiple The compound and / or its enantiomer reacts with one or more hydroxyl groups present on the substrate to graft one or more polymer chains from the resulting cellulose substrate, and

[0283] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) into a chromatographic medium suitable for use in an ion exchange or affinity chromatography method.

[0284] Typically, in even more preferred embodiments herein, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0285] Preferably, in this even more preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0286] In an alternative and even more preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0287] (i) providing a substrate formed of one or more cellulose acetate nanofibers,

[0288] (ii) subjecting the substrate to a reaction wherein the cellulose acetate is converted to cellulose and subsequently one or more neutral polymer chains are reacted through a plurality of formulas The compound and / or its enantiomers are grafted onto the resulting cellulose substrate by reacting with one or more hydroxyl groups present on the substrate under alkaline aqueous conditions, and

[0289] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) into a chromatographic medium suitable for use in an ion exchange or affinity chromatography method.

[0290] Typically, in this alternative even more preferred embodiment, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0291] Preferably, in this alternative even more preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0292] In a still more preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0293] (i) providing a substrate formed of one or more cellulose acetate nanofibers, treating the cellulose acetate to convert it into cellulose,

[0294] (ii) By making multiple The compound and its enantiomers react with one or more hydroxyl groups present on the substrate to graft one or more polymer chains from the resulting cellulose substrate, and

[0295] (iii) contacting the grafted product with a reagent that functionalizes the product of step (ii) into a chromatographic medium suitable for use in an ion exchange or affinity chromatography method, the reagent being glycidyl trimethylammonium chloride (GMAC); 1,4-butane sultone; sodium chloroacetate; NaIO4, followed by protein A; divinyl sulfone, followed by protein A; allyl glycidyl ether, followed by a halohydrin forming reagent (e.g., N-bromosuccinamide), followed by protein A; or allyl glycidyl ether, followed by an epoxide forming reagent, followed by protein A.

[0296] Typically, in this still more preferred embodiment, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0297] Preferably, in this still more preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0298] In an alternative yet more preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0299] (i) providing a substrate formed of one or more cellulose acetate nanofibers,

[0300] (ii) subjecting the substrate to a reaction wherein the cellulose acetate is converted to cellulose and subsequently one or more neutral polymer chains are reacted through a plurality of formulas The compound and / or its enantiomers are grafted onto the resulting cellulose substrate by reacting with one or more hydroxyl groups present on the substrate under alkaline aqueous conditions, and

[0301] (iii) contacting the grafted product with a reagent that functionalizes the product of step (ii) into a chromatographic medium suitable for use in an ion exchange or affinity chromatography method, the reagent being glycidyl trimethylammonium chloride (GMAC); 1,4-butane sultone; sodium chloroacetate; NaIO4, followed by protein A; divinyl sulfone, followed by protein A; allyl glycidyl ether, followed by a halohydrin forming reagent (e.g., N-bromosuccinamide), followed by protein A; or allyl glycidyl ether, followed by an epoxide forming reagent, followed by protein A.

[0302] Typically, in still more preferred embodiments of this alternative, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0303] Preferably, in this alternative yet more preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse contact points between the nanofibers of adjacent sheets.

[0304] In a still even more preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0305] (i) providing a substrate formed of one or more cellulose acetate nanofibers, treating the cellulose acetate to convert it into cellulose,

[0306] (ii) By making multiple The compound and its enantiomers react with one or more hydroxyl groups present on the substrate to graft one or more polymer chains from the resulting cellulose substrate, and

[0307] (iii) contacting the grafted product with a reagent that functionalizes the product of step (ii) into a chromatographic medium suitable for use in an ion exchange or affinity chromatography method, the reagent being divinyl sulfone followed by protein A; allyl glycidyl ether followed by a halohydrin forming reagent (e.g., N-bromosuccinamide) followed by protein A; or allyl glycidyl ether followed by an epoxide forming reagent followed by protein A.

[0308] Typically, in yet even more preferred embodiments herein, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0309] Preferably, in yet even more preferred embodiments herein, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0310] In an alternative yet even more preferred embodiment, the present invention provides a method for preparing a functionalized cellulose chromatography medium, the method comprising:

[0311] (i) providing a substrate formed of one or more cellulose acetate nanofibers,

[0312] (ii) subjecting the substrate to a reaction wherein the cellulose acetate is converted to cellulose and subsequently one or more neutral polymer chains are reacted through a plurality of formulas The compound and / or its enantiomers are grafted onto the resulting cellulose substrate by reacting with one or more hydroxyl groups present on the substrate under alkaline aqueous conditions, and

[0313] (iii) contacting the grafted product with a reagent that functionalizes the product of step (ii) into a chromatographic medium suitable for use in an ion exchange or affinity chromatography method, the reagent being divinyl sulfone followed by protein A; allyl glycidyl ether followed by a halohydrin forming reagent (e.g., N-bromosuccinamide) followed by protein A; or allyl glycidyl ether followed by an epoxide forming reagent followed by protein A.

[0314] Typically, in this alternative yet even more preferred embodiment, step (i) comprises providing a substrate formed from one or more nonwoven sheets, each nonwoven sheet comprising one or more cellulose acetate nanofibers.

[0315] Preferably, in this alternative yet even more preferred embodiment, step (i) comprises providing two or more nonwoven sheets stacked one upon another, each of said sheets comprising one or more cellulose acetate nanofibers, and simultaneously heating and pressing the stack of sheets to fuse the contact points between the nanofibers of adjacent sheets.

[0316] Chromatographic column of the present invention

[0317] The present invention also provides a chromatographic column. The chromatographic column of the present invention comprises one or more functionalized chromatographic media of the present invention. Alternatively, the chromatographic column of the present invention can be obtained by performing the method of the present invention and combining the product obtained thereby into the column.

[0318] There is also provided a method for preparing a chromatography column comprising carrying out the method of the invention and incorporating the product thus obtained into a column.

[0319] The chromatography column is typically suitable for use in a chromatography method, preferably a chromatography method as defined herein.

[0320] The chromatography column of the invention typically comprises one or more functionalised chromatography media of the invention in a reservoir, for example as defined above. The reservoir is typically cylindrical.

[0321] Typically, a chromatography column comprises one or more functionalized chromatography media of the invention stacked within a cylindrical reservoir.

[0322] Typically, the chromatography column comprises two or more functionalized chromatography media of the invention. Typically, the chromatography column comprises up to twenty functionalized chromatography media of the invention.

[0323] Typically, the chromatographic column also includes one or more frits in a generally cylindrical reservoir. Frits are well known to those skilled in the art and refer to rigid porous structures, typically porous structures of rigid metals, polymers or ceramics, preferably porous structures of rigid metals or ceramics. Frits are typically included in chromatographic columns to improve the flow distribution through the column and / or to support the one or more functionalized chromatographic media of the present invention. The pores in a typical frit have a diameter of 1 to 1000 μm, preferably 5 to 500 μm, more preferably 10 to 150 μm. Other suitable frit pore sizes include 1 to 20 μm, preferably 5 to 10 μm, more preferably 3 to 7 μm.

[0324] Typically, the column comprises two or more functionalized chromatographic media of the invention and one or more filters positioned between the functionalized chromatographic media.

[0325] In some embodiments, the column does not comprise a filter.

[0326] Instead of or in addition to the filter head, the column may comprise alternative spacer materials. Typical alternative spacer materials include nonwoven and woven materials.

[0327] Nonwoven polymer materials are known to those skilled in the art. Such nonwoven materials are porous, i.e. allow liquid to pass through, usually without significant pressure drop. Usually, the nonwoven polymer material is polypropylene. Usually, the surface density of the nonwoven material is 45-150gsm.

[0328] In some embodiments, the column comprises two or more functionalized chromatography media of the invention and one or more layers of nonwoven polymeric material as defined above, the one or more layers of nonwoven polymeric material being located between the functionalized chromatography media.

[0329] Woven materials are known to those skilled in the art. Such woven materials are porous, i.e. allow liquid to pass through, usually without significant pressure drop. Typically, the woven material is a woven polymer material, preferably woven polypropylene. Typically, the thickness of the woven material is less than 1 mm.

[0330] Typically, the chromatography column also comprises one or more inlet fluid distribution means and / or outlet fluid collection means. Such means are known to those skilled in the art.

[0331] Chromatographic method of the present invention

[0332] The invention also provides the use of a functionalized chromatography medium of the invention or a chromatography column of the invention in a chromatography method, in particular in a chromatography method as defined herein.

[0333] The present invention also provides a method for separating one or more biomolecules from a mobile phase, the method comprising contacting one or more biomolecules in the mobile phase with a functionalized chromatographic medium of the present invention or a chromatographic column of the present invention. The chromatographic medium or chromatographic column preferentially binds to the one or more biomolecules in the mobile phase, usually preferentially over other components (e.g., other biomolecules) also present in the mobile phase. This can be performed according to conventional methods known for the binding stage of such chromatographic methods.

[0334] Typically, therefore, the chromatography method is an ion (anion or cation) exchange, affinity capture, hydrophobic interaction or mixed mode chromatography method.

[0335] Preferably, the chromatography method is an anion exchange chromatography method and the chromatography medium is functionalized with DEAE or Q; the chromatography method is a cation exchange chromatography method and the chromatography medium is functionalized with SP or CM; the chromatography method is an affinity capture chromatography method and the chromatography medium is functionalized with protein A; or the chromatography method is a hydrophobic interaction chromatography method and the chromatography medium is functionalized with phenyl.

[0336] Thus, the present invention provides a chromatography method comprising the above steps. Typically, the chromatography method is performed according to the chromatography method as defined above.

[0337] One of the advantageous findings of the present invention is that the functionalized chromatography material produced by the method of the present invention has a high binding capacity and can be operated at high flow rates. Thus, typically in the chromatography method of the present invention, the one or more biomolecules in the mobile phase are in contact with the functionalized chromatography medium for a period of one minute or less, preferably 50 seconds or less, more preferably 40 seconds or less, still more preferably 30 seconds or less, still more preferably 20 seconds or less, or even 15 seconds or less, 12 seconds or less, 10 seconds or less, 8 seconds or less, 6 seconds or less, 4 seconds or less, 2 seconds or less, 1.5 seconds or less, or even 1 second or less.

[0338] The chromatographic method generally includes a further step of recovering the one or more biomolecules from the functionalized chromatographic medium or chromatographic column. This step can generally be achieved by contacting the functionalized chromatographic medium or chromatographic column to which the one or more biomolecules are adsorbed with an elution buffer. This can be performed according to conventional methods known for the elution phase of such chromatographic methods. Thus, this method is generally a bind-elute chromatographic method.

[0339] Between the binding step and the elution step, the method may further comprise a step of washing the functionalized chromatography medium or chromatography column of the invention to which the one or more biomolecules are adsorbed. This washing step is performed to remove any components that are not bound to the functionalized chromatography medium or chromatography column. This can be performed according to conventional methods known for the washing stage of such chromatography methods.

[0340] After the elution step, the method may further comprise a step of regenerating the functionalized chromatographic medium or chromatographic column of the invention. Typically, this is achieved by contacting the functionalized chromatographic medium or chromatographic column from which the one or more biomolecules have been eluted with a buffer. This can be performed according to conventional methods known for the regeneration phase of such chromatographic methods.

[0341] Typically, the one or more biomolecules are selected from cells, proteins, polypeptides, antibodies, amino acids, viruses and nucleic acids, including, for example, recombinant proteins, monoclonal antibodies, viral vaccines, viral vectors, RNA, exosomes, cells and plasmid DNA.

[0342] The monoclonal antibody can be a multispecific antibody (e.g., a bispecific antibody) or a domain-deficient antibody. Preferably, the monoclonal antibody is a humanized antibody or a human antibody. The antigen-binding fragment of the monoclonal antibody can be used. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments, diabodies and single-chain antibodies.

[0343] Typically, the one or more biomolecules are one or more monoclonal antibodies, or proteins engineered to present sites with affinity for Protein A binding, and optionally the functionalized chromatographic medium contained in the column carries at least one Protein A ligand group.

[0344] Typically, the chromatographic method employs a simulated or actual moving bed system. Thus, typically, the method comprises introducing the one or more biomolecules in a mobile phase into one or more simulated or actual moving bed chromatography devices having a plurality of connected chromatography columns containing the functionalized chromatographic media of the invention as an adsorbent.

[0345] The chromatography method may be carried out using any known simulated or actual moving bed apparatus, provided that it comprises the functionalized chromatography medium of the present invention as adsorbent.

[0346] Simulated and actual moving bed chromatography is a known technique familiar to those skilled in the art. The operating principle involves the countercurrent movement of a liquid eluent phase and a solid adsorbent phase. This operation allows for minimal use of solvents, which makes the process economically viable. Such separation techniques have been applied in different fields, including the purification of biomolecules using membrane adsorbents.

[0347] A simulated moving bed system consists of a number of individual columns containing adsorbent, which are connected together in series. The eluent is passed through the columns in a first direction. The injection points of the feed and eluent and the collection points of the separated components in the system are periodically changed by a series of valves. The overall effect is to simulate the operation of a single column containing a moving bed of solid adsorbent. Thus, a simulated moving bed system consists of columns which contain fixed beds of solid adsorbent through which the eluent is passed, as in a conventional fixed bed system, but in a simulated moving bed system the operation simulates a continuous countercurrent moving bed.

[0348] An actual moving bed system is similar in operation to a simulated moving bed system. However, instead of changing the injection points of the feed mixture and eluent and the collection points of the separated components by a valve system, a series of adsorption units (i.e., columns) are physically moved relative to the feed and discharge points. Again, the operation simulates a continuous countercurrent moving bed.

[0349] The materials of the present invention are also suitable for use in methods of immobilized enzyme biocatalysis, metal removal and water treatment. Thus, the present invention provides an enzyme biocatalysis method involving the functionalized chromatographic material of the present invention. The present invention also provides a metal removal method involving the functionalized chromatographic material of the present invention. The present invention also provides a water treatment method involving the functionalized chromatographic material of the present invention. It should be understood that some minor modifications may be required to the methods and products of the present invention in order to make the products suitable for use in these methods. Example

[0350] The following examples illustrate the invention.

[0351] Materials and Equipment

[0352] Unless otherwise noted, all chemicals were from or available from companies such as Fisher Scientific, Sigma-Aldrich, FluoroChem, Repligen, and VWR.

[0353] Washing program

[0354] Washing Solution A

[0355] The reaction medium was replaced with an equal volume of deionized water and circulated for 1 hour. The rinsing procedure was repeated again. Finally, the material was treated with an equal volume of aqueous ethanol (H2O: ethanol 2: 1) and then removed from the reaction vessel.

[0356] Washing plan B

[0357] The reaction medium was replaced with an equal volume of deionized water and circulated for 1 hour. Thereafter, the wash medium was replaced with 0.01M HCl, circulated for 1 hour, and then replaced with 0.001M HCl and circulated for 1 hour. Finally, the medium was replaced with a 2:1 mixture of H2O: EtOH and circulated for 1 hour. The derivatized nanofibers were then removed from the reaction vessel.

[0358] Washing Solution C

[0359] The reaction medium was replaced with an equal volume of a 1:1 mixture of warm (60°C) deionized water: acetone, which was circulated for 30 minutes. The washing procedure was repeated two more times. Finally, the medium was replaced with a 2:1 mixture of H2O: EtOH, which was circulated for 1 hour. The derivatized nanofibers were then removed from the reaction vessel.

[0360] Washing program D

[0361] 2 liters of ultrapure water were pumped through the nanofiber material.

[0362] Washing Solution E

[0363] The reaction medium was replaced with an equal volume of 1:1 deionized water: EtOH and circulated for 1 hour. The washing procedure was repeated twice more. Finally, the medium was replaced with a 2:1 mixture of H2O: EtOH and circulated for 1 hour. The derivatized nanofibers were then removed from the reaction vessel. Finally, the medium was replaced with a 2:1 mixture of H2O: EtOH and circulated for 1 hour. The derivatized nanofibers were then removed from the reaction vessel.

[0364] Washing program F

[0365] The reaction medium was replaced with ultrapure deionized water. The nanofiber material was gently stirred in the clean water for 30 minutes. Thereafter, the washing medium was replaced and the washing cycle was repeated.

[0366] Washing program G

[0367] The reaction medium was poured off and the beaker was replenished with water until the pH of the effluent was neutral or slightly acidic.

[0368] Washing Scheme H (for halohydrin nanofibers)

[0369] The reaction medium was replaced with an equal volume of deionized water: 1: 1 mixture of acetone, which was circulated for 1 hour. Thereafter, the washing solution was renewed and the fibers were washed for another 1 hour while stirring vigorously. This process was repeated 3 times, followed by a final wash of 1 hour with deionized water. The derivatized halohydrin nanofibers were then taken out from the reaction vessel and prepared for the next step.

[0370] I. Preparation of Materials

[0371] Preparation Example 1

[0372] A cellulose acetate solution with a relative molecular mass of 29,000 g / mol was dissolved in a common solvent and then electrospun to produce fibers with a diameter between 300-600 nm. Optimized conditions for nanofiber preparation can be found in, for example, O. Hardick et al., J. Mater. Sci. 46 (2011) 3890, the entire contents of which are incorporated herein by reference. Approximately 20 g / m 2 Sheets of material are stacked and subjected to a combination of heat and pressure.

[0373] Example 1 - Glycidol / Trimethylammonium Chloride Functionalization

[0374] The nanofiber material was derivatized according to the protocol outlined below:

[0375]

[0376] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0377]

[0378] A cellulose acetate sheet (44×32 mm×150 mm) obtained according to the method of Preparation Example 1 was placed in a large beaker containing 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was stirred at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0379] Step (ii): Glycidol polymerization

[0380]

[0381] The material from (i) was suspended in 1 L of 0.5 M NaOH. The reaction medium was allowed to circulate for 15 minutes, then different amounts of glycidol (15 mL, 30 mL, 60 mL, 120 mL, 180 mL) were carefully added in one go. The reaction medium was circulated at room temperature for 16 hours, and the material was subsequently washed according to washing scheme B.

[0382] Step (iii): Derivatization with Glycidyltrimethylammonium Chloride

[0383]

[0384] The material obtained in step (ii) is usually suspended in 1 L of 0.5 M NaOH. The reaction medium is allowed to circulate for 15 minutes, and then glycidyl trimethylammonium chloride (25 mL, 50 mL, 100 mL and 200 mL) is added in one go. The reaction medium is recirculated for 16 hours at room temperature. The material is then washed according to washing scheme C.

[0385] The trimethylammonium chloride content is measured by the following method. 50 mg of the material is washed with 100 mL of 0.1 M HCl solution on a Buchner filter funnel, and then washed with 100 mL of 0.01 M HCl solution. The material is then placed in a drying oven at 75 ° C and dried to constant weight, then torn into small pieces and then placed in a 50 mL centrifuge tube. A small magnetic stirring bar and 15 mL of deionized water and approximately 1 mL (added by a rubber-tipped pipette) of potassium chromate solution are then added, which turns the mixture yellow. The mixture is stirred vigorously for 20 minutes, and then titrated with 0.1 M silver nitrate. The endpoint of the titration is identified by the color changing from a clear yellow to a turbid brown.

[0386] The trimethylammonium chloride content (μmol / g) was calculated as micromoles of silver nitrate added to reach the endpoint per gram of nanofiber material used in the titration.

[0387] Example 2 - Glycidol / S functionalization

[0388] The nanofiber material was derivatized according to the protocol outlined below:

[0389]

[0390] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0391]

[0392] A cellulose acetate sheet (44×32 mm×150 mm) obtained according to the method of Preparation Example 1 was suspended in 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was circulated at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0393] Step (ii): Glycidol derivatization

[0394]

[0395] The material from (i) was suspended in 1 L of 0.5 M NaOH. The reaction medium was allowed to circulate for 15 minutes, then different amounts of glycidol (15 mL, 30 mL, 120 mL, 180 mL) were carefully added in one go. The reaction medium was circulated at room temperature for 16 hours, and the material was subsequently washed according to washing scheme B.

[0396] Step (iii): Derivatization of 1,4-butanesultone

[0397]

[0398] The reaction medium consisting of 150 ml of 1M NaOH (aqueous solution) is heated to 53° C. The glycidyl-functionalized material obtained in step (ii) is suspended therein together with 1,4-butanesultone (6 ml, 53° C., 58.6 mmol). The reaction medium is stirred at 60° C. for 15 minutes, 30 minutes and 60 minutes. The material is then washed according to washing scheme B.

[0399] The sulfonic acid (S) content (µmol / g) of the material was calculated by titration. The dried material from (iii) was washed with 0.1M HCL and 0.01M HCl. The material was then oven dried and weighed. After washing, the molarity of the material was determined from the amount of NaOH added to reach pH 7.

[0400] Example 3 - Glycidol / Carboxymethyl (CM) Functionalization

[0401] The nanofiber material was derivatized according to the protocol outlined below:

[0402]

[0403] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0404]

[0405] A cellulose acetate sheet (44×32 mm×150 mm) obtained according to the method of Preparation Example 1 was placed in a large beaker containing 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was stirred at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0406] Step (ii): Glycidol polymerization

[0407]

[0408] The material from (i) was suspended in 1 L of 0.5 M NaOH. The reaction medium was allowed to circulate for 15 minutes, then different amounts of glycidol (15 mL, 30 mL, 60 mL, 120 mL, 180 mL) were carefully added in one go. The reaction medium was circulated at room temperature for 16 hours, and the material was subsequently washed according to washing scheme B.

[0409] Step (iii): Carboxymethyl (CM) derivatization

[0410]

[0411] The material from (ii) was suspended in water (66 mL) at 62°C. A solution of sodium chloroacetate (12.8 g) in water (27.5 mL) was freshly prepared. A solution of KOH (6.2 g) in water (27.5 mL) was also freshly prepared. Both solutions were added to the reaction at a rate of 13.75 mL / hour while the temperature was maintained at 62°C. The reactants were stirred vigorously at 62°C for a total of 4 hours. After 4 hours at 62°C, the reaction medium was removed and the nanofiber material was washed under a continuous stream of water until the pH of the effluent was neutral. The beaker was then stirred in 0.01 M aqueous hydrochloric acid until a stable pH of 2-3 was reached.

[0412] Reference Example 1 - ATRP functionalization to provide dimethylamino-derivatized nanofiber materials

[0413] The nanofiber material was derivatized according to the protocol outlined below:

[0414]

[0415] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0416]

[0417] A cellulose acetate sheet (44×32 mm×150 mm) obtained according to the method of Preparation Example 1 was placed in a large beaker containing 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was stirred at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0418] Step (ii): Derivatization with divinyl sulfone

[0419]

[0420] The material obtained in step (i) was suspended in K2CO3 solution (24.4 g, 176.54 mmol K2CO3 in 275 mL H2O) and acetonitrile (75 mL). The mixture was stirred for 15 minutes, and then divinyl sulfone (50 mL, 498.1 mmol) was added dropwise over 2.5 hours. After the addition of divinyl sulfone, the reaction was stirred for another 1.5 hours. Thereafter, the nanofiber material was washed according to washing scheme C.

[0421] Step (iii)

[0422]

[0423] The material obtained in step (ii) was suspended in 375 mL of H2O containing ethylenediamine (125 mL, 1870 mmol) or 2-mercaptoethylamine hydrochloride (37.5 g, 167 mmol). The mixture was stirred at room temperature overnight. Thereafter, the derivatized nanofibrous material was washed according to washing scheme A.

[0424] Step (iv): Derivatization with α-bromoisobutyryl bromide:

[0425]

[0426] The dried material obtained in step (iii) is placed in a centrifuge tube and 2 mL of acetonitrile and 0.4 mL (3.24 mmol) of α-bromoisobutyryl bromide are added. The tube is gently stirred on an orbital shaker at room temperature for 5 minutes, followed by dropwise addition of triethylamine (0.4 ml, 2.86 mmol). The reaction mixture is stirred for 1 hour. Thereafter, the derivatized nanofiber material is washed according to washing scheme A.

[0427] Step (v): ATRP

[0428]

[0429] Typically, the derivatized nanofibrous material obtained in step (iv) is placed in a reaction vessel, to which 2.5 mL of saturated ascorbic acid, 0.33 ml of catalyst solution A and N-[3-(dimethylamino)propyl]methacrylamide (1.66 mL, 9.2 mmol) are added. The reaction mixture is gently agitated on an orbital shaker at room temperature for 4 hours. Thereafter, the derivatized nanofibrous material is washed according to washing scheme A.

[0430] Catalyst solution A

[0431] CuBr2 (0.3 g, 1.343 mmol) was dissolved in 10 ml H2O and 0.45 mL (1.650 mmol) 1,1,4,7,10,10-hexamethyltriethylenetetramine.

[0432] Example 4 - Glycidol-grafted, aldehyde-functionalized, protein A-coupled materials

[0433] The nanofiber material was derivatized according to the protocol outlined below:

[0434]

[0435] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0436]

[0437] A cellulose acetate sheet (44×32 mm×150 mm) obtained according to the method of Preparation Example 1 was placed in a large beaker containing 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was stirred at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0438] Step (ii): Glycidol polymerization

[0439]

[0440] The material from (i) is suspended in 1 L of 0.5 M NaOH. The reaction medium is allowed to circulate for 15 minutes and then 180 mL of glycidol are carefully added in one portion. The reaction medium is circulated at room temperature for 16 hours and the material is subsequently washed according to washing protocol B.

[0441] Step (iii): Oxidation to aldehyde

[0442]

[0443] The material from (ii) was suspended in 17.5 L of 0.05 M NaOAc buffer adjusted to pH 5.5. The reaction medium was circulated for 30 minutes and then NaIO4 (200 g, 0.94 mole, dissolved in 2 L of reaction medium) was added. The reaction medium was circulated for another 30 minutes. The material was then washed according to Washing Scheme C to provide an aldehyde functionalized material.

[0444] Step (iv): Protein A coupling

[0445] The material from step (iii) is added to 6 orifice plates. 2mL protein-A solution (rSPA, 50mg / ml protein A / deionized water) is added to each hole. Plate is gently stirred 1 hour on orbital oscillator. Thereafter, supernatant is removed and replaced with reducing buffer solution A (2.5ml per hole, to add 0.0762g NaCNBH3 to 10mL carbonate buffer, carbonate buffer is to add 0.0603g Na2CO3 (0.569mmol) and 0.337g NaHCO3 (4.012mmol) to 100mL deionized water) and stirred 15 minutes again. Thereafter, according to scheme D washing protein-A coupling material.

[0446] Example 5 - Glycidol-grafted, DVS-functionalized, Protein A-coupled Materials

[0447] The nanofiber material was derivatized according to the protocol outlined below:

[0448]

[0449] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0450]

[0451] A cellulose acetate sheet (44×32 mm×150 mm) obtained according to the method of Preparation Example 1 was placed in a large beaker containing 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was stirred at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0452] Step (ii): Glycidol polymerization

[0453]

[0454] The material from (i) is suspended in 1 L of 1 M NaOH. The reaction medium is allowed to circulate for 15 minutes and then 180 mL of glycidol are added in one portion. The reaction medium is circulated at room temperature for 16 hours and the material is subsequently washed according to washing protocol B.

[0455] Step (iii): Derivatization with divinyl sulfone

[0456]

[0457] The material from (ii) was suspended in a solution consisting of 550 mL H O and 150 mL acetonitrile in which K CO (48.8 g, 0.35 mol) was dissolved. The reaction medium was circulated for 15 minutes, then divinyl sulfone (100 ml, 0.86 mol) was added dropwise, after which the reaction medium was recirculated for 1.5 hours. The material was then washed according to washing scheme C.

[0458] Step (iv): Protein-A coupling:

[0459]

[0460] The material from step (iii) was suspended in 70 ml of Protein A solution (50 mg / mL Protein A suspension to which 668 mg (Na2CO3) and 58 mg NaHCO3 and NaOH were added to reach pH 11.1). The solution was circulated for 16 hours. The material was then washed according to Washing Protocol D.

[0461] Reference Example 2 - ATRP grafted, protein A coupled materials

[0462] The nanofiber material was derivatized according to the protocol outlined below:

[0463]

[0464] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0465]

[0466] Cellulose acetate sheets (5×65 cm×100 cm) were suspended in 17.5 L of 0.075 M NaOH (in 2:1 H2O:EtOH). The reaction medium was circulated at room temperature for 48 hours. The material was then washed according to washing protocol A.

[0467] Step (ii): α-bromoacetylation

[0468]

[0469] The dried material from (i) was suspended in 240 ml of tetrahydrofuran (THF) in which α-bromoisobutyryl bromide (6.24 mL, 50.4 mmol) was dissolved. The reaction mixture was cooled to 0°C and triethylamine (7.2 mL, 51.6 mmol) was added dropwise. The mixture was stirred at 0°C for 2 hours and then warmed to room temperature. The mixture was stirred at room temperature for another 16 hours. Thereafter, the derivatized nanofibrous material was washed according to washing scheme A.

[0470] Step (iii): ATRP polymerization

[0471]

[0472] 5 mL of acetone, 5 mL of saturated ascorbic acid, CuBr2 (30 mg, 0.134 mmol) and 11,4,7,10,10-hexamethyltriethylenetetramine (45 μL, 0.165 mmol) were added to the vessel. The material from (ii) was suspended in the reaction mixture along with divinyl sulfone (2.5 mL, 24.9 mmol). The reaction mixture was gently agitated using an orbital shaker at room temperature for 1 hour. Thereafter, the nanofiber material was washed according to Washing Scheme A.

[0473] Step (iv): Protein-A derivatization

[0474]

[0475] The 26 mm discs of material from step (iii) were placed in 6-well plates. 1 mL of protein-A solution (50 mg / mL solution as defined above) and 1 mL of carbonate buffer solution (as defined above) were added to each disc. The discs were gently agitated for 16 hours. Thereafter, the discs were washed using wash protocol D.

[0476] Reference Example 3 - Trimethylammonium chloride functionalization

[0477] The nanofiber material was derivatized according to the protocol outlined below:

[0478] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0479]

[0480] A cellulose acetate sheet (44×32 mm×150 mm) obtained according to the method of Preparation Example 1 was placed in a large beaker containing 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was circulated at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0481] Step (ii): Derivatization with Glycidyltrimethylammonium Chloride

[0482]

[0483] The material obtained from step (i) was suspended in 1 L of 0.5 M NaOH. The reaction medium was allowed to circulate for 15 minutes, and then glycidyl trimethylammonium chloride (25 mL, 50 mL, 100 mL) was added in one go. The reaction medium was recirculated at room temperature for 16 hours. The material was then washed according to washing scheme B.

[0484] The trimethylammonium chloride content is measured by the following method. 50 mg of the material is washed with 100 mL of 0.1 M HCl solution on a Buchner filter funnel, and then washed with 100 mL of 0.01 M HCl solution. The material is then placed in a drying oven at 75 ° C and dried to constant weight, then torn into small pieces and then placed in a 50 mL centrifuge tube. A small magnetic stirring bar and 15 mL of deionized water and approximately 1 mL (added by a rubber-tipped pipette) of potassium chromate solution are then added, which turns the mixture yellow. The mixture is stirred vigorously for 20 minutes, and then titrated with 0.1 M silver nitrate. The endpoint of the titration is identified by the color changing from a clear yellow to a turbid brown.

[0485] The trimethylammonium chloride content (μmol / g) was calculated as micromoles of silver nitrate added to reach the endpoint per gram of nanofiber material used in the titration.

[0486] Reference Example 4 - Glycidol Grafting

[0487] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0488]

[0489] A cellulose acetate sheet (44×32 mm×150 mm) obtained according to the method of Preparation Example 1 was placed in a large beaker containing 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was stirred at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0490] Step (ii): Glycidol polymerization

[0491]

[0492] The material from (i) was suspended in 1 L of 0.5 M NaOH. The reaction medium was allowed to circulate for 15 minutes, then different amounts of glycidol (15 mL, 30 mL, 60 mL, 120 mL, 180 mL) were carefully added in one go. The reaction medium was circulated at room temperature for 16 hours, and the material was subsequently washed according to washing scheme G.

[0493] Example 6 - Alternatives to Glycidyl-Grafted, DVS-Functionalized, Protein A-Conjugated Materials

[0494] The nanofiber material was derivatized according to the protocol outlined below:

[0495]

[0496] Protein=protein, the same below

[0497] Step (i): Glycidol polymerization and saponification

[0498] The glycidol polymerization and saponification of the CA nanofiber material was achieved by taking the CA nanofiber material (11×80mm×50mm) and suspending it in 1L of deionized water. The solvent was allowed to circulate for 3 hours and then 1L of deionized water was added again. After repeating this process 4 times, the nanofiber material was suspended in 350ml of 1M KOH. The reaction medium was allowed to circulate for 60 minutes and then different amounts of glycidol (100ml) were carefully added, of which 25% of the glycidol was added at once and the rest was added dropwise over 90 minutes. The reaction medium was circulated at room temperature for 4 hours and then the material was washed according to washing scheme B.

[0499] Step (ii): Derivatization with divinyl sulfone

[0500]

[0501] The material from (i) was suspended in a solution consisting of 550 mL H O and 150 mL acetonitrile in which K CO (48.8 g, 0.35 mole) was dissolved. The reaction medium was circulated for 15 minutes, then divinyl sulfone (100 ml, 0.86 mole) was added dropwise, after which the reaction medium was recirculated for 1.5 hours. The material was then washed according to washing scheme C.

[0502] Step (iii): Protein-A coupling:

[0503]

[0504] The material from step (ii) was suspended in 70 ml of Protein A solution (50 mg / mL Protein A suspension to which 668 mg (Na2CO3) and 58 mg NaHCO3 and NaOH were added to reach pH 11.1). The solution was circulated for 16 hours. The material was then washed according to Washing Protocol D.

[0505] Example 7 - Halohydrin Formation and Derivatization

[0506] The nanofiber material was derivatized according to the protocol outlined below:

[0507]

[0508] Step (i): Saponification of cellulose acetate (CA) to regenerated cellulose (RC)

[0509]

[0510] A cellulose acetate sheet (11×80 mm×50 mm) obtained according to the method of Preparation Example 1 was placed in a large beaker containing 5 L of 0.075 M sodium hydroxide solution (in 2:1 water:ethanol). The reaction mixture was stirred at room temperature for 48 hours. The material was then washed according to Washing Scheme A.

[0511] Step (ii): Glycidol polymerization

[0512]

[0513] The material from (i) was suspended in 350 1M KOH. The reaction medium was circulated for 60 minutes and then different amounts of glycidol (60 ml) were carefully added, 25% of which was added at once and the rest was added dropwise over 90 minutes. The reaction medium was circulated at room temperature for 4 hours and the material was subsequently washed according to washing scheme B.

[0514] Step (iii) Derivatization of Allyl Glycidyl Ether

[0515]

[0516] The material from (ii) was suspended in 1 L 1M KOH. Various amounts of allyl glycidyl ether (20, 30, 40, 50, 60, 70, 80, 90, 100 ml) were added by first adding 25% of allyl glycidyl ether and then adding the remainder dropwise over 90 minutes. The reaction was maintained by stirring at room temperature for 4 hours. Thereafter, the material was washed according to washing scheme B.

[0517] Step (iv): Halohydrin formation

[0518]

[0519] The material from (iii) was suspended in 1 L of 3:1 H2O:MeCN in which 25 g of N-bromosuccinimide was dissolved. The reaction medium was circulated through the material for 4 hours. Thereafter, the reaction medium was removed and the remaining material was washed according to washing scheme H.

[0520] Step (v): Protein immobilization

[0521]

[0522] The single strip of material from step (iv) was placed in a polyethylene pouch. 25 mL of Protein A solution (50 mg / mL Protein A suspension to which 668 mg (2CO3) and 58 mg NaHCO3 and NaOH were added to reach pH 11.1) was then added to the pouch. The pouch was sealed and the resulting mixture was slowly agitated on an orbital shaker for 16 hours. Thereafter, the derivatized material was removed from the pouch and washed according to Washing Protocol H.

[0523] Example 8 - Alternative Halohydrin Formation and Derivatization

[0524] The nanofiber material was derivatized according to the protocol outlined below:

[0525]

[0526] Step (i): Glycidol polymerization and saponification

[0527]

[0528] Glycidol polymerization and saponification of CA nanofiber material were achieved by taking CA nanofiber material (6×120mm×90mm) and suspending it in 1L of deionized water. The solvent was allowed to circulate for 3 hours and then 1L of deionized water was added again. After repeating this process 4 times, the nanofiber material was suspended in 350ml of 1M KOH. The reaction medium was allowed to circulate for 5 minutes and then 100mL of glycidol was carefully added, of which 25% of the glycidol was added at once and the rest was added dropwise over 90 minutes. The reaction medium was circulated at room temperature for 4 hours and then the material was washed according to washing scheme B.

[0529] Step (ii) Derivatization of Allyl Glycidyl Ether

[0530]

[0531] The material from (i) was suspended in 350 L 1M KOH. Various amounts of allyl glycidyl ether (20, 30, 40, 50, 60, 70, 80, 90, 100 ml) were added by first adding 25% of allyl glycidyl ether and then adding the remainder dropwise over 90 minutes. The reaction was maintained by stirring at room temperature for 6 hours. Thereafter, the material was washed according to washing scheme B.

[0532] The use of lower amounts of allyl glycidyl ether produces materials with particularly favorable flow properties. The use of higher amounts of allyl glycidyl ether produces materials with higher binding capacities.

[0533] Step (iii): Halohydrin formation

[0534]

[0535] The material from (ii) was suspended in 1 L of 3:1 H2O:MeCN in which 25 g of N-bromosuccinimide was dissolved. The reaction medium was circulated through the material for 4 hours. Thereafter, the reaction medium was removed and the remaining material was washed according to washing scheme H.

[0536] Step (iv): Protein immobilization

[0537]

[0538] The single strip of material from step (iii) was placed in a polyethylene pouch. To this pouch was then added 25 mL of a protein A solution (50 mg / mL protein A suspension to which 668 mg (NaCO) and 58 mg NaHCO and 1 M NaOH (to reach pH 11.1) were added). The pouch was sealed and the resulting mixture was slowly agitated on an orbital shaker for 16 hours. Thereafter, the derivatized material was removed from the pouch and washed according to wash protocol H.

[0539] II. Analytical methods

[0540] Example 9 - Determination of Dynamic Binding Capacity

[0541] The loading material was passed through the selected functionalized nanofiber disc contained in the reservoir on the AKTA Pure system (GE Healthcare). The material was loaded at a determined membrane volume flow rate per minute (mV / min) until the concentration after the reservoir outlet exceeded 10% of the loading amount, as determined by a UV flow unit. Taking into account the dead volume in the system and the reservoir equipment, the total amount of protein loaded onto the disc at 10% breakthrough was determined by analyzing the chromatogram in the Unicorn software (GE Healthcare).

[0542] For the anion exchange material, the loading material was 1 mg / mL BSA / 10 mM Tris (pH 8). For the cation exchange material, the loading material was 1 mg / mL lysozyme / sodium acetate pH 4.7 10 mM.

[0543] Example 10 - Determination of flow resistance

[0544] The pressure drop (∆P) across the selected functionalized nanofiber materials was determined using an AKTA Pure system (GE Healthcare). A buffer of 10 mM Tris (pH 8) was passed through the functionalized nanofiber disk contained in the reservoir. The flow rate at which the ∆ column pressure (∆P) was equal to 0.5 MPa was recorded.

[0545] III Analysis and comparison of specific materials

[0546] Example 11 - Charge density vs. trimethylammonium functionalization after adding a separate grafting step according to the invention The relationship between

[0547] Materials were prepared and tested according to Reference Example 3 to generate trimethylammonium functionality to enable the materials to act as anion exchangers. They were compared with grafted materials according to the invention prepared and tested according to Example 1.

[0548] The charge density obtained for the trimethylammonium functionalized material is shown in Figure 4 middle. Figure 4 The mean value of each data point in Figure 5 middle.

[0549] from Figure 4 and Figure 5 It is clearly seen that when a set amount of the functionalizing agent GMAC is used, increasing the amount of grafting does not significantly affect the charge density.

[0550] Reference Example 5 - Relationship between charge density and trimethylammonium functionalization

[0551] The material was prepared and tested according to Reference Example 3 to generate trimethylammonium functionality to enable the material to act as an anion exchanger. The results are shown in Figure 2 middle.

[0552] from Figure 2 It can be seen that increasing the amount of functionalizing agent (glycidyltrimethylammonium chloride) increases the charge density in the absence of a grafting step.

[0553] Example 12 - Dynamic Binding Capacity (DBC) and Trimethylammonium after adding a separate grafting step according to the present invention The relationship between functionalization

[0554] The method in Example 9 was used to determine the dynamic binding capacity (DBC) of the same material determined in Example 11.

[0555] The DBC diagram of the material of the present invention prepared in Example 1 is shown in Figure 6 middle. Figure 7 The DBC of these materials is compared with the DBC of the material which has not been subjected to the two-step grafting process of the present invention (ie the material of Reference Example 3).

[0556] It can be seen that the binding capacity of the grafted material produced according to the present invention is more than 4 times higher than that of the material without grafting. In contrast to the situation of the non-grafted material seen in Reference Example 6 (below), it is clear that for the grafted material, when the charge density remains constant, increasing the amount of grafting causes an increase in the binding capacity of the material.

[0557] Reference Example 6 - Charge Density vs. Dynamic Binding Capacity (DBC) for Trimethylammonium Chloride Material relation

[0558] Materials were made and tested according to Reference Example 3 to generate trimethylammonium functionality to enable the materials to act as anion exchangers. The dynamic binding capacity of these materials was determined according to Example 9.

[0559] The results are shown in Figure 3 middle.

[0560] As expected by some skilled artisans, Figure 3 It can be seen that increasing the amount of the functionalizing agent glycidyltrimethylammonium chloride increases the binding capacity in the absence of a grafting step.

[0561] Example 13 - Charge density vs. sulfonic acid (S) functionalization after adding a separate grafting step according to the present invention Relationship

[0562] The material of the present invention was given S functionality to enable the material to function as a cation exchange material and was tested as in Example 2.

[0563] The sulfonic acid content of the material was determined as described in Example 2. The results are shown in Figure 8 It can be observed that the grafting step rather than the functionalization step has a greater impact on the charge density of the material.

[0564] Example 14 - Dynamic Binding Capacity (DBC) vs. Sulfonic Acid (S) after adding a separate grafting step according to the present invention The relationship between functionalization

[0565] The dynamic binding capacity (DBC) of the material prepared according to Example 2 was determined using the method described in Example 9. The results are shown in Fig. 9 middle.

[0566] from Fig. 9 It can be seen that there is also a dependency between the grafting step and the functionalization step as seen in Example 13. These materials produced according to the present invention produced binding capacities between 47 and 207 mg / mL at a flow rate of 160 membrane volumes (mV) / min.

[0567] Example 15 - Dynamic Binding Capacity (DBC) and Carboxymethylation after adding a separate grafting step according to the present invention (CM) Relationship between functionalization

[0568] The materials of the present invention were given carboxymethyl (CM) functionality as in Example 3 to enable the materials to function as cation exchange materials. The dynamic binding capacity (DBC) of these materials relevant to the present invention was determined using the method in Example 9. The results are shown in Fig.10 middle.

[0569] from Fig.10 It can be seen that the material produced according to the present invention can achieve a binding capacity between 20 and 149 mg / mL at a flow rate of 160 mV / min.

[0570] Reference Example 7 - Binding Capacity of Dimethylamino Derivatized Materials Grafted Using ATRP

[0571] The materials according to the invention were given dimethylamino functionality as in Reference Example 1 to enable the materials to function as anion exchange membranes. The method in Example 9 was used to determine the dynamic binding capacity (DBC) of these materials according to the invention.

[0572] Example 16 - Relationship between polymer grafting degree and flow resistance at a set charge density

[0573] The method of Example 10 was used to determine the flow resistance of the same material measured in Example 11.

[0574] The results of this assay are shown in Fig.11 This shows that there is a clear relationship between the degree of polymer grafting of the charge functionalized material and the flow resistance. A clear relationship between charge density and flow resistance can also be seen.

[0575] Reference Example 8 - Determination of OH density after grafting

[0576] Take out the material sample produced according to Reference Example 4 and wash it with a large amount of water (using a Buchner funnel), then dry it to constant weight. Then weigh the sample and tear it into pieces and place it in a 50mL centrifuge tube. Add exactly 10mL of p-toluenesulfonyl isocyanate (20mL of p-toluenesulfonyl isocyanate in 500mL of acetonitrile) to the tube. Add a small stirring bar to the centrifuge tube, which is then sealed, placed in a 60°C water bath and stirred for 60 minutes. Thereafter, dilute the sample with 20ml of H2O and stir for another 10 minutes. Then add 40mL of isopropanol, stir the sample for another 10 minutes, and then titrate using 0.481M Bu4NOH on an automatic titrator (titration using method CA OH).

[0577] Two inflection points are observed in the titration curve. The first occurs at approximately pH 5.5 (VEP1) and the second occurs at approximately pH 9.5 (VEP2). From this the amount of free OH present in the sample is calculated:

[0578] OHV = ((VEP2-VEP1*f*c(TBAH)*MA)) / ms

[0579] OHV: Hydroxyl value of the sample (KOH / g sample)

[0580] VEP1: Titrant consumption up to the first equivalence point (mL)

[0581] VEP2: Titrant consumption until the second equivalence point (mL)

[0582] C(TBOAH): concentration of tetrabutylammonium chloride (mol / L)

[0583] F: correction factor (titer), unitless

[0584] Ma: molecular weight of KOH; here 56.11 g / mol

[0585] Ms: Sample size (g)

[0586] Analysis of the material produced according to Reference Example 5 showed that, for this particular substrate and grafting method, varying the amount of glycidol reagent produced materials with -OH group densities as high as 6000 μmol / g. Fig.13 Shown in.

[0587] Reference Example 9 - Effect of Adding Polymer Grafting on Flow Resistance of Uncharged Materials

[0588] The materials were treated with different amounts of glycidol as in Reference Example 4, and the flow resistance of each material (and the ungrafted RC material) was measured using the determination method described in Example 10. The results are shown in Fig.12 middle.

[0589] from Fig.12 It can be seen that for the uncharged material with different grafting degrees, there is no appreciable increase in pressure drop with respect to flow. It can therefore be concluded that there is no appreciable pore blockage in the material with increasing grafting degree. It can be clearly observed that once the material is functionalized with charged groups, it can be used as in Example 16 (above) and Fig.11 The relationship between grafting amount and flow resistance was reported in .

[0590] Example 17 - Productivity of Inventive Materials

[0591] The capacity (DBC) and productivity values ​​of several materials according to the invention were determined. The results obtained are listed in Table 1.

[0592] Table 1: Capacity and productivity of the claimed materials

[0593]

[0594] Comparative Example 1

[0595] Binding capacity, residence time and productivity values ​​for various commercially available porous bead materials were obtained from the manufacturers. The results obtained are listed in Table 2.

[0596] Table 2: Examples of porous bead capacity and productivity

[0597]

[0598] Comparative Example 2

[0599] Determination of the dynamic binding capacity as a function of residence time for A) Capto Q and bovine serum albumin (BSA) and B) Q Sepharose Fast Flow 9 The results are Figure 1 This shows that residence times below 2 minutes are not possible in these large-scale porous bead columns.

[0600] Comparative Example 3

[0601] The combined capacity, residence time and productivity values ​​for various commercially available membrane and monolith materials were determined from the manufacturer's data. The results obtained are listed in Table 3.

[0602] Table 3: Examples of membrane and monolith capacities and productivity

[0603]

[0604]

[0605]

[0606]

[0607] Other general aspects of the invention

[0608] 1. A method for preparing a functionalized polymer chromatography medium, the method comprising:

[0609] (i) providing a substrate formed of one or more polymer nanofibers,

[0610] (ii) grafting one or more neutral polymer chains from the substrate, and

[0611] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) into a chromatographic medium by introducing one or more charged groups onto the grafted product.

[0612] 2. The method according to aspect 1, wherein the amount of polymer introduced in step (ii) is between 500-60,000 μmol / g of grafted product.

[0613] 3. The method according to aspect 1 or 2, wherein the charge density of the product of step (iii) is between 100-2,000 μmol / g chromatographic medium.

[0614] 4. The method according to any one of the preceding aspects, wherein when the liquid phase passes through a functionalized polymer chromatography medium having a thickness of 0.05 to 10 mm at a flow rate of 1 to 640 membrane volumes per minute, the pressure drop across the medium is less than 2 MPa.

[0615] 5. The method according to any one of the preceding aspects, wherein the productivity of the functionalized polymer chromatography medium is from 50 mg / mL / min to 75,000 mg / mL / min.

[0616] 6. The method according to any one of the preceding aspects, wherein the productivity of the functionalized polymer chromatography medium is higher than 200 mg / ml / min.

[0617] 7. A method according to any one of the preceding aspects, wherein the grafting step (ii) has the effect of increasing the dynamic binding capacity (DBC) of the functionalized polymeric chromatography medium.

[0618] 8. The method according to any one of the preceding aspects, wherein grafting one or more neutral polymer chains from the substrate comprises growing one or more polymer chains from one or more functional groups present on the substrate, optionally in the presence of one or more catalysts.

[0619] 9. A method according to aspect 8, wherein the substrate is treated between steps (i) and (ii) to introduce the one or more functional groups, or the substrate is treated between steps (i) and (ii) to deprotect or activate any functional groups on the substrate, or the substrate is treated between steps (i) and (ii) to increase the number / density of functional groups on the substrate.

[0620] 10. The method according to aspect 8 or 9, wherein the functional group is a hydroxyl, amino or carboxylic acid group.

[0621] 11. The method according to any one of aspects 8 to 10, wherein growing one or more polymer chains comprises polymerizing a plurality of monomers from one or more functional groups present on the substrate, optionally in the presence of one or more catalysts.

[0622] 12. A method according to any one of aspects 8 to 11, wherein one or more of the polymer chains are branched.

[0623] 13. The method according to any one of aspects 8 to 12, wherein growing the polymer chain comprises polymerizing glycidol from one or more functional groups present on the substrate.

[0624] 14. The method according to any one of the preceding aspects, wherein the one or more polymer chains are one or more polyglycerol chains.

[0625] 15. A method according to any one of the preceding aspects, wherein step (ii) comprises making a plurality of formula The compound and its enantiomers react with one or more hydroxyl groups present on the nanofiber substrate.

[0626] 16. The method according to any one of the preceding aspects, wherein in step (iii) the reagent functionalizes the grafted product such that the resulting functionalized chromatography medium is suitable for use in an anion exchange chromatography method.

[0627] 17. The method according to aspect 16, wherein

[0628] - the chromatography method is a cation exchange method and the reagent functionalizes the chromatography medium with one or more charged groups including one or more carboxylate, sulfonate or phosphonate moieties; or

[0629] - The chromatography method is an anion exchange method and the reagent functionalizes the chromatography medium with one or more charged groups, including one or more quaternary amino or diethylamine moieties.

[0630] 18. The method according to any one of the preceding aspects, wherein the substrate is in the form of a film.

[0631] 19. The method according to any one of the preceding aspects, wherein the polymer is selected from cellulose, cellulose acetate, polysulfone, polyamide, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polyethylene oxide and mixtures thereof.

[0632] 20. The method according to any one of the preceding aspects, wherein the nanofibers have an average diameter of 10 nm to 1000 nm.

[0633] 21. A method according to any one of the preceding aspects, comprising

[0634] (i) providing a substrate formed of one or more cellulose nanofibers,

[0635] (ii) By making multiple The compound and its enantiomers react with one or more hydroxyl groups present on the substrate to graft one or more polyglycerol polymer chains from the substrate, and

[0636] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) into a chromatographic medium suitable for use in an ion exchange chromatography method.

[0637] 22. A functionalized chromatography medium obtainable by a method according to any of the preceding aspects.

[0638] 23. A method of preparing a chromatography column, the method comprising carrying out the method of any one of aspects 1 to 21 and combining the product obtained thereby into a column.

[0639] 24. A chromatography column, which (a) is obtainable by the method of aspect 23, or (b) comprises one or more functionalized chromatography media according to aspect 22.

[0640] 25. Use of the functionalized chromatography medium according to aspect 22 or the chromatography column according to aspect 24 in chromatography.

[0641] 26. A method of separating one or more biomolecules from a mobile phase, the method comprising contacting the one or more biomolecules in the mobile phase with the functionalized chromatography medium according to aspect 22 or the chromatography column according to aspect 24.

[0642] 27. The method according to aspect 26, wherein the one or more biomolecules in the mobile phase are contacted with the functionalized chromatographic medium for a period of one minute or less.

[0643] 28. A method for preparing a functionalized polymer chromatography medium, the method comprising

[0644] (i) providing a substrate formed of one or more polymer nanofibers,

[0645] (ii) grafting one or more neutral polymer chains from the substrate, and

[0646] (iii) contacting the grafted product with a reagent which functionalizes the product of step (ii) as a chromatographic medium.

[0647] 29. The method according to aspect 28, wherein the amount of polymer introduced in step (ii) is between 500-60,000 μmol / g of grafted product.

[0648] 30. The method according to aspect 28 or 29, wherein the step of contacting the grafted product with a reagent introduces one or more ligand groups into the grafted product, which renders the functionalized product comprising the one or more ligand groups suitable for use as a chromatographic medium.

[0649] 31. A method according to aspect 30, wherein the density of said ligand groups in the product of step (iii) is between 100 and 2,500 μmol / g chromatographic medium.

[0650] 32. The method according to any one of aspects 28 to 31, wherein when the liquid phase passes through the functionalized polymer chromatography medium having a thickness of 0.05 to 10 mm at a flow rate of 1 to 640 membrane volumes per minute, the pressure drop across the medium is less than 2 MPa.

[0651] 33. The method according to any one of aspects 28 to 32, wherein the productivity of the functionalized polymer chromatography medium is from 50 mg / mL / min to 75,000 mg / mL / min.

[0652] 34. The method according to any one of aspects 28 to 33, wherein the productivity of the functionalized polymer chromatography medium is higher than 200 mg / ml / min.

[0653] 35. A method according to any one of aspects 28 to 34, wherein the grafting step (ii) has the effect of increasing the dynamic binding capacity (DBC) of the functionalised polymeric chromatography medium.

[0654] 36. The method according to any one of aspects 28 to 35, wherein grafting one or more neutral polymer chains from the substrate comprises growing one or more polymer chains from one or more functional groups present on the substrate, optionally in the presence of one or more catalysts.

[0655] 37. A method according to aspect 36, wherein the substrate is treated between steps (i) and (ii) to introduce the one or more functional groups, or the substrate is treated between steps (i) and (ii) to deprotect or activate any functional groups on the substrate, or the substrate is treated between steps (i) and (ii) to increase the number / density of functional groups on the substrate.

[0656] 38. The method according to aspect 36 or 37, wherein the functional group is a hydroxyl, amino or carboxylic acid group.

[0657] 39. The method according to any one of aspects 36 to 38, wherein growing one or more polymer chains comprises polymerizing a plurality of monomers from one or more functional groups present on the substrate, optionally in the presence of one or more catalysts.

[0658] 40. A method according to any one of aspects 36 to 39, wherein one or more of the polymer chains are branched.

[0659] 41. The method according to any one of aspects 36 to 40, wherein growing the polymer chain comprises polymerizing glycidol from one or more functional groups present on the substrate.

[0660] 42. The method according to any one of aspects 28 to 41, wherein the one or more polymer chains are one or more polyglycerol chains.

[0661] 43. A method according to any one of aspects 28 to 42, wherein step (ii) comprises making a plurality of formula The compound and its enantiomers react with one or more hydroxyl groups present on the nanofiber substrate.

[0662] 44. A method according to any one of aspects 28 to 43, wherein in step (iii) the reagent functionalizes the grafted product such that the resulting functionalized chromatography medium is suitable for use in a chromatography method selected from ion exchange, affinity capture, hydrophobic interaction and mixed mode methods.

[0663] 45. A method according to aspect 44, wherein

[0664] - the chromatography method is a cation exchange method, the reagent functionalizing the chromatography medium with one or more charged groups, the charged groups comprising one or more carboxylate, sulfonate or phosphonate moieties;

[0665] - the chromatography method is an anion exchange method, the reagent functionalizing the chromatography medium with one or more charged groups, the charged groups comprising one or more quaternary amino or diethylamine moieties;

[0666] - the chromatography method is affinity capture chromatography, the reagent functionalizing the chromatography medium with one or more proteins, peptides, antibodies or fragments thereof, dyes, histidine groups or groups containing metal cations;

[0667] - the chromatography method is hydrophobic interaction chromatography and the reagent functionalizes the chromatography medium with one or more propyl, butyl, phenyl or octyl groups; or

[0668] - The chromatography method is a mixed mode chromatography method and the reagent functionalizes the chromatography medium with one or more MEP, octylamine, N-benzylmethylethanolamine or N-benzoyl-homocysteine ​​groups.

[0669] 46. ​​The method according to any one of aspects 28 to 45, wherein in step (iii), the reagent functionalizes the grafted product with one or more Protein A molecules.

[0670] 47. A method according to any one of aspects 28 to 46, wherein the substrate is in the form of a film.

[0671] 48. The method according to any one of aspects 28 to 47, wherein the polymer is selected from cellulose, cellulose acetate, polysulfone, polyamide, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polyethylene oxide and mixtures thereof.

[0672] 49. A method according to any one of aspects 28 to 48, wherein the nanofibers have an average diameter of 10 nm to 1000 nm.

[0673] 50. A method according to any one of aspects 28 to 49, comprising

[0674] (i) providing a substrate formed of one or more cellulose nanofibers,

[0675] (ii) By making multiple The compound and its enantiomers react with one or more hydroxyl groups present on the substrate to graft one or more polyglycerol polymer chains from the substrate, and

[0676] (iii) contacting the grafted product with an agent which functionalizes the product of step (ii) into a chromatography medium suitable for use in a chromatography method selected from the group consisting of ion exchange, affinity capture, hydrophobic interaction and mixed mode methods.

[0677] 51. A functionalized chromatography medium obtainable by a method according to any one of aspects 28 to 50.

[0678] 52. A method of preparing a chromatography column, the method comprising carrying out the method of any one of aspects 28 to 50 and combining the product obtained thereby into a column.

[0679] 53. A chromatography column, which (a) is obtainable by the method of aspect 52, or (b) comprises one or more functionalized chromatography media according to aspect 51.

[0680] 54. Use of the functionalized chromatography medium according to aspect 51 or the chromatography column according to aspect 53 in chromatography.

[0681] 55. A method of separating one or more biomolecules from a mobile phase, the method comprising contacting the one or more biomolecules in the mobile phase with a functionalized chromatography medium according to aspect 51 or a chromatography column according to aspect 53.

[0682] 56. The method according to aspect 55, wherein the one or more biomolecules in the mobile phase are contacted with the functionalized chromatographic medium for a period of one minute or less.

[0683] 57. A method according to aspect 55 or aspect 56, wherein the one or more biomolecules are one or more monoclonal antibodies, or proteins engineered to present sites with affinity for protein A binding, and the functionalized chromatographic medium carries at least one protein A ligand group.

[0684] 58. A method according to any one of aspects 28 to 50, wherein step (iii) of the method comprises contacting the grafted product with allyl glycidyl ether and a halohydrin forming agent.

[0685] 59. A method according to any one of aspects 28 to 50, wherein in step (iii):

[0686] (a) contacting the grafted product with allyl glycidyl ether;

[0687] (b) treating the product of step (a) with a halohydrin-forming reagent; and

[0688] (c) contacting the product of step (b) with Protein A.

[0689] 60. A method according to any one of aspects 28 to 50, wherein step (iii) comprises contacting the grafted product with a plurality of reagents which together functionalize the product of step (ii) into a chromatographic medium, and wherein the plurality of reagents comprises a halohydrin forming reagent.

[0690] 61. A method according to aspect 60, wherein the grafted product is contacted with the plurality of reagents sequentially.

Claims

1. A functionalized chromatographic medium comprising (i) at least one nonwoven sheet comprising one or more polymer nanofibers, said nanofibers having an average diameter of 10 to 1000 nm, wherein said nanofibers comprise one or more functional groups from which one or more neutral polymer chains grow, (ii) the one or more neutral polymer chains grafted onto the one or more polymer nanofibers, wherein the polymer chains are polyglycerol chains comprising glycidol monomer residues, and (iii) at least one ligand group introduced onto said one or more polymer chains, wherein the functionalized product comprising said ligand group is suitable for use as said chromatographic medium.

2. The functionalized chromatographic medium according to claim 1, wherein the at least one ligand group is charged.

3. The functionalized chromatographic medium of claim 2, wherein the at least one ligand group is one or more charged groups comprising one or more quaternary amino or diethylamine moieties.

4. The functionalized chromatography medium of claim 2, wherein the at least one ligand group is one or more charged groups comprising one or more carboxylate, sulfonate or phosphonate moieties.

5. The functionalized chromatographic medium of claim 1, wherein the chromatographic medium is used for affinity capture chromatography and the at least one ligand group comprises one or more moieties selected from one or more proteins, peptides, antibodies or fragments thereof, dyes, histidine groups or groups containing metal cations.

6. A functionalized chromatographic medium comprising (i) at least one nonwoven sheet comprising one or more polymer nanofibers, said nanofibers having an average diameter of 10 to 1000 nm, wherein said nanofibers comprise one or more functional groups from which one or more neutral polymer chains grow, (ii) the one or more neutral polymer chains grafted onto the one or more polymer nanofibers, wherein the polymer chains are polyglycerol chains comprising glycidol monomer residues, and (iii) at least one ligand group introduced onto said one or more polymer chains, wherein the functionalized product comprising said ligand group is suitable for use as said chromatographic medium, Wherein the at least one ligand group comprises one or more propyl, butyl, phenyl or octyl groups.

7. A functionalized chromatographic medium comprising (i) at least one nonwoven sheet comprising one or more polymer nanofibers, said nanofibers having an average diameter of 10 to 1000 nm, wherein said nanofibers comprise one or more functional groups from which one or more neutral polymer chains grow, (ii) the one or more neutral polymer chains grafted onto the one or more polymer nanofibers, wherein the polymer chains are polyglycerol chains comprising glycidol monomer residues, and (iii) at least one ligand group introduced onto said one or more polymer chains, wherein the functionalized product comprising said ligand group is suitable for use as said chromatographic medium, The medium comprises at least one hydrophobic group and at least one positively charged or at least one negatively charged portion selected from one or more of 4-mercapto-ethyl-pyridine, octylamine, N-benzylmethylethanolamine or N-benzoyl-homocysteine ​​groups.

8. The functionalized chromatography medium according to claim 7, wherein the medium comprises 4-mercapto-ethyl-pyridine groups and / or octylamine groups.

9. The functionalized chromatography medium according to any one of claims 1 to 8, wherein the polymer nanofibers have an average diameter of 200-800 nm or 200-400 nm.

10. The functionalized chromatography medium of any one of claims 1-8, wherein the chromatography medium comprises a stack of a plurality of nonwoven sheets, and wherein the chromatography medium comprises fused contact points between polymer nanofibers of adjacent sheets.

11. The functionalized chromatography medium according to any one of claims 1 to 8, wherein one or more of the polymer chains are branched.

12. The functionalized chromatography medium according to any one of claims 1 to 8, wherein the functional groups on the polymer nanofibers from which the neutral polymer chains grow are selected from hydroxyl, amino and carboxyl groups.

13. The functionalized chromatography medium according to claim 12, wherein the functional groups are hydroxyl groups.

14. The functionalized chromatography medium according to any one of claims 1 to 8, wherein 500-60,000 µmol / g of glycidol is introduced onto the polymer nanofibers.

15. The functionalized chromatography medium according to any one of claims 1 to 8, wherein 1000-60,000 µmol / g of glycidol is introduced onto the polymer nanofibers.

16. The functionalized chromatography medium according to any one of claims 1 to 8, wherein 1000-2000 µmol / g of glycidol is introduced onto the polymer nanofibers.

17. The functionalized chromatography medium according to any one of claims 1 to 8, wherein 5000-10,000 µmol / g of glycidol is introduced onto the polymer nanofibers.

18. The functionalized chromatography medium according to any one of claims 1 to 8, wherein 30,000-60,000 µmol / g of glycidol is introduced onto the polymer nanofibers.

19. The functionalized chromatography medium according to any one of claims 1 to 8, wherein the density of the ligand groups is 100-2,000 μmol / g functionalized chromatography medium.

20. The functionalized chromatography medium according to any one of claims 1 to 8, wherein the density of the ligand groups is 300-1,500 μmol / g functionalized chromatography medium.

21. The functionalized chromatography medium according to any one of claims 1 to 8, wherein the density of the ligand groups is 500-1,200 μmol / g functionalized chromatography medium.

22. The functionalized chromatography medium according to any one of claims 1 to 8, wherein the density of the ligand groups is 100-500 μmol / g functionalized chromatography medium.

23. The functionalized chromatography medium of any one of claims 1 to 8, wherein the density of the ligand groups is 1,200-2,000 μmol / g functionalized chromatography medium.

24. The functionalized chromatography medium of any one of claims 1-8, wherein the polymer nanofibers comprise a polymer selected from the group consisting of polyamide, polyacrylic acid, polymethacrylic acid, polyacrylonitrile, polystyrene, polysulfone, polycaprolactone, collagen, chitosan, polyethylene oxide, agarose, acetate agarose, cellulose, cellulose acetate, and combinations thereof.

25. The functionalized chromatography medium of claim 24, wherein the polyamide is nylon.

26. The functionalized chromatography medium of claim 24, wherein the polysulfone is polyethersulfone.

27. The functionalized chromatography medium of claim 24, wherein the polymer nanofibers comprise a polymer selected from the group consisting of polyethersulfone, cellulose, cellulose acetate, and combinations thereof.

28. The functionalized chromatography medium of any one of claims 1-8, wherein the polymer nanofibers are formed from two or more different polymers.

29. A chromatography column comprising the functionalized chromatography medium of any one of claims 1-28.

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