DE10237518A1 - Removing biomolecules from liquid media, useful for purification, product recovery or preparation of vectors, comprises retention on hydrotalcite of controlled particle size - Google Patents

Abstract

Removing at least one biomolecule (I) from a liquid or fluid medium using as absorption agent a double layered hydroxide (II), i.e. natural or synthetic hydrotalcite or similar compounds, where (II) is calcined before use and has mean particle size (D50) over 1 micron, is new. An Independent claim is also included for a composition comprising (I) and (II).

Description

The present invention relates to the use of layered double hydroxides in processes for Storage of biomolecules, especially for enrichment or depletion of biomolecules liquid or fluid media and a composition with a layered double hydroxide and at least one biomolecule, which in particular as an inorganic vector or pharmaceutical composition for the introduction of biomolecules can be used in cells.

From the state of the art Use of certain layered double hydroxides as anion exchangers or adsorbent known.

For example, the EP 0 206 799 B1 the use of mixed metal hydroxides as anion exchangers, particularly for controlling dye migration in a liquid.

The EP 0 566 260 B1 describes a process for the treatment of liquids with hydrotalcites as sorbent, it being essential to the invention that the sorbent is at least partially undried or freshly prepared or generated in situ during the treatment of a liquid medium.

WO 01/49869 A1 describes compositions with nucleic acids, which are embedded in bilaminar mineral particles. Prefers Hydrotalcites are used to make a drug that serves to transfect cells with the embedded nucleic acids.

Similarly, the EP 0 987 328 A2 bioinorganic hybrid compositions that can absorb biomaterials stably. The hybrid composition has the formula [M ²⁺ ₁ _ _x N ³⁺ _x (OH) ₂ ] [A _{Bi o} ^n– ] _{x / n} ⋅yH ₂ O. The use of layered double hydroxides as non-viral vectors for introducing DNA into cells is also described in Choy et al., Angew. Chem. 2000, 112 (22), pages 4208-4211. The Lotsch et al., Solid State Sciences 3 (2001), pages 883-886, deals with the separation of nucleoside monophosphates using preferential anion exchange intercalation in layered double hydroxides.

The intercalation of amino acids and sugar in layered double hydroxides is described, for example, in Aisawa and Narita, Zeoraito ( 2000 ) 17, pages 101-107.

Ulibarri et al., Applied Clay Science 18 (2001), pages 17-27, Parker et al., Ind. Eng. Are further concerned with the use of layered double hydroxides or hydrotalcites and hydrotalcite-like structures for the absorption of certain anions. Chem. Res. 34 (1995), pages 1196-1202, Pavan et al., Journal of Colloid and Interface Science 229 (2000), pages 346-352, and the JP-A-63-132898 ,

According to the prior art as an anion adsorbent Layered double hydroxides used, however, are particularly suitable for stable and reversible attachment or storage of biomolecules, e.g. for the enrichment or depletion of biomolecules from liquid or fluid media, not suitable. So there is a constant need for improved Materials in this field.

The object of the invention was therefore to To provide layered double hydroxides, which are particularly efficient Attachment or storage of biomolecules, especially for attachment or Depletion of biomolecules liquid or fluid media. The disadvantages of the prior art are to be avoided and a particularly effective interaction between the layered double hydroxide and the biomolecules depending on the type of use.

This task is done with the procedure according to claim 1 solved. Preferred embodiments can be found in the claims 2 to 24.

According to the invention, a layered double hydroxide, selected from the group of natural and synthetic hydrotalcites and hydrotalcite-like ones Compounds are used, such as in the literature Catalysis today, Vol. 11 (No.2) of December 2, 1991, pages 173 to 301, are described ("hydrotacite-like compounds). Such Compounds have a hydrotalcite-like structure.

In principle, any method familiar to the person skilled in the art can be used to produce the hydrotalcites, as described, for example, in the above reference Catalysis today (loc. Cit.) On pages 173 to 301, in particular 201 to 212, in the DE 20 61 114 , the US-A-5,399,329 and 5,578,286, the DE 101 19 233 or WO 01/12570.

A layered double hydroxide (LDH) of the general empirical formula is particularly preferred [M ₁ _ _x ²⁺ N _x 3+ (OH) ₂ ] ^{x +} [A ^n- ] _{x / n} ⋅yH ₂ O used, where M ^{2+ is} at least one divalent metal ion and N ^{3+ is} at least one trivalent metal ion, A ^{n -} at least one anion, x is a rational number between 0 and 1, n is a positive number and y is a positive number including 0.

In general, any divalent metal ion suitable for layered double hydroxides and known to the person skilled in the art or a combination of two or more such metal ions can be used as M ²⁺ . In particular, M ^{2+ represents} one or more from the group of Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Mn ²⁺ , Co ²⁺ , Ni ²⁺ , Fe ²⁺ , Sr ²⁺ , Ba ²⁺ and / or Cu ²⁺ .

In general, any trivalent cation suitable for layered double hydroxides and known to the person skilled in the art or a combination of two or more such cations can be used as N ³⁺ . In particular, N ^{3+ represents} one or more from the group of Al ³⁺ , Mn ³⁺ , Co ³⁺ , Ni ³⁺ , Ni ³⁺ , Fe ³⁺ , Ga ³⁺ , SC ³⁺ , B ³⁺ and / or trivalent cations of rare earth metals.

After a particularly advantageous one embodiment according to the invention is M²⁺ Magnesium and N³⁺ Aluminum. It can according to the invention also such Double layer hydroxides (LDHs) are used which are also monovalent Cations such as Li⁺, which contain the can partially or completely replace divalent cations. Consequently are also layered double hydroxides with monovalent cations from the present invention, especially those with the General formula below:[M₁__x ¹⁺N_x ³⁺(OH)₂]^{b +}[A^n-] X / n⋅yH₂O . where M¹⁺ a suitable monovalent cation, in particular Represents Li +, N³⁺, A^n-. x and y have the meaning given above, and b = 2x – 1.

The anion A ^{n -} is, in the non-calcined form of the layered double hydroxide, selected from carbonate, nitrate, halide, sulfate, phosphate and / or arsenate. The anion A ^{n -} in the non-calcined form carbonate ^- is particularly preferred.

According to a particularly advantageous embodiment of the invention, the layered double hydroxide is a hydrotalcite whose general empirical formula is between [Mg ₂ Al (OH) ₆ ] (CO ₃ ) _0.5 and [Mg _2.5 Al (OH) ₇ ] (CO ₃ ) _0.5 is and in particular the molecular formula of the formula [Mg _2.2 Al (OH) _6.46 ] (CO ₃ ) _0.5 is sufficient. With these hydrotalcites, particularly advantageous sorbents for biomolecules were obtained.

Within the scope of the present invention became surprising found that a particularly advantageous storage or storage of the biomolecule takes place when the layered double hydroxide is used in calcined form becomes. Ability is preferred the layered double hydroxides used by the process of calcination destroyed Brucite-like Form structure (double layer structure) again. This happens in a solution the anions contained therein are incorporated. That ability is commonly referred to as "memory effect". It was surprising found that the use of calcined layered double hydroxides leads to a particularly effective attachment or storage of biomolecules.

The for calcination of layered double hydroxides calcination conditions used are familiar to the person skilled in the art. Generally can be calcined in an oven at 350 to 650 degrees Celsius over a period of time from 0.5 to 15 hours. Calcination is preferred at 450 to 600 degrees Celsius over one Period of 0.5 to 5 hours.

According to an advantageous embodiment of the invention the calcined layered double hydroxide in dry form with the biomolecule (s) (e) containing liquid or fluid medium brought into contact, so that preferably during the Reconstitution of the double layer structure in the liquid or fluid medium contained biomolecules to the layered double hydroxide bound or stored therein and / or enveloped by it. Without being limited to this theoretical mechanism, it is believed that while the reconstitution of the double layer structure in addition to an intercalation into the interstices also an adsorption of the negatively charged biomolecules on the positively charged surface of the regenerated layered double hydroxide and a (partial) coating of the biomolecules takes place. The newly emerging double-layer structure is thereby due to the presence of the polyanionic biomolecules in the desired Way, i.e. in favor of a gentle and effective approach or Storage, influenced.

According to a further embodiment according to the invention However, the calcined layered double hydroxide can also be used prior to contacting with which the biomolecule (s) e) containing liquid or fluid medium are at least partially "reconstituted", for example by suspending the dry calcined material in watery or watery Media. However, it is also a reconstitution over humidity and carbon dioxide in the air. Surprisingly, it was found that also reconstituted before use as a sorbent Layered double hydroxides a more advantageous attachment or storage of biomolecules show as non-calcined layered double hydroxides. It is believed that this is due to the reconstitution of the calcined layered double hydroxides related influences is due to the structure of the individual layered double hydroxide particles.

In summary, it was found that it when using calcined double layer hydroxides as Sorbent, if necessary after at least partial reconstitution the double-layer structure, to a particularly gentle and at the same time efficient attachment or storage of biomolecules to or in the layered double hydroxide comes, also from an at least partial wrapping of the biomolecules is assumed by the (reconstituted) double layer hydroxide.

A layered double hydroxide is particularly preferred according to the invention used, which was in the carbonate form before the calcination, since this favors the "memory effect".

According to the invention, it was also found that a particularly efficient and at the same time gentle storage and storage of biomolecules is achieved when the layered double hydroxide used in calcined form an average particle size (D50) of more than about 1 μm having. It is assumed that if this particle size is observed the and storage of the biomolecules into the reconstituting or reconstituted layered double hydroxide in the liquid or fluid medium favors becomes.

It has been found that in particular larger or long chain biomolecules according to the invention particularly have it gently and efficiently stored or stored ("pack"). It is believed that these larger LDH particles become one Accumulation or storage only on one or a few particles comes and probably also a particularly advantageous addition or storage and serving is made possible by the (reconstituted) layered double hydroxide.

The further processing of the biomolecules contained liquid or fluid medium, especially when separating the composition with the layered double hydroxide and the biomolecules from the medium inevitable occurring shear forces, can lead to fragmentation of, in particular, larger or long-chain biomolecules, or make at least parts of these molecules susceptible to enzymatic cleavages. This becomes evident when using the layered double hydroxides according to the invention especially in calcined form with the above minimum particle size avoided efficiently.

According to the invention, particular preference is given to this average particle sizes (D50) between about 2 μm and 50 μm, in particular between 3 μm and 20 μm, particularly preferably between 5 μm and 15 μm. The upper limit of the average particle size is determined by the respective Use determined, with particle sizes of more than about 50 microns in the Most applications have proven to be impractical the D10 value of the particle size between about 1.0 and 5.0 μm, in particular between 1.5 and 4.0 μm, and preferably the D90 value the particle size between about 10 and 45 μm, in particular between 15 and 30 μm. If these particle sizes are observed themselves surprising particularly advantageous binding properties for biomolecules, especially larger biomolecules.

According to a preferred embodiment of the invention the calcined layered double hydroxide has an average pore diameter of more than about 10 nm.

According to a further preferred embodiment of the invention will be more than 50%, preferably more than 65%, and in particular more than 75% of the pore volume of pores with a diameter of formed more than 10 nm.

According to a further preferred embodiment of the invention, the calcined layered double hydroxide has a BET surface area (determined in accordance with DIN 66131) of at least 50 m ² / g, in particular at least 100 m ² / g, particularly preferably of at least 150 m ² / g. The high surface area obviously promotes the reconstitution or the advantageous attachment and storage of the biomolecules.

Biomolecule is used in the context of the present Invention a molecule understood that as building blocks nucleotides or nucleosides (nucleobases), Amino acids, Monosaccharides and / or fatty acids having. Count in this group thus the mono-, oligo- and polymers from these building blocks, especially the Oligonucleotides and nucleic acids, Oligopeptides and proteins, oligo- and polysaccharides and / or the Lipids as well as their above building blocks and respective derivatives. All chemical modifications of the protruding molecules understood that continue as polyanions on layered double hydroxides can be bound. As a rule, these will be modifications or derivatizations the functional (side) groups of one or more building blocks of the biomolecules act. However, derivatives are also included in the scope of the invention, where the "backbone" of the biomolecule is modified, such as in the case of peptide nucleic acids (PNA).

According to an advantageous embodiment of the invention the biomolecules are DNA or RNA molecules in double-stranded or more single-stranded Form with one or more nucleotide building blocks; Amino acids or Oligopeptides and proteins as well as mono-, oligo- or polymeric carbohydrates and Lipids, including of glycoproteins and glycolipids, as long as these as (poly) anions can be attached to or incorporated in layered double hydroxides.

As mentioned above, leave biomolecules are particularly gentle with the aid of the present invention attach to or into the layered double hydroxides. This advantage according to the invention comes naturally especially with larger or relatively long biomolecules like longer chain Oligonucleotides and nucleic acids or oligopeptides and proteins or long chain saccharides for Validity.

A preferred aspect of the present invention therefore relates to the use of the layered double hydroxides defined above for removing or obtaining biomolecules from liquid or fluid media which contain at least one larger or long-chain biomolecule as defined below. The larger or long-chain biomolecule, if it is an oligopeptide or protein, preferably has at least 5 amino acids, preferably at least 50 amino acids and particularly preferably at least 100 amino acids; if it is an oligonucleotide or a nucleic acid, at least 10 nucleotide building blocks, preferably at least 100 nucleotide building blocks and particularly preferably min at least 1000 nucleotide building blocks; if it is an oligosaccharide or polysaccharide, at least 3 monosaccharide components, preferably at least 30 monosaccharide components and particularly preferably at least 100 monosaccharide components.

After a particularly beneficial aspect of the invention contains the above composition thus has at least one biomolecule with a Molecular weight of at least 200 D, especially 10 kD, particularly preferably at least 100 kD. The size of the biomolecule are there are no upper limits in the context of the invention, so that also very large biomolecules with more than 500 kD or more than 1 MD or can be stored.

Regarding amino acids the inventive method or the use according to the invention the calcined layered double hydroxides, in particular for liquid or fluid media suitable, the peptides or proteins with at least 5 amino acids, preferably at least 50 amino acids and particularly preferred at least 100 amino acids exhibit.

With regard to nucleic acids the inventive method particularly advantageous for liquid or fluid media, the oligonucleotides or nucleic acids with at least 10 bases (pairs), preferably with at least 100 bases (pair), in particular contain at least 1000 bases (pair).

In terms of carbohydrates, that is inventive method particularly advantageous for liquid or fluid media, the oligonucleotides or nucleic acids with at least 3 monosaccharide units, preferably with at least 30 monosaccharide units and in particular at least 100 monosaccharide units contain.

Under liquid or fluid media all media are understood, which a contacting and thus a connection or Storage of the biomolecules allow in the layered double hydroxide. In practice, these are in particular aqueous or aqueous media in the form of a solution, suspension, dispersion, colloidal solution or emulsion.

Typical examples are industrial or non-industrial wastewater, Process process waters, Fermentation residues or media, biomolecules containing media from medical or biological research, with biomolecules contaminated, liquid or fluid contaminated sites and the like.

After another aspect concerns the invention a method comprising the following steps: a) bringing the liquid into contact or fluid medium containing at least one biomolecule as in any of the preceding claims defined, with the sorbent, optionally after at least partial reconstitution of the double layer structure of the calcined Schichtdoppelhydroxids; b) Enable the attachment or storage of the at least one biomolecule to or in the layered double hydroxide; c) separating the attached or stored biomolecule with the layered double hydroxide; d) if necessary separation or Desorption of the at least one biomolecule from the layered double hydroxide.

The method according to the invention for storage or storage of biomolecules on or in layered double hydroxides can be used both for enrichment (i.e. increase the concentration of the desired biomolecules) as well as depletion (i.e. reducing the concentration of the desired biomolecules) be used.

If the method according to the invention aimed at removing or disposing of biomolecules can in one a further step is the layered double hydroxide containing the biomolecules to be disposed of. The disposal can, for example, by thermal treatment for the removal of those containing the biomolecules Layered double hydroxide take place after thermal disintegration of the biomolecules that Layered double hydroxide can be disposed of particularly advantageously.

So it is according to a first aspect of the invention possible, biomolecules deliberately remove from media. This is an example wastewater treatment plays a big role here in most states strict legal regulations for the removal of biomolecules wastewater consist.

According to a further preferred embodiment The invention can also be the depletion or removal of biomolecules Culture media performed become. For example, in bioreactors due to the im Medium containing high concentration of biomolecules, in particular high molecular nucleic acids, to an undesirable Increase in viscosity come. Here can over the inventive method an efficient and biologically compatible removal of the disruptive biomolecules from the culture medium respectively. By adding the sorbent according to the invention to the Culture medium can reduce the viscosity also to a desired one Dimension set become.

Likewise, in many cases it is desirable that Concentration of biomolecules increase in a medium or these biomolecules preferably to win in pure form. For example, the extraction or purification falls desired Proteins, carbohydrates or nucleic acids from solutions to standard procedures in biological and medical research. According to the invention, in one next step is the biomolecule are desorbed or recovered from the double hydroxide layer, whereby the layered double hydroxide may be used again can.

According to a further aspect according to the invention, the layered double hydroxide particles can be connected to larger agglomerates, granules or shaped bodies by means of a suitable binder or to one Carrier are applied. The shape and size of such superordinate structures, which contain the primary layered double hydroxide particles, depends on the particular application desired. It is therefore possible to use all shapes and sizes familiar to the person skilled in the art and suitable in individual cases. For example, in many cases agglomerates with a diameter of more than 10 μm, in particular more than 50 μm, may be preferred. In the case of a bed for chromatography columns and the like, a spherical shape of the agglomerates can be advantageous. A possible carrier is, for example, calcium carbonate.

Any binder familiar to the person skilled in the art can also be used can be used as long as the biomolecules are deposited or embedded in or to the layered double hydroxide particles is not impaired too much or those for the stability of the particle agglomerates or the required application moldings guaranteed is. For example, without being limited to this, as Binding agents are used: agar-agar, alginates, chitosans, pectins, Gelatins, lupine protein isolates or gluten.

Another aspect of the present The invention relates to a composition with a layered double hydroxide as defined above and at least one biomolecule as above Are defined.

Another aspect of the present invention relates to the use of the compositions according to the invention as inorganic vectors for introducing biomolecules into cells or as a pharmaceutical composition, in particular as a reservoir for the storage and controlled release of biomolecules, preferably of nucleic acids. It has surprisingly been found that the gentle and effective attachment or incorporation of the biomolecules into or onto the layered double hydroxides is obtained by bringing the liquid or fluid medium containing the biomolecules into contact with the calcined or (partially) reconstituted layered double hydroxide can also be used for a particularly efficient introduction of these biomolecules into prokaryotic or eukaryotic cells. Apparently, according to the method according to the invention, biomolecules, in particular nucleic acids, can be packaged in a particularly advantageous manner for introduction into cells. The basic mechanism of such an introduction of DNA-LDH nanohybrids is described, for example, in the reference Choy et al., Angew. Chem. 2000, 112 (22), pages 4207-4211, and in EP 0 987 328 A2 to which reference is made and which are hereby incorporated by reference into the description. The use as a pharmaceutical composition, in particular as a reservoir for the storage and controlled release of biomolecules, preferably of nucleic acids, is described as such in WO 01/49869, to which reference is made and which is hereby incorporated by reference into the description.

The BET surfaces specified here were determined according to DIN 66131.

The specified (average) pore diameters, -volumes and areas were measured using a fully automatic nitrogen adsorption meter (ASAP 2000, Micrometrics) according to the manufacturer's standard range (BET, BJH, t-plot and DFT). The percentages of the Proportion of certain pore sizes the total pore volume of pores between 1.7 and 300 nm in diameter (BJH Adsorption Pore Distribution Report).

The invention is now based on the the following non-limiting Examples closer explained.

There were the following hydrotalcites used as starting materials, the production of corresponding Layered double hydroxides also by methods familiar to the person skilled in the art (see above) easily possible is. The setting of the porosimetry and the surfaces of the Layered double hydroxides can in particular be carried out through routine tests easy for hydrothermal after-treatment and for calcination.

• 1. HT-696, available from Sud-Chemie AG CERA TOFIX ^® NA (HT-696), a non-calcined Mg / Al hydrotalcite is carbonate.
• 2. HT-701, available from Sud-Chemie AG CERA TOFIX ^® NA (HT-701) is a Mg / Al hydrotalcite in calcined form.
• 3. HT-16, available under the trade name NA CERATOFIX ^® (HT-16) by Sud-Chemie AG is a nichtcalcinierter Mg / Al hydrotalcite in the nitrate form.

The calcined hydrotalcites were either directly to the liquid medium containing the biomolecules given, or previously suspended in water to the hydroxide form produce.

For comparative activation of HT-696 with acid became 0.1 g of the hydrotalcite in the form of a 10% suspension in distilled water 28.6 μl 99.8% acetic acid given, thoroughly mixed and then Centrifuged at 2500 rpm for 10 min. The residue was removed the acid residue suspended in 5 ml Tris buffer (10 mM Tris / HCl, pH 8.0) and again centrifuged.

As a further comparison, the Hydrotalcite HT-16 converted from the nitrate form to the chloride form by 0.1 g of the dry material in 5 ml buffer solution (10mM Tris / HCl, 1M NaCl, pH 8.0), centrifuged and to remove any remaining Washed NaCl in 5 ml Tris buffer (see above) and centrifuged again were washed (see Tables I and II; HT-16).

1. Determination the DNA binding capacity

0.1 g of the above hydrotalcite samples are added to 5 ml DNA solution of herring sperm-DNA sodium salt or acid, Sigma, Schnelldorf) of known concentration (2.5 mg / ml) and mixed until a uniform suspension is formed , The sample is then shaken at 250 rpm for 1 h or 16 h at room temperature. It is centrifuged at 2500 rpm for 15 min (the residue is used below in the elution experiments) and the supernatant is sterile filtered. If the buffer used does not contain EDTA, 10 μl 0.5 M EDTA solution is added to protect the samples from DNase contamination. The DNA concentration of the supernatant is determined photometrically on the basis of UV absorption at 260 nm (Sambrook et al., "Molecular Cloning - A Laboratory Manual", Volume III, page Cl, Cold Spring Harbor Laboratory Press, 2 ^nd Edition (1989) The binding capacity is calculated from the concentration difference.

It was described for the above Hydrotalcite samples the static binding capacities for both the DNA acid and also for the DNA salt (see above) is determined.

As a buffer for the preparation of the DNA solutions both a Tris buffer (10mM Tris / HCl, pH 8) and a TE buffer (10mM Tris / HCl, 1mM EDTA, pH 8) was used. The capacities determined were for both Buffer systems identical.

The capacities are always based on weight of the material used. When activated with acid the weight of the inactivated, dry form is given.

All experiments were carried out three times as The result is the average of the three measurements in the following Tables I and II given.

Table I: Binding capacities of various hydrotalcites and the comparison systems for DNA acid

Table II: Binding capacity of various hydrotalcites and the comparison systems for DNA salt

It turns out that when using the used according to the invention calcined hydrotalcits binding capacities for DNA both in the acid form as well as the salt form are considerably higher than not activated or acid activated Hydrotalcites and hydrotalcite in the nitrate form or in the chloride form of Case is.

The mean particle size (D50) of calcined HT-701 was 8.2 μm, the D10 2.7 μm and the D90 at around 20 μm. For comparison, the average pond size of HT-701 was fractionated by a dynamic classifying process using a classifying wheel. By adjusting the speed and the air speed, it is possible to set any medium pond sizes. The static binding capacities of particle size fractions of HT-701 thus prepared were determined by the above method. It was found that with an average particle diameter of less than 1 μm, a considerably poorer binding capacity for DNA salt or acid was obtained. The optimum binding capacity was found for medium pond sizes (D50) in the range between 2 μm and 50 μm, in particular between 3 μm and 20 μm. The highest binding capacities were achieved at D50 values between 5 μm and 15 μm.

2. Elution of the bound DNA:

Before the elution, the respective Residue (see point 1. above) with 5 ml Tris buffer (10mM Tris / HCl, pH 8) washed, then the indicated volume of the elution buffer below added and shaken at 250 rpm for 16 h. It is centrifuged for 15 min at 2500 rpm and the supernatant sterile. The DNA concentration of the elution fraction becomes determined photometrically and the recovery rate calculated.

For elution of the DNA from the layered double hydroxide the following buffer was used: 0.5 M Na₃PO₄, pH 7.3 with Na₃P O₄ set (elution buffer). With this buffer, a relative elution volume of 500ml / g the predominant Part of the DNA from the used according to the invention LDH eluted and in the supernatant be detected photometrically.

This is a reversible bond the DNA to the hydrotalcite and obtaining the desired one biomolecule in pure form possible. The separated double layer hydroxide can, if necessary, after Calcination again, can be used again.

3. Binding of Proteins using the example of BSA

BSA (fraction V, Sigma) used.

In each case 0.1 g of the hydrotalcite samples specified at the outset were added to 5 ml of BSA solution in TE buffer (c = 2 mg⋅ml ⁻¹ ) and shaken at 250 rpm for 16 h. After centrifuging for 10 min at 2500 rpm, the protein concentration in the supernatant was determined photometrically by measuring the absorption at 280 nm. This experiment was also carried out three times.

Here too it was shown that when used the calcined used in the present invention Hydrotalcite's binding capacities for protein significantly higher are as this with non-activated or acid-activated hydrotalcites and hydrotalcite in the nitrate or chloride form is the case.

4. Transfection of NIH-3T3 cells with hydrotalcite as an inorganic vector

a) Cell cultivation:

The NIH-3T3 cell line is used for the transfection experiments. These are adherently growing mouse embryo fibroblasts. The doubling time is approximately 20 hours. The cells are cultivated in the standard medium DMEM (with 4.5 g⋅l ^-1 glucose) with 10% NCS. The cultivation takes place in an incubator at 37 ° C under a humidified 5% CO ₂ atmosphere.

To prepare the stock culture, the thawed cell suspension is placed in a monolayer bottle (25 cm ² ) with 10 ml medium. A direct cell number determination is not possible with adherently growing cells; the growth rate is controlled via the degree of coverage of the culture vessel. At approx. 90% confluency - usually after 3-4 days - the culture is implemented in order to avoid the overgrowth of the cells (focal formation). For this purpose, the medium is decanted off, trypsin solution is added and incubated in the incubator for 10 min. The detached cell suspension is mixed with approximately 15 ml of serum-containing medium in order to block the trypsin. The centrifuged cells are resuspended in fresh medium and sown again at a dilution of 1:10.

b) Transfection:

As an inorganic vector for the transfection of NIH-3T3 cells, the hydrotalcite samples mentioned at the outset were "loaded" with the plasmid pQBI25-fC1 (company Qbiogene, Heidelberg) before the transfection. For this, 10 mg of the respective hydrotalcite sample were weighed out and washed with ethanol for sterilization. Then 2 ml of sterile, distilled water were added, vortexed briefly and centrifuged for 3 min at 5000 rpm. 1.5 ml of sterile plasmid DNA with a concentration of 1.45 ml-ml- ^{1 was} added to the residue and shaken at 250 rpm for 16 h at room temperature.

The DNA hydrotalcite hybrid was suspended in 10 ml of distilled, sterile water. The transfecti on with hydrotalcite as a vector was carried out with two different concentrations of DNA-hydrotalcite hybrid. The NIH-3T3 cells were seeded 24 h before the transfection with a density of 1–2⋅10 ⁵ cells⋅cm ^–2 in 6-hole plates and in the incubator at 37 ° C with humidified 5% CO ₂ - Atmosphere incubated. The stated quantities refer to one hole in the 6-hole plate. The analysis was carried out 48 hours after the start of the transfection with the fluorescence microscope. Transfected cells can be recognized by the GFP production, when excited with 474 nm they glow green when viewed under a fluorescence microscope.

Version 1:

That was right before the experiment Medium removed and 1.5 ml of new medium added. 25 or 100 μl of DNA hydrotalcite suspension was added and it was incubated for 3 h incubated. Subsequently the medium was changed and incubated for a further 45 h.

Variant 2 :

That was right before the experiment Medium removed and 1.5 ml of new medium added. 25 or 100 μl of Suspension was added and incubated for 24 hours. Then was the medium was changed and incubated for a further 24 h.

When using the hybrid with the (Reconstituted) calcined hydrotalcite (3rd, see above) could be compared to the Using the comparison materials significantly more successfully transfected cells are detected using fluorescence, so that the DNA hydrotalcite hybrids according to the invention are suitable as inorganic vectors.

Claims (36)

Method of removing or recovering at least one biomolecule from a liquid or fluid medium with the aid of a sorbent, where as Sorbent a layered double hydroxide selected from the group of natural and synthetic hydrotalcites and the hydrotalcite-like Compounds is used, and wherein the layered double hydroxide calcined before use, and an average particle size (D50) of more than 1 μm having. A method according to claim 1, characterized in that the at least one biomolecule selected is from the group of nucleic acids, Proteins, polysaccharides and / or lipids as well as their building blocks and respective derivatives. Method according to one of the preceding claims, characterized in that that the at least one biomolecule is selected from oligomers and polymers based on nucleotides, amino acids and carbohydrates. Method according to one of the preceding claims, characterized in that that the at least one biomolecule has a molecular weight of at least 200 D, preferably 10 kD, particularly preferably at least 100 kD, and in particular has at least 500 kD. Method according to one of the preceding claims, characterized in that that the at least one biomolecule if it is an oligopeptide or protein, at least 5 amino acids, preferably at least 50 amino acids and particularly preferably has at least 100 amino acids; if it is is an oligonucleotide or a nucleic acid, at least 10 Nucleotide building blocks, preferably at least 100 nucleotide building blocks and particularly preferably has at least 1000 nucleotide building blocks; if it is an oligosaccharide or polysaccharide, at least 3 monosaccharide building blocks, preferably at least 30 monosaccharide building blocks and particularly preferably has at least 100 monosaccharide building blocks. Method according to one of the preceding claims, characterized in that that the liquid or fluid medium around a colloidal solution, suspension, dispersion, solution or emulsion. Method according to one of the preceding claims, characterized in that it comprises the following steps: a) bringing the liquid or fluid medium containing at least one biomolecule as defined in one of the preceding claims into contact with the sorbent, optionally after at least partial reconstitution the double-layer structure of the calcined layered double hydroxide, b) Ermögli Chen the attachment or incorporation of the at least one biomolecule onto or into the layered double hydroxide, c) separating the attached or embedded biomolecule with the layered double hydroxide, d) optionally separating or desorption of the at least one biomolecule from the layered double hydroxide. Method according to one of the preceding claims, characterized in that that the mean particle size (D50) between about 2 μm and 50 μm, in particular between 3 μm and 20 μm, particularly preferably between 5 μm and 15 μm lies. Method according to one of the preceding claims, characterized in that M ²⁺ one or more from the group of Mg ²⁺ , Ca ²⁺ , Zn ²⁺ , Mn ²⁺ , Co ²⁺ , Ni ²⁺ , Fe ²⁺ , Sr ²⁺ , Ba ²⁺ and / or Cu ²⁺ . Method according to one of the preceding claims, characterized in that N ³⁺ one or more from the group of Al ³⁺ , Mn ³⁺ , Co ³⁺ , Ni ³⁺ , Cr ³⁺ , Fe ³⁺ , Ga ³⁺ , Sc ³⁺ , B ³⁺ and / or three valuable cations of rare earth metals. Method according to one of the preceding claims, characterized in that M ^{2+ is} magnesium and N ^{3+ is} aluminum. Method according to one of the preceding claims, characterized in that the anion A ⁿ - is selected in the non-calcined form from carbonate, nitrate, halide, sulfate, phosphate and / or arsenate. Method according to one of the preceding claims, characterized in that the anion A ^{n - is} carbonate in the non-calcined form. Method according to one of the preceding claims, characterized in that the layered double hydroxide is a hydrotalcite, the general empirical formula of which is between [Mg ₂ Al (OH) ₆ ] (CO ₃ ) _0.5 and [Mg _2.5 Al (OH) ₇ ] ( CO ₃ ) _0.5 and in particular I the general formula [Mg _2.2 Al (OH) ₆ , ₄ ] (CO ₃ ) _0.5 is sufficient. Method according to one of the preceding claims, characterized in that the calcined layered double hydroxide has a BET surface area of at least 50 m ² / g, in particular at least 100 m ² / g, particularly preferably of at least 150 m ² / g. Method according to one of the preceding claims, characterized in that that the calcined layered double hydroxide has an average pore diameter of more than about 10 nm. Method according to one of the preceding claims, characterized in that that more than 50%, preferably more than 65%, and especially more than 75% of the pore volume of pores with a diameter of more than 10 nm. Method according to one of the preceding claims, characterized in that that the biomolecule to be removed or obtained is bound, preferably at least partially between the layers of the layered double hydroxide is stored and / or enveloped by it. Method according to one of the preceding claims, characterized in that that the liquid or fluid medium around an aqueous Medium, preferably a fermentation medium, a fermentation residue from cell culture, process water or a waste water with at least a biomolecule like in one of the preceding claims acts in a defined manner. Method according to one of the preceding claims, characterized in that that in a further step the layered double hydroxide containing the biomolecules is disposed of. Method according to one of the preceding claims, characterized in that that in a further step the biomolecule from the layered double hydroxide is desorbed or recovered, whereby the layered double hydroxide 99f. can be used again. Method according to one of the preceding claims, characterized in that the layered double hydroxide has the following general formula: [M _1-x ²⁺ N _x ³⁺ (OH) ² ] ^{x +} [A ^{n -} ] x / n⋅yH ₂ O, where M2 + is at least one divalent metal ion and N ^{3 + is} at least one trivalent metal ion, A ^{n -} at least one anion, x is a rational number between 0 and 1, and y is a positive number including 0. Method according to one of the preceding claims, characterized in that the divalent metal ions in the layered double hydroxide are replaced in whole or in part by monovalent cations, in particular lithium, and the layered silicate preferably has the following general formula: [M _1-x ¹⁺ N _x 3 ⁺ (OH) ₂ ] ^{b +} [A ^{n -} ] _{x / n} ⋅yH ₂ O, where M ^{1+ is} a suitable monovalent cation, in particular lithium, and N ^{3 +} , A ^{n -} , x and y have the meaning given in claim 23, and b = 2x – 1. Method according to one of the preceding claims, characterized in that that the layered double hydroxide has a particle size (D10) between about 1.0 and 5.0 μm, in particular between 1.5 and 4.0 microns, and preferably the D90 values of particle size between about 10 and 45 μm, in particular between 14 and 30 μm lie. Composition with a layered double hydroxide as in one of the preceding claims defined and at least one biomolecule as in one of the preceding Expectations Are defined. Use of the composition according to claim 25 as an inorganic Vector for the introduction of biomolecules into cells, or as a pharmaceutical Composition, especially as a reservoir for storage and controlled release of biomolecules, preferably nucleic acids. Use of a layered double hydroxide as in one of claims 1 to 24 defined as a sorbent for enriching or depleting biomolecules according to a of the preceding claims 1 to 24 from liquid or fluid media, especially for the removal of biomolecules Fermentation media, fermentation residues from cell culture or wastewater or contaminated sites. Use according to claim 27 to reduce or adjust the viscosity of the medium. Use according to claim 27 or 28 in the manufacture or purification of biotechnologically manufactured Substances and active ingredients. Use according to a of claims 27 to 29 for the purification of nucleic acids. Use according to a of claims 27 to 30, characterized in that the layered double hydroxide particles combined with a binder to form particle aggregates or moldings or on a carrier are upset. Use according to claim 31, characterized in that the binder is Alginate, agar-agar, chitosans, pectins, gelatins, lupine protein isolates or gluten. Procedure according to a of claims 1 to 24, characterized in that the layered double hydroxide particles combined with a binder to form particle aggregates or moldings or on a carrier are upset. Method according to claim 33, characterized in that the binder Alginate, agar-agar, chitosans, pectins, gelatins, lupine protein isolates or gluten. Composition according to claim 25, characterized in that the layered double hydroxide particles combined with a binder to form particle aggregates or moldings or on a carrier are upset. Composition according to claim 35, characterized in that the binder Alginate, agar-agar, chitosans, pectins, gelatins, lupine protein isolates or gluten.