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

Abstract

Flexible and protease resistant (G4S) linkers are used extensively in protein engineering to connect various protein domains. Recently, several groups have observed xylose-based O-glycosylation at linker Ser residues that yield unwanted heterogeneity and may affect product quality. Because of this, an engineering effort was implemented to explore different linker sequence constructs. Here, we demonstrate the presence of an unexpected hydroxylation of a prolyl residue in the linker, made possible through the use of high-resolution mass spectrometry (HR-MS) and MS. The discovery started with the detection of a poorly resolved ∼+17 Da mass addition at the reduced protein chain level of an Fc-fusion construct by liquid chromatography-MS. Upon further investigation at the peptide level using HR-MS, the mass increase was determined to be +15.99 Da and was localized to the linker peptide SLSLSPGGGGGPAR [210-223]. This peptide corresponds to the C-terminus of Fc [210-216], the G4P linker [217-221], and first 2 amino acids of a growth factor [222-223]. The linker peptide was first subjected to MS with collision-induced dissociation (CID) activation. The fragmentation profile localized the modification to the GGGPA [218-222] portion of the peptide. Accurate mass measurement indicated that the modification is an addition of an oxygen and cannot be CH, thus eliminating several possibilities such as Pro→Leu. However, other possibilities cannot be ruled out. Higher-energy collision-induced dissociation (HCD)-MS and MS using CID/CID were both unable to differentiate between Ala222→ Ser222 or Pro221→ Hyp221. Finally, MS using high-resolution CID/HCD confirmed the mass increase to be a Pro221→Hyp221 post-translational modification.

Evidence for co-translational misincorporation of non-canonical amino acid hydroxyproline in recombinant antibodies produced in Chinese Hamster Ovary (CHO) cell lines

“…In addition to proteinogenic amino acids, non-proteinogenic amino acids and rare PTMs can also be detected in the protein sequence [ 19 – 21 ]. One of the more common mammalian non-proteinogenic amino acids is hydroxyproline, a known analog of proline that is found in collagen supporting its triple helix structure and stability [ 22 , 23 ]. Hydroxyproline is incorporated into collagen by post-translational modification [ 24 ].…”

“…Hydroxylation of proline occurs after protein synthesis and is catalyzed by the enzyme prolyl hydroxylase [ 25 ]. Tyshchuk et al [ 21 ] and Spahr et al [ 23 ] reported the presence of hydroxyproline and a corresponding hydroxylation site in recombinant therapeutics expressed using CHO expression systems. E .…”

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics

“…35,45 Recently, hydroxyproline was reported at a similar high level (40%) in a partially rigid G 4 P linker as part of an Fcgrowth factor fusion protein stably expressed in CHO cells. 9 Also, the level of sTyr recently reported in the Lc CDR-1 of a CHO-derived antibody was determined to a relatively high (~20% at the peptide level, corresponding to~40% at intact level). 7 Together, these observations and the data presented here demonstrate that CHO cells are generally capable of introducing high levels of hydroxyproline and sulfotyrosine in therapeutic proteins, which thereby significantly increase the heterogeneity of the molecules.…”

“…6,7 Further uncommon enzyme-catalyzed PTMs (based on the experience and knowledge from IgGs and monoclonal antibodies (mAbs)) have been reported in non-IgG domains and linkers of antibody-fusion proteins. [8][9][10][11][12] In therapeutic proteins, PTMs are especially critical if they negatively influence drug potency or drug safety. Even if PTMs just increase the product heterogeneity of the desired molecules, when identified they need to be diminished or removed by manufacturing process optimizations or further protein engineering, or they at least have to be monitored and controlled to demonstrate batch consistency or comparability of manufactured clinical material.…”

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

“…An aspartate in the first EGF‐like module of these factors needs to be hydroxylated for Ca 2+ binding and to maintain its activity . Recently, hydroxylation of proline in the GGGGP linker of a Fc‐fusion protein and hydroxylation of lysine on the heavy chain Fab of an IgG1 were identified …”