Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping.

Anahita D Eckard,Johnie K Young,David R Dupont

doi:10.1155/2018/3909674

Anahita D Eckard, Johnie K Young + Show 1 more

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https://doi.org/10.1155/2018/3909674

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Abstract

N-lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N-glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography–mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N-glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N-glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area.

Highlights

Posttranslational modifications (PTMs) which expand and create diverse protein functions in most cases include the introduction of a well-defined functional group to amino acids such as acetate or phosphate moiety
In this work we have developed two new simple analytical methods of reverse phase chromatography and reduced SDS-PAGE gel assay with offline software (GelAnalyzer) for quantitative assessment of deglycosylation yield in N-glycan mapping workflow
Reduced SDS-PAGE Gel Assay for Analysis of Deglycosylation Yield

Summary

Introduction

Posttranslational modifications (PTMs) which expand and create diverse protein functions in most cases include the introduction of a well-defined functional group to amino acids such as acetate or phosphate moiety. Separate from most PTMs, glycosylation is the enzymatic addition of different carbohydrate molecules in different structure to the peptide backbone of a protein. More than 50% of the proteins in human are glycoproteins. Glycosylation plays a crucial role in regulating or indicating key biological processes such as cell-cell signaling and protein folding [1, 2]. Two main types of glycosylation include N- and O-linked glycosylation. N-glycosylation is attached to Asparagine residue and the glycosylation site is identified from consensus sequence

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