HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals.
In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC-MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.
Melmer
Michael
M
Sandoz GmbH, Biochemiestrasse 10, 6250, Kundl, Austria.
Stangler
Thomas
T
Schiefermeier
Mark
M
Brunner
Werner
W
Toll
Hansjörg
H
Rupprechter
Alfred
A
Lindner
Wolfgang
W
Premstaller
Andreas
A
D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D062367:ortho-Aminobenzoates / Q000737:chemistry
D005456:Fluorescent Dyes; D006023:Glycoproteins; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C000219:anthranilamide