CHO.K1 vs. HEK-293 cell platforms to produce novel neurotherapeutic candidates

ITURRASPE, FRANCISCO; BURGI, MARÍA DE LOS MILAGROS; KRATJE, RICARDO; OGGERO EBERHARDT, MARCOS

Cuernavaca, Morelos

Congreso; 7th Latin American Glycobiology Congress; 2023

Red de glicociencia en salud, Univ. Autónoma del Estado de Morelos (México), Centro de Investigación en Dinámica Celular

Neurological disorders represent a group of pathologies characterized mainly by the loss of neurons in different areas of the brain, which defines the characteristic symptoms of each affection. There are some medicines currently approved for neurodegenerative disorders treatment that help with the associated symptoms without improving the patient´s condition. In an effort to develop new treatments, erythropoietin (EPO) has been proposed as a neurotherapeutic candidate. EPO is a 165 amino acids-consisting glycoprotein that bears three N-glycosylation sites and one O-glycosylation site. EPO is a biotherapeutic widely used for treating anemia since it is the main regulator of blood cell production. However, studies have shown that EPO exhibits neuroprotective, antiapoptotic, antioxidative and neuroplastic properties in the different tissues including the central nervous system. With the aim of blocking the erythropoietic activity (EA) but preserving its neurobiological action (NA), two hyperglycosylated human erythropoietin (hEPO) analogs were previously produced in our lab as neurotherapeutic candidates through glycoengeniering by hyperglycosylation. Thus, Mut 45_47 and Mut 104 were produced by CHO.K1 cells, affinity-purified and characterized, evidencing the incorporation of an extra N-glycan chain, the lack of their EA and the preservation of their NA. Considering that less sialylated hEPO is produced by the brain and that human cell lines produce glycoproteins with simpler glycosidic structures, lower sialic acid content and higher receptor binding, HEK-293 was chosen to produce EPO muteins that could resemble the properties of brain-produced hEPO. In this context, HEK-293 cells were transduced with mutein-coding lentiviral particles. The HEK-293-produced proteins were purified by immunoaffinity chromatography showing yields between 43% and 60% and purities higher than 90% in a single purification step. The partial N-deglycosylation procedure demonstrated a maximum expected occupancy degree of four N-glycosylation sites both in CHO.K1 and in HEK-293- produced variants. Besides, HEK-293 variants showed lower molecular mass than those produced by CHO.K1 cells. This difference could be due to less complex glycan structures as a consequence of a lower antennarity and/or a lesser sialic acid content, resembling the features of brain-produced hEPO. The increase in glycosidic content of CHO.K1 and HEK-293-derived muteins practically did not affect the temperature of thermal denaturation compared to hEPO confirming that tertiary structure was preserved after glycoengineering. In terms of biological activity, HEK-293-produced proteins did not show in vitro EA and preserved NA as the corresponding analogs produced in CHO.K1 cells. In summary, in this work we have produced, purified and characterized two hEPO variants which exhibited promising properties for treatment of neurological disorders.