WO2002032537A2 - Procede ameliore de chromatographie a lit etendu - Google Patents

Procede ameliore de chromatographie a lit etendu Download PDF

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WO2002032537A2
WO2002032537A2 PCT/US2001/027736 US0127736W WO0232537A2 WO 2002032537 A2 WO2002032537 A2 WO 2002032537A2 US 0127736 W US0127736 W US 0127736W WO 0232537 A2 WO0232537 A2 WO 0232537A2
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Prior art keywords
resin
recombinant protein
column
exchange resin
urea
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PCT/US2001/027736
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English (en)
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WO2002032537A3 (fr
Inventor
Walter Francis Junior Prouty
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Eli Lilly And Company
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Priority to AU2001296228A priority Critical patent/AU2001296228A1/en
Publication of WO2002032537A2 publication Critical patent/WO2002032537A2/fr
Publication of WO2002032537A3 publication Critical patent/WO2002032537A3/fr

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Classifications

    • 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
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
    • 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
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • 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
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57563—Vasoactive intestinal peptide [VIP]; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/58—Conditioning of the sorbent material or stationary liquid the sorbent moving as a whole

Definitions

  • Expanded Bed Chromatography is a method for recovering biomolecules directly from unclarified feedstocks such as fermentation broths or cell homogenates.
  • EBC is described in U.S. Patent No. 5,522,993 to Carlsson, et al . and Barnfield, et al . , Bioprocess Engineering 16: 51 (1997), the entire teachings of which are incorporated herein by reference.
  • EBC is a type of chromatography in which liquid is passed upwards through a bed of absorptive resin.
  • the resin particles are suspended in equilibrium due to the balance between particle sedimentation velocity and upward flow, thereby forming a stabilized bed that produces chromatographic plates. While it operates much as a packed chromatographic bed, the upward flow of liquid allows the bed to expand and create space between the suspended resin particles. Thus, it is possible to pass a stream of material through the column that contains substantial amounts of particulate matter.
  • An important advantage of this technology over downstream purification is that pre- filtration of the material going over the expanded bed is not required. Therefore, the process may be viewed as combining two steps (filtration and chromatographic capture) into one (EBC) .
  • the present invention is a method of separating a desired component (e.g., a biomolecule such as a recombinant protein) from a mixture such as an unclarified feedstock.
  • a desired component e.g., a biomolecule such as a recombinant protein
  • the mixture is solubilized in an aqueous urea solution to form a sample solution.
  • the sample solution is then passed upwards through a bed of resin, thereby binding the desired component to the resin and separating the desired component from the mixture.
  • the sample solution comprises a sufficiently high concentration of urea to reduce fouling of resin when the sample solution is passed through the resin.
  • an aqueous urea solution is then passed upwards through the column to elute undesired soluble and insoluble components.
  • the wash solution also contains a sufficient amount of urea to reduce fouling of the resin.
  • the method of the present invention utilizes urea to reduce resin fouling in EBC.
  • urea allows multiple runs with unclarified feedstocks (fifty runs and greater) without the need to change or regenerate the resin. Reduction in fouling is independent of column size. Thus, this improvement can be used when EBC is carried out on an industrial scale, for example, with 40 liter columns. Urea does not raise the solution conductivity or compete with solute for binding sites on the resin and therefore its use does not affect the yield of the recovered desired product.
  • the present invention is directed to an improved method of performing expanded bed chromatography (EBC) .
  • the improvement comprises maintaining a sufficiently high concentration of urea in the EBC column charge and wash solution to reduce or substantially prevent fouling of the resin during the chromatography.
  • EBC is a purification procedure that is well known in the art and is described in detail in US Patent No. 5,522,993 to Carlsson, et al . and and Barnfield, et al . , Bioprocess Engineering 16: 57 (1997) .
  • EBC can be used to separate a desired soluble component from an impure mixture, which can include insoluble particulate components.
  • EBC employs a column which contains a bed of solid phase polymer, also referred to as a "resin" . The resin selectively binds the desired component over other impurities.
  • EBC also works by differential binding, e.g., some bound contaminants can be eluted selectively by a wash step, leaving the desired component bound to the resin for later elution.
  • an elution step can selectively elute the desired component while leaving more tightly bound contaminants on the resin.
  • EBC can be used when a suitable solid phase resin is available that selectively or differentially binds the desired component over other impurity (ies) present in the mixture .
  • EBC differs from conventional packed bed chromatography in that the chromatography is run in an "upflow manner", i.e., the column charge and wash solutions are introduced or loaded sequentially onto the column from the bottom and are then pumped upwards through the column.
  • the desired component binds to the resin, while the impurities pass upwards and out of the top of the column.
  • the desired component can then be eluted from the resin by passing a suitable elution solution through the column in either a downflow or upflow manner.
  • EBC is particularly well suited for purifying biomolecules from unclarified feedstocks, e.g., eukaryotic or prokaryotic cell extracts, cell lysates or fermentation broths from, mammalian, fungal, plant or yeast cells.
  • EBC is therefore often used to separate recombinant proteins from insoluble cellular debris and soluble biomolecules (e.g., lipids, salts, amino acids, sugars, proteins and nucleic acids) in the cell homongenates or fermentation broths in which they are produced.
  • EBC has been described for ion exchange chromatography (anionic or cationic) as well as hydrophobic interaction, metal chelating and Protein A adsorption modes.
  • the method can be used to purify other biomolecules which adhere to the resin, including nucleic acids, carbohydrates and glycoproteins and the like.
  • the mixture from which the desired component is to be isolated is solubilized to form a column charge.
  • the term "solubilize” refers to combining the mixture with an aqueous solvent or a suitable organic/aqueous solvent mixture so that the desired component dissolves.
  • the column charge can contain other components such as detergents (non-ionic detergents, if ion exchange chromatography is being used) . Some or all of the other components in the mixture may also dissolve. Thus, solubilizing the mixture results in a solution that may contain some particulate matter.
  • the resulting solution also referred to as the "column charge” or “sample solution” is used to load or apply the mixture from which a desired component is to be isolated to a chromatography column; in the case of EBC, the column is loaded from the bottom.
  • concentration of the desired component in the column charge is chosen such that the desired component binds to the resin in the column and is typically between about 0.01 mg/mL and about 10.0 mg/mL.
  • the wash solution is loaded onto the column immediately after the column charge to elute both the soluble and insoluble impurities from the column. As the wash solution passes upward through the column, insoluble components and soluble components which bind to the resin less strongly than the desired component pass through the column and out the top, leaving the desired component bound to the resin.
  • wash solution and the column charge are typically the same solution, but for the presence of the impure mixture in the column charge.
  • the wash solution can differ from the column charge, provided that the desired component remains bound to the resin as the wash solution is being passed through the column.
  • the wash solution may contain salts or have a different pH such that the desired component remains bound to the resin while contaminants are selectively eluted.
  • the column charge and wash solution also contain a sufficiently large concentration of urea to reduce or substantially prevent column fouling.
  • Column fouling refers to reduced affinity of the resin for components which are normally absorbed onto the resin, increased clumping of the resin and increased channeling in the column. Column fouling results in changes in fluid dynamics wherein the purification of the desired component is adversely effected. When fouling reaches a certain level, the resin must be regenerated or replaced.
  • Maintaining the urea concentration above about 5.0 M preferably above about 6.0 M and preferably between about 6.0 M and about 8.0 M, more preferably between about 5.0 M and about 7.0 M, even more preferably between about 6.3 M and about 7.0 M in these solutions increases the longevity of the resin to greater than about 25 runs when the resin is used to isolate a recombinant protein or peptide from an unclarified solution. Although lesser concentrations of urea can also be used, the increase in resin longevity is not as great.
  • the concentration of urea is sufficient to reduce fouling when the resin lifetime is longer in the presence of urea than in its absence. Column lifetime can be measured, for example, by the number of runs before the resin needs to be regenerated or replaced.
  • the concentration of urea in the column charge and wash solution can be the same or different, but is preferably the same or higher in the wash solution. Most preferably the concentration of urea in the wash solution is higher than that used in the column charge. The higher urea concentration in the wash creates separate layers wherein there is very little mixing between the wash solution and the column charge. The concentration differential works to create a meniscus or plug that allows particulate matter to be pushed out of the top of the column much more efficiently.
  • the column charge has a concentration of urea between about 5 M and 6.5 M and the wash has a urea concentration greater than about 6.5 M.
  • the urea concentration in the wash is between about 7.0 M and about 8.0 M.
  • wash solution is passed through the column until substantially all of the non-bound components (soluble and insoluble) have eluted from the column.
  • wash solution is continued until the eluent is clear and colorless.
  • the refractive index or visible/ultraviolet absorption of the eluent can be monitored to determine when the non-bound components have completely washed off the column.
  • a "stable, fluidized bed” refers to resin beds in which there is little translational movement of the individual resin particles. Resin particles are suspended in equilibrium due to the balance between particle sedimentation velocity and upward flow. Thus, a given resin particle will remain within a limited volume that is a minute fraction of the total bed volume during the chromatography. In addition, there is little or no channeling.
  • the flow rate during EBC is selected so that a stable, fluidized bed is maintained.
  • suitable rates may vary according to the type of resin, charge solution density and column size and shape, one of ordinary skill in the art can select flow rates using routine experimentation.
  • the flow rate should not be so great that the bed expands and contacts the top adapter or top of the column. Flow rates from between about 5-3000 cm/hour are known in the art, typically between about 5-500 cm/hour and more typically between about 50-200 cm/hour.
  • the resins are selected so that a stable, fluidized bed is maintained.
  • the resin particles should be relatively small to allow short diffusion distances and have a high density.
  • resin particles with a diameter between about 100-1000 jura and a particle density between about 1.10-1.50 g/ml when hydrated are typical, although more dense particles are known in the art (e.g., zirconium-based particles).
  • the particles can be spherical or irregular in shape.
  • the resin particles typically comprise a polymer matrix into which glass, quartz, silica particles or zirconium are incorporated and should be large enough so as not to pass through the screen at the bottom of the column.
  • glass, quartz, silica particles or zirconium are incorporated and should be large enough so as not to pass through the screen at the bottom of the column.
  • spherical and irregular shaped glass and silica particles having a size in the range of from 100-300 jura are typical, although smaller particles are also known in the art.
  • the incorporated material is typically in the range of from about 5-50% of the weight of the wet final particle.
  • the polymer is derivatized to selectively bind the desired component.
  • suitable polymers include mono- or polyvinyl monomers such as acrylates, methacrylates or vinylbenzenes, or naturally-occurring polymers such as polysaccharides (e.g., agarose, starch, cellulose or derivatives thereof) .
  • the polymer is crosslinked for the desired rigidity and pore distribution.
  • Ion exchange resins contain groups which are suitable for binding ions, e.g., diethylamino ethyl or quarternary amine groups.
  • Suitable resin particles are available commercially from Amersha Pharmacia Biotech under the tradename STREAMLINE. Examples are found in the 2000 Pharmacia Biotech Catalog on pages 162-63, and include STREAMLINE CHELATING, Catolog No. 17-1280 (a metal chelating resin),
  • STREAMLINE DEAE Catolog No. 17-0994 (a weak anion exchange resin)
  • STREAMLINE HEPARIN catalog No. 17-1284 (a polysaccharide anticoagulant resin)
  • STREAMLINE PHENYL Catalog 17-5121 (a hydrophobic interaction resin)
  • STREAMLINE QXL Catolog No. 17-5075 (a strong, highly substituted anion exchange resin)
  • STREAMLINE RPROTEINA Catalog No. 17-1281 (an immunoglobulin binding)
  • STREAMLINE SP Catalog No. 17-0993 (a strong cation exchange resin)
  • STREAMLINE SPXL 17-5076 a highly substituted cation exchange resin
  • Ion exchange resins anionic or cationic are preferably used to separate recombinant proteins from unclarified feedstocks.
  • the precise amount of resin that is used can be readily determined by one of ordinary skill in the art, although there may be some variation depending upon the application and the feedstock. Typically, between about 10 to about 50 grams of crude product can be loaded on one liter of resin, more typically between about 20 to about 30 grams.
  • the desired component is desorbed from the column with an elution buffer.
  • the elution buffer can be applied in an upflow fashion as with the column charge and wash solution. Alternatively and preferably, the elution buffer is applied in a downflow fashion; the resin particles settle and the elution buffer is loaded from the top of the column as in conventional packed bed chromatography. Downflow elution has the advantage in that no movement of the top adapter is required, i.e., a fixed volume column can be used. This provides for greater ease of operation and the ability to use a less expensive, fixed head column.
  • elution buffer is a solution which, when passed through a chromatography column, results in decreased binding (or desorption) between a resin and a component that is bound to the resin.
  • elution buffers typically contain ionic components which compete with the desired component for the binding sites on the resin.
  • Salt solutions such as aqueous sodium chloride, potassium chloride and other physiologically acceptable salts are examples of suitable elution buffers for ion exchange resins.
  • salt concentrations of between about 0.3 M and about 1.0 M are used in the elution buffer. Changes in the pH can also change the binding affinity of the desired component to the resin.
  • the elution buffer is generally less dense than the wash solution, which contains urea.
  • the more dense wash solution will move down the column with a sharp band of the water elution buffer on top of it, resulting in "plug" flow.
  • Plug flow results in excellent chromatographic performance by providing a sharp elution band with little or no band spreading at the urea/water interface.
  • plug flow elutes the bound material in a smaller volume than would be attained in an upward flow mode, which is an asset to downstream processing.
  • Water is also used as an elution buffer with hydrophobic interaction chromatography, where high salt drives solutes onto the resin.
  • the desired component can be further purified or isolated by any suitable means such as lyophilization, spray drying or crystallization.
  • the invention described herein is preferably used as an early purification step in a series of purification steps that may be necessary to purify a particular peptide or protein to homogeneity such that the protein can be formulated and administered as a pharmaceutically acceptable drug product.
  • the purification step described herein is used as a first step to purify particulate matter from a solubilized suspension of prokaryotic or eukaryotic cells that have been engineered to express the peptide or protein of interest.
  • a pharmaceutically acceptable drug product may have the active protein or peptide combined with a pharmaceutically- acceptable buffer, and the pH adjusted to provide acceptable stability and solubility properties, and a pH acceptable for parenteral administration.
  • Pharmaceutically-acceptable anti-microbial agents may also be added. Meta-cresol and phenol are preferred pharmaceutically-acceptable antimicrobial agents.
  • One or more pharmaceutically-acceptable salts may also be added to adjust the ionic strength or tonicity.
  • One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin is an example of an isotonicity-adjusting excipient.
  • “Pharmaceutically acceptable” means suitable for administration to a human, and does not contain toxic elements, undesirable contaminants or the like, and does not interfere with the activity of the active compounds therein.
  • the present invention is suitable for use with any protein or peptide that is not irreversibly denatured when solubilized in the presence of the concentrations of urea described herein.
  • Most biologically active proteins and peptides are not overly sensitive to urea because urea typically does not cause breaks in disulfide bonds.
  • proteins or peptides that do unfold or change conformation in the presence of urea can generally be renatured by diluting with elution buffer that does not contain urea.
  • proteins and peptides suitable for use in the processes of the present invention include: Glucagon-like peptide-1 (GLP-1) and analogs thereof such as Val 8 -GLP-l(7-37)OH or Arg 34 -GLP-1 (7-37) OH.
  • GLP-1 Glucagon-like peptide-1
  • analogs thereof such as Val 8 -GLP-l(7-37)OH or Arg 34 -GLP-1 (7-37) OH.
  • U.S. Patent No. 5,118,666 U.S. Patent No 5,977,071; U.S. Patent No. 5,545,618; U.S. Patent No. 5,705,483; U.S. Patent No . 5,977,071; U.S. Patent No. 6,133,235; and Adelhorst, et al., " . Biol . Chem .
  • GLP-1 analogs that can be purified using the invention described herein.
  • Additional examples of proteins and peptides useful in the processes of the present invention include exendin-3, exendin-4, analogs of exendin-3 and exendin-4, human insulin, human insulin analogs such as LysB27ProB28-human insulin or AspB28-insulin, human growth human and analogs thereof, parathyroid hormone and analogs thereof.
  • Suitable peptides and proteins include but are not limited to, calcitonin, erythropoietin (EPO) , factor IX, factor VIII, 5-lipoxygenase and cyclooxygenase products and inhibitors, granulocyte colony stimulating factor (G-CSF) , granulocyte macrophage colony stimulating factor (GM-CSF) , macrophage colony stimulating factor (M-CSF) , nerve growth factor (NGF) , ciliary neurotrophic factor (CNF) , defensins, chemokines, growth hormone releasing factor (GRF), insulinlike growth factor (IGF-1) , growth hormone, heparins (regular and low molecular weight) , cyclosporin, insulin, leptin and its analogs and inhibitors interferon- .alpha.
  • EPO erythropoietin
  • factor IX factor IX
  • factor VIII 5-lipoxygenase and cyclooxy
  • interleukin-2 interleukin-2
  • IL-3 interleukin-3
  • IL-4 interleukin-4
  • IL-6 interleukin-6
  • interleukin-11 interleukin- 12
  • interleukin-1 receptor antagonist interleukin-1 receptor
  • IL-1R interleukin-1 receptor
  • LHRH luteinizing hormone releasing hormone
  • octreotide vasopressin analogs, peptide Y, gastrins, CCK peptides, thymosin- .alpha. -1, Ilb/IIIa inhibitors, .alpha. -1 antitrypsin, anti-RSV antibody, cystic fibrosis transmembrane regulator (CFTR) gene, integrins, selectins, deoxyribonuclease (DNase) , and FSH
  • the peptide or protein of interest may be expressed as a fusion protein wherein this fusion protein contains a cleavable component such as a carboxypeptidase leader sequence fused to the active protein or peptide.
  • a cleavable component such as a carboxypeptidase leader sequence fused to the active protein or peptide.
  • fusion proteins can be further purified and cleaved such that the active protein or peptide is released.
  • the active component then can be formulated such that is suitable as a pharmaceutically acceptable drug product.
  • the columns used for EBC typically have: a) a distributor in form of a bottom port that distributes an inlet flow- of liquid equally over the cross-sectional area of the column, (b) a top port, and (c) there between a void volume for holding resin particles.
  • STREAMLINE Columns suitable for use in EBC are commercially available from Pharmacia Biotech under the tradename STREAMLINE (see page 94 of the 2000 Pharmacia Biotech catalog), e.g., STREAMLINE 50, STREAMLINE 100, STREAMLINE 200 and STREAMLINE CD. Other columns are available from Upfront Chromatography A/S, Copenhagen, Denmark.
  • a system for carrying out EBC generally includes pumps for supplying the appropriate solutions, valves, as appropriate, to switch the flow of solvents into the column, a sample container, a column as described above, an adjustable upper adapter and a fraction collector.
  • the column is loaded with a resin suitable for selectively binding the desired component.
  • the wash solution is then pumped into the bottom of column with the outflow at the top of the column connected to waste.
  • the column charge is pumped into the column under conditions so that a stabilized fluidized bed is maintained.
  • a wash solution is pumped into the column. After the fluidized bed has been thoroughly washed, and unbound components such as cell particles have been eluted to waste through the top of the column, the first phase of the procedure is finished.
  • the flow through the column is reversed for elution of the bound sample components.
  • the valves are switched so that the flow is from a pump through the top of the column out of the bottom of the column to the fraction collector.
  • a suitable elution solution is introduced via pump into the bed whereby the desired component is released and collected.
  • the upper adapter which is of the traditional type found in a great number of columns for chromatography, is adjustable to a desired position in the column and contains a net to prevent beads from passing out the top of the column.
  • elution of the column is carried out in the fluidized mode by introducing the elution solution via a pump into the bottom of the column and collecting the sample constituents released from the resin with a fraction collector connected to the upper outlet of the column.
  • a fraction collector connected to the upper outlet of the column.
  • E. coli cells that were genetically engineered to express a fusion protein comprised of a carboxypeptidase leader sequence fused to the GLP-1 analog, Val 8 -GLP-1 (7-37 ) OH (a thirty-one amino acid peptide) were diluted with one volume of water and collected by centrifugation on a disk stack centrifuge. This cell paste containing the engineered fusion protein could be frozen for later use. To extract the fusion protein, the E. coli cell paste was suspended at up to 8% solids in 7 M urea containing 10 mM ethylendiamine, 0.5 M EDTA at pH 8.0. The pH of the mixture was raised to 11.5 using 10% NaOH. The mixture was incubated for 40 to 60 minutes at ambient temperature and then brought back to pH 8.0 using 10% HCl . Due to the volume of the cell paste, the concentration of the urea was between 5.5 and 6.3 M.
  • the column charge was prepared from extracted fusion protein as in Example 1 by dilution to 4% solids with 7 M urea (final volume 450 ml) containing 5.32 g fusion protein/ml.
  • the column tube (2.5 x 100 cm STREAMLINE column) was filled with 75 milliliters of STREAMLINE DEAE resin. The resin and column were treated sequentially according to the following table in the order as shown:
  • Example 2 This example was run to test the effect of repetitive runs on the performances of the EBC STREAMLINE DEAE column using starting material as prepared in Example 1 and the protocol essentially as indicated Example 2. Centrifuged cell paste (250 milliliters) was diluted with extraction buffer (7.0 M Urea, 20 mM Ethylene Diamine, 845 milliliters), pH adjusted to 11.5 for about 60 minutes, then back to 8.0. Finally, the charge material was diluted with additional urea (7.0 M, 1300 milliliters) to reduce conductivity to about 3 milliSiemens and bring urea concentration to about 6.3 M. The final ethanol and acetic acid washes were run after runs 7, 29 and 50. What is shown below is comparative data from runs number 5, 30 and 50.
  • Fusion protein charge was prepared as in Example 1.
  • the column was a STREAMLINE 200 and was filled with about 10 Liters if STREAMLINE DEAE resin, i.e., about 32 centimeter bed height.
  • Six successive runs were made on the column using running positions, buffers and relative buffer volumes as described in Example 2. Results of runs 4 and 6 are shown in the data box below to demonstrate reproducibility and scaleability of the process from a 2.5 centimeter diameter column to a 20 centimeter diameter column.
  • the ethanol wash described as the last step in Example 2 was not run in between any of these 6 column runs .
  • the charge solution was prepared essentially as described in Example 1.
  • the fusion protein solution was charged onto a STREAMLINE 400 column filled with about 40 Liters of STREAMLINE DEAE resin (40 x 32 cm of packed resin) .
  • buffers as described in Example 2 were run in the same order for this scaled up version of the EBC column run. The results of the run are described in Example 3.
  • Mainpeak pool was 82% pure as measured by high performance liquid chromatography.

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Abstract

L'invention concerne un procédé relatif à la séparation d'une composante visée (par exemple biomolécule du type protéine de recombinaison) dans un mélange qui peut être une charge non clarifiée. Selon une première étape, le mélange est solubilisé dans une solution d'urée aqueuse, donnant une solution échantillon. Selon une seconde étape, la solution est remontée vers un lit de résine, ce qui entraîne la liaison de la composante visée avec la résine et la séparation de cette composante par rapport au mélange. La concentration d'urée est suffisamment élevée pour réduire l'encrassement de la résine lorsque la solution traverse cette résine.
PCT/US2001/027736 2000-10-13 2001-09-28 Procede ameliore de chromatographie a lit etendu WO2002032537A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001296228A AU2001296228A1 (en) 2000-10-13 2001-09-28 Improved method of expanded bed chromatography

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US24045500P 2000-10-13 2000-10-13
US60/240,455 2000-10-13

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WO2002032537A2 true WO2002032537A2 (fr) 2002-04-25
WO2002032537A3 WO2002032537A3 (fr) 2002-07-11

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066734A1 (fr) * 2008-12-08 2010-06-17 Novo Nordisk A/S Purification à contre-courant de polypeptides
CN107782829A (zh) * 2017-11-01 2018-03-09 上海莱士血液制品股份有限公司 一种利用离子色谱法检测生物制品中三羟甲基氨基甲烷（Tris）的方法
US11369707B2 (en) * 2017-01-30 2022-06-28 Regeneron Pharmaceuticals, Inc. Compositions and methods for reducing bioburden in chromatography

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1042892A (ja) * 1996-08-01 1998-02-17 Higeta Shoyu Co Ltd タンパク質の回収精製法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1999 BROBJER MICHAEL: "Development and scale up of a capture step (expanded bed chromatography) for a fusion protein expressed intracellularly in Escherichia coli." Database accession no. PREV200000091626 XP002194008 & BIOSEPARATION, vol. 8, no. 1-5, 1999, pages 219-228, ISSN: 0923-179X *
DATABASE WPI Section Ch, Week 199817 Derwent Publications Ltd., London, GB; Class B04, AN 1998-131057 XP002194009 & JP 10 042892 A (HIGETA SHOYU KK), 17 February 1998 (1998-02-17) *
HJORTH R: "Expanded-bed adsorption in industrial bioprocessing: recent developments" TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 15, no. 6, 1 June 1997 (1997-06-01), pages 230-235, XP004064512 ISSN: 0167-7799 *
JOHANSSON H J ET AL: "Large scale recovery and purification of periplasmic recombinant protein from E. coli using expanded bed adsorption chromatography followed by new ion exchange media" JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 48, no. 1, 18 July 1996 (1996-07-18), pages 9-14, XP004037029 ISSN: 0168-1656 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066734A1 (fr) * 2008-12-08 2010-06-17 Novo Nordisk A/S Purification à contre-courant de polypeptides
US9441028B2 (en) 2008-12-08 2016-09-13 Novo Nordisk A/S Counter current purification of polypeptides
US11369707B2 (en) * 2017-01-30 2022-06-28 Regeneron Pharmaceuticals, Inc. Compositions and methods for reducing bioburden in chromatography
CN107782829A (zh) * 2017-11-01 2018-03-09 上海莱士血液制品股份有限公司 一种利用离子色谱法检测生物制品中三羟甲基氨基甲烷（Tris）的方法

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