CONICET | Buscador de Institutos y Recursos Humanos

Engineering by hyperglycosylation represents a strategy for improving the performance of biotherapeutics, being the use of glycosylated peptide tags an attractive alternative. Accordingly, two novel hGM-CSF-derived peptides, the native GMOP and a modified version of GMOP (mGMOP) with 4 and 6 potential O-glycosylation sites, respectively, were developed. Both tags contain a linear epitope (APAR) recognized by a monoclonal antibody (mAb CC1H7). The fusion of these peptides to hIFN-α2b (as case study), increased the molecular mass due to O-glycan attachment, and improved the pharmacokinetics and stability of GMOP-IFN and mGMOP-IFN chimeras, without significant differences among them. The goal of this work was to study the properties of novel peptide-IFN chimeras in a hyperglycosylated context. Also, the potentiality of both peptides to behave as tags for facilitating analytical techniques was evaluated. Two variants were designed by adding 3 copies of GMOP or mGMOP to the N-terminus and 1 copy to the C-terminus of hIFN-α2b, and expressed in CHO-K1 cells. Online predictors of potential O-glycosylation sites showed molecules with 17 and 27 putative sites, respectively. Both chimeras showed higher molecular masses (~45 and ~65 kDa) compared to wild type hIFN α2b, indicating the successful incorporation of O-linked glycans. Interestingly, GMOP3 IFN GMOP showed around 2-times higher antiviral activity than the corresponding for mGMOP3-IFN-mGMOP. Also, APAR/CC1H7 interaction was useful to develop an affinity tag system, allowing the successful purification of IFN chimeras. In conclusion, both peptides are susceptible to be used in a dual conceptualization: to improve biological properties and to enhance the production processes of therapeutic proteins.

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