Development of liquid chromatographic techniques and their possible hyphenation to mass spectrometry for therapeutic protein characterization

Monoclonal antibodies (mAbs) are indispensable in the therapeutic arsenal, particularly in oncology and auto-immune diseases. Due to its biological origin, a mAb-based drug represents a heterogeneous mixture of several variants, that result from its biological production method. Some variants can potentially influence quality of final drug product and are defined as critical quality of attributes (CQAs). To be able to ensure the efficacy, the safety and the quality of drugs, a rigorous control must be carried out. This thesis work is a part of a quality approach through research and development of new analytical methods. The goal is to ensure that the therapeutic proteins do not contain any CQAs, that could potentially affect the patient health. Therefore, liquid chromatographic (LC) methods and their potential hyphenation to mass spectrometry (MS) for therapeutic protein analytical characterization have been deeply studied. The major achievement accomplished during the thesis are presented in this manuscript

La désignation de suppléants par tirage au sort (ἐπιλαχών) dans l’Athènes classique

The procedure for appointment of substitutes is known through rare instances of the verb ἐπιλαγχάνω. The bibliography is mainly based on Harpocration’s late testimony, and tends to present it as a regular practice. Literary sources and epigraphic documents such as defective lists of bouleutai invite to take a much more balanced position.La procédure de désignation de suppléants est connue à travers de rares attestations du verbe ἐπιλαγχάνω. La bibliographie consacrée à cette question, qui se base sur le témoignage tardif d’Harpocration, tend à en faire une pratique générale, régulière et systématique. L’examen des sources littéraires et épigraphiques conduit à adopter une position beaucoup plus nuancée.Laffon Amarande. La désignation de suppléants par tirage au sort (ἐπιλαχών) dans l’Athènes classique. In: Revue des Études Grecques, tome 129, fascicule 1,2016. pp. 17-37

Ἀναρχία (anarchia) in Ancient Greece

L’anarchia désigne au sens propre la vacance du pouvoir dans la sphère militaire (la mort du chef sur le champ de bataille) et dans la sphère politique (la vacance de la magistrature, en particulier éponyme). La notion est vite devenue plus générale et exprime au figuré le manque ou le refus du commandement. Elle se rapproche des sens d’insubordination, insoumission, indiscipline, licence et désordre. L’expérience effective de la vacance du pouvoir dans les cités grecques, sa représentation et sa conceptualisation constituent les axes principaux de cette recherche. L’anarchia est envisagée à l’échelle de l’âme, de la famille, de la cité, voire de l’univers. Elle engage une réflexion sur l’articulation entre deux principes a priori antagonistes, l’aspiration à la liberté et la nécessité de l’ordre, et par conséquent sur les fondements de l’exercice légitime du pouvoir. L’étude repose sur l’analyse précise des emplois du terme anarchia dans les sources épigraphiques, historiques, littéraires et philosophiques. La première partie envisage les vacances effectives des magistratures dans le cadre du cours normal ou d’une rupture du fonctionnement des institutions ainsi que les palliatifs mis en place. Les occurrences du terme anarchia concernent les cités d’Athènes, Thasos, Téos, Syros et Bérénikè. S’ajoutent les emplois problématiques des termes akosmia à propos du régime crétois par Aristote et atagia dans les inscriptions thessaliennes. La deuxième partie envisage l’évolution sémantique du terme de l’absence de chef à l’anarchie chez les historiens et les poètes tragiques et la place de l’anarchia dans les théories du commandement élaborées par Xénophon, Platon et Aristote.The term anarchia refers literally to the absence of power, in the military sphere (that caused by the loss of a commander), and the political sphere (the absence of archontes, specifically the eponymous archon). The concept quickly generalised, coming to designate in the figurative sense the lack and want of power or the rejection and negation of power. It approaches the meanings of insubordination, rebelliousness, unruliness, licentiousness and disorder. The actual experience of power vacuum in the cities of Ancient Greece and how the Greeks represented it and conceptualised it are the three main lines of this research. Anarchia is conceived not only in the city but also in the soul of the individual, in the family, or even in the universe. It demands reflection on the articulation between two seemingly antagonistic principles, the desire for freedom and the necessity of order, and consequently upon the foundations of legitimate authority. This work relies on a precise analysis of the term anarchia in the epigraphic, historical, literary and philosophical sources. The first part deals with actual periods of power vacuum in the ordinary course of political life or in the context of institutional disruption and the implemented remedies. The term anarchia is employed in the cities of Athens, Thasos, Teos, Syros and Berenike. One must add the problematical use of the terms acosmia by Aristotle regarding the Cretan regime and atagia in the Thessalian inscriptions. The second part deals with the semantic evolution of the term from the absence of ruler to anarchy in the work of historians and tragic poets and the role of anarchia in the theory of leadership developed by Xenophon, Plato and Aristotle

Atelier Hésiode Biophilies antiques ? Manières d’expliquer et d’éprouver les rapports entre humain et nature dans l’Antiquité gréco-romaine

LUNDI 21 ET MARDI 22 NOVEMBRE 2022 UNIVERSITÉ D’ANGERS Amphithéâtre Germaine Tillion Maison de la Recherche Germaine Tillion 5 bis Bd de Lavoisier 49000 Angers En présentiel et en distanciel via Teams. Lundi 21 novembre : Rapports entre humain et nature en Grèce antique 9h30 Introduction Bertrand Guest, Amarande Laffon, Frédéric Le Blay et William Pillot, membres du programme HESIODE Présidence de séance : Adeline Grand-Clément, Université Toulouse Jean-Jaurès 10h Clément Baranger..

Propagation Of Partially Coherent Beams: Turbulence-Induced Degradation

A study of propagation of coherent beams through thermally induced turbulence was presented. Systematic experiments were conducted to assess the effect of phase turbulence on the intensity profiles of beams with different degrees of spatial coherence and the effects were compared with the distortion of a spatially coherent beam propagating in the same conditions. It was found that, a partially coherent beam was less affected than a spatially coherent beam, on propagation through thermally induced turbulence

Empirical correction of non-linear pH gradients and a tool for application to protein ion exchange chromatography

This concept article reports a practical solution to improve the linearity of effluent pH response as observed in pH gradient cation exchange chromatography (CEX). When performing pH gradient CEX, it is not easy to develop buffer systems that will universally provide pH response proportional with the mobile phase (buffer) composition. It is an especially challenging pursuit when exploring MS compatible buffers (e.g. ammonium-acetate, ammonium-carbonate). In addition to "non-proportional" behavior from the mobile phase composition, the chromatographic column itself will sometimes impose an unpredictable impact on the effluent pH. Here, we propose a simple approach based on the on-line measurement of effluent pH response, conversion of pH to mobile phase volume fraction (φ) and then generation of the inverse response function in the time domain. In the end, when setting the inverse function as the gradient program instead of a linear gradient, an improved - ideally linear - pH response can be produced. A simple Excel tool was developed to assist analysts with this correction procedure, and it has been made available by download for public use

The importance of being metal-free: The critical choice of column hardware for size exclusion chromatography coupled to high resolution mass spectrometry

The goal of the study was to evaluate the possibilities offered by a new generation of metal-free SEC column to perform direct SEC-MS of protein biopharmaceuticals using ammonium acetate as the main mobile phase additive. The prototype metal-free SEC column hardware used in this work was a polyether ether ketone (PEEK) infused stainless steel tube including PEEK frits. This PEEK-lined column provides a fully bioinert and metal-free fluidic path, while maintaining the stability of the metal hardware, and could be a good solution to limit possible undesired interactions between proteins and column wall/frits. This prototype metal-free SEC column was systematically compared with a conventional stainless-steel SEC column hardware packed with the same stationary phase material. Four different mAb products, namely trastuzumab, palivizumab, bevacizumab and NISTmAb, and one antibody drug conjugate (ADC), trastuzumab emtansine, were selected as test samples. It appears that peak symmetry, separation of low molecular weight species (LMWS), and the recovery of high molecular weight species (HMWS) were significantly improved for the different biopharmaceutical products on the metal-free SEC column. It has also been demonstrated that the largest differences between standard and metal-free SEC columns were observed for the most basic mAbs (high pI), which confirms that electrostatic interactions between the mAb and the metallic parts of the column (frits and inlet tube) could be responsible for the issues observed when performing SEC analysis with volatile mobile phase. Finally, it was feasible to perform SEC-MS analysis for a wide range of biopharmaceutical products using volatile mobile phase. Our results also highlight that an inappropriate column could bias the quantification of size variants when using MS-compatible mobile phases. Therefore, metal-free column, such as the PEEK-lined column, should be preferentially selected for SEC-MS analysis

Practical considerations on the particle size and permeability of ion-exchange columns applied to biopharmaceutical separations

The goal of this study was to better understand the possibilities and limitations of modern cation exchange chromatography (CEX) columns for the separation of protein biopharmaceuticals (typically mAbs and related products). Several commercial and research columns consisting of a non-porous polymeric core particle with a thin hydrophilic coating and grafted ion-exchanger sulfonate groups, were compared. The impact of particle size, porosity and packing pressure on the separation of therapeutic proteins was evaluated in a systematic way. First, it was shown that the porosity of modern CEX columns depends on the applied conditions, and lower apparent porosity as well as increased column pressures were observed when using low ionic strength mobile phase (less than 0.01 M NaCl), due to swelling. Column pressure seemed to be dependent on the 1/dp3 to 1/dp5 relationships with particle size, depending on whether 0.3 M NaCl or pure water was used as mobile phase, respectively. Using 5 cm long columns packed with 2 or 2.5 µm particles could easily result in higher than 1000 bar pressure drops when the mobile phase ionic strength is low. Therefore, it is recommended that particle size not be decreased to below 2.5 µm so that technologies can remain compatible with the current state of ultra-high pressure (UHPLC) instrumentation. This recommendation is underscored by the fact that a decrease in particle size does not produce improved separations, since the particles are non-porous (no intra-particle diffusion nor resistance to mass transfer) and that large solutes follow an on-off (bind and elute) type retention mechanism. The only advantage of CEX columns packed with small particles is that they can provide more specific surface area per unit length of column, and thus facilitate higher throughput methods. In conclusion, it appears that there is no need to further decrease the particle size in CEX since decreasing their particle size may result in more drawbacks than benefits

Current and future trends in reversed-phase liquid chromatography-mass spectrometry of therapeutic proteins

Reversed-phase liquid chromatography (RPLC) is one of the most widely used chromatographic approaches for the characterization of therapeutic proteins because it is able to answer several analytical queries and allow the fine characterization of the structural heterogeneity of these drugs. However, some issues may possibly occur when analysing therapeutic proteins by RPLC-MS, such as severe band broadening and peak tailing, strong adsorption, possible protein denaturation/degradation, limited MS sensitivity and poor chromatographic selectivity. The present review focuses on the latest advances in RPLC that have been adopted to overcome these issues, including the application of cutting-edge chromatographic conditions, alternative mobile phase compositions and critical discussions on recently commercialized bioinert instruments and RPLC stationary phases