Discovery and Investigation ofO-Xylosylation in Engineered Proteins Containing a (GGGGS)_nLinker

Abstract

Protein engineering is a powerful tool for designing or modifying therapeutic proteins for enhanced efficacy, greater safety, reduced immunogenicity, and better delivery. GGGGS [(G4S)n] linkers are commonly used when engineering a protein, because of their flexibility and resistance to proteases. However, post-translational modifications (PTMs) can occur at the Ser residue in these linkers. Here, we report, for the first time, the occurrence of O-xylosylation at the serine residue in (G4S)n>2 linkers. The O-xylosylation was discovered as a result of molecular mass determination, peptide mapping analysis, and MS/MS sequencing. Our investigation showed that (i) O-xylosylation is a common PTM for (G4S)(n>2) linkers; (ii) GSG is the motif for O-xylosylation; and (iii) the total amount of xylosylation per linker increases as the number of GSG motifs in the linker increases. Our investigation has also shown that the O-xylosylation level is clone-dependent, to a certain degree, but the xylosylation level varies considerably among the proteins examined-from <2% to >25% per linker-likely depending on the accessibility to the sites by the xylosyltransferase. Our work demonstrates that potential therapeutic proteins containing (G4S)n linkers should be closely monitored for O-xylosylation in order to ensure that drugs are homogeneous and of high quality. The strategies for elimination and reduction of O-xylosylation were also examined and are discussed.

Detection of a phosphorylated glycine-serine linker in an IgG-based fusion protein

“…Failure to detect PTMs represents a safety risk due to potential immunogenicity. Nevertheless, a complex PTM has been reported to be common for serine residues in the GSG sequence motifs of GS-linkers due to a series of xylose-containing O -glycans, with O -Xyl alone being the most abundant glycosidic substituent 18,21,31-32 . The relative level of O -xylosylated GS-linkers has been reported to differ between human embryonic kidney (HEK) cells and Chinese hamster ovary (CHO) cells, with approximately 30% present in fusion proteins expressed in HEK cells, and about 3% in the case of CHO cell expression 31 …”

O-Glycosylation of glycine-serine linkers in recombinant Fc-fusion proteins

“…Distribution of these forms relative to the unglycosylated form is shown in panel A of Table 1. From our previous work with xylose glycans in LCAT-Fc 17 and in other reports, 15,16 these observed forms with unique mass addition are clearly related to Ser xylosylation, which can be further characterized at the peptide level as described below.…”

“…5, bottom panel). Both 15 Da mass was confirmed to be the tetrasaccharide GAG core, Xyl-Gal-Gal-GlcA, based on the CID fragmentation pattern (Fig. 6, top panel).…”

“…[15][16][17] As reported by Wen et al, xylose attached at the GSG motif in the linker regions is the predominant modification in bispecific engineered antibodies produced by Chinese hamster ovary (CHO) cells, and the level of xylosylation is clone-dependent. 15 At the peptide level, very low levels of GAGs and intermediates, e.g., bi-, tri-, tetra-, and penta-saccharides together with a pentasaccharide sulfated at glucuronic acid (GlcA) can also be identified. Spencer et al reported O-glycosylation at two different Gly-Ser linker sequences [either (G4S) 3 or (G4S) 2 ] in an engineered protein containing linkers that connect two fibronectin Tn3 domains and a non-Tn3 domain.…”

“…The actual sites of Ser xylosylation were not determined because xylose may or may not be evenly incorporated, although localization was demonstrated by Wen using ETD. 15 Other glycosylated species of the tryptic linker peptide include masses 2442.07 Da Da may also contain sulfate or phosphorylated glycans that will be described in detail later in the results section. Table 1 (panel B) summarizes all of the observed xylose glycosylated species in the Fc-(G4S) 4 -fusion protein sample.…”