Host cell impact on pharmacokinetics and neurobiological activity of non-erythropoietic hyperglycosylated EPO variants

Abstract

Human erythropoietin (EPO), which is therapeutically used to treat anemia, has demonstrated neuroprotective and neuroplastic benefits. Three glycoengineered EPO muteins designed to reduce erythropoiesis while retaining neurobiological effects were expressed in CHO.K1 and HEK-293 cells and compared both physicochemically and biologically.Our findings demonstrated that two of the muteins preserved their tertiary structure integrity. Both HEK-293 variants exhibited lower molecular masses and fewer acidic isoforms compared to their CHO.K1 counterparts, due to their simpler glycan structures and lower sialic acid content. Disparity in the glycan profile impacted pharmacokinetics, causing HEK-derived muteins to have shorter half-lives and higher clearance rates than CHO-derived molecules.Regardless of the host cells, all variants enhanced neuritogenesis, filopodia formation, and synaptogenesis in cultured neurons. In vivo analyses assessing pharmacokinetics and the complexity of dendritic arbor revealed that HEK cell-derived variants possess dendritic growth comparable to that from CHO cells (42%-increase in dendrite length and a 41%-increase in the number of intersections per neuron), even though the former exhibited less favorable pharmacokinetic profiles.Since the improved pharmacokinetic properties of CHO.K1-derived EPO-based neurotherapeutics did not translate into enhanced in vivo hippocampal neuron arborization, HEK-293 cells represent a compelling alternative production platform, offering a more favorable glycosylation profile with simpler and less immunogenic glycans.