Presently, over 300 proteins or peptide based therapeutic medicines have been approved by the FDA owing to advances in protein engineering and technology. However, majority of these protein-based medications are unstable or have limited shelf life when in aqueous form. During pre-formulation and manufacturing, various technological processes including mixing, dissolving, filling (through pipes) can produce strong mechanical stresses on proteins. These stresses may cause the protein molecule to unfold, denature or aggregate. To improve stability upon formulation, they may be manufactured as freeze dried cakes that requires reconstitution with a buffer or water prior to administration. Although it has been successful in improving the stability of protein-based formulations, the freeze drying process itself also contributes to protein aggregation. This process introduces other stresses such as freezing, thawing and drying. In addition to these stresses, the agitation processes used during reconstitution may also destabilize the protein’s native structure. Two key processes used in preparation of protein based formulations were studied in this work; mechanical agitation and freeze drying. The aim of this project was to explore the aggregation of proteins that occur due to the various technological processes typical in the production of protein based formulations. The project has two parts that relates to liquid and solid formulations. In the first part, the effect of different methods of mechanical agitations on BSA protein was investigated. In the second part, the focus was on the effect of formulation (i.e. the application of amino acids) on aggregation of protein (BSA) in freeze dried formulations. Arginine and lysine were added individually into protein-based freeze-dried formulation to study their potential of improving the stability of the proteins during manufacturing, storage and reconstitution. In the formulation development, additional excipients were added to prevent moisture uptake due to the hygroscopic properties of the amino acids and to provide lyo- and cryo- protection for the protein molecule during freeze drying. Without further purification, BSA solutions prepared by using sonication, low shear rotor mixer or high shear tube/pipe mixing were studied using dynamic light scattering (DLS). Thioflavin T assay and turbidimetry analysis were used as complementary studies. In protein-based freeze dried formulations, at accelerated storage conditions, the presence of aggregates were studied in samples containing arginine or lysine using ThT assay and turbidimetry analysis. Characterisation of the freeze dried cakes was performed relative to their moisture sorption, cake shrinkage, mechanical properties and morphology using various analytical techniques. iv In the BSA solution studies, particle size analysis indicated two distributions for non-agitated BSA solution that corresponds to the average particle sizes of BSA molecules and their aggregates. Under mechanical stresses (all types), the intensity of distribution centered ≈ 7.8 nm reduces and broadens as the agitation time increases, indicating a reduction in the amount of “free” BSA macromolecules. The second distribution, as a result of increasing agitation time or shear intensity, reveals a significant shift towards larger sizes, or even splits into two particle size populations. These particle size growths reflect the formation of aggregates due to intensive collisions and, as a result, partial unfolding followed by hydrophobic interactions of exposed non-polar amino acids. UV spectra showed that aggregation in both low shear and mechanical vibration agitations were lower compared to the high shear stress. When compared to non-agitated BSA solution, ThT assay recorded ≈15 times higher fluorescence emission from the high shear samples, ≈2 times fluorescence emission from low shear and ≈6 times fluorescence emission from mechanical vibrations. Thus all the three agitation methods showed a good correlation between the results. The second part of this project was performed in three stages. In the initial 2 stages, 2- and 3-excipients component system were investigated to develop an optimal preliminary formulations which will be used in the final protein based 4-components formulations. From the 1st stage (ArgHCl/LysHCl + sugar/polyol), among 4 tested excipients (polyol and sugar), mannitol was observed to have resisted moisture uptake by the highly hygroscopic ArgHCl/LysHCl amino acids. However, mannitol is considered a good cryoprotector but has poor lyoprotection properties. Therefore, in the following stage, a 3rd excipient (in a 3-excipients component system) sucrose or trehalose, was introduced into the formulation. The formulation was made up of 20% ArgHCl (LysHCl), and various ratios of mannitol and sugar were explored. The criteria for selecting the best systems were based on ideal physicochemical properties i.e. moisture uptake, shrinkage, mechanical properties, matrix structure and appearance, and thermal properties. The final stage was the formulation of a 4-components system comprising the three excipients and combinations selected from the stage 2 studies, and the addition of BSA as the model protein. To study aggregation in this system, a freeze dried 4-components excipient/protein system was reconstituted and incubated at accelerated storage conditions over time. Fluorescence spectroscopy and turbidimetry were used to study aggregation of proteins, moisture uptake kinetics with gravimetric balance, and thermal analytical techniques were used to characterise the freeze dried cakes with and without BSA protein. This study represented a systematic analysis of aggregation of proteins in both liquid and solid formulations. Some of the novel aspects of this study include: v 1. The new experimental results obtained for aggregation of proteins in solution subjected to mechanical agitations. The high shear stress created by syringe agitation, simulated the real situation in post manufacturing process during filling through narrow pipes, and has been shown here to strongly affect the aggregation of protein macromolecules. 2. The development of a methodical approach for optimization of multi component (up to 4 excipients) protein based formulations. 3. The unexpected non-linear behavior of the physicochemical properties of the 3-excipients component system as a function of composition. To the best of my knowledge, this novel aspect has not been previously reported in literature. 4. Application of amino acid in protein based formulations has shown the inhibition of aggregation of BSA, with the highest effect observed with ArgHCl. The results of this study coincide with the conclusions published previously for aggregation of proteins in solution.

Applications of Hot Melt Extrusion (HME) in pharmaceuticals have become increasingly popular over the years but nonetheless a few obstacles still remain before wide scale implementation. In many instances these improvements are related to both processing and product performance. It is observed that HME process optimisation is majorly focused on the active pharmaceutical ingredient's (API) properties. Characterising polymeric properties for their suitability in HME should be equally studied since the impact of excipients on both product and process performance is just as vital. In this work, two well-established cellulose ester derivatives: Hydroxy Propyl Methyl Cellulose Acetate Succinate (HPMCAS) and Hydroxy Propyl Methyl Cellulose Phthalate (HPMCP) are studied for their HME suitability. Their thermal, thermodynamic, rheological, thermo-chemical and degradation kinetic properties were evaluated with model plasticisers and APIs. It was found the thermal properties of HPMCP are severely compromised whereas HPMCAS is more stable in the processing zone of 150 to 200 °C. Thermodynamic properties revealed that both polymers share an important solubility parameter range (20-30 MPa P1/2P) where the majority of plasticisers and BCS class II APIs lie. Thus, greater miscibility/solubility can be expected. Further, the processability of these two polymers investigated by rheometric measurements showed HPMCAS possesses better flow properties than HPMCP because HPMCP forms a weak network of chain interactions at a molecular level. However, adding plasticisers such as PEG and TEC the flow properties of HPMCP can be tailored. The study also showed that plasticisers have a major influence on thermo-chemical and kinetic properties of polymers. For instance, PEG reduced polymer degradation with reversal in kinetic parameters whereas blends of CA produced detrimental effects and increased polymer degradation with reduction in onset degradation temperatures. Further, both polymers are observed to be chemically reactive with the APIs containing free -OH, -SOR2RN- and -NH2 groups. Finally, these properties prove that suitability of HPMCP is highly debated for HME and demands great care in use while that of HPMCAS is relatively better than HPMCP in many instances.

Les protéines extraites des feuilles de luzerne sont une source importante de protéines. La filtration membranaire, technologie de séparation respectueuse de l’environnement avec une productivité élevée et de faible coût a été utilisée pour séparer et concentrer les protéines des feuilles de luzerne à partir de leur jus. Cependant le phénomène du colmatage de la membrane qui réduit sérieusement le flux et la séparation des protéines est un facteur limitant important dans l'application de la filtration membranaire. Pour améliorer la récupération des protéines et amenuiser le phénomène du colmatage, la filtration membranaire associée à fort cisaillement a été utilisée pour la filtration du jus de luzerne. Toujours dans l’objectif d'optimiser le processus de la filtration, "le mode de la filtration" et "les paramètres de fonctionnement" ont été étudiés pour réduire le colmatage de la membrane et améliorer le rendement de la filtration. Puis, l’effluent du jus de luzerne a été filtré par des membranes dans des conditions de fort cisaillement afin de recycler les protéines. En outre, le mécanisme du colmatage a été étudié et a permis d’évaluer les stratégies de contrôle du colmatage. L'optimisation du procédé membranaire, via l’étude du "mode de filtration" et des "paramètres de fonctionnement" a été conduit dans le but d’améliorer la séparation et la concentration des protéines et de réduire le colmatage. Trois types de « mode de filtration » ont été testés : la filtration frontale sur le module de la cellule amicon (DA), la filtration tangentielle dynamique sur le module à disque rotatif (CRDM) et la filtration frontale sur le module à disque rotatif (DRDM)). Les « paramètres de fonctionnement » qui ont été étudiés sont les suivants : le type de membranes (ultrafiltration (UF) et microfiltration (MF)), la vitesse de rotation, la température et la pression transmembranaire (TMP). Le comportement du débit (évolution du flux du perméat au cours de la filtration), les performances de la séparation (taux de clarification et de concentration), l’efficacité du nettoyage de la membrane (récupération de la perméabilité membranaire) et la productivité lors des tests de recyclage et de concentration ont été étudiés dans le but de définir des stratégies dans le contrôle du colmatage. Puis, l’effluent de luzerne a été filtré par UF afin de séparer et purifier les protéines. Le mécanisme du phénomène du colmatage des membranes lors de la filtration du jus de luzerne a été étudié. Le processus du colmatage de la membrane a montré une tendance d’un colmatage multi-site progressif. Le modèle du colmatage multisite progressif selon la loi de Darcy (SMDM) a été proposé afin de mieux décrire et comprendre le processus du colmatage. Les effets de la composition du fluide d’alimentation, du choix de la membrane et des conditions hydraulique ont joué un rôle important dans le processus progressif du colmatage. De plus, les coefficients de résistance et de compressibilité dans les différentes étapes et sites ont été calculés afin d’expliquer le processus complexe du colmatage et d’évaluer l'efficacité des stratégies du contrôle du colmatage. Une série d'essais avec de longues durées de filtration a été réalisée pour étudier le déclin du flux et le colmatage de la membrane à diverses étapes du processus. Ces résultats présentent une utilité pour améliorer la récupération des protéines et contrôler le colmatage dans le processus de la filtration membranaire à fort cisaillement du jus de luzerne. Ces résultats sont aussi utiles pour la conception et la mise en place des technologies membranaires dans les processus industriels. / Alfalfa leaf proteins extracted from plants are an important protein source. As an environmentally friendly separation technology with high productivity and low cost, membrane filtration was used to separate and concentrate leaf protein from alfalfa juice. However membrane fouling seriously reduces flux and protein separation and is an important limitation in the application of membrane filtration. To improve protein recovery and fouling control, dynamic shear-enhanced membrane filtration with high shear rate on membrane surface and excellent anti-fouling capacity was used for alfalfa juice filtration in this work. In order to optimize filtration process, filtration mode and operation parameters were investigated to reduce membrane fouling and improve separation performance. Then, alfalfa wastewater was also treated by dynamic shear-enhanced membrane filtration to recycle proteins. Furthermore, the fouling mechanism was studied and served as a valuable evaluation for fouling strategies. In this study, process optimization including “Filtration mode” and “Operation parameters” was studied to improve protein recovery and fouling control. In “Filtration mode”, three types of filtration modules (dead end filtration using laboratory Amicon cell (DA), dynamic cross filtration using rotating disk module (CRDM) and dead end filtration using rotating disk module (DRDM)) were used to investigate the filtration performance. As for “Operation parameters”, the operation parameters including membranes (ultrafiltration (UF) and microfiltration (MF)), rotating speed, temperature and transmembrane pressure (TMP) were studied to optimize the filtration process. Flux behavior (permeate flux and flux decline), separation performance (clarification and concentration capacity), membrane cleaning efficiency (permeability recovery) and productivity in full recycling tests and concentration tests were utilized to evaluate the various operation strategies. In addition, alfalfa wastewater was treated by UF membrane, while waste proteins were recycled. Fouling mechanism for alfalfa juice filtration was investigated. The fouling process showed significantly stepwise multisite patterns. Based on Darcy’s law, the stepwise multisite Darcy’s law model (SMDM) was proposed to better describe and understand the fouling process. The effects of feed composition, membrane and hydraulic conditions played an important role in stepwise fouling process. Moreover, the resistance coefficient and compressibility for different steps and sites were calculated to explain the complex fouling process and estimate the efficiency of flux decline control strategies. Besides, a series of long tests were utilized to study flux decline and membrane fouling at various fouling step process. These results can be used to understand the protein recovery and fouling control during shear-enhanced membrane filtration process of alfalfa juice. They have important implications for process design of membrane technology in industrial scale.

The thesis deals with study of mechnochemical activation of chemical reactions at interphase of MDF composites. MDF composites are high-perspective composite materials on polymer-cement base. They excel especially in terms of flexural strengths. High flexural stregth is caused by binding interactions between the polymer and the cement. This interactions arise due to mechanochemical activation of raw material mixture at the stage of production. This work focuses on the creation of artificial cement-polymer interphase by contacting the surfaces of two molded tablets (polyvinyl alcohol and monocalcium aluminate), activation of chemical reactions at interphase by means of specially designed appartus, which achieves good plane-parallelism of activation surfaces and good definability of activation conditions (shear rate, pressure). After experiments under different conditions, the activated surfaces are analyzed by SEM, EDS, XPS and FT-IR.

Cette thèse, qui s’inscrit dans le cadre du projet ANR Eco-Tech TRIPTIC, a eu comme objectif de contribuer au tri industriel en cadence de polymères contenant des traceurs détectables grâce à leurs propriétés en fluorescence X ou UV. Dans un premier temps, étant donné que le coût des traceurs choisis pour l’étude TRIPTIC est assez élevé, nous avons réalisé une étude préliminaire, avec des particules modèles de silice, visant à optimiser la dispersion de charges dans une matrice polypropylène/éthylène propylène rubber (PP/EPR). Nous avons montré que l’extrusion à taux de cisaillement élevé est une technologie efficace permettant une dispersion homogène de charges de tailles micro ou nanométriques. Dans un deuxième temps, après optimisation des conditions de dispersion, nous avons extrapolé nos résultats à la dispersion de particules de traceurs UV dans différentes matrices thermoplastiques. Nous avons montré que la dispersion de 1000 ppm de particules de traceurs, de tailles micrométriques, dans des matrices polypropylènes, en extrusion à haute vitesse (N= 800 rpm), n’a pas d’impact sur les propriétés mécaniques et physico-chimiques des mélanges tracés, ainsi que sur la photo-dégradation sous rayonnement UV. Cette fine dispersion a non seulement permis la conservation des propriétés des polymères tracés mais aussi une bonne détection dynamique, tant en fluorescence X qu’UV, sur un prototype conçu par des partenaires du projet (Pellenc Selective Technologies, CEA-LITT et ENSAM- LCPI). Enfin, nous avons validé l’extrusion à haute vitesse pour compatibiliser des mélanges de polymères ternaires (PP/EPR)/PE (polyéthylène) pouvant correspondre à la valorisation de polymères post-consommation, par exemple dans l’hypothèse où on souhaite recycler (PP/EPR) et PE ensembles. Les propriétés prometteuses des mélanges obtenus doivent leur permettre de trouver des applications dans l’industrie automobile, par exemple. Cette voie est d’un grand intérêt pour les applications industrielles, car elle permet d’envisager des propriétés mécaniques élevées pour les polymères recyclés. Elle ouvre aussi de nouvelles perspectives pour l’élaboration de matériaux allégés, obtenus à partir de matières vierges ou recyclées. / This thesis, which is part of the ANR Eco-Tech TRIPTIC project, had the objective of contributing to industrial sorting rate of polymers containing tracers detectable by their fluorescence X or UV properties. At first, given that the cost of tracers selected for TRIPTIC study is quite high, a preliminary study is conducted with models of silica particles to optimize the dispersion of fillers in polypropylene / ethylene-propylene rubber (PP / EPR) matrix. It was found that processing under high shear rate is an effective technology for accomplishing a homogeneous dispersion of micro or nanoscale fillers. In a second step, after optimization of dispersion conditions, our results are extrapolated to the dispersion of UV tracer in different thermoplastic matrices. It was shown that the dispersion of 1000 ppm of micrometer tracer particles, in polypropylene matrices, extruded at high shear rates (N = 800 rpm), has no impact on the mechanical and physico-chemical properties as well as in the photo-degradation of the polymer after UV irradiation exposure. This fine dispersion was beneficial not only for the conservation of the properties of traced polymers but also for achieving a good dynamic detection of UV or X tracers using a prototype developed by the project partners ( Pellenc Selective Technologies , CEA- LITT and ENSAM - RPI ). Finally, high shear processing has successfully used to the compatibilization of ternary blends ( PP / EPR ) / PE (polyethylene) which may correspond to the post-consumer polymers. This technique has proved to be an effective method to produce polymer blends with unique mechanical properties. This novel strategy of compatibilization is of a particular interest, especially for industrial application prospects. It also opens new perspectives for materials lightening as well as “high shear recycling” of immiscible polymers.

Applications of Hot Melt Extrusion (HME) in pharmaceuticals have become increasingly popular over the years but nonetheless a few obstacles still remain before wide scale implementation. In many instances these improvements are related to both processing and product performance. It is observed that HME process optimisation is majorly focused on the active pharmaceutical ingredient's (API) properties. Characterising polymeric properties for their suitability in HME should be equally studied since the impact of excipients on both product and process performance is just as vital. In this work, two well-established cellulose ester derivatives: Hydroxy Propyl Methyl Cellulose Acetate Succinate (HPMCAS) and Hydroxy Propyl Methyl Cellulose Phthalate (HPMCP) are studied for their HME suitability. Their thermal, thermodynamic, rheological, thermo-chemical and degradation kinetic properties were evaluated with model plasticisers and APIs. It was found the thermal properties of HPMCP are severely compromised whereas HPMCAS is more stable in the processing zone of 150 to 200 °C. Thermodynamic properties revealed that both polymers share an important solubility parameter range (20-30 MPa P1/2P) where the majority of plasticisers and BCS class II APIs lie. Thus, greater miscibility/solubility can be expected. Further, the processability of these two polymers investigated by rheometric measurements showed HPMCAS possesses better flow properties than HPMCP because HPMCP forms a weak network of chain interactions at a molecular level. However, adding plasticisers such as PEG and TEC the flow properties of HPMCP can be tailored. The study also showed that plasticisers have a major influence on thermo-chemical and kinetic properties of polymers. For instance, PEG reduced polymer degradation with reversal in kinetic parameters whereas blends of CA produced detrimental effects and increased polymer degradation with reduction in onset degradation temperatures. Further, both polymers are observed to be chemically reactive with the APIs containing free -OH, -SOR2RN- and -NH2 groups. Finally, these properties prove that suitability of HPMCP is highly debated for HME and demands great care in use while that of HPMCAS is relatively better than HPMCP in many instances.

Des procédés innovants basés sur l’extrusion à haute vitesse et l’irradiation sous rayonnement γ, ont été étudiés en vue d’améliorer la compatibilisation de deux polyoléfines non miscibles : le polypropylène PP et le polyéthylène PE. Dans un premier temps, l’étude de la distribution des temps de séjour de polypropylènes dans l’extrudeuse à haute vitesse a été réalisée. L’impact des conditions opératoires (débit et vitesse de rotation des vis) ainsi que l’impact du profil de vis et de la viscosité du polypropylène d’étude ont été mis en avant. Certains résultats expérimentaux sont souvent en accord avec ceux du logiciel de simulation d’extrusion bi-vis Ludovic, bien qu’il puisse y avoir des écarts pour les hautes vitesses de rotation des vis. Dans un deuxième temps, une fois l’écoulement caractérisé, nous avons réalisé des mélanges PP/PE, les paramètres qui ont varié sont la quantité d’énergie apportée durant le mélange (vitesse de rotation des vis) ainsi que la présence ou non de copolymère préformé et le type d’atmosphère utilisée durant l’extrusion. Faire varier le taux de cisaillement ne permet pas la modulation des propriétés finales du mélange surtout lorsque l’on extrude sous atmosphère inerte. L’utilisation de copolymère préformé, de type éthylène-octène, permet d’améliorer considérablement les propriétés de notre mélange en créant une interphase "cœur-couronne" entre la matrice PP, le compatibilisant et la phase dispersée PE. Pour terminer, une étape d’irradiation γ a été ajoutée au procédé de mise en œuvre classique de nos mélanges. En première partie de cette étude, la simulation de spectres RPE a permis d’identifier et quantifier les différentes espèces radicalaires créées. Dans la deuxième partie, l’influence de la place de la séquence d’irradiation a été étudiée : avant extrusion, entre extrusion et injection, après injection et avant recuit. Les meilleures améliorations ont été obtenues lorsque l’irradiation est suivie d’un traitement thermique. En conclusion, il est possible d’améliorer la compatibilité d’un mélange PP/PE en jouant essentiellement sur l’ordre des étapes de production sans avoir à modifier la formulation de ce dernier. / Innovative processes, based on high shear twin screw extrusion and γ-irradiation, have been used to increase the compatibility of two immiscible polyolefins: polypropylene PP and polyethylene PE. In a first part, the residence time distribution of polypropylenes in the twin screw extruder (TSE) has been investigated. The impact of the processing conditions (throughput and screw rotation speed), the screw profile and the polypropylene viscosity were underlined. Some of the experimental results are often in good agreement with those predicted by simulation software of twin screw extrusion (Ludovic), except for some experiments at high screw rotation rates. In a second part, once polymer flow was characterised in the TSE, we realized PP/PE blends. The impact of the mechanical energy (screw rotation speed), the presence or absence of copolymers and the nature of the extrusion atmosphere were analysed. Using high shear rate does not allow modulating the final properties of the blends particularly once inert atmosphere is used. The use of ethylene-octene copolymers increases significantly the final properties of the PP/PE blends by creating a “core-shell” morphology between the PP matrix, the copolymer and the PE dispersed phase. To finish, a γ-irradiation process step has been added to the classical processing of PP/PE blends. In a first part of this study, the simulations of ESR spectra have permitted to identify and quantify the different radicals created. In a second part, the sequence order of the γ-irradiation has been investigated: before extrusion, between extrusion and injection, and after injection followed by a thermal treatment. The best results were obtained once γ-irradiation is followed by a heat treatment. As a conclusion, the compatibility of immiscible polyolefins can be improved only by changing the order of the different processing steps without changing the formulation.

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