Purification of complex biopharmaceuticals with new processes, advanced analytics and computer-aided process design tools

Dissertação para obtenção do Grau de Mestre em Biotecnologia

Novel insights into the action of antimicrobial agents against human pathogens

Dissertation presented to obtain the Ph.D degree in Biochemistry

Phage display as a tool for development of novel therapeutics for breast cancer

Phage display technology is a powerful platform for the generation of highly specific human monoclonal antibodies (Abs) with potential use in clinical applications. Moreover, this technique has also proven to be a reliable approach in identifying and validating new cancer-related targets. For scientific or medical applications, different types of Ab libraries can be constructed. The use of Fab Immune libraries allows the production of high quality and affinity antigen-specific Abs. In this work, two immune human phage display IgG Fab libraries were generated from the Ab repertoire of 16 breast cancer patients, in order to obtain a tool for the development of new therapeutic Abs for breast cancer, a condition that has great impact worldwide. The generated libraries are estimated to contain more than 108 independent clones and a diversity over 90%. Libraries validation was pursued by selection against BSA, a foreign and highly immunogenic protein, and HER2, a well established cancer target. Preliminary results suggested that phage pools with affinity for these antigens were selected and enriched. Individual clones were isolated, however, it was not possible to obtain enough data to further characterize them. Selection against the DLL1 protein was also performed, once it is a known ligand of the Notch pathway, whose deregulation is associated to breast cancer, making it an interesting target for the generation of function-blocking Abs. Selection resulted in the isolation of a clone with low affinity and Fab expression levels. The validation process was not completed and further effort will have to be put in this task in the future. Although immune libraries concept implies limited applicability, the library reported here has a wide range of use possibilities, since it was not restrained to a single antigen but instead thought to be used against any breast cancer associated target, thus being a valuable tool.

New visualization model for large scale biosignals analysis

Benefits of long-term monitoring have drawn considerable attention in healthcare. Since the acquired data provides an important source of information to clinicians and researchers, the choice for long-term monitoring studies has become frequent. However, long-term monitoring can result in massive datasets, which makes the analysis of the acquired biosignals a challenge. In this case, visualization, which is a key point in signal analysis, presents several limitations and the annotations handling in which some machine learning algorithms depend on, turn out to be a complex task. In order to overcome these problems a novel web-based application for biosignals visualization and annotation in a fast and user friendly way was developed. This was possible through the study and implementation of a visualization model. The main process of this model, the visualization process, comprised the constitution of the domain problem, the abstraction design, the development of a multilevel visualization and the study and choice of the visualization techniques that better communicate the information carried by the data. In a second process, the visual encoding variables were the study target. Finally, the improved interaction exploration techniques were implemented where the annotation handling stands out. Three case studies are presented and discussed and a usability study supports the reliability of the implemented work.

Human activity recognition with accelerometry: novel time and frequency features

Human Activity Recognition systems require objective and reliable methods that can be used in the daily routine and must offer consistent results according with the performed activities. These systems are under development and offer objective and personalized support for several applications such as the healthcare area. This thesis aims to create a framework for human activities recognition based on accelerometry signals. Some new features and techniques inspired in the audio recognition methodology are introduced in this work, namely Log Scale Power Bandwidth and the Markov Models application. The Forward Feature Selection was adopted as the feature selection algorithm in order to improve the clustering performances and limit the computational demands. This method selects the most suitable set of features for activities recognition in accelerometry from a 423th dimensional feature vector. Several Machine Learning algorithms were applied to the used accelerometry databases – FCHA and PAMAP databases - and these showed promising results in activities recognition. The developed algorithm set constitutes a mighty contribution for the development of reliable evaluation methods of movement disorders for diagnosis and treatment applications.

Complex networks and data mining: toward a new perspective for the understanding of complex systems

Complex systems, i.e. systems composed of a large set of elements interacting in a non-linear way, are constantly found all around us. In the last decades, different approaches have been proposed toward their understanding, one of the most interesting being the Complex Network perspective. This legacy of the 18th century mathematical concepts proposed by Leonhard Euler is still current, and more and more relevant in real-world problems. In recent years, it has been demonstrated that network-based representations can yield relevant knowledge about complex systems. In spite of that, several problems have been detected, mainly related to the degree of subjectivity involved in the creation and evaluation of such network structures. In this Thesis, we propose addressing these problems by means of different data mining techniques, thus obtaining a novel hybrid approximation intermingling complex networks and data mining. Results indicate that such techniques can be effectively used to i) enable the creation of novel network representations, ii) reduce the dimensionality of analyzed systems by pre-selecting the most important elements, iii) describe complex networks, and iv) assist in the analysis of different network topologies. The soundness of such approach is validated through different validation cases drawn from actual biomedical problems, e.g. the diagnosis of cancer from tissue analysis, or the study of the dynamics of the brain under different neurological disorders.

Development of novel ionic liquids based on biological molecules

Ionic Liquids (ILs) belong to a class of compounds with unusual properties: very low vapour pressure; high chemical and thermal stability and the ability to dissolve a wide range of substances. A new field in research is evaluating the possibility to use natural chiral biomolecules for the preparation of chiral ionic liquids (CILs). This important challenge in synthetic chemistry can open new avenues of research in order to avoid some problems related with the intrinsic biodegradability and toxicity associated to conventional ILs. The research work developed aimed for the synthesis of CILs, their characterization and possible applications, based on biological moieties used either as chiral cations or anions, depending on the synthetic manipulation of the derivatives. Overall, a total of 28 organic salts, including CILs were synthesized: 9 based on L-cysteine derivatives, 12 based on L-proline, 3 based on nucleosides and 4 based on nucleotides. All these new CILs were completely characterized and their chemical and physical properties were evaluated. Some CILs based on L-cysteine have been applied for discrimination processes, including resolution of racemates and as a chiral catalyst for asymmetric Aldol condensation. L-proline derived CILs were also studied as chiral catalysts for Michael reaction. In parallel, the interactions of macrocyclic oligosugars called cyclodextrins (CDs) with several ILs were studied. It was possible to improve the solubility of CDs in water and serum. Additionally, fatty acids and steroids showed an increase in water solubility when ILs-CDs systems were used. The development of efficient and selective ILs-CDs systems is indispensable to expand the range of their applications in host-guest interactions, drug delivery systems or catalytic reactions. Novel salts derived from nucleobases were used in order to enhance the fluorescence in aqueous solution. Additionally, preliminary studies regarding ethyl lactate as an alternative solvent for asymmetric organocatalysis were performed.

Development of novel active pharmaceutical ionic liquids and salts based on antibiotics and anti-fungal drugs

Ionic Liquids (ILs) are class of compounds, which have become popular since the mid-1990s. Despite the fact that ILs are defined by one physical property (melting point), many of the potential applications are now related to their biological properties. The use of a drug as a liquid can avoid some problems related to polymorphism which can influence a drug´s solubility and thus its dosages. Also, the arrangement of the anion or cation with a specific drug might be relevant in order to: a) change the correspondent biopharmaceutical drug classification system; b) for the drug formulation process and c) the change the Active Pharmaceutical Ingredients’ (APIs). The main goal of this Thesis is the synthesis and study of physicochemical and biological properties of ILs as APIs from beta-lactam antibiotics (ampicillin, penicillin G and amoxicillin) and from the anti-fungal Amphotericin B. All the APIs used here were neutralized in a buffer appropriate hydroxide cations. The cation hydroxide was obtained on Amberlite resin (in the OH form) in order to exchange halides. The biological studies of these new compounds were made using techniques like the micro dilution and colorimetric methods. Overall a total of 19 new ILs were synthesised (6 ILs based on ampicillin, 4 ILs, based on amoxicillin, 6 ILs based on penicillin G and 4 ILs based on amphotericin B) and characterized by spectroscopic and analytical methods in order to confirm their structure and purity. The study of the biological properties of the synthesised ILs showed that some have antimicrobial activity against bacteria and yeast cells, even in resistant bacteria. Also this work allowed to show that ILs based on ampicillin could be used as anti-tumour agents. This proves that with a careful selection of the organic cation, it is possible to provoke important physico-chemical and biological alteration in the properties of ILs-APIs with great impact, having in mind their applications.

Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.

Bits, Atoms, and Information Sharing: new opportunities for participation

The particular characteristics and affordances of technologies play a significant role in human experience by defining the realm of possibilities available to individuals and societies. Some technological configurations, such as the Internet, facilitate peer-to-peer communication and participatory behaviors. Others, like television broadcasting, tend to encourage centralization of creative processes and unidirectional communication. In other instances still, the affordances of technologies can be further constrained by social practices. That is the case, for example, of radio which, although technically allowing peer-to-peer communication, has effectively been converted into a broadcast medium through the legislation of the airwaves. How technologies acquire particular properties, meanings and uses, and who is involved in those decisions are the broader questions explored here. Although a long line of thought maintains that technologies evolve according to the logic of scientific rationality, recent studies demonstrated that technologies are, in fact, primarily shaped by social forces in specific historical contexts. In this view, adopted here, there is no one best way to design a technological artifact or system; the selection between alternative designs—which determine the affordances of each technology—is made by social actors according to their particular values, assumptions and goals. Thus, the arrangement of technical elements in any technological artifact is configured to conform to the views and interests of those involved in its development. Understanding how technologies assume particular shapes, who is involved in these decisions and how, in turn, they propitiate particular behaviors and modes of organization but not others, requires understanding the contexts in which they are developed. It is argued here that, throughout the last century, two distinct approaches to the development and dissemination of technologies have coexisted. In each of these models, based on fundamentally different ethoi, technologies are developed through different processes and by different participants—and therefore tend to assume different shapes and offer different possibilities. In the first of these approaches, the dominant model in Western societies, technologies are typically developed by firms, manufactured in large factories, and subsequently disseminated to the rest of the population for consumption. In this centralized model, the role of users is limited to selecting from the alternatives presented by professional producers. Thus, according to this approach, the technologies that are now so deeply woven into human experience, are primarily shaped by a relatively small number of producers. In recent years, however, a group of three interconnected interest groups—the makers, hackerspaces, and open source hardware communities—have increasingly challenged this dominant model by enacting an alternative approach in which technologies are both individually transformed and collectively shaped. Through a in-depth analysis of these phenomena, their practices and ethos, it is argued here that the distributed approach practiced by these communities offers a practical path towards a democratization of the technosphere by: 1) demystifying technologies, 2) providing the public with the tools and knowledge necessary to understand and shape technologies, and 3) encouraging citizen participation in the development of technologies.

Novel antibiotic-loaded orthopaedic bone cements: insights on drug release profiles and biocompatibility

Acrylic bone cement (BC) is widely used as an anchor of artificial joints. Bacterial infection due to biofilm formation and inflammation are common and difficult to treat problems associated with commercial available BC formulations. Research on novel BC compositions is urgently needed. The main objective of this thesis was to develop a new biocompatible antibiotic-loaded BC with improved release profile. To achieve that aim several additives were incorporated, as an antibiotic (levofloxacin) to combat bacterial growth, an anti-inflammatory drug (diclofenac) to decrease the inflammatory process and two well-known and broadly used biopolymers, alginate and chitosan in order to increase matrix porosity, and in this way to intensify the amount of released drug. Novel BC formulations were tested in order to find the most suitable one that had potential to proceed to clinical application. Numerous tests were conducted as: a) evaluation of drug release profiles in different biomimetic media, b) mechanical and surface studies, c) microbiological activity testing against Staphylococcus aureus and d) in vitro biocompatibility assays (fibroblasts and osteoblasts). In general, the addition of biopolymers increased drug release, didn’t compromised BC mechanical properties and increased BC hydrophilicity. Microbiological testing revealed that Lev[BC]Chi was the only matrix that reduced significantly biofilm formation. On the contrary, alginate and diclofenac loading into BC seemed to increase biofilm growth. Biocompatibility studies showed some decrease in cell viability, in particularly on osteoblasts, mainly due to the high amounts of released drugs. In conclusion, the present work has shown that the matrix with more potential to proceed in further investigations was Lev[BC]Chi. Other conditions (namely additives and drugs concentrations) should be evaluated with the other tested BC matrices before being discharged.

Assessment of a novel Cu (II) complex as a potential anticancer agent

Widely used in cancer treatment, chemotherapy still faces hindering challenges, ranging from severe induced toxicity to drug resistance acquisition. As means to overcome these setbacks, newly synthetized compounds have recently come into play with the basis of improved pharmacokinetic/pharmacodynamic properties. With this mind-set, this project aimed towards the antiproliferative potential characterization of a group of metallic compounds. Additionally the incorporation of the compounds within a nanoformulation and within new combination strategies with commercial chemotherapeutic drugs was also envisaged. Cell viability assays presented copper (II) compound (K4) as the most promising, presenting an IC50 of 6.10 μM and 19.09 μM for HCT116 and A549 cell line respectively. Exposure in fibroblasts revealed a 9.18 μM IC50. Hoechst staining assays further revealed the compound’s predisposition to induce chromatin condensation and nuclear fragmentation in HCT116 upon exposure to K4 which was later demonstrated by flow cytometry and annexin V-FITC/propidium iodide double staining analysis (under 50 % cell death induction). The compound further revealed the ability to interact with major macromolecules such as DNA (Kb = 2.17x105 M-1), inducing structural brakes and retardation, and further affecting cell cycle progression revealing delay in S-phase. Moreover BSA interactions were also visible however not conclusive. Proteome profiling revealed overexpression of proteins involved in metabolic activity and underexpression of proteins involved in apoptosis thus corroborating Hoechst and apoptosis flow cytometry data. K4 nanoformulation suffered from several hindrances and was ill succeeded in part due to K4’s poor solubility in aqueous buffers. Other approaches were considered in this regard. Combined chemotherapy assays revealed high cytotoxicity for afatinib and lapatinib strategies. Lapatinib and K4 proteome profiling further revealed high apoptosis rates, high metabolic activity and activation of redundant proteins as part of compensatory mechanisms.