Mammalian cell lines addicted to a transcriptional state of high heterologous protein secretion

Abstract

The invention provides a mammalian production cell for synthesis of a heterologous product, further comprising a secreter-state-addiction genetic circuit whose expression confers a selective growth and / or survival advantage on those cells that synthesize and secrete the product or cells synthesizing and secreting products with desirable functional and structural characteristics (higher quality); while limiting proliferation of non / low-productive or low-quality productive escaper cells.

Description

[0001] TITLE: MAMMALIAN CELL LINES ADDICTED TO A TRANSCRIPTIONAL STATE OF HIGH HETEROLOGOUS PROTEIN SECRETION

[0002] FIELD OF THE INVENTION

[0003] The invention provides a mammalian production cell for synthesis of a heterologous product, futher comprising a genetic circuit whose activation confers a selective growth and / or survival advantage on those cells that synthesize and secrete the protein product or cells synthesizing products having the desired quality (i.e. desired structural and / or functional properties); while limiting proliferation of non- or low-productive mammalian production cells (such as genetic escaper cells or undesired variant cells).

[0004] BACKGROUND OF THE INVENTION

[0005] The biopharmaceutical industry manufactures biopharmaceutical proteins (biologies) with genetically engineered mammalian production cell lines e.g. Chinese hamster ovary (CHO), human and murine cell lines, which can synthesize and secrete pharmaceutically active, folded heterologous proteins with glycosylation profiles that support pharmacological application.

[0006] In the biopharmaceutical industry, secreted product purity is of importance, and is addressed during cell line development but also during production e.g. through a set of quality control tests including high-resolution liquid chromatography mass spectrometry (LC-MS). Host cell proteins (HCPs) that are synthesized and released from the cell may co-elute with the therapeutic protein product of interest, thereby becoming a specific concern, where current strategies of overcoming this involve the targeted deletion of genes encoding specific known HCPs.

[0007] Commercial production subsequently takes place preferably in batch or fed-batch suspension bioreactor cultures typically of up to 20,000 Liter volume, or, in smaller perfusion-based bioreactors. Here production usually takes place in durations ranging from 60 cell generations to more than 90 cell generations.

[0008] The development of stable, high-performing cell lines is a cornerstone of bioprocess optimization, particularly for recombinant protein production in CHO cells in which cell expansion in preparation for large-scale production is time-consuming, often resulting in titer decrease due to cell line instability. Despite the significant advantages of mammalian cell lines in general and CHO cell lines in particular as production cell lines, CHO cell lines are prone to high mutation rates. These include large chromosomal rearrangements due to the notoriously highly unstable genome(s) of the CHO cell lines (Lewis et al, 2013). The resulting high plasticity and aneuploidy of the CHO genome additionally complicates targeted gene insertions in which all alleles of a target gene must be edited to obtain a desired phenotypic change.

[0009] The secreted protein products are commonly encoded by transgenes usually under control of strong promoters such as CMV and EFla. However, in order to reach commercially relevant product titers, CHO production cell lines are usually developed using methods that amplify the number of transgene copies to levels of 10-100 copies in the chromosome. These random amplifications have traditionally used selectable markers such as glutamine synthetase (GS) or dihydrofolate reductase (DHFR) directly coupled to the transgene, allowing for the selection of clonal variants with an amplified transgene copy number. Usually, stability studies are required, in which candidate cell lines are serially passaged for durations of typically 60-90 generations of cell division, to experimentally assess stability for larger-scale production and a genetic profile inert to copy number loss, mutation and epigenetic silencing (e.g. histone acetylation). The random integration strategies are widely used due to high screening efficiency that generally allows the screening of several hundreds of clones for high production, typically in a deep-well cultivation system.

[0010] Heterologous proteins are synthesized and secreted through a range of integrated cellular activities starting with translation by ribosomes bound to the endoplasmic reticulum (ER) membrane. As the protein is translated, it is generally translocated into the ER lumen through the action of signal sequences at the protein's N-terminus. Within the ER lumen, the newly synthesized protein undergoes folding and post-translational modifications including glycosylation and disulfide bond formation. Chaperone proteins assist in proper folding, while quality control mechanisms ensure that only correctly folded proteins progress through the secretory pathway. Proteins destined for secretion are transported from the ER to the Golgi apparatus in vesicles. These vesicles bud off from the ER and fuse with the cis-Golgi network. Proteins sorted for secretion are packaged into secretory vesicles that bud off from the trans-Golgi network. These vesicles then traffic towards the plasma membrane. Upon reaching the plasma membrane, secretory vesicles undergo exocytosis, where they fuse with the plasma membrane and release their contents, including the heterologous proteins, into the extracellular space. The randomness of non-targeted transgene integration and amplification has been addressed by alternative approaches aiming to integrate transgenes into defined stable and transcriptionally active genomic loci e.g. in a process known as recombinase- mediated cassette exchange (RMCE) at pre-inserted landing pads. RMCE can result in cell lines stably expressing transgenes through 60-90 cell generations, yet the low achievable copy number of RMCE typically renders cell lines expressing a lower product titer.

[0011] Product heterogeneity has been detected from biologics-producing cell lines, which leads to low and high producing cells in a culture. Epigenetic silencing is usually stated to be a major source for this heterogeneity. Point-mutation (SNP) and copy number heterogeneity has however also been detected during construction of pharmaceutical cell lines, leading to amino acid substitution heterogeneity in fractions of the final product in test cell lines or declining productivity.

[0012] Due to the inherent plasticity of the genomes of cultured mammalian cells and in particular CHO cells, there is a need for stabilizing the production and secretion of product-resulting transgene expression over time. The present invention provides a solution to this problem.

[0013] SUMMARY OF THE INVENTION

[0014] Despite the high mutation rate in many mammalian production cell lines, cell variants producing and secreting less product, or secreting product variants having undesired quality (i.e. undesired structural and / or functional properties), are only significantly enriched in the production culture if allowed to grow faster than the high-secreting cell variants producing the desired product. We have found that such spontaneously formed lower-secreting, faster-growing cells exhibit a modified transcriptional signature compared to the higher-secreting, slower-growing cells (in a secreter state) that one can monitor and read-out in a mammalian cell through a native secreter-state responsive promoter.

[0015] A first aspect of the present invention provides a mammalian production cell genetically engineered to synthesize a product, wherein said cell further comprises:

[0016] (a) at least one essential gene operably linked to a secreter-state responsive promoter, wherein said secreter-state responsive promoter is heterologous with respect to said at least one essential gene; wherein production of the product confers a secreter state on said cell, wherein said secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said at least one essential gene is up-regulated when said secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said at least one essential gene when said secreter-state responsive promoter is not induced.

[0017] The mammalian production cell genetically engineered to synthesize a product may further comprise:

[0018] (b) a second essential gene operatively linked to a second secreter-state responsive promoter, wherein said second secreter-state responsive promoter is heterologous with respect to said second essential gene; wherein said second secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said second essential gene is up- regulated when said second secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said second essential gene when said second secreter-state responsive promoter is not induced.

[0019] A second aspect of the present invention provides a method of product biosynthesis comprising the steps of: i) providing the mammalian production cell according to the first aspect of the invention, ii) introducing the cell into a cultivation medium comprising substrate for production of said product, iii) cultivating said cell in said cultivation medium, iv) recovering said product, and v) optionally formulating said product.

[0020] A third aspect of the present invention provides for the use of at least one essential gene operably linked to a secreter-state responsive promoter to enhance product yield of a cultured population of mammalian production cells arising from a single cell, wherein said secreter-state responsive promoter is heterologous with respect to said at least one essential gene, wherein production of the product confers a secreter state on said cell, wherein said secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said at least one essential gene is up-regulated when said secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said at least one essential gene when said secreter-state responsive promoter is not induced.

[0021] A fourth aspect of the present invention provides for the use of the mammalian production cell according to the first aspect of the invention for producing a secreted heterologous protein product.

[0022] DESCRIPTION OF THE INVENTION

[0023] Brief description of the figures:

[0024] Figure 1: Titer of antibody secreting cell line CL003 (ancestor control) cultured in stability assay for about 45 cell divisions, and parallel pools from same ancestral cell line however having inserted secreter-state addiction based on pRAD51-Pah (i.e. cell lines CL102, WN486, and WN487) and cultured for about 75 cell divisions. Titers were measured on the third and fourth day of batch fermentation culture from frozen samples taken from the stability study.

[0025] Figure 2: Drawing showing a plasmid map of pCAS9 useful to express a programmable nuclease to direct targeted integration of a secreter-state responsive promoter-TIS into a mammalian cell genome to operably link to an essential gene.

[0026] Figure 3: Drawing of a plasmid map for pESP-RAD51-Pah useful to deliver the secreter- state promoter PRADSI to the Pah chromosomal essential gene through homologous recombination directed by the upstream and downstream homology arms, and isolated through fluorescent protein (by FACS enrichment) and / or neoR (by G418 selection).

[0027] Figure 4. Drawing showing a plasmid map of pSGRNA useful to express a guide RIMA to direct targeted cleavage with a programmable nuclease in order to insert a secreter- state responsive promoter-TIS into a mammalian cell genome with operably link to an essential gene.

[0028] Figure 5: Drawing of a plasmid map for pESP-MMP12-l_ASlL useful to deliver the secreter-state promoter PMMPI? to the Lasll chromosomal essential gene through homologous recombination directed by the upstream and downstream homology arms, and isolated through fluorescent protein (by FACS enrichment) and / or neoR (by G418 selection).

[0029] Figure 6: Drawing of a plasmid map for pESP-II-BCL21L-PAH useful to deliver the secreter-state promoter PBCL2IL to the Pah chromosomal essential gene through homologous recombination directed by the upstream and downstream homology arms, and isolated through fluorescent protein (by FACS enrichment) and / or HygR. (by hygromycin selection).

[0030] Figure 7: Specific antibody productivity of antibody secreting cell line CL003 (ancestor control) cultured for about 25 cell divisions, and same cell line with inserted secreter- state growth addiction WN258 (pMMP12-Lasll) cultured for about 50 cell divisions. Error bars depict standard error of the mean (n = 3).

[0031] Figure 8: Drawing of a plasmid map pESP-Empty_Vector useful to deliver the secreter- state promoters to the chromosomal essential gene when specific sequences for upstream and downstream homology arms and secreter-state responsive (SSR) promoters have been further inserted as indicated. Delivery is then performed through homologous recombination directed by the upstream (UP) and downstream (DW) homology arms (HA) and isolated through fluorescent protein (by FACS enrichment) and / or by utilizing the NeoR / KanR cassettes. Inner arrows indicate sequence insertion positions for HA-UP (1), specific secreter-state responsive promotor (SSR, 2) and HA- UP (3).

[0032] Figure 9: Antibody titer of single cell sorted cell lines from the antibody producing and secreting host cell line CL003 (CL003 CLD1-12) (n =12) and 15 different candidate SSA cell lines, single cell sorted from the same CL003 host cell line however having inserted secreter-state addiction chromosomally integrated based on various combinations of essential genes and promotors as listed in Table 7. Titers measured from a production model (Methods). Bars depict mean values and error bars depict standard error of the mean (n=2-5: CL303, CL304, CL387; n=6-10: CL303, CL311, CL362, CL400; n>10: all remaining SSA candidate cell lines and controls).

[0033] Figure 10: Titer of the antibody producing and secreting ancestral host CL003 control depicted against SSA candidate cell lines CL311, CL326, CL363, CL378, CL399, and CL400 single cell cloned from the same CL003 host cell line however having inserted secreter-state addiction chromosomally integrated based on the indicated various combinations of essential genes and secreter-state-responsive promotors (Table 7). All cultures were subjected to extended seed trains resulting in generations are depicted on the x-axis. Generation numbers were calculated from the CL003 antibody producing and secreting CHO host cell line thaw until production model seed, and calculations were based on each culture's growth rate during passage. Data points represent average titers from batch production models (Methods). Titers are means and error bars depict standard error of the mean, n=2-22 (n=22 for control CL003 cultures with a generation count of 9 and 22; n=2 for all remaining cultures).

[0034] Abbreviations, terms and definitions:

[0035] Secreter state refers to the cellular state of a mammalian cell (such as a cell genetically engineered to synthesize a product) during both efficient production and efficient secretion of a product under production conditions, and which results in a growth reduction attributable to the synthesis and secretion of said product, in particular in a cell that exhibits high level efficient synthesis of the product, compared to growth of a corresponding cell differing only in not producing said product. The secreter state can be monitored by detecting the direct and / or indirect response of native promoters in the cell to the production and secretion of the product. The secreter state results from the altered metabolic demand and adjusted resource allocation in such highly producing and secreting cells. The state is therefore characterized by a modified gene expression profile associated with a high intracellular target protein synthesis and secretion, as well as a relative growth reduction meaning that high-secreting cell lines grow at a relatively lower rate.

[0036] Growth reduction is a consequence of said secreter-state. It can be measured and quantified as a 'relative growth reduction' by measuring the percent-wise reduction in the specific growth rate of a mammalian production cell during synthesis of the product under production conditions as compared to a parent mammalian cell lacking at least one functional gene required for said synthesis when grown under comparable production conditions (wherein neither said mammalian production cell nor its parent comprises a secretor state promoter operably linked to an essential gene according to the present invention).

[0037] Secreter state responsive promoter refers to a promoter that is operably linked to an essential gene in a mammalian production cell, to form a genetic circuit. The secreter state promoter is induced directly or indirectly by said secreter state in said mammalian production cell. The secreter state promoter upregulates the expression of the essential gene in a mammalian production cell comprising the genetic circuit, when the production cell is cultured under production conditions as compared to a corresponding mammalian production cell differing only in either lacking the one or more genes encoding the protein product; or, and preferably, as compared to a mutant derivative of said mammalian production cell that synthesizes essentially none, or at least 50 % less, of the intended product of the production cell. Such mutant derivative includes a non / low-productive escape mutant isolated following long-term cultivation of a population of cells derived from said production cell. Accordingly, secreter-state responsive promoters are promoters that are upregulated intrinsically by the secreter state (quantified by a relative growth reduction) of cellular high product production and not extrinsic factors e.g. the environment of a batch, fed-batch or perfusion bioreactor. The secreter state responsive promoter is induced by the secreter state, thus not by the product per se. K secreter-state responsive promoter is a mammalian cell promoter or a synthetic promoter (such as a hybrid, scrambled or truncated version of such mammalian cell promoter) for said regulation of gene expression in a mammalian cell. A secreter state promoter, when operably linked to an essential gene in a secretor-state addicted mammalian production cell, is one that maintains a specific growth rate (doubling time) of the cells within a deviation of 15 % or less over the course of 50 or more cell divisions when cultured under production conditions. For example, said secreter state promoter is one that maintains a specific growth rate (doubling time) within a % deviation of no more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4 %, or less over the course of 50 cell divisions, under production conditions.

[0038] The term 'secreter state promoter' and 'secreter state responsive promoter' are used herein interchangeably.

[0039] Operably linked refers to the functional relationship between a promoter sequence and gene to which it is operably linked, whereby the promoter sequence is able to stimulate or modulate the transcription of the gene to which it is operably linked.

[0040] Secreter-state-addicted (SSA) mammalian cell refers to a mammalian cell engineered to comprise a genetic circuit comprising at least one 'secreter-state responsive promoter' operably linked to an essential gene of the cell, wherein said secreter-state responsive promoter is heterologous with respect to said essential gene. In one embodiment, said cell preferably has a genetic circuit comprising two, namely a first essential gene operably linked to a first secreter-state responsive promoter, and a second essential gene operably linked to a second secreter-state responsive promoter wherein said first secreter-state responsive promoter is heterologous with respect to said first essential gene and said second secreter-state responsive promoter is heterologous with respect to said second essential gene. A suitable secreter-state- addicted mammalian cell may however also comprise more than two different essential genes each controlled individually by at least two different secreter-state responsive promoters.

[0041] Secreter-state -addicted (SSA) mammalian production cell refers to a mammalian cell genetically engineered to synthesize a desired product, said cell being further genetically engineered to comprise a genetic circuit comprising said at least one 'secreter-state responsive promoter' operably linked to an 'essential gene' of the cell, as defined above. The desired product is one or several heterologous protein(s) (e.g. heavy and light chains of monoclonal antibodies) encoded by respective nucleic acid molecule(s) in said engineered mammalian cell. A secreter state-addicted mammalian production cell is one that under production conditions maintains a specific growth rate (doubling time) within a deviation of 15 % or less over the course of 50 or more cell divisions. For example, one that maintains a specific growth rate (doubling time) within a % deviation of no more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4 %, or less over the course of 60 cell divisions, under production conditions.

[0042] A secreter state-addicted mammalian production cell is one, wherein expression of the essential gene is up-regulated when the secreter-state responsive promoter is induced by the secreter-state under production conditions, relative to a basal level expression of the essential gene when the secreter state responsive promoter is not induced. The upregulation of the expression of the essential gene results in an increase in the specific growth rate of the secreter state-addicted mammalian production cell under production conditions, compared to a situation where the secreter state responsive promoter is not induced - i.e. where production and / or secretion of the product is reduced (e.g. measurable by reduced product titer). In other words, the genetic circuit of the present invention ensures that producing and secreting cells obtain a growth advantage over non- or low-producing cells.

[0043] Non-secreter-state addicted (non-SSA) mammalian production cell may be considered a "parent" cell of the secreter-state addicted mammalian production cell. It is a mammalian production cell comprising and expressing one or more genes encoding the protein product(s), but lacking the genetic circuit comprising said at least one secreter-state responsive promoters operably linked to an essential gene as disclosed in the present invention. The at least one essential gene in this non-secreter-state addicted mammalian production cell is operably linked to its native promoter.

[0044] Desired Products of the invention include - but are not limited to - heterologous (nonnative) products that, when synthesized by the genetically engineered mammalian production cell, results in a secreter state and confers a growth reduction upon the production cell as part of the secreter state, as compared to a parent or successor cell not producing said product. Non-limiting examples of such secreted product include a therapeutic protein and precursors thereof, such as human growth hormone, insulin, glucagon-like peptide-1, an antibody (such as a monoclonal or polyclonal antibody, bispecific antibody, a single-fragment antibody or nanobody), fusion protein, turoctocog alfa, antihemophilic factor (such as Factor VII, III and VIII), follitropin beta, etanercept, rituximab, adalimumab, bevacizumab, erythropoietin (EPO), infliximab, trastuzumab, daratumumab, ofatumumab, tocilizumab, growth differentiation factor 5, hepatitis B surface antigen, alpha-glucosidase, laronidase, interferon beta, Cl esterase inhibitor, tissue plasminogen activator, cameloid scFv / fragments. Some of these products may be expressed from several different coding nucleotide sequences. Additionally, some products may be subjected to a synthetic (chemical) modification process after production and harvest.

[0045] Quality of product according to the invention refers to the active form of the product (i.e. having the desired functional properties), its purity e.g. with regards to native host cell proteins (HCPs) and product-related impurities resulting from the host cell, and in certain embodiments it refers in particular to the profile of posttranslational modifications to the product such as a homogenous glycosylation profile with respect to the presence of N- and O-lined glycan structures (i.e. having the desired structural properties).

[0046] Landing pad according to the invention refers to a recombination-site-flanked temporary gene cassette in the genome, useful for introducing and substituting variants of secreter-state responsive promoters and translation rate regulating translation initiation sites [TISs] at a higher efficiency. Useful recombination sites can e.g. be variants of loxP for which recombination is mediated by the activity of a Cre recombinase, or FRT sites recombined by the activity of the Flp recombinase or att sites recombined by a Bxbl recombinase. The landing pad can be inserted using standard methods for targeted gene introduction such as CRISPR-based cleavage and homologous recombination guided by targeting homology arms of 1000-bp length upstream and downstream each, such sequence originating preferably directly from the cell line clone to be targeted (e.g. using PCR).

[0047] An essential gene refers to a gene in the mammalian production cell which, if down- regulated, leads to a reduction in the specific growth rate and / or maximum growth rate of the mammalian production cell under production conditions. In one embodiment, an essential gene is essential for growth irrespective of the nutrient composition of these production conditions, whereby sufficient expression of such essential gene to support cell growth is not dependent on the presence or absence of specific inhibitors or nutrients provided under the production conditions. In some embodiments, overexpression of the essential gene by up to 25, 50 or 100 % relative to wildtype does not lead to more than 3-10 % reduction in maximum specific growth rate. In some embodiments, synthetic downregulation of the essential gene using the secreter-state responsive promoter (defined as perturbation of expression level) by preferably >50 %, >75 %, >90 % does not trigger elevated levels of apoptosis or necrosis. The levels of apoptosis can e.g. be measured through various ELISAs e.g. such that are specific to caspases.

[0048] In some embodiments, a chosen essential gene is encoded on multiple chromosomes (non-haploid genes) of the cell genome; in which case, the same or heterologous secreter-state responsive promoters are operatively linked to one, several or all copies of said essential gene in the cell genome.

[0049] Non-conditionally essential gene refers to essential gene(s) that is essential for cell viability such that it is not possible to create viable cells with knock-outs of these genes under production conditions, such as Lasll, Atic, Pah, PccA, Eif2s2 and Ciaol . A secreter- state addiction system comprising said essential gene in mammalian production cells is "activated" by culturing the cells under production conditions, such that the level of expression of the non-conditionally essential gene is upregulated. A candidate non- conditionally essential gene can be experimentally verified as such by targeted deletion (e.g. using CRISPR / cas9 systems) and subsequent cultivation under known production conditions.

[0050] Conditionally essential gene refers to essential gene(s) that is conditionally essential for cell viability. A secreter state addiction system comprising said essential gene in mammalian production cells is "activated" by culturing the cells under production conditions that maintain conditional essentiality (e.g. being deficient for an essential nutrient), such that the level of expression of the conditionally essential gene is "upregulated". For example, the gene Glul encoding glutamine synthetase (GS) with enzyme classification number 6.3.1.2 is essential in environments deficient in L- glutamine. This gene provides an improved cell fitness in mammalian production cells grown under production conditions that are devoid of L-glutamine in a dosage-sensitive manner with regards to GS expression (e.g. through its gene copy number). Optionally, a specific inhibitor can be added to increase the requirement for the conditionally essential gene. In the case of GS, its inhibitor L-methionine sulfoximine (MSX) can be used. It is a requirement that the gene encoding GS is not being used to amplify transgene copy number in the mammalian production cell line. Another conditionally essential gene is L / mps encoding uridine monophosphate synthetase, essential in conditions devoid of L-uracil supply.

[0051] Essential gene basal expression level is the level of transcription of each 'essential gene' (as defined above) that is operably linked to a 'secreter-state responsive promoter' in a secreter-state-addicted mammalian production cell, when the 'secreter-state responsive promoter' is not induced. The secreter-state responsive promoter to which the essential gene is operably linked is heterologous with respect to said essential gene, in the sense that the promoter is not the native promoter of said essential gene (even though the promoter may be present in the same genome), and is thus not found operably linked to said essential gene in nature. Said basal (i.e. un-induced) level expression of an essential gene is a level sufficient to support growth of a cell under production conditions (or during a growth phase) at a level equal to or less than 10, 20, 50, 90 or 95% (for example from 0 - 5, 5- 10, 10 - 15, 15 - 20, 25 - 30, 30 - 40, 40 - 50, 50 - 75, or 75 - 90%) of the growth rate of a corresponding non-secreter-state addicted mammalian production cell wherein each said essential gene is operably linked to its native promoter. A lowered growth rate due to basal level essential gene expression in those cells where the secreter-state responsive promoter is not activated constitutes a selective disadvantage for non-productive cells, for example nonproductive mutants arising during product production.

[0052] Relative growth reduction is quantified by comparing the maximum growth rate of a non-secreter-state addicted mammalian production cell (i.e. lacking secreter-state responsive promoters operably linked to the at least one, or two essential genes), but comprising and expressing one or more genes encoding the heterologous product) relative to the maximum exponential growth rate of a parent mammalian cell either devoid or incapable of expressing gene(s) encoding the product, from which the mammalian production cell was derived when grown under comparable production conditions.

[0053] Production conditions refer to specific cultivation conditions chosen for production of the desired product, and can relate to the composition of the cultivation medium and / or other culture conditions (e.g. temperature, pH, stirring, aeration, induction, feeding of substrate etc.). The production conditions can therefore relate to both the early part of the manufacturing process where a cell is 'expanded' in numbers in seed trains to reach a desired volume, as well as what may be considered the final production phase (e.g. during feeding stage in fed-batch production). Propagation, perfusion, batch and fed- batch production may use various media and involve additions and control of carbon, pH, growth hormones, insulin, and other more complex growth and production promoting factors. The cells are preferably cultured in suspension (i.e. they are adapted for suspension culture), but adherent cell lines may also be used.

[0054] Translation initiation site (TIS), Ribosomal binding site (RBS), translation initiation region, Kozak, or translational strength element refer to the genetic region of the 5' untranslated region that control the translation strength of a particular messenger RNA. These regions can be genetically modified to yield a stronger or weaker rate of translation to protein.

[0055] Transgene describes the product coding gene(s) which typically encode the complete protein product or individual units of the protein product, all including relevant tags for secretion if needed.

[0056] Secretion relates to secretion of the target product from the intracellular environment to the medium. Intracellular production can be quantified by opening a cell pellet following centrifugation.

[0057] Growth rate refers to the specific growth rate of a cell culture.

[0058] Detailed description of the invention:

[0059] I. A secreter-state-addicted mammalian production cell

[0060] 1.1. A mammalian production cell genetically engineered to synthesize a product

[0061] A first aspect of the present invention provides a mammalian production cell genetically engineered to synthesize a product, wherein said cell further comprises: a) an essential gene operably linked to a secreter-state responsive promoter, wherein said secreter-state responsive promoter is heterologous with respect to said essential gene; wherein production of the product confers a secreter-state on said cell, wherein said secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said essential gene is up-regulated when said secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said essential gene when said secreter-state responsive promoter is not induced.

[0062] In a further aspect of the present invention, the mammalian production cell genetically engineered to synthesize a product comprises: a) a first essential gene operably linked to a first secreter-state responsive promoter, and b) a second essential gene operatively linked to a second secreter-state responsive promoter, wherein said first secreter-state responsive promoter is heterologous with respect to said first essential gene, and said second secreter-state responsive promoter is heterologous with respect to said second essential gene; wherein synthesis of the product confers a secreter-state on said cell, and wherein said first and second secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said first essential genes is up- regulated when said first secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said first essential gene when said first secreter-state responsive promoter is not induced, and expression of said second essential gene is up-regulated when said second secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said second essential gene when said second secreter-state responsive promoter is not induced.

[0063] In one embodiment, the first and second essential genes are different. In another embodiment, the first and second essential genes are identical. In a specific embodiment, the first and second essential genes are identical while the first and second secreter-state responsive promoters are different.

[0064] In one embodiment, the first and second secreter-state responsive promoters are different. In another embodiment, the first and second secreter-state responsive promoters are identical. In a specific embodiment, the first and second secreter-state responsive promoters are identical while the first and second essential genes are different.

[0065] In one embodiment, a secreter-state responsive promoter is inserted upstream of one allele of the target essential gene and at least one other allele of the target essential gene is not edited.

[0066] In one embodiment, a secreter-state addicted mammalian production cell is one that under production conditions maintains a specific growth rate (doubling time) within a deviation of 15 % or less over the course of 50 or more cell divisions. For example, one that maintains a specific growth rate (doubling time) within a % deviation of no more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4 %, or less over the course of 50 cell divisions, under production conditions.

[0067] In one embodiment, a secreter-state addicted mammalian production cell is cultured in suspension culture.

[0068] The secreter-state-addicted mammalian production cell according to the invention may be any mammalian cell. In one embodiment, the mammalian production cell of the invention belongs to, or is derived from, an industrially and regulatory well-known organism. A non-exhaustive list of suitable cell lines is given as follows: a cell line selected from among CHO (e.g. DG44, KI, S, XB11 ), mouse (Sp2 / 0), human embryonic kidney cells (HEK, e.g. HEK293), baby hamster kidney (BHK), mouse myeloma (NSO), insect cells (such as Spodoptera frugiperda Sf9 and Sf21 cell lines), chicken embryonic fibroblasts and human retinal cells.

[0069] The mammalian production cell genetically engineered to synthesize a product is one that comprises one or more genes encoding the product (or subunits of the product), wherein optionally said one or more genes are operably linked to a constitutive or inducible promoter. In one embodiment, the one or more genes encoding the product may be recombinant.

[0070] The product synthesis and secretion by the mammalian production cell of the invention is one that incurs a secreter state on the cell, reflected in a relative growth reduction, such product(s) include therapeutic proteins and their precursors, such as human growth hormone, insulin, glucagon-like peptide-1, an antibody (such as a monoclonal or polyclonal antibody, bispecific antibody, a single-fragment antibody or nanobody), turoctocog alfa, antihemophilic factor (such as Factor VII, III and VIII), follitropin beta, etanercept, rituximab, adalimumab, bevacizumab, erythropoietin (EPO), growth differentiation factor 5, Cl esterase inhibitor, tissue plasminogen activator, cameloid scFv fragments, preferably antibodies such as monoclonal and polyclonal antibodies, and single-fragment antibodies, and nanobodies. In one embodiment, the product is not a native product of the mammalian production cell of the invention, and thus not produced by a parent cell from which the mammalian production cell was derived. In one embodiment, the one or more genes encoding the product or encoding a metabolic pathway for synthesis of the product or parts of the product are heterologous with respect to the mammalian production cell of the invention; where said one or more genes may be transgenes.

[0071] The transgene(s) encoding the protein product can be expressed from strong constitutive and / or inducible promoters. Examples of suitable constitutive promoters driving transgene expression in CHO cells include a CMV promoter and EF-l-alpha promoter.

[0072] According to one embodiment, a mammalian cell not producing and secreting the product is first engineered to comprise a secreter-state addiction system as disclosed herein, which results in a > 5% reduced specific growth rate compared to its ancestor. Next, this cell is later genetically engineered for synthesis and secretion of a desired product, such as a heterologous protein product as disclosed herein.

[0073] According to one embodiment, said secreter state in said mammalian production cell of the invention, is quantified as a relative growth reduction by comparing the maximum growth rate of the mammalian production cell comprising one or more genes encoding the protein product(s) (but lacking secreter-state responsive promoters operably linked to the at least one essential gene), relative to the maximum growth rate of a parent mammalian cell lacking or incapable of producing and secreting one or more genes encoding the product or resulting as an escapee non-producing and non-secreting cell, and from which said mammalian production cell was derived, where the respective cells are cultured under essentially identical production conditions. The relative growth reduction of said mammalian production cell preferably corresponds to a percent reduction in the quantified maximum exponential growth rate selected from among > 5 %, >10%, >15%, >20%, >25%, >35%, and > 45 %.

[0074] In some embodiments the purity with regards to HCPs is affected by the invention. This may both relate to the presence of HCPs in early generations of culture of the cell bank and / or the stability of the profile of HCPs over time. Purity can be measured through mass spectrometry and through ELISA. In one embodiment, the production cell culture medium stably over at least 50 cell divisions comprises < 0.1%, <1%, <2%, <5%, or <20% HCPs relative to the total amount of product produced.

[0075] Due to the plasticity of the CHO genome sequence as well as in many general cancerous cell lines, nucleotide sequences and exon / intron junctions are typically putative. Therefore, the actual DNA sequence for the cell line of choice should be guided by PCR and possibly subsequent sequencing of genomic DNA purified from the target host cell line.

[0076] I. II. Essential genes of the secreter-state-addicted mammalian production cell

[0077] Non-limiting examples of CHO essential genes during production conditions include Glul, Lasll, Ciaol, LOC103163371, Eif2s2, Pcca, Pah, Limps, and Atic (see Table 1). An essential gene used in the context of this invention preferably does not encode a desired product to be expressed by the production cell line. In some embodiments, a chosen essential gene is not mitochondrial, and in some embodiments a suitable essential gene is encoded on multiple chromosomes (i.e. multiple alleles of non-haploid genes) of the cell genome; in which case, the same or heterologous secreter-state responsive promoters are operatively linked to one, several or all copies of said essential gene in the cell genome. Depending on the native expression level of the essential gene and the strength of the secreter-state responsive promoter, the secreter-state promoter need not be integrated on all copies of the allele as long as a relative growth reduction is observed upon down-regulation of said gene copy / copies. Hence, in one embodiment, a secreter-state responsive promoter is inserted upstream of one allele of the target essential gene and at least one other allele of the target essential gene is not edited.

[0078] A suitable essential gene can be found by insertion of an inducible promoter such as the TET ON promoter system (Gossen et al, 1995) directly upstream of the candidate essential gene and observing the growth-dependency for the inducer (e.g. doxycycline) in subsequent cultivation, while not observing pleiotropic metabolic effects compromising production of the target product. Candidate essential genes can also be found in CRISPR silencing screens where silencing of a candidate gene leads to loss of growth.

[0079] Table 1: Non-limiting examples of CHO essential genes and their murine homolog.

[0080] * https: / / www.ncbi.nlm.nih.gov

[0081] ** Essentiality conditional - requiring cultivation in absence of L-glutamine

[0082] *** Essentiality conditional - requiring cultivation in absence of uracil

[0083] I. Hi. Secreter-state responsive promoters of the secreter-state-addicted mammalian production cell Secreter-state responsive promoters are induced by the secreter state, not by the protein product per se. Suitable secreter-state responsive promoters that are induced by the secreter state in a mammalian production cell during production and secretion of a protein product can be selected from among the CHO genes and their cognate promoters disclosed in public databases known to the skilled person, for example chomine.com.

[0084] The secreter-state conferred on a mammalian production cell will depend on the product produced by the cell. Accordingly, the secretor state responsive promoter for a given (secretor-state addicted) mammalian production cell will be a promoter that is induced by the secreter-state of that specific cell. Hence transcript profiling provides a powerful tool to rapidly identify such secreter-state responsive promoter in the given production cell. Due to the increasing speed of cell line engineering, the direct introduction of candidate secreter-state promoters operably linked to an essential gene of choice in a mammalian production cell represents another efficient approach. Preferably a suitable secreter-state responsive promoter is one that maintains a specific growth rate (doubling time) of the mammalian production cells of the invention within a deviation of 15 % or less over the course of 50 or more cell divisions when cultured under production conditions. For example, said secreter state promoter is one that maintains a specific growth rate (doubling time) within a % deviation of no more than 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4 %, or less over the course of 50 cell divisions, under production conditions.

[0085] I.iv Matching a secreter-state responsive promoter to a mammalian production cell

[0086] Methods available for matching a secreter-state responsive promoter operably linked to an essential gene to a mammalian production cell in a product-specific manner, is experimentally fast and can be conducted in simple laboratory setups, as illustrated in the examples herein.

[0087] Furthermore, suitable secreter-state responsive promoters can be validated in a test assay in which a candidate secreter-state responsive promoter is operably linked to a reporter gene, e.g. encoding a fluorescent protein (e.g. green fluorescent protein) or luciferase and integrated into a mammalian production cell genetically engineered to synthesize a product, and integrated into a corresponding non-producing cell (control), which was isolated from the production cell following a serial dilution cultivation experiment in production medium for between 50-150 cell generations. Alternatively, the corresponding non-producing cell (control) can be a secreter-state-addicted mammalian cell (as defined herein) that lacks the gene(s) required to make the product. Secreter-state responsive promoters induce detectable fluorescent protein expression in producing cells compared to the non-producing cell (control) by at least 5 %, 7.5 %, 10 %, 15 %, 25 %, 60 %, 150 %, 300 %. Alternatively, instead of using a reporter gene, the transcript levels resulting from the candidate secreter-state responsive promoter can be validated using qPCR on the test and control cells. RNA sequencing can also be used to transcriptionally profile and identify suitable candidate secreter-state responsive promoters. In such tests, producing and corresponding (successor / ancestor) non / low-producing cell lines are cultured under identical conditions and the mRNA is extracted and sequenced. Suitable promoters are upregulated in the target producing cell line. Table 2 provides a non-limiting list of secreter-state responsive promoter candidates. Since many of the induced gene promoters will be related to, or correspond to those listed in Table 2, these provide a starting point for finding a matching secreter-state responsive promoter for a given mammalian production cell.

[0088] Table 2. Non-limiting examples of genes whose promoters can be useful to generate secreter state addicted mammalian cell lines.

[0089]

[0090] * A cognate promoter sequence of the indicated gene is found through its entry at https: / / www.ncbi.nlm.nih.gov / gene /

[0091] **https: / / www. kegg.jp / kegg /

[0092] Secreter-state responsive promoters may also be hybrids, scrambled or truncated versions of such natural mammalian cell promoters as long as such promoters still maintain a positive response to the secreter state. I. v Translation control elements

[0093] Regulating the growth of a mammalian production cell of the invention by means of essential gene expression, requires that the response threshold and curve of the secreter-state responsive promoter and the expression level of the essential gene are balanced; such that basal level essential gene expression only supports reduced growth, while in highly productive cells the induced secreter-state responsive promoter drives sufficient essential gene expression to support a significantly increased growth rate, preferably a growth rate similar to that of productive cells lacking the secreter-state responsive promoter of the invention or <5 % lower, when measured in the maximum growth phase.

[0094] One suitable approach to balancing the secreter-state responsive promoter's secreter- state-response to the expression level of the essential gene is to modify the translational strength of the essential gene. In mammalian cells TISs / Kozak elements can be used to modify translational strength. Examples of such elements in CHO conferring a broad range of translational strengths are provided in Table 3, while further examples can be found in the literature.

[0095] I. vi Engineering secreter-state responsive promoters and translational control element

[0096] K native secreter-state responsive promoter of choice is generally encompassed by the -1 to -1000 bp region upstream of the native regulated open reading frame (ORF) in mammalian organisms. In some embodiments the promoters of choice encompass the -1 to -1500 bp region upstream of the native regulated ORF. Core promoters that must be included and the sequence boundaries of their regulatory sites e.g. transcription factor binding sites, are common general knowledge. The translational control element / TIS can be added downstream of the promoter, to avoid alteration in the regulatory properties of the selected promoter sequence.

[0097] I.vii Improved production levels A surprising advantage of the secreter-state addicted mammalian production cell of the invention, in addition to having an increased initial growth rate as compared to non- secretory state addicted mammalian production cells, is that the cells retain significantly improved productivity during a large-scale fermentation (e.g. fed-batch or by perfusion), over many cell divisions, compared to mammalian production cells lacking engineered secreter-state-addiction. The surprising advantages conferred by secreter-state- addiction in a mammalian production cell of the invention are obtained irrespective of the type of protein product synthesized when such protein production confers a secretory state - as illustrated by the range of products synthesized by mammalian production cells and leading to a secretion state (e.g. in examples 1).

[0098] According to one embodiment, the mammalian production cell of the invention is characterized by improved product titer following at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 100, 150, 250, 300 or 400 generations of cell division from the start of culture, compared to a non-secreter-state-addicted production cell following the same generations of cell division. Product titer is measured as moles or grams of product produced per unit culture volume in the culture (i.e. secreted product). In one embodiment, production titers are increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% following at least 50 generations of cell division from single cell, compared to a reference non-secreter-state-addicted production cell following the same generations of cell division.

[0099] The number of generations of cell divisions are generally obtained during the cell line development and the culture expansion steps where the volumes and number of cells are increased (the seed train), while a few additional generations of cell division result during the final batch or fed-batch manufacturing process. Furthermore, continuous or semi-continuous manufacturing processes (e.g. by perfusion) additionally increase the number of cell generations.

[0100] Universal practice in the engineering of mammalian cell factories has aimed at minimizing the drawbacks of high secretion on cellular physiology and growth rate known to arise due to maintenance and expression of many product gene copies and / or aimed at randomly increasing transgene copy numbers through a directly coupled selection genes. The present invention provides a counter-intuitive solution to improving cell factory yields, since it makes mammalian cell growth and survival dependent on the cell producing and secreting its product while being subject to a constant secreter-state. While not wishing to be bound by theory, it is speculated that the selection pressure placed on a mammalian production cell of the invention and its descendants, where only productive cells in a secreter-state can grow / survive, may serve to progressively select for highly-secreter-stated cell sub-populations during long-term culture in a starting population of initially isogenic cells.

[0101] I. v / 77' Secreter-state responsive promoter and operatively linked essential gene combinations in the secreter-state-addicted mammalian production cell.

[0102] Suitable combinations of secreter-state responsive promoter and operatively linked essential gene in the secreter-state-addicted mammalian production cell are described below, with reference to Tables 1 and 2:

[0103] In one embodiment, an essential gene encoding a glutamine synthetase is operatively linked to a secreter state responsive promoter selected from any one of SEQ ID NO. 12- 79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes glutamatine synthetase as defined in SEQ ID NO. 2.

[0104] In one embodiment, an essential gene encoding a LAS1 like ribosome biogenesis factor is operatively linked to a secreter state responsive promoter selected from any one of SEQ ID NO. 12-79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes the LAS1 like ribosome biogenesis factor as defined in SEQ ID NO. 4.

[0105] In one embodiment, an essential gene encoding a cytosolic iron-sulfur protein is operatively linked to a secreter state responsive promoter selected from any one of SEQ ID NO. 12-79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes the cytosolic iron-sulfur protein as defined in SEQ ID NO. 5.

[0106] In one embodiment, an essential gene encoding an acetyl-CoA acetyltransferase is operatively linked to a secreter state responsive promoter selected from any one of SEQ ID NO. 12-79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes the acetyl-CoA acetyltransferase as defined in SEQ ID NO. 6.

[0107] In one embodiment, an essential gene encoding a eukaryotic translation initiation factor is operatively linked to a secreter state responsive promoter selected from any one of SEQ ID NO. 12-79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes the eukaryotic translation initiation factor as defined in SEQ ID NO. 7.

[0108] In one embodiment, an essential gene encoding a propionyl-CoA carboxylase is operatively linked to a secreter state responsive promoter selected from: any one of SEQ ID NO. 12-79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes the propionyl-CoA carboxylase as defined in SEQ ID NO. 8.

[0109] In one embodiment, an essential gene encoding a phenylalanine-4-hydroxylase is operatively linked to a secreter state responsive promoter selected from any one of SEQ ID NO. 12-79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes the phenylalanine-4-hydroxylase as defined in SEQ ID NO. 9.

[0110] In one embodiment, an essential gene encoding a uridine monophosphate synthetase is operatively linked to a secreter state responsive promoter selected from any one of SEQ ID NO. 12-79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes the uridine monophosphate synthetase as defined in SEQ ID NO.

[0111] 10.

[0112] In one embodiment, an essential gene encoding a 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase / IMP cyclohydrolase is operatively linked to a secreter state responsive promoter selected from any one of SEQ ID NO. 12-79. In one such embodiment, where the mammalian cell is a CHO cell, the essential gene encodes the 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase / IMP cyclohydrolase as defined in SEQ ID NO. 11.

[0113] 11. Methods for preparing and identifying cells of a secreter-state-addicted mammalian production cell line

[0114] In a preferred method of performing the invention, a secreter-state-addicted mammalian production cell line is prepared and identified by the following steps. To obtain the best working relation between secreter-state responsive promoter and essential gene, it may be relevant to prepare and test different candidate secreter-state responsive promoters with different TISs for regulating a selected essential gene.

[0115] Candidate secreter-state responsive promoters listed in Table 2 may be tested. Alternatively, unique, positively differentiating gene transcripts detected in a specific production cell line of interest may be identified; whose respective promoters provide a source of candidate secreter-state responsive promoters. Preferably such differentiating gene transcripts are identified by comparing the transcript profile of a mammalian production cell line (during production) with an isolated genetic or non-genetic escape mutant variant(s) derived from the corresponding production cell line, where such escape mutant variant is characterized by a 50 % lower production rate or more (e.g. 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% lower production rate, i.e. low- or nonproducing). Titer measures may equally be used to characterize the escape mutant - i.e. the escape mutant variant may be characterized by a 50 % lower titer or more (e.g. 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% lower titer, i.e. low- or non-producing). Positively differentiating gene transcripts can be identified by use of transcriptomics; for example by RIMA sequencing of the transcribed RNA extracted from productive mammalian production cell(s) compared to the non / low-producing escape mutant variant. The respective promoters of the identified positively differentiating gene transcripts can be tested in combination with a given essential gene in a mammalian production cell. Preferably the expression level of the identified positively differentiating gene transcripts in the mammalian production cell line (during production) is increased by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 fold, preferably by between 1.3-3 fold, when compared to the isolated genetic or non-genetic escape mutant variant(s).

[0116] A method for identifying one or more secreter-state responsive promoters is illustrated in example 3; where after said promoter(s) is operably linked to an essential gene in a chosen mammalian production cell for testing.

[0117] II. ii Introduction of secreter-state responsive promoter(s) in growth-regulating processes

[0118] One or more promoter(s) selected from the list of general candidate secreter-state responsive promoters (Table 2) or from identified specific secreter-state responsive promoter(s) (see II. i), are tested by operably linking it to an essential gene in the mammalian production cell. Different translation strengths of the essential gene can be simultaneously tested by providing alternative TIS sequences for the cognate essential gene; as well as testing different essential genes (as section II. i). The essential gene may be native or heterologous with respect to the mammalian production cell.

[0119] When the essential gene is a native gene, its cognate native promoter may be disrupted (i.e. made non-functional) by e.g. mutation or deletion events, and the secreter-state responsive promoter (that is heterologous with respect to the essential gene) is then operably linked to the native essential gene by targeted introduction. In another embodiment, the native essential gene promoter is replaced with the secreter-state responsive promoter by targeted introduction.

[0120] Suitable methods for targeted introduction of a genetic sequence in mammalian cells include homologous recombination in combination with a programmable nuclease such as CRISPR / cas9 or MAD7 to increase gene replacement efficiency. Alternative programmable nucleases well known for CHO include Cas-CLOVER and CRISPR / casl2a (Cpfl). To further increase efficiency of testing candidate promoters, a landing pad strategy using a recombinase such as Cre can be leveraged at a target essential gene. Homologous recombination is stimulated through routine use of 750-1250 bp long homologous flanking regions, carrying the nucleotide sequences to which the DNA is targeted. To account for cell line differences to the sequenced reference, genomic DNA is preferably amplified from the target cell line.

[0121] II. Hi Screening for balanced secreter-state and growth-regulation

[0122] Mammalian secreter-state addicted production cell clones comprising an inserted secreter-state responsive promoter operatively linked to an essential gene are then screened to identify clones where the secreter-state responsive promoter regulates an essential gene in a growth-controlling manner. For example, the growth of a number of such clones (e.g. 8-96 clones) is compared with cells of a corresponding non-secreter- state-addicted parental mammalian production cell line, under production conditions (e.g. using a parent mammalian production cell lines where the transgene(s) encoding the product are induced).

[0123] When cell growth is measured under conditions where product production by both cell lines is low / absent; a suitable secreter-state-addicted production clone is one that exhibits a significant, and preferably at least 5 %, lower growth rate than the non- secreter-state-addicted production cell line.

[0124] II. iv Validation of non-perturbed central and production metabolism

[0125] Changes in transcriptional regulation or expression of essential genes can lead to unwanted, indirect perturbation of the production genes and central carbon or nitrogen metabolism, compromising product formation and in turn reduced secreter-state in a secreter-state-addicted mammalian production cell and its progeny cell line. In order to exclude such cell lines, cell growth is measured under conditions where both cell lines synthesize product; where a suitable secreter-state-addicted production cell line is one that exhibits a growth rate equal to or lower than the non-secreter-state-addicted production cell line.

[0126] II. v Screening for growth -rate stability

[0127] Cell lines that fulfil the above criteria are tested for growth rate stability under production-mimicking conditions by cultivation for at least 20 generations of cell division, e.g. by serial passage and measurement of growth rates, or by taking samples from various steps in a scaled-up production process. Suitable secreter-state-addicted mammalian production cells maintain growth rates lower than the non-secreter-state- addicted production cell line over time. When the growth rate is measured as a function of generations of cell divisions (such as over 50 generations), the growth rate of the secreter-state-addicted production cell over time will remain within + / - 15% (such as + / - 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or 4%, more preferably within + / - 10%) of the starting point of culture, while the growth rate of the non-secreter-state-addiction production cell will increase more than the growth rate of the secreter-state-addicted production cell, such as increase by more than 15 % (or such as more than 10, 11, 12, 13, or 14%, or preferably more than 15%), from the starting point of culture. in. Methods for producing a desired product using cells of a secreter-state- addicted mammalian production cell line

[0128] A second aspect of the present invention concerns a method for producing a desired product

[0129] In one embodiment, the method of the present invention for producing a desired product comprises the steps of: a. providing a mammalian production cell genetically engineered to synthesize a product, wherein said cell further comprises an essential gene operably linked to a secreter-state responsive promoter, and wherein said secreter-state responsive promoter is heterologous with respect to said essential gene, b. introducing the genetically modified mammalian production cell into a cultivation medium comprising a substrate for production of said product, c. recovering said product synthesized by said culture of production cells in step b, and d. optionally formulating said product, wherein synthesis of the product confers a secreter-state on said cell, wherein said secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said essential gene is up-regulated relative to basal level expression of said essential gene when said secreter-state responsive promoter is induced by said secreter-state; and wherein a lack of said product synthesis in said secreter-state-addicted production cell line or progeny cell thereof reduces growth rate of said cell. In one embodiment, the method of the present invention for producing a desired product comprises the steps of: al. providing a mammalian production cell genetically engineered to synthesize and secrete a product, a2. further genetically engineer the cell to comprise an essential gene operably linked to a secreter-state responsive promoter, wherein said secreter-state responsive promoter is heterologous with respect to said essential gene, b. introducing this genetically modified mammalian production cell obtained from steps al and a2 into a cultivation medium comprising a substrate for production of said product, c. recovering said product synthesized by said culture of production cells in step b, and d. optionally formulating said product, wherein synthesis of the product confers a secreter-state on said cell, wherein said secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said essential gene is up-regulated relative to basal level expression of said essential gene when said secreter-state responsive promoter is induced by said secreter-state; and wherein a lack of said product synthesis in said secreter-state-addicted production cell line or progeny cell thereof reduces growth rate of said cell.

[0130] In one embodiment, the method of the present invention for producing a desired product comprises the steps of: al. providing a genetically engineered mammalian cell (secreter-sate addicted mammalian cell) comprising an essential gene operably linked to a secreter-state responsive promoter, wherein said secreter-state responsive promoter is heterologous with respect to said essential gene, a2. further genetically engineer the cell to synthesize and secrete a product, b. introducing this genetically modified mammalian production cell obtained from steps al and a2 into a cultivation medium comprising a substrate for production of said product, c. recovering said product synthesized by said culture of production cells in step b, and d. optionally formulating said product, wherein synthesis of the product confers a secreter-state on said cell, wherein said secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said essential gene is up-regulated relative to basal level expression of said essential gene when said secreter-state responsive promoter is induced by said secreter-state; and wherein a lack of said product synthesis in said secreter-state-addicted production cell line or progeny cell thereof reduces growth rate of said cell.

[0131] As the protein product is a secreted protein product, step c of the above disclosed embodiments of recovering said product is performed without opening the cells - i.e. without actively opening the cells.

[0132] A third aspect of the present invention concerns a method for producing a desired product comprising the steps of: a. providing a mammalian production cell genetically engineered to synthesize a product, wherein said cell further comprises a first essential gene operably linked to a first secreter-state responsive promoter, and a second essential gene operatively linked to a second secreter-state responsive promoter, wherein said first secreter-state responsive promoter is heterologous with respect to said first essential gene, and said second secreter-state responsive promoter is heterologous with respect to said second essential gene; b. introducing the genetically modified mammalian production cell into a cultivation medium comprising a substrate for production of said product, c. recovering said product synthesized by said culture of production cells in step b, and d. optionally formulating said product, wherein synthesis of the product confers a secreter-state on said cell, wherein said first and second secreter-state responsive promoter are induced by said secreter-state, and wherein expression of said first essential genes is up-regulated when said first secreter-state responsive promoter is induced by said secreter- state relative to a basal level expression of said first essential gene when said first secreter-state responsive promoter is not induced, and expression of said second essential genes is up-regulated when said second secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said second essential gene when said second secreter-state responsive promoter is not induced.

[0133] As the protein product is a secreted protein product, step c of recovering said product is performed without opening the cells - i.e. without actively opening the cells.

[0134] IV. Use of cells of a secreter-state-addicted mammalian production cell line for producing a desired product

[0135] A fourth aspect of the present invention concerns the use of a secreter-state-addicted mammalian production cell of the invention for producing a desired product, wherein a lack of product synthesis in said secreter-state-addicted production cell line or progeny cell thereof reduces growth rate of said secreter-state-addicted production cell line.

[0136] V. Use of cells of a secreter-state-addicted mammalian production cell line for producing a product with higher glycosylation quality

[0137] A fifth aspect of the present invention concerns the use of a secreter-state-addicted mammalian production cell of the invention for producing a desired product, wherein a lack of efficient N-linked glycosylation in said secreter-state-addicted production cell line or progeny cell thereof reduces growth rate of said secreter-state-addicted production cell line.

[0138] NUMBERED EMBODIMENTS OF THE INVENTION

[0139] Numbered embodiment 1: A mammalian production cell genetically engineered to synthesize and secrete a product, said mammalian cell further comprising: a. a first essential gene operably linked to a first secreter-state responsive promoter, wherein said first promoter is heterologous with respect to said first essential gene; and wherein synthesis of the product confers a secreter state on said cell, and wherein expression of said first essential gene is up-regulated when said secreter-state responsive promoter is induced by said secreter- state relative to a basal level expression of said first essential gene when said secreter-state responsive promoter is not induced.

[0140] Numbered embodiment 2: The mammalian production cell according to Numbered embodiment 1, wherein said cell further comprises: b. a second essential gene operatively linked to a second secreter- state responsive promoter, wherein said second secreter-state responsive promoter is heterologous with respect to said second essential gene; and wherein expression of said second essential gene is up-regulated when said second secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said second essential gene when said second secreter-state responsive promoter is not induced.

[0141] Numbered embodiment 3: The mammalian production cell according to Numbered embodiment 1 or 2, wherein said cell comprises c. one or more genes encoding the product, wherein said one or more genes are operably linked to a promoter.

[0142] Numbered embodiment 4: The mammalian production cell according to any one of Numbered embodiments 1 to 3, wherein said first secreter state promoter operably linked to an essential gene in said secretor-state addicted mammalian production cell, is one that maintains a specific growth rate (doubling time) of said cells within a deviation of 15 % or less over the course of 50 or more cell divisions when cultured under production conditions.

[0143] Numbered embodiment 5: The mammalian production cell according to any one of Numbered embodiments 1 to 4, wherein said secreter state conferred by synthesis and secretion of said product, when each of said first and / or second essential gene in the cell is operably linked to its native promoter, results in a relative growth reduction measured as a percent reduction in the exponential phase growth rate of the mammalian production cell selected from among > 5 %, >10%, >15%, >20%, >25%, >35% and > 45 % relative to a corresponding parent or derivative non-producing mammalian cell producing at least 50 % less product. Numbered embodiment 6: The mammalian production cell according to any one of Numbered embodiments 1 to 5, wherein the cell is a Chinese Hamster Ovary (CHO) cell.

[0144] Numbered embodiment 7: The mammalian production cell according to any one of Numbered embodiments 1 to 6, wherein the first and / or second essential gene is a non-conditional essential gene.

[0145] Numbered embodiment 8: The mammalian production cell according to any one of Numbered embodiments 1 to 7, wherein the cell is a CHO cell and the first and / or second essential gene encodes a protein sequence selected from SEQ ID NO. 2, 4, 5, 6, 7, 8, 9, 10, and 11, or a homologue thereof, preferably SEQ ID NO. 9 or a homologue thereof.

[0146] Numbered embodiment 9: The mammalian production cell according to any one of Numbered embodiments 1 to 8, wherein at least one of the first and / or second essential gene is operably linked to a synthetic transcription initiation site whose sequence is selected to modify the translational strength of the essential gene independent of induction of said secreter-state responsive promoter.

[0147] Numbered embodiment 10: The mammalian production cell according to any one of Numbered embodiments 1 to 9, wherein at least one essential gene allele is operably linked to said secreter-state-responsive promoter and at least one other essential gene allele remains operably linked to its native promoter.

[0148] Numbered embodiment 11: The mammalian production cell according to any one of Numbered embodiments 1 to 10, wherein the cell is characterized by an increased product yield after at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 generations of cell division from first culture, as compared to a parent mammalian production cell lacking said first and / or second essential gene operably linked to a secreter-state responsive promoter.

[0149] Numbered embodiment 12: The mammalian production cell according to any one of Numbered embodiments 1 to 11, wherein the cell is characterized by an increased product yield of at least 10, 25, 50, or 80% following at least 50 generations of cell division from a single cell as compared to a parent mammalian production cell lacking said first and / or second essential gene operably linked to a secreter-state responsive promoter.

[0150] Numbered embodiment 13: A method of product biosynthesis comprising the steps of: b. providing a mammalian production cell according to any one of Numbered embodiments 1 to 12, c. introducing the cell into a cultivation medium comprising substrate for production of said product, d. cultivating said cell in said cultivation medium, e. recovering said product from the cultivation medium without opening the cells, f. optionally chemically modifying said product, and g. optionally formulating said product.

[0151] Numbered embodiment 14: A method of product biosynthesis according to Numbered embodiment 13, wherein the product is selected from among a: peptide, enzyme, therapeutic protein and / or precursor thereof, human growth hormone, insulin, glucagon-like peptide-1, monoclonal antibody, polyclonal antibody, and singlefragment antibody.

[0152] Numbered embodiment 15: Use of at least one essential gene operably linked to a secreter-state responsive promoter to enhance product yield of a cultured population of mammalian production cells, wherein said promoter is heterologous with respect to said essential gene, wherein production of the product confers a secreter state on said cell, and wherein expression of said essential gene is up-regulated when said secreter- state responsive promoter is induced by said secreter state relative to a basal level expression of said essential gene when said secreter-state responsive promoter is not induced.

[0153] Numbered embodiment 16: Use of a mammalian production cell according to any one of Numbered embodiments 1 to 12 for producing a secreted heterologous protein product.

[0154] Numbered embodiment 17: Use of a mammalian production cell according to Numbered embodiment 16, wherein the product is selected from among a monoclonal or polyclonal antibody, and a single-fragment antibody.

[0155] Numbered embodiment 18: Use of a mammalian production cell according to Numbered embodiment 16 or 17, to enhance the degree of desired N-linked glycosylation of said product. EXAMPLES

[0156] EXAMPLE 1: Integration of secreter-state addiction and improvement of productivity and secretion

[0157] 1.1 Background of experiment

[0158] In this experiment, the native promoter of an essential gene Pah in a CHO antibody production cell line was replaced with candidate secreter-state promoter PRADSI by targeted insertion of an integration fragment featuring said promoter, and a fluorescent protein marker and antibiotic selection gene to isolate the cells (facilitated by integration fragment donor pESP-pRAD51-Pah). The RAD51 promoter (SEQ ID NO. 47) was chosen amongst candidate secreter state responsive promoters as it was found in an RNA-seq study to be upregulated by at least 1.5 fold under production conditions in an antibody high-producing cell line compared an antibody low-producing variant of such producer cell line.

[0159] Targeted gene introduction was mediated by CRISPR / Cas9 cleaving the targeted native promoter, facilitating efficient integration by flanking upstream and downstream homology arms (each 750 bp) directing targeted introduction of insert promoter / TIS- variable construct. Transfection was followed by pool sorting of correct integrants by fluorescence activated cell sorting (FACS) and could include an enrichment step in antibiotic selection.

[0160] 1.2 Materials and methods

[0161] Introduction of secreter-state responsive promoters in CHO production cell line through targeted integration:

[0162] The antibody-expressing CHO KI cell line was prepared for transfection in shake flask cultivation using the chemically-defined PowerCHO-2 CD (Lonza) according to standard procedures (Sergeeva, 2020).

[0163] To replace the native promoter of the targeted essential gene with the insert candidate secreter-state responsive promoter, DNA for expression of Cas9, sgRNA and integration fragment (Table 4) was mixed equimolarly for a total amount of 1 pg DNA. Freestyle MAX reagent (Thermo Fisher Scientific) was used as transfection agent and standard procedure was followed except for 105cells were used, washed with 0.5 mL CD CHO medium (Thermo Fisher Scientific) and cultivated in 1 mL CD CHO medium in 12-well plates for two hours before transfection. 2.7 pl Freestyle MAX reagent was used and total volume of the transfection mix was 50 pL. One hour later, the transfected cells were diluted with PowerCHO-2 CD to 3xl05cells / mL. Following 3 days of incubation at 37 °C, 5 % CO2, 120-rpm horizontal orbital shaking, an optional step of antibiotic selection (using G418) was initiated according to standard procedures (Sergeeva, 2020) based on repeated 3-4 days cultivations in selective medium of increasing concentration up to 4 pg / ml. Next, correctly targeted integrants were pool sorted through fluorescent protein expression (e.g. green fluorescent protein and red fluorescent protein) using a FACS according to standard and previously published methods but using PowerCHO-2 CD as cultivation media (e.g. Sergeeva, 2020). Finally, site-specific integration in clones was validated using PCR for at least one allele of the target site, and correct clones were expanded using previously published methods (e.g. Sergeeva, 2020).

[0164] Screening for improved short-term and long-term production: 105cells were thawed in 3 mL PowerCHO-2 CD medium in 6-well plates at 37 °C, 5 % CO2, 120-rpm horizontal orbital shaking. The cells were passaged to 2x 105cells / mL two times a week for a period of up to three months. Cells were cryopreserved every month: 1x10® cells were harvested, centrifuged at 200g and resuspended in fresh media with 10% DMSO. At select times, the selected samples were thawed and compared in batch cultivation based on PowerCHO-2 CD medium.

[0165] Small-scale batch fermentation: For batch fermentation, triplicates of each generated cell pool were seeded at 3 105VCD in 3 ml PowerCHO-2 CD supplemented with 2% Glutamine Synthetase Expression Medium (Sigma-Aldrich) cultivated in 6-well plates (Sartorius), shaken at 120 RPM with 1,9 cm orbit, at 37°C in a humidified incubator with 5% CO2. A 6-well plate filled with 3 ml PBS in each well was placed on top of every plate stack. Viability and VCD were monitored from day 3 - 7 using an NC-202 with Via2- Cassettes. Titer samples were taken between day 3 - 7, which were centrifuged at 200 G and supernatant stored at -20°C. Cultures were discontinued after sampling on day 7.

[0166] Titer measurement: Frozen titer samples were thawed along with an IgG standard (Sigma-Aldrich). A standard row was made between 400 and 0 pg / ml, and titer samples were diluted up to 4x. FQuant Titer (Cytena) plate was used to measure titer, following the manufacturer's protocol, on a SpectraMax ID5 (Molecular Devices), with the following modifications: Excitation was set to 640 nm and emission to 680. The FQuant plate was shaken on a ThermoMixer FP (Eppendorf) at 1800 RPM for 15 minutes and 5 minutes without shaking before measuring.

[0167] 1.3 Results

[0168] The batch cultivations of frozen cell samples from the stability assay showed that the secreter-state-addicted cell lines produced and secreted the heterologous antibody at a higher titer after 75 cell generations than the ancestral control cell line did after 45 cell generations in culture (Figure 1).

[0169] As presented in Figure 1, the specific productivity of the antibody secreting cell line was increased compared to the ancestor control when cultured in a batch production setup.

[0170] In summary, secreter-state-addicted transgene expressing CHO cell lines improve transgene expression with regards to titer, rate and / or carbon yield over time.

[0171] EXAMPLE 2: Improvement of CHO long-term production by introduction of secreter-state-responsive growth control using two essential genes

[0172] 2.1 Background of experiment

[0173] In this experiment, two essential genes are targeted for control by secreter state promoters.

[0174] 2.2 Materials and methods

[0175] First, the wildtype promoter of essential gene Lasll in a CHO antibody production cell line is replaced with candidate secreter-state promoter pMMP12 (SEQ ID NO. 39) by targeted insertion of an integration fragment featuring said promoter, a fluorescent protein marker and antibiotic selection gene, facilitated by integration fragment donor plasmid pESP-pMMP12-Lasll, see Table 5. Targeted gene introduction is mediated by CRISPR / Cas9 cleaving the targeted native promoter, as previously disclosed in example 1, facilitating efficient integration by flanking upstream and downstream homology arms (each 750 bp) directing targeted introduction insert promoter / TIS-variable construct. Transfection is followed by pool sorting of correct integrants by fluorescence activated cell sorting (FACS) optionally including an enrichment step in antibiotic selection. Next, this cell line can be used for the integration of a second secreter-state responsive promoter BCL21) operably linked to a second essential gene (Pah), using integration fragment donor plasmid pESP-II-pBcl2ll-Pah and guide-RNA plasmids pSGRNA-Pah, see Table 5. The integration fragment donor plasmid provides the secreter-state responsive promoter pBcl2ll for replacement of the wildtype promoter of essential gene Pah. Correct transfectants are identified as described in example 1 except that a hygromycin antibiotic and blue fluorescent protein excitation / emission setting is used suitable for the different integration vector. 2.3 Results

[0176] The batch cultivations of samples from the stability assay will show that the double secreter-state-addicted cell lines expressed their transgene over 50, 90 or 120 cell generations at a higher titer and / or at a higher carbon yield and / or at a higher volumetric rate.

[0177] In summary, double secreter-state-addicted transgene expressing CHO cell lines improve transgene expression with regards to titer, rate and / or carbon yield over time.

[0178] EXAMPLE 3: Identification of suitable secreter-state responsive promoter candidates

[0179] Different engineered production cell lines elicit different transcriptional responses indicative of the production process. In order to identify suitable secreter-state responsive promoter candidates, related cell lines with different degrees of target protein secretion are analysed in a time-course RNA-seq study.

[0180] 3.1 Methods

[0181] Typical genetic escaper cells are isolated from long-term cultivation with the genetically engineered mammalian production cells of interest in the intended cultivation medium. Suitable genetic escaper cells are characterized by having an at least 5 % higher exponential-phase growth rate and at least 30 % lower production rate or product titer (by secretion) than the original genetically engineered production cell.

[0182] Production cells and corresponding escaper cells are cultured under intended fermentation conditions, scaled down conditions or shake flask conditions mimicking the intended fermentation conditions. At time points across the fermentation, incl. such corresponding to the highest rate of production in the production cells, samples are taken for mRNA sequencing. Total RNA is purified using Purelink RNA Mini kit (Thermo Fischer) and prepared using TruSeq Stranded mRNA kit (Illumina) following the kit manufacturer's instructions. Reads are mapped and analyzed to the reference genome of the strain and next analyzed for differential expression between the production cells and corresponding non-secreting and / or lower-secreting (successor / ancestor) cells and over cultivation time. Promoters are chosen from genes with a stable or increasing upregulation in the high-secreting cell line over time.

[0183] 3.2 Results

[0184] Candidate suitable secreter-state responsive promoters are identified as those driving expression of genes that show a differential expression of >1.3 to 3 fold higher expression in the production cell relative to at least one isolated / comparable genetic escape cell line.

[0185] Example 4: Identification of secreter-state addicted cell populations with improved profile of HCPs

[0186] Production cell lines with stable profile of HCPs in culture are engineered by addicting the production cell lines to the secreter-state identified by RNA-seq analysis and reported by the RAD51 promoter (Table 2, SEQ ID NO. 47) operably linked to the Pah essential gene (Table 1).

[0187] Results: Following implementation of secreter state addiction, the profile of HCPs is monitored with mass spectrometry or ELISA specific to CHO HCPs in a stability study. The results will show a more stable profile compared to the ancestral control devoid of the invention.

[0188] EXAMPLE 5: Integration of secreter state addiction

[0189] 5.1 Background of experiment

[0190] In this experiment, the native promoter of an essential gene Lasli in a CHO antibody production cell line was replaced with candidate secreter-state promoter PMMPI? (SEQ ID NO. 39) by targeted insertion of an integration fragment featuring said promoter, and a fluorescent protein marker and antibiotic selection gene to isolate the cells. Targeted gene introduction was mediated by CRISPR / Cas9 cleaving the targeted native promoter, as disclosed previously in example 1, facilitating efficient integration by flanking upstream and downstream homology arms (each 750 bp) directing targeted introduction of insert promoter / TIS-variable construct. Transfection was followed by pool sorting of correct integrants by fluorescence activated cell sorting (FACS) and could include an enrichment step in antibiotic selection.

[0191] 5.2 Materials and methods

[0192] Introduction of secreter-state responsive promoters in CHO production cell line through targeted integration:

[0193] The antibody-expressing CHO KI cell line was prepared for transfection in shake flask cultivation using the chemically-defined PowerCHO-2 CD (Lonza) according to standard procedures (Sergeeva, 2020).

[0194] To replace the native promoter of the targeted essential gene with the insert candidate secreter-state responsive promoter, DNA for expression of Cas9, sgRNA, and integration fragment (Table 6) was mixed equimolarly for a total amount of 1 pg DNA. Freestyle MAX reagent (Thermo Fisher Scientific) was used as transfection agent and standard procedure was followed except for 105cells were used, washed with 0.5 mL CD CHO medium (Thermo Fisher Scientific) and cultivated in 1 mL CD CHO medium (Thermo Fisher Scientific) in 12-well plates for two hours before transfection. 2.7 pL Freestyle MAX reagent was used and total volume of the transfection mix was 50 pL. One hour later, the transfected cells were diluted with PowerCHO-2 CD to 3xl05cells / mL.

[0195] Following 3 days of incubation at 37 °C, 5 % CO2, 120-rpm horizontal orbital shaking, an optional step of antibiotic selection (using G418) was initiated according to standard procedures (Sergeeva, 2020) based on repeated 3-4 days cultivations in selective medium of increasing concentration up to 4 pg / mL. Next, correctly targeted integrants were pool sorted through fluorescent protein expression (e.g. green fluorescent protein and red fluorescent protein) using a FACS according to standard and previously published methods (e.g. Sergeeva, 2020). Finally, site-specific integration in clones was validated using PCR, and correct clones were expanded using previously published methods but using PowerCHO-2 CD as cultivation media (e.g. Sergeeva, 2020).

[0196] Screening for improved short-term and long-term production:

[0197] 105cells were thawed in 3 mL PowerCHO-2 CD medium in 6-well plates at 37 °C, 5 % CO2, 120-rpm horizontal orbital shaking (19mm throw). The cells were passaged to 2x 105cells / mL two times a week for a period of up to three months. Cells were cryopreserved every month: 1x10® cells were harvested, centrifuged at 200g and resuspended in fresh media with 10% DMSO. At select times, the selected samples were thawed and compared in batch cultivation (as in Example 1) or fed-batch bioreactor based on PowerCHO-2 CD medium.

[0198] Screening for improved secretion of the product

[0199] Product titers in the extracellular fraction of the developed SSA candidate cell lines and host cell line controls were evaluated in a 7-day batch production model. Cell lines were centrifuged at 200G for 2 minutes and seeded at 3xl05viable cells / mL in PowerCHO-2 CD medium containing 2% GSEM (Merck). The production models were incubated in 6- well plates at 37°C, 5% CO2, 125-rpm horizontal orbital shaking (19mm throw). Cultures were counted post-seed and cell density was adjusted to 3xl05±10%. Cultures were sampled on day 3, 4, 5, 6, and 7 for cell counting and determination of titer. Specific productivity was calculated by linear regression of the linear relationship between titer and integrated viable cell density.

[0200] 5.3 Results

[0201] The batch cultivations of frozen cell samples from the stability assay showed that the secreter-state-addicted cell lines produced and secreted the heterologous antibody at a higher titer than the non-secreter-state-addicted control cell line over at least 50 generations of cell division (Figure 7).

[0202] In summary, secreter-state-addicted transgene expressing CHO cell lines improve transgene expression with regards to titer, rate and / or carbon yield over time.

[0203] EXAMPLE 6: SECRETER-STATE ADDICTED CELL LINES HAVE IMPROVED TITERS

[0204] 6.1 Background of experiment

[0205] A large range of candidate secreter-state responsive promotors were selected from the list of generally suitable candidate secreter-state responsive promoters (Table 2) and experimentally tested by operably linking them to an essential gene in a mammalian antibody secreting cell line, similar to example 1 (see details in section 6.2 methods). Specifically, three different essential genes Pah, Lasll, and Eif2s2) were targeted and had their promoters replaced by candidate secreter-state promotors PGSS, PMMPI2, PRadsi, PCLU, Psili, PBCI2I1, PCAT, PGRP78, PGanab, or psecsia. A total of fifteen secreter-state addicted (SSA) candidate cell lines were generated as listed in Table 7. In brief, targeted gene introduction could be performed in several standard ways known in the art and was in this example mediated by a double-stranded cut from the programmable nuclease CRISPR / Cas9 as described herein, facilitating efficient integration by flanking upstream and downstream homology arms (each 750 bp) directing targeted introduction of the candidate secreter-state responsive promoter. Transfection was followed by pool sorting of cells exhibiting the signature fluorescence pattern of the transfection plasmids (GFP and RFP) using fluorescence activated cell sorting (FACS).

[0206] Titer improvement is a critical objective in bioprocess development. To evaluate the titer performance of SSA in the CHO cell lines, SSA candidate cell lines and ancestral CHO host control cell lines were compared in a batch production process. In this example, we demonstrate that SSA in engineered CHO cell lines increase titer.

[0207] Table 7: Overview of SSA candidate cell lines and genetic modifications

[0208] 6.2 Methods

[0209] 6.2.1 Cell Line Development The antibody-producing and secreting CHO KI host cell line CL003 was prepared for transfection in 6-well plate cultivation using the chemically-defined PowerCHO-2 CD (Lonza) and otherwise overall according to standard procedures (Sergeeva, 2020) with the following alterations: Passage before transfection was a 3-day passage, seeded at 2.5xl05viable cells / mL. To replace the native promoter of the targeted essential gene with the insert candidate secreter-state responsive promoter, DNA for expression of Cas9, sgRNA (consisting of three different candidate sgRNAs individually equimolar), and integration fragment (Table 8) was mixed equimolarly for a total amount of lpg DNA. Freestyle MAX reagent (Thermo Fisher Scientific) was used as transfection agent and standard procedure was followed with minor alterations: In brief, 105viable cells were harvested and washed in ImL CD CHO medium (Thermo Fisher Scientific). Cells were then cultivated in CD CHO medium in 12-well plates for two hours before transfection at 37°C, 5% CO2, 125-rpm horizontal orbital shaking (19mm throw). DNA (lpg total) was diluted in 25pL OptiPRO medium (Thermo Fisher Scientific). Freestyle MAX reagent (2.3pL) was diluted in 25pL OptiPRO and mixed with the diluted DNA (50pL total). Transfection mix was gently added to the cells, and the mix was allowed 1 hour incubation before diluting in PowerCHO-2 CD to 3xl05viable cells / mL in a 6-well plate and cultivated at previously stated conditions. Transfected cultures were cultivated for three days and then passaged for a 4 and 1-day passage seeded at 2xl05and 105viable cells / mL, respectively. Eight days post transfection, correctly targeted integrants were single cell sorted by means of fluorescent protein expression (e.g. green fluorescent protein and red fluorescent protein) using FACS (Sony SH800) according to standard methods. 50pL PowerCHO-2 CD medium with supplements (10% PowerCHO-2 CD, 7,5% InstiGRO (Advanced Instruments), 8 mM L-Glutamine (Thermo Fisher Scientific), 1,5% HEPES (Thermo Fisher Scientific), 1% Antibiotic-Antimycotic 100X (Thermo Fisher Scientific)) was used as cultivation medium. Single cell sorted cultures in U-bottom 96- well plates were cultivated at 37°C, 5% CO2, 125-rpm horizontal orbital shaking (19mm throw) and moved to stationary cultivation in plastic bags to limit volume loss to evaporation 1 day after single cell sorting. 50|j L PowerCHO-2 CD medium with previously stated supplements was added at 3, 6, and 9 days after single cell sorting. After 13-17 days, SSA candidate cell lines were expanded to 24-well plate format in PowerCHO-2 CD containing 8mM L-Glutamine and supplemented with PowerCHO-2 CD with previously stated supplement 3 days later. After 7 days, 24-well plate cultures were expanded to 12-well plates containing PowerCHO-2 CD. 1 to 4 days after 12-well plate expansion, cultures were transferred to 6-well plates containing PowerCHO-2 CD. Finally, sitespecific integration in clones was validated using PCR with primers flanking the 3' junction and correct clones were expanded in PowerCHO-2 CD using previously published methods (e.g. Sergeeva, 2020).

[0210] All cell lines were banked in PowerCHO-2 CD containing 10% DMSO and stored in the vapor phase of liquid nitrogen. 6.2.2 Recovery from storage and passage of the cell lines

[0211] 105cells were thawed in 3mL PowerCHO-2 CD medium in 6-well plates at 37°C, 5% CO2, 125-rpm horizontal orbital shaking (19mm throw). Cells were passaged for at least 1 week to recover before seeding in experiments. Two passaging schedules were followed: For cell line expansion seed trains during cell line development (described in section 6.2.1 above), passaging was performed in a 2-2-3 day schedule, and cells were seeded at 3xl05viable cells / mL (3-day passage) or 5xl05viable cells / mL (2-day passage). In preparation for seeding into batch cultivation, passaging schedule was adjusted to 3-3-1 in the final seed train before batch production. Cells were seeded at 7xl05viable cells / mL for a 1-day passage, and post-passage cell density was adjusted to 7xl05viable cells / mL ±10%.

[0212] 6.2.3 Screening for improved secretion of the product

[0213] Product titers in the extracellular fraction of the developed SSA candidate cell lines and host cell line controls were evaluated in a 6-day batch production model seeded at 3xl05viable cells / mL in PowerCHO-2 CD medium containing 2% GSEM (Merck). The production models were incubated in 6-well plates at 37°C, 5% CO2, 125-rpm horizontal orbital shaking (19mm throw). Cultures were counted post-seed and cell density was adjusted to 3xl05±10%. Cultures were sampled on day 6 for determination of titer.

[0214] All titer samples were frozen for at least 1 hour at -70°C before thawing at room temperature and analyzing using F. Quant Fc Titer kit (Cytena, CY.FQ.TIT.101) according to manufacturer's instructions. Human IgG (Sigma, I2511-10MG) was used as a standard.

[0215] As cell line development (CLD) controls undergoing the same cultivation treatment, twelve replicate product producing and secreting (non-SSA) clones were generated by single cell sorting of the ancestral CHO KI host cell line CL003 and expanding by the same procedure as the SSA candidate clones described above. Data points are averages obtained from 12 such sorted control clones (CL003 CLD1-12) used as reference controls.

[0216] 6.3 Results

[0217] In this experiment, secreter state-responsive promotors were operably linked to an essential gene in a mammalian antibody producing and secreting cell line as described above. A total of 15 different SSA candidate cell lines as listed in table 7 were generated and evaluated in batch production experiments. Titers were compared to titers obtained from 12 replicate host control cell lines (CL003 CLD1-12) in batch production experiments. The desuits demonstrate that batch cultivation of thawed cell samples from SSA candidate cultures achieve higher titers on day 6 of batch production than the CL003 CLD1-12 control cell lines (Figure 9).

[0218] Moreover, as presented in Table 7, the SSA candidate cell lines generated represent a broad range of candidate secreter-state responsive promotors and essential genes. As evident from Figure 9, it is demonstrated that the effects observed in SSA candidate cell lines are thus not limited to a single essential gene and promotor combination - but could be identified from various combinations of essential genes and promotor sequences.

[0219] EXAMPLE 7: SECRETER-STATE ADDICTED CELL LINES HAVE IMPROVED LONGTERM PRODUCTION

[0220] 7.1 Background of experiment

[0221] To demonstrate that SSA cell lines exhibit improved long-term production capability, a stability experiment was designed. Ancestral control cell line CL003 and SSA candidate cell lines generated as described previously (example 6) were thawed and exposed to an extended seed train.

[0222] In this example we demonstrate that SSA is a benefit for production cell lines when cultivated for prolonged time, as indicated by improved titer during batch production models.

[0223] 7.2 Methods

[0224] The antibody producing and secreting CHO KI host cell line CL003 was thawed and passaged for a total of 90 generations. After 9, 22, 61, and 90 generations, respectively, cultures were evaluated in production models and titers were measured.

[0225] A representative subset of 6 SSA candidate cell lines were thawed and subjected to an extended seed train, accumulating a total of 63 to 73 generations including cell line development from CL003, and seeded in batch production experiments as previously described.

[0226] Generation counts were calculated from CL003 antibody producing and secreting CHO KI host cell line thaw until production model seed, and calculations were based on each culture's growth rate during passage. Generation counts do not include the generations accumulated before thaw (during host cell line development), as these generations do not differ between the cell lines of our experiment. For SSA candidate cell lines, generation counts include cell line development of SSA candidate cell lines and single cell sorting, as described in example 6.

[0227] All batch production models were sampled on day 6 for titer analysis. All titer samples were frozen for at least 1 hour at -70°C before thawing at room temperature and analyzing using the F. Quant Fc Titer kit (Cytena, CY.FQ.TIT.101) according to manufacturer's instructions. Human IgG (Sigma, I2511-10MG) was used as a standard.

[0228] 7.3 Results

[0229] To evaluate the production performance of SSA candidate cell lines after extended cultivation, 6 SSA candidate cell lines were assessed in a study with extended cultivation and compared to CL003 host cell line controls in batch production models.

[0230] The results demonstrate that CL003 ancestral control titer (Figure 10) decline due to instability over the course of the passaging study ranging from 9 to 90 generations, whereas SSA candidate cell lines CL311, CL326, CL363, CL378, CL399, and CL400 consistently achieve significantly enhanced titers when compared to ancestral controls. Improved titers during long-term passaging highlights the efficacy of the invention and suitability for larger-scale bioprocess settings.

[0231] REFERENCES

[0232] Grav, L., Sergeeva, D., Lee, JS., Mas, IM., Kildegaard, HF. 2018. Minimizing Clonal Variation during Mammalian Cell Line Engineering for Improved Systems Biology Data Generation. ACS Synthetic biology 7, 2148-2159.

[0233] Lewis, N., Liu, X., Li, Y. et al. 2013. Genomic landscapes of Chinese hamster ovary cell lines as revealed by the Cricetulus griseus draft genome. Nat Biotechnol 31, 759-765. https: / / doi.org / 10.1038 / nbt.2624

[0234] Sergeeva, 2020. Targeted gene integration for improved production of therapeutic proteins in CHO cells. PhD thesis. DTU orbit

[0235] Gossen, Manfred, et al. 1995. Transcriptional activation by tetracyclines in mammalian cells. Science, 1766-1769.

Claims

CLAIMS1. A secreter-state addicted mammalian production cell genetically engineered to synthesize and secrete a protein product, said cell further comprising: a. a first essential gene operably linked to a first secreter-state responsive promoter, wherein the first essential gene is a gene in the mammalian production cell which, if down-regulated, leads to a reduction in the specific growth rate of the mammalian production cell under production conditions, wherein said first secreter-state responsive promoter is heterologous with respect to said first essential gene, wherein synthesis and secretion of the product confers a secreter state on said cell, wherein said first secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said first essential gene is up-regulated when said first secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said first essential gene when said first secreter-state responsive promoter is not induced.

2. The secreter-state addicted mammalian production cell according to claim 1, wherein said cell further comprises: b. a second essential gene operatively linked to a second secreter-state responsive promoter, wherein said second secreter-state responsive promoter is heterologous with respect to said second essential gene, wherein said second secreter-state responsive promoter is induced by said secreter-state, and wherein expression of said second essential gene is up-regulated when said second secreter-state responsive promoter is induced by said secreter-state relative to a basal level expression of said second essential gene when said second secreter-state responsive promoter is not induced.

3. The secreter-state addicted mammalian production cell according to claim 1 or 2 , wherein the secreter state responsive promoter is induced by said secreter-state, but not by the protein product per se.

4. The secreter-state addicted mammalian production cell according to any one of claims 1 to 3, wherein the first and / or second secreter state responsive promoter is selected from one or more candidate secreter-state responsive promoter(s) identifiable by (i) comparing a gene transcript profile of a non-secreter-state addicted mammalian production cell during production of the product with a gene transcript profile of an escape mutant variant cell derived from the non-secreter-state addicted mammalian production cell , wherein said escape mutant variant cell is characterized by having an at least 50 % lower production rate compared to the non-secreter-state addicted mammalian production cell, and (ii) identifying one or more promoter(s) associated with one or more positively differentiated gene transcript(s).

5. The secreter-state addicted mammalian production cell according to claims 4, wherein the one or more positively differentiating gene transcripts is identified by use of transcriptomics.

6. The secreter-state addicted mammalian production cell according to claims 4 or 5, wherein the expression level of the one or more positively differentiating gene transcript(s) in the mammalian production cell during production is increased by at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3 fold, compared to the escape mutant variant cell .

7. The secreter-state addicted mammalian production cell according to any one of claims 1 to 6, wherein said cell comprises c. one or more heterologous genes encoding the secreted protein product, wherein said one or more genes are operably linked to a promoter.

8. The secreter-state addicted mammalian production cell according to any one of claims 1 to 7, wherein said first secreter state responsive promoter is one that maintains a specific growth rate (doubling time) of said cell within a deviation of 15 % or less over the course of 40 or 50 or more cell divisions when cultured under production conditions.

9. The secreter-state addicted mammalian production cell according to any one of claims 1 to 8, wherein the first and / or second secreter state responsive promoter is a promoter of a gene selected from the list consisting of GSS, Gstpl, PRDX1, Sod2, G6PDX, CAT, Sodl, HTRA1, PDIA3, Por, CLU, USP18, Bcl2, Pdia4, P4hB, Pdia6, Hspa8, Fkbp2, Ube2k, Hspall, Hspala, GRP78, Manlal, B4galt6, Man2a2, Man2b2, Mgat5, MMP12, Bcl2ll, Ngb, RhoJ, Rsp6, Rp!3, Rpl27a, Oggl, Rad51, Rad52, Rad54l, Brea,Brcal, Ddit, Abcg2, Abcgl, Sls22al8, Pexl2, Atg7, Gls2, Creb3ll, Gsr, Erolb, Sec31a, Hsp90aal, Stipl, Clpb, Ppid, Pabpc4, Eif4g3, Eif4elb, Ernl, Mcm2, Pena, Priml, MrpH2, RpslO, Rpl32, Ganab, SHI, and Txnrdl, or homologous thereof.

10. The secreter-state addicted mammalian production cell according to any one of claims 1 to 9, wherein the first and / or second secreter state responsive promoter is a promoter of a gene encoding a protein selected from the list consisting of glutathione synthetase, glutathione S-transferase, glutathione reductase, peroxiredoxin, superoxide dismutases, glucose-6-phosphate dehydrogenase's, catalases, HtrA serine peptidases, protein disulfide isomerases, NADPH-cytochrome P450 reductases, clustering, ubiquitin specific peptidases, heat shock protein family A members, mannosidase alpha members, beta-1, 4-galactosyltransferases, matrix metallopeptidase 12, RAD51 recombinases, RAD52 DNA repair proteins, ATP binding cassette subfamily G members, peroxisomal biogenesis factors, and ATP-dependent Clp protease, or homologes thereof.

11. The secreter-state addicted mammalian production cell according to any one of claims 1 to 10, wherein the nucleotide sequence of the first and / or second secreter state responsive promoter is selected from the list consisting of SEQ ID NO. 12-79.

12. The secreter-state addicted mammalian production cell according to any one of claims 1 to 11, wherein the product is a secreted protein heterologous with respect to said production cell.

13. The secreter-state addicted mammalian production cell according to any one of claims 1 to 12, wherein said secreter state conferred by synthesis and secretion of said product, when each of said first and / or second essential gene in the cell is operably linked to its native promoter, results in a relative growth reduction measured as a percent reduction in the exponential phase growth rate of the mammalian production cell selected from among > 5 %, >10%, >15%, >20%, >25%, >35% and > 45 % relative to a corresponding parent or derivative non-producing mammalian cell producing at least 50 % less product.

14. The secreter-state addicted mammalian production cell according to any one of claims 1 to 13, wherein the first and / or second essential gene is a non-conditional essential gene.

15. The secreter-state addicted mammalian production cell according to any one of claims 1 to 14, wherein the first and / or second essential gene is essential for growth ofthe mammalian production cell irrespective of nutrient composition of said production condition.

16. The secreter-state addicted mammalian production cell according to any one of claims 1 to 15, wherein the first and / or second essential gene encodes a protein selected from the list consisting of a glutamine synthetase, a LAS1 like ribosome biogenesis factor, a cytosolic iron-sulfur protein, an acetyl-CoA acetyltransferase, a eukaryotic translation initiation factor, a propionyl-CoA carboxylase, a phenylalanine- 4-hydroxylase, a uridine monophosphate synthetase, and a 5-aminoimidazole-4- carboxamide ribonucleotide formyltransferase / IMP cyclohydrolase.

17. The secreter-state addicted mammalian production cell according to any one of claims 1 to 16, wherein the first and / or second essential gene encodes a protein sequence selected from the list consisting of SEQ ID NO. 2, 4, 5, 6, 7, 8, 9, 10, and 11, or a homologue thereof.

18. The secreter-state addicted mammalian production cell according to any one of claims 1 to 16, wherein the first and / or second essential gene encodes a protein sequence of SEQ ID NO. 4 or a homologue thereof.

19. The secreter-state addicted mammalian production cell according to any one of claims 1 to 16, wherein the first and / or second essential gene encodes a protein sequence of SEQ ID NO. 9 or a homologue thereof.

20. The secreter-state addicted mammalian production cell according to any one of claims 1 to 19, wherein at least one of the first and / or second essential gene is operably linked to a synthetic transcription initiation site whose sequence is selected to modify the translational strength of the essential gene independent of induction of said secreter-state responsive promoter.

21. The secreter-state addicted mammalian production cell according to any one of claims 1 to 20, wherein at least one essential gene allele is operably linked to said secreter-state-responsive promoter and at least one other essential gene allele remains operably linked to its native promoter.

22. The secreter-state addicted mammalian production cell according to any one of claims 1 to 21, wherein the cell is a Chinese Hamster Ovary (CHO) cell.

23. The secreter-state addicted mammalian production cell according to any one of claims 1 to 22, wherein the cell is characterized by an increased product titer after at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 generations of cell division fromfirst culture, as compared to a parent mammalian production cell lacking said first and / or second essential gene operably linked to said first and / or second secreter-state responsive promoter.

24. The secreter-state addicted mammalian production cell according to any one of claims 1 to 23, wherein the cell is characterized by an increased product titer of at least 10, 25, 50, or 80% following at least 50 generations of cell division from a single cell as compared to a parent mammalian production cell lacking said first and / or second essential gene operably linked to said first and / or second secreter-state responsive promoter.

25. The secreter-state addicted mammalian production cell according to any one of claims 1 to 24, wherein the product is selected from the list consisting of a peptide, enzyme, therapeutic protein and / or precursor thereof, human growth hormone, insulin, glucagon-like peptide-1, monoclonal antibody, polyclonal antibody, and singlefragment antibody.

26. The secreter-state addicted mammalian production cell according to any one of claims 1 to 24, wherein the product is a therapeutic protein selected from the list consisting of human growth hormone, insulin, glucagon-like peptide-1, an antibody (such as a monoclonal or polyclonal antibody, bispecific antibody, a single-fragment antibody or nanobody), turoctocog alfa, antihemophilic factor (such as Factor VII, III and VIII), follitropin beta, etanercept, rituximab, adalimumab, bevacizumab, erythropoietin (EPO), growth differentiation factor 5, Cl esterase inhibitor, tissue plasminogen activator, and cameloid scFv / fragments, or a precursor of said therepeutic protein.

27. The secreter-state addicted mammalian production cell according to any one of claims 1 to 24, wherein the product is a antibody, a monoclonal or polyclonal antibody, a single-fragment antibody, or nanobody, or a precursor thereof.

28. The secreter-state addicted mammalian production cell according to any one of claims 1 to 27 , wherein the product is a secreted protein heterologous with respect to said production cell; first and / or second essential gene is essential for growth of the mammalian production cell irrespective of nutrient composition of said production condition;wherein said secreter state conferred by synthesis and secretion of said product, when each of said first and / or second essential gene in the cell is operably linked to its native promoter, results in a relative growth reduction measured as a percent reduction in the exponential phase growth rate of the mammalian production cell selected from among > 5 %, >10%, >15%, >20%, >25%, >35% and > 45 % relative to a corresponding parent or derivative non-producing mammalian cell producing at least 50 % less product; wherein the secreter state responsive promoter is induced by said secreter- state, but not by the protein product per se; and wherein the first and / or second secreter state responsive promoter is selected from one or more candidate secreter-state responsive promoter(s) identifiable by (i) comparing a gene transcript profile of a non-secreter-state addicted mammalian production cell line during production of the product with a gene transcript profile of an escape mutant variant cell line derived from the non-secreter-state addicted mammalian production cell line, wherein said escape mutant variant cell is characterized by having an at least 50 % lower production rate compared to the non-secreter-state addicted mammalian production cell, and (ii) identifying one or more promoter(s) associated with one or more positively differentiated gene transcript(s).

29. A method of product biosynthesis comprising the steps of: a. providing a secreter-state addicted mammalian production cell according to any one of claims 1 to 28, b. introducing the cell into a cultivation medium comprising substrate for production of said product, c. cultivating said cell in said cultivation medium, d. recovering said product from the cultivation medium without opening the cells, e. optionally chemically modifying said product, and f. optionally formulating said product.

30. A method of product biosynthesis according to claim 29, wherein the product is a secreted heterologous protein product.

31. A method of product biosynthesis according to claim 29 or 30, wherein the product is selected from the list consisting of: peptide, enzyme, therapeutic protein and / or precursor thereof, human growth hormone, insulin, glucagon-like peptide-1, monoclonal antibody, polyclonal antibody, and single-fragment antibody.

32. A method of product biosynthesis according to claim 29 or 30, wherein the product is selected is a therapeutic protein selected from the list consisting of human growth hormone, insulin, glucagon-like peptide-1, an antibody (such as a monoclonal or polyclonal antibody, bispecific antibody, a single-fragment antibody or nanobody), turoctocog alfa, antihemophilic factor (such as Factor VII, III and VIII), follitropin beta, etanercept, rituximab, adalimumab, bevacizumab, erythropoietin (EPO), growth differentiation factor 5, Cl esterase inhibitor, tissue plasminogen activator, cameloid scFv / fragments, preferably antibodies such as monoclonal and polyclonal antibodies, and single-fragment antibodies, and nanobodies, or a precursor of said therepautic protein.

33. A method of product biosynthesis according to claim 29 or 30, wherein the product is a antibody, a monoclonal or polyclonal antibody, a single-fragment antibody, or nanobody, or a precursor thereof.

34. Use of at least one essential gene operably linked to a secreter-state responsive promoter to enhance product titer of a cultured population of mammalian production cells, wherein the essential gene is a gene in the mammalian production cell which, if down-regulated, leads to a reduction in the specific growth rate of the mammalian production cell under production conditions, wherein said secreter-state responsive promoter is heterologous with respect to said essential gene, wherein production of the product confers a secreter state on said cell, wherein said secreter-state responsive promoter is induced by said secreter state, and wherein expression of said essential gene is up-regulated, resulting in an increase in the specific growth rate of the mammalian production cell under production conditions, when said secreter-state responsive promoter is induced by said secreter state relative to a basal level expression of said essential gene when said secreter-state responsive promoter is not induced.

35. Use of a secreter-state addicted mammalian production cell according to any one of claims 1 to 28 for producing a secreted heterologous protein product.

36. Use of a secreter-state addicted mammalian production cell according to claim 35, wherein the product is selected from among a monoclonal or polyclonal antibody, and a single-fragment antibody.

37. Use of a secreter-state addicted mammalian production cell according to claim 35 or 36, to enhance the degree of desired N-linked glycosylation of said product.

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