Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Zhu, Lei; Guo, Qingcheng; Guo, Haipeng; Liu, Tao; Zheng, Yingxin; Gu, Peiming; Chen, Xi; Wang, Hao; Hou, Sheng; Guo, Yajun

doi:10.4161/mabs.36313

Cited by 43 publications

(52 citation statements)

References 39 publications

(41 reference statements)

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“…N‐ and O‐linked glycosylation of cytotoxic T‐lymphocyte‐associated antigen (CTLA4‐Ig) fusion protein was fully characterized by LC coupled to fluorescence detection and MS . Peptide mapping, using ETD fragmentation, determined three N‐ and four O‐linked glycosylation sites on the peptides and demonstrated the similarity of the CTLA4‐Ig fusion protein at the level of glycosylation patterns.…”

Section: New Glycomics and Glycoproteomics Biomedical Applicationsmentioning

confidence: 99%

Recent advances in mass spectrometric analysis of glycoproteins

et al. 2016

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Glycosylation is one of the most common post-translational modifications of proteins and plays essential roles in various biological processes, including protein folding, host-pathogen interaction, immune response, and inflammation and aberrant protein glycosylation is a well-known event in various disease states including cancer. As a result, it is critical to develop rapid and sensitive methods for the analysis of abnormal glycoproteins associated with diseases. Mass spectrometry in conjugation with different separation methods, such as capillary electrophoresis, ion mobility, and high performance liquid chromatography, has become a popular tool for glycoprotein analysis, providing highly informative fragments for structural identification of glycoproteins. This review provides an overview of the developments and accomplishments in the field of glycomics and glycoproteomics reported between 2014 and 2016.

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Section: New Glycomics and Glycoproteomics Biomedical Applicationsmentioning

confidence: 99%

Recent advances in mass spectrometric analysis of glycoproteins

et al. 2016

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“…6–9 For example, many of the marketed Fc-fusion proteins contain five or more glycosylation sites. 6–10 By solely characterizing these complex molecules by glycan release-based methods, all glycans from different sites become pooled together, thus information on glycosylation site-specificity is lost. Furthermore, O-glycans still prove to be difficult to remove from the protein by both enzymatic and chemical procedures.…”

Section: Introductionmentioning

confidence: 99%

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

Madsen

Farutin

Lin

et al. 2018

mAbs

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The characterization of glycosylation is required for many protein therapeutics. The emergence of antibody and antibody-like molecules with multiple glycan attachment sites has rendered glycan analysis increasingly more complicated. Reliance on site-specific glycopeptide analysis is therefore necessary to fully analyze multi-glycosylated biotherapeutics. Established glycopeptide methodologies have generally utilized a priori knowledge of the glycosylation states of the investigated protein(s), database searching of results generated from data-dependent liquid chromatography–tandem mass spectrometry workflows, and extracted ion quantitation of the individual identified species. However, the inherent complexity of glycosylation makes predicting all glycoforms on all glycosylation sites extremely challenging, if not impossible. That is, only the “knowns” are assessed. Here, we describe an agnostic methodology to qualitatively and quantitatively assess both “known” and “unknown” site-specific glycosylation for biotherapeutics that contain multiple glycosylation sites. The workflow uses data-independent, all ion fragmentation to generate glycan oxonium ions, which are then extracted across the entirety of the chromatographic timeline to produce a glycan-specific “fingerprint” of the glycoprotein sample. We utilized both HexNAc and sialic acid oxonium ion profiles to quickly assess the presence of Fab glycosylation in a therapeutic monoclonal antibody, as well as for high-throughput comparisons of multi-glycosylated protein drugs derived from different clones to a reference product. An automated method was created to rapidly assess oxonium profiles between samples, and to provide a quantitative assessment of similarity.

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“…The collected LC-MS/MS data were processed by BiopharmaLynx 1.3 software as described previously. 34 Differential scanning calorimetry DSC experiments were performed using VP-DSC (Microcal, Northampton, MA). The antibodies or their Fcs were tested individually at a protein concentration of 0.5 mg/mL; a buffer control without protein was used as reference.…”

Section: Reversed-phase Chromatography and Mass Spectrometrymentioning

confidence: 99%

Acid-induced aggregation propensity of nivolumab is dependent on the Fc

Liu

Guo

et al. 2016

mAbs

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Nivolumab, an anti-programmed death (PD)1 IgG 4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG 1 ), panitumumab (IgG 2 ) and atezolizumab (aglyco-IgG 1 ) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG 1 < aglyco-IgG 1 < IgG 2 < IgG 4 . This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG 4 backbone.

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Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

Cited by 43 publications

References 39 publications

Recent advances in mass spectrometric analysis of glycoproteins

Recent advances in mass spectrometric analysis of glycoproteins

Data-independent oxonium ion profiling of multi-glycosylated biotherapeutics

Acid-induced aggregation propensity of nivolumab is dependent on the Fc

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