The Spin-Off of Scientific Services of Novartis into a New, Independent Technology Company Offering Services to the Pharmaceutical, Chemical, and Nutrition Industry

Starting on October 1, 1999, the three sections 'Central Analytics', 'Physics', and 'Catalysis Synthesis Services' of the Scientific Services of Novartis will operate as an independent company. The new company will have about 180 employees and will offer services to customers in the pharmaceutical, chemical, and nutrition industry as well as to authorities and service firms active in these fields. The focus of activities for the new company is the chemical and physical characterization (analytics), optimization of products and processes, and the development and application of special synthetic methods, in particular by utilizing catalysis. Support is offered via single services, comprehensive service packages, or by taking over assignments for entire areas. The combination of a high scientific and technical standard built up on an ISO 9001 quality-management system, including cGMP and GLP, with an attractive working environment will be the basis for an innovative center of chemical and physical expertise

Step size of the rotary proton motor in single FoF1-ATP synthase from a thermoalkaliphilic bacterium by DCO-ALEX FRET

Thermophilic enzymes can operate at higher temperatures but show reduced activities at room temperature. They are in general more stable during preparation and, accordingly, are considered to be more rigid in structure. Crystallization is often easier compared to proteins from bacteria growing at ambient temperatures, especially for membrane proteins. The ATP-producing enzyme FoF1-ATP synthase from thermoalkaliphilic Caldalkalibacillus thermarum strain TA2.A1 is driven by a Fo motor consisting of a ring of 13 c-subunits. We applied a single-molecule F\"orster resonance energy transfer (FRET) approach using duty cycle-optimized alternating laser excitation (DCO-ALEX) to monitor the expected 13-stepped rotary Fo motor at work. New FRET transition histograms were developed to identify the smaller step sizes compared to the 10-stepped Fo motor of the Escherichia coli enzyme. Dwell time analysis revealed the temperature and the LDAO dependence of the Fo motor activity on the single molecule level. Back-and-forth stepping of the Fo motor occurs fast indicating a high flexibility in the membrane part of this thermophilic enzyme.Comment: 14 pages, 7 figure

Dielectric study of triton X100: a glass-forming liquid

Dissertação para obtenção do grau de mestre em Engenharia de MateriaisThe main purpose of this work was to realize an exhaustive study on the molecular mobility of a glass-forming liquid and evaluate the influence of thermal treatment in the phase transformations undergone by the material. It was also our goal to investigate it response when subjected to confinement in nano-porous inorganic materials. The liquid selected, Triton X100, is characterized by a high dielectric response and a high tendency to crystallize by coming from both molten and glassy states. However, it is possible to find the conditions under which crystallization is avoided and the material enters in the supercooled liquid state. This allowed us to study the molecular mobility in the liquid, supercooled liquid, glassy states and as well as the crystallization and investigate temperature driven phase transformations. To get a further insight in the crystallization behaviour, isothermal crystallization at different temperatures and from both glassy and molten states was promoted and monitored in real-time by Dielectric Spectroscopy Relaxation. This study gave information about the influence of the crystallization on the remaining amorphous phase. Motivated by the recent knowledge that molecular mobility and phases transformations can be significantly altered when a glass-forming liquid is confined in the nanometer scale, the molecular dynamics of the Triton X100 was evaluated when confined in mesoporous materials (SBA-15 and MCM-41; pore size, respectively, 5.7 and 3.4 nm). This study revealed that the confinement in SBA-15 is an effective strategy to avoid the crystallization of the Triton X100 independently of the thermal history. Dielectric Spectroscopy Relaxation (DRS) was the main technique used to obtain detailed information about the molecular mobility in a wide range of frequencies (10-2 – 106 Hz). As complementary techniques Differential Scanning Calorimetric (DSC) and polarized Optical Microscopy (POM) were used. Some of the results have been published in the Journal of Physical Chemistry B 2011, 115, (43), 12336-12347

Continuous crystallization of multicomponent materials

The challenges of developing continuous crystallization processes of multicomponent crystals are addressed within this thesis. Multicomponent crystals such as co-crystals and solid solutions, can be used to modify physical properties of active pharmaceuticals, agrochemicals and other materials. These can result in enhanced product properties such as higher solubility, faster dissolution, better stability or improved manufacturability in downstream processing through desirable morphology and better powder flowability. Continuous manufacturing is routinely used in many industries but is a new trend in the manufacture of pharmaceuticals driven by the potential to reduce plant footprint and intermediate inventory, improve yields, reduce lead time, implement real time monitoring and automation and make processes safer.Compared to crystallization of single component crystals, additional component and solid phases introduce additional complexity in the phase diagram. Co-crystal phase diagram measurement in a series of solvents can be very time consuming compared to a solubility curve of a single component. A semi-empirical approach of modeling phase diagrams as well as new methods of measuring phase diagrams of multicomponent materials are presented to accelerate the time to obtain a phase diagram compared to traditional approaches. Transitions from small scale batch crystallization to continuous crystallization is also demonstrated here for co-crystals and solid solutions with high selectivity and reproducibility with respect to the solid phase produced.The challenges of developing continuous crystallization processes of multicomponent crystals are addressed within this thesis. Multicomponent crystals such as co-crystals and solid solutions, can be used to modify physical properties of active pharmaceuticals, agrochemicals and other materials. These can result in enhanced product properties such as higher solubility, faster dissolution, better stability or improved manufacturability in downstream processing through desirable morphology and better powder flowability. Continuous manufacturing is routinely used in many industries but is a new trend in the manufacture of pharmaceuticals driven by the potential to reduce plant footprint and intermediate inventory, improve yields, reduce lead time, implement real time monitoring and automation and make processes safer.Compared to crystallization of single component crystals, additional component and solid phases introduce additional complexity in the phase diagram. Co-crystal phase diagram measurement in a series of solvents can be very time consuming compared to a solubility curve of a single component. A semi-empirical approach of modeling phase diagrams as well as new methods of measuring phase diagrams of multicomponent materials are presented to accelerate the time to obtain a phase diagram compared to traditional approaches. Transitions from small scale batch crystallization to continuous crystallization is also demonstrated here for co-crystals and solid solutions with high selectivity and reproducibility with respect to the solid phase produced

Dynamic Crystallization Pathways of Polymorphic Pharmaceuticals Revealed in Segmented Flow with Inline Powder X-ray Diffraction

Understanding the transitions between polymorphs is essential in the development of strategies for manufacturing and maximizing the efficiency of pharmaceuticals. However, this can be extremely challenging: crystallization can be influenced by subtle changes in environment, such as temperature and mixing intensity or even imperfections in the crystallizer walls. Here, we highlight the importance of in situ measurements in understanding crystallization mechanisms, where a segmented flow crystallizer was used to study the crystallization of the pharmaceuticals urea: barbituric acid (UBA) and carbamazepine (CBZ). The reactor provides highly reproducible reaction conditions, while in situ synchrotron powder X-ray diffraction (PXRD) enables us to monitor the evolution of this system. UBA has two polymorphs of almost equivalent free-energy and so is typically obtained as a polymorphic mixture. In situ PXRD analysis uncovered a progression of polymorphs from UBA III to the thermodynamic polymorph UBA I, where different positions along the length of the tubular flow crystallizer correspond to different reaction times. Addition of UBA I seed crystals modified this pathway such that only UBA I was observed throughout, while transformation from UBA III into UBA I still occurred in the presence of UBA III seeds. Information regarding the mixing-dependent kinetics of the CBZ form II to III transformation was also uncovered in a series of seeded and unseeded flow crystallization runs, despite atypical habit expression. These results illustrate the importance of coupling controlled reaction environments with in situ XRD to study the phase relationships in polymorphic materials

Experimental investigation on the applicability of FBRM in the control of batch cooling crystallization

Master'sMASTER OF ENGINEERIN

Structural and Functional Characterization of a New Bacterial Target Against Tuberculosis: The Phosphatase PtpA

Tuberculosis (TB) is one of the top causes of death remaining a major public health problem worldwide. Mycobacterium tuberculosis is the agent of TB, infecting the human respiratory tract. Its remarkable pathogenicity hinges upon the ability to challenge the immune system of the host by secreting phosphatases into macrophages. Among them, Protein Tyrosine Phosphatase A (PtpA) plays a key role on the infection process, preventing the phagosome-lysosome fusion and promoting the inhibition of phagosome acidification. Thus, PtpA becomes a promising target for the development of new anti-TB drugs. The aim of this work is to contribute to find new structure-based drug design approaches against TB, studying the inhibitory properties of three different families of compounds towards PtpA – chalcones, thiosemicarbazones and azaindoles. The protein was overexpressed in E. coli – final yield of 20 mg protein/ liter of culture – and successfully purified using affinity chromatography. To provide new insights into the binding mode of the studied compounds, molecular docking studies were performed suggesting thiosemicarbazones as non-competitive inhibitors and the chalcones and azaindoles with a preferential active site binding. The protein was also biophysically characterized. The oligomeric state was confirmed by SEC, proving that PtpA is a monomer in solution. The protein stability was assessed through TSA revealing that, with 10% glycerol, PtpA resists to the effects of 10% DMSO. TSA was also used to find a suitable protein storage condition (-80°C) and to confirm PEG400 as an alternative solvent for the inhibitors. In addition, distinct biophysical approaches – TSA, MST and urea-gel electrophoresis – were implemented to detect protein-ligand interactions but definitive evidence were not obtained. Ligand-free and co-crystallization assays were extensively explored and several crystals were tested at the ESRF, Diamond and MAX IV. Two crystal structures were obtained: a co-crystallization PtpA-Lap11 structure at 3.6 Å resolution and a soaking PtpA-C33 structure at 2.8 Å resolution. Despite the low/medium resolution obtained, both structures reveal the potential binding of the inhibitors with suspicious density blobs near His120B for Lap11 and at the active site for C33. The ligands were preliminarily modelled but further refinement cycles are required to elucidate the respective binding

Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)6 fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms

X-ray crystallographic studies of bovine serum albumin and helicobacter pylori thioredoxin-2

The initial motivation for crystallization of Bovine Serum Albumin (BSA) is an interest to understand how thiomolybdates interact with BSA and suppress copper intake from the food sources of cattle. The main objective of my research work is to determine the crystal structure of BSA using X-ray crystallography techniques. Once the tertiary structure of BSA is determined, its structural information can help us to study the interactions between BSA, copper, and thiomolybdates, and to understand the way in which thiomolybdates render copper unavailable in cattle. Many trials for the optimal crystallization conditions of BSA were attempted in order to grow high-quality BSA crystals. However, all crystals only diffract to 8 Å resolution limit. Such resolution is not sufficient to solve the tertiary structure of BSA. Another objective of my research was to crystallize Thioredoxin-2 (Trx-2) to obtain larger crystals which may lead to high resolution crystallographic data, better than 2.4 Å, for protein structure refinement. This is because Trx-2 diffraction data that had been collected are split at high resolution. The ambiguous data at high resolution might impede the structure refinement and even can cause the three-dimensional structure of Trx-2 to not be refined successfully. A number of attempts were conducted for crystallizing Trx-2 to grow bigger and higher quality of Trx-2 crystals. However, the improvement of crystal dimensions was not significant, the diffraction resolution limits are similar to previous published data, and the split data at high resolution was still observed