LC-MS analysis combined with principal component analysis and soft independent modelling by class analogy for a better detection of changes in N-glycosylation profiles of therapeutic glycoproteins.
Therapeutic proteins are among the top selling drugs in the pharmaceutical industry. More than 60 % of the approved therapeutic proteins are glycosylated. Nowadays, it is well accepted that changes in glycosylation may affect the safety and the efficacy of the therapeutic proteins. For this reason, it is important to characterize both the protein and the glycan structures. In this study, analytical and data processing methods were developed ensuring an easier characterization of glycoprofiles. N-glycans were (i) enzymatically released using peptide-N-glycosidase F (PNGase F), (ii) reduced, and (iii) analyzed by hydrophilic interaction liquid chromatography coupled to a high-resolution mass spectrometer (HILIC-HRMS). Glycosylation changes were analyzed in human plasma immunoglobulin G samples which had previously been artificially modified by adding other glycoproteins (such as ribonuclease B and fetuin) or by digesting with enzyme (neuraminidase). Principal component analysis (PCA) and classification through soft independent modelling by class analogy (SIMCA) were used to detect minor glycosylation changes. Using HILIC-MS-PCA/SIMCA approach, it was possible to detect small changes in N-glycosylation, which had not been detected directly from the extracted-ion chromatograms, which is current technique to detect N-glycosylation changes in batch-to-batch analysis. The HILIC-MS-PCA/SIMCA approach is highly sensitive approach due to the sensitivity of MS and appropriate data processing approaches. It could help in assessing the changes in glycosylation, controlling batch-to-batch consistency, and establishing acceptance limits according to the glycosylation changes, ensuring safety and efficacy. Graphical abstract N-glycosylation characterization using LC-MS-PCA approach.
Analytical Platform of the Faculty of Pharmacy and Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, Campus Plaine, CP 205/05B, 1050, Brussels, Belgium. aplaninc@ulb.ac.be.
Ana
A
Planinc
Laboratory of Instrumental Analysis and Bioelectrochemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, 1050, Brussels, Belgium.|Department of Analytical Chemistry and Pharmaceutical Technology (FABI), Center for Pharmaceutical Research (CePhaR), Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium.
Bieke
B
Dejaegher
Department of Analytical Chemistry and Pharmaceutical Technology (FABI), Center for Pharmaceutical Research (CePhaR), Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium.
Yvan Vander
YV
Heyden
Department of Analytical Chemistry and Pharmaceutical Technology (FABI), Center for Pharmaceutical Research (CePhaR), Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium.
Johan
J
Viaene
Pharmacy, Centre Hospitalier Universitaire Saint-Pierre, Brussels, Belgium.
Serge
S
Van Praet
Pharmacy, Centre Hospitalier Universitaire Saint-Pierre, Brussels, Belgium.
Florence
F
Rappez
Analytical Platform of the Faculty of Pharmacy and Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, Campus Plaine, CP 205/05B, 1050, Brussels, Belgium.
Pierre
P
Van Antwerpen
Analytical Platform of the Faculty of Pharmacy and Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, Campus Plaine, CP 205/05B, 1050, Brussels, Belgium.
Cédric
C
Delporte
D002626:Chemistry, Pharmaceutical / Q000379:methods* / Q000592:standards; D002853:Chromatography, Liquid; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000627:therapeutic use; D057230:Limit of Detection; D008956:Models, Chemical; D025341:Principal Component Analysis; D053719:Tandem Mass Spectrometry
D006023:Glycoproteins; D007074:Immunoglobulin G
High-resolution mass spectrometry (HRMS); Hydrophilic interaction liquid chromatography (HILIC); N-Glycans; Principal component analysis (PCA); Soft independent modelling by class analogy (SIMCA); Therapeutic glycoproteins