General Analytical Approaches for Full Glycoprotein Characterization
The general approaches for full characterization of glycoproteins focus on two aspects: carbohydrates and the protein itself. Methods that detects released carbohydrates are limited by the fact that it is almost impossible to achieve 100% release, therefore 85% is generally considered as an acceptable release extent.
In addition, challenges are associated with characterization of O-linked carbohydrates due to a lack of efficient release tools. From a products purity point of view, it is important to determine glycosylation site occupancy as well as their overall intact protein profiles. At present, site-specific glycosylation is routinely determined for mAb's, but it is more difficult to do so with other complex proteins. For example, capillary electrophoresis with sodium-dodecyl sulfate gel (CE-SDS) is often used to determine the degree of occupancy at light chain asparagine-107 and asparagine-297 of the heavy chain site in a monoclonal IgG1.
For more complex glycoproteins, currently available methods are not yet optimal for quantitative purposes. Using peptide mapping, we would be able to tell whether a site is occupied, but that is difficult to quantify even using top level of LC-MS. It is recognized that the ability to characterize intact glycoproteins is in its infancy and deeds further developments.
Generally, the purpose of testing is dictated by the methodology or instrumentation that is preferable to apply for detecting and quantifying glycoproteins. The characterization methods and routine lot release, in-process and stability testing are quite different. Before drafting a plan for testing strategies of mAb's and other recombinant therapeutic proteins, the key question should be addressed: does glycosylation significantly effects on bioactivity or stability? In any circumstance, glycan profiling is the best approach to obtain a “ fingerprint”. HPLC and CE are two most widely used techniques for routine lot release testing and labeling methods are generally more robust than electrochemical detection technologies. Selection of appropriate tools for lot release and stability testing should be based on appropriate characterization data, whether the main goal is to control a specific glycosylation feature or to control process consistency. It is important to note that product-specific exceptions are quite frequently observed.
Ultra-Performance Liquid Chromatography (UPLC) technology allows for an enhanced resolution of complex samples. UPLC amide of columns in HILIC mode, packed with sub-1.7 μm particle sorbents has enabled significant improvements in separation power, often defined as peak capacity for peptides, oligonucleotides, and other analytes.
Methodologies for a glycan analysis have been developed where N-linked glycans are released from IgG's and are labeled with 2-aminobenzamide (2-AB). The derivatized oligosaccharides are separated by HILIC using a 1.7 μm UPLC amide column. A fluorescence detector on a UPLC system with a low volume flow cell is specific for the 2-AB label and enhances sensitivity and selectivity.
Fluorescence labeling is an important element of this sensitive assay. In order to obtain maximum sensitivity, excess reagent needs to removed by SPE separation prior a sample inject to UPLC. In additional, UPLC-HILIC/TUV/MS detection can be used and is complementary to UPLC-HILIC/FLR
The same tryptic digest and peptide map would be used in this method. Normally the presence of high polarity glycan moiety greatly improves the retention of glycopeptides of interest and therefore, they are well resolved from the remaining non-glycosylated triptic peptides. This specific retention behavior has been confirmed in different glycoprotein digests and appears to be a generic behavior of all glycopeptides, including the O-linked ones. LC/MS detection would provide molecular weight conformation of glycans.
The proposed methodology is suitable for characterization of glycoproteins and in particular for monoclonal antibodies. The combination of UPLC-HILIC/FLR and UPLCHILIC/TUV/MS methods is useful for characterization of both N-linked and O-linked glycans.