Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity.
Many biopharmaceutical products exhibit extensive structural micro-heterogeneity due to an array of co-occurring post-translational modifications. These modifications often effect the functionality of the product and therefore need to be characterized in detail. Here, we present an integrative approach, combining two advanced mass spectrometry-based methods, high-resolution native mass spectrometry and middle-down proteomics, to analyse this micro-heterogeneity. Taking human erythropoietin and the human plasma properdin as model systems, we demonstrate that this strategy bridges the gap between peptide- and protein-based mass spectrometry platforms, providing the most complete profiling of glycoproteins. Integration of the two methods enabled the discovery of three undescribed C-glycosylation sites on properdin, and revealed in addition unexpected heterogeneity in occupancies of C-mannosylation. Furthermore, using various sources of erythropoietin we define and demonstrate the usage of a biosimilarity score to quantitatively assess structural similarity, which would also be beneficial for profiling other therapeutic proteins and even plasma protein biomarkers.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Yang
Y
Yang
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Fan
F
Liu
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Vojtech
V
Franc
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
Liem Andhyk
LA
Halim
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
Huub
H
Schellekens
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands.|Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, The Netherlands.
Albert J R
0000-0002-2405-4404
AJ
Heck
D000595:Amino Acid Sequence; D059451:Biosimilar Pharmaceuticals / Q000032:analysis*; D004921:Erythropoietin / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D011414:Properdin / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D059451:Biosimilar Pharmaceuticals; C554270:EPO protein, human; D006023:Glycoproteins; D011994:Recombinant Proteins; D011414:Properdin; D004921:Erythropoietin