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Molecular computations based on quantum mechanics and statistical mechanics have been applied to the understanding and quantification of processes leading to the degradation of therapeutic proteins. In particular, we focus on oxidation and aggregation. Specifically, two reactions, hydrogen transfer of hydrogen peroxide to form water oxide and the oxidation of dimethyl sulfide (DMS) by hydrogen peroxide to form dimethyl sulfoxide, were studied as models of these processes in general. Reaction barriers of the hydrogen transfer of H₂O₂ are in average of 10 kcal/mol or higher than the oxidation of DMS. Therefore, a two step oxidation mechanism in which the transfer of hydrogen atom occurs first to form water oxide and the transfer of oxygen to substrate occurs as the second step, is unlikely to be correct. Our proposed oxidation mechanism does not suggest a pH dependence of oxidation rate within a moderate range around neutral pH (i.e. under conditions in which hydronium and hydroxide ions do not participate directly in the reaction), and it agrees with experimental observations over moderate pH values. In the field of aggregation, we have developed a relatively simple approach for computing the change in chemical potential of a protein upon addition of an excipient (cosolute) to the protein solution. We have also developed a general approach to the design of excipients to prevent aggregation and are currently testing it experimentally. / Singapore-MIT Alliance (SMA)

PEGylation of protein side-chains has been used for more than 30 years to enhance the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, there are no structure- or sequence-based guidelines for selecting sites that provide optimal PEG-based pharmacokinetic enhancement with minimal loss to biological activity. Chapter 1 is a brief introduction to protein PEGylation. In chapter 2 we use the WW domain of the human protein Pin 1 (WW) as a model system to probe the impact of PEG on protein conformational stability. Using a combination of experimental and theoretical approaches, we develop a structure-based method for predicting which sites within WW are most likely to experience PEG-based stabilization, and show that this method correctly predicts the location of a stabilizing PEGylation site within the chicken Src SH3 domain. PEG-based stabilization in WW is associated with enhanced resistance to proteolysis, is entropic in origin, and likely involves disruption by PEG of the network of hydrogen-bound solvent molecules that surround the protein. Chapter 3 shows that PEG-based stabilization of the WW domain depends strongly on the identity of the PEG-protein linker, with the most stabilizing linkers involving conjugation of PEG to an Asn side-chain amide nitrogen. Chapter 4 investigates the interplay between structure-based guidelines for PEG-base stabilization developed in chapter 2 and the different chemistries explored in chapter 3.

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A method for extraction of therapeutic proteins from dried blood spots (DBS) followed by quantification on Gyrolab(TM) has been developed. The method makes it possible to measure the concentration of the analyte in the range 100-6000 ng/mL. The procedure can generate full analytical information from 15 μL blood originally sampled from a subject. The modest sample requirements allows for sampling a full pre-clinical pharmacokinetic profile from a single mouse. This may allow for reduced usage of animals during preclinical development of new therapeutic proteins in accordance with the 3R’s, replace, refine and reduce.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Au cours de cette thèse, nous avons été amenés à développer des méthodes analytiques afin de contrôler la qualité de protéines thérapeutiques en tant que produits finis ou d'évaluer le potentiel de certaines protéines à être des candidats médicaments. Deux protéines ont été étudiées : l'albumine de sérum humain (HSA) et l'antithrombine (AT). Ces protéines diffèrent par leur structure, leur glycosylation et leurs activités biologiques. Une méthode d'électrophorèse capillaire de zone (CZE) a permis de séparer neuf formes dans des préparations commerciales d'HSA plasmatique, dont des formes native, cystéinylée, glyquées et tronquées. Une autre méthode CZE a permis de séparer plus de huit formes dans des préparations commerciales d'AT plasmatique, dont des formes native, latente et hétérodimérique. Les deux méthodes CZE développées ont permis de mettre en évidence des différences significatives quant à la composition de préparations commerciales d'HSA ou d'AT produites par cinq fournisseurs différents. Des méthodes d'électrophorèse capillaire couplée à la spectrométrie de masse (CE-MS), avec un analyseur quadripôle-temps de vol (Q-TOF) et une ionisation électrospray (ESI), ont également été développées. Ces méthodes CE-MS ont permis d'identifier non seulement de nombreuses glycoformes et formes apparentées de l'AT, mais également des formes dimériques. Une méthode CE-MS en conditions non-dénaturantes a permis de montrer que la forme native et la forme latente de l'AT (deux conformères) présentaient un spectre de masse spécifique, permettant de les différencier sans ambiguïté. Afin d'évaluer l'affinité de variants d'AT pour l'héparine, une méthode d'électrophorèse capillaire d'affinité (ACE) a été développée. Cette méthode ACE a permis d'analyser les variants d'AT directement à partir des surnageants de culture cellulaire dans lesquels ils sont produits. Ainsi, il a été montré que certains variants présentaient une affinité très élevée pour l'héparine, en accord avec les mutations réalisées. Enfin, dans le cadre des études d'affinité entre l'AT et l'héparine, nous avons exploré une méthode nouvelle basée sur l'analyse de la dispersion de Taylor (TDA). / During this Ph.D., we developed analytical methods in order to check the quality of therapeutic proteins as finished products or to assess the potential of some proteins to be drug candidates. Two proteins were studied : human serum albumin (HSA) and antithrombin (AT). These proteins are very different regarding their structure, glycosylation, and biological functions. A capillary zone electrophoresis (CZE) method enabled to separate nine forms in commercial preparations of HSA issued from human plasma, among which native, cysteinylated, glycated, and truncated forms. Another CZE method allowed separating more than eight forms in commercial preparations of AT issued from human plasma, among which native, latent, and heterodimeric forms. Both CZE methods enabled to highlight significant differences in the composition of marketed preparations produced by five competitive pharmaceutical companies. Capillary electrophoresis-mass spectrometry (CE-MS) methods, using a quadrupole-time of flight (Q-TOF) analyzer and electrospray ionization (ESI), were also developed. These CE-MS methods enabled to identify not only a high number of glycoforms and related forms of AT, but also dimeric forms. A CE-MS method developed in non-denaturing conditions showed that native and latent forms of AT (two conformers) exhibited a specific mass spectrum, allowing to unambiguously distinguish them. With the aim to assess the binding affinity of AT variants toward heparin, we developed an affinity capillary electrophoresis (ACE) method. This ACE method enabled to analyze AT variants directly from the cell culture supernatants used to produce them. It has been shown that some AT variants had a very high affinity for heparin, in good agreement with the performed mutations. Finally, a new method based on Taylor Dispersion Analysis (TDA) was investigated to study the affinity between AT issued from plasma and heparin.

L’immunogénicité des biothérapies constitue une limitation majeure au traitement des patients atteints de maladies chroniques et se traduit par la production d’anticorps dirigés contre le biomédicament (anti-drug antibodies, ADA). La détection d’ADA de haute affinité et d’isotypes divers chez les patients suggère la mise en place d’une réponse immunitaire adaptative classique orchestrée par les cellules dendritiques. Par ailleurs, la présence d’agrégats protéiques dans les spécialités administrées serait un des facteurs favorisant l’immunogénicité, ces agrégats pouvant jouer le rôle de signal de danger.L’objectif de notre travail était de mieux comprendre les interactions des agrégats de protéines avec les cellules dendritiques et les lymphocytes T aboutissant au déclenchement d’une réponse immunitaire adaptative spécifique nécessaire à la production d’ADA.Dans un premier temps, nous avons montré que des agrégats d’infliximab, anticorps monoclonal anti-TNFa;, induisaient la maturation de cellules dendritiques humaines dérivées de monocytes (moDC). Celle-ci se traduit par l’augmentation de l’expression de marqueurs membranaires d’activation et de costimulation et la sécrétion de cytokines et chimiokines pro-inflammatoires. Nous avons également montré que ces modifications phénotypiques induites par les agrégats favorisaient la prolifération de lymphocytes T CD4+ et la production de cytokines. Par la suite, nous avons décrit les mécanismes cellulaires précoces impliqués dans l’activation de ces cellules en montrant que la neutralisation du récepteur FcgRIIa et de la tyrosine kinase Syk inhibait la maturation des moDC ainsi que l’activation des lymphocytes T CD4+.Par ailleurs, nous avons évalué le rôle des agrégats d’infliximab dans la génération de néo-épitopes, en mettant en évidence l’existence d’un répertoire de lymphocytes T CD4+ naïfs reconnaissant spécifiquement les agrégats d’infliximab, chez le sujet sain. Ainsi, grâce à des préparations d’agrégats bien caractérisées et d’un modèle de co-cultures autologues de lymphocytes T CD4+ naïfs et de moDC chargées avec les agrégats, nous avons montré que la fréquence de LT CD4+ naïfs spécifiques des agrégats d’infliximab est plus importante que celle retrouvée pour l’anticorps natif. Ces résultats suggèrent une présentation accrue d'épitopes et de néo-épitopes dérivant des agrégats d'infliximab.In fine, ce travail contribue à une meilleure compréhension des conséquences biologiques de l’agrégation des protéines à visée thérapeutique sur le déclenchement de la réponse immunitaire adaptative spécifique en mettant l’accent sur les rôles adjuvant et antigénique des agrégats protéiques. / Immunogenicity of biotherapeutic proteins is a major drawback in the treatment of patients with chronic diseases characterized by the production of anti-drug antibodies (ADA). The detection of ADA with high affinity and of various isotypes suggests a CD4 T cell-dependent adaptive immune response with a pivotal role for dendritic cells. Among other factors, the presence of protein aggregates in the administered products can promote immunogenicity, as aggregates seem to act as danger signals.The aim of our work is to better understand the interactions of protein aggregates with dendritic cells and T cells, leading to the establishment of a specific adaptive immune response needed for the production of ADA.We first showed that aggregation of infliximab, a monoclonal anti-TNFa; antibody, induced the maturation of human monocyte-derived dendritic cells (moDC) via an increase in the expression of activation and costimulatory surface markers and the secretion of pro-inflammatory cytokines and chemokines. Moreover, we showed that these phenotypic changes induced by aggregates promote CD4 T-cell proliferation and cytokine production. Subsequently, we described the early events involved in moDC and T-cell response by showing that the neutralization of the FcgRIIa receptor and the tyrosine kinase Syk inhibited moDC maturation and CD4 T-cell activation.Furthermore, we evaluated the involvement of infliximab aggregates in the generation of neo-epitopes by identifying a naïve CD4 T-cell repertoire recognizing infliximab aggregates in healthy subjects. By testing well characterized aggregate preparations and using an autologous co-culture model of naïve CD4 T cells and moDC loaded with aggregates, we showed that the frequency of naïve CD4 T cells specific for infliximab aggregates was higher than the one found for the native antibody. These results suggested an increased presentation of epitopes and neo-epitopes derived from infliximab aggregates.In resume, our work contributes to a better understanding of the biological consequences of therapeutic protein aggregation on the onset of the specific adaptive immune response by focusing on the adjuvant and antigenic role of protein aggregates.

The covalent attachment of poly(ethylene glycol) (PEG) to a protein surface (known as PEGylation), has been demonstrated to increase the serum half-life of therapeutic proteins by reducing kidney clearance and immunogenicity and by protecting against proteolysis. Theses beneficial effects could be further enhanced if PEGylation consistently increased protein conformational stability (i.e. the difference in free energy between the folded and unfolded states). However, the effects of PEGylation on protein conformational stability are unpredictable; PEGylation has been reported to increase, decrease, or have no effect on the conformational stability of medicinal proteins.This thesis details the results of two studies aimed at discovering the structural determinants which influence the thermodynamic impact of PEGylation on the WW domain, a small model protein. Chapter 1 is a brief introduction to protein therapeutics and protein PEGylation. Chapter 2 describes a study which demonstrates that the thermodynamic impact of PEGylation is strongly dependent on the site to which PEG is conjugated. The studies described in Chapter 3 elaborate on this site dependence, and demonstrate that PEG stabilizes the WW domain through interactions with the surface of the folded peptide, and that two factors – the orientation of the PEG chain (relative to the protein surface) and the identity of nearby side chains – play a critical role in determining the thermodynamic impact of PEGylation.