A NOVEL PEPTIDE-BASED PLATFORM FOR THE PRODUCTION OF O-GLYCOSYLATED THERAPEUTIC PROTEINS

NATALIA CEAGLIO; MARÍA DE LOS MILAGROS SALES; MARINA ETCHEVERRIGARAY; RICARDO KRATJE; MARCOS OGGERO

Lausanne

Congreso; 25th Meeting of the European Society for Animal Cell Technology (ESACT); 2017

European Society for Animal Cell Technology (ESACT)

"A current challenge of the biopharmaceutical industry is the improvement of the efficacy and stability of protein-based biotherapeutics, as well as their bioprocess optimization. In this work, we describe a new glycoengineering approach based in O-glycosylated peptide fusion technologies for enhancing the pharmacokinetic and pharmacodynamic properties of protein-based drugs. Moreover, since the peptides comprise a linear epitope recognized by a monoclonal antibody (CC1H7)1, this strategy could be useful to detect, quantify and purify the fusion protein.Experimental approachTwo peptide tags derived from the N-terminal end of human granulocyte and macrophage-colony stimulating factor (hGM-CSF) were developed: GMOP (comprising the first 14 amino acids of hGM-CSF and 4 potential O-glycosylation sites) and GMOPm (comprising the first 7 amino acids of hGM-CSF plus 8 additional residues, responsible of 6 potential O-glycosylation sites). Both peptides were fused to the N-terminus of human interferon-α2b (IFNwt), generating two different analogs that were produced in CHO-K1 cells and thoroughly characterized.Results and discussionGMOP-IFN and GMOPm-IFN exhibited increased molecular masses, more glycoforms and sialic acid than IFNwt, indicating that new O-glycans were attached to the IFN. Interestingly, both variants were strongly recognized by mAb CC1H7 in western blot assays. In vitro antiviral activity of IFN analogs was not affected, but antiproliferative activity of GMOP-IFN and GMOPm-IFN decreased 70% and 23% regarding IFNwt. Significant improvements in pharmacokinetics in rats were achieved, showing a 3.3 and 2.8-fold longer elimination half-life and a 3.7 and 4.1-fold decreased plasma apparent clearance in comparison with IFNwt, respectively. Also, O-glycans conferred a notable in vitro increment in thermal resistance and stability against plasma protease inactivation.Bibliography1Perotti et al., 2013, Prot Exp Purif 91:10-19."