Tablets comprising functionalized agarose particles, process for their production and use of the tablets

Abstract

A material in the form of a tablet, which is a three-dimensional body having an edge length and / or diameter of more than 1 mm, is described, which material comprises a plurality of agarose microparticles. Furthermore, a method for preparing the material and its use are described.

Description

Technical Field

[0001] This disclosure relates to a tablet comprising activated agarose (in particular, a tablet comprising a large number of magnetic particles), a method for producing these materials, and the use of these tablets for resuspension, functionalization of agarose, and biological applications. Background Technology

[0002] For biological applications, particularly in the field of protein science, agarose has become a recognized carrier medium. This is because, despite its relatively large particle diameter, the agarose network possesses high binding capacity and exhibits good processability in columns and when used with magnetic particles. For example, Ni-NTA agarose has been established for the purification of recombinant proteins with His tags. Other proteins can be purified using glutathione, maltose-binding proteins, or antibodies covalently attached to agarose. Functionalized agarose is also commercially available for covalently attaching biomolecules and, in some cases, for direct functionalization in an activated form without the need for additional, often toxic, reagents such as cyanogen bromide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, or carbonyldiimidazole.

[0003] The material used for this purpose is activated agarose particles, which can be functionalized by coupling with biomolecules such as proteins and antibodies. These products are sold in suspension form in inert solvents and in lyophilized powder form. Disadvantages in these cases include difficulty in metrology for manufacturers and users, and limited stability against hydrolysis. Examples of such products are CNBr-activated Sepharose 4B (Cytiva, Marlborough, USA) and NHS-activated MagBeads (CubeBiotech, Monheim, Germany).

[0004] Furthermore, agarose-based materials derived from suspensions also have some drawbacks. For example, agarose in suspensions must not be dried. Many activated agarose and magnetic particles must be stored in organic solvents, thus posing potential fire hazards and air transport problems. Additionally, dispensing requires pipettes, which can become clogged by agarose particles.

[0005] Alternatively, activated granules can be prepared by lyophilization, such as cyanogen bromide-activated agarose. However, this method has drawbacks due to the powder form and the time-consuming removal and dispensing process. Furthermore, the powder form promotes accelerated hydrolysis, limiting the storage stability of these products.

[0006] The manufacture of tablets for pharmaceutical purposes has been common for over a century. Brockedon invented the mechanical tablet press in 1843, which propelled the development of tablets, and Burroughs Wellcome & Co. began the industrial production of tablets in 1884.

[0007] JPH 029813 and JPS 5452719 describe the preparation of magnetic particles with a diameter of up to 2 mm for drug delivery purposes by adding magnetic materials, drugs (e.g., the antitumor agent fluorouracil) and hydrophilic substances (e.g., hydroxypropyl cellulose).

[0008] In DE-A-4406139, magnetic particles in the form of Nd-Fe-B permanent magnets are coated with carnauba wax and compressed into tablets together with a drug such as Eudragit or Levodopa. Optionally, excipients 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.

[0009] Another source of prior art is US5,939,470. Here, magnetic organic polymer particles are coated with soluble sugars, such as mannitol and trehalose. After adding water, the mixture is spray-dried, resulting in the precipitation of small, tablet-like particles.

[0010] US 6,274,386 describes a reagent formulation in tablet form comprising magnetic particles. The magnetic core particles comprise magnetite coated with glassy silica. Other possible components serve a stabilizing function.

[0011] Numerous reviews describe the uses of agarose for drug delivery. Thus, 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 for encapsulating and releasing drugs. Summary of the Invention

[0012] The subject of this disclosure is a reagent formulation in tablet form. Furthermore, the preparation of the tablet particles is based on agarose and, for example, magnetic agarose. Agarose and magnetic agarose are also referred to herein as solid phases.

[0013] In this application, the following definitions are used.

[0014] Chelating agents: Chelating agents consist of one or more functional groups that form two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom.

[0015] Diagnosis: Diagnosis as defined in this patent application refers to in vitro testing of a human, animal, or plant for medical care of a human, animal, or plant from which a sample is collected. “Medical care” shall include, for example but not limited to, diagnosis, prognosis, treatment, prevention, or monitoring of progression of any and all possible human, animal, or plant diseases (including infectious, genetic, traumatic, metabolic, degenerative, and neoplastic diseases) and tissue compatibility of donor and recipient. Agarose microparticles in tablet form according to this disclosure, after being resuspended in buffer, can be used for diagnosis by purifying 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 formed into tablets according to this disclosure). Other examples include synthesizing antibody or enzyme-functionalized agarose in tablet form by binding biomolecules to activated agarose or binding his-tagged biomolecules to INDIGO Ni-agarose, etc. Many other models for the use of agarose or magnetic beads according to this patent for diagnostic purposes are feasible, such as pull-down assays for protein-protein interactions [6].

[0016] Size: Most tablets produced according to this disclosure are spherical (e.g., produced by granulation) or cylindrical (e.g., produced by manual compression). However, other geometries are possible, such as cubic or cuboid, or granules with undefined structures, depending on the compression method or compression mold. Tablet sizes (including height, width, length, and diameter) range from about 1 mm to 5 cm. Suitable methods for measuring diameter and height are using a ruler and calipers, sieving with different sieve widths and collecting the individual fractions, or measuring using a glass slide with millimeter graduations under an optical microscope.

[0017] Using a Neubauer counting chamber with a size scale, the particle size distribution of agarose particles in the range of 10 µm to 500 µm can be easily determined by optical microscopy. Alternatively, a microscope or cell counter that generates an automated assessment of particle size distribution can be used. Agarose monoparticles that constitute part of a tablet are typically in spherical or ellipsoidal form; therefore, the size of an agarose monoparticle refers to the diameter of the bead.

[0018] Powder: According to Wikipedia, powder is a dry, bulk solid composed of many very fine particles that flow freely when shaken or tilted. Powder is a special subclass of particulate materials, although the terms powder and granules are sometimes used to distinguish different categories of materials. In particular, powder refers to particulate materials with a finer particle size and therefore more prone to clumping when flowing.

[0019] According to Whitten et al., [7] a covalent bond is a chemical bond that involves sharing electrons to form electron pairs between atoms. These electron pairs are called shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms when they share electrons is called a covalent bond. [7]

[0020] Iminodiacetic acid (Formula 1) coupled to the solid phase:

[0021]

[0022] Where X represents the solid phase, and the functionalization can be a carboxyl group before coupling (an amide group after coupling), an epoxy group (amine), or a halide group (amine).

[0023] Ammonitrilic acid (NTA) coupled to the solid phase (Formula 2):

[0024]

[0025] Where X represents the solid phase, and the functionalization can be a carboxyl group before coupling (an amide group after coupling), an epoxy group (amine), or a halide group (amine).

[0026] Ethylenediaminetetraacetic acid (EDTA) coupled to the solid phase (Formula 3):

[0027]

[0028] Where X represents the solid phase, and functionalization can be a carboxyl group before coupling (an amide group after coupling), an epoxy group (amine), or a halide group (amine).

[0029] Chelating agent, skeleton, EDTA (Formula 4):

[0030]

[0031] Where X: solid phase, functionalization can be carboxyl group before coupling (amide group after coupling), epoxy group (amine), halide group (amine), m: 0-3, n: 2-5.

[0032] Chelating agent, backbone, EDTA chain (Formula 5):

[0033]

[0034] Where X: solid phase, functionalization can be carboxyl group before coupling (amide group after coupling), epoxy group (amine), halide group (amine), m: 0-3, n: 2-5, o, p: 1-8.

[0035] According to this disclosure, tablets with a diameter and / or side length of at least 1 mm and, for example, at most 5 cm, preferably 2 mm to 2 cm, and most preferably 2 mm to 5 mm are prepared. The preferred form is cubic, spherical, or cylindrical, but other geometric shapes and irregular particles may also be used depending on the manufacturing method. The agarose particles are 2% to 10% agarose microparticles, with a diameter of 5 µm to 500 µm in an aqueous suspension, preferably 10 µm to 300 µm, and most preferably 25 to 200 µm. These particles are dried and can be regenerated when the tablets are resuspended in an aqueous medium.

[0036] According to this disclosure, the tablet contains a plurality of agarose particles, the diameter of which, after resuspending in an aqueous solution, is measured by an optical microscope to be 5 to 500 µm, preferably 20 to 150 µm, and more preferably 25 to 100 µm. The agarose particles can be crosslinked according to standard and known procedures using, for example, epichlorohydrin, 3-chloropropanediol, divinyl sulfone, etc.

[0037] According to this disclosure, the material (particularly agarose in tablet form as described in this disclosure) may be included in a kit, particularly in a kit for use selected from: purifying proteins with his tags, purifying proteins in mixtures of biomolecules, in diagnostics, covalently binding biomolecules, binding biotinylated biomolecules, and binding antibodies.

[0038] In one embodiment, the agarose particles can be magnetic, containing some magnetic particles within them, giving the agarose beads a magnetic attraction and allowing for magnetic separation upon resuspending. Examples of suitable magnetic materials are magnetite (Fe3O4) and maghematite (Fe2O3). Magnetic agarose-based materials are available from Cube Biotech (Monheim, Germany), Qiagen (Hilden, Germany), and Cytiva (Marlborough, USA).

[0039] According to this disclosure, in other methods, tablets can be produced by direct compression: this method involves directly compressing powders or powder blends using a manual tablet press (Micro-Tec MTB8, Micro-Tec, Haarlem, Netherlands) or an automatic tablet press (e.g., Prexima 80, IMA, Ozano del Emilia, Italy) to produce dense tablets with a size of 2 to 10 mm.

[0040] As an alternative, wet granulation can be used. Granules are formed with the aid of a binder (e.g., polyvinylpyrrolidone) and then compressed in a tableting machine (e.g., FlexiTab XL from Synthegon, Weiblingen, Germany) in the presence of a solvent (e.g., acetone, methanol, water, DMF) to obtain microtablets and tablets.

[0041] Dry granulation is a pharmaceutical manufacturing process that involves compacting powder into granules without the use of liquid binders or solvents. In this method, pressure is typically applied using a roller compactor (e.g., CCS220, Fitzpatrick, Waterloo, Canada) to compact the powder blend, producing granules. The resulting granules can then be further processed into tablets or capsules. Dry granulation is typically chosen when using liquids in the granulation process is impractical or unsuitable (e.g., for moisture-sensitive or heat-labile substances).

[0042] In melt extrusion, premixed powder is transferred to a melt extruder (e.g., the Pharma mini HME micro-mixer from ThermoScientific, Waltham, USA), where the material forms an extrudate, which is then ground and sieved to obtain tablets.

[0043] Another procedure is spray drying, in which the beads are used in an aqueous or organic suspension (preferably at 5-10%) and spray dried using a spray dryer such as the Büchi Mini Spray Dryer B-290 (Büchi, Switzerland), and the dried particles are collected in a glass sample collector. To obtain a particle size greater than 1 mm, an additional granulation step is necessary.

[0044] Alternatively, agarose in either magnetic or non-magnetic form can be resuspended in a polymer solution and then placed in a mold or suitable container.

[0045] Examples of suitable containers are Eppendorf tubes, Sarstedt screw-cap tubes, plastic bottles, Falcon tubes with a capacity of 15 or 50 ml, centrifuge tubes, the holes of microtiter plates, or glass tubes.

[0046] The solvent (e.g., water, methanol, ethanol, acetonitrile, DMF) is removed by drying, such as by heating, applying vacuum, or freeze drying, and the agarose is embedded in the polymer film.

[0047] Examples of suitable polymers include polyvinylpyrrolidone K30, K90, carboxymethyl cellulose, polyacrylic acid, polymethacrylic acid and esters, copolymers of methacrylic acid and ethyl acrylate such as Eudragit, polyvinyl alcohol, cellulose acetate phthalate, polyacrylamide, polyethyleneamine, polyallylamine, polyacrylamide-co-acrylic acid, polyhydroxy methacrylate, or polyvinyl acetate.

[0048] For biological applications, the polymer is absorbed in the above-mentioned solvent, water, or aqueous buffer, and then the agarose is resuspended in the biological buffer.

[0049] Priyanka, Kapil Kumar A good review of a method for producing microtablets that can be used to prepare tablets according to this disclosure is presented in Deepak Teotia, Journal of Drug Delivery & Therapeutics. 2018; 8(6):382-390.

[0050] In another embodiment, the agarose used can be activated using classical modifications such as cyanogen bromide, NHS (N-hydroxysuccinimide), epoxides, aldehydes, maleimide, or divinyl sulfone. For this purpose, commercially available activated agarose and magnetic bead materials are available, such as cyanogen bromide activated - sepharose 4 fast flow (C5338, Cytiva), NHS MagBeads (Cube Biotech), Epoxy Agarose (Cube Biotech), and Glyoxal Agarose (G303-100, Goldbio, St. Louis, Missouri, USA). These functionalizations allow the agarose to be used after resuspension of the tablet to react with biomolecules (e.g., substances produced by cells and living organisms) such as proteins, antibodies, nucleic acids, etc., without the need for condensing agents such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), DCC (dicyclohexylcarbodiimide), carbonyl diimidazole, etc. Many of these compounds are sensitive to water or oxygen, so the reduced contact area slows down hydrolysis or further functionalization, making functionalized agarose in tablet form easier to store and longer-lasting compared to storing in powder form.

[0051] In a further embodiment, agarose can be used with chelating agents (e.g., surface-derived IDA (iminodiacetic acid) [1] and NTA (aminotriacetic acid functionalized with coupling molecules Nα, Nα-bis(carboxymethyl)-L-lysine, catalog number 14580 Merck) [2]), or with ethylenediaminetriacetic acid, coupled to the solid phase via primary or secondary amino functional groups to carboxyl, epoxy, or halide groups.

[0052] In an alternative embodiment, ethylenediaminetetraacetic acid is coupled to an amino-functionalized solid phase via an amide group of one of the carboxylic acids.

[0053] Another method according to this patent application utilizes agarose covalently modified with a chelating agent comprising a backbone having solid-bound polyamines such as diethylenetriamine, triethylenetetramine, tetraethylenepentamine, or pentaethylenehexamine, wherein at least two amino functional groups are linked to the carboxyl group of EDTA via amide functional groups. These polyamines are bound to the solid phase via one or more amino functional groups not modified with the EDTA carboxyl group.

[0054] The synthesis of such materials is described in US20130072638, in which a pentadentate dimer is functionalized and covalently bonded to agarose or magnetic beads. Alternatively, allyl or epoxy-activated agarose can react with the polyamines listed above, and the polyamine agarose can further react with EDTA dianhydride, followed by hydrolysis of the remaining anhydride groups.

[0055] In a further embodiment, the agarose is covalently modified with a chelating agent comprising a backbone having a solid-bound polyamine such as diethylenetriamine, triethylenetetramine, tetraethylenepentamine, or pentaethylenehexamine, wherein at least two amino functional groups are linked to the carboxyl group of the EDTA chain via amide functional groups (wherein the EDTA group and the diamine are arranged alternately), and the EDTA chain is linked via an amide functional group from the EDTA carboxyl group and an amino group from the amine. These polyamines are bound to the solid phase via one or more unmodified amino functional groups.

[0056] The synthesis of such materials is described in WO2020109162. In this patent application, allyl or epoxy-activated agarose reacts with the polyamines listed above, and the polyamine agarose is further reacted with alternating EDTA and diamine EDTA chains linked via amide functional groups and prepared by reacting excess EDTA dianhydride with the diamine in a dry solvent (e.g., DMSO or DMF), followed by hydrolysis of the remaining anhydride groups.

[0057] The EDTA chain is an organic molecule of formula 1:

[0058]

[0059] Wherein, n: 1–12, X: solid phase, n: 0–7. They are prepared by reacting excess EDTA anhydride with ethylenediamine. The ratio of anhydride to diamine affects the chain length of the resulting adduct. The resulting product also has an anhydride group, which reacts to form an amide and a carboxylic acid when it is combined with an amino functional group, for example, in a polyamine (see Formula 2). The remaining anhydride group is hydrolyzed after combination.

[0060]

[0061] Metal ions, such as nickel, cobalt, copper, zinc, iron, titanium, and zirconium, can be loaded onto these chelating agents to form materials for the reversible binding and purification of his-tagged proteins, as well as phosphoproteins, phosphopeptides, and zinc finger proteins.

[0062] To obtain the tablets according to this disclosure, products such as dried Ni-NTA Agarose (CubeBiotech, Qiagen GmbH), Ni-IDA Agarose (Cube Biotech), or Ni-INDIGO Agarose (CubeBiotech) can also be used as the agarose component for tablet production. As alternatives, glutathione agarose and MagBeads (Cube Biotech), protein A agarose (Thermo Inc.), streptavidin agarose (Merck), Rho-1D4 antibody-functionalized agarose (Cube Biotech), and many other substances can be used in the tableting process to prepare a complex that releases the corresponding functionalized agarose particles during resuspension.

[0063] Other components of the tablets according to this disclosure may include fillers, binders, lubricants, or disintegration promoters. A good review of these substances can be found in Friedland, Arzneiformenlehre für PTA, 7th edition, 2013, ISBN 978-3-8047-3093-9.

[0064] Fillers are used in tablets with a certain amount of agarose to form the tablet body. This excipient should be inert and not affect the biological application of the agarose. Suitable fillers are starch, monosaccharides and disaccharides (e.g., corn starch, lactose), or sodium chloride, or mixtures of these substances.

[0065] Binders are responsible for the strength of tablets. They are used as wet binders to bind 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 its derivatives, mannitol, or sorbitol.

[0066] Examples of lubricants are magnesium soaps such as magnesium stearate, calcium stearate, higher fatty alcohols, or highly dispersed silica.

[0067] Disintegration promoters can function by increasing volume (pectin, alginate) or by forming gas (sodium bicarbonate, magnesium peroxide).

[0068] When the tablets are redissolved, polyethylene glycol or polypropylene glycol can have a positive effect on the swelling behavior of agarose.

[0069] Adding a solvent can be helpful in obtaining denser tablets. Possible substances include acetone, acetonitrile, heptane, or tetrahydrofuran, but water is also an option.

[0070] Surprisingly, extremely high stability was achieved in the production of the tablets disclosed herein. Therefore, despite exposure to a head-to-tail reversing oscillator for 24 hours, no cracking or chamfering was observed in the tablets produced according to this disclosure.

[0071] Meanwhile, when in contact with water, the resuspension time of the tablets into individual agarose particles in the suspension is surprisingly short. Therefore, the required resuspension time ranges from less than 5 seconds (without additional vortexing) to 5 minutes (with the aid of a vortex mixer).

[0072] The tablets produced according to this disclosure have advantages over prior art agarose. For example, they are very easy to dispense using individual tablets and can be used quickly due to rapid dissolution. As a further advantage, for moisture-sensitive products, such as cyanogen bromide-activated agarose, NHS-activated agarose, epoxy-activated agarose, or maleimide-activated agarose, harmful and anhydrous solvents can be eliminated, making transportation and handling easier and less costly.

[0073] A further advantage of the material according to this application is that, due to the use of stabilizers such as lactose, mannitol, polyethylene glycol, and subsequent removal of moisture during tablet production, the material in the tablet exhibits high stability against aggregation and chemical and biodegradation.

[0074] Tablets according to this disclosure are prepared for a wide range of applications after or in combination with resuspension.

[0075] One example is the use of tablets (comprising activated agarose and magnetic agarose beads) according to this disclosure for the covalent binding of biomolecules. Particles thus prepared with biomolecules on a solid phase can be used to bind, purify, or deplete molecules selectively bound to these biomolecules. For example, cyanogen bromide activated agarose resuspended from the tablet can bind antibodies, streptavidin, protein A, other proteins, and other biomolecules via its amino functional groups. Following the reaction, a suspension of functionalized biomolecules is obtained, which can be stored in a suitable buffer for up to several years.

[0076] NHS agarose and epoxide agarose can also covalently bind biomolecules via amino functional groups, resulting in greater stability of biomolecules bound to the solid phase. In addition, many biomolecules can bind to maleimide-activated agarose via thiol functional groups (e.g., from cysteine ​​functional groups). This reaction scheme allows covalent binding to different regions and functional groups of biomolecules. There are also many other functionalized particles that can be used for tablets, such as amino-activated, carboxyl-activated, and more particles. The covalent coupling schemes after resuspension are similar to those used for bead suspensions in solvents and can be found in references [3], [4], [5].

[0077] Furthermore, agarose and magnetic beads suitable for IMAC (immobilized metal affinity chromatography) (e.g., Ni-IDAMagBeads, Ni-NTA Agarose, and 100 Ni-INDIGO Agarose) can be directly used to purify his-tagged proteins after resuspending, in a manner similar to the purification of agarose bead suspensions that have been used as a standard for many years.

[0078] Additionally, glutathione agarose or Rho-1D4 MagBeads (which are antibody-functionalized particles) can be used to purify GHS fusion proteins or Rho-tagged proteins through reversible binding, and protein A, as a tablet component, can be used after resuspending from tablet form according to a standard protein A suspension. Furthermore, tablets containing streptavidin agarose can be resuspended and used to bind biotinylated biomolecules according to a protocol for streptavidin agarose suspensions. Example

[0079] Example 1:

[0080] Tablets synthesized from CNBr agarose

[0081] Activation of cyanogen bromide

[0082] Transfer 20 ml of pure agarose beads (Sepharose Fast Flow, catalog number 17015901, Cytiva) to a 400 ml beaker along with 160 ml of deionized water. Cool the beaker in a water bath at room temperature. Adjust the pH to approximately 11.0 using a pH meter and a magnetic stirrer with sodium hydroxide solution. Then, rapidly add 8.00 g of cyanogen bromide (catalog number 8201930050, Merck, Darmstadt, Germany) with stirring, maintaining the pH between 10 and 11 by continuously adding 1.75 M sodium hydroxide solution. The reaction is complete as long as the pH remains nearly constant.

[0083] dry

[0084] The reaction solution was vacuum filtered through a filter tank, washed four times with deionized water, and dried by suction. Activated agarose beads were suspended in the filter tank in 20 mM disodium hydrogen phosphate buffer (pH 4) and allowed to react for 4 minutes, then dried again by suction. The beads were then suspended in a filter tank (containing 20 mM disodium hydrogen phosphate buffer (pH 4) with 10% (m / v) polyethylene glycol 8000) and allowed to soak for 4 minutes, then dried by suction. The residue was resuspended in acetone, thoroughly dried by suction, transferred to a pre-weighed crystallizing dish, and dried overnight in a vacuum desiccator under high vacuum.

[0085] Granulation

[0086] The dried agarose beads were weighed, and the ratio of the mass of the dried beads to the volume of the suspended beads was determined.

[0087] Carefully grind the particles in a mortar. Place 500 mg of dried beads into a pre-weighed mixing bowl, add and mix 60 mg of finely powdered sodium chloride and 12 mg of lactose, along with 0.02 ml of a solution of 0.1 g polyethylene glycol (Mw: 8000) in 10 ml of acetone / 1 ml of deionized water. Once a homogeneous mixture is obtained, add 10 ml of acetone to the mixing container and mix until dry. Determine the weight of the dried particles and again correlate it with the volume of the suspension beads.

[0088] suppress

[0089] The treated cyanogen bromide-activated agarose beads are loaded into the die of a tablet press (Micro-Tec MTB3 tablet press, Microto Nano, Haarlem, Netherlands, with a Micro-Tec TPD3 Tablet Punch Die kit for Ø 3mm tablets, carbon steel) and replenished until the maximum number of granules are filled into the die. Excess granules are removed from the die, and the tablets are compressed under moderate manual pressure.

[0090] Example 2:

[0091] Synthetic tablets from NHS MagBeads

[0092] The solvent 2-propanol in 10 ml of pure NHS-activated MagBeads (Cube Biotech) was removed by magnetic separation and discarded. The residue was then resuspended in 50 ml of 2.5% polyethylene glycol (Mw: 1,000) in acetone and incubated for five minutes. After solvent removal and drying under vacuum, the agarose granules were mixed with 250 mg of mannitol and 10 ml of anhydrous acetone in a mortar using a pestle under a protective atmosphere and dried under vacuum. In the next step, the activated agarose was compressed into tablets using a manual tablet press according to Example 1.

[0093] Example 3:

[0094] Tablets synthesized from glyoxal agarose

[0095] 25 ml of pure glyoxal agarose beads (catalog number G303-100, Goldbio, St. Louis, Missouri, USA) were resuspended in 100 ml of a solution of 100 mM citrate, 50 mM sodium carbonate, 10% polyethylene glycol (Mw: 8000), and 10% lactose, pH 10.0, and incubated for five minutes. The supernatant was separated by filtration, and the residue was washed twice with acetone and twice with n-pentane. After drying under vacuum, the agarose was compressed into tablets using a manual tablet press according to Example 1 or 2.

[0096] Example 4:

[0097] Synthetic tablets from Ni-INDIGO MagBeads XL

[0098] 25 ml of pure Ni-INDIGO MagBeads XL (catalog number 55305, Cube Biotech) was dried by suction on a filter nudge, resuspended in 35 ml of acetone on the nudge, and dried by suction as much as possible. The agarose was then transferred to a pre-weighed crystallizing dish and dried in a vacuum desiccator. The dried beads can be carefully ground into a fine powder using a pestle in an evaporating dish.

[0099] Two possibilities for further processing:

[0100] 1. Water content

[0101] Prepare the solution in a sealed, partitioned bottle: 1 ml water, 10 ml acetone, 1 g polyethylene glycol 8000. Add 0.1 ml of the solution to 100 mg of finely ground, dried beads and grind until dry. Scrape the powder and mix with 10 mg of lactose. Add 0.1 ml of the solution to the powder mixture and mix again until dry. Homogenize the beads again with a mortar and pestle until uniform.

[0102] The powder contains approximately 500 mg of beads in a ratio of 50 mg of lactose and 87 mg of PEG 8000.

[0103] 2. Contains no water content

[0104] Prepare the solution in a sealed Schott bottle: 1 g polyethylene glycol 8000, 10 ml acetonitrile

[0105] (Note the tare weight of the mixing bowl) Mix 500 mg of finely ground dried beads with 100 mg of lactose. Add 3 ml of solution to the powder mixture and mix until dry. Dry the wet block overnight in a vacuum desiccator.

[0106] The powder contains approximately 500 mg of lactose in a ratio of 100 mg to 279 mg of PEG 8000.

[0107] After homogenization in Example 1 or drying in vacuum in Example 2, agarose can be compressed into tablets using a manual tablet press according to Example 1, 2 or 3.

[0108] Example 5:

[0109] Tablets were prepared from Ni-INDIGO MagBeads by spray drying.

[0110] The supernatant was removed from 25 ml of pure Ni-INDIGO MagBeads (Cube Biotech) and resuspended in 75 ml of deionized water containing 2 g of polyethylene glycol (Mw: 8,000) and 2 g of lactose. The mixture was stirred at a rate that prevented magnetic agarose sedimentation. A Büchi B 290 spray dryer (Büchi, Switzerland) was started according to the manual and allowed to pre-run for 30 minutes. The round-bottom flask was then connected to the spray dryer using silicone tubing, and the spray drying process was started with the following parameters: inlet temperature 100°C, aspirator 100%, and compressed air flow rate 50 mm. The pump was run at 20%, and the particle collection time was approximately 7 minutes. Magnetic tablets with an average diameter of 500 µm were produced, which released a large number of particles with an average particle size of 30 µm upon resuspension in the aqueous buffer.

[0111] To produce larger, more easily dispensed tablets, 200 ml of granules produced by a spray-drying process were placed in a micro-granule dryer (Jetboxx MiniSet 1L, Helios GmbH, Dachau, Germany), with 40 ml of deionized water added. The drying temperature was set to 60 °C and the drying time to 2.5 h, and the granulated tablets were collected in plasticized cylinders. This produced tablets with an average diameter of 5 mm, which, upon resuspending in an aqueous buffer, still produced particles with an average particle size of 30 µm.

[0112] Example 6

[0113] Tablets are prepared from Rho-1D4, StrepTactin, or Ni-NTA MagBeads by lyophilization.

[0114] Transfer 40 µl of a 50% suspension of Rho-1D4, StrepTactin, or Ni-NTA MagBeads in deionized water into a microtiter plate with round orifices, freeze and lyophilize at -80°C.

[0115] The lyophilized beads were mixed with 30-50 µl of 1 g of polyvinylpyrrolidone (PVP) K30 (molecular weight ~ 40,000). g / mol Mix the solution in 2 ml of methanol and mix thoroughly. After an incubation period of 60 minutes, dry the beads in a vacuum desiccator at 200 mbar and room temperature.

[0116] After drying, the beads are completely covered with a predominantly transparent PVP film. When the film is removed from the mold, a stable sheet is formed.

[0117] Under a microscope, the beads in the plate are visible through a transparent membrane. They are partially adhered to each other, but can be transformed into individual particles by dissolving them in, for example, deionized water or a buffer solution.

[0118] When the flakes were mixed with water or a buffer solution (e.g., 100 mM sodium phosphate, pH 6.0), the PVP dissolved and the beads swelled regularly and smoothly, returning to their approximate original size within 1–2 minutes after estimation. No undissolved aggregated beads were detected.

[0119] Example 7

[0120] Biological application: binding antibodies to CNBr agarose.

[0121] For this application, a tablet in cylindrical form with a diameter of 3 mm and a height of 3 mm, prepared according to Example 1, is used.

[0122] One tablet was placed in an Eppendorf tube and 1 ml of 1 mM hydrochloric acid was added. The tube was mixed by pulsed vortexing, and the agarose tablet was dissolved into a homogeneous suspension. After 5 minutes, the reaction mixture was aspirated to dryness using a filter screen. The dried agarose was then added to a solution of 500 µg Rho-1D4 antibody (catalog number #40020, Cube Biotech) in 500 µl of 100 mM NaCl, 100 mM Na2CO3 at pH 8.3, and pulsed vortexing was performed.

[0123] The mixture was incubated at 4 °C for 20 hours on a head-to-tail reversing shaker. Then, the supernatant was removed, and the functionalized agarose was washed five times with 100 mM NaCl and 100 mM Na2CO3 at pH 8.3 and dried by suction.

[0124] The concentration of Rho-1D4 in the sodium carbonate / NaCl buffer solution was monitored by measuring the OD at 280 nm before and after the reaction, using sodium carbonate / NaCl buffer solution as a blank control. This analysis showed that over 90% of the provided antibody bound to the functionalized agarose. The Rho-1D4-functionalized agarose prepared in this way can be used for the reversible binding and purification of rho-tagged proteins.

[0125] Example 8

[0126] Biological application: Binding GFP protein to glyoxal agarose.

[0127] A cylindrical tablet of Aldehyde MagBeads, 2 mm in diameter and 2 mm in height, prepared according to Example 3, was placed in an Eppendorf tube and mixed with 2 ml of 100 mM sodium citrate, 50 mM sodium carbonate, pH 10.0. The suspension was vortexed and incubated for five minutes, followed by magnetic separation and removal of the supernatant. The washing steps were then repeated. 2 ml of 50 mM sodium acetate, 25 mM sodium carbonate buffer (pH 9.0) containing 40 mg of GFP (green fluorescent protein) was added to the residue. The preparation of GFP is known to the experts, but GFP is commercially available (catalog number #29903, Cube Biotech).

[0128] The reaction mixture was incubated for two hours on a head-to-tail reversing shaker, and then the supernatant was removed by magnetic separation.

[0129] The supernatant was diluted 1:10 and measured at 488 nm, and compared with the protein solution used for binding. A blank control was used with 100 mM sodium citrate, 50 mM sodium carbonate, and pH 10.0.

[0130] As a result of this binding assay, magnetic beads from 2 mm cylinders were able to bind 1.7 to 2.0 mg of GFP, or optionally other proteins or other biomolecules.

[0131] Example 9

[0132] Biological application: Purification of his-tagged proteins using Ni-INDIGO MagBeads XL

[0133] For this application, a tablet in cylindrical form with a diameter of 3 mm and a height of 3 mm, prepared according to Example 4, is used.

[0134] Place one tablet in an Eppendorf tube and add 1 ml of PBS buffer. Vortex the tube with a pulse, dissolving the agarose tablet into a homogeneous suspension in less than five minutes. After sedimentation, adjust the magnetic bead suspension to 25% (v / v), meaning that 1 ml of the suspension after sedimentation contains 250 µl of pure magnetic beads.

[0135] As a source of recombinant proteins, cell pellets containing recombinant his-tagged proteins are used. These pellets can be prepared from bacteria such as *E. coli*, insect cells, and human cells, which is an existing technique in protein biology. Alternatively, a his-tagged GFP solution (Cube Biotech) can be used to demonstrate reversible binding to magnetic beads.

[0136] Thaw the *E. coli* cell pellet on ice. Resuspend the cell pellet in 1 mL of lysis buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 8.0, optionally containing up to 20 mM DTT and EDTA, supplemented with 1 mg / mL lysozyme). Then, add 6 U Benzonase® (3 units / mL bacterial culture) to the lysate to reduce viscosity caused by genomic DNA. If necessary, incubate the mixture on ice for 30 minutes. Otherwise, incubation at room temperature (20–25°C) may be more effective.

[0137] The lysate was then centrifuged at 10,000 xg and 4°C for 30 minutes, and the supernatant was collected.

[0138] Resuspend PureCube His Affinity MagBeads by vortexing and transfer 40 μL of the 25% magnetic bead suspension to a conical microcentrifuge tube. Add 500 µL of lysis buffer and mix the sample by vortexing. Place the tube on a magnetic microtube rack until the beads are separated and discard the supernatant. Pipe 1 mL of the clarified lysate onto the equilibrated magnetic beads and incubate the lysate-magnetic bead mixture at 4 °C on a head-to-tail oscillator for 1 hour.

[0139] Place the tube on a magnetic microtube rack until the beads separate and remove the supernatant. Then, remove the tube from the magnet, add 500 µL of wash buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0) and mix the suspension by vortexing. Place the tube back on the magnetic microtube rack and allow the beads to separate. Remove the supernatant and repeat the washing step twice. Elute the his-tagged proteins with 100 μL of elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 250 mM imidazole, pH 8.0) and repeat the elution step. Now collect each elution fraction in a separate tube, determine the protein concentration of each fraction, and analyze all fractions by SDS-PAGE. When a large excess of poly-his GFP (200 mg protein per ml of pure agarose or magnetic beads) is provided, the elution buffer can contain more than 800 µg of protein, which means that the capacity of each pure bead is approximately 80 mg of protein.

[0140] Example 10

[0141] Biological application: Purification of Rho-tagged proteins using Rho-1D4 Mag Beads according to Example 6.

[0142] Required buffer solution:

[0143] Binding buffer: 20 mM HEPES, 150 mM NaCl, 0.1% LMNG (Anatrace, Mommy, Ohio, USA), pH 7.0.

[0144] Elution buffer: Mix 3 to 6 mL of binding buffer with 5 mg of Rho 1D4 peptide (Cube Biotech, Monheim, Germany) and dissolve the peptide in the buffer.

[0145] program:

[0146] Centrifuge the desired aliquots of membrane proteins with the Rho tag at 100,000 g for one hour to remove any unwanted membrane components that subsequently appear.

[0147] According to Example 6, tablets corresponding to 50 µl of pure MagBeads were filled into 2 ml reaction tubes. The mixture was equilibrated three times with 2 ml of binding buffer. The supernatant was separated and removed by magnetic separation. The dried resin was then mixed with 2 mL of membrane protein and incubated overnight on a head-to-tail oscillator in a refrigerator.

[0148] Separate the supernatant from the protein solution and mix 25 µL of sample with 25 µL of sample blue buffer, then store in a cool place. Next, wash the remaining resin eight times with four or two mL of binding buffer. For this purpose, incubate the resin on a head-to-tail reverse shaker in a refrigerator for 10 minutes. Remove excess buffer. Now add 200 µL of elution buffer to each sample and elute at 2–8 °C for one hour (± 10 min) while rotating. After elution, mix 25 µL of each supernatant with 25 µL of sample blue buffer, label, and store in a refrigerator. Elute twice in total. Heat the sample prepared with blue buffer and shake at 46 °C for 30 min, then analyze by SDS-PAGE and Coomassie blue staining. The gel is recorded by photograph and shows single bands with the molecular weight of the purified membrane protein with the Rho tag.

[0149] Example 11

[0150] Mechanical storage test

[0151] The mechanical stability of CNBr agarose, NHS MagBeads, INDIGO MagBeads XL, and glyoxal agarose tablets (as cylinders with diameters and heights of 2 mm and 3 mm, respectively) produced according to Examples 1-4 was tested by placing individual tablets in eppendorf tubes and rotating them in a head-to-tail reverse mixer (Bio-RS 24Mini Rotator, Biosan, Riga, Latvia) at a speed of 25 rpm for 24 hours.

[0152] After 24 hours, the tablet remained completely intact. No fragments were found, and no traces of agarose or magnetic beads were visible on the Eppendorf tube wall.

[0153] Example 12

[0154] Tablet resuspension study

[0155] The resuspension of CNBr agarose, NHS MagBeads, INDIGO MagBeads XL, and glyoxal agarose tablets (as cylinders with diameters and heights of 2 and 3 mm, respectively) produced according to Examples 1-4 were tested by placing a single tablet in an Eppendorf tube and adding 1 ml of dd water. All tubes were vortexed for ten seconds, and the vortexing step was repeated up to five times. At least after the fifth vortexing step, all tablets were dissolved, and no visible aggregates were found in the suspension; all agarose beads existed as independent particles.

[0156] Example 13

[0157] Comparison of BrCN MagBeads tablets with lyophilized BrCN MagBeads powder in terms of long-term chemical stability

[0158] Regarding long-term stability, a comparison was made between BrCN agarose tablets in cylindrical form (2 mm in diameter and 2 mm in height) according to Example 1 and BrCN activated agarose in powder form according to Example 1 but without tableting. Therefore, after production, tablets were weighed and powder of equal weight was dispensed into Eppendorf tubes under a protective gas atmosphere and stored. Binding assays were then performed monthly using one tablet and one powder sample with Rho-1D4 antibody according to Example 5, with 100 µg provided for each sample, and the amount of binding capacity was measured by comparing the OD 280 of the Rho-1D4 solution before and after binding between samples. In the evaluation, the OD 280 value of the initial solution was set to 100%, with binding buffer used as a blank control. The antibody concentration (in percentage) of the supernatant after the reaction was then subtracted from 100%.

[0159]

[0160]

[0161] The results of this comparison indicate that tablets made from cyanogen bromide-activated agarose have much better long-term stability compared to CNBr agarose powder.

[0162] literature

[0163] 1. Porath, J., et al. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258: 598-599.

[0164] 2. Hochuli, E., Dobeli, H., and Schacher, A. 1987. New metal chelateadsorbent for proteins and peptides containing neighbouring histidineresidues. J Chromatogr 411: 177-184.

[0165] 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 ChromatographyA, Volume 639, Issue 1, 1993, Pages 23-31, ISSN 0021- 9673, https: / / doi.org / 10.1016 / 0021-9673(93)83084-6.

[0166] 4. Richard F. Murphy, J.Michael Conlon, Ashraf Imam, Gregory J.C.Kelly, Comparison of nonbiospecific effects in immunoaffinity chromatographyusing cyanogen bromide and bifunctional oxirane as immobilising agents,Journal of Chromatography A, Volume 135, Issue 2, 1977, Pages 427-433, ISSN0021-9673, https: / / doi.org / 10.1016 / S0021- 9673(00)88384-3.

[0167] 5. Zhang X, Duan Y, Zeng X. Improved Performance of RecombinantProtein A Immobilized on Agarose Beads by Site-Specific Conjugation. ACSOmega. 2017 Apr 30;2(4):1731-1737. doi: 10.1021 / acsomega.7b00362. Epub 2017Apr 28. PMID: 30023643; PMCID: PMC6044777.

[0168] 6. Einarson MB, Orlinick JR (2002) Identification of Protein-ProteinInteractions 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.

[0169] 7. Whitten, Kenneth W., Kenneth D. Gailey, and Raymond E. Davis. "7-3Formation ofcovalent bonds." General Chemistry (1992): 264.

Claims

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

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

3. A material in tablet form, said tablet being a three-dimensional body with a side length and / or diameter of 1 mm to 10 mm, said material comprising a plurality of agarose particles with an average size of 10 µm to 250 µm.

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

5. The material in tablet form according to any one of claims 1-4, wherein, The tablets also contain lactose.

6. The material in tablet form according to any one of claims 1-5, wherein, The tablets also contain mannitol.

7. The material in tablet form according to any one of claims 1-6, wherein, The tablets also contain polyethylene glycol, which has a molecular weight of 200 to 20,000, preferably 1,000 to 8,000.

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

9. The material according to any one of claims 1-7, wherein, The tablet contains agarose that is functionalized for use in the immobilized metal ion affinity chromatography (IMAC) method.

10. The material according to any one of claims 1-7 and 9, wherein, The tablet contains agarose that is covalently bonded with a chelating agent.

11. The material according to any one of claims 1-7 and 9-10, wherein, Agarose is functionalized with iminodiacetic acid (IDA), NTA as Nα,Nα-bis(carboxymethyl)-L-lysine, or ethylenediaminetriacetic acid, wherein the IDA, NTA, or ethylenediaminetriacetic acid is coupled to a solid-phase group, such as an epoxy, carboxyl, or halide group, via their secondary and primary amino functional groups. Alternatively, agarose is functionalized with ethylenediaminetetraacetic acid (EDTA), wherein the EDTA is covalently coupled to the agarose, and a carboxylic acid group is covalently coupled to a solid-phase amino group via an amide group.

12. The material according to any one of claims 1-7 and 9-11, wherein, Agarose is functionalized with a chelating agent comprising a backbone having a solid-bound polyamine such as diethylenetriamine, triethylenetetramine, tetraethylenepentamine, or pentaethylenehexamine, wherein at least two amino functional groups of the polyamine are linked to the carboxyl group of EDTA via amide functional groups, and the polyamine is bound to the solid phase via one or more amino functional groups not modified with EDTA.

13. The material according to any one of claims 1-7 and 9-11, wherein, The chelating agent comprises a backbone having a solid-bound polyamine such as diethylenetriamine, triethylenetetramine, tetraethylenepentamine, or pentaethylenehexamine, wherein at least two amino functional groups of the polyamine are covalently bonded to the carboxyl group of an EDTA chain, the EDTA chain consisting of alternating EDTA molecules and diamines linked via amide functional groups, and the polyamine being bonded to the solid phase via one or more amino functional groups not modified with EDTA.

14. The material according to any one of claims 10-13, wherein, The chelating agent is loaded with metals such as nickel, cobalt, copper, zinc, iron, titanium, and zirconium.

15. The material according to any one of claims 1-14, wherein, The tablets also contain agarose functionalized with biomolecules.

16. The material according to any one of claims 1-15, wherein, The tablets also contain agarose functionalized with protein A or G.

17. The material according to any one of claims 1-16, wherein, The tablets also contain agarose functionalized with streptavidin.

18. The material according to any one of the preceding claims, wherein, The tablet also contains magnetic agarose microparticles, which are magnetic particles embedded in an agarose network.

19. The material according to any one of the preceding claims, wherein, The diameter of the magnetic particles is in the range of 8 to 5000 nm, preferably 25 to 2000 nm, and more preferably 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, the agarose having individual agarose particles with an average particle size between 10 and 500 µm, the method comprising applying pressure to a dry powder or a powder thickened with water or a 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 are added.

22. A method for preparing agarose in tablet form, wherein, Agarose, in either magnetic or non-magnetic form, is suspended in a polymer solution, then placed in a mold or container to remove the solvent, resulting in agarose embedded in a polymer film.

23. Use of the tablet form of agarose according to any one of claims 1 to 19 for the purification of proteins with his tag or Rho tag.

24. Use of agarose in tablet form according to any one of claims 1 to 19 for purifying proteins in a mixture of biomolecules.

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

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

27. Use of agarose in tablet form as described in any one of claims 1-19 for binding biotinylated biomolecules.

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

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

30. Use of the kit of claim 29 for purifying proteins with a his tag or proteins with a Rho tag.

31. Use of the kit of claim 29 for purifying proteins in a mixture of biomolecules.

32. Use of the kit according to claim 29 in diagnostics.

33. Use of the kit of claim 29 for covalently binding biomolecules.

34. Use of the kit of claim 29 for binding biotinylated biomolecules.

35. Use of the kit of claim 29 for binding antibodies.

36. Use of the material of any one of claims 1 to 19 for purifying proteins in a mixture of biomolecules.

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

38. Use of the material according to any one of claims 1-19 for covalently binding biomolecules.

39. Use of the material of any one of claims 1-19 for binding biotinylated biomolecules.

40. Use of the material according to any one of claims 1-19 for binding antibodies.

Images