One of the major concerns regarding administration of protein biotherapeutics for disease treatment lies in their low stability and short plasma half-life. Glycosylation is the most important co/postranslational event carried out by eukaryotic cells. Its manipulation through glycoengineering represents an effective tool in order to improve plasma half-life, as well as solubility, bioactivity, secretion and antigenicity of therapeutic proteins. For these reasons, we have developed a novel glycoengineering approach to generate proteins bearing O-glycans through their fusion to a 15-mer peptide tag named GMOPm. That sequence comprises the first 7 amino acids of the N-terminal region of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) together with 8 more residues that have been added with the aim of generating 6 potential O-glycosylation sites. The goal of this work
was to study the ability of GMOPm to improve the biological properties of a widely used human biotherapeutic, human interferon-α2b (hIFN-α2b). Five chimeras were constructed by adding GMOPm to the N- and/or C-terminal ends of hIFN-α2b in different proportions, to obtain variants with 7 to 29 potential O-glycosylation sites as predicted in silico: GMOPm-IFN; (GMOPm)2-IFN; (GMOPm)3-IFN; (GMOPm)2- IFN-GMOPm and (GMOPm)3-IFN-GMOPm. CHO-K1 recombinant cell lines were cultured for IFN variants production, achieving concentrations between 0.7 and 8 μg.ml-1 in culture harvests. The chimeras were purified from culture supernatant by affinity chromatography, using a monoclonal antibody anti-hIFN-α2b, with yields ranging from 40 to 100%. SDS-PAGE analysis of purified proteins demonstrated that the more the number of GMOPm tags added, the higher the molecular mass of the fusion protein. Moreover, increases in the molecular masses were higher than those expected considering the GMOPm peptide portion alone, suggesting that Oglycans have been attached at least to some of the potential O-glycosylation sites. Interestingly, all IFN variants retained in vitro antiviral activity, although it decreased concomitantly with the number of fused tags. However, it is widely known that in vitro activity generally does not reflect in vivo efficacy of a biotherapeutic. Indeed, improvement of pharmacockinetic properties is considered so vital to a protein´s in vivo activity that many times it is achieved at the expense of in vitro activity. Thus, the increment in glycosylation achieved by GMOPm addition may result in an improved pharmacokinetic profile and, consequently, an improved in vivo bioactivity. This will be analyzed in a near future. In summary, in this work we have produced, purified and in vitro characterized five GMOPm/IFN chimeras which exhibited promising properties for the in vivo evaluation of their pharmacokinetic and biological activity.