Production of new hyperglycosylated EPO-analogs using CHO.K1 and HEK-293 cell lines

ITURRASPE, F.; BURGI, M. M.; KRATJE, R.; OGGERO EBERHARDT, M.

Santa Fe

Simposio; IX SLATCC; 2022

Centro Biotecnológico del Litoral

Three hyperglycosylated human erythropoietin (hEPO)-analogs were produced in our laboratory as neurotherapeutic candidates through glycoengeniering by hyperglycosylation to block the erythropoietic activity (EA) but preserving its neurobiological action (NA). Mut 45_47, Mut 104 and Mut 151_153 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.Furthermore, the HEK-293 cell line produces glycoproteins with simpler glycosidic structures, lower sialic acid content and higher receptor binding. Therefore, HEK-293 cells-derived-hEPO could be similar to the hEPO produced in the brain. Likewise, human cell lines are widely considered to produce glycoproteins as biotherapeutics due to their ability to introduce post-translational modifications comparable to those found in proteins naturally secreted by humans.To obtain hEPO-variants with a similar glycan profile to the hEPO produced in the brain, HEK-293 cell lines were transduced with lentiviral particles. The new proteins were purified by immunoaffinity chromatography showing yields between 43% and 60% and purities higher than 90% in a single purification step. Also, the HEK-produced proteins did not show in vitro EA as the corresponding analogs produced in CHO.K1 cells.Besides, HEK-produced variants showed lower molecular mass than those produced by CHO.K1 cells. This difference could be due to less complex glycan structures produced by HEK-293 cells as a consequence of a lower antennarity and/or a lesser sialic acid content, resembling the hEPO produced in the brain.