Development of orthogonal chromatographic methods for the purity analysis of therapeutic oligonucleotides

This application note presents the results obtained for the development of different HPLC methods on model therapeutic oligonucleotides, including R&D-grade Nusinersen, an approved 18-mer phosphorothioate oligonucleotide with 2’-O-(2-methoxyethyl) modifications.
Authors
Arnaud Delobel
Claire Butré
Morgane Lescut
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Absolute Quantification of Oligonucleotides by ICP-MS/MS

A method was developed for the absolute quantification of purified therapeutic oligonucleotides, based on phosphorus determination by ICP-MS/MS.
Authors
Arnaud Delobel
Juliusz Bianga
Magali Perez
Damien Mouvet
Caroline Cajot
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New 2D-LC/MS approaches for the analysis of in-process samples and for the characterisation of mAbs in a regulated environment

Two applications are presented here and describe the use of a 2D-LC system for MS characterisation of mAbs without sample preparation.
Authors
Arnaud Delobel
Claire I. Butré
Camille Alain
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Charge variants analysis of monoclonal antibodies

Charge variants are considered as a Critical Quality Attribute of therapeutic monoclonal antibodies, as they can have a significant impact on the efficacy of the drug. State-of-the-art analytical techniques are required to both quantify and identify the variants. In this application note, we present the portfolio proposed by Quality Assistance, including capillary isoelectric focusing (cIEF) and ion-exchange chromatography (IEX), with applications to commercial monoclonal antibodies. Approaches based on (2D-)LC/MS are presented for the identification of the variants.
Authors
Arnaud Delobel
Claire I. Butré
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Development, optimisation and validation of a TNFα neutralising assay using a Quality by Design approach

Using a QbD approach, we developed a TNFα neutralising assay capable of fulfilling the Analytical Target Profile. Statistical Design of Experiment was applied to evaluate the effect of the different factors and to choose the optimal parameters.
Authors
Fabian Vandermeers
Capucine Lepers
Florence Dusserre-Bresson
Christelle Plennevaux
Stéphanie Pennincx
Julie Nicaise
Estelle Lara
Vincent Bertholet
Arnaud Delobel
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Innovative ICP-MS/MS approach for the determination of phosphodiester to phosphorothioate ratio in phosphorothioate oligonucleotides

Phosphodiester to phosphorothioate ratio (P=O/P=S) is a critical quality attribute of phosphorothioate oligonucleotides. The determination of this ratio is commonly performed by P-NMR, which requires specific equipment and cannot be easily applied in a regulated (GxP) environment. For the first time, we present in this application note the preliminary results obtained for the development of an ICP-MS/MS method to determine P=O/P=S ratio.
Authors
Juliusz Bianga
Caroline Cajot
Philippe De Raeve
Arnaud Delobel
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Stress testing of Humira (adalimumab) monitored by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

In this application note, we present how this technology can be used to monitor structural changes at the amino acid level during stress testing studies of a monoclonal antibody. This appreach can be applied similarly to stability studies. 
Authors
Eric Largy
Arnaud Delobel
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Epitope mapping of an interleukin receptor for three therapeutic antibodies by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

In this application note, we present a case study in which we performed epitope mapping for three monoclonal antibodies binding to an interleukin receptor. 
Authors
Eric Largy
Caroline Cajot
Arnaud Delobel
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Analytical Characterisation of Cell-Based Medicinal Products

Ensuring the characterisation of in vitro differentiated cells for therapeutic purposes brings many challenges. These include the difficulty of controlling cell variability and complexity of the steps required to generate a stable product.
Authors
Fabian Vandermeers
Sandra Thys
Valérie Deffontaine
Estelle Lara
Arnaud Delobel
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Analytical approaches for the evaluation of ADCC activity of mAbs

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) is amechanism of action of antibodies through which virus-infected or cancer cells are targeted for destruction by components of the cell-mediated immune system, such as natural killer cells.
Authors
Arnaud Delobel
Florence Dusserre-Bresson
Eric Largy
Capucine Lepers
Cléa Mantini
Christelle Plennevaux
Fabian Vandermeers
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Customised labelling of antibodies: A powerful tool for multi-parameters analyses in Flow Cytometry

The characterisation of biomarkers by flow cytometry is an attractive approach for Quality Control applications of cell products. Unfortunately, the design of a customised panel remains challenging, especially for stem cell products.
Authors
Valérie Deffontaine
Fabian Vandermeers
Capucine Lepers
Géry Van Vyncht
Arnaud Delobel
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Characterisation of the glycosylation of Humira (adalimumab) and Erbitux (Cetuximab) by high resolution mass spectrometry

Due to their mode of production, therapeutic mAbs present diverse and heterogeneous glycosylations. Their analysis may require the use of orthogonal techniques in order to fully characterize their nature and location. In this application note, the use of mass spectrometry as a powerful tool for Adalimumab and Cetuximab glycosylation characterization is presented.
Authors
Eric Largy
Fabrice Cantais
Géry Van Vyncht
Arnaud Delobel
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Characterisation of Anti-HCP antibodies by 2D differential Gel Electrophoreses

Host-cell proteins are a major class of process-related impurities in biotherapeutics that can impact the safety and efficacy of  the product. Despite the advent of advanced analytical techniques, ELISA remains as the industry gold standard for the detection and quantification of HCPs during process development and for routine analyses.
Authors
Arnaud Delobel
Alix Verret
Nicolas Trinchero
Géry Van Vyncht
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Characterisation of Antibody-Drug Conjugates by High-Resolution Mass Spectrometry

Due to their mode of production and conjugation chemistry, antibody-drug conjugates are complex and heterogeneous molecules. Their analysis usually requires the use of many orthogonal analytical techniques in order to fully characterise their different variants and drug-to-antibody ratio.
Authors
Arnaud Delobel
Eric Largy
Fabrice Cantais
Anicet Catrain,
Géry Van Vyncht
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Characterisation of the glycosylation of Enbrel: Etanercept by high resolution mass spectrometry

Due to their mode of production, therapeutic glycoproteins present complex and heterogeneous glycosylations. Their analysis usually requires the use of many orthogonal analytical techniques in order to fully characterise their nature and location.
Authors
Arnaud Delobel
Eric Largy
Fabrice Cantais
Géry Van Vyncht
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Human platelet lysate as additive for Wharton's Jelly-MSC culture

This application note presents the results obtained during a study performed to evaluate the use of human Platelet Lysates (hPL) as an alternative to Fetal Bovine Serum (FBS) in culture media for Mesenchymal Stem Cells (MSC)
Authors
Arnaud Delobel
Fabian Vandermeers
Sandra Thys
Géry Van Vyncht
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Surface Plasmon Resonance

Biotherapeutics are a class of complex molecules that require the use of innovative techniques for their characterisation. Biacore technology offers label-free assays that provide precise and reliable concentration and kinetic measurements.
Authors
Arnaud Delobel
Fabian Vandermeers
Valérie Deffontaine
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Characterisation of Humira Adalimumab by high resolution mass spectrometry

Due to their mode of production, monoclonal antibodies are complex and heterogeneous molecules. Their analysis usually requires the use of many orthogonal analytical techniques in order to fully characterise the different variants.
Authors
Fabrice Cantais
Anicet Catrain
Eric Largy
Géry Van Vyncht
Arnaud Delobel
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Fast and accurate absolute-quantification of proteins and antibodies using Isotope Dilution-Triple Quadrupole ICP-MS

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is used increasingly in metallomic studies to analyze metals and metal species and their interactions within biological and ecological systems.
Authors
Agilent Technologies
Philippe De Raeve
Juliusz Bianga
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