Protein Protocols Protein Protocols

Monitoring Glycoprotein Heterogeneity 905
126
Analysis of Glycoprotein Heterogeneity
by Capillary Electrophoresis and Mass Spectrometry
Andrew D. Hooker and David C. James
1. Introduction
The drive toward protein-based therapeutic agents requires both product quality and
consistency to be maintained throughout the development and implementation of a
production process. Differences in host cell type, the physiological status of the cell,
and protein structural constraints are known to result in variations in posttranslational
modifications that can affect the bioactivity, receptor binding, susceptibility to proteolysis,
immunogenicity, and clearance rate of a therapeutic recombinant protein in
vivo (1). Glycosylation is the most extensive source of protein heterogeneity, and many
recent developments in analytical biotechnology have enhanced our ability to monitor
and structurally define changes in oligosaccharides associated with recombinant proteins.
Variable occupancy of potential glycosylation sites may result in extensive
glycosylation macroheterogeneity in addition to the considerable diversity of carbohydrate
structures that can occur at individual glycosylation sites, often referred to as
glycosylation microheterogeneity. Variation within a heterogeneous population of
glycoforms may lead to functional consequences for the glycoprotein product. Therefore,
regulatory authorities such as the US Food and Drug Administration (FDA) and
the Committee for Proprietary Medical Productions demand increasingly sophisticated
analysis for biologics produced by the biotechnology and pharmaceutical industries
(2). The FDA has described a “well-characterized biologic” as “a chemical entity whose
identity, purity, impurities, potency and quantity can be determined and controlled.”
The glycosylation of a recombinant protein product can be examined by:
1. Analysis of glycans released by chemical or enzymatic means.
2. Site-specific analysis of glycans associated with glycopeptide fragments following proteolysis
of the intact glycoprotein.
3. Direct analysis of the whole glycoprotein.
A number of techniques are currently available to provide rapid and detailed analysis
of glycan heterogeneity:
1. High-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
2. Enzymatic analysis methods such as the reagent array analysis method (RAAM; 3,4).
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
905