US20110045574A1 - Chromatography medium - Google Patents

Chromatography medium Download PDF

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US20110045574A1
US20110045574A1 US12/988,707 US98870709A US2011045574A1 US 20110045574 A1 US20110045574 A1 US 20110045574A1 US 98870709 A US98870709 A US 98870709A US 2011045574 A1 US2011045574 A1 US 2011045574A1
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Prior art keywords
separation medium
hydrophobic
ligands
separation
lid
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Inventor
Jan Bergstrom
Gunnar Glad
Bo-Lennart Johansson
Jean-Luc Maloisel
Nils Norrman
Tobias E. Soderman
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Cytiva Bioprocess R&D AB
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Assigned to GE HEALTHCARE BIO-SCIENCES AB reassignment GE HEALTHCARE BIO-SCIENCES AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JOHANSSON, BO-LENNART, MALOISEL, JEAN-LUC, SODERMAN, TOBIAS E., GLAD, GUNNAR, NORRMAN, NILS, BERGSTROM, JAN
Publication of US20110045574A1 publication Critical patent/US20110045574A1/en
Assigned to GE HEALTHCARE BIOPROCESS R&D AB reassignment GE HEALTHCARE BIOPROCESS R&D AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GE HEALTHCARE BIO-SCIENCES AB
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    • 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
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B01D15/327—Reversed phase with hydrophobic interaction
    • 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
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
    • B01J20/28009—Magnetic properties
    • 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
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • 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
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/287—Non-polar phases; Reversed phases
    • 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
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • 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
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291—Characterised by the shape of the carrier, the coating or the obtained coated product
    • B01J20/3293—Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
    • 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
    • B01J39/00—Cation exchange; Use of material as cation exchangers; Treatment of material for improving the cation exchange properties
    • B01J39/26—Cation exchangers for chromatographic processes
    • 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
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material

Definitions

  • the present invention is within the field of chromatography. More precisely, it relates to a novel chromatography medium, namely a hydrophobic medium provided with different lids excluding molecules over a certain size due to the porosity of the hydrophobic medium and/or the porosity of the lid.
  • the chromatographic methods suggested up to date are based on different modes of interaction with a target.
  • the functional groups are permanently bonded ionic groups with their counter ions of opposite charge
  • hydrophobic interaction chromatography HIC
  • the interaction between the stationary phase and the component to be separated is based on hydrophobicity.
  • HIC Chromatography based on hydrophobic interaction is generally divided in to two types named HIC and RPC.
  • HIC refers to hydrophobic interaction chromatography of proteins using very hydrophilic matrixes to which hydrophobic ligands are immobilized to a very low degree in order to avoid denaturation of separated proteins.
  • hydrophobic ligands are immobilized to a very low degree in order to avoid denaturation of separated proteins.
  • RPC refers to chromatography using matrixes usually silica based functionlized to a very high degree with hydrophobic non polar molecules/ligands usually aliphatic carbon chains containing 4 to 18 carbon atoms. Adsorption is normally achieved without the need of added salt. Desorption or elution is most often done by gradually increasing the content of an organic solvent in the elution solution.
  • RPC media with 18 carbon chains binds proteins strongly.
  • the proteins are also denaturated to different degree during the binding process. In order to elute the proteins it is necessary to use extraordinary conditions.
  • RPC media with shorter chains (4 Carbons) may be used together with water/acetonitrile based eluents containing additives such as triflouracetic acid for protein separations.
  • the stationary phase also known as the separation matrix, comprises a support, which is commonly a plurality of essentially spherical particles, and ligands coupled to the support.
  • the support is porous to allow a larger amount of ligand and consequently more bound target compound in each particle.
  • the support is most often a natural or synthetic polymer and the spherical particles may be produced in a number of different ways. Silica and glass beads are also used. Natural polymers often used for this purpose are the polysaccharides dextran and agarose.
  • the porosity of the matrix is very important.
  • Advantages of polymeric media are that it is easy to vary pore size over broad ranges and their high chemical stability e.g. tolerance of high pH:values.
  • a general rule, which is accepted throughout the literature, is to use media with large pore sizes for large molecules. Mass transfer in these pores is a result of diffusion processes and not of convection.
  • mAbs Humanized monoclonal antibodies hold significant promise as biopharmaceuticals.
  • HCPs host cell proteins
  • a wide variety of mAbs isolation technique is available today that include affinity chromatography on Protein A, ion exchange chromatography and hydrophobic interaction chromatography (HIC). All these techniques involve the use of a mobile phase (adsorption buffer) that must be adjusted to accomplish interaction between mAb-molecules and the ligands.
  • a chromatographic medium which has a pore size distribution and surface properties that prevents large molecules, such as mAbs, from entering the beads and which internally can adsorb proteins and other peptides or biomolecules independently of buffer conditions.
  • Ion exchangers aimed for separation of biomolecules are able to adsorb most charged biomolecules at low ionic strength but when the ionic strength is increased over a certain level these media loose the ability to adsorb the sample molecules.
  • the present invention provides a medium having such a high hydrophobicity in the core entity that proteins can interact or be adsorbed both at very low and high ionic strengths in a broad pH range.
  • the invention in a first aspect, relates to a separation medium comprising a porous, hydrophobic core entity; and a porous, hydrophilic lid covering the whole exterior of the core entity, wherein the lid only allows molecules under a certain size to penetrate and interact with the internal part of the core entity of the separation medium.
  • the hydrophobic core entity only allows molecules under a certain size to penetrate and interact with the internal part of the core entity of the separation medium.
  • the porous hydrophilic lid is a porous outer layer or jacket/shell surrounding the core entity.
  • the separation medium is preferably provided with a highly hydrophobic core in order to bind proteins and other hydrophobic molecules independently of the properties of the sample and of running conditions such as ionic strength and pH, in case of chromatography, or the supernatant, in case of a batch-wise procedure.
  • the separation medium is preferably bead-shaped but may also be a membrane.
  • the size exclusion property of the medium is determined by the porosity of one or both of its porous constituents.
  • the certain size referred to above is below the size that excludes the target molecules/organism/particles such as cells, cell particles, virus, virus like particles, plasmids, any type of antibodies, lipids, proteins, peptides and nucleic acids. This medium will efficiently separate low size molecules from larger molecules of the type mentioned above.
  • the hydrophobic core entity may be hydrophobic per se and may be based on a hydrophobic polymer.
  • a hydrophobic polymer For example, styren/ethylstyren/DVB, vinylethers and acrylates containing hydrophobic substituents as well as fluoroalkane-containing polymers.
  • the hydrophobic core entity is hydrophilic per se and is based on a hydrophilic polymer, functionalized with hydrophobic interaction ligands.
  • the hydrophobic interaction ligands may comprise aliphatic hydrocarbons, such as C1-C30 alkyl, preferably C4-C16 alkyl, and/or aromatic hydrocarbons, such as phenyl, antracene, naphtalene.
  • the ligand density should always be optimized for adsorption at low ionic strength and in the purpose of obtaining the desired highly hydrophobic nature of the medium.
  • the ligand density should be above normal HIC-levels, i.e. >90 ⁇ mole/ml core entity.
  • the hydrophobic core entity allows adsorption of the molecules (with the lowest molecular weight and/or smallest size) in the sample that are able to penetrate the lid at low ionic strength, such as 0-1 M, preferably 0-0.4 M, most preferably 0-0.1 M, and in the pH interval 2-11.
  • the core entity comprises further ligands besides said hydrophobic ligands which may be located on and/or associated with the core entity and/or on the hydrophobic ligands.
  • the further ligands may be hydrophobic and may contain a non dominating charged group.
  • said further ligands are electrostatic interaction ligands, i.e. a positive and/or a negative charge ligand.
  • these further ligands may be octylamine ligands, provided with a positive charge, which increases the binding of small negatively charged molecules to the core entity.
  • the amount of further added charged ligands should be adjusted so that they do not interfere too much with the hydrophobic interaction to the main hydrophobic ligand.
  • the core entity and lid are made of agarose and the hydrophobic ligands are hydrocarbon ligands comprising 4-16 carbons, preferably octyl ligands.
  • the core entity has a pore size preventing molecules over a certain size from entering the pores.
  • a polymeric hydrogel e.g. dextran is provided in the lid to fill the pores and thereby further decrease and adjust the pore size to prevent high molecular weight molecules from entering the pores.
  • the hydrophilic lid comprises any type of chromatography ligands that reversibly bind the molecules within the sample with the highest molecular weight(s) and/or largest size(s). Some of the small molecules will probably also bind to the lid but during the elution step they most probably will penetrate the lid and bind to the core.
  • magnetic particles may be incorporated into the core entities of the separation medium.
  • the invention also relates to a separation medium which comprises the lid bead medium described above in mixture with conventional chromatography media, such as HIC, IE or affinity media.
  • chromatography media such as HIC, IE or affinity media.
  • the lid bead medium comprises up to 10% of the total media volume.
  • the lid bead medium comprises octyl ligands and the chromatography media comprises a cation exchange media. Benefits of this mixed media are increased resolution between large and small molecules.
  • the invention in a second aspect, relates to use of the separation medium as described above, for adsorbing molecules under a certain size from a sample at low ionic strength at pH 2-11, preferably in one single step.
  • the procedure may be used to purify molecules over or under said size.
  • this aspect involves a method of using the separation medium comprising a step of adsorbing molecules and optional further step(s).
  • the pore size referred to above varies with, and is determined by, the size of the target molecule in the specific application.
  • This certain size range preferably corresponds to the size range of molecules/organisms/particles selected from cells, cell particles, bacteria, virus, virus like particles, plasmids, antibodies, lipids, proteins, peptides, nucleic acid.
  • the sample may be, for example, a culture supernatant and the high molecular weight molecule may be a monoclonal antibody.
  • the separation medium will be very useful for addition at cell harvest to rapidly remove undesired enzymes, like proteases, peptidases and nucleases, such as trypsin, chymotrypsin, DNase and RNase.
  • the separation medium may be used in chromatographic or batch mode. Any column or batch format may be used. For some applications, such as purification of biopharmaceuticals for clinical phase I and II studies, disposable columns in RTP (ready to process) format are preferred.
  • the adsorbed low molecular weight molecules may be the desired molecules, such as biomarkers or drug markers, which are eluted, for example by decreased polarity in the eluent, from the core entity and further analysed.
  • the target molecule is adsorbed in the core entity but not used for further analysis, such as for lipid removal.
  • the target molecule is a large molecule/organism/particle, such as a cell, cell particles, bacteria, virus, virus like particles, plasmids, antibodies, proteins, it may obtained in the flow-through and thus not adsorbed by the medium.
  • a large molecule/organism/particle such as a cell, cell particles, bacteria, virus, virus like particles, plasmids, antibodies, proteins
  • the target molecule is a large molecule/organism/particle, such as a cell, cell particles, bacteria, virus, virus like particles, plasmids, antibodies, proteins, which is adsorbed on the lid.
  • the target molecule is then eluted by a technique appropriate for the chosen absorption mode (e.g. ion exchange: salt and/or a pH gradient, IMAC: imidazol gradient, boronate: sugar or pH gradient, HIC: lowering the salt concentration).
  • the preferred use of the separation medium according to the invention is for purification of monoclonal antibodies.
  • Another preferred use is wherein the target molecule is a molecule or particle ⁇ 1 000 kD.
  • This embodiment is suitable for purification of virus, such as influenza virus, but also for other large molecules such as IgM antibodies.
  • FIG. 1 is an illustration of three different bead designs that have been tested.
  • FIG. 1A SEPHAROSETM 20 Fast Flow (20% agarose particle) and Spinning disc and
  • FIG. 1B lid-dextran SEPHAROSETM 6 Fast Flow.
  • the present invention relates to a chromatographic medium which has both hydrophilic and hydrophobic regions in the matrix and may be based on a matrix which is hydrophobic per se, or on a hydrophilic matrix with hydrophobic ligands, and is provided with a hydrophilic outer layer.
  • the medium may also be based on a hydrophilic matrix internally functionalized with hydrophobic ligands (e.g. a hydrophobic internal core in a bead).
  • Hydrophobic per se start material e.g. DVB beads covered by a hydrophilic and porous layer.
  • hydrophilic start material fully functionalized with hydrophobic functional groups and then covered by a hydrophilic and porous layer. Layer activation of a hydrophilic matrix and internally couple hydrophobic ligands ( FIG. 1 ).
  • the invention also provides a method of separating large molecular weight molecules, such as antibodies, from other components of a liquid, which requires less time and process steps than the prior art methods. This is achieved by a method wherein the liquid comprising the desired high molecular weight molecules, such as antibodies, is contacted with the chromatographic medium, and substantially pure high molecular weight molecules are recovered in non-binding or reversible binding mode.
  • Another advantage is that low molecular weight molecules, such as host cell proteins, adsorb to the internal parts of the medium even at very low ionic strengths.
  • the support matrix and core entity of the chromatographic medium can be based on organic or inorganic material. It is preferably in the form of an organic polymer, which is insoluble but may be more or less swellable in water.
  • Suitable polymers are polyhydroxy polymers, e.g. based on polysaccharides, such as agarose, dextran, cellulose, starch, pullulan, etc. and completely synthetic polymers, such as polyacrylic amide, polymethacrylic amide, poly (hydroxyalkylvinyl ethers), poly(hydroxyalkylacrylates) and polymethacrylates (e.g.
  • polyglycidylmethacrylate polyvinylalcohols and polymers based on styrenes and divinylbenzenes, and copolymers in which two or more of the monomers corresponding to the above-mentioned polymers are included.
  • Polymers, which are soluble in water, may be derivatized to become insoluble, e.g. by cross-linking and by coupling to an insoluble body via adsorption or covalent binding.
  • Hydrophilic groups can be introduced on hydrophobic polymers (e.g.
  • Suitable inorganic materials to be used in support matrices are silica, glass, zirconium oxide, graphite, tantalum oxide etc.
  • Preferred support matrices lack groups that are unstable against hydrolysis, such as silanol, ester, amide groups and groups present in silica as such.
  • the support matrix is in the form of irregular or spherical particles with sizes in the range of 1-1000 ⁇ m, preferably 5-50 ⁇ m for high performance applications and 50-300 ⁇ m for preparative purposes.
  • An interesting form of support matrix has densities higher or lower than the liquid sample or buffer solutions to be used, such as fermentation feeds.
  • This kind of matrices is especially applicable in large-scale operations for fluidised or expanded bed chromatography as well as for different batch wise procedures, e.g. in stirred tanks. Fluidised and expanded bed procedures are described in WO 92/18237 and WO 92/00799. The most practical use of these matrices has been to combine particles/beads with a density higher than the density of a fluidising liquid with an upward flow.
  • This kind of support matrices in expanded bed mode is particularly beneficial in case the sample solution contains particulate and sticky materials.
  • hydrophilic support matrix in practice means that the accessible surface of the matrix is hydrophilic and protein friendly, i.e. that the surface not irreversibly adsorbs and denaturates proteins.
  • accessible surfaces on a hydrophilic base matrix expose a plurality of polar groups for instance comprising oxygen and/or nitrogen atoms. Examples of such polar groups are hydroxyl, amino, carboxy, sulphonate (S and SP ligands) ester, ether of lower alkyls (such as (—CH 2 CH 2 O—) n H where n is an integer 2, 3, 4 and higher).
  • a hydrophilic surface coat, possible in the form of hydrophilic extenders belongs conceptually to the support matrix.
  • This invention relates to a medium which maximizes the interaction with molecules small enough to penetrate into the hydrophobic core of the separation matrix, such as HCPs, and minimizes the interaction with the largest proteins in the samples, such as MAb-molecules and MAb-dimers in a MAb purification process. Furthermore, one embodiment of the invention enables that mobile phases with no extra addition of salt can be used and that no elution buffer (desorption buffer) is needed to recover the desired high molecular weight molecules or organisms for embodiments with neutral or almost neutral lids (non-binding lids).
  • elution buffer desorption buffer
  • agarose beads based on high contents of agarose (20%) was designed to obtain a bead with a porosity that excludes large proteins (monoclonal antibodies) ( FIG. 1A ).
  • a prototype based on SEPHAROSETM 6 Fast Flow and a gel filtration lid was produced. The lid was obtained by coupling dextran to the outer segment of the beads. The pore sizes obtained in the dextran filled segment is designed to prevent large molecules, such as mAbs, to enter the core of the beads ( FIG. 1B ).
  • the third approach is based on the spinning disc media described in WO2009/099375. Also in this case the beads were designed to exclude large proteins ( FIG. 1A ) by the introduction of a neutral outer segment achieved as described by commonly owned layer activation patents EP 0 966 488 B1 (Process for introducing a functionality.)
  • a synthesis procedure was used to prevent ligands to be coupled to the surface of the beads and in that way also eliminate adsorption due to hydrophobic interaction of monoclonal antibodies to the outer part of the beads.
  • the prototypes are named lid-OH core-octyl media meaning that the octyl ligands only are attached in the core of the beads.
  • the amount of ligands in the core of the beads is adjusted so that host cell proteins are adsorbed using adsorption buffers with very low ionic strengths adjusted to any pH relevant for chromatography of biomolecules.
  • Volumes of matrix refer to settled bed volume and weights of matrix given in gram refer to suction dry weight.
  • stirring is referring to a suspended, motor-driven stirrer since the use of magnet bar stirrer is prompt to damage the beads.
  • Small-scale reactions (up to 20 mL of gel) were performed in closed vials and stirring refers to the use of a shaking table.
  • Agarose (50 g) was dissolved in water (250 g) by heating at 95° C. for approximately 10 hours.
  • the solution was added to toluene (375 mL) and ethyl cellulose (35 g) in an emulsification vessel, the temperature was kept at 70° C.
  • the emulsification vessel was equipped with a blade stirrer. The speed of the stirrer was increased step by step from 150 rpm to 340 rpm, maintaining the temperature at 70° C. When the agarose particles were judged by a microscope to have a desired size the speed was decreased to 150 rpm. Thereafter the emulsion was cooled and the beads were allowed to gel. The beads were washed with ethanol and water.
  • Allyl activated SEPHAROSETM 20 Fast Flow 50 g of drained SEPHAROSETM 20 Fast Flow (SEPHAROSETM 6 Fast Flow, GE Healthcare, Uppsala, Sweden) was mixed with 15 mL of 50% NaOH solution, 6 g of Na 2 SO 4 and 0.1 g of NaBH 4 . The mixture was stirred at 50° C. for 1 h, followed by addition of 30 mL allyl glycidyl ether (AGE). The reaction slurry was stirred at 50° C. for 17 h. Then the gel was washed on a glass filter with distilled water, ethanol and finally with distilled water again.
  • SEPHAROSETM 6 Fast Flow was allylated according to above and the allyl content was determined by titration to 259 ⁇ mol/mL.
  • the Spinning Disc apparatus was manufactured by ABB Industriservice according to given specification (see below):
  • the agarose solution was fed to six discs via needles. By using six discs instead of one, there is an increase in capacity.
  • the agarose flow was the same to each of the six discs. This means that the bead size originating from each disc is the same.
  • the speed range of the discs was adjusted within 3001-3010 rpm and the relative humidity in the dome was 100%. If the relative humidity is less than 100% there is a risk that water will be evaporated from the agarose drops.
  • the porosity of the spinning disc beads after cross-linking with epichlorohydrin is presented in Table 1.
  • the porosity was estimated with different dextrans and the void volume was obtained with blue dextran 2000.
  • the spinning disc prototype was produced to obtain a porosity that not allows immunoglobulins to penetrate the beads. This means that molecules with a molecular weight larger than ca. 150 000 g/mol should not diffuse into the beads.
  • the particle size was 190 ⁇ m ⁇ 5 ⁇ m.
  • the spinning disc prototype was used to produce media for capture of proteins with a molecular weight less than approximately 70 000 g/mol while larger molecules such as immunoglobulins (human IgG) should not be able to diffuse into the beads and interact with the ligands in the core of the beads.
  • Allyl activated spinning disc medium Spinning disc medium was washed with distilled water on a glass filter. The gel, 25 mL, was drained on the filter and weighed into a 3-necked round bottomed flask. NaOH (20 mL, 50%-solution) was added and mechanical stirring started. Sodium borohydride, 0.1 g, and sodium sulphate, 2.9 g, were added to the flask and the slurry heated to 50° C. on a water bath. After approximately one hour 27.5 mL of allyl glycidyl ether was added. The slurry was then left under vigorously stirring over night.
  • the slurry was transferred to a glass filter and the pH adjusted to around 7 with acetic acid (60%). The gel was then washed with distilled water ( ⁇ 4), ethanol ( ⁇ 4) and distilled water ( ⁇ 4). The allyl content was then determined by titration to 321 ⁇ mol/mL.
  • the three different octyl media to be investigated (Prototypes: lid-OH core-octyl SEPHAROSETM 20 Fast Flow, lid-OH core-octyl spinning disc and lid-dextran octyl core SEPHAROSETM Fast Flow), with respect to breakthrough capacity, were packed in HR 5/5 columns and the sample solution was pumped at a flow rate of 0.3 or 1.0 mL/min through the column after equilibration with buffer solution.
  • the breakthrough capacity was evaluated at 10% of the maximum UV detector signal (280 nm). The maximum UV signal was estimated by pumping the test solution directly into the detector.
  • the breakthrough capacity at 10% of absorbance maximum (Q b10% ) was calculated according to the formula:
  • T R10% is the retention time (min) at 10% of absorbance maximum
  • T RD the void volume time in the system (min)
  • C concentration of the sample (4 mg protein/mL)
  • V C column volume (mL).
  • the adsorption buffer used at breakthrough capacity measurements was 25 mM TRIS (pH 8.0) or 50 mM acetate (pH 4.75).
  • Prototype lid-OH core-octyl SEPHAROSETM 20 Fast Flow was also tested with a clarified feed in flow-through mode.
  • the feed 32 mL was pumped through a HR 5/5 column packed with the prototype and the flow-through fraction (36 mL) was analysed with respect of the amount of host cell proteins (HCP) and the amount of monoclonal antibody recovered.
  • the adsorption buffer used at these experiments was 25 mM phosphate buffer adjusted to pH 7.2.
  • the samples used for breakthrough measurements were human immunoglobulin (IgG, Gammanorm), ovalbumin and lysozyme.
  • the proteins were dissolved in the adsorption buffers at a concentration of 4 mg/mL and only one protein at a time was applied into the column
  • the monoclonal antibody was an IgG1 (based on IEF its pI is in the range 7.5 to 8.4), expressed in NS0 cells.
  • the filtered non-purified cell culture supernatant was used as sample.
  • the concentration of the mAb in the sample was 1.3 mg/mL and 32 mL of the sample were applied to the column (HR 5/5 column packed with prototype lid-OH core-octyl SEPHAROSETM 20 Fast Flow).
  • the growth medium used at production of the monoclonal antibody was DMEM (Gibco) and 10% fetal calf serum (FCS, Gibco).
  • HCP Host Cell Protein
  • Samples for ELISA were pre-treated by addition of 10% 2.0 M Tris, 1% BSA, 0.5% TWEENTM 20, pH 8.0 (50 ⁇ L BSA solution to 450 ⁇ L sample).
  • the samples were diluted in “Sample Diluent Buffer” (catalogue number F223A, Cygnus Technologies) and analyzed by a NS/0 HCP ELISA kit (catalogue number F220, Cygnus Technologies) using the “High sensitivity protocol” specified in the kit.
  • the spectrophotometer VERSAMAXTM and the software SOFTMAX® Pro both from Molecular Devices, was used for reading and evaluation of the plates.
  • Lid-OH core-octyl SEPHAROSETM 20 Fast Flow is based on SEPHAROSETM 20 Fast Flow with octyl as core ligand.
  • the base matrix SEPHAROSETM 20 Fast Flow is designed with high content of agarose in order to obtain a porosity that prevents monoclonal antibodies to penetrate the beads.
  • SEPHAROSETM 20 Fast Flow was activated with a high degree of allyl groups (0.37 mmol/mL) meaning that high ligand content was obtained in the core of the beads.
  • This type of media is mainly aimed to be used for only one cycle. However it is possible to elute adsorbed host cell proteins. To verify this adsorbed lysozyme was eluted by using 1.0 M NaOH+30% isopropanol as desorption buffer.
  • This prototype is based on SEPHAROSETM 6 Fast Flow that has a porosity that makes it possible for IgG to diffuse into the matrix. Therefore, according to FIG. 1 the pore sizes in the outer part of the beads have been reduced by attaching dextran and in that way prevent IgG to diffuse into the beads. According to Table 2 the breakthrough capacity of IgG was 0 mg/mL which clearly proves that IgG not diffuse into the beads. However, a relatively high breakthrough capacity was observed for ovalbumin (16 mg/mL). The molecular weight of ovalbumin and IgG is approximately 43 000 and 150 000 g/mol, respectively. This means that the dextran lid has a high “size-selectivity” and can allow ovalbumin to diffuse into the beads while IgG is prevented to interact with the core ligands.
  • Lid-OH core-octyl Spinning disc is based on a Spinning disc beads with octyl as core ligand.
  • the Spinning disc medium was designed to obtain a porosity that prevents monoclonal antibodies to penetrate the beads.
  • the spinning disc prototype was activated with a high degree of allyl groups (0.32 mmol/mL) meaning that high ligand content was obtained in the core of the beads. Furthermore, no ligands were coupled in the outer part of the beads (see the preparation of the beads). According to Table 1 the IgG breakthrough capacity was very low (0.6 mg/mL) while the capacity of ovalbumin was 23 times higher. These results also show that the porosity of the spinning disc prototype means that very small amounts of IgG are able to diffuse into the core of the beads.
  • Table 3 are the results from recovery and HCP measurements presented.
  • the HCP content was reduced by more than 50% and the recovery of the mAb was 89%.
  • influenza virus When producing influenza virus at large scale aiming at influenza vaccines it is important to reduce the levels of protein and DNA in the final preparation.
  • the particles of the present invention are well suited for the purification of viruses since viral particles are significantly larger in size than most of the contaminants.
  • the DotBlot HA assay was used according to a standard protocol.
  • the PICOGREEN® DNA assay was used according to the manufacturers instruction (available from Invitrogen).
  • the Bradford protein assay was used according to the manufacturers instruction. (Available from Bio-Rad)
  • the DNA ladder used was 1 kb Plus DNA marker (Invitrogen)
  • influenza virus sample produced in-house was used in the study.
  • the virus was propagated in MDCK cells until lysis occurred.
  • Influenza virus strain A/H1N1/Solomon Islands was used.
  • the material was concentrated ⁇ 10 ⁇ in an ultrafiltration (UF) step and another ⁇ 2 ⁇ in a diafiltration (DF) step (Hollow Fiber Cartridge 500 kDa).
  • the diafiltration buffer was 50 mM Tris-HCl, 150 mM NaCl pH 7.3 and the sample was frozen until used.
  • the starting material and flow-through fraction was analysed for virus concentration, DNA concentration and protein concentration. The virus recovery, DNA depletion and protein depletion was calculated. The results are shown in Table 4 and Table 5.
  • DNA with molecular weight up to around 500 base pairs are efficiently removed while the larger DNA cannot enter the pore structure and bind to the positively charged octyl amine ligands situated in the inner part of the particles.
  • a second purification step such as a conventional anion exchange step could remove the larger DNA in this case.
  • the sample can be treated with a nuclease such as BENZONASE® to reduce the molecular weight to well below 500 base pairs and in that way obtain almost complete DNA and protein depletion when the influenza virus sample is passed through a column packed with particles of the present invention.
  • a nuclease such as BENZONASE®

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018063981A1 (fr) * 2016-09-29 2018-04-05 Bio-Rad Laboratories, Inc. Procédés de purification de conjugués protéine-nanoparticule
US20180273909A1 (en) * 2015-09-22 2018-09-27 Sanofi Pasteur Biologics, Llc Purification of respiratory syncytial virus
CN111420644A (zh) * 2020-03-27 2020-07-17 北京石油化工学院 一种复合型混合模式聚合物层析介质的制备方法
US10814305B2 (en) 2016-09-29 2020-10-27 Bio-Rad Laboratories, Inc. Agarose-filled ceramic apatite
US12409441B2 (en) 2020-02-19 2025-09-09 Bio-Rad Laboratories, Inc. Method of preparing polymer-filled chromatography resin

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102015094B (zh) 2008-04-22 2016-11-09 通用电气医疗集团生物工艺研发股份公司 层析介质
US20110142863A1 (en) * 2009-12-16 2011-06-16 Millipore Corporation Flow through purification processes for large biomolecules
IN2012DN06243A (fr) 2010-02-19 2015-09-25 Ge Healthcare Bio Sciences Ab
CN103502807B (zh) * 2011-03-31 2016-06-29 通用电气健康护理生物科学股份公司 用于样本制备的方法和装置
WO2013015738A1 (fr) * 2011-07-22 2013-01-31 HJERTÉN, Maria Capture de bioparticules et de biopolymères pathogènes et non pathogènes
US9580692B2 (en) 2012-02-29 2017-02-28 Ge Healthcare Bioprocess R&D Ab Method for endotoxin removal
US10041050B2 (en) 2012-02-29 2018-08-07 Ge Healthcare Bioprocess R&D Ab Method for endotoxin removal
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JP6702587B2 (ja) * 2014-03-14 2020-06-03 ジーイー・ヘルスケア・バイオプロセス・アールアンドディ・アクチボラグ 生物粒子の精製用分離マトリックス
US10626164B2 (en) 2014-07-25 2020-04-21 Csl Limited Purification of VWF
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GB201506113D0 (en) 2015-04-10 2015-05-27 Ge Healthcare Bio Sciences Ab Method for chromatography
EP3439780B1 (fr) * 2016-04-06 2024-07-03 Cytiva BioProcess R&D AB Matrice de séparation
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GB201806736D0 (en) * 2018-04-25 2018-06-06 Ge Healthcare Bioprocess R&D Ab Method for virus purification
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JP2026503238A (ja) 2022-12-27 2026-01-28 サイティバ・バイオプロセス・アールアンドディ・アクチボラグ 生物学的粒子の精製
JP2026512897A (ja) 2023-04-12 2026-04-21 サイティバ・バイオプロセス・アールアンドディ・アクチボラグ クロマトグラフィーシステム、その使用及びエンベロープ又は膜状生物学的粒子を分離するための方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039094A1 (fr) * 1997-03-04 1998-09-11 Amersham Pharmacia Biotech Ab Matrices de separation et separation utilisant ces matrices
US6531523B1 (en) * 2000-10-10 2003-03-11 Renal Tech International, Llc Method of making biocompatible polymeric adsorbing material for purification of physiological fluids of organism
US20030125656A1 (en) * 2001-12-31 2003-07-03 Vadim Davankov Hemo-and biocompatible beaded polymeric material for purification of physiological fluids of organism, method of producing the material, as well as method of and device for purification of physiological fluids of organism with use of the material
WO2004020994A1 (fr) * 2002-08-27 2004-03-11 Amersham Biosciences Ab Particules chromatographiques bicouches
US20050181378A1 (en) * 2004-02-18 2005-08-18 Applera Corporation Polyelectrolyte-coated size-exclusion ion-exchange particles
US20050267295A1 (en) * 1999-11-25 2005-12-01 Makonnen Belew Method for selective removal of a substance from samples containing compounds having nucleic acid structure
WO2007054548A2 (fr) * 2005-11-09 2007-05-18 Nanosep Ab Particules

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS61287444A (ja) * 1985-06-13 1986-12-17 Toyo Soda Mfg Co Ltd 二重構造担体
DK165090D0 (da) 1990-07-09 1990-07-09 Kem En Tec As Konglomererede partikler
SE9101149D0 (sv) 1991-04-17 1991-04-17 Pharmacia Lkb Biotech Beads for down stream processing
SE9700768D0 (sv) 1997-03-04 1997-03-04 Pharmacia Biotech Ab Förfarande för att introducera funktionalitet
CN1250329C (zh) * 2003-09-27 2006-04-12 南开大学 内毒素吸附剂及其制备方法
US20050079984A1 (en) 2003-10-08 2005-04-14 Miles Samuel Lee Method for stripping cured paint from plastic, steel, aluminum, brass, magnesium and non-ferrous substrates with surfactants low in volatile organic compounds
JP2007522807A (ja) * 2004-02-18 2007-08-16 アプレラ コーポレイション 高分子電解質コーティングされたサイズ排除イオン交換粒子
BRPI0514435B8 (pt) * 2004-08-20 2021-05-25 American Nat Red Cross isolamento sequêncial de proteínas e esquemas de purificação através de cromatografia por afinidade
SG131016A1 (en) * 2005-09-19 2007-04-26 Millipore Corp Asymmetric porous adsorptive bead
US20080299671A1 (en) * 2005-12-02 2008-12-04 Ge Healthcare Bio-Sciences Ab Hydrophobic Interaction Chromatography
CN101765458B (zh) * 2007-07-25 2014-05-28 通用电气健康护理生物科学股份公司 分离基质
CN101939094B (zh) * 2008-02-05 2013-11-20 通用电气健康护理生物科学股份公司 分离介质的制备方法
CN102015094B (zh) 2008-04-22 2016-11-09 通用电气医疗集团生物工艺研发股份公司 层析介质
KR100889904B1 (ko) 2008-12-16 2009-03-23 주식회사 효민산업 수중직립 관절이완 및 척추교정 장치

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998039094A1 (fr) * 1997-03-04 1998-09-11 Amersham Pharmacia Biotech Ab Matrices de separation et separation utilisant ces matrices
US20050267295A1 (en) * 1999-11-25 2005-12-01 Makonnen Belew Method for selective removal of a substance from samples containing compounds having nucleic acid structure
US6531523B1 (en) * 2000-10-10 2003-03-11 Renal Tech International, Llc Method of making biocompatible polymeric adsorbing material for purification of physiological fluids of organism
US20030125656A1 (en) * 2001-12-31 2003-07-03 Vadim Davankov Hemo-and biocompatible beaded polymeric material for purification of physiological fluids of organism, method of producing the material, as well as method of and device for purification of physiological fluids of organism with use of the material
WO2004020994A1 (fr) * 2002-08-27 2004-03-11 Amersham Biosciences Ab Particules chromatographiques bicouches
US20050181378A1 (en) * 2004-02-18 2005-08-18 Applera Corporation Polyelectrolyte-coated size-exclusion ion-exchange particles
WO2007054548A2 (fr) * 2005-11-09 2007-05-18 Nanosep Ab Particules

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
Amersham. Reversed phase chromatography principles and methods. Amersham Biosciences. 1999;1-86. *
Bruker Daltonics. Purification Kits MB-HIC. 2006. p1-6. *
Maa et al. Mixed-bed ion-exchange columns for protein high-performance liquid chromatography. Journal of Chromatography. 1988;452:331-345. *
Mahoney P. DNA molecular weight. Newton's Home Page. 2003;1-2. *
Supelco. Discovery DSC-MCAX (Mixed-Mode Cation Exchange) SPE Products. Sigma-Aldrich. 2003. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180273909A1 (en) * 2015-09-22 2018-09-27 Sanofi Pasteur Biologics, Llc Purification of respiratory syncytial virus
WO2018063981A1 (fr) * 2016-09-29 2018-04-05 Bio-Rad Laboratories, Inc. Procédés de purification de conjugués protéine-nanoparticule
US10814305B2 (en) 2016-09-29 2020-10-27 Bio-Rad Laboratories, Inc. Agarose-filled ceramic apatite
US10934366B2 (en) 2016-09-29 2021-03-02 Bio-Rad Laboratories, Inc. Protein-nanoparticle conjugate purification methods
US11905335B2 (en) 2016-09-29 2024-02-20 Bio-Rad Laboratories, Inc. Protein-nanoparticle conjugate purification methods
US12409441B2 (en) 2020-02-19 2025-09-09 Bio-Rad Laboratories, Inc. Method of preparing polymer-filled chromatography resin
CN111420644A (zh) * 2020-03-27 2020-07-17 北京石油化工学院 一种复合型混合模式聚合物层析介质的制备方法

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