Bioactive peptides have high biological activity, but generally have low stability in plasma, are sensitive to proteases, and can be cleared from the circulation in minutes. Glycosylation is often required for optimal biologic bioactivity, especially for protein therapeutics. Selective glycosylation has the potential to improve drug potency through such effects as enhanced receptor selectivity or prolonged half-life. For example, Amgen’s Aranesp® has a three-fold longer half-life than its Epogen®, since it has 5 N-linked oligosaccharide chains compared to 3 for Epogen, permitting less frequent, more convenient dosing. Conventional mammalion, CHO-based cell culture is typically utilized to manufacture biologics that require glycosylation, but it yields heterogeneously-glycosylated species and is complex and expensive. Chemical synthesis of proteins, such as via solid-phase or solution-phase synthesis, can be used to manufacture non-glycosylated peptides and other small proteins, but has been unable to generate glycosylated peptides and proteins.
GlyTech has established a scalable manufacturing process for human type N-glycans and succeeded in producing several kilograms of common N-glycan structures per year. Using these N-glycans, they have found that glycosylation can be applied to peptides to improve their physicochemical properties. These glycosylation technologies enable them to add several glycans to specific positions in the target peptides and proteins, so they are able to tailor their PK profiles, solubility, etc., for improved drug development. These chemically synthesized glycopeptides and glycoproteins have a single glycoform and are homogeneous, unlike CHO-produced versions. In addition, the human type N-glycans are biodegradable and considered to be less toxic because of their nature. Therefore chemical glycosylation is potential technology for generating promising novel peptide and protein drugs.
This presentation will describe a method for manufacturing glycosylated peptides and proteins via chemical synthesis. As an example glycopeptide, GlyTech has created several glycosylated GLP-1 analogs. Their optimized GLP-1 analogs had potent efficacy and good plasma stability and pharmacokinetics in mice. They also have chemically glycosylated somatostatin analogues and shown markedly improved binding selectivity and half life versus marketed therapeutics. Thus, GlyTech has demonstrated that glycosylating bioactive peptides can be useful to optimize their PK/PD and other properties.
For larger peptides, which are commonly called proteins, Bachem and GlyTech have selected interferon-beta 1a as an example glycoprotein that they produced synthetically. Interferon-beta 1a (INF-beta-1a) is a glycosylated 166 amino acid protein with an approximate molecular weight of 22.5 kD. It is currently approved and widely used for the treatment of multiple sclerosis, and also has oncologic activity. Commercial INF-beta-1a is produced by mammalian cell culture and is a mixture of at least 10 glycoforms. We describe a method for manufacturing selectively-glycosylated INF-beta-1a via a synthetic, chemical route. In this method, the protein structure of INF-beta 1a was analyzed to determine optimal glycosylation points, glycan number, and glycan structure. Three fragments of the protein were selected for manufacturing, one of which was glycosylated. These fragments were ligated to form the final product. Each fragment synthesis was optimized and scaled up. Purification of the final product yielded a monodisperse and well-characterized product. Comparison to commercially-available INF-beta 1a demonstrated that the synthesized INF-beta 1a had one glycoform, whereas the commercial product had more than 10. In vitro bioactivity assays showed that synthesized INF-beta 1a had markedly increased bioactivity compared to commercial INF-beta 1a. Additionally, in animal models the terminal half-life of synthesized INF-beta 1a was about 2.5 times longer than commercial INF-beta 1a. Finally, the AUC of subcutaneous administration of synthesized INF-beta 1a in rats was substantially increased over that of commercial INF-beta 1a. Taken together, this optimized, fully-synthetic INF-beta 1a demonstrated numerous advantages over commercial IFN-beta 1a.
In conclusion, this presentation will demonstrate that optimizing glycosylated peptides’ and proteins’ physicochemical properties by a fully-synthetic manufacturing route is possible and that these optimized molecules can possess improved biologic activity. This technology for chemically synthesizing glycosylated peptides and proteins could be widely applicable to generate novel drugs.
Keywords: Peptides, Glycosylation, Chemical Synthesis