An Integrated Platform of Analytical Chemical Techniques for Profiling of Therapeutic Glycoproteins

Author/Creator

Author/Creator ORCID

Date

2016-01-01

Department

Chemistry & Biochemistry

Program

Chemistry

Citation of Original Publication

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Abstract

Glycans are extensively dispersed in biological systems in "free state" and in attached forms such as glycoproteins, glycolipids, and proteoglycans. Glycans are commonly investigated as critical species in therapeutic glycoprotein drug development. Therapeutic glycoprotein drugs have high specificities granting precise action and long half-lives allowing infrequent dosing compared to low-molecular-mass chemical drugs. Strong evidence linking bioactivity and efficacy to glycosylation necessitate understanding, measuring, and controlling glycosylation in glycoprotein-based drugs. The oligosaccharide content of glycoprotein products, in addition to the thorough characterization of biosimilars, has become increasingly important. Glycans are involved in a wide range of biological and physiological processes including regulatory and recognition functions, cellular immunity, growth, and development. The functions of glycans are often dependent on the structure and types of oligosaccharides attached to the proteins. The structures of glycans are highly diverse, complex, and heterogeneous due to post-translational modifications and physiological conditions, making it remarkably challenging to comprehensively characterize glycan profiles and determine their structures. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and liquid chromatography with mass spectrometry (LC-MS) methods are compared based on their sensitivity to glycan detection and efficiency to separate a simple glycan mixture. Liquid chromatography with high-resolution mass spectrometry (LC-HRMS) and multi-enzyme digestions are used to develop a list of analytical controls needed for the primary sequence characterization of biosimilar therapeutics compared to their original innovator compounds. Analytical figures of merit determined for a standard glycan using LC-MS revealed detection limits in the sub parts per million range. Chromatographic figures of merit affirmed higher column efficiency with HPAEC-PAD. Multi-enzyme analysis dramatically increased sequence coverage for three protein models, two of which are highly used classes of therapeutic biologics. A critical list of analytical controls was developed for primary sequence characterization of biosimilar therapeutics highlighting consistency as an absolute necessity in the characterization process.