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1. Adsorption and recovery issues of recombinant monoclonal antibodies in reversed‐phase liquid chromatography†

Author:
Beck, Alain,   et al.   ; Fekete, Szabolcs; Wagner, Elsa; Vuignier, Karine; Guillarme, Davy;     Show all 5 Authors
Source:
Journal of separation science 2015 v.38 no.1 pp. 1-8
ISSN:
1615-9306
Subject:
adsorption; ferritin; glycosylation; human serum albumin; hydrophobicity; immunoglobulin G; isoelectric point; molecular weight; monoclonal antibodies; ovalbumin; reversed-phase liquid chromatography; silica; transferrin
Abstract:
... The poor recovery of large biomolecules is a well‐known issue in reversed‐phase liquid chromatography. Several papers have reported this problem, but the reasons behind this behavior are not yet fully understood. In the present study, state‐of‐the‐art reversed‐phase wide‐pore stationary phases were used to evaluate the adsorption of therapeutic monoclonal antibodies. These biomolecules possess mol ...
DOI:
10.1002/jssc.201400996

2. Advantages of Extended Bottom-Up Proteomics Using Sap9 for Analysis of Monoclonal Antibodies

Author:
Beck, Alain,   et al.   ; Srzentić, Kristina; Fornelli, Luca; Laskay, Ünige A.; Monod, Michel; Ayoub, Daniel; Tsybin, Yury O.;     Show all 7 Authors
Source:
Analytical chemistry 2014 v.86 no.19 pp. 9945-9953
ISSN:
1520-6882
Subject:
aspartic proteinases; deamidation; liquid chromatography; mass spectrometry; monoclonal antibodies; peptides; post-translational modification; proteolysis; proteomics
Abstract:
... Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence c ...
DOI:
10.1021/ac502766n

3. Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. II- Identification of sub-units for the characterization of even and odd load drug species B Analytical technologies in the biomedical and life sciences

Author:
Beck, Alain,   et al.   ; Sarrut, Morgan; Fekete, Szabolcs; Janin-Bussat, Marie-Claire; Colas, Olivier; Guillarme, Davy; Heinisch, Sabine;     Show all 7 Authors
Source:
Journal of chromatography 2016 v.1032 pp. 91-102
ISSN:
1570-0232
Subject:
drugs; hydrophobic interaction chromatography; isomers; mass spectrometry; reversed-phase liquid chromatography
Abstract:
... This paper is the second part of a two-part series dedicated to the development of an on-line comprehensive HICxRPLC-UV/MS method for the characterization of a commercial inter-chain cysteine-linked ADC (brentuximab vedotin, Adcetris®). The first part focused on the optimization of the chromatographic conditions. In the second part of this series of papers, the structural characterization of the B ...
DOI:
10.1016/j.jchromb.2016.06.049

4. Analysis of antibody-drug conjugates by comprehensive on-line two-dimensional hydrophobic interaction chromatography x reversed phase liquid chromatography hyphenated to high resolution mass spectrometry. I − Optimization of separation conditions B Analytical technologies in the biomedical and life sciences

Author:
Beck, Alain,   et al.   ; Sarrut, Morgan; Corgier, Amélie; Fekete, Szabolcs; Guillarme, Davy; Lascoux, David; Janin-Bussat, Marie-Claire; Heinisch, Sabine;     Show all 8 Authors
Source:
Journal of chromatography 2016 v.1032 pp. 103-111
ISSN:
1570-0232
Subject:
drugs; hydrophobic interaction chromatography; manufacturing; mass spectrometry; mixing; quality control; reversed-phase liquid chromatography; solvents
Abstract:
... Antibody-drug-conjugates (ADCs) manufacturing leads to a mixture of species which needs to be characterized during development and for further quality control. The coupling of on-line HIC x RPLC to high resolution mass spectrometry can be considered as a very efficient analytical method, providing extensive information on ADC sample, within a reduced time scale. Our intention in this first paper i ...
DOI:
10.1016/j.jchromb.2016.06.048

5. Analysis of monoclonal antibody by a novel CE‐UV/MALDI‐MS interface

Author:
Beck, Alain,   et al.   ; Biacchi, Michael; Bhajun, Ricky; Saïd, Nassur; François, Yannis Nicolas; Leize‐Wagner, Emmanuelle;     Show all 6 Authors
Source:
Electrophoresis 2014 v.35 no.20 pp. 2986-2995
ISSN:
0173-0835
Subject:
electrophoresis; glycosylation; monoclonal antibodies; peptide mapping; peptides; proteins; quality control
Abstract:
... mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product‐ and time‐consuming. CE‐MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samp ...
DOI:
10.1002/elps.201400276

6. Analytical characterization of biosimilar antibodies and Fc-fusion proteins

Author:
Beck, Alain,   et al.   ; Diemer, Hélène; Ayoub, Daniel; Debaene, François; Wagner-Rousset, Elsa; Carapito, Christine; Van Dorsselaer, Alain; Sanglier-Cianférani, Sarah;     Show all 8 Authors
Source:
Trends in analytical chemistry 2013 v.48 pp. 81-95
ISSN:
0165-9936
Subject:
markets; mass spectrometry; monoclonal antibodies; proteins; spectrometers
Abstract:
... Mass spectrometry (MS) is one of the key analytical techniques to detect and to identify primary sequence differences, to assess similarity and to evaluate batch variability of reference monoclonal antibodies (mAbs). The next generation of high-resolution mass spectrometers and the early use of improved MS-based methodologies will help bring biosimilar mAbs and Fc-fusion proteins into highly regul ...
DOI:
10.1016/j.trac.2013.02.014

7. A new anti-human Fc method to capture and analyze ADCs for characterization of drug distribution and the drug-to-antibody ratio in serum from pre-clinical species B Analytical technologies in the biomedical and life sciences

Author:
Beck, Alain,   et al.   ; Excoffier, Mélissa; Janin-Bussat, Marie-Claire; Beau-Larvor, Charlotte; Troncy, Lysiane; Corvaia, Nathalie; Klinguer-Hamour, Christine;     Show all 7 Authors
Source:
Journal of chromatography 2016 v.1032 pp. 149-154
ISSN:
1570-0232
Subject:
Macaca fascicularis; antibody specificity; antigens; biotin; blood serum; clinical trials; drugs; enzyme-linked immunosorbent assay; humans; ligands; liquid chromatography; mass spectrometry; mice; monoclonal antibodies; neoplasm cells; patients; precipitin tests; rats; therapeutics; toxicity
Abstract:
... Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. They combine monoclonal antibody specificity for over-expressed tumor antigens and the high cytoxicity of small molecular drugs (SMDs) and can therefore selectively kill tumor cells while minimizing toxicity to normal cells. Nevertheless, the premature deconjugation of ADCs in the circulation may trigger off targe ...
DOI:
10.1016/j.jchromb.2016.05.037

8. An Online Four-Dimensional HIC×SEC-IM×MS Methodology for Proof-of-Concept Characterization of Antibody Drug Conjugates

Author:
Beck, Alain,   et al.   ; Ehkirch, Anthony; D’Atri, Valentina; Rouviere, Florent; Hernandez-Alba, Oscar; Goyon, Alexandre; Colas, Olivier; Sarrut, Morgan; Guillarme, Davy; Heinisch, Sabine; Cianferani, Sarah;     Show all 11 Authors
Source:
Analytical chemistry 2018 v.90 no.3 pp. 1578-1586
ISSN:
1520-6882
Subject:
antibodies; drugs; gel chromatography; hydrophobic interaction chromatography; mass spectrometry; proteins; quality control; salts
Abstract:
... There are currently two main techniques allowing the analytical characterization of interchain cysteine-linked antibody drug conjugates (ADCs) under native conditions, namely, hydrophobic interaction chromatography (HIC) and native mass spectrometry (MS). HIC is a chromatographic technique allowing the evaluation of drug load profile and calculation of average drug-to-antibody ratio (DAR) in quali ...
DOI:
10.1021/acs.analchem.7b02110

9. A Novel Online Four-Dimensional SEC×SEC-IM×MS Methodology for Characterization of Monoclonal Antibody Size Variants

Author:
Beck, Alain,   et al.   ; Ehkirch, Anthony; Goyon, Alexandre; Hernandez-Alba, Oscar; Rouviere, Florent; D’Atri, Valentina; Dreyfus, Cyrille; Haeuw, Jean-François; Diemer, Hélène; Heinisch, Sabine; Guillarme, Davy; Cianferani, Sarah;     Show all 12 Authors
Source:
Analytical chemistry 2018 v.90 no.23 pp. 13929-13937
ISSN:
1520-6882
Subject:
ammonium acetate; drugs; gel chromatography; mass spectrometry; molecular weight; monoclonal antibodies; phosphates; product safety; therapeutics
Abstract:
... The determination of size variants is a major critical quality attribute of a therapeutic monoclonal antibody (mAb that may affect the drug product safety, potency, and efficacy. Size variant characterization often relies on size-exclusion chromatography (SEC), which could be hampered by difficult identification of peaks. On the other hand, mass spectrometry (MS)-based techniques performed in nond ...
DOI:
10.1021/acs.analchem.8b03333

10. Characterization of an antibody-drug conjugate by hydrophilic interaction chromatography coupled to mass spectrometry

Author:
Beck, Alain,   et al.   ; D’Atri, Valentina; Fekete, Szabolcs; Stoll, Dwight; Lauber, Matthew; Guillarme, Davy;     Show all 6 Authors
Source:
Journal of chromatography 2018 v.1080 pp. 37-41
ISSN:
1570-0232
Subject:
Hodgkin disease; drugs; hydrophilic interaction chromatography; mass spectrometry; reversed-phase liquid chromatography
Abstract:
... Brentuximab vedotin (Adcetris) is a cysteine-linked antibody-drug conjugate (ADC) used in the treatment of Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (ALCL). In this study, the drug payload and glycan modifications of this ADC were simultaneously characterized using a unique LC-MS middle-up analysis, involving hydrophilic interaction chromatography (HILIC). This work demonst ...
DOI:
10.1016/j.jchromb.2018.02.026

11. Characterization of antibody drug conjugate positional isomers at cysteine residues by peptide mapping LC–MS analysis

Author:
Beck, Alain,   et al.   ; Janin-Bussat, Marie-Claire; Dillenbourg, Marina; Corvaia, Nathalie; Klinguer-Hamour, Christine;     Show all 5 Authors
Source:
Journal of Chromatography B 2015 v.981-982 pp. 9-13
ISSN:
1570-0232
Subject:
antibody specificity; cysteine; drug toxicity; drugs; hydrophobicity; isomers; mass spectrometry; monoclonal antibodies; neoplasm cells; peptide mapping; peptides; reversed-phase high performance liquid chromatography
Abstract:
... Antibody-drug conjugates (ADCs) are becoming a major class of oncology therapeutics. Because ADCs combine the monoclonal antibody specificity with the high toxicity of a drug, they can selectively kill tumor cells while minimizing toxicity to normal cells. Most of the current ADCs in clinical trials are controlled, but heterogeneous mixtures of isomers and isoforms. Very few protocols on ADC chara ...
DOI:
10.1016/j.jchromb.2014.12.017

12. Characterization of cetuximab Fc/2 dimers by off-line CZE-MS

Author:
Beck, Alain,   et al.   ; François, Yannis-Nicolas; Biacchi, Michael; Said, Nassur; Renard, Charly; Gahoual, Rabah; Leize-Wagner, Emmanuelle;     Show all 7 Authors
Source:
Analytica chimica acta 2016 v.908 pp. 168-176
ISSN:
0003-2670
Subject:
analytical chemistry; chemical structure; drugs; glycosylation; lysine; mass spectrometry; molecular weight; monoclonal antibodies; pH; therapeutics
Abstract:
... Monoclonal antibody (mAb) therapeutics attract the largest concern due to their strong therapeutic potency and specificity. The Fc region of mAbs is common to many new biotherapeutics as biosimilar, antibody drug conjugate or fusion protein. Fc region has consequences for Fc-mediated effector functions that might be desirable for therapeutic applications. As a consequence, there is a continuous ne ...
DOI:
10.1016/j.aca.2015.12.033

13. Characterization of the N-Terminal Heterogeneities of Monoclonal Antibodies Using In-Gel Charge Derivatization of α-Amines and LC-MS/MS

Author:
Beck, Alain,   et al.   ; Ayoub, Daniel; Bertaccini, Diego; Diemer, Hélène; Wagner-Rousset, Elsa; Colas, Olivier; Cianférani, Sarah; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine;     Show all 9 Authors
Source:
Analytical chemistry 2015 v.87 no.7 pp. 3784-3790
ISSN:
1520-6882
Subject:
derivatization; mass spectrometry; monoclonal antibodies; peptide mapping; polypeptides; post-translational modification; proteins; proteomics; signal peptide
Abstract:
... The bioproduction of recombinant monoclonal antibodies results in complex mixtures of a main isoform and numerous macro- and microvariants. Monoclonal antibodies therefore present different levels of heterogeneities ranging from primary sequence variants to post-translational modifications. Among these heterogeneities, the truncation and fragmentation of the primary amino-acid sequence result in s ...
DOI:
10.1021/ac504427k

14. Characterization of 30 therapeutic antibodies and related products by size exclusion chromatography: Feasibility assessment for future mass spectrometry hyphenation B Analytical technologies in the biomedical and life sciences

Author:
Beck, Alain,   et al.   ; Goyon, Alexandre; D’Atri, Valentina; Colas, Olivier; Fekete, Szabolcs; Guillarme, Davy;     Show all 6 Authors
Source:
Journal of chromatography 2017 v.1065-1066 pp. 35-43
ISSN:
1570-0232
Subject:
Food and Drug Administration; ammonium acetate; biopharmaceutical industry; biopharmaceuticals; gel chromatography; hydrophobic interaction chromatography; mass spectrometry; molecular weight; monoclonal antibodies; proteins; salts
Abstract:
... Despite the popularity of targeted and immune therapies, the number of studies dealing with the quantitation of aggregates for Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAb and related products are still very scarce in literature. In this work, 30 therapeutic proteins including monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), Fc-fusion proteins a ...
DOI:
10.1016/j.jchromb.2017.09.027

15. Characterization of therapeutic antibodies and related products by two-dimensional liquid chromatography coupled with UV absorbance and mass spectrometric detection B Analytical technologies in the biomedical and life sciences

Author:
Beck, Alain,   et al.   ; Stoll, Dwight; Danforth, John; Zhang, Kelly;     Show all 4 Authors
Source:
Journal of chromatography 2016 v.1032 pp. 51-60
ISSN:
1570-0232
Subject:
absorbance; drug formulations; glycosylation; ion exchange; liquid chromatography; mass spectrometry; monoclonal antibodies; proteins
Abstract:
... The development of analytical tools for the characterization of large biomolecules is an emerging and rapidly evolving area. This development activity is motivated largely by the current trend involving the increase in development and use of large biomolecules for therapeutic uses. Given the inherent complexity of these biomolecules, which arises from their sheer size and possibilities for chemica ...
DOI:
10.1016/j.jchromb.2016.05.029

16. Characterizing various monoclonal antibodies with milder reversed phase chromatography conditions

Author:
Beck, Alain,   et al.   ; Bobály, Balázs; D'Atri, Valentina; Lauber, Matthew; Guillarme, Davy; Fekete, Szabolcs;     Show all 6 Authors
Source:
Journal of chromatography 2018 v.1096 pp. 1-10
ISSN:
1570-0232
Subject:
Food and Drug Administration; hydrolysis; mass spectrometry; monoclonal antibodies; reversed-phase liquid chromatography; risk reduction; temperature; therapeutics
Abstract:
... Reversed phase liquid chromatography (RPLC) of therapeutic monoclonal antibodies (mAbs) is often performed at elevated temperatures (80–90 °C) and in the presence of relatively high concentrations of TFA (0.1%). Under such conditions, it is possible to achieve suitable performance in terms of peak shapes and recoveries. Yet, it is also possible, to cause on-column hydrolysis and the generation of ...
DOI:
10.1016/j.jchromb.2018.07.039

17. Cutting-edge capillary electrophoresis characterization of monoclonal antibodies and related products B Analytical technologies in the biomedical and life sciences

Author:
Beck, Alain,   et al.   ; Gahoual, Rabah; Leize-Wagner, Emmanuelle; François, Yannis-Nicolas;     Show all 4 Authors
Source:
Journal of chromatography 2016 v.1032 pp. 61-78
ISSN:
1570-0232
Subject:
biopharmaceuticals; capillary zone electrophoresis; chromatography; isoelectric focusing; mass spectrometry; monoclonal antibodies; proteins
Abstract:
... Out of all categories, monoclonal antibodies (mAbs), biosimilar, antibody-drug conjugates (ADCs) and Fc-fusion proteins attract the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide comprehensive in-depth characterization of these molec ...
DOI:
10.1016/j.jchromb.2016.05.028

18. Cutting‐edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies

Author:
Beck, Alain,   et al.   ; Debaene, François; Diemer, Hélène; Wagner‐Rousset, Elsa; Colas, Olivier; Dorsselaer, Alain Van; Cianférani, Sarah;     Show all 7 Authors
Source:
Journal of mass spectrometry 2015 v.50 no.2 pp. 285-297
ISSN:
1076-5174
Subject:
antibodies; deuterium; dissociation; electron transfer; electrophoresis; liquid chromatography; mass spectrometry
Abstract:
... The approval process for antibody biosimilars relies primarily on comprehensive analytical data to establish comparability and high similarity with the originator. Mass spectrometry (MS) in combination with liquid chromatography (LC) and electrophoretic methods are the corner stone for comparability and biosimilarity evaluation. In this special feature we report head‐to‐head comparison of trastuzu ...
DOI:
10.1002/jms.3554

19. Determination of isoelectric points and relative charge variants of 23 therapeutic monoclonal antibodies B Analytical technologies in the biomedical and life sciences

Author:
Beck, Alain,   et al.   ; Goyon, Alexandre; Excoffier, Melissa; Janin-Bussat, Marie-Claire; Bobaly, Balazs; Fekete, Szabolcs; Guillarme, Davy;     Show all 7 Authors
Source:
Journal of chromatography 2017 v.1065-1066 pp. 119-128
ISSN:
1570-0232
Subject:
cation exchange chromatography; isoelectric focusing; isoelectric point; monoclonal antibodies
Abstract:
... Despite the popularity of therapeutic monoclonal antibodies (mAbs), data relative to their ionic physico-chemical properties are very scarce in the literature. In this work, isoelectric points (pIs) of 23 Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved mAbs were determined by imaged capillary isoelectric focusing (icIEF), and ranged from 6.1 to 9.4. The obtained val ...
DOI:
10.1016/j.jchromb.2017.09.033

20. Development of a fast workflow to screen the charge variants of therapeutic antibodies A

Author:
Beck, Alain,   et al.   ; Wagner-Rousset, Elsa; Fekete, Szabolcs; Morel-Chevillet, Laura; Colas, Olivier; Corvaïa, Nathalie; Cianférani, Sarah; Guillarme, Davy;     Show all 8 Authors
Source:
Journal of chromatography 2017 v.1498 pp. 147-154
ISSN:
0021-9673
Subject:
cation exchange chromatography; drug therapy; drugs; immunoglobulin G; monitoring; monoclonal antibodies; peptide mapping; research and development; risk
Abstract:
... Chemical or enzymatic modifications of therapeutic monoclonal antibodies (mAbs) having high risk towards safety and efficacy are defined as critical quality attributes (CQAs). During therapeutic mAbs process development, a variety of analytical techniques have to be used for the thorough characterization and quantitative monitoring of CQAs. This paper describes the development of a rapid analytica ...
DOI:
10.1016/j.chroma.2017.02.065