WO2010043701A1 - Clarification process - Google Patents
Clarification process Download PDFInfo
- Publication number
- WO2010043701A1 WO2010043701A1 PCT/EP2009/063566 EP2009063566W WO2010043701A1 WO 2010043701 A1 WO2010043701 A1 WO 2010043701A1 EP 2009063566 W EP2009063566 W EP 2009063566W WO 2010043701 A1 WO2010043701 A1 WO 2010043701A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cell
- desired biological
- cells
- supernatant layer
- cell pellet
- Prior art date
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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
Definitions
- the present invention relates to a method for the clarification of a cell broth and to a method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance.
- Fermentative production of biological substances delivers a complex cell broth from which the biological substances should be isolated and purified by a great number of steps.
- solid material such as the cells and cell debris is to be separated from the cell broth fluid - a step called clarification.
- the biological substances are present extracellularly and will thus be present in the cell broth fluid.
- clarification methods used to-date include centrifugation, filtration (such as microfiltration, depth filtration and filtration through absolute pore size membranes) and expanded bed chromatography. Flocculation may be employed in order to enhance any of these clarification methods, in particular in combination with filtration.
- Known flocculation agents for this purpose can range from simple electrolytes to synthetic poly-electrolytes (such as DEAE dextran, acryl-based polymers, polyethylene amine) or inorganic materials (such as diatomaceous earth or perlites).
- synthetic poly-electrolytes such as DEAE dextran, acryl-based polymers, polyethylene amine
- inorganic materials such as diatomaceous earth or perlites.
- An objective of the present invention is to provide a cost-effective method for the clarification of a cell broth harvested from a bioreactor and which contains mammalian cells as well as secreted desired biological substances.
- a further object of the present invention is to provide a method for the recovery of secreted desired biological substances from a cell broth containing mammalian cells producing the secreted desired biological substances
- the present invention relates to a method for the clarification of a cell broth containing mammalian cells and culture medium as well as a secreted desired biological substance having an overall positive charge in the cell broth by the following steps: a. contacting the cell broth with a particulate anion exchange material having a specific density of the particles of between 1.4 and 3 g/ml, b. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, and c. separating the resulting cell pellet from the supernatant layer.
- cell broth means a cell culture inoculated with intact mammalian cells, and which may further contain culture medium as defined below, as well as secreted biological substances.
- cell density particularly when the cell density is extremely high, it may also be desirable to dilute the starting material from the bioreactor to a preferred cell density.
- the so diluted material is still covered by the term cell broth.
- the process according to the present invention may be carried out with cell densities up to 175x10 6 cells/ml, more preferably up to 130x10 6 cells/ml.
- the cell density can be measured using a cell counter such as Vi-CELLTM (with the trypan blue exclusion method) but other suitable methods include cytometry, packed cell volume determination, or Coulter counters (with the Electrical Sensing Zone Method).
- a cell counter such as Vi-CELLTM (with the trypan blue exclusion method) but other suitable methods include cytometry, packed cell volume determination, or Coulter counters (with the Electrical Sensing Zone Method).
- the initial cell density is above 130x10 6 cells/ml it is advisable to first dilute the cell broth.
- a cell broth with an initial cell density above 100x10 6 cells/ml be first diluted. Dilution preferably may be done to a cell density of not more than 80x10 6 cells/ml.
- the cell broth may be diluted with a solution that does not greatly change the environment of the cell so as to not cause lysis of the mammalian cells, i.e. an isotonic solution such as PBS.
- secreted biological substances biological substances which upon the production thereof by the mammalian cells are predominantly released (actively or passively) into the culture medium.
- desired biological substance is intentionally being produced making use of the mammalian cells.
- all positive charge of the secreted desired biological substances is meant here that the electrostatic contribution of positive and negatively charged ionogenic groups on the biological substance under the solvent conditions in the cell broth results in a net positive charge.
- the overall charge of a biological substance is based on the pK a of the acidic and basic residues and the pH of the solution - in this case the pH of the cell broth.
- the pi the pH where the net charge is zero
- the substance must be higher than the pH of the cell broth.
- culture medium is meant here the extracellular environment of the cells, which contains the nutrients and other constituents supporting the growth and production of cells, but may also contain waste products or host cell proteins (HCP) or material from lysed cells.
- HCP host cell proteins
- the composition of the culture medium may vary in time during the course of the culturing of cells and at the stage of clarification may be depleted of one or more of the original constituents.
- contacting is meant here introduction of anion exchange to cell broth and settling of cells (e.g. under gravity or with centrifugation).
- anion exchange material is meant here particulate weak or strong anion exchange chromatography media.
- the anion exchange material generally comprises a carrier, which may be organic material or inorganic material or a mixture of organic and inorganic material. Suitable organic materials are agarose based media and metacrylate. Suitable inorganic materials are silica, ceramics and metals.
- the particles preferably may have a size of between 15 and 150 ⁇ m. More preferably their size is between 15 and 70 ⁇ m. The particles may have a density suitable for effecting relatively rapid sedimentation of the cells from the cell broth, but not too high as it was observed that too dense particles did not affect the sedimentation.
- the method according to the present invention applies particulate anion exchanger material having a specific density of the particles of between 1.4 and 3 g/ml. Preferably, the particle density is about 2 g/ml.
- a method suitable for determination of the particle density of the anion exchange material is described in the Examples section. Suitable anion exchange materials which fulfill this requirement are e.g. materials with particles made of or containing silica, ceramic material or a metal core. - A -
- equate incubation time is meant here the time in which the precipitation of the cells results in a distinct cell pellet volume and a supernatant layer.
- separating is meant here any method to remove the supernatant from the cell pellet, such as by decanting or drawing out the supernatant or e.g. by draining the pellet from the vessel through a port at the bottom.
- the "supernatant layer” is the liquid overlying volume as a result of the settling.
- the supernatant layer may (and generally will) still contain cells, be it at a cell density significantly lower than the initial cell density.
- the present invention relates to a method for the recovery of secreted desired biological substances from a cell broth containing mammalian cells and culture medium as well as a secreted desired biological substance having an overall positive charge in the cell broth by a. contacting the cell broth with particulate anion exchange material having a specific density of the particles of between 1.4 and 3 g/ml, b. allowing an adequate incubation time to result in the formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer, and d. isolating the secreted desired biological substances from the supernatant layer With “recovery” is meant here obtaining the desired product from by-products and waste.
- the present invention further relates to a method for the recovery of secreted desired biological substances from a cell broth containing mammalian cells and culture medium as well as a secreted desired biological substance having an overall positive charge in the cell broth, wherein the resulting precipitate is further processed by e. re-suspending the resulting precipitate, f. allowing an adequate incubation time to result in the formation of a cell pellet and a supernatant layer, g. separating the resulting cell pellet from the supernatant layer and h. isolating the secreted desired biological substances from the supernatant layer.
- step e. through h. of the above process are repeated one or more times.
- the resulting cell pellet is re-suspended in a solution that does not greatly change the environment of the cell so as to not cause lysis of the mammalian cells, such as an aqueous (preferably isotonic) salt solution, more preferably in PBS.
- aqueous (preferably isotonic) salt solution preferably in PBS.
- the supernatant layers are collected and the secreted desired biological substance is extracted from the pooled supernatants.
- mammalian cells include CHO (Chinese Hamster Ovary) cells, hybridomas, BHK (Baby Hamster Kidney) cells, myeloma cells, human cells, for example HEK-293 cells, human lymphoblastoid cells, E1 immortalized HER cells, mouse cells, for example NSO cells. More preferably, E1 immortalized HER cells are used, most preferably PER.C6 cells.
- the cells in the process of the present invention are E1 -immortalized HER cells, more preferably PER.C6 cells (see U.S.
- Patent 5,994,1208 the content of which is incorporated by reference here.
- PER.C6 cells are exemplified by cells as deposited under ECACC No. 96022940 (see, e.g., U.S. Patent 5,994,128, EP 0833934 B1 , the contents of which are incorporated by reference here).
- the cell broth for clarification may be obtained by any cell culturing method suitable for attaining a cell density of the mammalian cells of at least 15x10 6 cells/ml. Suitable methods in this respect are described in e.g. WO2005095578, WO2004099396 and WO2008006494. The contents thereof are incorporated herein by reference.
- Biological substances which may be produced by the cells, for example by expressing a (recombinant) gene coding therefore are for example viruses or (recombinant) proteins, in particular receptors, enzymes, fusion proteins, blood proteins such as proteins from the blood coagulation cascade, multifunctional proteins such as for instance erythropoietin, virus or bacterial proteins for instance for use in vaccines; immunoglobulins such as for example IgG or IgM, and the like; Preferably a protein, more preferably an immunoglobulin or a part thereof is produced by the cells.
- the biological substances such as proteins or vaccines produced by the cells can be used as an active ingredient in a pharmaceutical preparation.
- the terms 'product' and 'biological substance' are interchangeable.
- Suitable methods for extracting the secreted desired biological substances from the supernatant layer are for example filtration (such as depth filtration, microfiltration, ultrafiltration, diafiltration), chromatography (such as size exclusion chromatography, affinity chromatography, cation exchange chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography), aqueous two-phase extraction, precipitation or centrifugation.
- the desired biological substance can be extracted very efficiently by cation exchange chromatography.
- affinity chromatography in particular protein A chromatography, and cation exchange chromatography are especially suitable separation methods.
- Figure 1 Supernatant cell density as a function of time for various anion exchange materials. The cell densities were measured by Vi-CELL.
- X 1 Total cell density, cells/ml
- Si-PEI Bakerbond Wide-Pore PEI (PolyEthylenelmine) Prep LC Packing grafted silica beads (JT Baker).
- DEAE Hyper D diethylaminoethyl grafted ceramic beads (Pall).
- TP DEAE diethylaminoethyl functionality on a methacrylate support (Tosoh).
- PER.C6® cells were producing an antibody.
- the particle density (g dry/ml) was determined pycnometrically.
- the volume of a 10 mL pycnometer (#15123R-10 Kimble Glass, Inc., Vineland, NJ) was determined as follows:
- volume of pycnometer is determined by: v _ [w f -w t )
- P H2O (T) is the density of water as a function of temperature.
- the particle density was then determined as follows: a. Weigh clean, dry, empty pycnometer (W 1 ) assembly. b. Weigh dry (dried at 50 0 C for 3h) anion exchange material in pycnometer (W r ). c. Completely fill pycnometer with water at room temperature d. Insert thermometer and wipe off excess water at over flow tube. Cap overflow tube. e. Re-weigh pycnometer (Wf) assembly. f. Particle density ( p d ) is determined by:
- Particle densities of various anion exchange materials were determined using the method described above.
- Si-PEI (15 and 40 ⁇ m), ToyoPearl Super Q (35 and 65 ⁇ m),
- ToyoPearl DEAE (35 and 65 ⁇ m) and DEAE Hyper D were evaluated with a cell broth containing PER.C6 ® cells producing a monoclonal antibody (MAb).
- the PER.C6 ® cells were prepared according to the procedure outlined in WO20088006494.
- the X t was 98.8x10 6 cells/ml on day 13 of the reactor.
- the material was diluted to 75x10 6 cells/ml with Dulbecco's PBS (5 mS/cm) to a final volume of 20 ml.
- the anion exchanger was added as a 50/50 slurry (total volume was 24 ml) and the cells were allowed to settle until the pellet volume was constant (60 min). Aliquots of the harvest and supernatant were analyzed by analytical protein A chromatography to determine the product recovery. The recovery was determined by:
- Mass of MAb in Ha- vest The analyses were corrected for the biomass when necessary, i.e. when the cell density is extremely high, the cells contribute significantly to the working volume.
- Figure 1 shows the supernatant cell density as a function of time for each anion exchange material as well as the control where no anion exchange material was added. Accelerated cell settling was observed in each case compared with the control. The smaller particle size appears to decrease the supernatant cell density below 10x10 6 cells/ml, where as the larger particle size decreases the cell density to 11-15 ⁇ 10 6 cells/ml.
- Figure 2 shows the supernatant volume versus time for each anion exchange material. Addition of the Si-PEI material results in the largest amount of supernatant volume which corresponds to the most compact pellet. The pellet accounted for 40% of the total volume in this case, whereas the pellet accounted for 63% of the total volume in the control.
- the Si-PEI materials have a greater density than the methacrylate and agarose based materials, which apparently allows for more compact pellets and faster settling rates.
- the ceramic Hyper D materials have an intermediate density with corresponding intermediate settling rates and pellet volumes.
- An cell culture harvest with initial cell density of 175 x 10 6 cells/mL was diluted to ⁇ 75 * 10 6 cells/mL with PBS(lnitial volume of 1.7 L). Following dilution Si-PEI chromatography media were added to the harvest (0.1 L of Si-PEI resin per L of diluted harvest). The cells were allowed to settle for ⁇ 60 minutes. The product containing supernatant was decanted and the settled cells were washed twice with PBS. The initial supernatant was pooled together with the two washes to maximize product recovery ( ⁇ 95%). The combined pool contains less than 5 x 10 6 cells/ml, and the HCP content is reduced by 59%.
- the product recovered after the Si-PEI settling is further purified by depth filtration.
- Depth filtration consist of a primary filter (typically 10 or 5 ⁇ m pore size) used for further reduction of the cell mass, followed by a secondary filter (typically 3 or 1 ⁇ m pore size) that removes smaller particles and prepares the clarified harvest for sterile filtration typically through a gradient 0.8/0.2 ⁇ m filter.
- the depth filtration train can be Millipore Millistak+HC filters containing media such as DOHC (primary) followed by XOHC (secondary) or CUNO ZetaPlus filters containing media such as 10M02 (primary) followed by 60ZA05A (secondary). In either case the clarified harvest is further filtered through 0.8/0.2 ⁇ m filters (Supor, Pall).
- the conditions used for immobilizing the antibody are slightly acidic (pH ⁇ 5.3) and conductivity of ⁇ 4.5 mS/cm. After binding the antibody is washed with equilibration buffer and finally eluted with a buffer step containing 100 mM sodium chloride. An additional reduction in HCP content (78%) is obtained by this step.
- the eluted antibody can be further purified by a combination of chromatography and filtration techniques until the required purity specifications are met.
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- 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)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
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Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2009305344A AU2009305344A1 (en) | 2008-10-17 | 2009-10-16 | Clarification process |
JP2011531502A JP2012505864A (en) | 2008-10-17 | 2009-10-16 | Clarification method |
CA2739392A CA2739392A1 (en) | 2008-10-17 | 2009-10-16 | Clarification process |
EP09784045A EP2337787A1 (en) | 2008-10-17 | 2009-10-16 | Clarification process |
CN200980140506XA CN102203113A (en) | 2008-10-17 | 2009-10-16 | Clarification process |
US13/083,538 US20110184154A1 (en) | 2008-10-17 | 2011-04-09 | Cell broth clarification and host cell protein removal |
Applications Claiming Priority (4)
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 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2009/063565 Continuation-In-Part WO2010043700A1 (en) | 2008-10-17 | 2009-10-16 | Clarification process at higher cell density |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2009/063568 Continuation-In-Part WO2010043703A1 (en) | 2008-10-17 | 2009-10-16 | Removal of host cell proteins |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2010043701A1 true WO2010043701A1 (en) | 2010-04-22 |
Family
ID=40377277
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2009/063566 WO2010043701A1 (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) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012030512A1 (en) | 2010-09-03 | 2012-03-08 | Percivia Llc. | Flow-through protein purification process |
US10695744B2 (en) | 2015-06-05 | 2020-06-30 | W. R. Grace & Co.-Conn. | Adsorbent biprocessing clarification agents and methods of making and using the same |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0160520A2 (en) * | 1984-05-02 | 1985-11-06 | Celltech Limited | A Process for separating animal cells from a liquid culture |
US20030170810A1 (en) * | 2001-09-18 | 2003-09-11 | Masoud Vedadi | Methods and apparatuses for purification |
EP1581644B1 (en) * | 2003-01-09 | 2007-06-06 | Genentech, Inc. | Purification of polypeptides |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2525945T3 (en) * | 2001-11-28 | 2015-01-02 | Sandoz Ag | 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 |
-
2009
- 2009-10-16 WO PCT/EP2009/063566 patent/WO2010043701A1/en active Application Filing
- 2009-10-16 KR KR1020117008314A patent/KR20110085981A/en not_active Application Discontinuation
- 2009-10-16 CA CA2739392A patent/CA2739392A1/en not_active Abandoned
- 2009-10-16 AU AU2009305344A patent/AU2009305344A1/en not_active Abandoned
- 2009-10-16 JP JP2011531502A patent/JP2012505864A/en active Pending
- 2009-10-16 CN CN200980140506XA patent/CN102203113A/en active Pending
- 2009-10-16 EP EP09784045A patent/EP2337787A1/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0160520A2 (en) * | 1984-05-02 | 1985-11-06 | Celltech Limited | A Process for separating animal cells from a liquid culture |
US20030170810A1 (en) * | 2001-09-18 | 2003-09-11 | Masoud Vedadi | Methods and apparatuses for purification |
EP1581644B1 (en) * | 2003-01-09 | 2007-06-06 | Genentech, Inc. | Purification of polypeptides |
Non-Patent Citations (3)
Title |
---|
ABHINAV A. SHUKLA, JAGANNADHA RAO KANDULA: "Harvest and Recovery of Monoclonal Antibodies from Large-Scale Mammalian Cell Culture", BIOPHARM INTERNATIONAL, vol. 21, 1 May 2008 (2008-05-01), Duluth, MN, pages 2034 - 2045, XP002521695, ISSN: 1542-166X, Retrieved from the Internet <URL:http://biopharminternational.findpharma.com/biopharm/article/articleDetail.jsp?id=513386> [retrieved on 20090330] * |
AUNINS J G ET AL: "INDUCED FLOCCULATION OF ANIMAL CELLS IN SUSPENSION CULTURE", BIOTECHNOLOGY AND BIOENGINEERING, WILEY & SONS, HOBOKEN, NJ, US, vol. 34, no. 5, 1 January 1989 (1989-01-01), pages 629 - 638, XP002493859, ISSN: 0006-3592 * |
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, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 128, no. 4, 6 March 2007 (2007-03-06), pages 813 - 823, XP005916242, ISSN: 0168-1656 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012030512A1 (en) | 2010-09-03 | 2012-03-08 | Percivia Llc. | Flow-through protein purification process |
US10695744B2 (en) | 2015-06-05 | 2020-06-30 | W. R. Grace & Co.-Conn. | Adsorbent biprocessing clarification agents and methods of making and using the same |
Also Published As
Publication number | Publication date |
---|---|
EP2337787A1 (en) | 2011-06-29 |
AU2009305344A1 (en) | 2010-04-22 |
JP2012505864A (en) | 2012-03-08 |
KR20110085981A (en) | 2011-07-27 |
CA2739392A1 (en) | 2010-04-22 |
CN102203113A (en) | 2011-09-28 |
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