title: Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein.
abstract: Molecular mass determination by electrospray ionization mass spectrometry of a recombinant IgG-based fusion protein (mAb1-F) produced in human embryonic kidney (HEK) cells demonstrated the presence of a dominant +79 Da product variant. Using LC-MS tryptic peptide mapping analysis and collision-induced dissociation (CID) and electron-transfer/higher-energy collision dissociation fragmentations, the modification was localized to the C-terminal serine residue of a glycine-serine linker [(G4S)2] of a fused heavy chain containing in total 2 (G4S)2-linkers. The modification was identified as a phosphorylation (+79.97 Da) by the presence of a 98 Da neutral loss reaction with CID, by spiking a synthetic phosphoserine peptide, and by dephosphorylation with alkaline phosphatase. A thermolysin digest combined with higher-energy collision dissociation (HCD) positioned the phosphoserine to one specific glycine-serine linker of the fused heavy chain, and the relative level of phosphorylated linker was determined to be 11.3% and 0.4% by LC-MS when the fusion protein was transiently expressed in HEK or in stably transformed Chinese hamster ovary cells, respectively. This observation demonstrates that fusions with glycine-serine linker sequences should be carefully evaluated during drug development to prevent the introduction of a phosphorylation site in therapeutic fusion proteins.
authors.affiliation: a Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Roche Diagnostics GmbH , Penzberg , Germany.
authors.forename: Oksana
authors.initials: O
authors.lastname: Tyshchuk
authors.affiliation: a Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Roche Diagnostics GmbH , Penzberg , Germany.
authors.forename: Hans Rainer
authors.initials: HR
authors.lastname: Völger
authors.affiliation: b Oncology Discovery & Translational Area, Roche Innovation Center Zurich , Schlieren , Switzerland.
authors.forename: Claudia
authors.initials: C
authors.lastname: Ferrara
authors.affiliation: c Roche Pharma Technical Development Penzberg, Roche Diagnostics GmbH , Penzberg , Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Bulau
authors.affiliation: a Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Roche Diagnostics GmbH , Penzberg , Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Koll
authors.affiliation: a Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Roche Diagnostics GmbH , Penzberg , Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Mølhøj
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000737:chemistry*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005998:Glycine / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry*; D010766:Phosphorylation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*
chemical_list: D018033:Antibodies, Bispecific; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D012694:Serine; D005998:Glycine
keywords: Alkaline phosphatase; CID; EThcD; Glycine-serine linker; HCD; fusion protein; mass spectrometry; phosphorylation; phosphoserine; post-translational modification