Fig 1: Generation of an EPO producing CHO cell line using recombinase-mediated cassette exchange (RMCE) system. (A) Overview of the RMCE-based cell line development. A CHO-K1 host cell line with an integrated, FRT and F5 flanked CMV-eGFP cassette24 was used as a master host. The master host was transfected with an RMCE donor plasmid (encoding for EPO) together with a plasmid encoding Flp recombinase for a site-specific recombination between FRT and F5 sites, respectively. In the resulting cell line the CAG-EPO cassette is integrated in the target locus. Dual FACS strategy was used for selecting targeted, eGFP-negative cells and FITC-positive cells for EPO antibody-based cold capture. (B) Viable cell density. (C) Cell-specific growth rate. (D) EPO titre profile. (E) Cell-specific productivity. Experimental values represent the mean of two biological replicates ± SEM.
Fig 2: Comparison of the CHOEPO and CHOCXEPO cell lines in controlled bioreactors with 40 mM glucose and at 37C. (black) CHOEPO, (blue) CHOCXEPO. (A) Integral of viable cell concentration (IVCC, filled circle) and cell viability percentage (light circle). (B) EPO titres. (C) Glucose (filled circle) and lactate profiles (light circle). (D) Glutamine (filled circle) and ammonia profiles (light circle). Experimental values represent the mean of two biological replicates ± SEM.
Fig 3: Development of a MYC and XBP1s overexpressing CHO cell line. (black) CHOEPO cell line; (red) CHOCEPO cell line; (green) CHOXEPO cell line; (blue) CHOCXEPO cell line. (A) Overview of the cell engineering strategy to overexpress MYC and XBP1s in the CHOEPO cell line. The CHOXCEPO cell line was generated by transfecting cells with the CMV-MYC-PuroR and the CMV-XBP1s-HygroR plasmid, and isolating cells resistant to puromycin and hygromycin in the culture medium. Additionally, the CHOCEPO and CHOXEPO cell lines were generated as control cell lines. (B) Integral of viable cell concentration (IVCC). (C) cell-specific growth rate (µ). (D) EPO titre. (E) cell-specific EPO productivity (qEPO). (F) Glucose consumption rate (qGLC). (G) Lactate production rate (qLAC). (H) Ratio of lactate produced to glucose consumed (YLAC/GLC). (I) Glutamine consumption rate (qGLN). (J) Ammonia production rate (qNH3). (K) Ratio of ammonia produced to glutamine consumed (YNH3/GLN). Each dot corresponds to the duplicates (n = 2) of the three isolated cell clones. p values (One-way ANOVA) below to 0.05 indicate the statistical significance of parameter variation.
Fig 4: Comparison of glucose and temperature effect on the culture performance of CHOEPO and CHOCXEPO cell lines. (black) CHOEPO in 20 mM glucose, (grey) CHOEPO in 40 mM glucose, (dark blue) CHOCXEPO in 20 mM glucose; (light blue) CHOCXEPO in 40 mM glucose. (A) Overview of the culture conditions evaluated for both cell lines. (B) Integral of viable cell concentration. (C) Cell-specific growth rate. (D) EPO titre. (E) Cell-specific productivity. Experimental values represent the mean of two biological replicates ± SEM. (*), (**) and (***) indicated p < 0.05, 0.001, 0.0001 (Two-way ANOVA). Culture of CHOEPO at 37 °C and 20 mM glucose was the control condition.
Fig 5: Relative mRNA expression of recombinant EPO, MYC and XBP1 and their relationship with recombinant EPO production. (black) CHOEPO cell line; (red) CHOCEPO cell line; (green) CHOXEPO cell line; (blue) CHOCXEPO cell line. (A,B) and C) mRNA expression of EPO, MYC and XBP1, respectively, normalised using GAPDH as internal standard. Samples for mRNA expression analysis correspond to day 3. (D–F) Comparison between normalised EPO titres (x-axis) and mRNA levels of EPO, MYC and XBP1s (y-axis), respectively. (G–I) Comparison between normalised IVCC titres (x-axis) and mRNA levels of EPO, MYC and XBP1s (y-axis), respectively. Each dot corresponds to a replicate derived from CHOEPO cells as well as from three independent single-cell derived CHOCEPO , CHOXEPO and CHOCXEPO cell lines.
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