Concerted properties of two novel hyperglycosylated peptide tags: from improving biological actions to facilitating the detection and purification of biotherapeutics

LEOPOLD, MARÍA JESÚS; KRATJE, RICARDO; OGGERO, MARCOS; CEAGLIO, NATALIA

Lisboa

Congreso; 27th ESACT Meeting; 2022

Background and noveltyDevelopment of effective biotherapeutics with adequate physicochemical and functional properties has been a concern during the last decades. Glycosylation engineering has become one of the reference strategies for improving the performance of biotherapeutics, being the use of glycosylated peptide tags an attractive alternative.In our laboratory we have developed two novel peptides, GMOP and mGMOP, derived from the human granulocyte and macrophage-colony stimulating factor (hGM-CSF). These peptides have 4 and 6 potential O-glycosylation sites, respectively, and they contain a linear epitope (APAR) recognized by a monoclonal antibody (mAb CC1H7). Fusion of both peptides to a model protein, hIFN-α2b, increased the molecular mass due to O-glycan attachment, and improved the pharmacokinetics and stability of GMOP-IFN and mGMOP-IFN chimeras. However, despite mGMOP-IFN exhibited a higher O-glycosylation degree, no significant differences were found between the pharmacokinetic parameters of both proteins. In another aspect, the chimeras retained the capacity to bind to CC1H7 mAb.Experimental approachThe goal of this work was to study the properties of the peptides fused to hIFN-α2b in a hyperglycosylated context, and to evaluate their potentiality to behave as tags for facilitating the purification of the fusion proteins. For this, 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 to obtain molecules with 17 and 27 potential O-glycosylation sites, respectively. The corresponding CHO-K1-producer cell lines were obtained by transduction and subsequent puromycin selective pressure. Results and discussionThe chimeras presented higher molecular masses (~45 and ~65 kDa) than the wild type hIFN-α2b, indicating the successful incorporation of O-linked glycans. Interestingly, GMOP3-IFN-GMOP showed around 2-times higher antiviral activity than mGMOP3-IFN-mGMOP. Also, APAR/CC1H7 interaction was useful to develop an affinity tag system, allowing the successful purification of IFN chimeras. Considering that we have previously found that the affinity constant between APAR/CC1H7 increased in a high ionic strength environment, optimal binding conditions for 3 different salts and pH were identified through an experimental design: KCl 3.75M, pH 7.5; NaCl 4M, pH 7.5; Na2SO4 1M, pH 8, which caused a 60-, 70- and 30-fold increase in the apparent binding affinity, respectively. Importantly, Na2SO4 1M improved the dynamic binding capacity of the immunoaffinity matrix. In conclusion, we have set the basis to demonstrate the bifunctionality of both GMOP and mGMOP peptides for enhancing both the properties and the production processes of therapeutic proteins.