N-linked glycosylation, the process of attaching complex carbohydrates known as glycans to the side-chain of asparagine residues in proteins, is widely involved in human health and disease. The importance of this essential life process stems in part from the fact that N-linked glycans have crucial impacts on the functional and structural properties of proteins, including their biological activity, solubility, stability, aggregation resistance, circulatory half-life, and immunogenicity. Biopharmaceutical glycoproteins have therefore been developed with recombinant N-glycosylation patterns designed to enhance therapeutic properties such as half-life and stability. However, these glycoengineering strategies have been challenging to implement as the rational design of glycosylation sites for improved properties relies on three-dimensional structural data and the knowledge of whether a given site of glycan attachment will affect therapeutic activity. Additionally, there is no guarantee that a designed glycosylation site will be occupied by a glycan with the desired N-glycan structure or with an N-glycan at all. This is important because even subtly different glycan structures (i.e., presence/absence of a single monosaccharide unit) can have significantly different effects on glycoprotein structure and function. Here, we investigated the use of glycoengineered Escherichia coli strains for the creation of new-to-nature glycoproteins bearing strategically attached N-glycans that improve the therapeutic properties of the protein. To this end, we leveraged E. coli strains equipped with heterologous glycosylation machinery that enables biosynthesis and conjugation of human-like glycans onto target proteins of interest. As proof-of-concept, we focused on recombinant human growth hormone (rhGH), which is used clinically to treat growth-related diseases but requires daily subcutaneous injections due to its short half-life and propensity to aggregate, resulting in decreased efficacy and safety. We first uncovered all glycosylation permissive sites within rhGH using a technique called shotgun scanning glycomutagenesis (SSGM), which enables comprehensive glycosite profiling of virtually any target protein. By coupling SSGM with an in vitro activity assay, we identified all sites within rhGH that could be glycosylated without a significant reduction in biological activity. Importantly, several well-expressed and glycosylated rhGH variants harboring human-like glycan structures at different internal sites exhibited significantly reduced aggregation and increased in-vivo half-life. Taken together, our glycoprotein engineering platform based on glycosylation-competent E. coli represents an exciting strategy for the development of novel biopharmaceuticals that are better tolerated by and last longer in the patient.
