We present results of molecular simulations, quantum mechanical calculations, and experimental data aimed towards the rational design of solvent formulations. In particular, we have found that the rate limitation of oxidation of methionine groups is determined by the breaking of O-O bonds in hydrogen peroxide, not by the rate of acidic catalysis as previously thought. We have used this understanding to design molecular level parameters which are correlated to experimental data. Rate data has been determined both for G-CSF and for hPTH(1-34). / Singapore-MIT Alliance (SMA)

No description available.

Recently reported data demonstrate that Vav3, a Rho Guanine Nucleotide Exchange Factor (Rho GEF) is overexpressed in breast tumors, coexpressed with ER, necessary for proliferation in breast cancer cells, and predictive of response to neoadjuvant endocrine therapies in patients with ER+ tumors. Such data beg the question as to what roles Vav3 plays in modulation of steroid receptor activity in breast cancer and in resistance to current hormonal therapies. Using reporter assays, I provide novel evidence that Vav3 potentiates Estrogen Receptor activity and represses Androgen Receptor activity in breast cancer cells. Vav3 potentiates ligand-dependent estrogen receptor activity in the MCF-7. A truncated, constitutively active form of Vav3, caVav3 potentiates ligand dependent ER activity in both MCF-7 and T47D. Vav3 activates Rho GTPases through its GEF domain. ER potentiation by caVav3 is dependent upon GEF activity. A caVav3 mutant with defective GEF function represses basal and ligand-mediated ER activity in T47D. Although other studies have shown that Vav3 could activate various Rho GTPases, only constitutively active Rac1 mutants potentiated ER activity in both cell lines. Contrastingly, reporter assays were used to show that caVav3 inhibits ligand-mediated AR activity in the AR+ T47D cell line by both R1881 and DHT stimulation. caVav3-mediated repression of AR activity is GEF-dependent, as caVav3 GEF mutants potentiate AR activity. Constitutively active forms of Rho GTPases were found to repress AR activity to different extents, but R1881-mediated AR activity was only significantly repressed by caCdc42. My studies of the effect of androgens on AR protein by western blot show that androgens downregulate AR protein in the highly Vav3 positive T47D cell line. Previous studies have demonstrated that androgens stabilize AR protein in MCF-7, and I now provide evidence that overexpression of Vav3 or caVav3 reverses hormone-mediated AR protein stabilization in MCF-7. These data are especially relevant given recently published data that decreased AR protein levels contributed to failure of response to MPA in patients with metastatic breast cancer. Further breast cancer studies may prove Vav3 to be a potential drug target in hormone dependent, hormone independent, and metastatic disease.

L'utilisation d'antibiotiques pour lutter contre les infections bactériennes a favorisé l'émergence de souches résistantes. Comprendre les mécanismes de résistance est crucial pour lutter contre ces pathogènes. Cette thèse propose une étude structurale d'une pompe à efflux multidrogues de Pseudomonas aeruginosa qui se compose d'un transporteur MexB, d'une protéine canal OprM et d'une protéine adaptateur MexA. Les partenaires du complexe tripartite stabilisés en nanodisques ont permis la formation du complexe in vitro. L'optimisation des conditions de production du complexe a permis de cribler les différents paramètres régissant son assemblage. L'étude structurale par cryo-ME révèle un complexe de 30 nm de long en conformation de repos. L'étude de la stabilisation des protéines membranaires par nanodisques a conduit au développement d'un système minimal, débarrassé des lipides. Ce système minimal a révélé la nécessité d'une phase lipidique autour de MexB pour l'assemblage du complexe. / Antibiotics use against bacterial infections has led to the emergence of resistance. Understanding the mechanisms underlying resistance to antibiotics is critical to fight against these pathogens. This thesis presents a structural study of a multidrug efflux pump in Pseudomonas aeruginosa, composed of a transporter MexB, an exit duct OprM and an adaptor protein MexA. The proteins reconstituted in nanodiscs allowed tripartite complex formation in vitro. Optimization of yield led to the identification of key parameters governing complex assembly. Structural cryo-EM study revealed a 30 nm long complex in a resting state. The study of membrane protein stabilization by nanodisks led to the development of a minimal system devoid of lipids. This system showed a lipid phase around MexB is required for complex formation.

Control and analysis of protein aggregation is an increasing challenge to biopharmaceutical research and development. Therefore it is important to understand the interactions, causes and analysis of particles in order to control protein aggregation to enable successful biopharmaceutical formulations. This work investigates the role of different non-ionic surfactants on protein conformational stability, as assessed by HSDSC, and on protein size stability as assessed by Dynamic Light Scattering (DLS), HIAC and MFI. BSA and IgG2 were used as model proteins. Thermal unfolding experiments indicated a very weak surfactant-immunoglobulin IgG2 interaction, compared to much stronger interactions for the BSA surfactant systems. The DLS results showed that BSA and IgG2 with different surfactants and concentration produced different levels of particle size growth. The heat treatment and aging of samples in the presence of Tween 20, Tween 80, Brij 35 and Pluronic F-68 surfactants led to an increase in the populations of larger particles for BSA samples, whereas IgG2 systems did not notably aggregate under storage conditions MFI was shown to be more sensitive than HIAC technique for measuring sub-visible particles in protein surfactant systems. Heat treatment and storage stress showed a significant effect on BSA and IgG2 protein sub-visible particle size stability. This work has demonstrated that both proteins with different Tween 20, Tween 80, Brij 35 and Pluronic F-68 concentrations, have different level of conformational and size stability. Also aging samples and heating stress bears the potential to generate particles, but this depends on surfactant type. Poor predictive correlations between the analytical methods were determined.

Owing to their key role in mucosal functioning as surface barriers with biospecific interaction potentials, the mucins are interesting candidates for use as surface modifiers in biomaterials applications. In this work, “mild” fractionation procedures were used to prepare mucins of bovine (BSM), porcine (PGM), and human (MG1) origin. Biophysicochemical analysis showed the prepared mucins to differ in size, charge, conformation, and composition. In turn, these factors were shown to govern mucin adsorption on hydrophilic and hydrophobic model surfaces. To enable for detailed coating analysis, methods for the qualitative and quantitative analysis of mucin-based coatings were developed. Of particular interest, a method for the determination of the fraction of surface-exposed, presumed bioactive proteins in a complex mucin coating was described. It was shown, using microscopy and activation assays, that mucin precoating effectively suppresses the neutrophil response towards a polymeric model biomaterial. Under optimal coating conditions, all mucins performed equally well, thus indicating them to be functionally similar. Coating analysis suggested that efficient mucin surface-shielding is critical for good mucin coating performance. Following a study on the complexation of albumin with preadsorbed mucin, we investigated the effect of mucin precoating on the conformation and neutrophil-activating properties of adsorbed host proteins. We found that mucin precoating greatly reduces the strong immune-response normally caused by adsorbed proinflammatory proteins (IgG and sIgA). Detailed coating analysis revealed that the fraction of surface-exposed protein in the mucin-protein composite influences the neutrophil response. Unexpectedly low neutrophil activation for composites containing near-monolayer concentrations of exposed IgG, suggested IgG to act synergistically with mucin on the surface. Conformational analysis supported this by showing that a preadsorbed mucin layer could stabilize adsorbed IgG through complexation. Our findings link well to the complex in vivo situation and suggest that functional mucosal mimics can be created in situ for improved biomaterials performance.

<p>  </p> <p>With the recent growth in demand for biologics across the globe, it remains critical to manufacture these biologics in solid-state to improve stability as well as to increase the ease of transportation across the world. To meet these increased demands, it is of paramount importance to use various processing methods that have shorter processing times. It is also important to understand the impact of the processing methods and various formulation components on the stability of the proteins.  In Chapter 1, a review of the various processing methods that are used in the industry along with additional processing methods that are being investigated will be discussed. The common drying methods such as lyophilization and spray drying along with the novel techniques as well as specific examples of processing parameters to improve the processing conditions that better suit the protein formulations will be mentioned. </p> <p>The studies in Chapter 2 examined the effects of processing methods (freeze drying and spray freeze drying) and the excipients on the protein structure and physical stability. Protein solids containing one of two model proteins (lysozyme or myoglobin) were produced with or without excipients (sucrose or mannitol) using freeze drying or spray freeze drying (SFD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), circular dichroism spectrometry (CD), size exclusion chromatography (SEC), BET surface area measurements, and solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS). ssFTIR and CD could identify little to no difference in the structure of the proteins in the formulation. ssHDX-MS was able to identify the population heterogeneity, which was undetectable by conventional characterization techniques of ssFTIR and CD. ssHDX-MS metrics such as Dmax and peak area showed a good correlation with the protein physical instability (loss of the monomeric peak area by size exclusion chromatography) in 90-day stability studies conducted at 40oC for lysozyme.  The higher specific surface area was associated with greater loss in monomer content for myoglobin-mannitol formulations as compared to myoglobin-only formulations. Spray freeze drying seems a viable manufacturing technique for protein solids with appropriate optimization of formulations. The differences observed within the formulations and between the processes using ssHDX-MS, BET surface area measurements, and SEC in this study provide an insight into the influence of drying methods and excipients on protein physical stability.</p> <p>Based on this work, it was identified that spray freeze drying can be a viable alternative to produce solid-state protein formulations with similar stability as the freeze drying process. However, due to the long processing times and scale-up issues involved in the spray freeze drying process, there is a necessity to explore additional drying processes. Chapter 3 focuses on using another novel technique known as electrostatic spray drying (ESD) to produce solid-state protein formulations at lower drying temperatures than conventional spray drying and its effect on protein stability. A mAb formulation was dried by either conventional spray drying or electrostatic spray drying with charge (ESD). The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), and solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Conventional spray drying of the mAb formulation at the inlet temperature of 70oC failed to generate dry powders due to poor drying efficiency; electrostatic spray drying at the same temperature at 5kV enabled the formation of powder formulation with satisfactory moisture contents. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a good correlation with the loss of the monomeric peak area measured by size exclusion chromatography in the 90-day accelerated stability study conducted at 40oC. Low-temperature (70oC inlet temperature) drying with an electrostatic charge (5kV) led to better protein physical stability as compared with the samples spray-dried at the high temperature (130oC inlet temperature) without charge.</p> <p>This study shows that electrostatic spray drying can produce solid monoclonal antibody formulation at a lower inlet temperature than traditional spray drying with better physical stability. While ESD can be a viable option for thermal-sensitive formulations, it is important to understand the impact of various formulation components on the stability of the proteins while using spray drying. Based on our previous studies, a good understanding of the effect of different sugars and the presence of surfactants on the spray-dried proteins has been established. However, the impact of the selection of buffer on protein stability has not been studied. In Chapter 4, the effect of buffer salts on the physical stability of spray dried and lyophilized formulations of a model protein, bovine serum albumin (BSA) were examined. BSA formulations with various buffers were dried by either lyophilization or spray drying. The protein powders were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). Particle characterizations such as BET surface area, particle size distribution, and particle morphology were also performed. Results from conventional techniques such as ssFTIR did not exhibit correlations with the physical stability of studied formulations. Deconvoluted peak areas of deuterated samples from the ssHDX-MS study showed a satisfactory correlation with the loss of the monomeric peak area measured by SEC (R2 of 0.8722 for spray-dried formulations and 0.8428 for lyophilized formulations) in the 90-day accelerated stability study conducted at 40oC. PXRD was unable to measure phase separation in the samples right after drying. In contrast, ssNMR successfully detected the occurrence of phase separation between the succinic buffer component and protein in the lyophilized formulation, which results in a distribution of microenvironmental acidity and the subsequent loss of long-term stability. In summary, this study demonstrated that buffer salts have less impact on physical stability for the spray-dried formulations than the lyophilized solids.</p> <p>The study in Chapter 5 looked at examining the physical stability of spray freeze dried (SFD) bovine serum albumin (BSA) solids produced using the radio frequency (RF)-assisted drying technique. BSA formulations were prepared with varying concentrations of trehalose and mannitol, with an excipient-free formulation as control. These formulations were produced using traditional spray freeze drying (SFD) or RF-assisted spray freeze drying (RFSFD). The dried formulations were then characterized using solid-state Fourier transform infrared spectroscopy (ssFTIR), Karl Fischer moisture content measurement, powder X-ray diffraction (PXRD), size exclusion chromatography (SEC), solid-state hydrogen/deuterium exchange with mass spectrometry (ssHDX-MS). Traditional characterization tools such as ssFTIR and moisture content did not have a good correlation with the physical stability of the formulations measured by SEC. ssHDX-MS metrics such as the maximum deuterium uptake (Dmax) (R2 = 0.791) and deconvoluted peak areas of the deuterated samples (R2 = 0.914) showed a satisfactory correlation with the SEC stability data. RFSFD improved the stability of formulations with 20 mg/ml of trehalose and no mannitol and had similar stability with all other formulations as compared to SFD. This study demonstrated that the RFSFD technique can significantly reduce the duration of primary drying cycle from 48 h to 27.5 h while maintaining or improving protein physical stability as compared to traditional lyophilization.</p> <p>Lastly, Chapter 6 consists of a summary of the conclusions formed from the work presented in this thesis. Furthermore, suggestions for future work are provided based on observations of results, less-explored areas of formulation and processing conditions as well as characterization tools to understand effects on protein physical stability.</p> <p><br></p>

No description available.

Control and analysis of protein aggregation is an increasing challenge to biopharmaceutical research and development. Therefore it is important to understand the interactions, causes and analysis of particles in order to control protein aggregation to enable successful biopharmaceutical formulations. This work investigates the role of different non-ionic surfactants on protein conformational stability, as assessed by HSDSC, and on protein size stability as assessed by Dynamic Light Scattering (DLS), HIAC and MFI. BSA and IgG2 were used as model proteins. Thermal unfolding experiments indicated a very weak surfactant-immunoglobulin IgG2 interaction, compared to much stronger interactions for the BSA surfactant systems. The DLS results showed that BSA and IgG2 with different surfactants and concentration produced different levels of particle size growth. The heat treatment and aging of samples in the presence of Tween 20, Tween 80, Brij 35 and Pluronic F-68 surfactants led to an increase in the populations of larger particles for BSA samples, whereas IgG2 systems did not notably aggregate under storage conditions MFI was shown to be more sensitive than HIAC technique for measuring sub-visible particles in protein surfactant systems. Heat treatment and storage stress showed a significant effect on BSA and IgG2 protein sub-visible particle size stability. This work has demonstrated that both proteins with different Tween 20, Tween 80, Brij 35 and Pluronic F-68 concentrations, have different level of conformational and size stability. Also aging samples and heating stress bears the potential to generate particles, but this depends on surfactant type. Poor predictive correlations between the analytical methods were determined.

Living cells accumulate compensatory solutes for protection against the harmful effects of extreme environmental conditions such as high salinity, temperature and desiccation. Even at high concentrations these solutes do not disrupt the normal cellular functions and at times counteract by stabilizing the cellular components. These properties of compensatory solutes have been exploited for stabilizing proteins and cells in vitro. Betaines are widespread natural compensatory solutes that have also been used in other applications such as therapeutic agents and polymerase chain reaction (PCR) enhancers. Some biomedical applications of novel synthetic analogues of natural betaines were investigated. Natural compensatory solutes are either dipolar zwitterionic compounds or polyhydroxyl compounds, and the physical basis of compensation may differ between these, so one focus was on synthetic betaines with hydroxyl substituents. The majority of the synthetic solutes stabilized different model proteins against stress factors such as high and low temperatures. The presence of hydroxyl groups improved protection against desiccation. The observed stabilization effect is not just on the catalytic activity of the enzyme, but also on its structural conformation. Synthetic compensatory solutes have a potential application as protein stabilizers. Dimethylthetin was evaluated as a therapeutic agent and found to be harmful in a sheep model. However, from the study we were able to generate a large-animal continuous ambulatory peritoneal dialysis (CAPD) model and showed that glycine betaine could be added to the dialysis fluid in chronic renal failure. Some synthetic compensatory solutes reduce the melting temperatures of DNA better than most natural solutes. Synthetic solutes were identified that have potential to enhance PCR and could replace some reagents marketed by commercial suppliers. Density, viscosity and molecular model data on the solutes showed correlations with the biochemical effects of the solutes, but no physical measurements were found that reliably predicted their potential for biotechnological applications.