Grenoble, France
Grenoble, France

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Patent
Promise Advanced Proteomics | Date: 2017-05-10

The present invention relates to a method for quantifying an anti-TNF antibody in a sample of a human individual comprising a step of adding to a test sample which may contain therapeutic anti-TNF antibodies a known amount of two or more labeled forms of said anti-TNF antibodies.


Patent
Promise Advanced Proteomics | Date: 2017-05-10

The present invention relates to a method for quantifying a therapeutic antibody in a sample of a human individual comprising a step of adding to a test sample which may contain therapeutic antibodies to be quantified a known amount of two or more labeled forms of said therapeutic antibodies.


Lebert D.,Promise Advanced Proteomics | Louwagie M.,University Grenoble Alpes | Louwagie M.,CEA Grenoble | Louwagie M.,French Institute of Health and Medical Research | And 19 more authors.
Journal of Proteome Research | Year: 2015

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications. © 2015 American Chemical Society.


Lebert D.,PROMISE Advanced Proteomics | Picard G.,PROMISE Advanced Proteomics | Beau-Larvor C.,Pierre Fabre | Troncy L.,Pierre Fabre | And 12 more authors.
Bioanalysis | Year: 2015

Background: In preclinical studies, monoclonal antibodies (mAbs) are traditionally assayed by ligand-binding-assays. Recently, quantitative liquid chromatography mass spectrometry (MS)-based assays have emerged which circumvent a number of challenges. These assays may also be multiplex, making them potentially compatible with pharmacokinetic assays for combined antibody therapies. Materials & methods: We combined a quantitative MS-based approach with the protein standard for absolute quantification (PSAQ™) strategy to simultaneously quantify three mAb variants presenting minor sequence differences. Stable isotopically labeled mAbs were produced and used as quantification standards. Titration curves were performed to assess the analytical performances of the method. LC-MS/MS and ELISA data were cross-compared. Results: The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development. © 2015 Future Science Ltd.


PubMed | Rennes University Hospital Center, Grenoble Alpes University Hospital and PROMISE Advanced Proteomics
Type: | Journal: Analytical and bioanalytical chemistry | Year: 2016

Infliximab (IFX) is a chimeric monoclonal antibody targeting tumor necrosis factor-alpha. It is currently approved for the treatment of certain rheumatic diseases or inflammatory bowel diseases. Clinical studies have suggested that monitoring IFX concentrations could improve treatment response. However, in most studies, IFX was quantified using ELISA assays, the resulting discrepancies of which raised concerns about their reliability. Here, we describe the development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for IFX quantification in human plasma. Full-length stable-isotope-labeled antibody (SIL-IFX) was added to plasma samples as internal standard. Samples were then prepared using Mass Spectrometry Immuno Assay (MSIA) followed by trypsin digestion and submitted to multiple reaction monitoring (MRM) for quantification of IFX. The chromatographic run lasted 13min. The range of quantification was 1 to 26mg/L. For two internal quality controls spiked with 6 and 12mg/L of IFX, the method was reproducible (coefficients of variation (CV%): 12.7 and 2.1), repeatable (intra-day CV%: 5.5 and 5.0), and accurate (inter-day and intra-day deviations from nominal values: +6.4 to +3.7% and 5.5 to 9.2%, respectively). There was no cross - contamination effect. Samples from 45 patients treated with IFX were retrospectively analyzed by LC-MS/MS and results were compared to those obtained with an in-house ELISA assay and the commercial Lisa Tracker method. Good agreement was found between LC-MS/MS and in-house ELISA (mean underestimation of 13% for in-house ELISA), but a significant bias was found with commercial ELISA (mean underestimation of 136% for commercial ELISA). This method will make it possible to standardize IFX quantification between laboratories. Graphical Abstract Interassay comparison of the three methods: LC-MS/MS vs inhouse ELISA assay or vs Lisa Tracker ELISA assays, Passing & Bablok (a) and Bland & Altman (b) for the comparison of LC-MS/MS vs in-house ELISA assay; Passing & Bablok


PubMed | Promise Advanced Proteomics
Type: Journal Article | Journal: Journal of proteome research | Year: 2015

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.

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