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.

Nutrient Optimization Reduces Phosphorylation and Hydroxylation Level on an Fc-Fusion Protein in a CHO Fed-Batch Process

“…Phosphorylation also occurred on the last serine of a glycine‐serine linker, although the linker had only two repeats of GGGGS, (GGGGS) 2 , and the linker was on the C‐terminus of an Fc domain. The authors also found that the phosphorylation level was much higher for proteins expressed by HEK293 cells (11.3%) than that expressed by CHO cells (0.4%) . In our study, we found that CHO cells can also give high phosphorylation (>20%) on the last serine of a glycine‐serine linker, Ser46.…”

“…For a CHO cell line developed in‐house that produces a GLP1‐Fc fusion protein, we identified partial phosphorylation of the last serine of the (GSSSS) 3 linker. There was only one previous report on the phosphorylation of fusion proteins . Phosphorylation also occurred on the last serine of a glycine‐serine linker, although the linker had only two repeats of GGGGS, (GGGGS) 2 , and the linker was on the C‐terminus of an Fc domain.…”

“…In contrast, phosphorylation could be undesirable. Tyshchuk et al recently identified a phosphorylation site on the glycine‐serine linker of an IgG‐fusion protein, where phosphorylation of the last serine residue of a flexible (GGGGS) 2 linker to the C‐terminal of an IgG was observed. Phosphorylation levels were much more pronounced for proteins expressed through transient expression in HEK293 cells (11.3%) than those expressed through stable expression in CHO cells (0.4%).…”

“…Phosphorylation levels were much more pronounced for proteins expressed through transient expression in HEK293 cells (11.3%) than those expressed through stable expression in CHO cells (0.4%). Tyshchuk et al suggested that sequence optimization should be considered at the +1, +2, and +3 positions of a serine residue to avoid potential phosphorylation motifs.…”

High-resolution mass spectrometry confirms the presence of a hydroxyproline (Hyp) post-translational modification in the GGGGP linker of an Fc-fusion protein

“…14 Even short non-repeating linkers may contain PTMs, such as the phosphorylated serine residue identified in a G4S linker of a fusion protein by Tsyhchuk. 15 This undesirable product heterogeneity prompted us to explore new linker sequences that have better properties.…”