Rational design of glycoengineered interferon-β analogs with improved aggregation state: experimental validation

Abstract

Recombinant human interferon-β (rhIFN-β) used clinically has lower efficacy than expected due to protein instabilities such as aggregation. Increasing molecular stability, glycoengineering has been used to improve clinical efficacy for a number of therapeutics; however, often labor-intensive trail-and-error approaches are used to identify additional glycosylation sites. In this study two rhIFN-β analogs with one additional glycosylation site, L6T and S75N, identified by a rational in silico approach, were characterized. These rhIFN-β analogs were synthesized in parallel with a Chinese hamster ovary (CHO) codon-optimized natural human IFN-β (Opt-IFN-β) and expressed in CHO cells using the same expression system. The molecular weights for both analogs were observed to be higher than Opt-IFN-β, consistent with hyper-glycosylation. The in vitro biological assay showed the hyper-glycosylated analogs and the Opt-IFN-β had similar activity. The aggregation studies demonstrated that both analogs had lower tendencies to aggregate compared to the Opt-IFN-β. These experimental studies validate the in silico strategy to predict suitable glycosylation sites that would be glycosylated, while maintaining biological function. Moreover, this work describes hyper-glycosylated rhIFN-β analogs with improved solubility (i.e. lower aggregation). These findings, together with the rational in silico design, will allow us to increase protein glycosylation with the goal to enhance therapeutic efficacy.

Keywords: aggregation; glycoengineering; human interferon-β; in silico design; therapeutic proteins.