EP1539321A1 - Procede pour determiner des conditions de chromatographie adequates pour la separation de molecules biologiques - Google Patents

Procede pour determiner des conditions de chromatographie adequates pour la separation de molecules biologiques

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Publication number
EP1539321A1
EP1539321A1 EP03757672A EP03757672A EP1539321A1 EP 1539321 A1 EP1539321 A1 EP 1539321A1 EP 03757672 A EP03757672 A EP 03757672A EP 03757672 A EP03757672 A EP 03757672A EP 1539321 A1 EP1539321 A1 EP 1539321A1
Authority
EP
European Patent Office
Prior art keywords
chromatography
biological sample
buffer
protein
chromatography media
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Withdrawn
Application number
EP03757672A
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German (de)
English (en)
Inventor
Hartmut SCHLÜTER
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Merck Patent GmbH
Original Assignee
Merck Patent GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Merck Patent GmbH filed Critical Merck Patent GmbH
Publication of EP1539321A1 publication Critical patent/EP1539321A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8658—Optimising operation parameters
    • G01N30/8662—Expert systems; optimising a large number of parameters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/069—Absorbents; Gels to retain a fluid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N2030/009—Extraction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
    • G01N30/463—Flow patterns using more than one column with serial coupling of separation columns for multidimensional 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
    • Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction

Definitions

  • the invention relates to a multiparallel method for quickly finding suitable chromatography parameters for the separation of biological molecules.
  • the invention relates in particular to a multiparallel chromatography system for developing methods for the purification of proteins and other biomolecules.
  • the system uses cavities from multi-well plates (e.g. 96-well plates) that are filled with chromatographic gels and consists of various start buffers in which the samples to be chromatographed are dissolved and with which the gels are equilibrated and solutions for desorbing ( Eluting) of the biomolecules bound to the gels.
  • WO 99/24138 A1 describes a method in which one in the cavities
  • Multiwell plate different chromatography media are arranged. These media are used for the analysis of biological substances in the context of so-called “asays”.
  • biological sample means purified or unpurified proteins, peptides, nucleic acids of all kinds, carbohydrates, lipids, low molecular weight metabolites or mixtures thereof. This includes - but not exclusively - complex protein mixtures of human, animal or vegetable Tissues or cells as well as cells of microorganisms.
  • biomolecules The “biological sample” is also referred to below as “biomolecules”.
  • chromatography media are understood to mean two types:
  • ion exchangers anion ion exchangers, cation exchangers
  • metal affinity chromatography media reversed-phase materials
  • gels for hydrophobic interaction chromatography (HIC), hydroxyl pathite media (HAP) affinity chromatography media with all kinds of ligands but also gels with magnetic properties (magneto -Beads, coated or uncoated).
  • HIC hydrophobic interaction chromatography
  • HAP hydroxyl pathite media
  • a special feature of such gels is the ability to bind the biological sample, which must also be able to be freed from the binding substance again by means of suitable elution agents (solutions).
  • elution solutions are the ability to desorb (elute) the biological sample from the chromatography gel, that is to say to shift the equilibria of the binding of the biological sample to the chromatography gel in the Such that the biomolecules bound to the chromatography gel have no or only low affinities for the chromatography gel after addition of the elution solution.
  • the invention is suitable for the automated search for suitable chromatography media and the associated buffer and elution conditions for the purification of peptides and proteins, but also other biomolecules from homogenates (raw extracts).
  • the invention is based on the absorption of biomolecules on gel particles, on the so-called stationary phase.
  • a biological sample consisting of biomolecules such as proteins, peptides, etc., dissolved in the start buffer (mobile phase)
  • gel particles so-called batch process
  • the liquid supernatant in which the biomolecules are found, which are not Have affinity for the chosen gel under the chosen conditions, removed from the gel particles.
  • the gel particles are then with
  • the invention can be carried out automatically, so that the results from e.g. from a 96-well chromatography can be evaluated manually or by means of a computer program, clearly presented and prepared for interpretation.
  • the data obtained are interpreted in such a way that the results of the interpretation result in suggestions for the chromatographic purification of (for example) proteins from protein extracts of a biological sample.
  • Frontal chromatography, displacement chromatography or gradient chromatography are possible as chromatographic modes.
  • Purification of the target protein can also include several different, contiguous chromatographies (combinations of chromatographies).
  • the invention further provides a kit in which the method according to the invention
  • the multi-well plates can already be prefabricated with chromatography media for binding the biological sample (group B materials) and a set of different chromatography media (solutions of any composition) for elution (group NB materials).
  • the system contains protocols for carrying out the experiments in multi-well format with n cavities (n> 1), commercially available chromatography media distributed on multi-well plates, and eluents distributed on multi-well plates are adapted to the respective chromatography media.
  • the system is used for the systematic search for reproducible chromatographic purification steps for biomolecules.
  • the system is designed for the combination of pipetting robots, but can also be used manually. Due to the selected different chromatography parameters (e.g. pH value, ion concentration, etc.) different populations of biomolecules will bind to the gels in the different cavities. If, for example, an anion exchange gel is used as the chromatographic gel, biomolecules which are still sufficient at pH 4 are bound to the gel in B1 (FIG. 1, the solution in the cavity B1 has a pH of 4 and an NaCl concentration of 50 mmol / l) have many negative charges, so they have a low isoelectric point.
  • the low NaCI concentration means that biomolecules with low affinity for the
  • Bind gel In the G11 cavity (FIG. 1, the solution in the G11 cavity has a pH of 7 and a NaCl concentration of 750 mmol / l), biomolecules with a significantly higher isoelectric point can be expected. At the same time, due to the high NaCI concentration, only biomolecules with very high affinity will bind to the gel particles, the vast majority of the biomolecules will remain in solution.
  • the system will also contain protocols to further analyze the biomolecules concentrated on the gel particles, e.g. about protein determination or electrophoresis.
  • the invention enables the finding in the multi-well format of the parallel suitable chromatography media and the associated eluents in a much shorter time than before.
  • the system can be used for the development of chromatographic purification steps of biomolecules.
  • the data obtained with the system can be used for the development of chromatography steps in frontal chromatography mode, in displacement chromatography mode or in gradient chromatography mode.
  • the system can be used (e.g. in combination with mass spectrometry) for comparative studies of the biomolecule profiles ("profiling") of two different states (“differential display”) in order to be able to identify molecules that are relevant to a defined state (e.g. sick ) are characteristic.
  • the system can be used as sample preparation for 2D electrophoresis strategies.
  • Figure 1 is a schematic representation of a 96 well
  • FIG. 2 shows the results of a multiparallel chromatography of a protein mixture to find suitable parameters for the chromatographic purification of 4 different target proteins.
  • the graphic shows the absolute yields of the various target proteins (in%, based on the amount of the respective protein used), namely ribonuclease A (top left), cytochrome C (top right), lysozyme (bottom left) and myoglobin ( bottom right), in the eluates of the multiparallel
  • FIG. 3 shows the results of a multiparallel chromatography of a protein mixture to find suitable parameters for the chromatographic purification of 4 different target programs. teinen.
  • the graphic shows the specific yields of the various target proteins (in%, based on the total amount of all proteins in each cavity).
  • Protein concentration of ribonuclease A, cytochrome C, lysozyme and myoglobin dependence of the peak area detected on the amount of protein injected.
  • FIG. 6 a chromatogram of the cation exchange
  • FIG. 7 a chromatogram of the cation exchange
  • FIG. 8 a chromatogram of the cation exchange
  • Lysozyme and myoglobin dissolved in a sample application buffer with pH 6 and 0 mM NaCI (parameter set from the experiments with the multiparallel chromatography system).
  • Enzymes with angiotensin conversion enzyme-like activity from a protein extract of porcine kidney tissue Specific enzyme activities (quotient of absolute enzyme activity (determined
  • FIG. 11 shows the results of a
  • FIG. 12 shows a chromatogram of a hydrophobic
  • FIG. 13 shows the protein concentrations of the fractions
  • FIG. 1 shows an example of a 96-well plate (multi-well plate), which is defined as a matrix by columns (X direction) and rows (Y direction), with different chromatography media depending on the location of those defined by the matrix Matrix points of the plate are arranged, the individual cavities of which are each coated with the same amount of chromatography gel (eg with a cation exchange gel), but have individual combinations of pH values and saline concentrations.
  • cavity B1 shows a pH of 4.5 and an NaCl concentration of 0.05 mol / l.
  • Cavity G12 would have a pH of 7.0 and a NaCI concentration. of 1 mol / l.
  • FIG. 4 shows a typical reversed-phase HPLC separation of the system with which the quantification was carried out.
  • the chromatogram comes from the separation of the protein mixture before processing with the multiparallel chromatography system.
  • the system creates a calibration line for each protein ( Figure 5).
  • To determine the calibration line for quantifying the individual proteins different defined amounts of the individual proteins are injected.
  • To create the calibration line the peak areas of the proteins are plotted against the protein concentrations.
  • some parameter sets for the sample loading buffer are selected (pH 3 mM + NaCI; pH 3 + 0 mM NaCI; pH 4 + 0 mM NaCI; pH 6 + 0 mM NaCI; pH 7 + 200 mM NaCI) and the protein mixture was chromatographed under these conditions (Fig. 6-
  • the yield can be obtained if the protein mixture is applied to the cation exchanger in a pH 3 and 0 mM NaCl buffer.
  • a look at the result in FIG. 2 reveals that a co-elution of cytochrome C and lysozyme can be expected under these conditions.
  • FIG. 7 shows the chromatogram of the separation of the protein mixture with a pH 4 buffer and 500 mM NaCl as start and sample application buffer. Under this
  • Well plate distributed in the cavities each equal amounts of a cation exchange gel.
  • the individual cavities differ in terms of pH and ionic strength.
  • the result of the separation is obtained by determining the protein concentration of the proteins of the individual cavities bound to the gel.
  • FIG. 10 shows the results of the determination of the specific activity of the
  • the chosen chromatography (here cation exchange chromatography) can then be used as a favorable initial step for concentration if the protein sought, here detected via its enzymatic activity, elutes in a fraction in which a protein concentration which is low compared to the other fractions can be found is (for example, Fig. 10 in the fraction pH 3, 500 mmol / l NaCl;), if the specific activity reaches a particularly high value.
  • FIG. 11 shows specific enzyme activities of the third embodiment
  • FIG. 12 shows the chromatogram of a hydrophobic interaction (HIC) gradient chromatography, which was derived from the parameter sets of the multiparallel chromatography of the experiment shown in FIG. 11 (phenyl-HIC chromatography).
  • HIC hydrophobic interaction
  • the enzymatically active fraction elutes in the region of the gradient, which shows that the prediction is correct that the target enzyme sought can be bound to a phenyl-HIC column and chromatographed under the conditions described in FIG. 11.
  • FIG. 13 shows protein concentrations of the fractions of a multi-well cation exchange chromatography with step gradient elution. Gradient level 1: 0.5 mol / l NaCl; Gradient level 2: 2 mol / l NaCl. UCE: Urotensin-generating activity.
  • urotensin-forming activity was only detectable in the fraction in which the protein extract was applied to the gel at pH 8 and which was eluted with 2 mol / l NaCl. It can be clearly seen that the highest protein amounts can be eluted at 0.5 mol / l (at pH 4.5 to 8). However, the UCE activity eluted in chromatography with the pH 8 buffer only after elution with a 2 molar salt concentration. This result is of great advantage for the purification of the UC enzyme, since the UCE elutes in a fraction with a low protein concentration and in this way a large amount of the accompanying proteins (approx. 95% of the originally applied amount of protein) can be separated off.
  • Multiparallel cation exchange chromatography of a mixture of 4 model proteins in 32 wells with different starting conditions variation of the pH values (row of numbers, “rows") and the salt concentration (rows of letters, “columns”)) and one-stage elution.
  • Ribonuclease A, cytochrome C, lysozyme and myoglobin are mixed together as model proteins (1250 ⁇ g per protein).
  • 100 ⁇ l of cation exchange gel (Fractogel EMD (M) SO 4 " (Merck) are distributed over 32 cavities.
  • the following buffers are prepared as the equilibration buffer (40 mM each) for the cation exchanger (X direction of the deep well matrix ):
  • NaCL NaCL is added to the buffers in the Y direction of the deep well matrix, so that NaCI concentrations of 0 mM NaCI, 100 mM NaCI, 200 mM NaCI, 500 mM NaCI are formed in the cavitates.
  • a 32-well matrix buffer plate with the corresponding buffers without gel is set up in parallel. The gels are washed 3 times with 300 ⁇ L of the respective equilibration buffer (from the 32-well matrix buffer plate, Table 1). Aliquots of the protein mixture are dissolved in the respective (200 ⁇ l) sample application buffer (Table 1) and applied to the gels in the different cavities. After washing with the appropriate buffers (Table 1), the proteins are eluted with 3 ⁇ 100 ⁇ l of a 2 M NaCl solution.
  • the composition of the eluates is quantified using a reversed phase HPLC system (FIG. 4 and FIG. 5).
  • a reversed phase HPLC system 100 ⁇ l of the protein mixture (50 ⁇ g each protein), dissolved in 0.1% TFA, was passed through a reversed phase column (TSKgel Super-Octyl 4.6 mm ID x 5.0 cm L; Tosohaas Biosep) separated.
  • a SMART system (from Amersham) is used as the HPLC system.
  • the mobile phase consists of 0.1% TFA in distilled water (solution A) and 0.1% TFA in acetonitrile (solution B). Chromatography is performed with a flow of 1 ml / min and a gradient of 23% to 44% solution
  • Fig. 5 shows the calibration lines for determining the protein concentration of ribonuclease A, cytochrome C, lysozyme and myoglobin.
  • the mixtures are chromatographed using the same cation exchange gel used for multiparallel chromatography in gradient chromatography mode.
  • a self-packed cation exchange column (HR10 / 30 with 2 ml Fractogel EMD SO 3 " (M), Merck) was used.
  • the mobile phase consisted of 40 mM citric acid buffer, pH 3 without NaCl (buffer A) and 40 mM citric acid buffer, pH3 with 2 M NaCI (Buffer B). matography was performed at a flow rate of 2.0 ml / min. The gradient had a gradient of 0% to 75% buffer B in 90 min.
  • Multi-well cation exchange chromatography with different starting conditions variation of the pH values (row of digits, "rows") and the salt concentration (rows of letters, “columns”)) and single-stage elution.
  • Pig kidneys are used for the production of protein extracts.
  • kidney tissue is cut (at temperatures from 4 to 6 ° C) into approximately 1 cm 3 large pieces, filled into pre-cooled lyophilization tubes, frozen in liquid nitrogen and stored at -80 ° C overnight.
  • the lyophilization plant type 2040 from Snijders Tilbug, Holland
  • the tissue pieces are completely dried for about a week 1 week.
  • the water-free tissue pieces are then pulverized with a grain mill (Varius, Messerschmidt) at the finest level. 2 g of the powder are dissolved in 20 ml of buffer (10 mM phosphate buffer, pH 7.3).
  • a homogenizer Ultra Turrax T 25 from Jahnke-Kunkel
  • the gel (300 ⁇ l / cavity) is distributed over 32 cavities of a 96 deep well plate (2.2 ml) and 1000 ⁇ l buffer (40 mmol / l) each with A to H in Table 2, in the cavities from rows 1 to 8 are added, so that one row (labeled with a letter) of the 96-well plate contains one and the same buffer with an identical pH value.
  • 400 ⁇ l of the salt solutions (NaCl, in mmol / l (final concentration): 1: 0; 2: 100; 3: 200 and 4: 500) are then pipetted into the cavitates, so that, for example, all cavitates with the designation 2 have the salt concentration of Contain 100 mmol / l.
  • one or more copies of the buffers are created according to the above-mentioned pipetting scheme, with which the individual cavities are later washed.
  • the sample and gel are suspended and incubated for 10 minutes.
  • the 96-well plate is then centrifuged (Speed-Vac centrifuge: 1 min).
  • the supernatant is copied to a 96-well plate and saved for analysis (protein concentration, activity, etc.).
  • the 32 different individual sample gel suspensions in the 96-well plate are combined with the corresponding, individual
  • Buffer solutions 300 ⁇ l each, the buffers are copied from a 96 deepwell plate (in which the individual solutions are located according to the scheme given above), to wash the 32 gels, then centrifuged and the supernatant solution pipetted off and discarded) to remove the non-binding proteins. This process is repeated 2 times. After this
  • the binding proteins are eluted by washing by adding a 2 molar NaCl solution (100 ⁇ l per cavity) to the cavities. After centrifugation (1 min), the eluates are copied onto one or more 96-well plates in order to make the individual samples available for subsequent analysis.
  • the protein extract was then chromatographed on a cation exchange column using the self-displacement method.
  • the parameters determined in FIG. 10 were used as equilibration and sample application buffers: the pobe was dissolved in a 40 mM buffer with a pH of 3 and an addition of 500 mM NaCl and applied to the self-
  • the gels (50 ⁇ l / cavity) are then divided into cavities 1A to 4A (HIC gels), 5A (HAP gel) and 6A-12A (chelate gel) of a 96 deep well plate (2, 2 ml) distributed.
  • 1000 ⁇ l buffer (Table 2) are added to the cavities of the rows 1A to 12A to the gels, so that the buffers according to Table 2 are located in the cavities of the 96-well plate, numbered 1 to 12.
  • 50 ⁇ l of the protein-containing sample protein concentration 0.3 ⁇ g / ⁇ l; protein extract extraction, see above under A.) are added to each of the 8 cavities.
  • the gels are washed twice with the equilibration buffer and then the binding proteins are eluted with 3 times the gel volume of the elution buffer.
  • the eluates are copied onto a 96-well plate and used for analyzes (protein concentration, activity, etc.).
  • the angiotensin conversion enzyme-like enzyme activity was determined as in Jankowski et al. 2001 (Jankowski, J. et al. (2001) Anal Biochem. 290, 324-9). The results are shown in Figure 3.
  • additives to stabilize the biomolecules such as: glycerol, sucrose, sodium molybdate, ethylene glycol, urea, guanidinium chloride, betaine, taurine, DTE, DTT, monothioglycerol, detergents, polyethylene glycol (PEG), chloroform, methanol, H 2 O, protease inhibitors (EDTA , EGTA, PMSF, DFP, Benzamidine, Aprotinin, Pefabloc SC, TLCK, TPCK, Phosphoramidon, Antipain, Leupeptin, Pepstatin A, Hirudin).
  • hydrophobicity of the gel e.g. butyl Sepharose ⁇ octyl Sepharose ⁇ phenyl Sepharose
  • chelate complexing group of the gel
  • imino-diacetic acid IDA
  • tris carboxymethyl ethylenediamine
  • NTA nitrilotriacetic acid
  • start buffer e.g.: 0.5 to 2 M NaCI.
  • Variation of the gel matrix • Variation of the elution: a) pH gradient (variation of the pH values in the direction of decreasing pH values), b) elution with a competitive ligand (eg ammonium chloride, sulfate, imidazole, or histamine). c) Elution with chelates (EDTA, EGTA).
  • a competitive ligand eg ammonium chloride, sulfate, imidazole, or histamine.
  • Elution with chelates EDTA, EGTA.
  • TFA triethylammonium acetate
  • TEAA triethylammonium acetate
  • the homogenization of the freeze-dried kidney extract was carried out here in the respective start buffer:
  • the start buffer (20 mmo / l each) was used: (1) citrate buffer, pH 3; (2) citrate buffer pH 3.5; (3) formate buffer pH 4; (4) succinate buffer pH 4.5; (5) acetic acid pH 5; (6) malonate buffer pH 5.5; (7) malonate buffer pH 6; (8) phosphate buffer pH 7; (9) HEPES buffer pH 7.5; (10) HEPES buffer pH 8.
  • Approx. 200 mg kidney tissue powder was dissolved in 3 ml buffer for each batch.
  • Suspensions in the 96-well plate are suspended with the corresponding, individual buffer solutions (500 ⁇ l each, the buffers are copied from a 96-deepwell plate in which the individual solutions are located according to the scheme given above) to the gels wash in the 10 cavities, then centrifuged and the supernatant solution pipetted off and discarded to remove the non-binding proteins. This process is repeated 5 times.
  • the binding proteins are eluted by pipetting 600 ⁇ l of a 0.5 molar NaCl solution into the cavities. After centrifugation (1 min), the eluates are copied onto a 96-well plate to determine the individual
  • the still binding proteins are eluted by pipetting 600 ⁇ l of a 2 molar NaCl solution into the cavities. After centrifugation (1 min), the eluates are copied onto a 96-well plate in order to make the individual samples available for subsequent analysis.
  • a protocol has been developed for this purpose to enable samples.
  • a 96 deepwell plate with a filter (20 ⁇ m, Macherey & Nagel) is filled with a size exclusion gel (1000 ⁇ l / cavity, Biogel P6, BioRad).
  • the size exclusion gel should be equilibrated with a buffer that prevents electrostatic interactions between proteins and the gel.
  • a salting of a protein solution from 2 mol / l NaCI to 0.29 mol / l NaCI succeeds when 350 ⁇ l sample is applied to the cavities filled with 1000 ⁇ l gel. Desalination is done by centrifuging the sample through the size exclusion gel. With this test approach, a protein yield of 79% was achieved.
  • the salt concentration in the eluate can be reduced to 0.2 mol / l if the sample volume of the sample to be buffered is reduced to 300 ⁇ l. In this case, however, the protein yield drops to 67%.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un système de chromatographie multiparallèle pour développer des méthodes de nettoyage de protéines et d'autres biomolécules. Ce système comprend des plaques mutipuits (par ex. des plaques à 96 puits), dont les cavités sont remplies de gels pour chromatographie, plusieurs tampons d'amorçage, dans lesquels les échantillons à chromatographier sont dissous et qui servent à l'équilibrage des gels, et des solutions pour désorber (éluer) les biomolécules liées aux gels. Ledit système comporte également des aides pour l'interprétation des résultats des analyses (par ex. des déterminations de concentration protéinique, des dosages biologiques, etc.) subséquentes à la chromatographie et portant sur les excédents des biomolécules et/ou des produits d'élution non liés. Ces aides peuvent aussi être des programmes, dans lesquels les résultats sont entrés et qui, après traitement de ces résultats, fournissent des interprétations et des suggestions pour élaborer les opérations de nettoyage d'une biomolécule.
EP03757672A 2002-09-19 2003-09-19 Procede pour determiner des conditions de chromatographie adequates pour la separation de molecules biologiques Withdrawn EP1539321A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10243529 2002-09-19
DE10243529 2002-09-19
PCT/DE2003/003108 WO2004028658A1 (fr) 2002-09-19 2003-09-19 Procede pour determiner des conditions de chromatographie adequates pour la separation de molecules biologiques

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EP1539321A1 true EP1539321A1 (fr) 2005-06-15

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US (1) US20060096924A1 (fr)
EP (1) EP1539321A1 (fr)
AU (1) AU2003273737A1 (fr)
DE (1) DE10393819D2 (fr)
WO (1) WO2004028658A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2004097403A1 (fr) * 2003-04-29 2004-11-11 Sigmol, Inc. Definition et identification de profils
DE102006027496B3 (de) * 2006-06-14 2008-01-10 Boehringer Ingelheim Pharma Gmbh & Co. Kg Verfahren zur Optimierung chromatographischer Reinigungsverfahren für Biomoleküle
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