CN102203113A - Clarification process - Google Patents

Clarification process Download PDF

Info

Publication number
CN102203113A
CN102203113A CN200980140506XA CN200980140506A CN102203113A CN 102203113 A CN102203113 A CN 102203113A CN 200980140506X A CN200980140506X A CN 200980140506XA CN 200980140506 A CN200980140506 A CN 200980140506A CN 102203113 A CN102203113 A CN 102203113A
Authority
CN
China
Prior art keywords
cell
supernatant layer
biological agents
culture fluid
cell culture
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.)
Pending
Application number
CN200980140506XA
Other languages
Chinese (zh)
Inventor
G·扎尔比斯-帕帕斯托伊特西斯
M·C·库策乌斯基
E·贝尔彻席尔默
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.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
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 DSM IP Assets BV filed Critical DSM IP Assets BV
Publication of CN102203113A publication Critical patent/CN102203113A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The present invention relates to a method for the clarification of a cell broth containing cells by the following steps: contacting the cell broth with a particulate anion exchange material having a specific density of the particles of between 1.4 and 3, allowing an adequate incubation time, and separating the resulting cell pellet from the supernatant layer. The present invention further relates to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance by contacting the cell broth with particulate anion exchange material having a specific density of the particles of between 1.4 and 3 g/ml, allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, separating the resulting cell pellet from the supernatant layer and extracting the secreted desired biological substance from the supernatant layer. Optionally, residual biological substance can be further extracted from the resulting cell pellet by one or more washing step.

Description

Defecation method
The present invention relates to be used to clarify the method for cell culture fluid (broth) and reclaim the method that excretory is expected biological agents from cell culture fluid, described cell culture fluid contains the cell that produces described excretory expectation biological agents.
Fermentation method for producing such as the biological agents of medicine, particularly monoclonal antibody provides complicated cell culture fluid, should separate with purifying biological by many steps from this cell culture fluid and learn material.
The first step is that this step is called clarification from the liquid separation solid material of cell culture fluid such as cell and cell debris.In many cases, biological agents is present in the extracellular and therefore is present in the liquid of cell culture fluid.
Up to now the example of the defecation method of Shi Yonging comprise centrifugal, filter (as micro-filtration, depth type filtration and by absolute aperture membrane filtration) and expanded bed chromatography.Can use flocculation to strengthen any of these defecation method, particularly with the filtration combination.
Known flocculation agent for this purpose can be that simple ionogen is to synthetic polyelectrolyte (as deae dextran, the polymkeric substance based on acryl, polyvinylamine) or inorganic materials (as diatomite or perlite).The progress of this respect is to use chitosan in recent years.
The use shortcoming of many flocculation agents is: they may be in conjunction with the expectation biological agents of being paid close attention to, the expectation biological agents that their may inactivation be paid close attention to, flocculence takes a long time and/or flocculation agent may be difficult to or spend that higher just to be prepared into medical use desired high-quality.In addition, need remove throw out from final purifying molecule, this is to need expensive time-consuming assay determination to confirm the process of removing of this throw out usually.
Method of the present invention does not have any of these shortcoming.
An object of the present invention is to provide cost-effective method to be used to clarify the cell culture fluid from the bio-reactor results, this cell culture fluid contains mammalian cell and excretory expectation biological agents.
Another object of the present invention provides the method that reclaims excretory expectation biological agents from cell culture fluid, and described cell culture fluid contains the mammalian cell that produces described excretory expectation biological agents.
In specific embodiment, the present invention relates to be used to clarify the method for cell culture fluid, the excretory expectation biological agents that described cell culture fluid contains mammalian cell and substratum and have total positive charge in described cell culture fluid, described method is carried out as follows:
A. making described cell culture fluid and particle proportion is that the particulate anion-exchange material of 1.4-3g/ml contacts,
B. hatch the enough time so that form cell mass (pellet) and supernatant layer, and
C. separating obtained cell mass and described supernatant layer.
In the present invention, " cell culture fluid " finger kind the cell culture of complete mammalian cell, it can also contain substratum and excretory biological agents as hereinafter definition.In the method for the invention, particularly when cell density is very high, also can be desirably the parent material of bio-reactor be diluted to preferred cell density.For purpose of the present invention, so the material of dilution still is encompassed in the scope of term cell culture fluid.
As actual upper bound, can use up to 175 * 10 6Individual cell/ml, more preferably up to 130 * 10 6The cell density of individual cell/ml carries out method of the present invention.
Cell density can use cell counter such as Vi-CELL TM(use trypanblue exclusion method) measured, but can use other appropriate method, comprises cytometry, packed cellvolume determines or Coulter counter (using electrical sensing zone method (Electrical Sensing Zone Method)).
If initial cell density is higher than 130 * 10 6Individual cell/ml, then diluting cells nutrient solution at first preferably.In practice, preferably at first dilute initial cell density and be higher than 100 * 10 6The cell culture fluid of individual cell/ml.Dilution can preferably proceed to cell density and be no more than 80 * 10 6Individual cell/ml.Thereby cell culture fluid can not cause the mammalian cell cracking with the solution dilution of not obvious change cellular environment, promptly uses the isotonic solution such as PBS to dilute.
" excretory biological agents " mainly discharges (active or passive) by mammalian cell and goes into the biological agents in the substratum when this paper refers to that it produces.
" expectation " refers to that at this paper biological agents is wittingly by using mammalian cell to produce.
" total positive charge " of excretory expectation biological agents is under this paper refers to solvent condition in cell culture fluid, and the electrostatic contribution of positive charge and negatively charged ions group causes clean positive charge on the biological agents.The total charge of biological agents is based on the pK of acid and alkaline residue aAnd the pH of solution, be the pH of cell culture fluid in this case.For the biological agents that has clean positive charge in cell culture fluid, the pI of this material (wherein net charge is 0 pH) must be higher than the pH of cell culture fluid.
" substratum " at born of the same parents' external environment of this paper phalangeal cell, nutrition and other compositions that it contains the sustenticular cell growth and produces, but also can contain waste product or host cell proteins (HCP) or from the material of cracked cell.The composition of substratum can change during cell cultures, and can exhaust one or more original composition in the clarification stage.
" contact " refers in cell culture fluid to introduce anionresin and makes cell settlement (for example under gravity or use centrifugal) at this paper.
" anion-exchange material " is a little less than this paper refers to particulate or the reinforcing yin essence ion-exchange chromatography media.Anion-exchange material comprises carrier usually, and it can be the mixture of organic materials or inorganic materials or organic and inorganic materials.Suitable organic materials is based on the medium and the methacrylic ester (metacrylate) of agarose.Suitable inorganic materials is silica, pottery and metal.The preferred size of particle is 15-150 μ m.Their preferred sizes are 15-70 μ m.Particulate density can be suitable for making cell from cell culture fluid sedimentation quickly, but density can not be too high, because too intensive according to observations particle does not cause sedimentation.
It is the particulate anion-exchange material of 1.4-3g/ml that method of the present invention is used particle proportion.Preferably, pellet density is about 2g/ml.The method that is suitable for the pellet density of definite anion-exchange material is described in the embodiment part.The suitable anion exchange material that meets this requirement is a particulate material of for example being made or contained silica, stupalith or metal core by silica, stupalith or metal core.
" enough incubation times " precipitation of inner cell between this paper refers at this moment causes distinct cell mass volume and supernatant layer.
" separation " as by decantation or extraction supernatant, perhaps for example extracts agglomerate by the relief outlet of bottom out from container in any method that this paper refers to remove from cell mass supernatant.
" supernatant layer " is the liquid that is positioned at the top owing to sedimentation.The supernatant layer can (and usually) still contain cell, and its cell density significantly is lower than initial cell density.
According to another embodiment, the present invention relates to reclaim the method for excretory expectation biological agents from cell culture fluid, the excretory expectation biological agents that described cell culture fluid contains mammalian cell and substratum and have total positive charge in described cell culture fluid, described method is carried out as follows:
A. making described cell culture fluid and particle proportion is that the particulate anion-exchange material of 1.4-3g/ml contacts,
B. hatch the enough time so that form cell mass and supernatant layer,
C. separating obtained cell mass and described supernatant layer, and
D. separate described excretory expectation biological agents from described supernatant layer.
" recovery " refers to expect product from the acquisition of by product and refuse at this paper.
The invention still further relates to the method that reclaims excretory expectation biological agents from cell culture fluid, the excretory expectation biological agents that described cell culture fluid contains mammalian cell and substratum and have total positive charge in described cell culture fluid, wherein the gained precipitation is further handled as follows:
E. gained is precipitated resuspending,
F. hatch the enough time so that form cell mass and supernatant layer,
G. separating obtained cell mass and described supernatant layer, and
H. separate described excretory expectation biological agents from described supernatant layer.
In other method of the present invention, the step e to h of aforesaid method is repeated one or many.
In another preferred method of the present invention, thereby gained cell mass resuspending is not caused the mammalian cell cracking in the solution of not obvious change cellular environment, as resuspending in water-based (preferred wait ooze) salts solution, more preferably PBS.
Preferably, collect supernatant layer and extract excretory expectation biological agents from the blended supernatant.
The example of mammalian cell comprises CHO (Chinese hamster ovary) cell, hybridoma, BHK (young hamster kidney) cell, myeloma cell, the people's cell such as the HEK-293 cell, human lymphoblastoid, E1 immortalization HER cell, such as the mouse cell of NS0 cell.More preferably use E1 immortalization HER cell, most preferably the PER.C6 cell.
In preferred embodiments, the cell in the inventive method is an E1-immortalization HER cell, more preferably PER.C6 cell (see United States Patent (USP) 5,994,128, its content is incorporated this paper by reference into).
The PER.C6 cell is an exemplary cells, and it carries out preservation with ECACC preserving number 96022940, and preservation date is on February 29th, 1996 (see for example United States Patent (USP) 5,994,128, EP 0833934 B1, its content is incorporated this paper by reference into).
Being used for clarifying cell culture fluid can be by being suitable for realizing at least 15 * 10 6Any cell culture processes of the mammalian cell density of individual cell/ml obtains.Appropriate method in this respect is described in for example WO2005095578, WO2004099396 and WO2008006494.Its content is incorporated this paper by reference into.
Can by cell of the present invention for example by to express biological agents that its (reorganization) gene of coding produces be for example viral or (reorganization) albumen, particularly acceptor, enzyme, fusion rotein, such as from the proteic blood protein of coagulation cascade, such as the multifunctional protein of erythropoietin, for example be used for the virus or the bacterioprotein of vaccine; Immunoglobulin (Ig) such as IgG or IgM etc.Optimization protein, more preferably immunoglobulin (Ig) or its part are produced by cell.Preferably, the biological agents such as albumen or vaccine that is produced by cell can be as the activeconstituents in the pharmaceutical preparation.In the present invention, term " product " and " biological agents " are used interchangeably.
The appropriate method of extracting excretory expectation biological agents from the supernatant layer is for example to filter (as depth type filtration, micro-filtration, ultrafiltration, diafiltration), chromatography (as size exclusion chromatography, avidity chromatography, cation-exchange chromatography, hydrophobic interaction chromatography, fixing metal avidity chromatography), double water-phase extraction, precipitation or centrifugal.Advantageously, the expectation biological agents can be extracted very effectively by cation-exchange chromatography.In the expectation biological agents is under the situation of immunoglobulin (Ig), and avidity chromatography, particularly albumin A chromatography and cation-exchange chromatography are particularly suitable separation methods.
Description of drawings
Fig. 1: for various anion-exchange materials, as the supernatant cell density of the function of time.Cell density is measured by Vi-CELL.
Fig. 2: for various anion-exchange materials, as the supernatant volume of the function of time.Cumulative volume is 24ml under every kind of situation.
Embodiment
Symbol:
X t=total cell density, individual cell/mL.
Si-PEI=Bakerbond Wide-Pore PEI (polymine) Prep LC Packing grafting silica beads (Prep LC Packing grafted silica bead) (JT Baker)
DEAE Hyper D=diethylamino ethyl grafting ceramic bead (diethylaminoethyl grafted ceramic bead) (Pall)
Season amino functionality (Tosoh) on the Super Q=methacrylic ester upholder
Diethylamino ethyl functionality (Tosoh) on the TP DEAE=methacrylic ester upholder
The PBS=phosphate-buffered saline
According to the described method of WO2008006494, use PER.C6
Figure BPA00001347636100051
Cell carries out and prepares following clarification and test.These PER.C6
Figure BPA00001347636100052
Cell produces antibody.
Determine the method for the pellet density of anion-exchange material
Determine pellet density (g dry weight/ml) by pycnometric method.Definite 10mL specific gravity flask as described below (#15123R-10 Kimble Glass, Inc., Vineland, volume NJ):
1. combination weighing cleaning, the empty specific gravity flask (W of exsiccant i).
2. at room temperature water is filled specific gravity flask fully.
3. insert thermometer and wipe the excessive water of upflow tube.Cover upflow tube.
4. the recombinant specific gravity flask (W that weighs f).
5. determine the volume of specific gravity flask by following formula:
V pyc = ( W f - W i ) ρ H 2 O ( T )
ρ wherein H2O(T) be water-mass density as temperature function.
Definite pellet density as described below then:
A. combination weighing cleaning, the empty specific gravity flask (W of exsiccant i).
B. anion-exchange material (the W of dry in the specific gravity flask (50 ℃ dry 3h) down weighs r).
C. at room temperature water is filled specific gravity flask fully.
D. insert thermometer and wipe the excessive water of upflow tube.Cover upflow tube.
E. recombinant weigh specific gravity flask (W ' f).
F. pellet density (ρ d) determine by following formula:
ρ d = ( W r - W i ) [ V pyc - ( W f ′ - W r ) ρ H 2 O ( T ) ]
Measure in triplicate.
The pellet density of anion-exchange material
Use aforesaid method to determine the pellet density of various anion-exchange materials.
The results are summarized in following table.
Figure BPA00001347636100071
Embodiment 1: use the clarifying comparison of the anion-exchange material of different densities
Use contains the PER.C6 that produces monoclonal antibody (MAb)
Figure BPA00001347636100072
The cell culture fluid of cell is estimated Si-PEI (15 and 40 μ m), ToyoPearl Super Q (35 and 65 μ m), ToyoPearl DEAE (35 and 65 μ m) and DEAE Hyper D.PER.C6 The described method preparation of cell such as WO20088006494.X at the 13rd day reactor tBe 98.8 * 10 6Individual cell/ml.Material (5mS/cm) is diluted to 75 * 10 with Da Erbaike PBS (Dulbecco ' s PBS) 6Individual cell/ml, final volume is 20ml.Adding makes cell settlement keep constant (60min) until the agglomerate volume as the anionresin thing (exchanger) (cumulative volume is 24ml) of 50/50 slurries.
The equal portions of cutting and supernatant are passed through analyzing proteins A chromatographic analysis to determine the product rate of recovery.The rate of recovery is determined by following formula:
Figure BPA00001347636100074
When needs, promptly when cell density very high, when cell is obviously facilitated (contribute) working volume, then to the biomass correction analysis.
Fig. 1 illustrates for every kind of anion-exchange material, as the supernatant cell density of the function of time and the contrast that does not add anion-exchange material.All observe the cell settlement that quickens compared with the control in each case.Less granularity seems the supernatant cell density is reduced to and is lower than 10 * 10 6Individual cell/ml, and bigger granularity is reduced to 11-15 * 10 with cell density 6Individual cell/ml.
Fig. 2 illustrates for every kind of anion-exchange material, the supernatant volume of relative time.Add Si-PEI material production maximum supernatant volume, this is corresponding to the agglomerate of consolidation.In this case, agglomerate accounts for 40% of cumulative volume, and agglomerate accounts for 63% of cumulative volume in contrast.The Si-PEI material has bigger density than methacrylic ester with based on the material of agarose, and this obviously allows the agglomerate of consolidation more and subsidence rate faster.Pottery Hyper D material has intermediate density, and this is corresponding to medium subsidence rate and agglomerate volume.
Embodiment 2: the purifying of expectation biological agents
With initial cell density is 175 * 10 6The cell culture harvest thing of individual cell/mL is～75 * 10 with the PBS dilution 6Individual cell/mL (original volume is 1.7L).After the dilution, add Si-PEI chromatography media (cutting of the Si-PEI resin of 0.1L/L dilution) to cutting.Make cell settlement～60 minute.Pour out the supernatant that contains product, and with settled cell PBS washed twice.Initial supernatant is mixed with twice washing to increase the product rate of recovery (～95%).The mixture (pool) that merges contains and is lower than 5 * 10 6Individual cell/ml, and HCP content reduces by 59%.
The product that reclaims after the Si-PEI sedimentation is further purified by depth type filtration.Depth type filtration is made up of elementary filter (being generally 10 or 5 μ m apertures) and secondary filter subsequently (being generally 3 or 1 μ m aperture), described elementary filter is used for further reducing cell lump (cell mass), and described secondary filter is removed littler particle and prepared clarifying cutting to carry out sterilising filtration by gradient 0.8/0.2 μ m filter usually.Depth type filtration row (train) can be Millipore Millistak+HC filters, and it contains medium, as D0HC (elementary) and X0HC (secondary) subsequently; Perhaps CUNO ZetaPlus filter, it contains medium, as 10M02 (elementary) and 60ZA05A (secondary) subsequently.Under any circumstance, (Supor Pall) further filters by 0.8/0.2 μ m filter with clarifying cutting.
In addition, observing 85% HCP by secondary filter during depth type filtration reduces.HCP can be owing to the charged character of these filters by the minimizing of secondary filter, existing report (Yigzaw Y in this former document, Piper R, Tran M, Shukla AA.2006. Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal during Mo
Clarifying material is further purified by the cation-exchange chromatography such as GigaCap S (Tosoh).Monoclonal antibody (product) is fixed on the resin, and capacity (capacity) is＞the 95g/L chromatography media.The condition that is used for fixing antibody is slightly acid (pH～5.3), and specific conductivity is～4.5mS/cm.In conjunction with after, antibody is washed with level pad, at last with containing the buffer step wash-out of 100mM sodium-chlor.Obtain HCP content by this step and further reduce (78%).
The antibody of wash-out can be by the single step purification that is combined into of chromatography and filtering technique, until the purity requirement that suits the requirements.
By the CEX step, total minimizing of host cell proteins is as shown in the table in the cell culture harvest thing.
HCP(μg/mg MAb) The %HCP clearance rate
The cell culture harvest thing 200 0
Behind the PEI cell settlement 81 59
After the depth type filtration 12 85
After CEX catches 2.8 78
Amount to 99

Claims (8)

1. be used to clarify the method for the cell culture fluid that contains cell, described method is carried out as follows:
A. making described cell culture fluid and particle proportion is that the particulate anion-exchange material of 1.4-3g/ml contacts,
B. hatch the enough time so that form cell mass and supernatant layer, and
C. separating obtained cell mass and described supernatant layer.
2. reclaim the method for excretory expectation biological agents from cell culture fluid, described cell culture fluid contains the cell that produces described excretory expectation biological agents, and described method is carried out as follows:
A. making described cell culture fluid and particle proportion is that the particulate anion-exchange material of 1.4-3g/ml contacts,
B. hatch the enough time so that form cell mass and supernatant layer,
C. separating obtained cell mass and described supernatant layer, and
D. extract described excretory expectation biological agents from described supernatant layer.
3. the method for claim 21, wherein the gained cell mass is further handled as follows:
E. with gained cell mass resuspending,
F. hatch the enough time so that form cell mass and supernatant layer,
G. separating obtained cell mass and described supernatant layer, and
H. extract described excretory expectation biological agents from described supernatant layer.
4. the method for claim 3, wherein repeating step e to h.
5. claim 3 or 44 method, wherein with gained cell mass resuspending in aqueous saline solution.
6. the method for claim 5, wherein said aqueous saline solution is PBS.
7. claim 3 or 4 method wherein merged each supernatant layer before extracting described expectation biological agents.
8. each method among the claim 3-7 wherein uses cation-exchange chromatography to extract described expectation biological agents.
Each method in the aforementioned claim, wherein said expectation biological agents are immunoglobulin (Ig) or its part.
CN200980140506XA 2008-10-17 2009-10-16 Clarification process Pending CN102203113A (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US10632408P 2008-10-17 2008-10-17
US61/106,324 2008-10-17
EP08167508 2008-10-24
EP08167508.4 2008-10-24
PCT/EP2009/063566 WO2010043701A1 (en) 2008-10-17 2009-10-16 Clarification process

Publications (1)

Publication Number Publication Date
CN102203113A true CN102203113A (en) 2011-09-28

Family

ID=40377277

Family Applications (1)

Application Number Title Priority Date Filing Date
CN200980140506XA Pending CN102203113A (en) 2008-10-17 2009-10-16 Clarification process

Country Status (7)

Country Link
EP (1) EP2337787A1 (en)
JP (1) JP2012505864A (en)
KR (1) KR20110085981A (en)
CN (1) CN102203113A (en)
AU (1) AU2009305344A1 (en)
CA (1) CA2739392A1 (en)
WO (1) WO2010043701A1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012030512A1 (en) 2010-09-03 2012-03-08 Percivia Llc. Flow-through protein purification process
EP3302784B1 (en) 2015-06-05 2021-10-06 W.R. Grace & Co.-Conn. Adsorbent bioprocessing clarification agents and methods of making and using the same

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003045996A1 (en) * 2001-11-28 2003-06-05 Sandoz Gmbh Chromatographic purification of recombinant human erythropoietin
CN1686850A (en) * 2005-04-08 2005-10-26 山东金佰生物技术有限公司 Downstream extraction technique for production flocculant of microbe

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8411192D0 (en) * 1984-05-02 1984-06-06 Celltech Ltd Separating animal cells from liquid culture
US20030170810A1 (en) * 2001-09-18 2003-09-11 Masoud Vedadi Methods and apparatuses for purification
ZA200504990B (en) * 2003-01-09 2006-08-30 Genentech Inc Purification of polypeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003045996A1 (en) * 2001-11-28 2003-06-05 Sandoz Gmbh Chromatographic purification of recombinant human erythropoietin
CN1686850A (en) * 2005-04-08 2005-10-26 山东金佰生物技术有限公司 Downstream extraction technique for production flocculant of microbe

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FRANK RISKE ET AL.: "The use of chitosan as a flocculant in mammalian cell culture dramatically improves clarification throughput without adversely impacting monoclonal antibody recovery", 《JOURNAL OF BIOTECHNOLOGY》 *

Also Published As

Publication number Publication date
AU2009305344A1 (en) 2010-04-22
KR20110085981A (en) 2011-07-27
WO2010043701A1 (en) 2010-04-22
CA2739392A1 (en) 2010-04-22
JP2012505864A (en) 2012-03-08
EP2337787A1 (en) 2011-06-29

Similar Documents

Publication Publication Date Title
WO2010043703A1 (en) Removal of host cell proteins
US11504646B2 (en) Depth filters for disposable biotechnological processes
EP1175931A1 (en) Integration of high cell density bioreactor operation with ultra fast on-line downstream processing
RU2751506C2 (en) Methods for processing fluid containing recombinant therapeutic protein and their application
CN105358572A (en) Continuous multistep process for purifying antibodies
TW200835542A (en) Method of isolating biomacromolecules using low pH and divalent cations
JP2022526965A (en) Improved immunoglobulin affinity chromatography by using pre-capture aggregation
CN102178952A (en) Method for extracting human TIG (Tetanus Immune Globulin) based on chromatography
CN109320611B (en) Purification method of human-mouse chimeric monoclonal antibody biological similar drug
NL8902351A (en) BIOCHEMICAL SEPARATION OF MATERIAL FROM A LIQUID ENVIRONMENT.
CN102203113A (en) Clarification process
CN102186875A (en) Clarification process at higher cell density
US20110184154A1 (en) Cell broth clarification and host cell protein removal
CA2832341C (en) Separation of genomic dna from a target molecule using cation exchange
WO2021064244A1 (en) Purification process based on magnetic beads
CN105579572B (en) Method for purifying high density granular cell culture cutting
RU2794431C1 (en) Improving immunoglobulin affinity chromatography by the application of pre-capture floculation
CN116685383A (en) Virus vector purification device and method
JPWO2021046443A5 (en)

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1161601

Country of ref document: HK

C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20110928

REG Reference to a national code

Ref country code: HK

Ref legal event code: WD

Ref document number: 1161601

Country of ref document: HK