Decoding of O-Linked Glycosylation by Mass Spectrometry.

Protein glycosylation (N- and O-linked) plays an important role in many biological processes, including protein structure and function. However, the structural elucidation of glycans, specifically O-linked glycans, remains a major challenge and is often overlooked during protein analysis. Recently, mass spectrometry (MS) has matured as a powerful technology for high-quality analytical characterization of O-linked glycans. This review summarizes the recent developments and insights of MS-based glycomics technologies, with a focus on mucin-type O-glycan analysis. Three main MS-based approaches are outlined: O-glycan profiling (structural analysis of released O-glycan), a "bottom-up" approach (analysis of an O-glycan covalently attached to a glycopeptide), and a "top-down" approach (analysis of a glycan attached to an intact glycoprotein). In addition, the most widely used MS ionization techniques, i.e., matrix-assisted laser desorption ionization and electrospray ionization, as well as ion activation techniques like collision-induced dissociation, electron capture dissociation, and electron transfer dissociation during O-glycan analysis are discussed. The MS technical approaches mentioned above are already major improvements for studying O-linked glycosylation and appear to be valuable for in-depth analysis of the type of O-glycan attached, branching patterns, and the occupancy of O-glycosylation sites.

Reduction of product-related species during the fermentation and purification of a recombinant IL-1 receptor antagonist at the laboratory and pilot scale.

Through a parallel approach of tracking product quality through fermentation and purification development, a robust process was designed to reduce the levels of product-related species. Three biochemically similar product-related species were identified as byproducts of host-cell enzymatic activity. To modulate intracellular proteolytic activity, key fermentation parameters (temperature, pH, trace metals, EDTA levels, and carbon source) were evaluated through bioreactor optimization, while balancing negative effects on growth, productivity, and oxygen demand. The purification process was based on three non-affinity steps and resolved product-related species by exploiting small charge differences. Using statistical design of experiments for elution conditions, a high-resolution cation exchange capture column was optimized for resolution and recovery. Further reduction of product-related species was achieved by evaluating a matrix of conditions for a ceramic hydroxyapatite column. The optimized fermentation process was transferred from the 2-L laboratory scale to the 100-L pilot scale and the purification process was scaled accordingly to process the fermentation harvest. The laboratory- and pilot-scale processes resulted in similar process recoveries of 60 and 65%, respectively, and in a product that was of equal quality and purity to that of small-scale development preparations. The parallel approach for up- and downstream development was paramount in achieving a robust and scalable clinical process.