US20110155668A1 - Separation medium for chromatography of various biomolecules - Google Patents

Separation medium for chromatography of various biomolecules Download PDF

Info

Publication number
US20110155668A1
US20110155668A1 US13/002,878 US200913002878A US2011155668A1 US 20110155668 A1 US20110155668 A1 US 20110155668A1 US 200913002878 A US200913002878 A US 200913002878A US 2011155668 A1 US2011155668 A1 US 2011155668A1
Authority
US
United States
Prior art keywords
lid
inner core
separation medium
porosity
biomolecules
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US13/002,878
Inventor
Gunnar Glad
Bo-Lennart Johansson
Jean-Luc Maloisel
Nils Norrman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytiva Sweden AB
Original Assignee
GE Healthcare Bio Sciences AB
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 GE Healthcare Bio Sciences AB filed Critical GE Healthcare Bio Sciences AB
Assigned to GE HEALTHCARE BIO-SCIENCES AB reassignment GE HEALTHCARE BIO-SCIENCES AB ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MALOISEL, JEAN-LUC, GLAD, GUNNAR, JOHANSSON, BO-LENNART, NORRMAN, NILS
Publication of US20110155668A1 publication Critical patent/US20110155668A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/26Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804Affinity chromatography
    • B01D15/3828Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/26Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28054Solid 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 surface properties or porosity
    • B01J20/28088Pore-size distribution
    • B01J20/28092Bimodal, polymodal, different types of pores or different pore size distributions in different parts of the sorbent
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282Porous sorbents
    • B01J20/285Porous sorbents based on polymers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30Processes for preparing, regenerating, or reactivating
    • B01J20/32Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3291Characterised by the shape of the carrier, the coating or the obtained coated product
    • B01J20/3293Coatings on a core, the core being particle or fiber shaped, e.g. encapsulated particles, coated fibers
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/16Extraction; Separation; Purification by chromatography
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/16Extraction; Separation; Purification by chromatography
    • C07K1/22Affinity chromatography or related techniques based upon selective absorption processes
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/10Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166Fluid composition conditioning, e.g. gradient

Definitions

  • the present invention relates to a novel separation medium intended for chromatography of various biomolecules.
  • the separation medium has an inner core and a lid which are differently charged. Large molecules are separated from the medium by charge repulsion and small molecules are eluted from inner core of the separation medium.
  • chromatography Within biotechnology, one of the most widely used separation methods is chromatography.
  • the term chromatography embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile.
  • the sample mixture introduced into the mobile phase, undergoes a series of interactions i.e. partitions between the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase.
  • Separated components emerge in a certain order, depending on their interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
  • 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
  • Other chromatographic separation principles are well known to the skilled person in the art.
  • 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. Natural polymers often used for this purpose are the polysaccharides dextran and agarose.
  • the porosity of the beads is very important.
  • One advantage of polymeric media is the opportunity of pore size variation over broad ranges.
  • a general rule, which is accepted throughout the literature, is to use media with large pore sizes for large molecules.
  • the present invention provides novel media and methods for separation of large as well as small biomolecules which combines several separation principles and parameters into one and the same medium.
  • the invention provides a separation medium, comprising an inner core of a porous material provided with charged ligands, and an outer lid comprising a porous material provided with charged ligands, wherein the charge of the ligands in the inner core is opposite that of the charge of the ligands in the lid.
  • the ligands in the core are negatively charged then the ligands in the lid are positively charged, and vice versa.
  • the porous material is preferably a sieving material, such as a gel filtration material commonly used for chromatography.
  • the porous material in the inner core may have the same porosity as the porous material in the lid. In this case the amount of charged ligands is higher in the lid than in the core.
  • the ligand density in the lid should be >10%, such as >25% or >50% or >100%, of the ligand density in the core.
  • the separation medium may be designed for any type of biomolecule(s) and/or portions thereof.
  • the porosity of the lid and the inner core allows penetration of a biomolecule with a molecular weight of less than approximately 100 000 g/mol.
  • This limit is very useful for separation of relatively large molecules, such as MAbs, which do not enter the inner core but are obtained in the flow through if a column format is used.
  • the separation medium may be used in any format, such as a column format or batch format.
  • the porosity may be chosen to exclude molecules of certain sizes or weights and to allow penetration of those molecules that are smaller and/or lighter than these.
  • the invention is not restricted to any specific porosity ranges, on the contrary, the porosity of the lid and inner core is to be decided in dependence on the size and or molecular weight of the biomolecule(s) intended to be purified.
  • the porosity of the inner core and lid allows penetration of a biomolecule with a molecular weight of less than 50 000 g/mol. This limit may be very useful for separation of small molecules which have entered the inner core and may be eluted therefrom.
  • the porous material in the inner core has the larger porosity than the porous material in the lid.
  • the porosity of the lid is such that biomolecules larger or equal to a molecular weight of 20 000, preferably 50 000 or 100 000 g/mol are excluded, and the porosity of the inner core is larger than the lid porosity.
  • the porous material is derived from a synthetic polymer material, such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters and vinylamides, or from a natural polymer material, such as carbohydrate material selected from agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan and alginate.
  • a synthetic polymer material such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters and vinylamides
  • a natural polymer material such as carbohydrate material selected from agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan and alginate.
  • the charged ligands may be selected from, for example are —SO 3 ⁇ , —COO ⁇ , —N + (CH 3 ) 3 or —NH + (C 2 H 5 ) 2
  • the invention in a second aspect, relates to a method for biomolecule separation comprising applying a sample to the above described separation medium, wherein large molecules are prevented from entering the medium by charge repulsion from the medium and small molecules are captured in the inner core.
  • the charge repulsion is caused by the charged ligands in the lid.
  • the small molecules may be eluted with a salt gradient.
  • a typical desorption means that ionic strength is increased compared to that used during adsorption and in many cases corresponds to at least 0.4-0.6 M NaCl.
  • the small molecules are eluted with a salt gradient and/or a pH gradient, in such a way that a portion of the small molecules gradually adhere to the ligands in the lid on their way out from the separation medium.
  • the method may be run in any column format and the desired biomolecules may be obtained in the flow-through. This is the case when it is desired to purify or separate large molecules such as cells, cell particles, virus, plasmids or immunoglobulins.
  • the method is especially suitable for MAb purification.
  • the method may be run in column format and the desired molecules may be obtained by elution from the inner core of the separation medium. This is the case when it is desired to purify or separate small molecules such as small proteins and peptides.
  • the method is especially suitable for biomarker purification.
  • FIG. 1 shows a schematic illustration of the chromatographic properties and the construction of SEPHAROSETM 6 Fast Flow bead with a DEAE-dextran lid and a negatively charged ligand in the core of the bead.
  • P is host cell proteins and IgG is human immunoglobulin.
  • FIG. 2 is a schematic illustration of the chromatographic properties and the construction of SEPHAROSETM 6 Fast Flow bead with positively charged groups ( ⁇ N + (CH 3 ) 3 — prototype Q) in the lid and a negatively charged ligand (—SO 3 ⁇ ) in the core of the bead.
  • P is host cell proteins and IgG is human immunoglobulin.
  • the present invention may be used for separation of humanized monoclonal antibodies (mAbs) which hold significant promise as biopharmaceuticals.
  • mAbs humanized monoclonal antibodies
  • HCPs host cell proteins
  • the separation medium according to the invention may be used for separation of almost any molecule but will be exemplified by a medium having a lid and an inner core wherein said medium maximizes the HCPs interaction and minimizes the interaction between the IgG molecules (monoclonal antibodies) and the core-ligands.
  • This new chromatography medium of the invention makes it possible to apply adsorption conditions so that most of the HCPs interact with core-ligands.
  • FIG. 1 is a schematic illustration of a bead constructed with a lid comprised of both a gel filtration lid and a positively charged ligand.
  • the mobile phase To accomplish charge repulsion the mobile phase must be adjusted to a pH-value where the monoclonal antibodies are positively charged ( FIG. 1 ).
  • the interior of the beads are occupied with core-ligands that are negatively charged ( FIG. 1 ).
  • the host cell proteins also must be positively charged to be able to interact with the negatively charged core ligands.
  • IgG is hindered to penetrate into the beads by charge repulsion and a steric effect (due to smaller pore sizes in the lid compare to the pore sizes in the core of the beads).
  • FIG. 2 is a bead construction depicted that excludes IgG to penetrate the core of the bead only by charge repulsion.
  • the pore size distribution of the bead means that IgG can penetrate into the core of the bead but the charge repulsion lid prevent this to happen.
  • the charge repulsion lid is designed in a way that smaller proteins than IgG are not influenced by charge repulsion and will therefore have access to the interior of the beads.
  • Volumes of matrix refer to settled bed volume.
  • Weights of matrix given in gram refer to suction dry weight. It is understood that these matrices are still water solvated material.
  • 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.
  • DEAE Dextran Lid Prototypes (DEAE-DX1, DEAE-DX2 And DEAE-DX3) Based on SEPHAROSETM 6 Fast Flow
  • SEPHAROSETM 6 Fast Flow was washed with distilled water on a glass filter. The gel, 100 mL, was drained on the filter and weighed into a 3-necked round bottomed flask. NaOH (50 mL, 50%-solution) was added and mechanical stiffing started. Sodium borohydride, 0.4 g, and sodium sulphate, 11.4 g, were added to the flask and the slurry heated to 50° C. on a water bath. After approximately one hour, 100 mL of allyl glycidyl ether (AGE) was added. The slurry was then left under vigorously stirring over night.
  • AGE allyl glycidyl ether
  • 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; 270 ⁇ mol/mL.
  • DEAE-dextran Coupling of DEAE-dextran in the lid. 10 mL portions of the partially brominated gel were transferred to flasks and mixed with DEAE-dextran solutions (see below). DEAE-dextran was obtained from GE Healthcare, Uppsala, Sweden. Three prototypes with different amount of DEAE-dextran applied were constructed:
  • Prototype DEAE-DX1 7.2 g DEAE-Dextran in 15 mL of water.
  • Prototype DEAE-DX2 6.0 g DEAE-Dextran in 15 mL of water.
  • Prototype DEAE-DX3 4.8 g DEAE-Dextran in 15 mL of water.
  • SEPHAROSETM 6 Fast Flow was washed with distilled water on a glass filter. The gel, 100 mL, was drained on the filter and weighed into a 3-necked round bottomed flask. NaOH (50 mL, 50%-solution) was added and mechanical stiffing started. Sodium borohydride, 0.4 g, and sodium sulphate, 11.4 g, were added to the flask and the slurry heated to 50° C. on a water bath. After approximately one hour, 100 mL of AGE 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; 270 ⁇ mol/mL.
  • the charge repulsion lid media to be investigated (Prototypes: DEAE-DX1, DEAE-DX2, DEAE-DX3 and Q), 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 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 RS 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 50 mM acetate (pH 4.0).
  • the samples used were human immunoglobulin (IgG, Gammanorm), bovine serum albumin (BSA), 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 fractionation range of SEPHAROSETM 6 Fast Flow for globular proteins is 1 ⁇ 10 4 -4 ⁇ 10 6 according to the manufacture (GE Healthcare, Uppsala, Sweden).
  • this invention has proved that human immunoglobulin can be selectively be hindered to diffuse into the beads by charge repulsion lid and a combination of charge repulsion and a “gel filtration lid” ( FIGS. 1 and 2 ) while smaller proteins have access to the interior of the beads.
  • the breakthrough capacity of all four prototypes was determined for a number of proteins (IgG, BSA, ovalbumin and lysozyme).
  • the molecular weight of the proteins is shown in Table 2.
  • the mobile phase used 50 mM acetate, pH 4.0
  • the isoelectric point of the proteins is presented in Table 2).

Abstract

The present invention relates to a separation medium, comprising an inner core of a porous material provided with charged ligands, and an outer lid comprising a porous material provided with charged ligands, wherein the charge of the ligands in the inner core is opposite that of the charge of the ligands in the lid. The present invention also relates to a method for biomolecule separation comprising applying a sample to the above separation medium, wherein large molecules are prevented from entering the medium by charge repulsion from the medium and small molecules are captured in the inner core.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a filing under 35 U.S.C. §371 and claims priority to international patent application number PCT/SE2009/050666 filed Jun. 4, 2009, published on Jan. 14, 2010 as WO 2010/005364, which claims priority to application number 0801640-4 filed in Sweden on Jul. 8, 2008.
  • FIELD OF THE INVENTION
  • The present invention relates to a novel separation medium intended for chromatography of various biomolecules. The separation medium has an inner core and a lid which are differently charged. Large molecules are separated from the medium by charge repulsion and small molecules are eluted from inner core of the separation medium.
  • BACKGROUND OF THE INVENTION
  • Within biotechnology, one of the most widely used separation methods is chromatography. The term chromatography embraces a family of closely related separation methods. The feature distinguishing chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions i.e. partitions between the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through a column containing the stationary phase. Separated components emerge in a certain order, depending on their interaction with the stationary phase. The least retarded component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
  • The chromatographic methods suggested up to date are based on different modes of interaction with a target. Thus, for example, in ion-exchange chromatography, the functional groups are permanently bonded ionic groups with their counter ions of opposite charge, while in hydrophobic interaction chromatography (HIC), the interaction between the stationary phase and the component to be separated is based on hydrophobicity. Other chromatographic separation principles are well known to the skilled person in the art.
  • 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. In most separation matrices, 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. Natural polymers often used for this purpose are the polysaccharides dextran and agarose.
  • For separation of biomolecules, by chromatographic and batch-wise procedures, the porosity of the beads is very important. One advantage of polymeric media is the opportunity of pore size variation over broad ranges. A general rule, which is accepted throughout the literature, is to use media with large pore sizes for large molecules.
  • It would be desirable to have alternative ways of separating large molecules without having to use large porosity and which are not limited to separation of large molecules but may also be used to separate molecules of any size.
  • SUMMARY OF THE INVENTION
  • The present invention provides novel media and methods for separation of large as well as small biomolecules which combines several separation principles and parameters into one and the same medium.
  • In a first aspect, the invention provides a separation medium, comprising an inner core of a porous material provided with charged ligands, and an outer lid comprising a porous material provided with charged ligands, wherein the charge of the ligands in the inner core is opposite that of the charge of the ligands in the lid.
  • If the ligands in the core are negatively charged then the ligands in the lid are positively charged, and vice versa.
  • The porous material is preferably a sieving material, such as a gel filtration material commonly used for chromatography.
  • The porous material in the inner core may have the same porosity as the porous material in the lid. In this case the amount of charged ligands is higher in the lid than in the core. The ligand density in the lid should be >10%, such as >25% or >50% or >100%, of the ligand density in the core.
  • The separation medium may be designed for any type of biomolecule(s) and/or portions thereof.
  • Preferably, the porosity of the lid and the inner core allows penetration of a biomolecule with a molecular weight of less than approximately 100 000 g/mol. This limit is very useful for separation of relatively large molecules, such as MAbs, which do not enter the inner core but are obtained in the flow through if a column format is used. The separation medium may be used in any format, such as a column format or batch format. The porosity may be chosen to exclude molecules of certain sizes or weights and to allow penetration of those molecules that are smaller and/or lighter than these. The invention is not restricted to any specific porosity ranges, on the contrary, the porosity of the lid and inner core is to be decided in dependence on the size and or molecular weight of the biomolecule(s) intended to be purified.
  • In another embodiment the porosity of the inner core and lid allows penetration of a biomolecule with a molecular weight of less than 50 000 g/mol. This limit may be very useful for separation of small molecules which have entered the inner core and may be eluted therefrom.
  • In an alternative embodiment, the porous material in the inner core has the larger porosity than the porous material in the lid. For example, the porosity of the lid is such that biomolecules larger or equal to a molecular weight of 20 000, preferably 50 000 or 100 000 g/mol are excluded, and the porosity of the inner core is larger than the lid porosity.
  • The porous material is derived from a synthetic polymer material, such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters and vinylamides, or from a natural polymer material, such as carbohydrate material selected from agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan and alginate.
  • The charged ligands may be selected from, for example are —SO3 , —COO, —N+(CH3)3 or —NH+(C2H5)2
  • In a second aspect, the invention relates to a method for biomolecule separation comprising applying a sample to the above described separation medium, wherein large molecules are prevented from entering the medium by charge repulsion from the medium and small molecules are captured in the inner core.
  • In this case, the charge repulsion is caused by the charged ligands in the lid.
  • The charge repulsion, and thereby the prevention of entering of large molecules, may be accompanied by a lower porosity (=steric hindrance) in the lid compared to the inner core.
  • In the method according to the invention large molecules are obtained in the flow through by charge repulsion and small molecules adsorbed in the inner core are eluted from the separation medium.
  • The small molecules may be eluted with a salt gradient. A typical desorption means that ionic strength is increased compared to that used during adsorption and in many cases corresponds to at least 0.4-0.6 M NaCl.
  • Alternatively, the small molecules are eluted with a salt gradient and/or a pH gradient, in such a way that a portion of the small molecules gradually adhere to the ligands in the lid on their way out from the separation medium.
  • The method may be run in any column format and the desired biomolecules may be obtained in the flow-through. This is the case when it is desired to purify or separate large molecules such as cells, cell particles, virus, plasmids or immunoglobulins. The method is especially suitable for MAb purification.
  • The method may be run in column format and the desired molecules may be obtained by elution from the inner core of the separation medium. This is the case when it is desired to purify or separate small molecules such as small proteins and peptides. The method is especially suitable for biomarker purification.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows a schematic illustration of the chromatographic properties and the construction of SEPHAROSE™ 6 Fast Flow bead with a DEAE-dextran lid and a negatively charged ligand in the core of the bead. P is host cell proteins and IgG is human immunoglobulin.
  • FIG. 2 is a schematic illustration of the chromatographic properties and the construction of SEPHAROSE™ 6 Fast Flow bead with positively charged groups (−N+(CH3)3— prototype Q) in the lid and a negatively charged ligand (—SO3 ) in the core of the bead. P is host cell proteins and IgG is human immunoglobulin.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention may be used for separation of humanized monoclonal antibodies (mAbs) which hold significant promise as biopharmaceuticals. One of the most challenges faced in the purification of mAbs is their separation from host cell proteins (HCPs) in the cell culture media.
  • The separation medium according to the invention may be used for separation of almost any molecule but will be exemplified by a medium having a lid and an inner core wherein said medium maximizes the HCPs interaction and minimizes the interaction between the IgG molecules (monoclonal antibodies) and the core-ligands. This was accomplished by modification of chromatography beads with a charge repulsion lid or a lid composed of both a “gel filtration lid” and a charge repulsion lid. This new chromatography medium of the invention makes it possible to apply adsorption conditions so that most of the HCPs interact with core-ligands.
  • In FIG. 1 is a schematic illustration of a bead constructed with a lid comprised of both a gel filtration lid and a positively charged ligand. To accomplish charge repulsion the mobile phase must be adjusted to a pH-value where the monoclonal antibodies are positively charged (FIG. 1). Furthermore, the interior of the beads are occupied with core-ligands that are negatively charged (FIG. 1). This means that the host cell proteins also must be positively charged to be able to interact with the negatively charged core ligands. With the construction presented in FIG. 1 IgG is hindered to penetrate into the beads by charge repulsion and a steric effect (due to smaller pore sizes in the lid compare to the pore sizes in the core of the beads).
  • In FIG. 2 is a bead construction depicted that excludes IgG to penetrate the core of the bead only by charge repulsion. The pore size distribution of the bead means that IgG can penetrate into the core of the bead but the charge repulsion lid prevent this to happen. The charge repulsion lid is designed in a way that smaller proteins than IgG are not influenced by charge repulsion and will therefore have access to the interior of the beads.
  • EXAMPLES
  • The present examples are provided for illustrative purposes only, and should not be construed as limiting the scope of the present invention as defined by the appended claims. All references given below and elsewhere in the present specification are hereby included herein by reference.
  • Preparation of Charge Repulsion Media Based on SEPHAROSE™ 6 Fast Flow and Designed for Capture of Host Cell Proteins General
  • Volumes of matrix refer to settled bed volume.
  • Weights of matrix given in gram refer to suction dry weight. It is understood that these matrices are still water solvated material. For large scale reaction 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.
  • Conventional methods were used for the analysis of the functionality and the determination of the degree of allylation, epoxidation, or the degree of substitution of ion exchanger groups on the beads.
  • Two different ways to prepare a separation matrix according to the invention are exemplified below, starting from a cross linked agarose gel (SEPHAROSE™ 6 Fast Flow, GE Healthcare, Uppsala, Sweden).
  • Example 1 Preparation of DEAE Dextran Lid Prototypes (DEAE-DX1, DEAE-DX2 And DEAE-DX3) Based on SEPHAROSE™ 6 Fast Flow Preparation of DEAE Dextran Lid
  • Allyl activation of SEPHAROSE™ 6 Fast Flow. SEPHAROSE™ 6 Fast Flow was washed with distilled water on a glass filter. The gel, 100 mL, was drained on the filter and weighed into a 3-necked round bottomed flask. NaOH (50 mL, 50%-solution) was added and mechanical stiffing started. Sodium borohydride, 0.4 g, and sodium sulphate, 11.4 g, were added to the flask and the slurry heated to 50° C. on a water bath. After approximately one hour, 100 mL of allyl glycidyl ether (AGE) was added. The slurry was then left under vigorously stirring over night. After about 20 hours 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; 270 μmol/mL.
  • Partial bromination. Allylated gel, 41 mL, was weighed into a flask and 260 mL of distilled water and 0.5 g sodium sulphate was added. 0.53 equivalents of bromine, 300 μl, were then added with a pipette during vigorous stirring. After approximately 5 minutes (when the bromine had been consumed) the gel was washed with distilled water on a glass filter.
  • Coupling of DEAE-dextran in the lid. 10 mL portions of the partially brominated gel were transferred to flasks and mixed with DEAE-dextran solutions (see below). DEAE-dextran was obtained from GE Healthcare, Uppsala, Sweden. Three prototypes with different amount of DEAE-dextran applied were constructed:
  • Prototype DEAE-DX1: 7.2 g DEAE-Dextran in 15 mL of water.
    Prototype DEAE-DX2: 6.0 g DEAE-Dextran in 15 mL of water.
    Prototype DEAE-DX3: 4.8 g DEAE-Dextran in 15 mL of water.
  • To each slurry 2 g of NaOH and 0.1 g NaBH4 were added. The slurries were heated to 50° C. and left stirring over night. After approximately 18 hours the pH was adjusted to approximately 7 with acetic acid (60% solution). The gel was then washed with distilled water on a glass filter. The amount of DEAE-dextran attached (estimated by measure the dry weight before and after coupling of DEAE-dextran) to the prototypes are presented in Table 1 (see the section chromatographic evaluation).
  • Core Coupling
  • Coupling of —SO3 groups in the core of the beads. 10 mL of DEAE-DX prototype (see above) were put with distilled water into a beaker and vigorous overhead stirring was begun. Bromine was added until the slurry had a remaining deeply orange/yellow colour. After 10 minutes of stirring sodium formiate (approximately 1.5 g) was added until the slurry was completely discoloured. The gel was then washed with distilled water on a glass filter.
  • Drained brominated gel was transferred to a flask with 4 g of sodium sulphite, dissolved in 10 mL of 2 M NaOH. The mixture was then left stirring in 50° C. over night. After 20 hours the gel was washed with distilled water on a glass filter.
  • Example 2 Preparation of Prototype Q Based on SEPHAROSE™ 6 Fast Flow Preparation of Q (—N+(CH3)3-groups) Lid
  • Allyl activation of SEPHAROSE™ 6 Fast Flow. SEPHAROSE™ 6 Fast Flow was washed with distilled water on a glass filter. The gel, 100 mL, was drained on the filter and weighed into a 3-necked round bottomed flask. NaOH (50 mL, 50%-solution) was added and mechanical stiffing started. Sodium borohydride, 0.4 g, and sodium sulphate, 11.4 g, were added to the flask and the slurry heated to 50° C. on a water bath. After approximately one hour, 100 mL of AGE was added. The slurry was then left under vigorously stirring over night. After about 20 hours 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; 270 μmol/mL.
  • Partial bromination. Allylated gel, 41 mL, was weighed into a flask and 260 mL of distilled water and 0.5 g sodium sulphate was added. 0.53 equivalents of bromine, 300 μl, were then added with a pipette during vigorous stirring. After approximately 5 minutes (when the bromine had been consumed) the gel was washed with distilled water on a glass filter.
  • Q-coupling (trimethyl ammonium chloride, TMA-chloride). 10 mL of the partially brominated SEPHAROSE™ 6 Fast Flow was transferred to a flask and mixed with 5 mL of TMA-chloride solution. 5 mL of 2 M NaOH was then added and the slurry was heated to 50° C. and left stirring over night. After approximately 18 hours the pH was adjusted to approximately 7 with acetic acid (60% solution). The gel was then washed with distilled water on a glass filter. The total amount of coupled TMA-groups was determined by chloride titration and the Cl capacity was estimated to 105 μmol/mL.
  • Core Coupling
  • Coupling of —SO3 groups in the core of the beads. 10 mL of the Q-lid prototype (see above) were put with distilled water into a beaker and vigorous overhead stirring was begun. Bromine was added until the slurry had a remaining deeply orange/yellow colour. After 10 minutes of stirring sodium formiate (approximately 1.5 g) was added until the slurry was completely discoloured. The gel was then washed with distilled water on a glass filter.
  • Drained brominated gel was transferred to a flask with 4 g of sodium sulphite, dissolved in 10 mL of 2 M NaOH. The mixture was then left stirring in 50° C. over night. After 20 hours the gel was washed with distilled water on a glass filter.
  • Experiment 3 Chromatographic Evaluation of the Four Prototypes (DEAE-DX1, DEAE-DX2, DEAE-DX3 and Q)
  • The charge repulsion lid media to be investigated (Prototypes: DEAE-DX1, DEAE-DX2, DEAE-DX3 and Q), 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 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 (Qb10%) was calculated according to the formula:

  • Q b10 %=(T R10% −T RDC/V c
  • where TR10% is the retention time (min) at 10% of absorbance maximum, TRS the void volume time in the system (min), C the concentration of the sample (4 mg protein/mL) and VC the column volume (mL). The adsorption buffer used at breakthrough capacity measurements was 50 mM acetate (pH 4.0).
  • Sample
  • The samples used were human immunoglobulin (IgG, Gammanorm), bovine serum albumin (BSA), 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.
  • Instrumental Apparatus
  • LC System: ÄKTAexplorer 10 XT or equal
  • Software: UNICRON™ Column: HR 5/5 Instrument Parameters
  • Flow rate: 0.3 mL/min
    Detector cell: 10 mm
  • Wavelength: 280 nm UNICORN™ Method
  • The main method used is depicted below:
  • 0.00 Base CV 1.00 {mL} #Column volume {mL} Any
  • 0.00 Block Start Conditions
      • 0.00 Base SameAsMain
      • 0.00 Wave length 280 {nm} 254 {nm} 215 {nm}
      • 0.00 AvaragingTime 2.56 {sec}
      • 0.00 Alarm Pressure Enable 3.00 {MPa} 0.00 {MPa}
      • 0.00 End Block
    0.00 Block Column Position 0.00 Block Equilibration
      • 0.00 Base SameAsMain
      • 0.00 PumpAInlet A1
      • 0.00 BufferValveA1 A11
      • 0.00 Flow 0.3 {mL/min}
      • 1.00 Set Mark ( )#column name
      • 3.9 AutoZeroUV
      • 5.0 #Equilibration volume End Block
    0.00 Block Sample Loading
      • 0.00 Base volume
      • 0.00 Flow (1)#flow rate {mL/min}
      • 0.00 Set Mark ( )#sample
      • 0.00 InjectionValve Inject
      • 0.00 Watch UV Greater Than (100) #20 percent maxabs {mAu} END BLOCK
      • 49.00 InjectionValve Load
      • 49.00 End Block
    0.00 Block Column Wash
      • 0.00 Base SameAsMain
      • 0.00 InjectionValve Load
      • 0.00 Watch Off UV
      • 0.00 PumpAInlet A1
      • 0.00 BufferValveA1 A11
      • 0.00 Watch UV Less Than (20) #5 percent {mAu} END BLOCK
      • 20.00 End Block
    0.00 Block Gradient Elution
      • 0.00 Base SameAsMain
      • 0.00 PumpBInlet B1
      • 0.00 Gradient 100 {% B} 2.00 {base}
      • 0.00 Flow 0.30 {mL/min}
      • 10.00 Gradient 0.00 {% B} 0.00 {base}
      • 10.00 End Block
    0 Block Reequilibration
      • 0.00 End Method
    Results and Discussion
  • The base matrix used for the different prototypes where SEPHAROSE™ 6 Fast Flow. The fractionation range of SEPHAROSE™ 6 Fast Flow for globular proteins is 1×104-4×106 according to the manufacture (GE Healthcare, Uppsala, Sweden). This means that human immunoglobulin and smaller proteins can diffuse into the beads. However, this invention has proved that human immunoglobulin can be selectively be hindered to diffuse into the beads by charge repulsion lid and a combination of charge repulsion and a “gel filtration lid” (FIGS. 1 and 2) while smaller proteins have access to the interior of the beads.
  • Three different prototypes (DEAE-DX1, DEAE-DX2, DEAE-DX3) with a lid composed of charged dextran (DEAE dextran) have been tested and all three prototypes were substituted with —S03 groups in the core of the beads (FIG. 1). Different amounts of DEAE dextran was tested (Table 1) in order to accomplish charge repulsion and a gel filtration lid for large positively charged molecules (IgG) while smaller positively charged proteins are allowed to penetrate the lid. To test if attachment of only positively charged groups (—N+(CH3)3) in the lid segment can hinder IgG to diffuse into the SEPHAROSE™ 6 Fast Flow beads the Q-prototype was produced (FIG. 2 and Table 1). This prototype was also substituted with —S03 groups in the core of the beads (FIG. 2).
  • TABLE 1
    The amount of ligands coupled in the lid and the ion exchange
    capacity in the core for DEAE-dextran lid prototypes (FIG. 1) and
    for one prototype with Q-groups (—N+(CH3)3) in the lid (FIG. 2).
    Prototype Lid concentration —SO3 conc. in the core
    DEAE-DX1  37 mg/mL 80 μmol/mL
    DEAE-DX2  34 mg/mL 71 μmol/mL
    DEAE-DX3  24 mg/mL 71 μmol/mL
    Q 105 μmol/mL 80 μmol/mL
  • The breakthrough capacity of all four prototypes (DEAE-DX1-3 and Q) was determined for a number of proteins (IgG, BSA, ovalbumin and lysozyme). The molecular weight of the proteins is shown in Table 2. The mobile phase used (50 mM acetate, pH 4.0) means that all investigated proteins are positively charged (the isoelectric point of the proteins is presented in Table 2).
  • TABLE 2
    Molecular weight of the proteins used for breaktrough determination.
    Molecular weight Isoelectric
    Protein (g/mol) point
    IgG 150 000 5.5
    BSA  68 000 5.1
    Ovalbumin 43500 4.7
    Lactalbumin 14400 5.2
  • The results from the breakthrough measurements are presented in Table 3 and the prototype DEAE-DX3, with the lowest amount of dextran in the lid (24 mg/mL), resulted in low breakthrough capacity of IgG but relatively high for the other investigated proteins (Table 3). Table 3 also shows that a higher amount of DEAE dextran (prototypes DEAE-DX1-2) in the lid resulted in a low capacity of both IgG and BSA. Prototype DEAE-DX1 with the highest content of DEAE-dextran in the lid resulted also in a low breakthrough capacity of ovalbumin. The result clearly shows that IgG can be excluded from entering the beads by a lid composed of DEAE-dextran. High amounts of DEAE-dextran in the lid can be used to exclude smaller proteins such as BSA and ovalbumin. To test if only charge repulsion effects can exclude IgG from entering the beads prototype Q was designed (FIG. 2). Prototype Q with (—N+(CH3)3) in the lid segment (no dextran) gave very low breakthrough capacity of IgG and high capacities of the other proteins (Table 3).
  • TABLE 3
    Breakthrough capacity of IgG, BSA, ovalbumin and lysozyme for three
    prototypes (DEAE-DX1-3) composed of a DEAE dextran lid and
    —S03 groups in the core of the beads (SEPHAROSE ™ 6 Fast Flow)
    and one prototype (Q) with a charged lid composed of —N+(CH3)3-
    groups (no dextran) and —S03 groups in the core of the beads.
    Breakthrough
    capacity (mg/mL)
    Prototype IgG BSA Ovalbumin Lysozyme
    DEAE-DX1 0 0.1 1 8
    DEAE-DX2 0 0.2 15 36
    DEAE-DX3 0.5 12 34 52
    Q 2.7 32 41 56
  • Even though the present invention has been described above in terms of specific embodiments, many modification and variations of this invention can be made as will be obvious to those skilled in the art, without departing from its spirit and scope as set forth in the following claims.

Claims (17)

1. A separation medium, comprising an inner core of a porous material provided with charged ligands, and an outer lid comprising a porous material provided with charged ligands, wherein the charge of the ligands in the inner core is opposite that of the charge of the ligands in the lid.
2. The separation medium of claim 1, wherein the porous material in the inner core has the same porosity as the porous material in the lid.
3. The separation medium of claim 2, wherein the porosity of the inner core and lid allows penetration of a biomolecule with a molecular weight of less than 100 000 g/mol.
4. The separation medium of claim 3, wherein the porosity of the inner core and lid allows penetration of a biomolecule with a molecular weight of less than 50 000 g/mol.
5. The separation medium of claim 2, wherein the amount of charged ligands is higher in the lid than in the core.
6. The separation medium of claim 1, wherein the porous material in the inner core has the larger porosity than the porous material in the lid.
7. The separation medium of claim 6, wherein the porosity of the lid is such that biomolecules larger or equal to a molecular weight of 20 000, preferably 50 000 or 100 000 g/mol are excluded, and the porosity of the inner core is larger than the lid porosity.
8. The separation medium of claim 1, wherein the porous material is derived from a synthetic polymer material, or from a natural polymer material.
9. The separation medium of claim 1, wherein the charged ligands are —SO3 , —COO, —N+(CH3)3 or —NH+(C2H5)2.
10. A method for biomolecule separation comprising applying a sample to the separation medium of claim 1, wherein large biomolecules are prevented from entering the medium by charge repulsion from the medium and small biomolecules are captured in the inner core.
11. The method of claim 10, wherein the charge repulsion is caused by the charged ligands in the lid.
12. The method of claim 11, wherein the charge repulsion, and thereby the prevention of entering of large biomolecules, is accompanied by a lower porosity (=steric hindrance) in the lid compared to the inner core.
13. The method of claim 10, wherein large biomolecules are obtained in the flow through by charge repulsion and small biomolecules adsorbed in the inner core are eluted from the separation medium.
14. The method of claim 13, wherein the small biomolecules are eluted with a salt gradient.
15. The method of claim 13, wherein the small biomolecules are eluted with a salt gradient and/or a pH gradient, in such a way that a portion of the small biomolecules gradually adhere to the ligands in the lid on their way out from the separation medium.
16. The method of claim 10, wherein the method is run in column format and the desired biomolecules are obtained in the flow-through.
17. The method of claim 10, wherein the method is run in column format and the desired biomolecules are obtained by elution from the inner core of the separation medium.
US13/002,878 2008-07-08 2009-06-04 Separation medium for chromatography of various biomolecules Abandoned US20110155668A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
SE0801640-4 2008-07-08
SE0801640 2008-07-08
PCT/SE2009/050666 WO2010005364A1 (en) 2008-07-08 2009-06-04 Separation medium for chromatography of various biomolecules

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE2009/050666 A-371-Of-International WO2010005364A1 (en) 2008-07-08 2009-06-04 Separation medium for chromatography of various biomolecules

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/771,302 Continuation US9155980B2 (en) 2008-07-08 2013-02-20 Separation medium for chromatography of various biomolecules

Publications (1)

Publication Number Publication Date
US20110155668A1 true US20110155668A1 (en) 2011-06-30

Family

ID=41507285

Family Applications (2)

Application Number Title Priority Date Filing Date
US13/002,878 Abandoned US20110155668A1 (en) 2008-07-08 2009-06-04 Separation medium for chromatography of various biomolecules
US13/771,302 Active 2030-03-26 US9155980B2 (en) 2008-07-08 2013-02-20 Separation medium for chromatography of various biomolecules

Family Applications After (1)

Application Number Title Priority Date Filing Date
US13/771,302 Active 2030-03-26 US9155980B2 (en) 2008-07-08 2013-02-20 Separation medium for chromatography of various biomolecules

Country Status (2)

Country Link
US (2) US20110155668A1 (en)
WO (1) WO2010005364A1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120309618A1 (en) * 2010-02-19 2012-12-06 Ge Healthcare Bio-Sciences Ab Method for production of chromatography media
WO2013048330A1 (en) * 2011-09-30 2013-04-04 Ge Healthcare Bio-Sciences Ab Method for purification of cleaved pro-insulin
WO2016162310A1 (en) * 2015-04-10 2016-10-13 Ge Healthcare Bioprocess R&D Ab Method for chromatography
US11352388B2 (en) 2015-04-10 2022-06-07 Cytiva Bioprocess R&D Ab Method for chromatography

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102958602A (en) * 2010-07-09 2013-03-06 通用电气健康护理生物科学股份公司 Chromatography method and media used therefore
US8802448B2 (en) * 2011-07-27 2014-08-12 Pall Corporation Mixed mode ligands
AU2014270076A1 (en) * 2013-05-20 2015-11-12 Shantha Biotechnics Private Limited Purification of polysaccharide protein conjugates
US10266563B2 (en) 2014-06-24 2019-04-23 Ge Healthcare Bio-Process R&D Ab Chromatography method
US9960620B2 (en) 2014-09-16 2018-05-01 Navitas Semiconductor, Inc. Bootstrap capacitor charging circuit for GaN devices
CN109803736B (en) 2016-09-29 2022-02-08 生物辐射实验室股份有限公司 Agarose filled ceramic apatite
US20220266169A1 (en) * 2019-04-23 2022-08-25 Rensselaer Polytechnic Institute Product agnostic tool for quantifying separability and orthogonality for individual and sequential separation processes
US20230087779A1 (en) * 2020-02-19 2023-03-23 Bio-Rad Laboratories, Inc. Method of preparing polymer-filled chromatography resin

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5436161A (en) * 1988-11-10 1995-07-25 Pharmacia Biosensor Ab Matrix coating for sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems
US6426315B1 (en) * 1997-03-04 2002-07-30 Amersham Pharmacia Biotech Ab Process for introducing a functionality
US6852230B2 (en) * 2000-12-31 2005-02-08 Amersham Biosciences Ab Method for the manufacture of compositions containing low concentrations of salts
US20050196856A1 (en) * 2004-02-18 2005-09-08 Applera Corporation Polyelectrolyte-coated size-exclusion ion-exchange particles
US20070125711A1 (en) * 2003-10-31 2007-06-07 Ge Healthcare Bio-Sciences Ab Novel separation matrix
US20070212540A1 (en) * 2005-09-19 2007-09-13 Millipore Corporation Asymmetric porous adsorptive bead

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5436161A (en) * 1988-11-10 1995-07-25 Pharmacia Biosensor Ab Matrix coating for sensing surfaces capable of selective biomolecular interactions, to be used in biosensor systems
US6426315B1 (en) * 1997-03-04 2002-07-30 Amersham Pharmacia Biotech Ab Process for introducing a functionality
US6852230B2 (en) * 2000-12-31 2005-02-08 Amersham Biosciences Ab Method for the manufacture of compositions containing low concentrations of salts
US20070125711A1 (en) * 2003-10-31 2007-06-07 Ge Healthcare Bio-Sciences Ab Novel separation matrix
US20050196856A1 (en) * 2004-02-18 2005-09-08 Applera Corporation Polyelectrolyte-coated size-exclusion ion-exchange particles
US20070212540A1 (en) * 2005-09-19 2007-09-13 Millipore Corporation Asymmetric porous adsorptive bead

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120309618A1 (en) * 2010-02-19 2012-12-06 Ge Healthcare Bio-Sciences Ab Method for production of chromatography media
US8574437B2 (en) * 2010-02-19 2013-11-05 Ge Healthcare Bio-Sciences Ab Method for production of chromatography media
WO2013048330A1 (en) * 2011-09-30 2013-04-04 Ge Healthcare Bio-Sciences Ab Method for purification of cleaved pro-insulin
CN103827136A (en) * 2011-09-30 2014-05-28 通用电气健康护理生物科学股份公司 Method for purification of cleaved pro-insulin
CN103827136B (en) * 2011-09-30 2016-07-06 通用电气健康护理生物科学股份公司 Purify the insulinogenic method of cracking
US9409967B2 (en) 2011-09-30 2016-08-09 Ge Healthcare Bioprocess R&D Ab Method for purification of cleaved pro-insulin
WO2016162310A1 (en) * 2015-04-10 2016-10-13 Ge Healthcare Bioprocess R&D Ab Method for chromatography
CN107466298A (en) * 2015-04-10 2017-12-12 通用电气医疗集团生物工艺研发股份公司 Method for chromatogram
CN107466298B (en) * 2015-04-10 2022-03-11 思拓凡生物工艺研发有限公司 Method for chromatography
US11352388B2 (en) 2015-04-10 2022-06-07 Cytiva Bioprocess R&D Ab Method for chromatography
US11542300B2 (en) * 2015-04-10 2023-01-03 Cytiva Bioprocess R&D Ab Chromatographic methods for purification of proteins from plasma
US11807666B2 (en) * 2015-04-10 2023-11-07 Cytiva Bioprocess R&D Ab Chromatographic methods for purification of proteins from plasma

Also Published As

Publication number Publication date
US9155980B2 (en) 2015-10-13
US20130153499A1 (en) 2013-06-20
WO2010005364A1 (en) 2010-01-14

Similar Documents

Publication Publication Date Title
US9155980B2 (en) Separation medium for chromatography of various biomolecules
US11439926B2 (en) Chromatography medium
Pfaunmiller et al. Affinity monolith chromatography: a review of principles and recent analytical applications
Li et al. Affinity monolith chromatography: A review of general principles and applications
Zou et al. Monolithic stationary phases for liquid chromatography and capillary electrochromatography
US10562007B2 (en) Separation matrix
JP5596560B2 (en) Method for manufacturing separation medium
JP5826180B2 (en) Separation matrix
EP1715948B1 (en) Chromatographic material for the absorption of proteins at physiological ionic strength
Vergara-Barberan et al. Current trends in affinity-based monoliths in microextraction approaches: A review
Hahn Methods for characterization of biochromatography media
Poddar et al. Affinity monolith chromatography: A review of general principles and recent developments
Lacki et al. High throughput screening techniques in protein purification
WO2011035282A1 (en) Dual capture separation
EP1455920B1 (en) Separation method
US9044740B2 (en) Purification of immunoglobulins
Fernández‐Lahore et al. Ceramic‐Based Adsorbents in Bioproduct Recovery and Purification
Gong et al. Synthesis of zwitterionic stationary phase based on hydrophilic non‐porous poly (glycidymethacrylate‐co‐ethylenedimethacrylate) beads and their application for fast separation of proteins
Mallik Development of monolithic supports and improved immobilization methods for high-performance affinity chromatography and free drug analysis
Morbidelli et al. Preparation of macroporous methacrylate-based monoliths for chromatographic applications
Sedzik et al. Gels Mimicking Antibodies in Their Selective Recognition of Proteins and Its Potential Use for Protein Crystallization
Sines et al. ESSENTIAL GUIDES TO METHOD DEVELOPMENT IN AFFINITY CHROMATOGRAPHY

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION