CHO.K1 and HEK-293 cell platforms: towards the production of erythropoeitin analogs as neurotherapeutic candidates

ITURRASPE, F.; BURGI, M. M.; SCORTICATI, C.LA; KRATJE, R. B.; OGGERO, M. R.

Viña del Mar. Valparaíso

Simposio; X Simposio Latinoamericano de Tecnología de Cultivo de Células Animales (X SLATCC); 2024

Pontificia Universidad Católica de Valparaíso. Universidad de Chile. Universidad de Concepción.

Background: Human erythropoietin (hEPO), a biotherapeutic used for treating anemia, has beenproposed as a neurotherapeutic considering its neuroprotective and neuroplastic properties. Aiming to block the erythropoietic activity (EA) but preserving its neurobiological action (NA), twohyperglycosylated hEPO muteins were designed through glycoengineering and produced usingCHO.K1 cells. To obtain hEPO variants with properties that resemble the naturally brain-derived EPO,HEK-293 cells were chosen as host cells due to their ability to produce simpler glycosidic structures, lacking residues considered immunogenic. Aiming to compare CHO vs. HEK-hEPO derived muteins in terms of their biological performance, an extensively characterization was carried out.Methods: EPO derivatives were produced in both cell lines, immunoafnity puried and characterizedusing SDS-PAGE, isoelectric focusing, thermal shift, circular dichroism, partial deglycosylation andglycan analysis. Pharmacokinetic (PK) parameters were studied in Wistar rats and NA was evaluated in vitro by assessing neuritogenesis and lopodia formation and in vivo by studying the dendritic tree of hippocampal pyramidal neurons.Results: N-deglycosylation procedure demonstrated a maximum expected occupancy degree of four N-glycosylation sites of variants produced in both cell lines. HEK-293 variants showed lowermolecular mass and lesser acidic isoforms than those produced by CHO.K1 cells. These differences are probably due to less complex glycan structures with lower antennarity and a lesser sialic acid content. Also, the increase in glycosidic content of hEPO muteins or the amino acid mutations practically did not affect the temperature of thermal denaturation compared to hEPO, conrming that tertiary structure was preserved after glycoengineering. Disparities in the glycosylation proles resulted in signicant differences in the PK parameters. Thus, the plasmatic clearance and half-life of CHO-derived muteins were 14-fold lower and 2-fold higher, respectively than the corresponding HEK293-derived molecules. In terms of biological activity, all variants preserved in vitro neuritogenesis and filopodia formation, similar to hEPO. Remarkably, the in vivo NA was signicantly improved regards to hEPO using both muteins produced in both cell lines. As a result, 42%-higher dendrite length and a 41%-higher number of intersections per neuron were observed.Conclusion: Despite the fact that variants produced in HEK-293 cells displayed less favorable PKparameters than their CHO.K1 counterparts, they enhanced in vivo neuroplasticity as regards to EPOwith no statistical differences between muteins. The contribution of the PK and receptor interaction to the in vivo biological activity should be compared in the future in order to propose them as candidates for treatment of neurological disorders.