EP2209543A1 - Verwendung von schichtdoppelhydroxiden zur an- bzw. abreicherung von biomolekülen aus flüssigen oder fluiden medien - Google Patents
Verwendung von schichtdoppelhydroxiden zur an- bzw. abreicherung von biomolekülen aus flüssigen oder fluiden medienInfo
- Publication number
- EP2209543A1 EP2209543A1 EP08802738A EP08802738A EP2209543A1 EP 2209543 A1 EP2209543 A1 EP 2209543A1 EP 08802738 A EP08802738 A EP 08802738A EP 08802738 A EP08802738 A EP 08802738A EP 2209543 A1 EP2209543 A1 EP 2209543A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- layered double
- double hydroxide
- biomolecule
- hydrotalcite
- biomolecules
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/06—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04
- B01J20/08—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising oxides or hydroxides of metals not provided for in group B01J20/04 comprising aluminium oxide or hydroxide; comprising bauxite
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02C—CAPTURE, STORAGE, SEQUESTRATION OR DISPOSAL OF GREENHOUSE GASES [GHG]
- Y02C20/00—Capture or disposal of greenhouse gases
- Y02C20/40—Capture or disposal of greenhouse gases of CO2
Definitions
- the present invention relates to a method for enrichment or depletion of biomolecules, in particular peptides, depspeptides, Peptidnuklemsauren (PNAs) and proteins from liquid media by means of a Schichtdoppelhydroxids.
- the present invention relates to the use of certain layered double hydroxides for the addition or incorporation of at least one compound selected from the above group of biomolecules.
- Biomolecules such as enzymes, hormones or antibodies, belong to the group of proteins on which almost every biological reaction depends.
- the large-scale production of these biomolecule is done today with the help of cell culture technology in microorganisms or Saugetlerzellen in so-called fermenters.
- the target proteins are purified in a series of steps from the rest of the cell mass. This purification can involve many steps and thus can cost up to 80% of the total cost of the process.
- chromatographic methods are usually used to enrich the target protein in the process stream on the one hand and on the other hand to separate foreign proteins and other contaminants such as endotoxins, viruses or DNA. If the chromatography materials are used repeatedly, they must be continually checked for their separation performance or any microbial contamination.
- Hydrotalcite belongs to the class of so-called Schichtdoppel- hydroxide (layered double hydroxides, LDH), represented by the general formula [M 2+ i x N 3+ x (OH) 2] [A n "] x / n • y H 2 O.
- Schichtdoppel- hydroxide layered double hydroxides, LDH
- M 2+ is a divalent alkaline earth or transition metal ion such as Mg 2+ , Ni 2+ , Cu 2+ or Zn 2+
- N 3+ is a trivalent main group or transition metal ion such as Al 3+ , Cr 3 + , Fe 3+ or Ga 3+
- x is a rational number between 0 and 1 and y is a positive number including 0.
- Hydrotalcite is usually composed of magnesium and aluminum ions and carbonate as an anion Hydrotalcite has a cationic layer structure and could therefore be used as an ion exchanger for anionic proteins
- An overview of common chromatographic materials for ion exchangers is available in: Protein purification: principles, high resolution methods, and application, 1998 2 nd edition, pages 164-165.
- hydrotalcite in the activated, calcined form as anion exchanger or adsorbent is known from the prior art. When calcining hydrotalcite in the Usually treated for several hours at 600 0 C, with water and carbon dioxide escape. The calcined hydrotalcites have basic properties and are very heat stable.
- US 2005/0196850 A1 describes the immobilization of the enantioselective enzyme lipase from the organism Candida rugosa on conventional Mg-Al hydrotalcite.
- DE 102 37 518 A1 and DE 102 37 517 A1 describe a process for the treatment of biomolecules, in particular DNA, in liquid or fluid media using a calcined layered double hydroxide as sorbent.
- the layer double hydroxides used according to the prior art as Anionenadsorptionsmittel are especially for the stable and reversible attachment or storage of biomolecules from the group of proteins, peptides, depsipeptides, Peptidnuklem- acids (PNAs), especially in active form, eg to obtain enzymatic Activity in enzymes or binding speci fi cations, eg in the case of antibodies, etc., or for accumulation or depletion of biomolecules from liquid or fluid media is not sufficiently suitable.
- PNAs Peptidnuklem- acids
- the object of the invention was therefore to provide stable and inexpensive layered double hydroxides, which enable a particularly efficient accumulation or incorporation of proteins, in particular for accumulation or depletion of proteins from liquid or fluid media.
- the attached or embedded biomolecules from the group of proteins, peptides, depsipeptides, peptide nucleic acids (PNAs) should be immobilized in native or active form, if possible, in order to keep the desired properties of the biomolecule as unchanged as possible relative to the non-immobilized starting biomolecule allow. In a preferred task, it should also be possible to recover the separated biomolecules.
- the disadvantages of the prior art should be avoided and a particularly effective interaction between the layered double hydroxide and the proteins should be ensured depending on the type of use.
- certain layered double hydroxides with a specific ratio of Mg 2+ to Al 3+ cations represent a particularly favorable accumulation or incorporation of biomolecules selected from the group of proteins, peptides, depsipeptides, peptide nucleic acids (PNAs). and unexpectedly better suited for the immobilization of such biomolecules in "active" form.
- Erfmdungsgelien is therefore the molar ratio of Mg 2+ to Al 3+ in Schichtdoppelhdydroxid at a maximum of 2.5: 1.
- the present invention relates to the use of a layered double hydroxide selected from the group consisting of natural and synthetic hydrotalcites and compounds having a hydrotalcite-like structure, wherein the layered double hydroxide is in uncalcined form and m is carbonate form, and wherein the molar ratio of Mg 2+ to Al 3+ in Schichtdoppelhdydroxid at a maximum of 2.5: 1, for on or Incorporation of a compound selected from the group consisting of biomolecules, in particular peptides, depsipeptides, peptide-nuclear acids (PNAs) and proteins.
- a layered double hydroxide selected from the group consisting of natural and synthetic hydrotalcites and compounds having a hydrotalcite-like structure, wherein the layered double hydroxide is in uncalcined form and m is carbonate form, and wherein the molar ratio of Mg 2+ to Al 3+ in Schichtdoppelhdydroxid at a maximum of 2.5: 1, for
- the layered double hydroxides used according to the invention are thus selected from the group consisting of natural and synthetic hydrotalcites and compounds having a hydrotalcite-like structure.
- the person skilled in the art is familiar with what is meant by hydrotalcites and compounds having a hydrotalcite-like structure.
- a layered double hydroxide selected from the group of natural and synthetic hydrotalcites and hydrotalcite-like compounds can be used, as described, for example, in US Pat Catalysis Today, Vol. 11 (No.2) of December 2, 1991, pages 173 to 301. (“hydrotacite-like compounds”) Such compounds have a hydrotalcite-like structure.
- the molar ratio of Mg 2+ to Al 3+ is at most 2.5: 1, preferably at most 2.3: 1, more preferably at most 2, 1: 1, in particular at most 2.0: 1, and / or the weight loss ratio of M 2+ to N 3+ , calculated as the weight ratio of the oxides MO to N 2 O 3 , at a maximum of 2.2: 1, preferably at a maximum of 2.0: 1, in particular a maximum of 1.85: 1.
- the molar ratio of Mg 2+ to Al 3+ is at least 0.25: 1, in particular at least 0.3: 1, and / or the weight ratio of Mg 2+ to Al 3+ , calculated as the weight ratio of the oxides MgO to Al 2 O 3 , at least 0.22: 1, in particular at least 0.26: 1.
- M 2+ is at least one divalent metal ion (Mg 2+ ) and N 3+ is at least one trivalent metal ion (Al 3+ ),
- a n is at least one anion (carbonate),
- x is a rational number between 0 and 1
- n a positive number and
- y is a positive number including 0.
- the layered double hydroxide does not contain Zn.
- LDHs double-layer hydroxides
- monovalent cations such as, for example, Li +
- monovalent cation layered double hydroxides are also covered by the present 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 O / 28 alo, 72 (OH) 2 ] (CO 3 ) 0 , 72 lies
- materials which, in addition to a hydrotalcite phase, also have a boehmite phase are particularly well suited.
- Such a boehmite phase often occurs at high aluminum contents of the hydrotalcites.
- both mixed phases and mixtures of materials with hydrotalcite phase and materials with boehmite phase can be used.
- the hydrotalcite portion is 55% by weight or more.
- the layer double hydroxides used according to the invention are present in non-calcined form. Calcination is understood in particular to mean a temperature treatment in which the double-layer structure is completely or partially lost. According to a preferred aspect, the layered double is considered uncalci- mert when there is no temperature treatments at greater than about 150 0 C, in particular more than 250 0 C, in particular more than 350 0 C, in particular more than 450 0 C, in particular more than 500 0 C has learned.
- the interlayer anion A n ⁇ is preferably selected from the group consisting of carbonate, nitrate, halide, sulfate and phosphate or mixtures thereof, where n is a positive integer.
- a n ⁇ (see empirical formula above) is particularly preferably carbonate or the layered double hydroxide is in carbonate form, with preferably at least 50%, more preferably at least 75% and especially preferably at least 90% of the interlayer anions A n being carbonate ions.
- the layered double hydroxide used here has the fact that it is stable in air and thus can be easily stored.
- the biomolecules do not comprise serum ruminal (BSA).
- BSA serum ruminal
- the layered double hydroxides used according to the invention can bind biomolecules in amounts which are of interest for industrial use.
- the layered used is shown in comparison with a commercially available strong anion exchange material Macro-Prep 25 Q ® (Bio-Rad, Kunststoff) both in static and dynamic, as the system a higher Bmdungskapazitat.
- the biomolecule could be eluted again from the layered double hydroxide in the dynamic system.
- Static capacity is the maximum load in the equilibrium state of a static, ie non-perfused system with a long contact time between biomolecule solution and sorbent (layered double hydroxide).
- the biomolecule in the fluid medium is incubated together with the layered double hydroxide in a suitable vessel, then centrifuged off and the amount of bound biomolecule determined in the layered double hydroxide free supernatant.
- the capacity of an ion exchanger as a function of the flow rate is called dynamic capacity.
- the hydrotalcite is packed into a chromatography column, the protein-containing liquid medium is pumped through the column and the breakthrough curve of the biomolecule is determined.
- the recording of breakthrough curves serves to characterize the adsorption of the target biomolecule on the chosen sorbent. For this purpose, breakthrough curves are recorded at different fluid velocities and used to determine the dynamic capacity.
- the column is loaded continuously with a biomolecule solution (1 mg / ml) with increasing flow rates.
- the dynamic capacity results from the calculation of the volume at the beginning of the loading until the breakthrough of the curve (UV detector). This will be explained in more detail below in the test descriptions.
- the layered double hydroxide used in the process according to the invention preferably has a BET surface area of more than 15 m 2 / g, more preferably more than 20 m 2 / g, particularly preferably more than 55 m 2 / g, preferably more than 65 m 2 / g, and more preferably also a pore volume of more than 0.30 ml / g, more preferably of more than 0.4 ml / g, particularly preferably in the range of 0.45-0.6 ml / g (determined according to the BJH method; cumulative pore volume for pores having a diameter in the range of 1.7 to 300 nm).
- the layered double hydroxide has an average pore diameter of more than about 10 nm.
- layered double hydroxide particles having a diameter of from about 0.5 to 100 ⁇ m, particularly preferably from 1 to 80 ⁇ m, particularly preferably from 4 to 60 ⁇ m.
- the layered double hydroxide in the form of granules, in particular with an average particle size of 0.08 - 2.5 mm.
- the Schichtdoppelhydroxid is equilibrated before the task of the biomolecule in the liquid medium to a pH of 5.0 to 10.0, particularly preferably from 6.0 to 9.0.
- the Schichtdoppelhydroxid m a suitable buffer, for. B. HEPES, slurried or applied to a filter package prepared from the Schichtdoppelhydroxid with such a buffer.
- the concentration of the buffer is preferably between 30 and 100 mmol / l.
- the use is carried out in a chromatography process.
- the layered double hydroxide is packed into a chromatography autoclave and the biomolecule-containing liquid medium is added to the column.
- the column can then be flowed through by an eluent, whereby a separation of the proteins into different fractions is possible by adsorption effects. This is of particular interest for the separation of biomolecules produced in the fermentation process.
- the elution of the biomolecule adsorbed on the column can be carried out by various methods. Via a salt gradient, different biomolecules can be obtained at different salt concentrations. The use of salt gradients reduces the electrostatic interactions between the layered double hydroxide and the biomolecule. Consequently, the biomolecule is released from the layered double hydroxide.
- the biomolecules are eluted by changing the pH.
- the eluent has a different pH than the liquid medium.
- proteins are understood to mean molecules which have amino acids as building blocks. The amino acids have charged residues that can interact with charged groups on the surface of the layered double hydroxide.
- biomolecules in particularly high concentrations on or in the layered double hydroxides.
- biomolecules can be re-isolated or purified by elution.
- this advantage according to the invention is particularly evident in the case of larger or long-chain oligopeptides and proteins.
- a preferred aspect of the present invention therefore relates to the use of the above-defined layered double hydroxides for the removal or recovery of biomolecules, in particular proteins, 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, at least 5 amino acids, preferably at least 50 amino acids and more preferably at least 100 amino acids.
- biomolecules are enzymes and antibodies, components for gene therapy, etc.
- the purification and / or separation according to the invention takes place while retaining the biological function.
- the biomolecules also include nucleic acids, e.g. Oligo- and polynucleotides, ribonucleic acids (RNA) or deoxyribonucleic acids (DNS).
- nucleic acids e.g. Oligo- and polynucleotides, ribonucleic acids (RNA) or deoxyribonucleic acids (DNS).
- the above composition thus contains at least one biomolecule having a molecular weight of at least 200 D, in particular 10 kD, particularly preferably at least 40 kD.
- biomolecules have no upper limits, so that even very large biomolecules with more than 500 kD or more than 1 MD can be deposited or stored in a particularly advantageous manner. According to one embodiment of the invention, however, it is also possible to deposit or store individual amino acids.
- Liquid or fluid media are understood here to mean all media which make it possible to bring them into contact and thus to deposit or store the biomolecules in the layered double hydroxide.
- aqueous or water-containing media in the form of a solution, suspension, dispersion, colloidal solution or emulsion.
- liquids derived from biological sources or contain proteins such.
- the samples must be prepared depending on the method, such as. B. by coarse filtration or buffering of the biomolecules. These processes are familiar to the person skilled in the art.
- the invention relates to a method comprising the following steps:
- the use according to the invention for the attachment or incorporation of biomolecules onto or in layered double hydroxides can be used both for enrichment (i.e., increasing the concentration of the desired biomolecule) and depletion (i.e., decreasing the concentration of the desired biomolecule).
- the layered double hydroxide containing the biomolecule can be disposed of in a further step.
- the disposal can take place, for example, by thermal treatment of the layered double hydroxide in order to remove the biomolecule contained, wherein after the thermal disintegration of the biomolecule, the layered double hydroxide can be disposed of in a particularly advantageous manner.
- biomolecules from media it is possible to selectively remove or purify biomolecules from media. This plays an important role, for example, in wastewater treatment, since in most countries there are strict legal regulations on the removal of biomolecules from wastewater.
- biomolecules in many cases it is desirable to increase the concentration of biomolecules in a medium or to obtain these biomolecules in pure form as far as possible.
- recovery or purification of desired biomolecules is one of the standard procedures in biological and medical research.
- the biomolecule in a further step, can be desorbed again from the layered double hydroxide or can be won, whereby the Schichtdoppelhydroxid can be used again if necessary.
- the layer Doppelhydroxidhydroxidchen over a z. B. suitable binder to larger agglomerates, granules or molded articles are connected or applied to a carrier.
- the shape and size of such parent structures containing the primary layered double hydroxide particles will depend on the particular application desired. It is thus possible to use all forms and sizes which are familiar to the person skilled in the art and which are suitable in individual cases. For example, agglomerates with a diameter of more than 10 .mu.m, in particular more than 50 .mu.m, may be preferred in many cases. In the case of a bedding for chromatography columns and the like, a spherical shape of the agglomerates may be advantageous.
- a possible carrier is calcium carbonate, for example.
- any binder which is familiar to the person skilled in the art as long as the attachment or incorporation of the proteins into or onto the layered double hydroxide particles is not excessively impaired or the stability of the particle agglomerates or shaped bodies required for the particular application is ensured.
- binders agar-agar, alginates, chitosans, pectins, gelatins, lupine protein isolates or gluten.
- compositions having a layered double hydroxide as defined above and at least one biomolecule as defined above.
- Figure 1 Representation of the pH-dependent adsorption of the protein ovalbumin in an inventive Schichtdoppel- hydroxide (hydrotalcite 1) compared to the commercially available Anion (2004) (Macro-Prep 25 Q ®; Bio-Rad, Kunststoff).
- hydrotalcite 1 Representation of the pH-dependent adsorption of the protein ovalbumin in an inventive Schichtdoppel- hydroxide (hydrotalcite 1) compared to the commercially available Anion (2004).
- hydrotalcite 1 a 1 mg / ml ovalbumin solution in the respective buffers were used per test batch;
- Fig. 2 Adsorption isotherm of ovalbumin on hydrotalcite 1 at pH 8, taken in 50 mM Tris buffer. 20 mg of adsorbent were incubated with 15 ml of protein solution at concentrations of 0.53-8 mg / ml for 3 hours at room temperature;
- FIG. 3 shows a graph in which the loading of the hydrotalcite 1 used in the process according to the invention is shown as a function of the application rate for the model protein BSA (A) in comparison with a chromatography material (B) commercially available from Bio-Rad.
- A model protein BSA
- B chromatography material
- Fig. 4 a graph showing the results of adsorption of the protein ovalbumin to the hydrotalcites 1, 4 and 7 in 0.05 M HEPES buffer (pH 7). For the adsorption, 7 ml of a 1 mg / ml ovalbumin solution were used per test batch;
- Fig. 5 a graph showing the results of the desorption of the protein ovalbumin of hydrotalcite 1, 5 and 6. Zur Desorption 5 ml of 1 M NaCl in 0.05 M HEPES solution, pH 8.5 were used per test batch.
- the reported (average) pore volumes and areas were calculated using a fully automatic nitrogen adsorption instrument (ASAP 2000, Micrometrics) according to the manufacturer's standard program (BET, BJH (IP Barrett, LG Joyner, PP Haienda, J. Am. Chem. Soc 73 (1951), 373), t-plot and DFT).
- the percentage data on the proportion of certain pore sizes refers to the total pore volume of pores between 1.7 and 300 nm diameter (BJH Adsorption Pore Distribution Report).
- the D90 value indicates the value at which 90% of the particles in the measured sample have a smaller or equal particle diameter.
- the D100 value, the D50 value or the D10 value indicate the value at which 100%, 50% or 10% of the particles in the measured sample have a smaller or the same particle diameter.
- the water content of the products at 120 ° C was determined using the method DIN / ISO 787/2.
- This analysis is based on the total effect of the layered double hydroxide or the corresponding product. After dissolution of the solids, the individual components are analyzed and quantified using specific analytical methods such as ICP.
- the proportion of the crystalline hydrotalcite phase or boehmite phase can be determined by quantitative X-ray diffraction. Details of this method are e.g. described in "Handbook of Clay Science", F. Bergaya, B.K. G. Therry, G. Lagaly (ed.), Elsevier, Oxford, Amsterdam, 2006, ch. 12.1: I. Srodon, Identification and Quantitative Analysis of Clay Minerals; "X-Ray Diffraction and the Identification and Analysis of Clay Minerals", D.M. Moora and R.C. Reaynolds, Oxford University Press, New York, 1997, p. 765, incorporated herein by reference.
- Quantitative X-ray diffraction is based on Rietveld's formalization of refinement.
- This algorithm was originally developed by HM Rietveld to refine crystal structures. The process is commonly used in mineralogy and eg the cement industry for the quantification of mineral phases in unknown samples. In the more general case, this method can be used for the quantitative determination of the contents of crystalline compounds (phases) in mixtures of different crystalline compounds (phases).
- the Rietveld refinement algorithm is based on a calculated fit of a simulated diffraction pattern to a measured diffraction pattern. First, the existing crystalline phases (compounds) are determined by assigning peaks of the diffraction diagram.
- the diffraction pattern is then calculated based on the crystal structure of the minerals present in the sample as well as the equipment and the sample specific parameters.
- the parameters of the model are adjusted in order to obtain a good approximation of the calculated and the measured diffraction diagram, eg using the "least square fit" method. Details of the method are described, for example, in RA Young: “The Rietveld Method", Oxford University Press, 1995. The Rietveld method can also be used for strongly overlapping reflections in the diffraction diagram.
- the pH of hydrotalcite is determined with a pH meter in an aqueous / ethanolic suspension. For this a 1: 1 mixture of water and Ethanaol (95%) is produced. In a vessel, 50 g of the water / ethanol mixture are weighed to the nearest 0.1 g. 1 g of hydrotalcite is weighed to the nearest 1 mg and then added quantitatively to the beaker containing the etha- transferred nol / water mixture. It is then stirred for 5 min and then read the pH on the instrument.
- hydrotalcites were used as starting material, wherein the respective commercial reference source is indicated or the production of corresponding layered double hydroxides is also easily possible by methods familiar to the person skilled in the art (see above).
- Al / Mg hydrotalcite in carbonate form available from Sud-Chemie AG under the name Synthal ®.
- the Zn hydrotalcite used was prepared according to EP 1 381 567 B1, test no. 4, without a subsequent hydrothermal treatment. Chemical analyzes :
- the hydrotalcite used in the chloride form was prepared according to Example 1 a) of DE 198 36 580 A1, wherein the amounts of MgCl 3 '6H 2 O and A1C1 3 ' 6H 2 O were varied so that a molar ratio of Mg: Al of 1.71.
- the hydrotalcite used in the carbonate form was prepared according to Example 2 a) of DE 198 36 580 A1, wherein the amounts of MgCl 3 -OH 2 O and AlCl 3 '6H 2 O were varied so that a molar ratio of Mg: Al of 2.35.
- Ovalbumin (Sigma-Aldrich GmbH, Taufkirchen) is the main protein of the white component of chicken egg (60 to 65%). The molecular mass is 45 kDa and the isoelastic point is 4.6.
- Bovine serum albumin (BSA):
- Bovine serum albumin (Sigma-Aldrich GmbH, Taufkirchen) belongs to the group of globular proteins. In particular, it ensures the maintenance of colloidotic pressure in the human organism. The molecular weight is 66 kDa and the isoelectric point is 4.6. Alkaline phosphatase:
- This enzyme is derived from calf intestinal mucosa and is commercially available from Fluka, DE.
- the isoeletric point is about 6 and the molecular weight is 140 kDa.
- hydrotalcite 25 mg are equilibrated with 10 ml of the appropriate buffer at pH 5-9 (see Table I).
- the adsorbent material suspended in the buffer is sonicated for 30 minutes and shaken at 15 rpm for one hour in an overhead mixer. The material is then centrifuged off at 4000 g for 10 min and the supernatant discarded. For washing, 10 mL redist. Water was added, the adsorbent resuspended and then shaken for 5 min at 15 rpm. The supernatant was discarded after recentrifugation at 4000 g / 10 min.
- the photometric quantitation of proteins is based on the measurement of the UV absorption of the aromatic amino acids tyrosine (phenol group, 275 nm), tryptophan (indole group, 279 nm) and to a lesser extent phenylalanine (257 nm) at 280 nm. Depending on the amino acid composition Concentrations between 20 and 3000 ⁇ g / ml protein are detected.
- the absorption of a calibration series of solutions of known protein concentration at 280 nm is first determined. From the linear relationship between absorption and protein concentration, the unknown protein concentration can be calculated
- This method combines the reduction of Cu 2+ to Cu + by proteins in an alkaline medium, with the highly sensitive and selective colorimetric detection of Cu + through the use of a dichloroquinonic acid-containing reagent.
- FIG. 1 shows the results of the pH-dependent adsorption of the protein ovalbumin onto the hydrotalcite 1 in comparison to Macro-Prep 25 Q® from Bio-Rad.
- the hydrotalcite adsorbs about three times more ovalbumin than Macro-Prep 25 Q ® .
- the uptake of an adsorption isotherm is investigated as a function of the equilibrium loading of the hydrotalcite on the protein concentration in the supernatant.
- the experiment was carried out at a pH of 8 in a 50 mM Tris buffer.
- hydrotalcite 1 was equilibrated with Tris buffer at pH 8 as described above.
- a protein strain solution of 10 mg / ml in 50 mM Tris buffer is prepared and diluted according to the instructions given in Table 2 with the respective buffer and then added to 20 mg of the prepared sorbent (Doppeltikhydroxid).
- 100 mg of the corresponding adsorbent material (hydrotalcite in comparison with Macro-Prep 25 Q® ) are suspended in a 1 mL Eppendorf vessel with 1 mL of the corresponding 50 mM buffer (see Table 2) and 15 mm in a sealed column with a punch down x 50 mm (Omnifit, Cambridge, England).
- the second column end piece is placed and the column is connected to the HPLC pump SFD SDS 9404 (Schambeck SFD GmbH, Bad Honnef) so that it is flowed through by the mobile phase from bottom to top.
- the pump is set to the flow rate for which the capacity of the adsorbent is to be determined.
- the moving column of the column is slowly hand-tightened during equilibration with 20 mL of the appropriate buffer and then relieved by a quarter turn of the thread.
- Example 3 adsorption of alkaline phosphatase to double tikhydroxiden with different ratio of M 2+ to 3+ N
- adsorbents 25 mg are equilibrated with 10 ml of the appropriate buffer (50 mM) at the appropriate pH (see Table I).
- the adsorber material suspended in the buffer is treated for 30 minutes in an ultrasonic bath and shaken at 15 rpm for 30 mm in an overhead mixer. The material is then centrifuged off at 3219 g, 10 0 C for 10 min and the supernatant discarded.
- alkaline phosphatase The activity determination of alkaline phosphatase is based on the enzymatic cleavage of p-nitrophenyl phosphate into p-nitrophenol and inorganic phosphate. The resulting p-nitrophenol causes a yellowing, which can be detected at 405 nm.
- the activity of the stored alkaline phosphatase is determined in the falcon tube.
- the stored enzyme is suspended in 2.7 ml of 1.0 M diethanolamine buffer solution (pH 9.8) and treated with 0.3 ml of 150 mM p-nitrophenyl phosphate solution, a yellowing occurs. After two minutes, stop the reaction by adding 1 ml of 1 M NaOH.
- the suspension is centrifuged off at 3219 g (10 ° C.) for 10 min and pipetted from the supernatant 34.2 ⁇ l together with 273.3 ⁇ l of buffer solution into a well of a 96-well plate. The absorbance is measured at 405 nm.
- One unit converts 1.0 ⁇ mol of p-nitrophenyl phosphate per minute at pH 9.8 and 37 ° C.
- hydrotalcites 1, 2 and 3 show the best adsorption performance.
- the enzymatic activity of the alkaline phosphatase is detectable in the immobilized form in contrast to the reference material 4.
- Example 5 Comparison of protein adsorption and desorption of ovalbumin on hydrotalcites of different molar ratios
- hydrotalcite 1 adsorbs about 1 ⁇ more ovalbumin than the hydrotalcite 7, whereas hydrotalcite 4 (not according to the invention) shows no adsorption, see FIG. 4.
- the samples were incubated 2x for 45 min in 1 M NaCl in 0.05 M HEPES buffer, pH 8.5. Subsequently, the adsorber material is centrifuged off at 4000 g for 10 min and the protein content in the supernatant determined on the basis of the BCA protein test. All experiments were performed in duplicate.
Abstract
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP08802738A EP2209543A1 (de) | 2007-10-01 | 2008-10-01 | Verwendung von schichtdoppelhydroxiden zur an- bzw. abreicherung von biomolekülen aus flüssigen oder fluiden medien |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP07019288A EP2044992A1 (de) | 2007-10-01 | 2007-10-01 | Verwendung von Schichtdoppelhydroxiden zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien |
EP08802738A EP2209543A1 (de) | 2007-10-01 | 2008-10-01 | Verwendung von schichtdoppelhydroxiden zur an- bzw. abreicherung von biomolekülen aus flüssigen oder fluiden medien |
PCT/EP2008/008325 WO2009046918A1 (de) | 2007-10-01 | 2008-10-01 | Verwendung von schichtdoppelhydroxiden zur an- bzw. abreicherung von biomolekülen aus flüssigen oder fluiden medien |
Publications (1)
Publication Number | Publication Date |
---|---|
EP2209543A1 true EP2209543A1 (de) | 2010-07-28 |
Family
ID=38941805
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP07019288A Withdrawn EP2044992A1 (de) | 2007-10-01 | 2007-10-01 | Verwendung von Schichtdoppelhydroxiden zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien |
EP08802738A Withdrawn EP2209543A1 (de) | 2007-10-01 | 2008-10-01 | Verwendung von schichtdoppelhydroxiden zur an- bzw. abreicherung von biomolekülen aus flüssigen oder fluiden medien |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP07019288A Withdrawn EP2044992A1 (de) | 2007-10-01 | 2007-10-01 | Verwendung von Schichtdoppelhydroxiden zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien |
Country Status (2)
Country | Link |
---|---|
EP (2) | EP2044992A1 (de) |
WO (1) | WO2009046918A1 (de) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102274754B (zh) * | 2011-06-16 | 2013-04-10 | 北京化工大学 | 一种提高多相不对称催化活性和不对称选择性的方法 |
CN102516991B (zh) * | 2011-11-30 | 2013-11-27 | 北京化工大学 | 一种核苷酸与水滑石纳米片复合发光薄膜材料及其制备方法 |
WO2017164121A1 (ja) * | 2016-03-22 | 2017-09-28 | 一般財団法人阪大微生物病研究会 | 標的蛋白質の精製方法 |
CN110522732A (zh) * | 2019-09-04 | 2019-12-03 | 中国科学院上海硅酸盐研究所 | 一种超小纳米水滑石-肽核酸复合材料及其制备方法和应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE10237517B4 (de) * | 2002-08-16 | 2012-04-12 | Süd-Chemie AG | Verfahren zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien unter Verwendung von Schichtdoppelhydroxiden und Verwendung von an einem Schichtdoppelhydroxid an- oder eingelagerten Biomolekülen als anorganischer Vektor |
DE10237518A1 (de) * | 2002-08-16 | 2004-02-26 | Süd-Chemie AG | Verwendung von Schichtdoppelhydroxiden zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien |
-
2007
- 2007-10-01 EP EP07019288A patent/EP2044992A1/de not_active Withdrawn
-
2008
- 2008-10-01 EP EP08802738A patent/EP2209543A1/de not_active Withdrawn
- 2008-10-01 WO PCT/EP2008/008325 patent/WO2009046918A1/de active Application Filing
Non-Patent Citations (1)
Title |
---|
See references of WO2009046918A1 * |
Also Published As
Publication number | Publication date |
---|---|
EP2044992A1 (de) | 2009-04-08 |
WO2009046918A1 (de) | 2009-04-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1968722B1 (de) | Verfahren zur abtrennung von proteinen aus flüssigen medien | |
Mikutta et al. | Biodegradation of forest floor organic matter bound to minerals via different binding mechanisms | |
EP1890804B1 (de) | Verwendung von stevensit zur mykotoxinadsorption | |
Gasser et al. | Humic acid adsorption onto Mg/Fe layered double hydroxide | |
WO2006077074A2 (de) | Verfahren zur herstellung von kationisierten adsorbentien, danach erhältliche sorptionsmittel sowie deren bevorzugte verwendung | |
Abu-Eishah | Removal of Zn, Cd, and Pb Ions from water by Sarooj clay | |
EP2006367B1 (de) | Hilfsmittel zur Filterung und/oder Flockung für die Reinigung flüssiger Nahrungsmittel | |
EP2209543A1 (de) | Verwendung von schichtdoppelhydroxiden zur an- bzw. abreicherung von biomolekülen aus flüssigen oder fluiden medien | |
EP2648839B1 (de) | Granulierte zeolithe mit hoher adsorptionskapazität zur adsorption von organischen molekülen | |
WO2005121791A1 (de) | Sorptionsmittel für nukleinsäuren, enthaltend säureaktiviertes schichtsilicat | |
EP1960519A1 (de) | Verfahren zur sorption von mindestens einem nukleinsäuremolekül unter verwendung säureaktivierter schichtsilicate | |
EP1858639B1 (de) | Verfahren zur abtrennung von biomolekülen aus flüssigen medien | |
EP2040562B1 (de) | Verfahren zur abtrennung von proteinen aus flüssigen medien unter verwendung thermisch modifizierter tonmaterialien | |
DE10237518A1 (de) | Verwendung von Schichtdoppelhydroxiden zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien | |
EP2377612A1 (de) | Adsorptionsmittel zum Adsorption von basischen Mischungen | |
Sohling et al. | Natural mixture of silica and smectite as a new clayey material for industrial applications | |
DE10237517B4 (de) | Verfahren zur An- bzw. Abreicherung von Biomolekülen aus flüssigen oder fluiden Medien unter Verwendung von Schichtdoppelhydroxiden und Verwendung von an einem Schichtdoppelhydroxid an- oder eingelagerten Biomolekülen als anorganischer Vektor | |
Vreysen et al. | Influence of the synthesis method of Al-hydroxy intercalated clays on their fulvic acid sorption capacity | |
JP7306961B2 (ja) | 塩基性アミノ酸用吸着剤 | |
EP2437618B1 (de) | Verfahren zum auftrennen von pflanzenproteinen | |
DE102011105909A1 (de) | Prozess zur Herstellung eines Rapsproteinisolats | |
WO2008151824A1 (de) | Immobilisierung von enzymen auf bleicherden |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20100430 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR |
|
AX | Request for extension of the european patent |
Extension state: AL BA MK RS |
|
DAX | Request for extension of the european patent (deleted) | ||
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: RALLA, KATHRIN Inventor name: KASPER, CORNELIA Inventor name: SCHEPER, THOMAS Inventor name: SOHLING, ULRICH Inventor name: RUF, FRIEDRICH Inventor name: SUCK, KIRSTIN |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: SUED-CHEMIE IP GMBH & CO. KG |
|
17Q | First examination report despatched |
Effective date: 20140107 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20150501 |