Fig 1: Characterization of EPO expression in GLUL-MSX-mediated HEK293 producer cell pool. (a) Endogenous (light gray bars) and exogenous (dark gray bars) GLUL and EPO genomic DNA copy count analyzed via droplet digital PCR (ddPCR). Values represent mean ± SD (b) Endogenous (light gray bars) and exogenous (dark gray bars) GLUL and EPO mRNA copy count analyzed via ddPCR. Values represent mean ± SD (c) Immunoblot of GLUL and EPO protein in HEK293 wildtype (WT), GLUL-KO (KO #24) and cell pool #8 (CP#8). Actin was used as a loading control.
Fig 2: Production and stability of EPO of HEK293 producer cells. GLUL-KO clones were transfected with bicistronic vector expressing human GLUL and human EPO cultured in glutamine-deficient media. Post-methionine sulfoximine (MSX) selection, nine cell pools were generated and characterized for (a) viable cell density and (b) EPO production. Values represent mean ± SD (c) MSX selection was subsequently removed from culture for stability testing over 12 weeks with specific productivity measured at day 4 cultures. (n = 2).
Fig 3: Productivity of HEK293 EPO producer cell pool in a 2 L fed-batch culture. Cell pool #8 was cultured in 2 L stirred-tank bioreactors over 10 days and characterized daily for (a) viable cell density (VCD, diamonds) and viability (%, squares). (b) EPO production was measured by ELISA over period of culture together with (c) glucose (g/L), (d) glutamine (mM), (e) glutamate (mM), (f) lactate (g/L), (g) ammonia (mM), and (h) osmolality (mOsm/kg).
Fig 4: HEK293-derived EPO site-specific N-glycosylation. Glycopeptides from all three N-glycan sites were acquired with LCMS/MS Orbitrap HCD (normalized collisional energy 30%) and identified using the Byonic software. Label-free quantitation was done using OpenMS/KNIME.
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