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

Spahr, Chris; Shi, Stone D.-H.; Lu, Hsieng S.

doi:10.4161/mabs.28763

Cited by 21 publications

(8 citation statements)

References 32 publications

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“…The identification of xylose-containing glycans in (G4S) n and similar linkers and phosphorylated serine in a G4S linker demonstrate that even commonly used linkers may be susceptible to undesirable post-translational modification. [12][13][14][15] In screening various new linker candidates, including G4P and (G4P) 2 , additional PTMs were observed. By combining highresolution tandem MS with various MS n techniques, an unexpected Pro!Hyp PTM was confidently identified in the G4P linker of our Fc-fusion protein.…”

Section: Discussionmentioning

confidence: 99%

“…The HPLC column, gradient, and running conditions, as well as the ESI-TOF instrumental parameters, are identical to those described previously. 14 The resulting Full MS spectra were summed, then deconvoluted to [30,000-50,000 Da] using the MaxEnt algorithm in the Agilent Mass Hunter software. A mass step of 1 Da, a S/N threshold of 30.0, and an average mass % peak height of 50 were used.…”

Section: Lc-ms Of Reduced Protein Chainsmentioning

confidence: 99%

“…2 Xylose-containing glycans have recently been observed at Ser residues in (G4S) n and similar linkers. [12][13][14] Following initial placement of xylose on Ser by a xylosyltransferase, rampant extension of the glycan by various other enzymes may occur. More than 20 different species of heterogeneous xylose glycans have been identified, including intermediates of the glycosaminoglycan biosynthetic pathway, and species containing phosphorylation, sulfation, and sialylation.…”

Section: Introductionmentioning

confidence: 99%

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

Spahr

Gunasekaran

Walker

et al. 2017

mAbs

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Lc-ms Of Reduced Protein Chainsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Spahr

Gunasekaran

Walker

et al. 2017

mAbs

Self Cite

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“…By combining IdeS and EndoS digestion, the latter removing the distal glycan heterogeneity, fucosylation yield could easily be determined on Fc/2. Spahr et al recently characterized the O-glycosylation of glycine–serine linkers in recombinant Fc-fusion proteins measured using TOF-MS following their reduction . Glycoforms were successfully measured before and after the removal of the Fc N-glycans using PNGase F. A heterogeneous population of O-glycans with a xylose core decorated with galactose, glucuronic acid, N-acetylglucosamine, N-acetylneuraminic acid, as well as phosphate was observed.…”

Section: Mass Spectrometric Approachesmentioning

confidence: 99%

Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals

et al. 2015

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“…Glycine, the smallest amino acid is unique because unlike all others, it contains hydrogen as its side chain rather than a carbon (24), permitting much more conformational flexibility. (Gly 4 Ser) n is often used as a linker for different polypeptides because it is not prone to misfolding errors, and Gly plays an irreplaceable role in this structure (25).…”

Section: Introductionmentioning

confidence: 99%

Reducing Hinge Flexibility of CAR-T Cells Prolongs Survival In Vivo With Low Cytokines Release

Zhang

Sun

Du³

et al. 2021

Front. Immunol.

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Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in B cell malignancies. However, high tumor burden limits clinical efficacy and increases the risk of cytokine release syndrome and neurotoxicity, which is associated with over-activation of the CAR-T cells. The hinge domain plays an important role in the function of CAR-T cells. We hypothesized that deletion of glycine, an amino acid with good flexibility, may reduce the flexibility of the hinge region, thereby mitigating CAR-T cell over-activation. This study involved generating a novel CAR by deletion of two consecutive glycine residues in the CD8 hinge domain of second-generation (2nd) CAR, thereafter named 2nd-GG CAR. The 2nd-GG CAR-T cells showed similar efficacy of CAR expression but lower hinge flexibility, and its protein affinity to CD19 protein was lower than that of 2nd CAR-T cells. Compared to the 2nd CAR-T cells, 2nd-GG CAR-T cells reduced proinflammatory cytokine secretion without diminishing the specific cytotoxicity toward tumor cells in vitro. Furthermore, 2nd-GG CAR-T cells prolonged overall survival in an immunodeficient mouse model bearing NALM-6 when tumor burden was high. This study demonstrated that a lower-flexibility of CD8α hinge improved survival under high tumor burden and reduced proinflammatory cytokines in preclinical studies. While there is potential for improved safety and efficacy, yet this needs validation with clinical trials.

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

Cited by 21 publications

References 32 publications

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

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

Chromatographic, Electrophoretic, and Mass Spectrometric Methods for the Analytical Characterization of Protein Biopharmaceuticals

Reducing Hinge Flexibility of CAR-T Cells Prolongs Survival In Vivo With Low Cytokines Release

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