Protein Glycosylation: Methods for Determination

Abstract

Glycoproteins are produced in eukaryotes as pools of different glycoforms with varying glycan structures attached to a single invariant peptide backbone. The characteristic glycoform profile is dependent upon the glycan structures added during co‐translation and modified post‐translationally. The glycosylation process is important to ensure the full efficacy of any glycoproteins used therapeutically. As the number of therapeutic increases there is a requirement to apply rapid analysis of glycans to glycoproteins that are produced as biopharmaceuticals for human therapy. Such methods are required as quality control to ensure that there is minimal variability between multiple batches in a bioprocess. Similarly, protocols adopted for diagnostic screening need to be rapid in order to handle multiple samples. Full structural analysis is possible by a combination of techniques including NMR, mass spectrometry and HPLC using exoglycosidase arrays for sequential breakdown of glycan structures. However there are other techniques that allow rapid screening that do not necessarily include full structural analysis. High though‐ put techniques are available for mass spectrometry, HPLC as well as lectin arrays, FACE or capillary electrophoresis Rapid analysis using such techniques may provide profiles that are adequate at least for ensuring consistency in production. The choice of method therefore will depend upon the degree of analytical information required. Several of the methods described in this chapter may be acceptable as providing glycan profiles but may not provide unambiguous structural assignments. The analysis of glycosylation patters are important to ensure consistency during a biopharmaceutical production process. Here we present the basis for analysis by several techniques that might be suitable during bioprocessing.

Glycosylation and Characterization of Human Transferrin in an End-Stage Kidney Disease

“…In general, the change in glycosylation may arise from alterations in the expression and/or activity of different transferases and glycosidases, as well as the availability of sugar substrates [37]. Mass spectrometry is regarded as a "gold standard" in the determination of glycan structures [38,39], and no decisive conclusions can be made without this method. It is not usually used as a high-throughput approach that allows comparisons between large groups of samples.…”

A Holistic 4D Approach to Optimize Intrinsic and Extrinsic Factors Contributing to Variability in Microarray Biosensing in Glycomics

“…In this context, analytical tools ranging from one-dimensional, high-throughput glycoprofiling methods to approaches yielding an accurate structural elucidation of glycans from a sample are available. Most of these methods are principally based on chromatographic, electromigratory, optical or mass spectrometric approaches [ 11 , 12 , 13 , 14 ]. This implies (a) the detection and quantification of all types of glycans; (b) the segregation of individual isomers; and (c) the assignment of components to a particular structure, including the overall topology of the molecule and appropriate linkages.…”