Tablets containing fuctionalized agarose particles, method for producing them and use of the tablets

Abstract

Described is a material in form of a tablet as a three-dimensional body with a side length and / or diameter of more than 1 mm, comprising a plurality of agarose microparticles. Furthermore, a method for preparing this material and its use are described.

Description

[0001] TABLETS CONTAINING FUCTIONALIZED AGAROSE PARTICLES, METHOD FOR PRODUCING THEM AND USE OF THE TABLETS

[0002] Brief description of the disclosure

[0003] The present disclosure relates to a tablet containing activated agarose and in particular comprising a multitude of magnetic particles, a method for the production of these materials, as well as the use of these tablets for resuspension, functionalization of the agarose, and biological applications.

[0004] Background of the disclosure

[0005] For biological applications, especially in the field of proteins, agarose has become the accepted carrier medium. Reasons for this are the high binding capacity of the agarose network despite the large particle diameter, as well as the good processability in columns and with magnetic particles. For example, the use of Ni-NTA agarose has become established for the purification of His-tagged recombinant proteins. Other proteins can be purified with glutathione covalently attached to agarose, maltose-binding protein, or antibodies. Functional agarose is also commercially available to covalently attach biomolecules, in some cases in activated form for direct functionalization, without additional, often toxic, reagents, such as cyanogen bromide, l-ethyl-3 -(3 -dimethyl - aminopropyljcarbodiimide, or carbonyl diimidazole.

[0006] Materials used for this purpose are activated agarose particles, which can be functionalized by coupling of biomolecules, such as proteins and antibodies. These products are marketed in the form of suspensions in inert solvents and as lyophilized powders. The disadvantages in these cases are the difficult dosing for the manufacturer and user, as well as the limited stability to hydrolysis. Examples for such products are CNBr activated Sepharose 4B (Cytiva, Marlborough, USA) and NHS activated MagBeads (Cube Biotech, Monheim, Germany).

[0007] And agarose-based materials from suspensions also have some disadvantages. For example, agarose in suspension must not dry out. Many activated agaroses and magnetic particles must be stored in organic solvents, so that, for example, fire hazards and problems with air shipment can occur. Also, pipettes are needed for portioning, which can be clogged by the agarose particles.

[0008] Alternatively, activated particles, e.g., cyanogen bromide-activated agaroses, are prepared in lyophilized form. There are disadvantages here due to the dusty form and the time-consuming removal and portioning. In addition, the powder form favors accelerated hydrolysis, so that the storage stability of these products is limited.

[0009] The preparation of tablets for pharmaceutical purposes is common since more than hundred years. The mechanical tablet press has been invented in 1843 by Brockedon allowed the development of tablets, while in 1884 the industrial production of tablets was started by Burroughs Wellcome & Co.

[0010] JPH 029813 and JPS 5452719 describe the preparation of magnetic granules of up to 2 mm diameter by adding a magnetic material, a drug, such as the antitumor agent fluorouracil and a hydrophilic substance, such as hydroxypropyl cellulose for drug delivery purposes.

[0011] In DE-A-4406139 magnetic particles in form of Nd-Fe-B permanent magnets are coated with carnauba wax and pressed to tablets with drugs, such as Eudragit or Lavodopa. Optionally, auxiliary substances, such as polyethylene glycol, dibutyl phthalate, lactose, magnesium stearate, sodium chloride and 2-propanol are added. The application of these composite particles is drug delivery.

[0012] Another source of state of the art is US5,939,470. Here, magnetic organic polymer particles are coated with a soluble sugar, such as mannite and trehalose. After addition of water the mixture is spray-dried, resulting in the precipitation of small, tablet-like particles.

[0013] In US 6,274,386, a reagent preparation containing magnetic particles in tablet form is described. The magnetic core particles are comprising magnetite, which is coated by glass-like silica. Other possible components are stabilizing

[0014] Many reviews are describing the use of agarose for drug delivery. So, in Jiang et al., "Extraction, Modification and Biomedical Application of Agarose Hydrogels: A Review”, Mar. Drugs 2023, 21, 299. https: / / doi.org / 10.3390 / md21050299 agarose is used to encapsulate and release drugs.

[0015] Description of the disclosure

[0016] Subject matter of the present disclosure is a reagent preparation in the form of tablets. Moreover, the preparation of the tablet particles, is based on agarose and for example magnetic agarose. Agarose and magnetic agarose are also herein referred to as solid phase. In the present application, the following definitions are used.

[0017] Chelator: A chelator is consisting of one or more functional groups, which forms two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom.

[0018] Diagnostics: Diagnostics as defined in this patent application means the in vitro testing of human animal or plant for the purpose of medical care of the human, animal or plant from whom the specimen was taken. "Medical care” shall include, by way of example and not of limitation, diagnosis, prognosis, treatment, prevention, or monitoring the progress of any and all possible human, animal or plant disease (including infectious, genetic, traumatic, metabolic, degenerative, and neoplastic disease) as well as compatibility of donor and recipient with respect to tissue. The agarose microparticles in tablet form according to this disclosure can be used for diagnostics after resuspension in buffer by purification of the analytes according to standard protocols. As an example, antibody concentrations can be monitored with Protein A or antigen-functionalized agarose or magnetic beads, which are forming a tablet, according to the present disclosure. Other examples contain the synthesis of antibody- or enzyme functionalized agarose in tablet form by binding of the biomolecules onto activated agarose or binding of his-tagged biomolecules onto INDIGO Ni-Agarose or the like. Many other models for diagnostic use of agarose or magnetic beads according to this patent are feasible, such as pull-down assays [6] for protein-protein interactions.

[0019] Size: Most tablets produced according to the present disclosure are spheres, as produced by granulation, or cyclic cylinders, as produced by manual tablet compression. However, depending on the tableting method or tablet compression mold, other geometric shapes are also possible, such as cubes or cuboids, or particles with a non-de fined structure. The size, consisting of height, width, length and diameter of the tablets, ranges from about 1 mm to 5 cm. Suitable methods for measuring the diameter and height are a ruler and calipers, sieving with different sieve widths and collection of the individual fractions, or measuring in light microscopes using microscope slides with a millimeter scale.

[0020] The particle size distribution of agarose particles in the range from 10 pm to 500 pm can be easily determined by light microscopy using Neubauer counting chambers with a size scale. Alternatively, microscopes or cell counters that generate an automatic evaluation of the particle size distribution can be used. Agarose single particles, which are part of the tablets, normally have the form of spheres or ellipsoids, so size of agarose single particles means diameter of the beads.

[0021] Powder: According to Wikipedia a powder is a dry, bulk solid composed of many very fine particles that may flow freely when shaken or tilted. Powders are a special sub-class of granular materials, although the terms powder and granular are sometimes used to distinguish separate classes of material. In particular, powders refer to those granular materials that have the finer grain sizes, and that therefore have a greater tendency to form clumps when flowing.

[0022] According to Whitten et al., [7] a covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms, when they share electrons, is known as covalent bonding. [7]

[0023] Iminodiacetic acid, coupled onto solid phase (formula 1):

[0024] With X: Solid phase, functionalization could be carboxy before coupling (amide after coupling), epoxy (amine), halide (amine)

[0025] Nitrilotriacetic acid (NTA) coupled onto solid phase (formula 2):

[0026] With X: Solid phase, functionalization could be carboxy before coupling (amide after coupling), epoxy (amine), halide (amine) Ethylenediamine tetraacetic acid (EDTA) coupled onto solid phase (formula 3):

[0027] With X: Solid phase, functionalization could be carboxy before coupling (amide after coupling), epoxy (amine), halide (amine)

[0028] Chelator, scaffold, EDTA (formula 4):

[0029] With X: Solid phase, functionalization could be carboxy before coupling (amide after coupling), epoxy (amine), halide (amine), m: 0-3, n: 2-5

[0030] Chelator, scaffold, EDTA chains (formula 5):

[0031] With X: Solid phase, functionalization could be carboxy before coupling (amide after coupling), epoxy (amine), halide (amine), m: 0-3, n: 2-5, o, p: 1-8

[0032] According to the present disclosure, tablets with a diameter and / or a side length of at least 1 mm and for example up to 5 cm, preferably 2 mm to 2 cm, and most preferably 2 mm to 5 mm are prepared. The form is preferably cubes, spheres or cylindrical bodies, but also other geometrical forms and irregular particles, depending of the production method. The agarose particles are 2% to 10% agarose microparticles with a diameter in aqueous suspension of 5 pm to 500 pm, preferably 10 pm to 300 pm, most preferably 25 to 200 pm. These particles are dried and can be regenerated, when the tablet is resuspended in aqueous media.

[0033] According to the present disclosure, the tablets contain a plurality of agarose particles, which have a diameter of 5 to 500 pm, preferably 20 to 150 pm, more preferably 25 to 100 pm, determined with a light microscope after resuspension in aqueous solutions. The agarose particles can be crosslinked, following the standard and known procedures with for example epichlorohydrin, 3- chloro propanediol, divinyl sulfone, and the like. According to the present disclosure, the material (in particular the agarose in tablet form as described in the present disclosure) may be comprised in a kit, in particular in a kit for a use selected from the group consisting of the purification of his-tagged proteins, purify proteins from a mixture of biomolecules, in diagnostics, to covalently bind biomolecules, to bind biotinylated biomolecules and to bind antibodies.

[0034] In one embodiment the agarose particles could be magnetic, with some magnetic particles inside, making the agarose beads magnetically attractable and usable for magnetic separation after resuspension. Examples for suitable magnetic materials are magnetite (FesCU), and maghemite (Fe2O3). Magnetic agarose-based materials can be obtained from Cube Biotech (Monheim, Germany), Qiagen (Hilden, Germany), and Cytiva (Marlborough, USA).

[0035] According to the present disclosure, among other ways, tablets can be produced via direct compression: This method involves a direct compression of powders or powder blends with a manual (Micro-Tec MTB8, Micro-Tec, Haarlem, Netherlands) or automatic tablet press (e.g., Prexima 80, IMA, Ozzano dell’emilia, Italy), yielding compact tablets with the size of 2 to 10 mm.

[0036] As an alternative, wet granulation can be used. By means of a binder, such as polyvinyl pyrrolidone, granules are formed and then compressed in a compression machine (e.g. FlexiTab XL from Synthegon, Waiblingen, Germany) in presence of a solvent, such as acetone, methanol, water, DMF to obtain mini tablets and tablets.

[0037] Dry granulation is a pharmaceutical manufacturing process that involves the compaction of powders into granules without the use of liquid binders or solvents. In this method, a powder blend is compacted by applying pressure, typically through a roller compactor (e.g., CCS220, Fitzpatrick, Waterloo, Canada), to create granules. The resulting granules can then be further processed into tablets or capsules. Dry granulation is often chosen when the use of liquids in the granulation process is impractical or undesirable, such as with moisture-sensitive or heat-labile substances.

[0038] In melt extrusion the pre-mixed powder is transferred into a melt-extruder (for instance Pharma mini HME micro compounder from Thermo Scientific, Waltham, USA, where the material forms extrudates, which are milled and sieved to obtain tablets.

[0039] Another procedure is spray-drying, where the beads are used in an aqueous or organic suspension, preferred at 5-10%, and spray-dried with a spray dryer, such as a Biichi Mini Spray Dryer B-290 (Biichi, Switzerland), and the dried particles are collected in the glass sample collector. In order to obtain a resulting particle size of more than 1 mm diameter, an additional granulation step is mandatory.

[0040] Alternatively, agarose in magnetic or non-magnetic form is resuspended in a polymer solution and then placed in a mold or in an appropriate container.

[0041] Examples of suitable containers are Eppendorf tubes. Sarstedt screw-top tubes, plastic bottles, Falcon tubes with 15- or 50-ml capacity, centrifuge tubes, wells of a microtiter plate, or glass tubes.

[0042] The solvent, e.g. water, methanol, ethanol, acetonitrile, DMF, is removed by drying, e.g. by heating, applying a vacuum or freeze-drying, and the agarose is embedded in a polymer film.

[0043] Examples for suitable polymers include polyvinylpyrrolidone K30, K90, carboxymethylcellulose, polyacrylic acid, polymethacrylic acid and esters, copolymers of methacrylic acid and ethyl acrylate, e.g. Eudragit, polyvinyl alcohol, cellulose acetate phthalate, polyacrylamide, polyvinylamine, polyallylamine, polyacrylamide-co-acrylic acid, poly-hydroxy-methacrylate, or polyvinyl acetate.

[0044] For biological application, the polymers are taken up in the above-mentioned solvents, water or aqueous buffers and the agarose is then resuspended in biological buffers.

[0045] A good overview of methods for producing mini tablets, which can be used to prepare tablets according to the present i disclosure is demonstrated in Priyanka, Kapil Kumar* Deepak Teotia, Journal of Drug Delivery & Therapeutics. 2018; 8(6):382-390.

[0046] In another embodiment the agarose used can be activated with classical modifications, such as cyanogen bromide, NHS (N-hydroxy succinimide), epoxide, aldehyde, maleimide or divinyl sulfone. For this purpose, activated agarose and magnetic bead materials are commercially avalilable, like cyanogen.bromide-activated-sepharose 4 fast flow (cat. No. C5338, Cytiva), NHS MagBeads (cat. No. Cube biotech), Epoxy Agarose (cat No. Cube Biotech), and Glyoxal Agarose (cat No. G303-100, Goldbio, St Louis, MO, USA) These functionalizations allow the use of the agarose after resuspension of the table to react with biomolecules (for example substances produced by cells and living organism), such as proteins, antibodies, nucleic acids, and the like without the necessity of a condensing agent, such as EDC (l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide), DCC (dicyclohexyl carbodiimide), carbonyl diimidazole, and the like. Many of these compounds are sensitive against water or oxygen, and so the reduced contact area slows hydrolysis or further degradation of functionality, making the functionalized agaroses easier and longer to store in tablet form, compared to storage of the powder.

[0047] In a further embodiment agarose can be used with a chelator-functionalization like IDA (iminodiacetic acid) [1] of the surface, as well as NTA (nitrilotriacetic acid functionalization, with the coupled molecule Na, Na-Bis(carboxymethyl)-L-lysine, cat No. 14580 Merck) [2], or with ethylenediamine triacetic acid, coupled via primary or secondary amino function onto carboxy, epoxy or halogenide groups to solid phase.

[0048] In an alternative embodiment, ethylenediamine tetraacetic acid is coupled via one of the carboxylic acids onto amino-functionalized solid phase via an amide group.

[0049] Another way according to this patent application is to utilize agarose, covalently modified with a chelator comprising a scaffold, with a solid-phase bound polyamine like diethylene triamine, triethylene tetramine, tetraethylene pentamine, or pentaethylene hexamine, in which at least two amino functions are linked to carboxy groups of EDTA via amide functionality. These polyamines are bound to the solid phase via one or more amino functions, which are not modified with EDTA carboxy groups.

[0050] A synthesis of such a material is described in US20130072638, where a pentadentate dimer is functionalized and covalently bound onto agarose or magnetic beads. Alternatively, allyl- or epoxy- activated agarose can react with a polyamine as listed above, and the polyamine agarose can further react with EDTA dianhydride, followed by hydrolysis of the remaining anhydride groups.

[0051] In a further embodiment, agarose is covalently modified with a chelator comprising a scaffold, with a solid-phase bound polyamine like diethylene triamine, triethylene tetramine, tetraethylene pentamine, or pentaethylene hexamine, in which at least two amino functions are linked via amide functionality to carboxy groups of EDTA chains, where EDTA groups and diamines are alternating, connected via amide functionality from EDTA carboxy groups and amino groups from the amines. These polyamines are bound to the solid phase via one or more amino functions, which are not modified with EDTA.

[0052] A synthesis of such a material is described in W02020109162. In this patent application, allyl- or epoxy- activated agarose is reacted with a polyamine as listed above, and the polyamine agarose can further react with EDTA chains of alternate EDTA and diamines, linked via amide functions and prepared by reacting a surplus of EDTA dianhydride with diamines in dry solvents, such as DMSO or DMF, followed by hydrolysis of the remaining anhydride groups.

[0053] EDTA chains are organic molecule of the formula 1

[0054] With n: 1 - 12, X: solid phase, n: 0-7. They are prepared by a reaction of a surplus of EDTA anhydride with ethylene diamine. The ratio of anhydride to diamine influences the chain length of the resulting adducts. The resulting products also have acid anhydride groups, which react to form acid amide and carboxylic acid when bound to amino functions, e.g. in polyamines (see formula 2). Remaining anhydride groups are hydrolyzed after binding. To these chelators metal ions, such as nickel, cobalt, copper, zinc, iron, titanium and zirconium can be loaded to form a material for reversible binding and purification of his-tagged proteins, as well as phosphoproteins, phosphopeptides and zinc finger proteins.

[0055] In order to obtain tablets according to the present disclosure, also products like dried Ni-NTA Agarose (Cube Biotech, Qiagen GmbH) Ni-IDA Agarose (Cube Biotech) or Ni-INDIGO Agarose (Cube Biotech) can be used as agarose components for tablet production. As additional alternatives, Glutathione Agarose and MagBeads (Cube Biotech), protein A agarose (Thermo Inc.), streptavidin agarose (Merck), Rho-1D4 antibody- functionalized agarose (Cube Biotech) and many more can be used in the tableting process to prepare composites, which are capable of releasing the corresponding functionalized agarose particles during resuspension.

[0056] Other components of the tablets according to the present disclosure may include fillers, binders, lubricants, or disintegration accelerators. A good overview of these substances can be found in Friedland, Arzneiformenlehre fur PTA, 7thedition, 2013, ISBN 978-3-8047-3093-9.

[0057] Fillers are used in tablets together with amounts of agarose to form the tablet body. The excipient should be indifferent and not affect the biological application of the agarose. Suitable fillers are starches, mono- and disaccharides, such as corn starch, lactose, or sodium chloride or mixtures of these substances.

[0058] Binders are responsible for the strength of the tablets. They are used as wet binders to cement the powder particles during granulation or as dry binders. Wet binders include polyvinylpyrrolidone, starch paste, gelatin or cellulose derivatives, while dry binders can be cellulose and derivatives, mannitol or sorbitol.

[0059] Examples of lubricants are magnesium soaps such as magnesium stearate, calcium stearate, higher fatty alcohols or highly dispersed silicon dioxide.

[0060] Disintegration accelerators can act by volume increase (pectin, alginates) or gas formation (sodium hydrogen carbonate, magnesium peroxide).

[0061] Polyethylene glycol or polypropylene glycol can have a positive effect on the swelling behavior of the agarose when the tablets are redissolved. In order to get a more compact tablet, it can be helpful to add a solvent Possible substances could be acetone, acetonitrile, heptane, or tetrahydrofurane, but also water.

[0062] Surprisingly, very high stability was achieved in the production of the tablets of the present disclosure. Thus, despite 24 hours of exposure to an end-over-end head shaker, no splitting or rounding of the corners was observed in the tablets produced according to the disclosure.

[0063] At the same time, when getting into contact with water, the resuspension time of the tablets to single agarose microparticles in suspension was surprisingly short. Thus, the resuspension times required ranged from less than 5 seconds without additional vortexing to 5 minutes with the assistance of a vortex mixer.

[0064] The tablets produced according to the present disclosure have advantages over the prior art agaroses. For example, they are very easy to portion via the use of individual tablets and are quickly available due to rapid dissolution. As a further advantage, hazardous and water-free solvents can be dispensed with for moisture-sensitive products such as cyanogen bromide- activated agarose, NHS-activated agarose, epoxy-activated agarose or maleimide-activated agarose, which makes shipping and handling easier and less expensive.

[0065] A further advantage of the materials according to this application is the high stability of the materials in the tablet against aggregation and chemical and biological degradation, due to the use of stabilizers like lactose, mannitol, polyethylene glycol, and subsequent moisture removal during tablet production.

[0066] The tablets according to the present disclosure are ready to use for a wide range of applications after resuspension or in combination with resuspension.

[0067] One example is the use of tablets according to the present disclosure, containing activated agaroses and magnetic agarose beads, for covalent binding of biomolecules. The so produced particles with biomolecules on solid phase can be used for binding, purification or depletion of molecules, which selectively bind onto these biomolecules. For example, cyanogen bromide activated agarose, resuspended from tablets, can bind antibodies, streptavidin, Protein A, other proteins, and other biomolecules via their amino function. After reaction, a suspension of functionalized biomolecules is obtained, which can be stored for years in appropriate buffer. NHS Agarose and Epoxy Agarose also can covalently bind biomolecules via amino function, which results in biomolecules, bound on solid phase, with an even higher stability. In addition to that, many biomolecules can be bound onto maleimide-activated agarose via thiol functions, e.g. from cysteine functionalities. This reaction scheme allows the covalent binding onto different regions and functional groups of the biomolecules. And there are many other functionalized particles, which can be used for tablets, such as amino-activated, carboxy-activated, and many more. The protocols for covalent coupling after resuspension are similar to them used for bead suspensions in solvents and can be found in the literature [3], [4], [5],

[0068] In addition, agaroses and magnetic beads, which are suitable for IMAC (immobilized metal affinity chromatography), like Ni-IDA MagBeads, Ni-NTA Agarose, and 100 Ni-INDIGO Agarose can directly be used after resuspension for the purification of his-tagged proteins with protocols, which are similar to the purification with agarose bead suspension, which is used as a standard for many years.

[0069] Also, glutathione agarose or Rho-1D4 MagBeads, which are antibody-functionalized particles, can be used to purify GHS fusion proteins or Rho-tagged proteins via reversible binding, and Protein A as a tablet component can be used according to standard Protein A suspension, after resuspension from the tablet form. And a tablet containing Streptavidin Agarose can be resuspended and used to bind biotinylated biomolecules, according to the protocol for Streptavidin Agarose suspension.

[0070] Examples

[0071] Example 1:

[0072] Synthesis of a tablet from CNBr Agarose

[0073] Cyanogen bromide activation

[0074] Transfer 20 ml pure agarose beads (Sepharose Fast Flow, cat. no. 17015901, Cytiva) with 160 ml deionized water into a 400 ml beaker. The beaker is placed in a water bath at room temperature for cooling. Using a pH meter and magnetic stirrer, the pH is adjusted to approx. 11.0 with sodium hydroxide solution. Then 8.00g of cyanogen bromide (cat. no. 8201930050, Merck, Darmstadt, Germany) is added rapidly with stirring and the pH is kept between pH 10 and 11 by continuous addition of 1.75 M sodium hydroxide solution. The reaction is completed as soon as the pH value remains almost constant. Drying

[0075] The reaction solution is vacuum-filtrated through a filter chute and washed four times with deionized water and aspirated dry. The activated agarose beads are suspended in the filter chute in a 20mM disodium hydrogen phosphate buffer, pH 4 and allowed to act for 4 minutes before the beads are again aspirated dry. The beads are then suspended in the filter chute with a 20mM disodium hydrogen phosphate buffer pH 4 containing 10% (m / v) polyethylene glycol 8000, allowed to soak for 4 minutes, and dry aspirated. The residue is resuspended in acetone, thoroughly aspirated dry, transferred to a pre-weighed crystallization dish and dried in a vacuum desiccator overnight at high vacuum.

[0076] Granulation

[0077] The dried agarose beads are weighed and the ratio of the mass of the dried beads to the volume of the suspended beads is determined.

[0078] The particles are carefully ground in a mortar. 500 mg of the dried beads are placed in a preweighed mixing bowl, 60 mg of finely powdered sodium chloride and 12 mg of lactose and 0.02 ml of a solution of 0.1g Polyethylene glycol, mw 8000, in 10 ml acetone / lml deionized water are added and mixed. Once a homogeneous mixture is obtained, 10 ml of acetone is added to the mixing vessel and mixed until dryness. The weight of the dry granules is determined and again related to the volume of suspended beads.

[0079] Pressing

[0080] The treated cyanogen bromide activated agarose beads are loaded into a die mould from a tablet press (Micro-Tec MTB3 tablet press, Micro to Nano, Haarlem, the Netherlands, with Micro-Tec TPD3 Tablet Punch Die kit for 0 3mm tablets, carbon steel) and replenished until the maximum of the particles is filled to the die mould. Excess particles are removed from the die mould, and the tablet pressed with moderate manual pressure.

[0081] Example 2:

[0082] Synthesis of a tablet from NHS MagBeads

[0083] The solvent of 10 ml pure NHS activated MagBeads (Cube Biotech), 2-propanol, is removed by magnetic separation and discarded. Then the residual is resuspended in 50 ml 2.5% polyethylene glycol, mw 1.000, in acetone, and incubated for five minutes. After removal of the solvent and drying in vacuum, the agarose particles are mixed together with 250 mg mannitol and 10 ml dry acetone in a mortar with pestle under protective gas, and dried under vacuum. In the next step the activated agarose can be pressed to tablets with a manual tablet press according to example 1.

[0084] Example 3:

[0085] Synthesis of a tablet from Glyoxal Agarose

[0086] Resuspend 25 ml pure Glyoxal Agarose beads (cat No. G303-100, Goldbio, St. Louis, MO, USA) in 100 ml 100 mM citrate, 50 mM sodium carbonate, 10% Polyethylene glycol, mw 8000, 10% lactose, pH 10.0 and let five minutes incubate. The supernatant is separated by means of filtration, and the residue is washed two times with acetone and two times with n-pentane. After drying in vacuum, the agarose can be pressed to tablets with a manual tablet press according to example 1 or 2.

[0087] Example 4:

[0088] Synthesis of a tablet from Ni-INDIGO MagBeads XL

[0089] Aspirate 25 ml pure Ni-INDIGO MagBeads XL (cat. No. 55305, Cube Biotech) dry on a filter nudge, resuspend with 35 ml acetone in the nudge and aspirate as dry as possible. Then transfer the agarose to a crystallizing dish (pre-weighed) and dry in a vacuum desiccator. Dry beads can be carefully triturated to a fine powder with a pestle in an evaporating dish.

[0090] Two possibilities for further processing:

[0091] 1. with water content

[0092] Prepare a solution in a sealed bulkhead bottle: 1 ml water, 10 ml acetone, 1 g polyethylene glycol 8000. Add 0.1 ml of the solution to 100 mg of dry beads finely powdered and triturate until dry. The powder is scraped together and mixed with 10 mg lactose. To the powder mixture add 0.1 ml of the solution and mix again to dryness. The beads are homogenized with mortar and pestle again until homogeneous.

[0093] The powder has a ratio of approximately 500 mg beads to 50 mg lactose and 87 mg PEG 8000. 2. without water content

[0094] Prepare a solution in a sealed Schott bottle: 1 g polyethylene glycol 8000, 10 ml acetonitrile (Note mixing bowl tare) 500 mg dry beads finely powdered are mixed with 100 mg Lactose. Add 3 ml solution to the powder mixture and mix until dry. The wet mass is dried overnight in a vacuum desiccator.

[0095] The powder has a ratio of approx. 500 mg to 100 mg lactose and 279 mg PEG 8000.

[0096] After homogenization in example 1 or drying in vacuum in example 2, the agarose can be pressed to tablets with a manual tablet press according to example 1, 2 or 3.

[0097] Example 5:

[0098] Preparation of tablets from Ni-INDIGO MagBeads via spray-drying

[0099] Remove the supernatant from 25 ml pure Ni-INDIGO MagBeads (Cube Biotech) and resuspend in 75 ml deionized water, in which 2 g polyethylene glycol mw 8,000 and 2 g lactose are dissolved. Stir at a speed so that the magnetic agarose does not settle. The Biichi B 290 spray dryer (Biichi, Buchs, Switzerland) is started according to the manual and allowed to pre-run for 30 minutes. Then connect the round-bottom flask to the spray dryer with a silicone tube and start the spray drying process with the following parameters: Inlet temperature 100°C, aspirator 100%, compressed air flow 50 mm. The pump is operated at 20%, the collection time of the particles is about 7 minutes. Magnetic tablets with an average diameter of 500 pm are produced, which release a large number of particles with an average particle size of 30 pm when re-suspended in aqueous buffers.

[0100] To produce larger, more portionable tablets, 200 ml of the particles produced from the spray drying procedure are placed in a mini granulate dryer (Jetboxx MiniSet IL, Helios GmbH, Dachau, Germany), 40 ml deionized water is added, the drying temperature is set to 60 °C and the drying time to 2.5 h, and the granulated tablets are collected in the plasticizing cylinder. This produces tablets with an average diameter of 5 mm, still giving particles with an average particle size of 30 pm when re-suspended in aqueous buffers.

[0101] Example 6

[0102] Preparation of Tablets from Rho-1D4, StrepTactin or Ni-NTA MagBeads via Lyophilization with

[0103] Polymer 40 pl of a 50% suspension of Rho-lD4, StrepTactin, or Ni-NTA MagBeads in deionized water are pipetted into a roundwell microtiter plate, frozen at-80°C and lyophilized.

[0104] The lyophilized beads are mixed with 30 - 50 pl of a solution of 1 g polyvinylpyrrolidone (PVP) K30 (M ~ 40,000 g / mol) in 2 ml methanol and mixed well. After an incubation period of 60 minutes, the beads are dried in a vacuum desiccator at 200 mbar and room temperature.

[0105] After drying, the beads are completely covered by a predominantly transparent film of PVP. When the film is removed from the mold, a stable platelet is formed.

[0106] Under the microscope, the beads in the plate are visible in a transparent film. They partially adhere to each other, but can be transformed into individual particles by dissolving them in e.g. deionized water or buffer solution.

[0107] When the platelet is mixed with water or buffer (e.g. 100 mM sodium phosphate, pH 6.0), the PVP dissolves and the beads swell regularly, round and, after estimation, return to their approximate original size within 1-2 minutes. No non-dissolving, aggregated beads can be detected.

[0108] Example 7

[0109] Biological Application: Binding of antibodies onto CNBr Agarose

[0110] For this application tablets in form of a cylindrical body with a diameter of 3 mm and a height of 3 mm, prepared according example 1, are used.

[0111] One tablet is placed in an Eppendorf tube, and 1 ml 1 mM hydrochloric acid is added. The tube is mixed by pulse-vortexing, and the agarose tablet is getting dissolved to a homogenous suspension. After 5 minutes the reaction mixture is suction dried with a filter nudge. Then the dried agarose is added to a solution of 500 pg Rho-1D4 antibody (cat # 40020, Cube Biotech) in 500 pl 100 mM NaCl, 100 mM Na2COs pH 8.3, and pulse-vortexed.

[0112] The mixture is incubated on an end-over end shaker for 2 Oh at 4 °C. Then the supernatant is removed, the functionalized agarose is washed five times with 100 mM NaCl, 100 mM Na2COs pH 8.3 and suction-dried.

[0113] The concentration of Rho-1D4 in the sodium carbonate / NaCl buffer solution is monitored by measuring the OD at 280 nm wavelength before and after the reaction, with sodium carbonate / NaCl buffer solution as blank. By using the described analytics, it can be shown that more than 90% of the provided antibody is bound to the functionalized agarose. The so prepared Rho-lD4-functionalized agarose can be used to reversibly bind and purify rho-tagged proteins. Example 8

[0114] Biological Application: Binding of GFP protein onto Glyoxal Agarose

[0115] Tablets in the form of cylindrical bodies of Aldehyde MagBeads with a diameter of 2 mm and a height of 2 mm, prepared according to example 3, are placed in an Eppendorf tube and mixed with

[0116] 2 ml 100 mM sodium citrate, 50 mM sodium carbonate, pH 10.0. The suspension is mixed by vortexing, and incubated for five minutes, separated magnetically and the supernatant removed. Then the washing step is repeated. Add 2 ml of a 50 mM sodium acetate, 25 mM sodium carbonate buffer, pH 9.0, containing 40 mg GFP (Green Fluorescent Protein), to the residue. The preparation of GFP is known to the expert, but GFP is also commercially available (cat # 29903, Cube Biotech). The reaction mixture is incubated for two hours on an end-over-end shaker, and then the supernatant is removed by magnetic separation.

[0117] The supernatant is diluted 1:10 and measured at 488 nm and compared with the protein solution for binding. 100 mM sodium citrate, 50 mM sodium carbonate, pH 10.0 is used as blank.

[0118] As a result from this binding assay, the magnetic beads from a 2 mm cylindrical body can bind 1.7 to 2.0 mg GFP, or alternatively a different protein or other biomolecule.

[0119] Example 9

[0120] Biological Application: Purification of his-tagged proteins with Ni-INDIGO MagBeads XL

[0121] For this application tablets in form of a cylindrical body with a diameter of 3 mm and a height of

[0122] 3 mm, prepared according example 4, are used.

[0123] One tablet is placed in an Eppendorf tube, and 1 ml PBS buffer is added. The tube is mixed by pulse-vortexing, and the agarose tablet is getting dissolved to a homogenous suspension within less than five minutes. After settling, the magnetic bead suspension can be adjusted to 25% (v / v), which means, that 1 ml suspension contains 250 pl pure magnetic beads after settling.

[0124] As a source of recombinant protein, a cell pellet with a recombinant his-tagged protein is used. This pellet can be prepared from e-coli as bacteria, insect cells, as well as human cells, which is state of the art in protein biology. As an alternative a solution of his-tagged GFP (Cube Biotech) can be used to demonstrate reversible binding onto the magnetic beads.

[0125] Thaw the E. coli cell pellet on ice. And resuspend the cell pellet in 1 mL Lysis Buffer (50 mM sodium phosphate, 300 M sodium chloride, 10 mM imidazole, pH 8.0, optionally with up to 20 mM DTT and EDTA, supplemented with 1 mg / mL lysozyme). Then 6 U Benzonase® (3 units / mL bacterial culture) are added to the lysate to reduce viscosity caused by genomic DNA. The mixture is incubated for 30 min on ice, if necessary. Otherwise, incubating at room temperature (20-25°C) may be more efficient.

[0126] Then the lysate is centrifuged for 30 min at 10,000 x g and 4 °C, and the supernatant collected.

[0127] The PureCube His Affinity MagBeads are resuspended by vortexing, and 40 pL of the 25% magnetic bead suspension are transferred into a conical microcentrifuge tube. 500 pL Lysis Buffer are added and the sample is mixed by vortexing. The tube is placed on a magnetic microtube stand until the beads are separated and the supernatant is discarded. 1 mL of the cleared lysate is pipetted onto the equilibrated magnetic beads, and the lysate-magnetic bead mixture is incubated at 4°C for 1 h on an end-over-end shaker.

[0128] The tube is placed on the magnetic microtube stand until the beads separate and the supernatant is removed. Then the tube is removed from the magnet, 500 pL wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0) are added and the suspension is mixed by vortexing. The tube is placed again on the magnetic microtube stand and the beads are allowed to separate. The supernatant is removed and the washing step is repeated twice. The his- tagged protein is eluted using 100 pL elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 8.0), and the elution step is being repeated. Each elution fraction is now collected in a separate tube, the protein concentration of each fraction is determined, and all fractions are analyzed by SDS-PAGE. When a large surplus ofpoly-his GFP is offered (200 mg protein per ml pure agarose or magnetic beads) the eluates can contain more than 800 pg protein, which means a capacity of about 80 mg protein per ml pure beads. Example 10

[0129] Biological Application: Purification of Rho-tagged protein with Rho-1D4 Mag Beads according to Example 6

[0130] Buffers required:

[0131] Binding buffer: 20 mM HEPES, 150 mM NaCl, 0.1 % LMNG (Anatrace, Maumee, OH, USA), pH 7.0 Elution buffer: Mix 3 to 6 mL Binding Buffer with 5 mg Rho 1D4 peptide (Cube Biotech, Monheim, Germany) and dissolve the peptide in it.

[0132] Procedure:

[0133] The required aliquots of the Rho-tagged membrane protein are centrifuged for one hour at 100,000 g to remove any unwanted membrane components that subsequently arise.

[0134] Tablets corresponding to 50 pl of pure MagBeads are filled into 2 ml reaction tubes according to example 6. Equilibrate three times with two milliliters of binding buffer. The supernatant is separated by magnetic separation and removed. The dry resin is then mixed with 2 mL of membrane protein and left to incubate overnight on the end-over-end head shaker in the refrigerator.

[0135] The supernatant of the protein solution is separated and a 25 pL sample is mixed with 25 pL sample blue buffer, which is stored in a cool place. The remaining resin is then washed eight times with four or two milliliters of binding buffer. For this purpose, the resin is incubated for 10 minutes on the end over end head shaker in the refrigerator. The excess buffer is removed. Now add 200 pL volume of the elution buffer to each sample and elute for one hour (± 10 min) at 2 - 8 °C while rotating. After elution, 25 pL volume of each supernatant is mixed with 25 pL sample blue buffer, labeled and stored in the refrigerator. Elute twice in total. The prepared samples with blue buffer are heated and shaken at 46 °C for 30 minutes, then analyzed by SDS PAGE and Comassie Blue staining. The gel is documented by photo and shows a single band with the molecular weight of the purified Rho-tagged membrane protein. Example 11

[0136] Mechanical Storage Tests

[0137] CNBr Agarose, NHS MagBeads, INDIGO MagBeads XL and Glyoxal Agarose tablets produced according examples 1-4 as cylinders with a diameter and height of 2 and 3 mm are tested for their mechanical stability by putting a single tablet into an eppendorf tube and letting them rotate in an end-over end mixer (Bio-RS 24 Mini Rotator, Biosan, Riga, Latvia) with 25 rotations per minute and 24 hours rotation time.

[0138] After 24 hours, the tablets were still completely intact. There was no chipping, and no traces of agarose or magnetic beads were visible on the walls of the Eppendorf Tubes.

[0139] Example 12

[0140] Investigation of Resuspendability of the Tablets

[0141] CNBr Agarose, NHS MagBeads, INDIGO MagBeads XL and Glyoxal Agarose tablets produced according examples 1-4 as cylindrical bodies with a diameter and height of 2 and 3 mm are tested for their resuspendability by putting a single tablet into Eppendorf tubes and addition of 1 ml dd water. All tubes are mixed by vortexing for ten seconds, and the vortexing step is repeated up to five times. After the fifth vortexing step at the latest, all tablets are dissolved and no aggregates are visible in the suspension; all agarose beads are present as individual particles.

[0142] Example 13

[0143] Comparison of BrCN MagBeads Tablets with lyophilized BrCN MagBeads powder in terms of chemical long-term stability

[0144] Some BrCN Agarose tablets, cylindrical bodies with a diameter of 2 mm and a height of 2 mm according to example 1 and BrCN activated agarose in powder form, according to example 1, but without tablet preparation, are compared in terms of long-term stability. So, after production, tablets are weighted and powder with identical weight is portioned in a Eppendorf tube under protective gas and stored. Then eveiy month a binding assay with Rho-1D4 antibody according to example 5 has been done with one tablet and one powder sample, with 100 pg offered per sample, and the amount of binding capacity is measured by OD 280 of the Rho-1D4 solution before and after the binding, compared between the samples. In the evaluation, the 280 value of the initial solution is set as 100%, whereby the binding buffer is taken as a blank. The antibody concentration of the supernatant after the reaction (in percent) is then subtracted from the 100%.

[0145] The results of this comparison show, that tablets from cyanogen bromide-activated agarose have a much better long-time stability, compared to CNBr agarose powder.

[0146] Literature

[0147] 1. Porath, J., et al. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258: 598-599.

[0148] 2. Hochuli, E., Dobeli, H., and Schacher, A. 1987. New metal chelate adsorbent for proteins and peptides containing neighbouring histidine residues. J Chromatogr 411: 177-184.

[0149] 3. A.P.G. van Sommeren, P.A.G.M. Machielsen, T.C.J. Gribnau, Comparison of three activated agaroses for use in affinity chromatography: Effects on coupling performance and ligand leakage journal of Chromatography A, Volume 639, Issue 1, 1993, Pages 23-31, ISSN 0021- 9673, https: / / doi.org / 10.1016 / 0021-9673(93)83084-6.

[0150] 4. Richard F. Murphy, J.Michael Conlon, Ashraf Imam, Gregory J.C. Kelly, Comparison of nonbiospecific effects in immunoaffinity chromatography using cyanogen bromide and bifunctional oxirane as immobilising agents, Journal of Chromatography A, Volume 135, Issue 2, 1977, Pages 427-433, ISSN 0021-9673, https: / / doi.org / 10.1016 / S0021- 9673(00)88384-3.

[0151] 5. Zhang X, Duan Y, Zeng X. Improved Performance of Recombinant Protein A Immobilized on Agarose Beads by Site-Specific Conjugation. ACS Omega. 2017 Apr 30;2(4):1731-1737. doi: 10.1021 / acsomega.7b00362. Epub 2017 Apr 28. PMID: 30023643; PMCID: PMC6044777.

[0152] 6. Einarson MB, Orlinick JR (2002) Identification of Protein-Protein Interactions with Glutathione S-Transferase Fusion Proteins. In: Protein-Protein Interactions: A Molecular Cloning Manual. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press, pp 37- 57.

[0153] 7. Whitten, Kenneth W., Kenneth D. Gailey, and Raymond E. Davis. "7-3 Formation ofcovalent bonds." General Chemistry (1992): 264.

Claims

Claims1. A material in form of a tablet as a three-dimensional body with a side length and / or diameter of more than 1 mm, comprising a plurality of agarose microparticles.

2. A material in form of a tablet as a three-dimensional body with a side length and / or a diameter of 1 mm to 20 mm, comprising a plurality of agarose particles with a mean size of 5 pm to 500 pm.

3. A material in form of a tablet as a three-dimensional body with a side length and / or a diameter of 1 mm to 10 mm, comprising a plurality of agarose particles with a mean size of 10 pm to 250 pm.

4. A material in form of a tablet as a three-dimensional body with a side length and / or diameter of 1 mm to 5 mm, comprising a plurality of agarose particles with a mean size of 25 pm to 150 pm.

5. The material in form of a tablet according to any of claims 1-4, where the tablet also contains lactose.

6. The material in form of a tablet according to any of claims 1-5, where the tablet also contains mannitol.

7. The material in form of a tablet according to any of claims 1-6, where the tablet also contains polyethylene glycol, with a molecular weight of 200 to 20.000, preferably from 1.000 to 8.000.

8. The material according to any of claims 1-7, wherein the agarose, which the tablet contains, is activated agarose, which can react with biomolecules directly without additional reagents to form covalent bonds, such as cyanogen bromide-activated, NHS- activated, maleimide-activated and epoxy-activated agarose.

9. The material according to any of claims 1-7, wherein the agarose, which the tablet contains, is agarose, functionalized for IMAC (Immobilized Metal Ion Affinity Chromatography).

10. The material according to any of claims 1-7 and 9, wherein the agarose, which the tablet contains, is agarose, to which a chelator is bound via covalent bonds.

11. The Material according to any of claims 1-7 and 9-10, where agarose is functionalized with IDA (iminodiacetic acid), NTA as Na, Na-Bis(carboxymethyl)-L-lysine, or ethylene diamine triacetic acid, which are coupled via their secondary and primary amino function onto solid phase groups, such as epoxy, carboxy or halogenide, or functionalized with ethylenediamine tetraacetic acid, which is covalently coupled to agarose with one carboxylic acid group via amide groups onto solid phase amino groups.

12. The material according to claim to claim 1-7 and 9-11, where agarose is functionalized with a chelator, comprising a scaffold with a solid-phase bound polyamine such as diethylene triamine, triethylene tetramine, tetraethylene pentamine, or pentaethylene hexamine, in which at least two amino functions of the polyamine are linked to carboxy groups of EDTA via amide functionality, and the polyamine is bound to the solid phase via one or more amino functions, which are not modified with EDTA.

13. The material according to anyone of claims 1-7 and 9-11, wherein a chelator comprising a scaffold with a solid phase bound polyamine such as diethylenetriamine, triethylenetetramine, tetraethylene pentamine or pentaethylene hexamine, in which at least two amino functions of the polyamine are covalently bound to carboxy groups of EDTA chains, which consist of alternating EDTA molecules and diamines linked via amide functions, and the polyamine is bound to the solid phase via one or more amino functions, which are not modified with EDTA.

14. The Material according to any of claims 10-13, where the chelator is loaded with a metal, such as nickel, cobalt, copper, zinc, iron, titanium, and zirconium.The material according to any of claims 1-14, wherein the tablet further contains agarose, functionalized with biomolecules.The material according to any of claims 1-15, wherein the tablet further contains agarose, functionalized with Protein A or G.

17. The material according to any of claims 1-16, wherein the tablet further contains agarose, functionalized with Streptavidin.

18. The material according to any of the previous claims, wherein the tablet further contains magnetic agarose microparticles with magnetic particles, embedded by the agarose network.

19. The material according to any of the previous claims, wherein the magnetic particles have a diameter in the range from 8 to 5000 nm, preferably from 25 to 2000 nm, more preferably from 100 to 1000 nm.

20. A method for producing agarose in tablet form with a diameter or side length of 1 mm to 20 mm with single agarose particles of a mean particle size between 10 and 500 pm, by applying pressure to a dry powder or a powder thickened with water or solvent, by means of a tablet press.

21. The method according to claim 20, wherein during the method one or more solvents selected from acetone, acetonitrile, ethanol, or 2 -propanol is added.

22. A method for preparing agarose in tablet form, wherein agarose in magnetic or nonmagnetic form is suspended in a polymer solution, then placed in a mold or in a container, and the solvent is removed to obtain the agarose embedded in a polymer film.

23. Use of agarose in tablet form according to any of claims 1 to 19 for purification of his- tagged proteins or Rho-tagged proteins.

24. Use of agarose in tablet form according to any of claims 1 to 19 to purify proteins from a mixture of biomolecules.

25. Use of agarose in tablet form according to any of claims 1-19 in diagnostics.

26. Use of agarose in tablet form according to any of claims 1-19 to covalently bind biomolecules.

27. Use of agarose in tablet form according to any of claims 1-19 to bind biotinylated biomolecules.

28. Use of agarose in tablet form according to any of claims 1 -19 to bind antibodies.

29. A kit comprising the material according to any one of claims 1 -19.

30. Use of the kit according to claim 29 for the purification of his-tagged proteins or Rho- tagged proteins.

31. Use ofthe kit according to claim 29 to purify proteins from a mixture of biomolecules.

32. Use ofthe kit according to claim 29 in diagnostics.

33. Use ofthe kit according to claim 29 to covalently bind biomolecules.

34. Use of the kit according to claim 29 to bind biotinylated biomolecules.

35. Use ofthe kit according to claim 29 to bind antibodies.

36. Use ofthe material according to any of claims 1 to 19 to purify proteins from a mixture of biomolecules.

37. Use of the material according to any of claims 1-19 in diagnostics.

38. Use of the material according to any of claims 1-19 to covalently bind biomolecules.

39. Use of the material according to any of claims 1-19 to bind biotinylated biomolecules.

40. Use of the material according to any of claims 1 -19 to bind antibodies.

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