AU2009305344A1 - Clarification process - Google Patents

Clarification process Download PDF

Info

Publication number
AU2009305344A1
AU2009305344A1 AU2009305344A AU2009305344A AU2009305344A1 AU 2009305344 A1 AU2009305344 A1 AU 2009305344A1 AU 2009305344 A AU2009305344 A AU 2009305344A AU 2009305344 A AU2009305344 A AU 2009305344A AU 2009305344 A1 AU2009305344 A1 AU 2009305344A1
Authority
AU
Australia
Prior art keywords
cell
cells
desired biological
cell pellet
supernatant layer
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
AU2009305344A
Inventor
Emily Belcher Schirmer
Michael Christopher Kuczewski
Grigorios Zarbis-Papastoitsis
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.)
Percivia LLC
Original Assignee
Percivia LLC
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 Percivia LLC filed Critical Percivia LLC
Publication of AU2009305344A1 publication Critical patent/AU2009305344A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • 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
    • C07K1/30Extraction; Separation; Purification by precipitation
    • 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
    • C07K1/18Ion-exchange 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
    • C07K1/36Extraction; Separation; Purification by a combination of two or more processes of different types
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24Methods 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)

Description

WO 2010/043701 PCT/EP2009/063566 CLARIFICATION PROCESS 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 5 a cell broth containing cells producing the secreted desired biological substance. Fermentative production of biological substances, such as pharmaceuticals and in particular monoclonal antibodies, delivers a complex cell broth from which the biological substances should be isolated and purified by a great number of steps. 10 As a first step solid material such as the cells and cell debris is to be separated from the cell broth fluid - a step called clarification. In many instances the biological substances are present extracellularly and will thus be present in the cell broth fluid. Examples of clarification methods used to-date include centrifugation, 15 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 20 electrolytes to synthetic poly-electrolytes (such as DEAE dextran, acryl-based polymers, polyethylene amine) or inorganic materials (such as diatomaceous earth or perlites). A recent development is the use of chitosan for this purpose. Disadvantages of the use of many flocculating agents are amongst others that they may bind the desired biological substance of interest, that they may 25 inactivate the desired biological substances of interest, that the flocculation process takes too long and/or that the flocculation agent may be hard or expensive to prepare in the high quality needed for medical use. In addition the flocculent needs to be removed from the final purified molecule a process that often requires expensive and time consuming analytical assays to verify its removal. 30 The method according to the present invention does not have any of these disadvantages. 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.
WO 2010/043701 PCT/EP2009/063566 -2 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 In a specific embodiment, the present invention relates to a method 5 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, 10 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. In the context of the present invention "cell broth" means a cell culture inoculated with intact mammalian cells, and which may further contain culture medium 15 as defined below, as well as secreted biological substances. In the process of the present invention, 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. For the purpose of the present invention the so diluted material is still covered by the term cell broth. 20 As a practical upper limit, the process according to the present invention may be carried out with cell densities up to 175x1 06 cells/ml, more preferably up to 130 x106 cells/ml. The cell density can be measured using a cell counter such as Vi-CELL T M (with the trypan blue exclusion method) but other suitable methods include 25 cytometry, packed cell volume determination, or Coulter counters (with the Electrical Sensing Zone Method). If the initial cell density is above 130x10 6 cells/ml it is advisable to first dilute the cell broth. In practice it is preferred that a cell broth with an initial cell density above 1 00x1 06 cells/ml be first diluted. Dilution preferably may be done to a cell density 30 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. With "secreted biological substances" is meant here biological substances which upon the production thereof by the mammalian cells are 35 predominantly released (actively or passively) into the culture medium.
WO 2010/043701 PCT/EP2009/063566 -3 With "desired" is meant here that the biological substance is intentionally being produced making use of the mammalian cells. With "overall positive charge" of the secreted desired biological substances is meant here that the electrostatic contribution of positive and negatively 5 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 pKa of the acidic and basic residues and the pH of the solution - in this case the pH of the cell broth. For the biological substance to have a net positive charge in the cell broth, the pl (the pH where the net charge is zero) of the 10 substance must be higher than the pH of the cell broth. With "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. The composition of the culture medium may vary in time 15 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. With "contacting" is meant here introduction of anion exchange to cell broth and settling of cells (e.g. under gravity or with centrifugation). With "anion exchange material" is meant here particulate weak or 20 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 pm. More preferably their 25 size is between 15 and 70 pm. 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 30 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.
WO 2010/043701 PCT/EP2009/063566 -4 With "adequate 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. With "separating" is meant here any method to remove the supernatant from the cell pellet, such as by decanting or drawing out the supernatant or 5 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. According to a further embodiment the present invention relates to a 10 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, 15 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 20 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 25 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 30 h. isolating the secreted desired biological substances from the supernatant layer. In a further method according to the present invention step e. through h. of the above process are repeated one or more times. In a further preferred method according to the present invention the 35 resulting cell pellet is re-suspended in a solution that does not greatly change the WO 2010/043701 PCT/EP2009/063566 -5 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. Preferably the supernatant layers are collected and the secreted desired biological substance is extracted from the pooled supernatants. 5 Examples of 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, El immortalized HER cells, mouse cells, for example NSO cells. More preferably, El immortalized HER cells are used, most preferably PER.C6 cells. 10 In a preferred embodiment, the cells in the process of the present invention are El-immortalized HER cells, more preferably PER.C6 cells (see U.S. Patent 5,994,128, 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 15 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. W02005095578, W02004099396 and W02008006494. The contents thereof are incorporated herein by 20 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 25 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. Preferably, the biological substances such as proteins or vaccines produced by the cells can be used as an active ingredient in a pharmaceutical preparation. In the 30 context of the present invention, 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 35 exclusion chromatography, affinity chromatography, cation exchange chromatography, WO 2010/043701 PCT/EP2009/063566 -6 hydrophobic interaction chromatography, immobilized metal affinity chromatography), aqueous two-phase extraction, precipitation or centrifugation. Advantageously, the desired biological substance can be extracted very efficiently by cation exchange chromatography. In case of immunoglobulins as the desired biological substances 5 affinity chromatography, in particular protein A chromatography, and cation exchange chromatography are especially suitable separation methods. Short description of the figures Figure 1. Supernatant cell density as a function of time for various anion 10 exchange materials. The cell densities were measured by Vi-CELL. Figure 2. Supernatant volume as a function of time for various anion exchange materials. The total volume in each case was 24 ml. EXAMPLES 15 Symbols: Xt = Total cell density, cells/ml Si-PEI = Bakerbond Wide-Pore PEI (PolyEthylenelmine) Prep LC Packing grafted silica beads (JT Baker). 20 DEAE Hyper D = diethylaminoethyl grafted ceramic beads (Pall). Super Q = quaternary amino functionality on a methacrylate support (Tosoh). TP DEAE = diethylaminoethyl functionality on a methacrylate support (Tosoh). PBS = Phosphate Buffered Saline The following experiments were carried out using PER.C6@ cells 25 which were prepared according to the procedure outlined in W02008006494. These PER.C6@ cells were producing an antibody. Method for the determination of particle density of anion exchange materials. The particle density (g dry/ml) was determined pycnometrically. The 30 volume of a 10 mL pycnometer (#15123R-10 Kimble Glass, Inc., Vineland, NJ) was determined as follows: 1. Weigh clean, dry, empty pycnometer (W) assembly. 2. Completely fill pycnometer with water at room temperature. 3. Insert thermometer and wipe off excess water at overflow tube. Cap overflow 35 tube.
WO 2010/043701 PCT/EP2009/063566 -7 4. Re-weigh pycnometer (Wf) assembly. 5. Volume of pycnometer is determined by: where pH20(T) is the density of water as a function of temperature. 5 The particle density was then determined as follows: a. Weigh clean, dry, empty pycnometer (W) assembly. b. Weigh dry (dried at 50'C for 3h) anion exchange material in pycnometer (W). c. Completely fill pycnometer with water at room temperature 10 d. Insert thermometer and wipe off excess water at over flow tube. Cap overflow tube. e. Re-weigh pycnometer (Wf) assembly. f. Particle density ( Pd) is determined by: 15 The measurements were done in triplicate Particle density of anion exchange materials Particle densities of various anion exchange materials were determined using the method described above.
WO 2010/043701 PCT/EP2009/063566 -8 The results are summarized in the following table: Mean Anion Particle Manufacturer/ Chemistry/ particle mexhae Catalog No. Backbone size densit (g materialdr/L (pm) Bakerbond JT Baker/ 10, 20, 30 amine/ 40 2.02 ± 0.03 WP PEI 7368 Silica Bakerbond JT Baker/ 1', 2', 30 amine/ 15 2.06 ± 0.1 WP PEI 7180 Silica DEAE Pall/ 3' amine/ 50 1.44 ± 0.08 HyperD 20067 Ceramic Toyopearl Tosoh/ 4' amine/ 65 1.22 ± 0.02 SuperQ- 43205 Methacrylate 650M Toyopearl Tosoh/ 3' amine/ 65 1.17 ± 0.04 DEAE-650M 43201 Methacrylate Toyopearl Tosoh/ 4' amine/ 35 1.17 ± 0.01 SuperQ- 19823 Methacrylate 650S Toyopearl Tosoh/ 3' amine/ 35 1.25 ± 0.05 DEAE-650S 19804 Methacrylate Example 1 Comparison of clarification with anion exchanger materials of various densities 5 Si-PEI (15 and 40 pm), ToyoPearl Super Q (35 and 65 pm), ToyoPearl DEAE (35 and 65 pm) 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 W020088006494.The Xt was 98.8x10 6 cells/ml on day 13 of the reactor. The material was diluted to 75x10 6 cells/ml 10 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).
WO 2010/043701 PCT/EP2009/063566 -9 Aliquots of the harvest and supernatant were analyzed by analytical protein A chromatography to determine the product recovery. The recovery was determined by: Mas of MAb in Supematant Recovery = _____2)________ Mass of IIAb in Harvest 5 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 10 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 1O X106 cells/ml, where as the larger particle size decreases the cell density to 11-15x106 cells/ml. Figure 2 shows the supernatant volume versus time for each anion 15 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 20 compact pellets and faster settling rates. The ceramic Hyper D materials have an intermediate density with corresponding intermediate settling rates and pellet volumes. Example 2 Purification of desired biological substance An cell culture harvest with initial cell density of 175 x 106 cells/mL 25 was diluted to - 75 x 106 cells/mL with PBS(Initial 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 30 product recovery (- 95%). The combined pool contains less than 5 x 106 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 ptm pore size) WO 2010/043701 PCT/EP2009/063566 -10 used for further reduction of the cell mass, followed by a secondary filter (typically 3 or 1 tm pore size) that removes smaller particles and prepares the clarified harvest for sterile filtration typically through a gradient 0.8/0.2 tm filter. The depth filtration train can be Millipore Millistak+HC filters containing media such as DOHC (primary) followed 5 by X0HC (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 tm filters (Supor, Pall). In addition an 85% HCP reduction was observed through the secondary filter during depth filtration. Reduction in HCP through the secondary filter 10 could be attributed to the charged nature of these filters and has been previously reported in the literature (Yigzaw Y, Piper R, Tran M, Shukla AA. 2006. Exploitation of the Adsorptive Properties of Depth Filters for Host Cell Protein Removal during Monoclonal Antibody Purification. Biotechnology Progress 22(1):288-296.). The clarified material is further purified by Cation Exchange 15 Chromatography such as GigaCap S (Tosoh). The monoclonal antibody (product) is immobilized on the resin at a capacity of > 95 g/L of chromatography media. 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 20 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.
WO 2010/043701 PCT/EP2009/063566 - 11 The overall reduction in Host Cell Proteins from the cell culture harvest through the CEX step is summarized below: HCP (pg/mg MAb) % HCP Clearance Cell Culture Harvest 200 0 Post-PEI Cell Settling 81 59 Post Depth Filtration 12 85 Post CEX Capture 2.8 78 Overall 99

Claims (8)

1. A method for the clarification of a cell broth containing cells by the following steps: 5 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. 10
2. A method for the recovery of secreted desired biological substances from a cell broth containing cells producing the secreted desired biological substance 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, 15 b. allowing an adequate incubation time to result in formation of a cell pellet and a supernatant layer, c. separating the resulting cell pellet from the supernatant layer and d. extracting the secreted desired biological substances from the supernatant layer 20
3. Method according to claim 21, wherein the resulting cell pellet is further processed by e. re-suspending the resulting cell pellet, f. allowing an adequate incubation time to result into formation of a cell pellet and a supernatant layer, 25 g. separating the resulting cell pellet from the supernatant layer and h. extracting the secreted desired biological substances from the supernatant layer
4. Method according to claim 3, wherein step e through h are repeated
5. Method according to claim 3 or 44 wherein the resulting cell pellet is re 30 suspended in an aqueous salt solution
6. Method according to claim 5, wherein the aqueous salt solution is PBS.
7. Method according to claim 3 or 4 wherein the respective supernatant layers are combined prior to extracting the desired biological substance.
8. Method according to any of claims 3 to 7 wherein the desired biological 35 substance is extracted using cation exchange chromatography. WO 2010/043701 PCT/EP2009/063566 -13 Method according to any of the claims above, wherein the desired biological substances are immunoglobulins or parts thereof.
AU2009305344A 2008-10-17 2009-10-16 Clarification process Abandoned AU2009305344A1 (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
AU2009305344A1 true AU2009305344A1 (en) 2010-04-22

Family

ID=40377277

Family Applications (1)

Application Number Title Priority Date Filing Date
AU2009305344A Abandoned AU2009305344A1 (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

Family Cites Families (5)

* 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
KR20040065567A (en) * 2001-11-28 2004-07-22 산도즈 게엠베하 Chromatographic Purification of Recombinant Human Erythropoietin
ZA200504990B (en) * 2003-01-09 2006-08-30 Genentech Inc Purification of polypeptides
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

Also Published As

Publication number Publication date
KR20110085981A (en) 2011-07-27
WO2010043701A1 (en) 2010-04-22
CA2739392A1 (en) 2010-04-22
CN102203113A (en) 2011-09-28
JP2012505864A (en) 2012-03-08
EP2337787A1 (en) 2011-06-29

Similar Documents

Publication Publication Date Title
WO2010043703A1 (en) Removal of host cell proteins
US11590486B2 (en) Solid phase for mixed-mode chromatographic purification of proteins
US20040033562A1 (en) Integration of high cell density bioreactor operation with ultra fast on-line downstream processing
US20200369718A1 (en) Sterile chromatography and manufacturing processes
JP2017036316A (en) Improved depth filters for disposable biotechnological processes
WO2013158279A1 (en) Protein purification methods to reduce acidic species
CA2751000A1 (en) Methods of purifying small modular immunopharmaceutical proteins
TW201313735A (en) Methods of reducing level of one of more impurities in a sample during protein purification
WO2008100578A2 (en) Method of isolating antibodies by precipitation
TW200835542A (en) Method of isolating biomacromolecules using low pH and divalent cations
WO2015198320A1 (en) Methods of purifying antibodies
US20150133643A1 (en) Low Organic Extractable Depth Filter Media Processed with Solvent Extraction Method
US20110184154A1 (en) Cell broth clarification and host cell protein removal
CN109320611B (en) Purification method of human-mouse chimeric monoclonal antibody biological similar drug
AU2009305344A1 (en) Clarification process
CA2739501A1 (en) Clarification process at higher cell density
Schirmer et al. Primary clarification of very high-density cell culture harvests by enhanced cell settling
CN105579572B (en) Method for purifying high density granular cell culture cutting
WO2016153978A1 (en) Use of dextran to enhance protein purification by affinity chromatography
CA2832341A1 (en) Separation of genomic dna from a target molecule using cation exchange
Jungbauer 4.1 Principles of Product Extraction from Cell Culture and Purification for Pharmaceutical Proteins

Legal Events

Date Code Title Description
MK4 Application lapsed section 142(2)(d) - no continuation fee paid for the application