EP2257620A1 - Methode zur steigerung der reklonierungseffizienz - Google Patents
Methode zur steigerung der reklonierungseffizienzInfo
- Publication number
- EP2257620A1 EP2257620A1 EP09723350A EP09723350A EP2257620A1 EP 2257620 A1 EP2257620 A1 EP 2257620A1 EP 09723350 A EP09723350 A EP 09723350A EP 09723350 A EP09723350 A EP 09723350A EP 2257620 A1 EP2257620 A1 EP 2257620A1
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- European Patent Office
- Prior art keywords
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- cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/105—Insulin-like growth factors [IGF]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/24—Genital tract cells, non-germinal cells from gonads
- C12N2502/243—Cells of the female genital tract, non-germinal ovarian cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Definitions
- the present invention relates to the field of cell culture technology and relates to methods for propagating / cloning cells, preferably cell lines, that are of importance for the production of biopharmaceuticals. Furthermore, the invention relates to methods for the production of proteins using cells that have been obtained and propagated by means of single cell deposition and compositions that allow an increase of single cells.
- biopharmaceuticals For the biotechnological production of biologically active or therapeutic proteins in mammalian cells, so-called “biopharmaceuticals”, the corresponding
- Mammalian cells are stably transfected with DNA specific for the respective biologically active
- the use of serum-free or chemically defined media in the recloning step results in limited recloning efficiency, i. only a small percentage of the deposited cells survive and grow into a monoclonal cell line.
- the cell suspension is serially diluted and the cells are then placed in a microtiter plate at different cell counts per well In high cell count wells, sufficient or no cell will survive, due to sufficient secretion of autocrine growth factors. the fewer cells survive, so that in this way the dilution can be adjusted so that statistically only one single cell survives per well and grows into a monoclonal line
- These single cell clones are detected by visual and / or imaging techniques and the cell ions are cultured further into larger culture vessels ,
- FACS technology In the FACS technology, a flow cytometer is used to generate single-cell clones. For this purpose, the cells are brought into a laminar flow and individually deflected into the wells of the microtiter plates. This ensures that the surviving colonies are indeed individual clones. Therefore, FACS technology is the preferred method over limited dilution.
- a particularly disadvantageous effect of low recloning efficiency is when the subsequent analysis of the individual cell clones is to take place via an automated system:
- An analysis robot can not usually differentiate between wells with living or dead cells and automatically measures all wells of the microtiter plate. With a recloning efficiency of only 10%, this means that in 90% of cases, the robot analyzes an empty well - and uses the same amount of reagents as it does for the analysis of a living cell clone. In case of example! 90% of the time as well as 90% of the material costs are wasted without data gain.
- Serum contains an undefined mixture of different soluble proteins and growth factors that support the survival and proliferation of cells.
- serum-free Zeillinienher ein therefore corresponds to the state-of-the-art in regulatory terms.
- Another approach is to carry out a "limited dilution" for recloning, since this method only statistically leads to the formation of single-cell clones, but it is also possible for several clones to grow up in one well, is a multiple (usually 2-3-fold) repetition of this Necessary to ensure that the cell line obtained is truly a single clone
- feeder lines Another approach is the use of "feeder” lines, the term comes from the English word “feed” and refers to a co-cultivation with cells that are mostly unable to divide, which serve to nourish the cells of the culture that are actually desired and to supply secreting growth factors.
- Feeder cells can significantly increase the efficiency of recloning.
- a disadvantage of this method is the high expense of generating the feeder cells parallel to the actual single cell deposit. Furthermore, the
- the object of the present invention is to increase the efficiency of recloning in the serum-free FACS-based cloning of production cells.
- the invention is based on the increase of the recloning efficiency without the use of feeder cells. 5
- the recloning efficiency and thereby the quantity of the clones obtained can be significantly increased. This effect could be observed from concentrations of 100-500 ⁇ g / L IGF or 50-100 mg / L insulin in the cloning medium.
- the effect of IGF on the recloning efficiency is significantly higher than that of insulin.
- the use of IGF in insulin-free medium is therefore a preferred embodiment.
- Optimum curves are observed with respect to recloning efficiency in both insulin and IGF.
- the optimum for insulin is 100mg / L and for IGF 500 ⁇ g / L.
- the level of recloning efficiency when using IGF is higher.
- iGF optionally with feeder cells and albumin, in particular HSA
- HSA histone deacetylase
- albumin especially serum albumin (HSA) into the culture medium causes an increase in the recloning efficiency.
- HSA serum albumin
- a clear effect is already visible at low albumin, in particular HSA concentrations from 100 mg / L and can be increased by higher Albumi ⁇ - / HSA concentrations of 400-1000 mg / L even more.
- albumin / HSA addition 500 mg / L.
- Bumin / HSA concentration optimum in IGF-containing medium (without insulin supplement) is below 1000 mg / l (see Figures 2, 3 and 4).
- the optimum concentration of albumin / HSA is in the range of 300-600 mg / l or 400-600 mg / l, preferably in the range of 400-500 mg / l, particularly preferably 400 mg / l. Also particularly preferred is an albumin / HSA concentration of 500 mg / l.
- -6- Purified human material, recombinant material (from prokaryotes or eukaryotes) or recombinant plant material may be used as albumin / HSA-Que!
- the highest recloning efficiency can be achieved by a combination of these approaches, i. the addition of IGF and Aibumin / HSA to a serum and insulin-free culture medium.
- additional feeder cells can be used.
- the primary target of recloning is the identification of high-producing cell clones.
- Higher recloning efficiency due to the broader base of clones obtained and due to a normal distribution of clones in terms of productivity, leads to a higher probability of obtaining high-producing cell clones.
- a further advantage of the method described is that the addition of IGF or insulin and optionally albumin / HSA increases the recloning efficiency to such an extent that feeder cells can be dispensed with. This reduces on the one hand the expense in the production and preculture of the feeder cells and at the same time increases the reproducibility of Einzelzeliklontechnik. Furthermore, it allows the use of technologies for the visual detection of the living cell count as a tool for clone analysis. Since the amount of product in the culture medium correlates linearly with the number of producing cells, the technological development is to record both the amount of product and the number of cells in order to determine the highest-producing cell clones.
- feeder cells from the growing production cells are difficult to distinguish visually, they are included in themayl cell count, which leads to an inaccurate calculation of the total cell count and thus the specific productivity.
- a method without feeder cell is therefore advantageous and represents a preferred embodiment.
- the process further increases the cost-effectiveness of this process step and also enables the use of automated clone analysis technology in high-throughput.
- the prior art describes the use of special media compositions to increase the recloning efficiency (WO2006047380).
- Insulin is described as an essential component.
- the use of IGF in place of insulin is explicitly pointed out as an alternative and superior possibility.
- the data of the present application show that in particular by the combination of iGF ⁇ preferably ⁇ 800 ⁇ g / l or 100-500 ⁇ g / l) and albumin, in particular HSA (preferably ⁇ 1000 mg / l or 300-600 mg / l, in particular preferred 400-500mg / L) in an insulin-free medium, a further increase in recloning efficiency can be achieved.
- the albumin (in particular HSA) concentrations used are in the present patent application below 1000 mg / l and thus significantly below those in the cited WO2006047380 patent.
- DG44 cells are cultured for at least five passages in five varying media containing 5 different concentrations of IGF or insulin
- FIGURE 2 is a diagrammatic representation of FIGURE 1
- DG44 cells are cultured for at least five passages in BI-own medium (containing 50 ⁇ g / l IGF but no insulin) supplemented with different HSA concentrations (40, 400 and 1000 mg / l) and feeder cells.
- Anschuessend individual cells are cloned using FACS. After 21 days, the growing clones are counted and the recloning efficiency is determined. For simplicity, the recloning efficiency is normalized, i. the highest value received is set to 100%.
- DG44 and NSO cells are cultured in the appropriate media for at least five passages. Anschuessend individual cells are cloned using FACS. After 21 days, the growing clones are counted and the
- Recloning efficiency normalized, i. the highest value received is
- DG44 cultured in Bl-own medium without insulin supplement
- 50 ⁇ g / l or 500 ⁇ g / l IGF supplement and with or without 500mg / l HSA supplement in the medium DG44 cells are cultured in the appropriate media for at least five passages. Subsequently, individual cells are cloned using FACS. After 21 days, the growing clones are counted and the recloning efficiency is determined. For simplicity, the recloning efficiency is normalized, i. the highest value received is set to 100%.
- insulin is known to those skilled in the art There are a number of different insulin molecules (Gilman, AG et al., Eds., The Pharmacological Basis of Therapeutics, Pergamon Press, New York, 1990, pp. 1463-1495) is Z, B. Zinc insulin Commonly used in cell culture is human zinc insulin or recombinant insulin The concentration of insulin
- -11- can be measured in culture medium using routine experiments such as a commercially available insulin-specific ELISA.
- insulin-free means that the culture medium contains no insulin, in particular no recombinant insulin and no insulin is added to it.
- Albumin is the most abundant protein in plasma. It is produced in the liver and contributes to the maintenance of osmotic pressure in the blood. Albumin binds to nutrients and metabolites, helping to transport them.
- albumin In serum-containing cell culture media, albumin is often the dominant serum component.
- a preferred embodiment of the present invention is a biochemically defined serum-free and insulin-free single-cell cloning medium containing serumaibumin.
- the term "albumin” in the present invention means a polypeptide component having the biological activity of albumin, which generally refers to any animal albumin, in particular a mammalian, such as human, bovine, equine, murine, rat and swine albumin, as well as albumin from birds, in particular
- the albumin is human serum albumin (HSA)
- HSA human serum albumin
- the albumin is not derived from a natural animal source (serum).
- recombinant or synthetic albumin especially recombinant HSA.
- Recombinant HSA The production of recombinant HSA is well known in the art and may e.g. with the help of genetically modified yeasts (U.S.Patent No. 5,612,197).
- Recombinant HSA can be obtained commercially from various suppliers, for example via Sigma-Aldrich (Recombinant HSA, Cat. No, A-7223).
- HSA concentration of HSA can be determined by routine methods such as a commercially available ELISA (eg "Human Albumin ELISA Quantitation Kit", Bethyl Laboratories, Montgomery, TX).
- the cell culture medium contains recombinant aibumin in a concentration sufficient to promote the growth of a single cell, especially a single CHO
- recombinant albumin is in one
- cloning / recloning in the context of Zeilkuitur means a technique by means of which one can obtain a cell population of identical cells from a cell of origin.
- cell cloning or “single cell cloning” thus means a method in which individual cells are identified from a cell pool with cells of different genotype, isolated and then propagated to a cell population consisting of a multiplicity of genetically identical cells. If the cells are stored individually for this purpose, i.
- Single clones or “single cell clones” or “clones” for short are genetically identical cells derived from one (1) single cell
- a pool of stably transfected cells is not a cell clone of the same lineage, ie not a monoclonal cell population, even if genetically identical starting cells are transfected with an identical nucleic acid.
- subclones / subcultures refers to different generations of cells originating from a cell of origin / culture by single or multiple passaging of the dividing cells, for example, “subclones / subcultures” when identical cells / cell cultures are cultured over several generations and be propagated.
- cloning efficiency or “recloning efficiency” is defined as the percentage of cells that survive, divide, and form vital cell populations after storage. If, for example, 100 cells are distributed to 100 culture vessels in the case of cell sorting and 25 of these 100 individually deposited cells grow to cultures, the cloning efficiency is 25%.
- effective or efficient recloning is meant a cloning efficiency of at least 10%, preferably at least 20%, more preferably at least 30%, and even more preferably at least 40%, according to a particularly preferred embodiment of the present invention effective recloning, a cloning with an efficiency of at least 50%, preferably of at least 60%, more preferably of at least 70% and even more preferably of at least 80%.
- divisible / expandable in the sense of the present invention describes the potential of a tents / cell population to be able to divide endlessly, but at least over 2, preferably 4. This potential can be achieved, for example, by irradiation with 11371 Cs or mitomycin C treatment be reduced or completely disturbed.
- derivative / derivative refers to cells genetically derived from a particular parent cell, such as by subculturing (with or without selection pressure) and / or generated by genetic manipulation, re-iso-cellizations of cells of the same cell type are also from the term "derivative / derivative".
- all CHO cell lines are derivatives / derivatives of the hamster ovary cells isolated from Cricetulus griseus by Puck et al., 1958, regardless of whether they were obtained by subculturing, re-isolation or genetic manipulations.
- feeder cell comes from the English word “feed” and means a co-cultivation with mostly divisive cells, which serve to supply the actually desired times of the culture with nutrients and sekret mandat growth factors.
- living cells are arrested by irradiation with UV or gamma rays or treatment with mitomycin C in growth.
- the resulting feeder lines live and produce and secrete growth factors but are unable to divide.
- autologous feeder cell means that both the toll ⁇ ter cell and the row to be cultured in the presence of this feeder cell originate taxonomically from the same origin: for example, if the cell to be cultured is a hamster cell (subfamily Cricetinae), preferably around a cell of the genus Cricetulus or Mesocricetus, for example, a CHO or BHK cell, so each of this subfamily originally isolated
- the term "autoimmune feeder line” means that both feeder cell and the cell to be cultivated originate taxonomically from the same genus or were originally isolated from the same genus (cell from Cricetulus or Mesocricetus, respectively) the cell to be cultivated, for example, a hamster cell of the genus Cricetulus or Mesocricetus, preferably a CHO or a BHK cell, so each of the respective genus originally isolated fairofer cell represents an autoimmune feecfer cell in the context of this invention.
- an "auto-renal feeder cell” is present if the feeder cell and the cell to be cultivated originate from the same species, for example Cricetulus griseus or Mesocricetus auratus
- there is an "auto-oxygenated feeder cell” if feeder cell and the cell to be cultivated come from the same species and have the same tissue tropism (eg Cricetulus griseus ovarian cells - CHO cells).
- a feeder cell is an autoimmune feeder cell when both the feeder cell and the cell to be cultivated are from the same basic cell, for example, when both cells are originally CHO-DG44 cells or Descendants of this cell is.
- the feecfer line conveys the same resistances, e.g. to antibiotics, such as the cell to be cultured. This is particularly advantageous if the cell deposition in the presence of a selection agent, e.g. an antibiotic.
- limited dilution refers to an alternative method for recloning, in which a cell suspension is serially diluted and the cells are subsequently deposited in a microtiter plate with different cell counts per well, in wells with high cell numbers several or all cells become due to sufficient secretion of autocrine growth factors
- FACS technology In the FACS technology, a fluorescence-activated cell sorter (Flow Cytometer) is used to generate single cell clones. For this purpose, the cells are brought into a laminar flow and individually deflected into the wells of the microtiter plates. This ensures that the surviving colonies are indeed individual clones. Therefore, FACS technology is the preferred method over limited dilution.
- Flow Cytometer Fluorescence-activated cell sorter
- serum refers to the cell-free constituent of the blood Serum contains an undefined mixture of different soluble proteins and growth factors, which support the survival and proliferation of cells.
- FBS bovine serum
- Typical concentration ranges are 10-20% FCS or FBS as an addition to the culture medium.
- serum-free means culture media as well as culturing conditions, characterized in that cells in the absence of animal and / or human serum, preferably in the absence of any
- Serum isolated proteins preferably in the absence of non-recombinant proteins are cultivated.
- the term "cells adapted to serum-free conditions" means those cells which can be propagated in the absence of animal or human serum or serum proteins.
- protein-free means that the culture medium contains no animal proteins, with proteins isolated from bacteria, yeasts or fungi not being understood as animal proteins.
- chemically defined describes a cell culture medium which is serum-free, preferably also protein-free, and which consists of chemically defined substances Chemically defined media thus consist of a mixture of predominantly pure individual substances
- An example of a chemically defined medium is, for example, the CD CHO medium from Invitrogen (Carlsbad, CA, US).
- serum-cultured cell refers to cells that are adapted to growth in liquid cultures (“suspension cultures”) and whose ability to adhere to vascular surfaces, such as cell culture dishes / bottles, has been reduced or lost.
- Cells that are adapted to both serum-free growth and growth in suspension are referred to as "serum-free medium-adapted and non-adherent cells.” When these cells are produced from feeder cells, these cells are by definition by-products "Feeder lines adapted to serum-free medium and not adherent".
- Conditioned medium means medium from a culture of living cells which is filtered and added to the fresh medium in the seeding microtiter plates.
- the effect of the conditioned medium is based on its content of growth factors which sericinate from the cells of the preculture into the medium
- Conditioned medium clearly has a positive effect on recloning efficiency, but the gains achieved are insignificant and insufficient as a sole measure of practical application.
- Biopharmaceutically significant proteins / polypeptides include e.g. Antibodies, enzymes, cytokines, lyrnphokines, Adscosionsmoieküle, receptors and their derivatives or fragments, but are not limited to these. In general, all polypeptides that act as agonists or antagonists and / or find therapeutic or diagnostic use are important.
- Other proteins of interest are, for example, proteins / polypeptides which are used for altering the properties of host cells in the context of so-called "cell engineering", such as anti-apoptotic proteins, chaperones, metabolic enzymes, glycosylation enzymes and their derivatives or fragments, but are not limited to these.
- polypeptides is used for amino acid sequences or proteins and refers to polymers of amino acids of any length. This term also includes proteins which are posttranslationally modified by reactions such as cytosylation, phosphorylation, acetylation or protein processing , Deletions or insertion of amino acids, fusion with other proteins, while retaining its biological activity, and the polypeptides may multimerize to form homo- and heteromers.
- Recombinant proteins are proteins which are produced by recombinant expression in host cells. Such recombinant proteins are produced under highest purity conditions in order to minimize the risk of contamination. Recombinant proteins are usually produced in appropriate host cells, e.g. Yeast cells, animal cells or prokaryotic cells (E. coli or other bacterial strains)
- an expression vector such as a purified plasmid, bacteriophage, isolated DNA, mRNA, virus or other nucleic acid is used to introduce the recombinant protein into the host cell and integrate it into the host cell chromosome.
- Eukaryotic expression systems are preferred because they typically possess the necessary cellular machinery to properly modify, process, and fold complex mammalian proteins such as antibodies.
- Recombinant HSA is available, for example, from various commercial suppliers such as Sigma Aldrich.
- therapeutic proteins are insulin, insulin-like growth factor, human growth hormone (hGH) and other growth factors, receptors, tissue plasminogen activator (tPA), erythropoietin (EPO), cytokines, for example, interleukins (IL) such as IL-1, IL-2, IL -3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-Q 1 IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 , IL-16, IL-17, 1L-18, interferon (fFN) -a-pha, beta, gamma, omega or tau, tumor necrosis factor (TNF) such as TNF-alpha, beta or gamma, TRAIL, G-CSF, GM- CSF, M-CSF, MCP-1 and VEGF.
- TNF tumor necrosis factor
- antibodies are monoclonal, polyclonal, multispecific and single chain antibodies and fragments thereof, such as Fab, Fab ', F (ab') 2, Fc and Fc 'fragments, light (L) and heavy (H) immunoglobulin chains and their constant, variable or hypervariable regions as well as Fv and Fd fragments.
- the antibodies may be of human or non-human origin. Humanized and chimeric antibodies are also considered.
- fragment antigen-binding Fab
- Fab fragments consist of the variable regions of both chains, which are held together by the adjacent constant regions. You can e.g. by treatment with a protease, such as papain, are generated from conventional antibodies or by DNA cloning. Other antibody fragments are
- -20- F (ab ') 2 fragments which can be prepared by proteolytic digestion with pepsin.
- VH variable region of the heavy
- variable part Since covalent attachment via the cysteine residues of the constant chains is not possible with these Fv fragments, these Fv fragments are often otherwise stabilized.
- variable region of heavy and light chain often by means of a short
- scFv Single-chain Fv fragment
- multimeric scFv derivatives In recent years, various strategies have been developed to produce multimeric scFv derivatives. The intention is to produce recombinant antibodies with improved pharmacokinetic properties and enhanced binding avidity. To achieve the suckerimerization of the scFv fragments, these are prepared as fusion proteins with multimerization domains. As the multimerization domains, e.g. the CH3 region of an IgG or coiled coil structures, such as the leucine zipper domains, in other strategies, the interaction between the VH and VL regions of the scFv fragment is used for multimerization (eg, , Tri- and pentabodies).
- multimerization domains e.g. the CH3 region of an IgG or coiled coil structures, such as the leucine zipper domains
- the interaction between the VH and VL regions of the scFv fragment is used for multimerization (eg, , Tri- and pentabodies).
- a shortening of the peptide linker in the scFv molecule to 5-10 amino acids results in the formation of homodimers by superimposition of VH / VL chains .
- the diabodies can additionally be stabilized by introducing disulfide bridges Examples of diabodies can be found in the literature.
- mi ⁇ ibody refers to a bivalent, homodimeric scFv derivative which consists of a fusion protein which contains the CH3 region of an immunoglobulin, preferably IgG, particularly preferably IgGI, as the dimerization region which links the scFv fragments via a hinge protein. Region, also of IgG, and an unker region ..
- trimers in the fragments designated bi-, tri- or They are also derivatives of scFv fragments, with multimerization being achieved via di-, tri-, or tetrameric coiled-coil structures.
- antibody fusion or “antibody fusion protein” refers to the fusion / coupling of a protein with an antibody or part of a protein
- Antibody Specifically, these include genetically engineered fusion proteins in which a therapeutic protein is coupled to the Fc portion of an antibody to thereby increase serum half-life / stability of the protein.
- the term also includes antibody fusions consisting of a peptide and an antibody or part of an antibody.
- preferred host cells are hamster cells, such as BHK21, BHK TK “, CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1, and CHO-DG44 cells or derivatives / descendants of these Zeilünien.
- Particularly preferred are CHO-DG44, CHO DUKX, CHO-K1 and BHK21 cells, in particular CHO-DG44 and CHO-DUKX cells
- mouse myeloma cells preferably NSO and Sp2 / 0 cells, as well as derivatives / derivatives of these cell lines.
- hamster and mouse cells which can be used according to the invention are given in the following Table 1.
- other mammalian cells including, but not limited to, human, mouse, rat, monkey, rodent, or eukaryotic cells, including, but not limited to, yeast, insect, avian, and plant cells, may also be used be used as host cells for the production of biopharmaceutical proteins.
- Recombinant mammalian cells preferably rodent cells, more preferably hamster cells, e.g. CHO or BHK cells.
- Host cells are preferred when established, adapted, and cultured completely under serum-free conditions. Particularly preferred are host cells which are additionally established in medium, adapted and completely cultured, which is not only serum-free, but also free of any proteins / peptides of animal origin.
- production cell or “producer cell” or “production clone” refers to a cell that is used in a process to produce a protein.
- these include ge ⁇ etisch-modified cells that are used for the industrial production of recombinant proteins.
- the term primarily refers to genetically-altered eukaryotic host cells which express a recombinant protein and are used for the production of this protein. These include in particular monoclonal cell lines for the production of therapeutic proteins.
- the present invention relates to a method / method for cultivating a single line (1) comprising the following steps: a) cultivating a cell population, b) isolating one (1) single cell from said cell pool, and c) cultivating said single cell in a serum-free one and insulin-free medium containing insulin-like growth factor (IGF).
- IGF insulin-like growth factor
- the method according to the invention is characterized in that the medium IGF is contained in a concentration of less than 800 ⁇ g / l. Further preferred concentration ranges for IGF are 50-750 ⁇ g / L, and particularly preferred are 250-750 ⁇ g / L. ideally the IGF concentration is 500 ⁇ g / L.
- the method according to the invention is characterized in that the isolation of the individual cell takes place by means of FACS.
- the method according to the invention is characterized in that the cell originates from an established, immortalized cell line.
- the cell is a recombinant cell, that is, it is a genetically engineered cell.
- said cell is a cell which produces recombinant or heteroioge proteins.
- it is a production cell used in biopharmaceutical protein production.
- the present invention thus relates in particular to a method / a method for culturing a (1) individual recombinant production cell comprising the following steps: a) culturing a cell population, b) isolating one (1) individual recombinant production cell from said cell pool using FACS and
- IGF insulin-like growth factor
- the method according to the invention is characterized in that the cell is a non-human cell, preferably a hamster or mouse cell.
- the method according to the invention is characterized in that the cell is a rodent cell, preferably a hamster or mouse cell.
- the method according to the invention is characterized in that the cell is a Chinese hamster ovary (CHO) cell Another preferred cell is the NSO cell.
- the method according to the invention is characterized in that said row is a eukaryotic cell such as a yeast, plant, worm, insect, avian, fish, reptile or mammalian cell.
- the cell is a chicken or duck bird cell.
- a eukaryotic cell if it is a vertebrate cell, especially if it is a mammalian cell.
- the method according to the invention is characterized in that said mammalian cell is a Chinese hamster ovary (CHO) 1 a monkey kidney cell CV1, a monkey kidney cell COS, a human lens epithelium PER.C6 TM, a human embryonic kidney cell HEK293 or a human Myeloma cell, a human amniocyte cell line, a baby hamster ovary, an African green monkey kidney cell, a human cervical carcinoma cell line, a canine kidney cell, a rat liver cell, a human lung cell, a human liver cell, a mouse breast cancer cell or if it has a myeloma cell, a canine, Pig or Macaquenzelle is or a cell from the rat, rabbit, rabbit, cat, goat.
- the method according to the invention is characterized in that said cell is a CHO cell type cell, a CHO K1 cell, a CHO DG44 cell, a CHO
- CHO mutants Led to Lec35 Especially preferred is the CHO DG44 line.
- the method according to the invention is characterized in that the medium additionally contains albumin.
- albumin Preference is given here to recombinant albumin or human serum albumin (HSA). Particularly preferred is human recombinant serum albumin.
- the method according to the invention is characterized in that albumin is contained in a concentration of less than 1 g / l.
- the albumin / HSA concentration optimum in IGF-containing medium (without insulin supplement) is below 1000 mg / l (see Figures 2, 3 and 4).
- Other preferred albumin / HSA concentration ranges are 250-999 mg / L, 250-900 mg / I and 250-750 mg / L.
- a concentration of 400 mg / l and of 500 mg / l albumin in particular of recombinant human serum albumin.
- the concentration optimum of albumin / HSA is in the range of 300-600 mg / l and 400-600 mg / l, preferably in the range of 400-500 mg / l.
- the method according to the invention is characterized in that the single cell is cultured in the presence of feeder cells.
- feeder cells are preferably autologous feeder cells, more preferably autologous CHO feeder cells.
- the method according to the invention is characterized in that the isolation of one (1) individual cell in step b) is achieved by "limited dilution" or by means of a fluorescence activated cell sorting (FACS) device. Particularly preferred is the isolation of one (1) single cell by means of FACS.
- FACS fluorescence activated cell sorting
- the method according to the invention is characterized in that the line ( ⁇ ) in step a), b) and c) expresses a protein of interest.
- the method according to the invention is characterized in that the protein of interest is a therapeutic protein, preferably an antibody, an antibody fusion protein or an antibody fragment.
- the method according to the invention is characterized in that the protein of interest is a membrane-bound or a secreted protein, preferably an antibody, an antibody fusion protein or an antibody fragment.
- Said antibody is preferably a monoclonal, polyclonal, mammalian, murine, chimeric, humanized, primatized, primate, human or antibody fragment or derivative thereof such as immunoglobulin light chain, immunoglobulin heavy chain, immunoglobulin light and heavy chain , a Fab fragment, an F (ab ') 2 fragment, an Fc part, an Fc-Fc fusion protein, an Fv fragment, a "single chai ⁇ " Fv fragment, a "single domain” Fv fragment, a tetravalent "single chain” Fv fragment, a disulfide-linked Fv fragment, a domain of deleted antibody or fragment, a "minibody", "diabody”, or a fusion polypeptide of one of said fragments with another peptide or polypeptide, an Fc-peptide fusion, a Fc-toxin infusion or a "scaffold" protein.
- a preferred embodiment of the invention consists in the use of conditioned medium in order to increase the recloning efficiency, in particular after FACS-based single cell deposition.
- the method according to the invention is characterized in that conditioned medium is used.
- Conditioned medium is used in particular as a base medium or as a media additive in steps a) and / or b) and / or c). Preference is given to conditioned medium
- conditioned medium is used in step c) as the base medium or as a media additive.
- the proportion of conditioned medium may be from 10 to 100% of the total volume of medium, preferably a proportion of 30-75%, more preferably 50% conditioned medium.
- the present invention also relates to a cell which is generated by a method according to the invention.
- the present invention further relates to a method for producing a protein of interest in a cell, preferably a CHO line, under serum-free and insulin-free culture conditions comprising the steps of: a) producing a cell population containing a gene of interest which (c) culturing these cells under culture conditions allowing growth of the cells, c) isolating and depositing a single cell into a vessel, preferably into a 96 well plate, d) cultivating said single cell in Serum-free and
- Insulin-free medium containing IGF optionally in the presence of albumin and / or feeder cells, e) selection of a cell according to its expression level
- Protein of interest f) harvesting the protein of interest, for example by separating the cell portion from the supernatant; and g) purifying the protein of interest.
- the method according to the invention is characterized in that preferably autologous feeder cells are used in step d).
- the IGF concentration of less than 800 ⁇ g / l have.
- the IGF concentration is preferably in the range between 50-750 ⁇ g / L, and particularly preferably in the range between 250-750 ⁇ g / L. Ideally, the IGF concentration is 500 ⁇ g / L.
- Albumin (especially HSA) concentration of less than 1000mg / l include.
- Albumin concentration is preferably in the range between 250-999 mg / L,
- the optimum concentration of albumin, in particular HSA, is in the range of 300-600 mg / l or 400-600 rng / l, preferably in the range of 400-500 mg / l.
- the method according to the invention is characterized in that the isolation of a single cell in step c) is performed by FACS.
- DG44 cells are used.
- step e) is automated.
- an analysis robot is preferably used in e).
- Such an automated method is particularly characterized in that the automated system consists of: i) (robot) station performing FACS-based single-cell cloning, ii) incubating to cultivate the lines after single cell cloning, linked to iii) robot station containing a protein -,
- antibody-detecting assay performs, such as ELISA or HTRF ® (homogenous time resolved fluorescence) assay.
- ELISA ELISA
- HTRF ® homogenous time resolved fluorescence
- the method according to the invention is characterized in that the protein of interest is a recombinant protein.
- the method according to the invention is characterized in that the protein of interest is a therapeutic protein, preferably an antibody, an antibody fusion protein or an antibody fragment.
- the method according to the invention is characterized in that the protein of interest is a membrane-bound or a secreted protein, preferably an antibody, an antibody fusion protein or an antibody fragment.
- Said antibody is preferably a monoclonal, polyclonal, mammalian, murine, chimeric, humanized, primatized, primate, human or antibody fragment or derivative thereof such as immunoglobulin light chain, immunoglobulin heavy chain, immunoglobulin light and heavy chain , a Fab fragment, an F (ab ') 2 fragment, an Fc part, an Fc-Fc fusion protein, an Fv fragment, a "single chai ⁇ " Fv fragment, a "single domain” Fv fragment, a tetravalent "single chain” Fv fragment, a disulfide-linked Fv fragment, a domain of deleted antibody or fragment, a "minibody", "diabody”, or a fusion polypeptide of one of said fragments with another peptide of one
- the present invention thus relates in particular to a method for producing a protein of interest, preferably an antibody, in a recombinant production time, preferably a CHO cell, under serum-free and insulin-free culture conditions comprising the following steps: a) Preparation of a cell population which a gene of interest which codes for a protein of interest; b) cultivation of these cells under serium-free culture conditions permitting growth of the cells; c) isolation and deposition of a single cell into a vessel, preferably into a 96-well plate , by FACS, d) cultivation of said single cell in serum-free and insulin-free medium containing IGF in a concentration below 800 ⁇ g / l or in a range of 250-750 ⁇ g / [, preferably 500 ⁇ g / l, preferably in the presence of Albumin in a concentration below 1000 mg / l and / or feeder cells, e) selection of a cell corresponding to i HREM level of expression of protein of interest, this selection is preferably
- the present invention further relates to a protein which is produced by a method according to the invention.
- the present invention also relates to a method for selecting a production cell, wherein a method according to the invention as described above in various embodiments is applied.
- the production cell according to the invention is characterized in that it is selected by one of the methods described according to the invention.
- the production cell according to the invention is characterized in that the host cell is a hamster or mouse cell.
- the production cell according to the invention is characterized in that the host cell is a hamster cell or a mouse myeloma cell, preferably a CHO or BHK cell. ZeIIe or an NSO cell.
- a CHO DG44 cell is especially preferred.
- the present invention further relates to the use of a production cell for biopharmaceutical protein production according to the invention.
- the present invention also relates to a serum-free and insulin-free culture medium which enables the cultivation of one (1) single cell containing IGF (preferably ⁇ 800 ⁇ g / l or 250-750 ⁇ g / l, in particular 500 ⁇ g / l) and optionally Albumin (preferably ⁇ 1000 mg / l or 300-600 mg / l or 400-500 mg / l).
- the single cell here is a CHO cell, particularly preferably a CHO DG44 cell.
- the cells CHO-DG44 / dhfr (, Urlaub et al. 1983) " ' ⁇ are constantly supplemented as suspension lines in serum-free and with hypoxanthine and thymidine ex-Ceii medium (JRH, USA) or B -private medium Zelikulturflaschen! .
- the cells NSO can be permanently cultured as suspension cells in serum-free hybridoma medium, Animal component-free medium (Sigma, Aldrich, St. Louis, USA) in ZeI Iku Itu rf flaschen at 37 0 C in a humid atmosphere and 5% CO 2 .
- the celizahia and viability can be determined with a CEDEX Cell Counter (Innovatis, DE) or trypanebiau staining and the cells are then seeded at a concentration of 1-3 x 10 5 / mL and passaged every 2-3 days.
- the cells are irradiated with a radioactive radiation source (Cs137 Strahier, Gammacell 2000, Molsgaard Medical
- the cells are seeded with ca, 10000 cells / well in 96-well microtiter plates in the cell-specific Ex-Ceil medium (JRH, USA) or Bl-own medium (eg TH-9) medium and at about 37 ° C and 5% CO 2 stored in Brutraumatmospreheat.
- JRH, USA cell-specific Ex-Ceil medium
- Bl-own medium eg TH-9
- the process is carried out in accordance with NSO cells, wherein the feeder cells are kept / seeded in each specific for the cell medium.
- the conditioned medium is obtained from the supernatant of a CHO cell culture.
- the CHO cells are seeded at a seeding density of 0.3 ⁇ 10 6 cells / ml and cultured for two to four days.
- the supernatant is separated from the cells by centrifugation and then sterile filtered through an O, 2 ⁇ m filter.
- the resulting filtrate is used as a conditioned medium.
- Cells expressing a fluorescent protein can alternatively be sorted according to their fluorescence intensity with respect to the intracellularly expressed fluorescent protein, and the cells are individually deposited in (optionally) 96 well microtiter plates equipped with feeder cells the cell deposition in Ex-CeII medium (JRH, USA) or in Bl-own medium (eg TH-9) with the appropriate supplements to IGF or Insulin and optionally or preferably HSA.
- Ex-CeII medium JRH, USA
- Bl-own medium eg TH-9
- the cell deposit is carried out according to Hybridoma medium, Animal component free medium (Sigma, Aldrich, St. Louis, USA).
- CALCULATION OF THE RECONCILIATION EFFICIENCY Recloning efficiency is calculated from the quotient of positive wells per plate / total wells per plate. Positive wells are considered to be wells containing exactly one clone.
- CHO DG44 cells are adapted for 5 passages to B [s own, serum-free medium. This is followed by a FACS-based single cell deposit with different insulin and IGF concentrations in medium supplemented with 500 mg / L HSA. After 21 days, the tall clones are counted and calculated from the proportion of grown clones in the total number of wells used, the recloning efficiency. Optimum curves can be observed for both insulin and IGF. The optimum for insulin is 100mg / L and for IGF 500 ⁇ g / L. ( Figure 1) The level of recloning efficiency when using IGF is higher than with insulin.
- CHO DG44 cells are adapted for 5 passages the basal medium ExCeII 302 or lead medium (containing 50 ⁇ g / l IGF, but no insulin supplement). This is followed by a FACS-based single cell deposit with different HSA concentrations with and without feeder cells. After 21 days, the tall clones are counted and from this the proportion of grown clones in the total amount! the wells used calculated the recloning efficiency. It shows a positive effect of
- Figure 2 shows that the HSA concentration optimum in IGF-containing medium (without insulin supplementation) at less than 100.0 mg /! lies.
- CHO DG44 and NSO cells are adapted for 5 passages to Bl's own medium (CHO DG44) or CD hybridoma medium (Invitrogen). Thereafter, a
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AUPN442295A0 (en) * | 1995-07-26 | 1995-08-17 | Commonwealth Scientific And Industrial Research Organisation | Regulated autocrine growth of mammalian cells |
AT409379B (de) * | 1999-06-02 | 2002-07-25 | Baxter Ag | Medium zur protein- und serumfreien kultivierung von zellen |
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DE10338531A1 (de) * | 2003-08-19 | 2005-04-07 | Boehringer Ingelheim Pharma Gmbh & Co. Kg | Verfahren zur Reklonierung von Produktionszellen |
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