Glycan variability on a recombinant IgG antibody transiently produced in HEK-293E cells

Nallet, Sophie; Fornelli, Luca; Schmitt, Simone; Parra, Julien; Baldi, Lucia; Tsybin, Yury O.; Wurm, Florian Μ.

doi:10.1016/j.nbt.2012.02.003

Cited by 33 publications

(37 citation statements)

References 21 publications

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“…When producing recombinant antibodies either transient gene expression (TGE) or stable gene expression (SGE) is used . The antibodies studied in this article are produced using both types of expression systems to evaluate possible differences in IgG glycosylation pattern by the two procedures.…”

Section: Introductionmentioning

confidence: 99%

Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated from Transiently as well as Permanently Transfected Cell Lines

et al. 2013

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The effector functions of IgG depend on the presence of carbohydrates attached to asparagine 297 in the Fc-portion. In this report, glycosylation profiles of recombinant wild-type and mutant IgG1 and IgG3 antibodies produced from three cell lines were analysed using LC-ESI-Orbitrap. Clear differences were detected between IgG1 and IgG3 glycoforms, where IgG1 generally contained fucosylated glycoforms, whilst IgG3 mainly were non-fucosylated. When using NS-0 and J558L cells for permanent transfection, IgG1 wt glycoforms differed between the two cell lines, whilst IgG3 wt glycoforms did not. Transiently transfected HEK 293E cells were used to produce IgG1 and IgG3 wt and mutants, affecting complement activation. Cell supernatants were harvested at early and late time points and analysed separately. IgGs harvested late showed simpler and less developed glycosylation structure compared to those harvested early. The IgG harvested early was slightly more effective in complement activation than those harvested late, whilst the antibody-dependent cellmediated cytotoxicity was unaltered. Generally, the glycosylation pattern of the mutants tested, including a hinge truncate mutant of IgG3, did not differ significantly from the wild-type IgGs. The striking difference in glycosylation pattern of IgG1 compared to IgG3 therefore appears not to be due to the long hinge region of IgG3 (62 amino acids) relative to the IgG1 hinge region (15 amino acids). Furthermore, mutation variants at or near the C1q binding site showed similar glycosylation structure and difference in their complement activation activity observed earlier is thus most likely due to differences in protein structure only.

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Section: Introductionmentioning

confidence: 99%

Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated from Transiently as well as Permanently Transfected Cell Lines

et al. 2013

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“…Typically, qualitative and quantitative analysis of the glycosylation profile are performed in a bottom-up fashion-for instance, by quantifying tryptic glycopeptides (38). However, and more commonly, in order to reduce the high complexity of all possible combinations of different glycans that can be found on recombinant IgGs, glycans are released via enzymatic reaction with PNGase F (39), with or without combination with physical methods such as microwave-assisted digestion (40), for both qualitative and quantitative analysis.…”

Section: Intact Protein Ms-mentioning

confidence: 99%

Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS

Fornelli

Damoc

Thomas

et al. 2012

Molecular & Cellular Proteomics

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The primary structural information of proteins employed as biotherapeutics is essential if one wishes to understand their structure-function relationship, as well as in the rational design of new therapeutics and for quality control. Given both the large size (around 150 kDa) and the structural complexity of intact immunoglobulin G (IgG), which includes a variable number of disulfide bridges, its extensive fragmentation and subsequent sequence determination by means of tandem mass spectrometry ( Top-down mass spectrometry (MS) 1 (1-3) has continued to demonstrate its particular advantages over traditionally employed bottom-up MS strategies (4). Specifically, top-down MS allows the characterization of specific protein isoforms originating from the alternative splicing of mRNA that code single nucleotide polymorphisms and/or post-translational modifications (PTMs) of protein species (5). Intact protein molecular weight (MW) determination and subsequent gasphase fragmentation of selected multiply charged protein ions (referred to as tandem MS or MS/MS) theoretically might result in complete protein sequence coverage and precise assignment of the type and position of PTMs, amino acid substitutions, and C-or N-terminal truncations (6), whereas the bottom-up MS approach allows only the identification of a certain protein family when few or redundant peptides are found for a particular protein isoform. At a practical level, however, top-down MS-based proteomics struggles not only with the single-or multi-dimensional separation of undigested proteins, which demonstrates lower reproducibility and repeatability than for peptides, but also with technical limitations present in even state-of-the-art mass spectrometers. The outcome of a top-down MS experiment depends indeed on the balance between the applied resolution of the mass spectrometer and its sensitivity. The former is required for unambiguous assignment of ion isotopic clusters in both survey and MS/MS scans, whereas the latter is ultimately dependent on the scan speed of the mass analyzer, which determines the number of scans that can be accumulated for a given analyte ion on the liquid chromatography (LC) timescale to enhance the resulting signal-to-noise ratio (SNR). Until recently, the instrument of choice for top-down MS has been the Fourier transform ion cyclotron resonance (FT-ICR) mass From the ‡Biomolecular Mass

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“…Finally, intact protein mass spectrometry results presented in Figure S1 in the Supporting Information allow direct quantification of the relative abundances of the different glycoforms, whereas relative quantification of protein glycoforms by bottom-up mass spectrometry is typically performed on one or several enzymatically derived peptides that contain these glycoforms. 39,41 Top-Down Mass Spectrometry of Murine IgG. To increase the performance of protein fragmentation by ETD in a single LC-MS experiment we explored the possibility that the LC-MS system might provide a substantially long protein elution time.…”

Section: Articlementioning

confidence: 99%

Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

et al. 2011

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Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.

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Glycan variability on a recombinant IgG antibody transiently produced in HEK-293E cells

Cited by 33 publications

References 21 publications

Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated from Transiently as well as Permanently Transfected Cell Lines

Different Glycosylation Pattern of Human IgG1 and IgG3 Antibodies Isolated from Transiently as well as Permanently Transfected Cell Lines

Analysis of Intact Monoclonal Antibody IgG1 by Electron Transfer Dissociation Orbitrap FTMS

Structural Analysis of Intact Monoclonal Antibodies by Electron Transfer Dissociation Mass Spectrometry

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