Understanding the rheology of liquid protein formulations

The work described in this thesis had the main aim of understanding protein solution rheology. This was from a biopharmaceutical perspective, with account of the biophysical properties of proteins and in particular their level of aggregation. Molecular interactions influencing the rheology of a range of protein solutions were studied. Proteins were selected to relate directly to the diversity of protein types used in biopharmaceuticals. In addition, the roles of a surfactant formulation additive and synthetic amphiphilic polymers in the flow behaviour of protein solutions were studied. The effect of protein concentration on solution viscosity in a commercially available biopharmaceutical formulation of a recombinant albumin (rAlbumin) was studied. The effect of the level of protein aggregation, variation in protein concentration and its impact on solution viscosity was revealed. Theoretical models predicting the increase of viscosity with concentration were applied to these data. A recent model that accounts for multiple protein species in solution, predicted the experimental data best. The rAlbumin study, although a relatively simple system, represented a 'real-life' formulation with results highlighting the need to account for heterogeneity in the level of aggregation when addressing the increase of viscosity observed at high concentration of protein solutions. Beta-lactoglobulin (b-LG) excipient-free solutions were characterised by bulk and interfacial shear rheology. Solutions at various concentrations, characterised using conventional rheology instrumentation, evidenced an apparent yield stress behaviour at a low shear rate range (0.01 - 10 1/s), whilst showing constant viscosities throughout higher shear rates. Comparing interfacial shear rheology, air-water interface-free bulk rheology measurements, and tensiometry results, it was demonstrated that the complexity of this protein's solution rheology was due to the formation of a protein viscoelastic film at the air-water interface, as present in conventional rheometry. This is in agreement with literature. Further studies considered the effect of insoluble b-LG aggregates on the solutions' rheology, linking with their characterisation in size and quantication. The presence of insoluble proteinaceous particles was suggested to have an impact on the solution's flow behaviour, particularly at the lower shear rates. Excipient-free monoclonal antibody (mAb) solutions were studied with the aim of generating protein aggregates (soluble and insoluble) to explore their impact on solution rheology. mAb samples were subjected to thermal stress and were characterised for their purity, aggregate content and size. The change in species content did not alter the original protein's yield-stress behaviour at low shear rates. An increase in aggregate content was related to the increase of viscosities observed at high shear rates. Establishing a relationship between species content (in volume fraction) and viscosities, as for the rAlbumin study, was not possible due to this mAbs specific aggregation behaviour. However, from the b-LG and mAb case studies, our results highlight the importance of detailed characterisation of protein solutions with orthogonal biophysical techniques so as to better understand protein solution rheology. An additional study looking at the effect of polysorbate-80 upon protein rheology was made. In agreement with literature, this commonly used excipient in biopharmaceuticals was demonstrated to affect the rheological measurements of globular protein solutions. Amphiphilic brush-like poly(ethylene glycol) methacrylate polymers were also synthesised and tested as novel additives with b-LG and mAb solutions, for their potential effects on protein solution rheology, similar to those observed with polysorbate-80. Preliminary results showed that the effects of these polymers are likely related to competition for the air-water interface, between these and the proteins involved. This competition leads to changes in the yield-like behaviour at low shear rates

Understanding the rheology of liquid protein formulations

The work described in this thesis had the main aim of understanding protein solution rheology. This was from a biopharmaceutical perspective, with account of the biophysical properties of proteins and in particular their level of aggregation. Molecular interactions influencing the rheology of a range of protein solutions were studied. Proteins were selected to relate directly to the diversity of protein types used in biopharmaceuticals. In addition, the roles of a surfactant formulation additive and synthetic amphiphilic polymers in the flow behaviour of protein solutions were studied. The effect of protein concentration on solution viscosity in a commercially available biopharmaceutical formulation of a recombinant albumin (rAlbumin) was studied. The effect of the level of protein aggregation, variation in protein concentration and its impact on solution viscosity was revealed. Theoretical models predicting the increase of viscosity with concentration were applied to these data. A recent model that accounts for multiple protein species in solution, predicted the experimental data best. The rAlbumin study, although a relatively simple system, represented a 'real-life' formulation with results highlighting the need to account for heterogeneity in the level of aggregation when addressing the increase of viscosity observed at high concentration of protein solutions. Beta-lactoglobulin (b-LG) excipient-free solutions were characterised by bulk and interfacial shear rheology. Solutions at various concentrations, characterised using conventional rheology instrumentation, evidenced an apparent yield stress behaviour at a low shear rate range (0.01 - 10 1/s), whilst showing constant viscosities throughout higher shear rates. Comparing interfacial shear rheology, air-water interface-free bulk rheology measurements, and tensiometry results, it was demonstrated that the complexity of this protein's solution rheology was due to the formation of a protein viscoelastic film at the air-water interface, as present in conventional rheometry. This is in agreement with literature. Further studies considered the effect of insoluble b-LG aggregates on the solutions' rheology, linking with their characterisation in size and quantication. The presence of insoluble proteinaceous particles was suggested to have an impact on the solution's flow behaviour, particularly at the lower shear rates. Excipient-free monoclonal antibody (mAb) solutions were studied with the aim of generating protein aggregates (soluble and insoluble) to explore their impact on solution rheology. mAb samples were subjected to thermal stress and were characterised for their purity, aggregate content and size. The change in species content did not alter the original protein's yield-stress behaviour at low shear rates. An increase in aggregate content was related to the increase of viscosities observed at high shear rates. Establishing a relationship between species content (in volume fraction) and viscosities, as for the rAlbumin study, was not possible due to this mAbs specific aggregation behaviour. However, from the b-LG and mAb case studies, our results highlight the importance of detailed characterisation of protein solutions with orthogonal biophysical techniques so as to better understand protein solution rheology. An additional study looking at the effect of polysorbate-80 upon protein rheology was made. In agreement with literature, this commonly used excipient in biopharmaceuticals was demonstrated to affect the rheological measurements of globular protein solutions. Amphiphilic brush-like poly(ethylene glycol) methacrylate polymers were also synthesised and tested as novel additives with b-LG and mAb solutions, for their potential effects on protein solution rheology, similar to those observed with polysorbate-80. Preliminary results showed that the effects of these polymers are likely related to competition for the air-water interface, between these and the proteins involved. This competition leads to changes in the yield-like behaviour at low shear rates

Environmental pH influences Candida albicans biofilms regarding its structure, virulence and susceptibility to fluconazole

Candida albicans colonizes sites with different environmental pH. However, it is unclear how these conditions can interfere on biofilms. This study aimed to evaluate the influence of environmental pH on behavior of C. albicans regarding its structure, virulence and susceptibility to fluconazole (FLZ). Minimal inhibitory concentration, minimal fungicidal concentration and time kill were used to evaluate the susceptibility to FLZ in planktonic cells under three pH values (4.0, 5.5, 7.0). These pH values were used for biofilms analysis. C. albicans ATCC 90028 was developed on poly(-methlymethacrylate) resin for 48 h. Then, 2.56 mu g/mL of FLZ was added to experimental groups for 24 h, and biofilms were analyzed by cell quantification, bioactivity, secretion of proteinases and phospholipases and structure. All data were analyzed by two-way ANOVA, followed by Tukey's test (alpha = 0.05). For planktonic cells, changes in environmental pH decreased the susceptibility to FLZ. C. albicans biofilms developed at pH 5.5 showed higher cell counts, bioactivity, bio-volume, average thickness and roughness coefficient (p 0.05), but increased secretion of proteinase and phospholipase (p < 0.05). Within the conditions studied, it was shown that environmental pH modulates the structure, virulence and susceptibility of C albicans to FLZ69-703944CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ132399/2012-1; 2006-7/30814

Three-dimensional instability of a ow past a sphere: Mach evolution of the regular and Hopf bifurcations

A fully three-dimensional linear stability analysis is carried out to investigate the unstable bifurcations of a compressible viscous fluid past a sphere. A time-stepper technique is used to compute both equilibrium states and leading eigenmodes. In agreement with previous studies, the numerical results reveal a regular bifurcation under the action of a steady mode and a supercritical Hopf bifurcation that causes the onset of unsteadiness but also illustrate the limitations of previous linear approaches, based on parallel and axisymmetric base flow assumptions, or weakly nonlinear theories. The evolution of the unstable bifurcations is investigated up to low-supersonic speeds. For increasing Mach numbers, the thresholds move towards higher Reynolds numbers. The unsteady fluctuations are weakened and an axisymmetrization of the base flow occurs. For a sufficiently high Reynolds number, the regular bifurcation disappears and the flow directly passes from an unsteady planar-symmetric solution to a stationary axisymmetric stable one when the Mach number is increased. A stability map is drawn by tracking the bifurcation boundaries for different Reynolds and Mach numbers. When supersonic conditions are reached, the flow becomes globally stable and switches to a noise-amplifier system. A continuous Gaussian white noise forcing is applied in front of the shock to examine the convective nature of the flow. A Fourier analysis and a dynamic mode decomposition show a modal response that recalls that of the incompressible unsteady cases. Although transition in the wake does not occur for the chosen Reynolds number and forcing amplitude, this suggests a link between subsonic and supersonic dynamics

Methods for network meta-analysis of continuous outcomes using individual patient data : a case study in acupuncture for chronic pain

Background: Network meta-analysis methods, which are an extension of the standard pair-wise synthesis framework, allow for the simultaneous comparison of multiple interventions and consideration of the entire body of evidence in a single statistical model. There are well-established advantages to using individual patient data to perform network meta-analysis and methods for network meta-analysis of individual patient data have already been developed for dichotomous and time-to-event data. This paper describes appropriate methods for the network meta-analysis of individual patient data on continuous outcomes. Methods: This paper introduces and describes network meta-analysis of individual patient data models for continuous outcomes using the analysis of covariance framework. Comparisons are made between this approach and change score and final score only approaches, which are frequently used and have been proposed in the methodological literature. A motivating example on the effectiveness of acupuncture for chronic pain is used to demonstrate the methods. Individual patient data on 28 randomised controlled trials were synthesised. Consistency of endpoints across the evidence base was obtained through standardisation and mapping exercises. Results: Individual patient data availability avoided the use of non-baseline-adjusted models, allowing instead for analysis of covariance models to be applied and thus improving the precision of treatment effect estimates while adjusting for baseline imbalance. Conclusions: The network meta-analysis of individual patient data using the analysis of covariance approach is advocated to be the most appropriate modelling approach for network meta-analysis of continuous outcomes, particularly in the presence of baseline imbalance. Further methods developments are required to address the challenge of analysing aggregate level data in the presence of baseline imbalance

Characterization Of Commercial Magnetorheological Fluids At High Shear Rate: Influence Of The Gap

This paper reports the experimental tests on the behaviour of a commercial MR fluid at high shear rates and the effect of the gap. Three gaps were considered at multiple magnetic fields and shear rates. From an extended set of almost two hundred experimental flow curves, a set of parameters for the apparent viscosity are retrieved by using the Ostwald de Waele model for non-Newtonian fluids. It is possible to simplify the parameter correlation by making the following considerations: the consistency of the model depends only on the magnetic field, the flow index depends on the fluid type and the gap shows an important effect only at null or very low magnetic fields. This lead to a simple and useful model, especially in the design phase of a MR based product. During the off state, with no applied field, it is possible to use a standard viscous model. During the active state, with high magnetic field, a strong non-Newtonian nature becomes prevalent over the viscous one even at very high shear rate; the magnetic field dominates the apparent viscosity change, while the gap does not play any relevant role on the system behaviour. This simple assumption allows the designer to dimension the gap only considering the non-active state, as in standard viscous systems, and taking into account only the magnetic effect in the active state, where the gap does not change the proposed fluid model

Limites da participação de cidadãos na política pública de saúde:: proposta de uma metodologia de avaliação

A gestão participativa nos conselhos locais de saúde&nbsp; é uma possibilidade de se estabelecer democraticamente as regras, as normas e&nbsp; acima de tudo, a possibilidade de melhorar o desempenho e a efetividade da política pública de saúde. Os conselhos de saúde no Brasil&nbsp; têm atribuições deliberativas e funcionam em caráter permanente, com os mais diversos segmentos da sociedade. No entanto, esses mesmos conselhos apresentam limitações no que diz respeito à deliberação e fiscalização da política pública. Este artigo discute portanto as limitações que os conselhos de saúde apresentam, a partir da identificação de determinantes da gestão participativa. A pesquisa é de natureza qualitativa e descritiva. Os resultados da pesquisa apontaram que a gestão participativa é fato estimulador de criação de espaços de mobilização, apesar das dificuldades e limitações. Estas dificuldades são entendidas como a interrupção de processos, falta de novas lideranças, não controle dos recursos, interferência da opinião técnica e apresentação de projetos que não se ajustam às demandas da população. Portanto, à medida que o modelo de gestão desses conselhos avança em direção à maior participação, o desempenho da política pública local de saúde tende a ser mais eficiente

The Novel Collagen Matrikine, Endotrophin, is Associated with Mortality and Cardiovascular Events in Patients with Atherosclerosis

Background: Rupture of atherosclerotic plaques is the major cause of acute cardiovascular events. The biomarker PRO-C6 measuring Endotrophin, a matrikine of collagen type VI, may provide valuable information detecting subjects in need of intensified strategies for secondary prevention. Objective: In this study, we evaluate endotrophin in human atherosclerotic plaques and circulating levels of PRO-C6 in patients with atherosclerosis, to determine the predictive potential of the biomarker. Methods: Sections from the stenotic human carotid plaques were stained with the PRO-C6 antibody. PRO-C6 was measured in serum of patients enrolled in the Carotid Plaque Imagining Project (CPIP) (discovery cohort, n = 577) and the innovative medicines initiative surrogate markers for micro- and macrovascular hard end-points for innovative diabetes tools (IMI-SUMMIT, validation cohort, n = 1,378). Median follow-up was 43 months. Kaplan–Meier curves and log-rank tests were performed in the discovery cohort. Cox proportional hazard regression analysis (HR with 95% CI) was used in the discovery cohort and binary logistic regression (OR with 95% CI) in the validation cohort. Results: PRO-C6 was localized in the core and shoulder of the atherosclerotic plaque. In the discovery cohort, PRO-C6 independently predicted future cardiovascular events (HR 1.089 [95% CI 1.019 −1.164], p = 0.01), cardiovascular death (HR 1.118 [95% CI 1.008 −1.241], p = 0.04) and all-cause death (HR 1.087 [95% CI 1.008 −1.172], p = 0.03). In the validation cohort, PRO-C6 predicted future cardiovascular events (OR 1.063 [95% CI 1.011 −1.117], p = 0.017). Conclusion: PRO-C6 is present in the atherosclerotic plaque and associated with future cardiovascular events, cardiovascular death and all-cause mortality in two large prospective cohorts

The CD14+CD16+ inflammatory monocyte subset displays increased mitochondrial activity and effector function during acute Plasmodium vivax malaria

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax-infected patients display significant increase in circulating monocytes, which were defined as CD14(+)CD16- (classical), CD14(+)CD16(+) (inflammatory), and CD14loCD16(+) (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16(+) cells, in particular the CD14(+)CD16(+) monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14(+) were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14(+)CD16(+) monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-alpha and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14(+)CD16(+) cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection