Functional analysis of glycosylation in Etanercept: Effects over potency and stability

Etanercept is a biotechnological product that has a complex glycosylation profile. To elucidate Etanercept glycosylation effect over biological activity and stability, we deglycosylated sequentially this molecule. Sequential deglycosylation was performed to understand which glycans are critical for Etanercept folding and activity. Extended study showed that gross glycosylation differences, affect thermal stability, hydrodynamic radius, pI, CDC, ADCC, protection against oxidation and charge surface exposition with any effect (within biological assay dispersion) over TNF alpha neutralization, indicating which glycoforms have a critical effect over Etanercept ADCC, CDC and stability. In this regard, complete remotion of sialic acids have a predominant importance over pI, ADCC, CDC and surface charge while N and O glycosylation over thermal stability, hydrophobicity, aggregation and protection against oxidation. Our research suggest that gross differences in the glycosylation profile are relevant for the stability and biological main activities of Etanercept, and that significant differences that affect the activities related to this fusion protein could be detected with proper analytical methods and stability studies.