Marcos Oggero

Human erythropoietin (hEPO) is the principal regulator of erythropoiesis and has been used therapeutically as recombinant hEPO (rhEPO) since 1989. Beyond this hematopoietic role, hEPO is also a tissue-protective cytokine with neurotrophic and neuroprotective activities. Clinically, rhEPO is widely used to treat anemia, although even in that setting its approved labeling carries boxed warnings for an increased risk of death, myocardial infarction, stroke, venous thromboembolism, vascular access thrombosis, and tumor progression or recurrence. Neuroprotective applications, however, require substantially higher and more frequent doses to overcome its limited blood–brain barrier permeability, and at these doses rhEPO produces dose-related adverse effects. Some of these, such as polycythemia and thrombotic events, are attributable to its erythropoietic activity, whereas others, including the excess mortality reported in acute ischemic stroke, remain mechanistically unexplained. This safety profile, together with the absence of benefit on the primary endpoint of three large randomized trials, is the main reason why rhEPO has not been approved for neurodegenerative disorders or neuronal tissue injury, and it has motivated the search for non-erythropoietic hEPO analogues. This review examines the dual biological functions of hEPO and their implications for biopharmaceutical innovation through two complementary cases originating from the Universidad Nacional del Litoral (UNL), Argentina. Zelltek emerged from academic research on recombinant glycoprotein production and developed a manufacturing platform for rhEPO, subsequently commercializing the product and contributing to the development of Argentina’s biopharmaceutical industry. BioSynaptica arose from research into the non-erythropoietic activities of hEPO and is advancing engineered, hyperglycosylated hEPO variants designed to dissociate erythropoietic activity from neuroprotective and other tissue-protective effects. Considered together, these university spin-offs illustrate two distinct translational trajectories arising from the biology of a single molecule: the industrial production of an established therapeutic protein and the preclinical development of engineered candidates targeting unmet medical needs. Their experiences also highlight the complementary roles of academic research, technology transfer, intellectual property, and sustained development efforts in translating biological discoveries into biopharmaceutical innovation.

Neurological disorders affect millions of people causing behavior‐cognitive disabilities. Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non‐sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N‐glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO‐derivatives were produced by CHO.K1 cells, affinity‐purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.

Purpose IFN4N is a glycoengineered version of recombinant human interferon alpha 2 (rhIFN-α2) that was modified to exhibit four N-glycosylation sites. It shows reduced in vitro specific biological activity (SBA) mainly due to R23 mutation by N23. However, it has improved pharmacokinetics and led to a high in vivo antitumor activity in mice. In order to prepare a new IFN-based biobetter, this work compares the influence of glycosylation (affecting pharmacokinetics) with the in vitro antiproliferative SBA on the in vivo efficacy. Methods Based on IFN4N, three groups of muteins were designed, produced, and characterized. Group A: variants with the same glycosylation degree (4N) but higher in vitro antiproliferative SBA (R23 restored); group B: muteins with higher glycosylation degree (5N) but similar in vitro antiproliferative activity; and group C: variants with improved glycosylation (5N and 6N) and in vitro antiproliferative bioactivity. Results Glycoengineering was successful for improving pharmacokinetics, and R23 restoration considerably increased in vitro antiproliferative activity of new muteins compared to IFN4N. Hyperglycosylation was able to improve the in vivo efficacy similarly to or even better than R23 restoration. Additionally, the highest glycosylated mutein exhibited the lowest immunogenicity. Conclusions Hyperglycosylation constitutes a successful strategy to prepare a novel IFN biobetter.

Objectives The influence of glycosylation on the antigen-neutralizing ability of two potential biotherapeutic anti-human IFN-α2b antibodies composed by murine and humanized single-chain Fv fused to human Fcγ1 (chimeric and humanized scFv-Fc, respectively) was studied. Results Chimeric antibodies produced in CHO-K1 and HEK293 mammalian cells showed no differences in the antigen–antibody affinity but demonstrated differences in the in vitro neutralization of IFN-α2b activity. On the other hand, the humanized antibodies produced in the same cell types showed differences in both the antigen–antibody affinity and the antigen-neutralizing ability. These differences are due to the scFv domain, as evidenced by its expression in CHO-K1 and HEK293 cells. In order to determine if the Fc glycosylation influences the antigen binding ability, both parameters were analyzed on chimeric and humanized deglycosylated scFv-Fc. Surprisingly, no differences in the antigen–antibody affinity were observed, but differences in the antigen-neutralizing ability of both chimeric and humanized antibodies, and their respectively deglycosylated glycoforms were found. Conclusions Fc glycosylation influences the antigen neutralization ability of two anti-rhIFN-α2b recombinant antibodies. Although affinity is the widely accepted parameter to analyze antibody antigen binding, it does not appear to be sufficient to describe the behavior of recombinant antibodies in vitro. This work contributes with a high impact knowledge to develop therapeutic recombinant antibodies where glycosylation and producer cell lines must be taken into account for their influence on the antigen binding capacity and not only for their impact on the effector properties as it has been historically considered for antibodies.