Classifications

• • 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
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor

Definitions

• the present invention relates to a Drosophila melanogaster S2 cell line, methods for obtaining such a cell line, and methods of using such a cell line for, e.g., recombinant production of polypeptides.
• Protein production systems for expressing polypeptides or proteins of interest in recombinant organisms or cells are the backbone of commercial biotechnology.
• Prokaryotic cell culture systems are easy to maintain and cheap to operate.
• prokaryotic cells are not capable of post-translational modification of eukaryotic proteins.
• many proteins are incorrectly folded, requiring specific procedures to refold them, which adds to the cost of production.
• mammalian cells are capable of post-translational modification, and generally produce proteins, which are correctly folded and soluble.
• the chief disadvantages of mammalian cell systems include the requirement for specialised and expensive culture facilities, the risk of infection, which can lead to loss of the whole culture, and the risk of contaminating the end product with potentially hazardous mammalian proteins.
• Insect cells are also used for polypeptide expression.
• Drosophila melanogaster S2 cells available from, for example, depositories such as Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DMSZ) and the American Type Culture Collection (ATCC).
• S2 cells have the advantage of not being dependent on anchorage to a solid substrate, thus allowing for easy culture in cell suspension.
• a high growth rate is essential.
• Moraes et al. (2012) it was reported that S2 cells in specific studies have achieved maximum specific cell growth rates of 0.084 h(-l) and maximum cell concentrations of about 5.0E7 cells/mL in an optimized bioreactor system.
• the present inventors have identified a new Drosophila melanogaster S2 cell line which has advantageous properties, including a high growth rate and the ability to achieve a high cell density in culture. These properties allow for a faster and more economical cell line development.
• the present invention provides a native cell line derived from S2 cells and characterized by a growth rate and/or cell density higher than that of standard, reference S2 cells as represented by, e.g., CRL-1963 S2 cells from the ATCC.
• the invention further provides for cells of such a cell line and their progeny, mutants, derivatives and/or transfectants, as well as methods for preparing and cultivating such a cell line and methods for recombinant production of polypeptides and proteins using such cells or cell lines.
• Fig. 1 shows specific dilution and seeding steps of the clonal selection method used for establishing the novel cell line. For more details, see Example 1.
• Fig. 2 shows a comparison between the cell density achieved by an S2 cell line according to the present invention
• Drosophila melanogaster S2 cell line which has a higher growth rate and achieves a higher cell density than S2 cells from the CRL-1963 ATCC deposit. When seeded at about 8E6 cells per mL, the novel cell line can grow to up to 67E6 cells in 3-4 days in normal shake flask culture.
• the novel cell line, designated ExpreS2+ was generated through cloning of S2 cells using limiting dilution in 96-well plates, and has been deposited at DMSZ as DSM ACC 3146.
• the novel cell line does not comprise foreign or heterologous DNA, but can advantageously serve to be transfected into a recombinant production cell line for any polypeptide of interest.
• the novel cell line can also be used in a variety of cellular assays, for antigen display for antibody screening assays, functional presentation of membrane proteins for small molecule screening assays, for antigen display for dendritic cell based anti-cancer vaccines, as source of Drosophila S2 proteins for use in a host cell protein assays, flow cytometry assays, and other uses. So, in a first aspect, the invention relates to a native cell line or clone derived from
• Drosophila melanogaster S2 parent cells which cell line has one or more properties selected from:
• the cell line or clone has the properties of both (a) and (b).
• the cell line or clone has a growth rate from about 17% to about 69% higher than the S2 reference cell line. In one embodiment, the cell density is from about 17% to about 113% higher than that of the S2 reference cell line. In one embodiment, the cell line or clone has a growth rate in the range of about 25% to about 35% higher, such as about 31% higher, than that of CRL-1963 cells and a cell density in the range of about 50% to about 60% higher, such as about 55% higher, than that of CRL-1963 cells, on average.
• the S2 parent and reference cells can be obtained from any source, including depositories or other cell banks, commercial or non-commercial sources, in possession of non-genetically modified S2 cells.
• the S2 parent cell line is selected from the group consisting of CRL-1963 (ATCC), DSM ACC 130 (DMSZ), RBC1153 (RIKEN Bio Resource Center), Stock No. 6 (DGRC) and 90070546 (ECACC), and a progeny or mixture of any thereof.
• the S2 reference cell line is CRL-1963 from ATCC.
• the growth rate is calculated as the difference in cell numbers after 3 days of suspension culture at a cultivation temperature of about 25 °C, with an initial cell concentration of about 8E6 cells/mL.
• the cell density is the cell concentration after 3 or 4 days, such as 3 days, of suspension culture at a cultivation temperature of about 25 °C, with an initial cell concentration of about 8E6 cells/mL.
• Suitable standard S2 culture media are commercially available, or can be prepared from standard ingredients.
• the standard S2 cell culture medium comprises about 10% Foetal Bovine Serum (FBS).
• the invention relates to the cell line deposited as DSM ACC 3146 or a cell thereof.
• the DSM ACC 3146 cell line was deposited at the DSMZ, InhoffenstraBe 7 B, 38124 Braunschweig, Germany, on 30 November 2011.
• the invention further relates to a progeny, derivative or mutant of the DSM ACC3146 cell line or cell thereof, the progeny, derivative or mutant having one or more properties selected from : (a) a growth rate at least about 15 % higher than S2 cells (ATCC CRL-1963) in a standard S2 cell culture medium, and
• one or more genes have been mutated, down-regulated, deleted or overexpressed in the progeny, derivative or mutant as compared to the DSM ACC 3146 cell line.
• the progeny, derivative or mutant has a growth rate from about 17% to about 69% higher than the S2 reference cell line.
• the cell density is from about 17% to about 113% higher than that of the S2 reference cell line.
• the invention relates to a recombinant cell or cell line, which is the DSM ACC 3146 cell line or cell thereof which further comprises one or more heterologous
• such a heterologous polynucleotide is operably linked to an inducible, a regulated or a constitutive promoter.
• such a heterologous polynucleotide is comprised in an expression vector, such as a viral expression vector.
• the invention relates to a cell of any previous aspect for use in a method of producing a polypeptide.
• the invention relates to a method for the production of a polypeptide of interest, the method comprising the steps of
• the method optionally comprises culturing the cell or cell line before obtaining the polypeptide.
• the invention provides a process for obtaining the cell line or clone of any preceding aspect, comprising the steps of:
• the native S2 parent cell line, the S2 reference cell line, or both are independently selected from the group consisting of CRL-1963 (ATCC), DSM ACC 130 (DMSZ), RBC1153 (RIKEN Bio Resource Center), Stock No. 6 (DGRC) and 90070546 (ECACC), and a progeny or mixture of any thereof.
• the invention relates to a cell line or clone obtained by the process of the preceding aspect or embodiment.
• S2 cell refers to a cell from a Schneider-2 embryonic Drosophila melanogaster cell line.
• the S2 cell line is, for example, available from DSMZ, InhoffenstraBe 7 B, 38124
• a cell line "derived from” another cell line refers to a cell line obtained by selecting a subpopulation, typically a clone, of the original, parent cell line (e.g., S2) . This can be achieved, e.g., by clonal selection, isolating individual cells, which are then separately expanded and optionally tested for desired properties.
• a "native” cell line as used herein refers to a cell line, which is not recombinant, e.g., it has not been subjected to transformation or transfection with heterologous polynucleotides.
• a cell "clone” as used herein refers to a group of identical cells that share a common ancestry, meaning they are derived from the same parent cell by cell division. Unless otherwise indicated or contradicted by context, a "progeny" of another cell or cell line as used herein refers to a cell or to a cell population obtained from a parent cell or cell line, typically by cell division in culture over a period of time where slight differences in genotype and/or phenotype may occur between some cells and/or the parent.
• a “mutant" cell or cell line refers to a cell or cell line whose DNA differs from that of its parent cell or cell line, e.g., by the insertion, deletion, substitution or overexpression of nucleotides or polynucleotides in the DNA.
• E6 refers to millions, i.e., 10 5 , typically in the context describing the number of millions of cells.
• a polynucleotide or polypeptide which is "heterologous" in relation to a host cell refers to a polynucleotide not normally comprised in the host cell, typically introduced by transfection or transformation, or to a polypeptide not normally expressed and secreted by the host cell.
• operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
• a promoter is operably linked to a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the
• the invention provides a native, non-transfected cell line which is derived from a Drosophila melanogaster S2 parent cell line, and which has a higher growth rate and/or a higher cell density than that of a S2 reference cell line, such as CRL-1963 (ATCC).
• a native, non-transfected cell line which is derived from a Drosophila melanogaster S2 parent cell line, and which has a higher growth rate and/or a higher cell density than that of a S2 reference cell line, such as CRL-1963 (ATCC).
• the cell line of the invention is DSM ACC 3146. In one embodiment, the cell line of the invention is a progeny, derivative or mutant of DSM ACC 3146. Also provided are cells, clones and subpopulations of such cell lines.
• Suitable S2 parent cell lines can be obtained from various sources, including numerous depositories and other commercial and non-commercial sources.
• Exemplary S2 cells include those available from the ATCC (CRL-1963), DSMZ (DSM ACC 130), the Cell Bank at the RIKEN Bio Resource Center (RBC1153), the Drosophila Genetics Research Center (DGRC) (Stock number 6), the Drosophila RNAi Screening Center at Harvard Medical School; the Russian Academy of Sciences (MWIGG, Moscow), the European Collection of Cell Cultures (ECACC) (cat. code 90070546), and InVitrogen (Catalog no. R690-07).
• the parent S2 cell line can be a progeny of any one of the cell lines from the aforementioned sources, obtained by cultivating the S2 cell line.
• the S2 parent cell line is selected from the group consisting of CRL-1963 (ATCC), DSM ACC 130 (DMSZ), RBC1153 (RIKEN Bio Resource Center), Stock No. 6 (DGRC) and 90070546 (ECACC), and a progeny of any thereof.
• the S2 parent cell line is CRL-1963 or a progeny thereof.
• the S2 parent cell line is RBC1153 or a progeny thereof.
• the S2 parent cell line is Stock No. 6 or a progeny thereof.
• the S2 parent cell line is 90070546 or a progeny thereof.
• the parent S2 cell line comprises cells from the ACC 130 S2 cell line, the CRL-1963 S2 cell line, or a mixture thereof.
• any one of the aforementioned deposited S2 cell lines can also be used as an S2 reference cell line.
• the S2 reference cell line is CRL-1963.
• the growth rate of the cell line is at least about 5%, at least about 10%, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 85%, at least about 90%, or at least about 100% higher than that of the reference cell line.
• the growth rate of the cell line may be no more than about 500%, no more than about 150%, no more than about 100%, no more than about 90%, no more than about 80%, no more than about 75%%, no more than about 70%, no more than about 69%, or no more than about 55% higher than that of the reference cell line.
• the growth rate of the cell line is from about 10% to about 200%, from about 10% to about 100%, from about 15% to about 100%, from about 15% to about 70%, from about 17% to about 69%, or from about 23% to about 55% higher than that of the reference cell line.
• the growth rate of the cell line is, on average from a series of measurements, about 25 to about 35% higher than that of the reference cell line. In a specific embodiment, the growth rate of the cell line is, on average from a series of measurements, about 31% higher than that of the reference cell line. In a specific embodiment, the growth rate of the cell line is, on average from a series of measurements, about 27% higher than that of the reference cell line.
• the cell density of the cell line is at least about 5%, at least about 10%, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or at least about 110% higher than that of the reference cell line.
• the cell density may be no more than about 500%, no more than about 200%, no more than about 150%, no more than about 113%, or no more than about 92% higher than that of the reference cell line.
• the cell density of the cell line is from about 10% to about 300%, from about 10% to about 200%, from about 15% to about 150%, from about 17% to about 113%, from about 27% to about 113%, or from about 27% to about 92% higher than that of the reference cell line.
• the cell density is, on average from a series of measurements, about 45% to about 60% higher, such as about 50% to about 60% higher than that of the reference cell line.
• the growth rate of the cell line is, on average from a series of measurements, about 55% higher than that of the reference cell line. In a specific embodiment, the growth rate of the cell line is, on average from a series of measurements, about 49% higher than that of the reference cell line.
• the cell line or clone has the properties of both a higher growth rate and a higher cell density than those of the reference cell line.
• the growth rate and cell density of the cell line are in the range of from about 10% to about 200% and from about 10% to about 300%, from about 10% to about 100% and from about 10% to about 200%, from about 15% to about 100% and from about 15% to about 150%, from about 15% to about 70% and from about 15% to about 120%, from about 17% to about 69% and from about 17% to about 113%, or from about 23% to about 55% and from about 27% to about 92% higher than those of the reference cell line, respectively.
• the growth rate and cell density are, on average from a series of measurements, about 25 to about 35% and about 50% to about 60% higher than those of the reference cell line, respectively. In a specific embodiment, the growth rate and cell density of the cell line are, on average from a series of measurements, about 31% and 55% higher than those of the reference cell line, respectively. In a specific embodiment, the growth rate and cell density of the cell line are, on average from a series of measurements, about 27% and 49% higher than those of the reference cell line, respectively.
• the growth rate of the progeny, derivative or mutant of the DSM ACC3146 cell line is at least about 5%, at least about 10%, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 85%, at least about 90%, or at least about 100% higher than that of the reference cell line.
• the growth rate of the progeny, derivative or mutant of the DSM ACC3146 cell line may be no more than about 500%, no more than about 150%, no more than about 100%, no more than about 90%, no more than about 80%, no more than about 75%%, no more than about 70%, no more than about 69%, or no more than about 55% higher than that of the reference cell line.
• the growth rate of the progeny, derivative or mutant of the DSM ACC3146 cell line is from about 10% to about 200%, from about 10% to about 100%, from about 15% to about 100%, from about 15% to about 70%, from about 17% to about 69%, or from about 23% to about 55% higher than that of the reference cell line.
• the growth rate of the progeny, derivative or mutant of the DSM is the growth rate of the progeny, derivative or mutant of the DSM
• the ACC3146 cell line is, on average from a series of measurements, about 25 to about 35% higher than that of the reference cell line. In a specific embodiment, the growth rate of the cell line is, on average from a series of measurements, about 31% higher than that of the reference cell line. In a specific embodiment, the growth rate of the progeny, derivative or mutant of the DSM ACC3146 cell line is, on average from a series of measurements, about 27% higher than that of the reference cell line.
• the cell density of the progeny, derivative or mutant of the DSM ACC3146 cell line is at least about 5%, at least about 10%, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or at least about 110% higher than that of the reference cell line.
• the cell density may be no more than about 500%, no more than about 200%, no more than about 150%, no more than about 113%, or no more than about 92% higher than that of the reference cell line.
• the cell density of the progeny, derivative or mutant of the DSM ACC3146 cell line is from about 10% to about 300%, from about 10% to about 200%, from about 15% to about 150%, from about 17% to about 113%, from about 27% to about 113%, or from about 27% to about 92% higher than that of the reference cell line.
• the cell density of the progeny, derivative or mutant of the DSM is the cell density of the progeny, derivative or mutant of the DSM
• the growth rate of the progeny, derivative or mutant of the DSM ACC3146 cell line is, on average from a series of measurements, about 45% to about 60% higher, such as about 50% to about 60% higher than that of the reference cell line.
• the growth rate of the progeny, derivative or mutant of the DSM ACC3146 cell line is, on average from a series of measurements, about 55% higher than that of the reference cell line.
• the growth rate of the progeny, derivative or mutant of the DSM ACC3146 cell line is, on average from a series of measurements, about 49% higher than that of the reference cell line.
• the progeny, derivative or mutant of the DSM ACC3146 cell line or clone has the properties of both a higher growth rate and a higher cell density than those of the reference cell line.
• the growth rate and cell density of the progeny, derivative or mutant of the DSM ACC3146 cell line are in the range of from about 10% to about 200% and from about 10% to about 300%, from about 10% to about 100% and from about 10% to about 200%, from about 15% to about 100% and from about 15% to about 150%, from about 15% to about 70% and from about 15% to about 120%, from about 17% to about 69% and from about 17% to about 113%, or from about 23% to about 55% and from about 27% to about 92% higher than those of the reference cell line, respectively.
• the growth rate and cell density are, on average from a series of measurements, about 25 to about 35% and about 50% to about 60% higher than those of the reference cell line, respectively.
• the growth rate and cell density of the progeny, derivative or mutant of the DSM ACC3146 cell line are, on average from a series of measurements, about 31% and 55% higher than those of the reference cell line, respectively.
• the growth rate and cell density of the progeny, derivative or mutant of the DSM ACC3146 cell line are, on average from a series of measurements, about 27% and 49% higher than those of the reference cell line,
• the respective growth rate and cell density of the cell line of the invention, its parent S2 cell line and its progeny, derivative or mutant can be measured using standard assays known in the art.
• cells in cell culture grow (i.e., multiply) until some nutrient in the culture medium is exhausted. Before this occurs, distinct phases in the growth curve can typically be observed.
• a lag phase which essentially is a period of no growth during which the cells adapt to the new environment. The length of this phase varies with organism and cultivation conditions.
• This phase is often negligible, due to culture transfer between identical media, and it can also be minimized by providing a sufficient inoculum concentration (e.g., 0.5E6 cells/mL or higher).
• a sufficient inoculum concentration e.g., 0.5E6 cells/mL or higher.
• the establishment of exponential growth depends on a number of factors, including a viable inoculum, inoculum concentration, a suitable energy source, the presence of excess nutrients and growth factors, the absence of inhibitors and a suitable environment (i.e. temperature, dissolved oxygen).
• a suitable environment i.e. temperature, dissolved oxygen.
• Suitable conditions for optimized growth of S2 and S2-related cells are known in the art, and are extensively reviewed in, e.g., Moraes et al. (2012).
• the temperature should be in the range of about 25°C to about 28°C, and agitation in the range of 100 to 140 rpm.
• Example 6 An exemplary assay for testing cell growth is provided in Example 6. According to this assay, cells from the cell line to test and reference S2 cells are separately seeded into 250 mL culture flasks at a concentration of 8E6 cells/mL, cultured in suspension for 3-4 days at a temperature of 25°C and at 110 rpm, counted, and then re-seeded at the same
• the growth rate is measured after 3 days of culture as this may approximate the growth rate at exponential growth better than 4-day measurements.
• the growth rate is calculated as the difference in cell numbers after 3 days of suspension culture at a cultivation temperature of about 25 °C, with an initial cell concentration of about 8E6 cells/mL.
• the cell density is the cell concentration after 3 or 4 days, such as 3 days, of suspension culture at a cultivation temperature of about 25 °C, with an initial cell concentration of about 8E6 cells/mL.
• Suitable standard S2 culture media for cell cultivation or for use in testing cell growth rate and cell density are commercially available and/or known in the art.
• Exemplary media include Schneider's medium, D22, M3, M3: D22 a 1 : 1 volume ratio, Sf-900 II, serum-free EX-CELL 400 series, including EX-CELL 420®, DES expression, HyQ SFX, Drosophila SFM, IPL-41, Grace's, TC-100, TNM-FH, TNM-FH :Sf-900 II at a 1 : 1 volume ratio and Insect Xpress.
• the standard S2 cell culture medium comprises about 10% Foetal Bovine Serum (FBS).
• the S2 culture media employed for growth rate comparison is EX-CELL 420®, optionally comprising 10% FBS. In one embodiment, the S2 culture media employed for growth rate comparison is Schneider's media, optionally comprising 10% FBS. In one embodiment, the S2 culture media employed for growth rate comparison is IPL-41, optionally comprising 10% FBS. In one embodiment, the S2 culture media employed for growth rate comparison is TC-100, optionally comprising 10% FBS.
• the culture medium comprises the following components (Schneider's) supplemented with 10% FBS, as set out in Table 1 :
• the standard S2 culture media comprises the following components (TC- 100) supplemented with 10% FBS or a combination of yeastolate (e.g ., 3 g/L), lipid emulsion (e.g ., 1%), glucose (10 g/L), glutamine (3.5 g/L) and Pluronic F68 (0.1%) (see Galesi et al ., 2007), as set out in Table 2: TABLE 2
• the standard S2 culture media comprises the followi ng components (IPL- 41) supplemented with 10% FBS or a combination of yeastolate ultrafiltrate (e.g., 6 g/L), glucose (e.g., 10 g/L), glutamine (e.g., 3.5 g/L), fructose (e.g., 0.5 g/L), lactose (e.g., 2 g/L), tyrosine (e.g., 0.6 g/L), methionine (e.g., 1.48 g/L) and lipid emulsion (e.g., 1% (v/v)) (see Batista et al, 2008), as set out in Tables 3 and 4: TABLE 3
• yeastolate ultrafiltrate e.g., 6 g/L
• glucose e.g., 10 g/L
• glutamine e.g., 3.5 g/L
• fructose e.g.,
• the invention also provides a process for obtaining the cell line of the invention, comprising the steps of:
• the native S2 parent cell line, the S2 reference cell line, or both are independently selected from the group consisting of CRL-1963 (ATCC), DSM ACC 130 (DMSZ), RBC1153 (RIKEN Bio Resource Center), Stock No. 6 (DGRC) and 90070546
• the native S2 parent cell line is from the cell line deposited as ATCC CRL-1963 or DSMZ ACC 130.
• the S2 reference cell line is CRL-1963.
• the S2 parent and S2 reference cell line are from the same source.
• the dilution series can be performed in, e.g., 15 ml centrifuge tubes. The dilution is done in order to achieve a series of very low concentrations of cells to seed in 96 well plates in order to find an optimum dilution of cells that give 30 or less clones per plate, e.g., 5-30 clones per 96-well plate (corresponding to about 0.05 to 0.3 clones per well). Statistically, there should be less than one clone per well on average.
• the dilution series is performed in many steps and with a low dilution rate in order to minimize propagation of errors in the dilution series. For additional details, see Example 1. Well comprising clones can be identified by visual analysis using, e.g., a microscope.
• Assays suitable for testing the cells in step (e) are described elsewhere herein, and include, for example, the assay described in Example 6.
• the growth rate of the selected cell line is at least about 5%, at least about 10%, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 85%, at least about 90%, or at least about 100% higher than that of the reference cell line.
• the growth rate of the selected cell line may be no more than about 500%, no more than about 150%, no more than about 100%, no more than about 90%, no more than about 80%, no more than about 75%%, no more than about 70%, no more than about 69%, or no more than about 55% higher than that of the reference cell line.
• the growth rate of the selected cell line is from about 10% to about 200%, from about 10% to about 100%, from about 15% to about 100%, from about 15% to about 70%, from about 17% to about 69%, or from about 23% to about 55% higher than that of the reference cell line.
• the growth rate of the selected cell line is, on average from a series of measurements, about 25 to about 35% higher than that of the reference cell line. In a specific embodiment, the growth rate of the selected cell line is, on average from a series of measurements, about 31% higher than that of the reference cell line. In a specific embodiment, the growth rate of the selected cell line is, on average from a series of measurements, about 27% higher than that of the reference cell line.
• the cell density of the selected cell line is at least about 5%, at least about 10%, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 27%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% or at least about 110% higher than that of the reference cell line.
• the cell density may be no more than about 500%, no more than about 200%, no more than about 150%, no more than about 113%, or no more than about 92% higher than that of the reference cell line.
• the cell density of the selected cell line is from about 10% to about 300%, from about 10% to about 200%, from about 15% to about 150%, from about 17% to about 113%, from about 27% to about 113%, or from about 27% to about 92% higher than that of the reference cell line.
• the cell density of the selected cell line is, on average from a series of measurements, about 50% to about 60% higher than that of the reference cell line.
• the growth rate of the selected cell line is, on average from a series of measurements, about 55% higher than that of the reference cell line.
• the growth rate of the selected cell line is, on average from a series of measurements, about 49% higher than that of the reference cell line.
• the selected cell line or clone has the properties of both a higher growth rate and a higher cell density than those of the reference cell line.
• the growth rate and cell density of the selected cell line are in the range of from about 10% to about 200% and from about 10% to about 300%, from about 10% to about 100% and from about 10% to about 200%, from about 15% to about 100% and from about 15% to about 150%, from about 15% to about 70% and from about 15% to about 120%, from about 17% to about 69% and from about 17% to about 113%, or from about 23% to about 55% and from about 27% to about 92% higher than those of the reference cell line, respectively.
• the growth rate and cell density of the selected cell line are, on average from a series of measurements, about 25 to about 35% and about 45% to about 50% higher than those of the reference cell line, respectively.
• the growth rate and cell density of the selected cell line are, on average from a series of measurements, about 31% and 55% higher than those of the reference cell line,
• the growth rate and cell density of the selected cell line are, on average from a series of measurements, about 27% and 49% higher than those of the reference cell line, respectively.
• the invention provides a cell line obtained or obtainable by the process of any preceding embodiment.
• the invention further relates to a progeny, derivative or mutant of the DSM ACC3146 cell line or cell thereof, the progeny, derivative or mutant preserving one or more characterizing features of the DSM ACC 3146 cell line, such as a higher growth rate than native S2 cells (ATCC CRL-1963), a higher cell density than native S2 cells (ATCC CRL-1963), or both.
• Progeny includes cell populations obtained by cultivating DSM ACC 3146 cells under suitable growth conditions described herein (see, e.g., Examples 3 to 5) or otherwise known in the art, as well as cell preparations of DSM ACC 3146 which are frozen for the purpose of storage.
• Derivatives of the DSM ACC 3146 cell line include individual cells, clones and subpopulations of the cell line, e.g., obtained by clonal selection methods.
• Mutants of the DSM ACC 3146 cell line include cells where one or more genes have been mutated, down-regulated, deleted from the genome or overexpressed, either by exposing the cell line to a mutagen or radiation, by directed mutagenesis, as well as cells where mutations have occurred naturally.
• the invention also provides a recombinant cell or cell line, which is the DSM ACC3146 cell line or cell thereof which further comprises one or more heterologous polynucleotides.
• the heterologous polynucleotide is operably linked to an inducible, a regulated or a constitutive promoter.
• the heterologous polynucleotide can be comprised in a cloning and/or an expression vector, such as a viral expression vector. Cultured host cells are transfected with the expression vector, and the protein produced thereby can be recovered from the cells themselves or from the culture medium.
• a "cloning vector” means a plasmid DNA which can be used to insert a DNA fragment of interest into a host cell, normally in order to produce multiple copies of the fragment and hence the vector.
• “Expression vector” means a plasmid or viral DNA containing necessary regulatory signals for the synthesis of mRNA derived from gene sequences, which can be inserted into the vector.
• the term “promoter” as used herein means a nucleotide sequence that provides a cell with the regulatory sequences for expression of a coding sequence operably linked thereto.
• a coding sequence is located 3' to a promoter sequence.
• the promoter typically a DNA polynucleotide
• Suitable promoters used in Drosophila melanogaster S2 cells for protein expression include the pMT promoter, the P2ZOp2F (OPIE2 promoter) as well as promoters described in WO 2009/150222 (ExpreS2ion Biotechnologies ApS).
• polyadenylation sequence refers to a DNA sequence which when transcribed is recognized by the expression host to add polyadenosine residues to transcribed mRNA. It is operably linked to the 3'-end of the DNA encoding the polypeptide to be expressed. Suitable polyadenylation sequences includes the OPIE2 polyA tail and the late
• SV40 polyA tail as described in Angelichio et a/. 1991, Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured Drosophila cells, Nucleic Acids Research, Vol. 19, No. 18 5037-5043.
• a “protein export signal polynucleotide sequence” refers to a sequence that directs or facilitates the translocation of an expressed protein across the membrane of the host expression cell.
• a protein export signal polynucleotide sequence may be present in the N- terminus of a precursor polypeptide (a pre-peptide or pre-pro-peptide) to directs its translocation across a membrane.
• a precursor polypeptide is processed by cleavage of the signal sequence to generate a mature peptide or a pro-peptide. If the product of off- cleavage of the signal peptide is a pro-peptide, the mature peptide is the product of subsequent post-translational modifications that involve further removal of amino acids.
• Suitable protein export signal polynucleotide sequences within this definition include the Drosophila BiP signal sequence for secretion and CPY.
• the Drosophila BiP protein encodes an immunoglobulin-binding chaperone protein. This secretion signal efficiently targets high levels of BiP into the secretory pathway of S2 cells (see Kirkpatrick, R.B. et a/. (1995) J. Biol. Chem. 270: 19800-19805).
• the terms "ubiquitous chromatin opening element” or "UCOE” as used herein refers to a DNA sequence which opens chromatin or maintains chromatin in an open state and facilitates reproducible expression of an operably-linked gene in cells.
• matrix attachment region or "MAR” or “Scaffold/matrix attachment region” as used herein refers to a nucleotide sequence in the DNA of eukaryotic chromosomes where the nuclear matrix attaches. MARs mediate structural organization of the chromatin within the nucleus. These elements constitute anchor points of the DNA for the chromatin scaffold and serve to organize the chromatin into structural domains. Studies on individual genes led to the conclusion that the dynamic and complex organization of the chromatin mediated by S/MAR elements plays an important role in the regulation of gene expression.
• amplification control element or “Ace” as used herein refers to an element, which is involved in initiating amplification of DNA from the replication origin.
• Deletion analyses of transgenic constructs derived from the third chromosome cluster have identified a 320-bp amplification control element (ACE3) required for amplification. This includes the D.
• ACE3 320-bp amplification control element
• AERs melanogaster ACE3 element and amplification enhancing regions
• Order-beta element refers to an origin of replication.
• the origin of replication (also called the replication origin) is a particular sequence in a genome or plasmid at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double- stranded RNA viruses. DNA replication may proceed from this point bidirectionally or unidirectionally.
• the origin of replication binds the pre-replication complex, a protein complex that recognizes, unwinds, and begins to copy DNA.
• One specific "Ori-beta element" within this definition is the D. melanogaster Ori-beta element.
• the invention relates to a method for the production of a polypeptide of interest, the method comprising the steps of
• expression vector comprising a polynucleotide sequence encoding the polypeptide; and (b) obtaining the polypeptide.
• the method optionally comprises culturing the cell or cell line before obtaining the polypeptide.
• Suitable cell culture methods include those described in Examples 3 to 5 or, e.g., in Moraes et al. (2012).
• the cell or cell line of the invention is transfected so as to become stably transfected, meaning that transfection with a polynucleotide produces permanent lines of cultured cells with a new gene inserted into their genome. Usually this is done by linking the desired gene with a "selectable marker", such as a selectable gene. This refers to a genetic element present in an expression vector, which, when expressed, provides an indication of successful transformation of the host cell.
• the selectable marker may provide the transformed host cell with resistance to an antibiotic (a dominant type marker) one or with the ability to metabolise a particular nutrient (an auxotrophic type of selectable marker, i.e. a marker that "cures" a deficiency in the host).
• an auxotrophic type of selectable marker i.e. a marker that "cures" a deficiency in the host.
• the selectable marker is under the control of a promoter that is separate from the promoter that controls expression of the gene to be expressed by the vector.
• An exemplary selectable marker can be a gene which confers resistance to an antibiotic (like Zeocin). Upon putting the antibiotic into the culture medium, only those cells which incorporate the resistance gene will survive, and essentially all of those will also have incorporated the DNA polynucleotide according to the invention.
• EXAMPLE 1 This Example describes the development of the ExpreS2+ cell line by cloning of S2 cells and subsequent picking of clones. The protocols described below were used.
• EX-CELL 420® (SIGMA, Cat. No. 14420) + added 10% FBS.
• Materials 96-well cell culture plates (Nunc), Centrifuge tubes (Greiner) 10 mL and 50 mL. Inspect and count S2 cells using a CASY cell counter (Roche). Comment the inspection and record cell number and viability.
• Preparation for incubation Incubate at ca. 25 °C until distinct clones are visible. Record start and end date. Renewal of medium: During the incubation period, examine the plates twice weekly. Add fresh medium when needed.
• EX-CELL 420® 12-well plates (Nunc). Cells to use: Use untransfected cells.
• Clone transfer Inspect the 96-well plate for clones and mark the lid to indicate in which wells clones have been identified. Transfer the cell suspension from the well to a well in a 12-well plate and add medium to a final volume of 1.5 ml (EX-CELL 420® + 10% FBS). Record date for inspection and clone transfer. Name the plates with a unique name, which should be the name of the plate that the clones are transferred from followed by a number.
• This example describes protocols used for expanding Drosophila S2 cells. Expansion of Drosophila S2 cells from 12-well plates to T25s
• Medium renewal Transfer cells from each well into individual T25s. Add 3 ml EX-CELL 420® + 10% FBS. Incubate at 25 degrees C until cells reach a density of 6E6-9E6 cells/ml. Record start and end time of incubation. Renew the medium every 3-4 days by centrifugation and resuspension in approximately 5 ml fresh medium. Record dates where medium has been replaced and record batch number of medium. Inspect and count the cells regularly by using the CASY instrument. Record date for inspection and cell counting, comment the inspection and record cell number and viability.
• the cell suspension ⁇ 25 ml, transfer to a R125. If the cell suspension is 25 ml - 50 ml, transfer to a R250. If the cell suspension > 50 ml, transfer up to 50 ml to a R250 and discard the rest. Record amount of cell suspension, what kind of shake flask used and time.
• This example describes a protocol for maintenance of S2 cells in T75 in serum-containing medium.
• This example describes initiation of ExpreS2+ cell culture from frozen stock followed by expansion in shake flasks with or without FBS (Fetal Bovine Serum).
• FBS Fetal Bovine Serum
• EX-CELL 420® SAFC cat.no. 14420; Fetal Bovine Serum, FBS: Life Technologies cat.no. 10099-158; Shake flasks, 250ml with 0.2 ⁇ vented cap: Corning cat.no. 431144 (Optimal working volume: 25-75ml); Shake flasks, 1000ml with 0.2 ⁇ vented cap: Corning cat.no. 431147; (Optimal working volume: 100-250ml); Tissue Culture Flasks, 25cm2 (T25) : Greiner cat.no. 690160 (Optimal working volume: 5-7ml); Tissue Culture Flasks, 75cm2 (T75) :
• Cells can also be routinely kept in a T75 (see protocol for maintenance of ExpreS2+ cultures in T- flasks, see Example 3. Cells can be cultured in shake flasks with or without FBS. Cells for transfection can routinely be kept in EX-CELL 420® + 10% FBS.
• EX-CELL 420® SAFC cat.no. 14420; Fetal Bovine Serum, FBS: Life Technologies cat.no. 10099-158; Tissue Culture Flasks, 25cm2 (T25) : Greiner cat.no. 690160 (Optimal working volume: 5-7ml); Tissue Culture Flasks, 75cm2 (T75) : Greiner cat.no. 658170 (Optimal working volume: 10-20ml).
• This example describes evaluation of S2 cell lines in shake flask culture. The following protocol was used to compare the respective growth rates and cell densities of ExpreS2+ and S2 cells (ATCC CRL-1963).
• ExpreS2+ and S2 cells were maintained in 250 ml shake flasks containing 50 ml EX-CELL 420®. They were seeded at 8E6 cells/ml. Every 3-4 days the cells were counted, centrifuged and re-suspended again to 8E6 cells/ml. This continued for 19 such passages. The results are shown in Table 5 and 6 below, and in Fig. 2.
• Table 6 shows average growth rates over the entire batch culture from day 0 to day 3 of S2 cells and ExpreS2+ cells. An average increase in average growth rates of ca. 30% was obtained for ExpreS2+ cells as compared to S2 cells. The growth rate was calculated as described above. For S2 the growth rate varied from 0.0153 to 0.0197 (h(-l) and for ExpreS2+ from 0.0197 to 0.0257 h(-l). The average growth rates were 0.0183 h(-l) and 0.0241 h(-l) for S2 and ExpreS2+, respectively.
• S2 cells grow from an initial concentration of about 8E6 cells per mL to on average about 30E6 cells per mL (ranging from 24E6 to 47E6 cells per mL), while
• ExpreS2+ grows from about 8E6 to on average about 45E6 cells per mL (ranging from 29E6 to 67E6 cells per mL.
• An average of 40% increase in final cell density of ExpreS2+ cells was obtained compared to S2 cells, using all data points. If considering only data from 3-day culture times, the increase in final cell density was 55% for ExpreS2+ as compared to S2 cells.

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