Particles are omnipresent in biopharmaceutical products. In protein-based therapeutics such particles are generally associated with impurities, either derived from the drug product itself (e.g.... Show moreParticles are omnipresent in biopharmaceutical products. In protein-based therapeutics such particles are generally associated with impurities, either derived from the drug product itself (e.g. protein aggregates), or from extrinsic contaminations (e.g. cellulose fibers). These impurities can affect product stability, as well as cause adverse effects once introduced into the human body. Particulate impurities are present over a wide range of sizes (from nanometers to millimeters) making them difficult to characterize by using a single method.Novel drug products may also contain particles that act as the active pharmaceutical ingredient (e.g., living cells) or a drug delivery vehicle (e.g., lipid nanoparticles). Unwanted immunotoxicity and inconsistent in vivo functionality can result from particle instability and aggregate formation. Therefore, the efficacy and safety of these therapeutics is dependent on the particle composition, quantity and size distribution.Consequently, well-established methods are required to quantify and characterize particles in the submicron- and micron-size ranges. In this thesis, we developed new approaches which allow for comprehensive characterization of the particle populations present in biopharmaceutical products, both as impurities or as API. Furthermore, the performed work focused on comparing different particle characterization techniques to allow a better understanding of the limitations and strengths of each method applied. Show less
Cells, their fragments and secreted factors may all be transported to distant sites via the blood circulatory system and exert their action far away from the site of origin. Identification of these... Show moreCells, their fragments and secreted factors may all be transported to distant sites via the blood circulatory system and exert their action far away from the site of origin. Identification of these mediating factors and unraveling of the pathogenesis resulting in organ damage will contribute to our general understanding and may ultimately result in new treatment strategies. The studies included in this thesis focus on mechanisms of coagulopathy and other blood abnormalities in patients with cancer or inflammation. Show less
Complex injectable formulations, such as protein therapeutics, controlled release systems and cell therapy products, are gaining a paramount position in the therapy of many life-threatening and... Show moreComplex injectable formulations, such as protein therapeutics, controlled release systems and cell therapy products, are gaining a paramount position in the therapy of many life-threatening and chronic diseases. Most of these products have in common that sub-visible particles (SVP), i.e., particulate matter in the size range of about 1 – 100 µm, are critical quality attributes. Most protein therapeutics are liquid or freeze-dried formulations in which the presence of SVP is unwanted; many injectable controlled release systems are based on particulate drug delivery systems in the sub-visible size range; and cell concentration and viability are important characteristics of cell therapy products. With the continuous improvement of existing and emerging particle analysis techniques, the potentials of these tools in addressing current characterization challenges in the field of complex injectable formulations have to be investigated. Therefore, the aim of this thesis was to develop methods, based on a set of state-of-the-art particle analysis techniques, for characterization of pharmaceutically relevant sub-visible particles and to study the value of these methods in the characterization of complex injectable formulations. Show less
Synthetic long peptides (SLPs) derived from cancer antigens hold great promise as well-defined antigens for immunotherapy of cancer. However, the formulation of SLPs for in vivo administration... Show moreSynthetic long peptides (SLPs) derived from cancer antigens hold great promise as well-defined antigens for immunotherapy of cancer. However, the formulation of SLPs for in vivo administration still needs to be improved. So far, SLPs have been formulated in Montanide-based water-in-oil emulsions in (pre-)clinical trials. However, the use of Montanide as an adjuvant has some important limitations, such as: non-biodegradability; significant local side effects; poor control of release rate; lack of specific dentritic cell (DC)-activating capacity; and the presence of organic solvents (needed to dissolve the peptides prior to mixing with the adjuvant) in the final formulation. Therefore, alternative formulations containing an effective delivery system for peptide-based cancer vaccines are highly needed. Among the numerous vaccine delivery systems, poly(lactic-co-glycolic acid) (PLGA) biodegradable particulate delivery systems are particularly interesting because they are biocompatible; can protect soluble antigens from degradation and rapid clearance once administered; allow for co-encapsulation of (multiple) antigens and adjuvants; and mimic the size and structure of a pathogen, being more efficiently taken up by DCs than soluble antigen. This thesis describes fundamental studies on the design and applicability in a preclinical setting of PLGA-based particulate formulations for the delivery of SLP-based cancer vaccines. Show less
An active pool of __Tissue Factor__ (TF) is required to initiate blood coagulation, but the mechanism that regulates the activation of TF is highly debated. The reduced coagulant activities of both... Show moreAn active pool of __Tissue Factor__ (TF) is required to initiate blood coagulation, but the mechanism that regulates the activation of TF is highly debated. The reduced coagulant activities of both human and mouse disulfide-mutated TF indicates that the formation of a Cys186-Cys209 or Cys190-Cys213 disulfide bond in the extracellular domain of TF controls its procoagulant function. Also the strong evolutionary conservation of this allosteric disulfide bond supports previous assumption. Procoagulant active TF is also found intravascularly on small vesicles (microparticles), and high levels of microparticle-associated TF is thought to increase the risk for thrombosis in cancer patients. Despite the fact that chemotherapy is an independent risk factor for thrombosis, chemotherapy-induced tumor destruction was not associated with an increase in microparticle-associated TF in testis cancer patients. The soluble isoform of TF, alternatively spliced TF (asTF), induces angiogenesis through interaction with integrins and independent of intracellular protease-activated receptor (PAR)-mediated signaling. PAR-2 but not PAR-1 plays a role in arteriogenesis in vivo. Furthermore, PAR-2 is involved in the anti-inflammatory response that may promote arteriogenesis. Show less
Regulatory processes are responsible for the organization, division and death of cells in multicellular organisms such as humans. Additionally, cells are highly regulated internally, able to... Show moreRegulatory processes are responsible for the organization, division and death of cells in multicellular organisms such as humans. Additionally, cells are highly regulated internally, able to survive and respond in vastly different micro-environments. Many types of interactions of cells with their environment can be distinguished, and need to be controlled in experiments aimed at unravelling and predicting cellular behavior in vivo. The in vivo microenvironment is mimicked by exposing cells to complex and changing environments. To describe the stochastic differences between cells and the local experimental conditions in sufficient detail and to obtain statistically relevant results, high-throughput experimentation is required. In this thesis four new research methods are developed, aimed at a deeper understanding of cellular regulation in vivo. Show less
Microparticles (MPs) have important physiological and pathological roles in blood coagulation, inflammation and tumor progression. In recent years MPs also have been recognized to participate in... Show moreMicroparticles (MPs) have important physiological and pathological roles in blood coagulation, inflammation and tumor progression. In recent years MPs also have been recognized to participate in important biological processes, such as in signaling and in the horizontal transfer of their specific proteins and mRNAs. However, studies of MPs have been hampered by the lack of methods for the sensitive detection and accurate quantification of MPs. Thus, we have developed a new methodology by using atomic force microscopy (AFM) and cryo-electron microscopy (cryo-EM) to detect, quantify and characterize MPs in plasma. We have shown that AFM detects 1000-fold more platelet derived-MPs than a conventional flow cytometry does. These MPs have diameters ranging from 10-475 nm with a peak at 67.5 nm, which is clearly far below the detection limit of flow cytometry. By using cryo-EM we found that the number of lipoprotein particles exceeds that of MPs or exosomes in plasma. We also demonstrated that by using immuno-magnetic beads selected subset of MPs could directly be captured/depleted from plasma and assessed for MP-associated tissue factor activity. In the future the measurement of MPs will perhaps serve as a diagnostic tool to identify and predict diseases, like cancer. Show less
This thesis elaborates the occurrence of venous thrombosis in cancer patients. Cancer is known to be associated with venous thrombosis with a spectrum of clinical manifestations varying from deep... Show moreThis thesis elaborates the occurrence of venous thrombosis in cancer patients. Cancer is known to be associated with venous thrombosis with a spectrum of clinical manifestations varying from deep vein thrombosis of the leg and pulmonary embolism, recurrent thrombophlebitits saltans et migrans (also called Trousseau__s syndrome) to disseminated intravascular coagulation and arterial embolism. The causes of venous thrombosis can be divided in environmental risk factors such as bed rest, surgery, plaster cast, trauma, long-distance travel, oral contraceptives or pregnancy and puerperium and genetic risk factors such as factor V Leiden and prothrombin 20210A mutation. Various factors may contribute to the development of venous thrombosis in cancer patients, and circulating mucins as well as circulating microparticles which express active TF on their surface may provide a missing link between cancer and thrombosis in (adeno) carcinoma patients. Treatment options include vitamin K antagonists and low-molecular-weight heparins, and the long-term use of these heparins in prevention of venous thrombosis may improve the outcome in comparison with oral anticoagulants. Further research is needed to better understand the morbidity and mortality associated with thrombosis in cancer patients and to optimize strategies of prevention and treatment Show less
