Rapid Fc glycosylation analysis of Fc fusions with IdeS and liquid chromatography mass spectrometry

Lynaugh, Heather; Li, Huijuan; Gong, Beini

doi:10.4161/mabs.25302

Cited by 35 publications

(26 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For example, there appeared to be no effect on IdeS digestion when the hinge region cysteines were replaced with serine or conjugated to small molecules. 25,30 It is thus not surprising that engineering of wild type IgG4 CPSC segment to that of IgG1 and IgG2 (CPPC) does not affect the cleavage, as shown in the current study. Domain mapping of IgG molecules by IdeS proteolysis provides a useful alternative to peptide mapping for antibody characterization.…”

Section: Discussionsupporting

confidence: 63%

“…[17][18][19] Application of IdeS has been demonstrated in many literature reports on IgG1 and IgG2 molecules, [20][21][22][23][24][25][26][27][28] as well as Fc fusion proteins. 29,30 Almost all the reports to date used primarily LC-MS techniques in subsequent analysis of the resulting antibody fragments. This is because the size of the antibody fragments produced by IdeS is well-suited for so-called middle-up or middle-down structural characterization.…”

Section: Introductionmentioning

confidence: 99%

“…[20][21][22][23][24][25]30,33,34,63 In most reported studies, antibody fragments were analyzed by LC-MS techniques directly to determine glycoform distributions. Whereas MS-based methods are superior in speed and sample consumption, the complexity in operating MS instruments and in data analysis often prevent them from being used in routine quality tests.…”

mentioning

confidence: 99%

See 2 more Smart Citations

A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

129

View full text Add to dashboard Cite

Section: Discussionsupporting

confidence: 63%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A new tool for monoclonal antibody analysis

Zhang

Mueller

et al. 2014

mAbs

129

View full text Add to dashboard Cite

“…This has led to its widespread use for analytical characterization of these molecules, 12,14,19 but its utility in providing monomeric products from digested Fc-fusion proteins is handicapped by the cleavage below the disulfide-containing hinge region. In an effort to tailor the Fc fragment for optimal pairing with IdeS, we found that a truncated fragment lacking the hinge sequence and proximal CH2 residues is a perfectly adequate fusion partner for expression of extracellular proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Several examples of Fcfusion proteins, where the Fc was derived from human IgG1, have indeed been reported to be cleaved by IdeS. [12][13][14] In each of these examples, the Fc partner contained the entire hinge region, as is typical for Fc-fusion proteins. The presence of the IgG hinge region complicates the practical usage of proteins released by IdeS cleavage, as the cleaved product retains this sequence and forms F(ab 0 )2-like disulfide-linked homodimers.…”

Section: Introductionmentioning

confidence: 99%

A hingeless Fc fusion system for site-specific cleavage by IdeS

Novarra¹,

Grinberg²,

Rickert³

et al. 2016

mAbs

View full text Add to dashboard Cite

Fusion of proteins to the Fc region of IgG is widely used to express cellular receptors and other extracellular proteins, but cleavage of the fusion partner is sometimes required for downstream applications. Immunoglobulin G-degrading enzyme of Streptococcus pyogenes (IdeS) is a protease with exquisite specificity for human IgG, and it can also cleave Fc-fusion proteins at a single site in the Nterminal region of the CH2 domain. However, the site of IdeS cleavage results in the disulfide-linked hinge region partitioning with the released protein, complicating downstream usage of the cleaved product. To tailor the Fc fragment for release of partner proteins by IdeS treatment, we investigated the effect of deleting regions of IgG-derived sequence that are upstream of the cleavage site. Elimination of the IgGderived hinge sequence along with several residues of the CH2 domain had negligible effects on expression and purity of the fusion protein, while retaining efficient processing by IdeS. An optimal Fc fragment comprising residues 235-447 of the human IgG1 heavy chain sufficed for efficient production of fusion proteins and minimized the amount of residual Ig-derived sequence on the cleavage product following IdeS treatment. Pairing of this truncated Fc fragment with IdeS cleavage enables highly specific cleavage of Fc-fusion proteins, thus eliminating the need to engineer extraneous cleavage sequences. This system should be helpful for producing Fc-fusion proteins requiring downstream cleavage, particularly those that are sensitive to internal miscleavage if treated with alternative proteases.Abbreviations: EPOR, erythropoietin receptor; Fc, fragment crystallizable; IdeS, Immunoglobulin G-degrading enzyme of Streptococcus pyogenes; mAb, monoclonal antibody; PBS, phosphate-buffered saline; SEC, size exclusion chromatography; scFv, single chain variable fragment; TRAILR2, TNF-related apoptosis inducing ligand receptor 2

show abstract

Sample Preparation Strategies for LC–MS Bioanalysis of Proteins

Yuan

2017

Protein Analysis Using Mass Spectrometry

View full text Add to dashboard Cite

Rapid Fc glycosylation analysis of Fc fusions with IdeS and liquid chromatography mass spectrometry

Cited by 35 publications

References 17 publications

A new tool for monoclonal antibody analysis

A new tool for monoclonal antibody analysis

A hingeless Fc fusion system for site-specific cleavage by IdeS

Sample Preparation Strategies for LC–MS Bioanalysis of Proteins

Contact Info

Product

Resources

About