Biosensors are detection devices that couple biological recognition elements to physiochemical transducers to generate quantifiable signals. Immunosensors are biosensors that use antibodies as the recognition element. The highly specific nature of antibody-antigen binding is exploited to create immunosensors that are sensitive to analytes in complex mixtures and demonstrate a rapid response. Fiber optical immunosensors based on long-period gratings have limited sensitivity at the refractive index of ordinary aqueous solutions (~1.33). A composite film was designed to raise the local refractive index of the sensor, thus increasing sensitivity. Titanium dioxide deposition raised the refractive index of the sensor to ~1.42. Bovine serum albumin was immobilized onto a dextran hydrogel and attached to the LPG element via reductive amination. The thickness of the hydrogel was estimated to be 500 nm using Environmental Scanning Electron Microscopy. The affinity film was probed by an evanescent wave to detect changes in refractive index due to the binding of anti-BSA IgG. Under these conditions, the sensor yielded a signal ratio of approximately 10-4 refractive index units per nm signal. Reproducible binding was shown over multiple exposures, with no cross reactivity for non-specific antibodies and other proteins. Anti-BSA IgG (20 µg/mL) in whole serum was recycled through the fiber holder with an accompanying peak wavelength shift that averaged 2 nm on an Optical Spectrum Analyzer with a noise level of 0.1 nm. The BSA affinity film was regenerated 50 times and showed a baseline shift of -1.3 nm. / Master of Science

Many of the industrial processes, used by manufacturers to produce biologics, have not been significantly updated since their original design and conception. And thus, there is a great opportunity to update and optimize manufacturing processes. Downstream purification is often considered the bottleneck of the manufacturing process and when biologics are being purified for clinical applications, the final purity is paramount. As a result, pharmaceutical products are subjected to multiple concentration, conditioning, and chromatographic steps. The pharmaceutical industry is constantly and slowly evolving and is always looking to improve efficiency. Simulations and modeling are becoming more commonly used in the pharmaceutical industry as a tool to strategically design and test new production and separation processes developed at the research and development scale. In this thesis, computational fluid dynamics (CFD) modeling was used to develop more efficient bioseparation processes by (1) using a cuboid module geometry and (2) chromatographic medium with product-specific affinity ligands. The laterally-fed class of chromatography modules has a unique cuboidal geometry, with lateral feeding of the sample in the channel above the bed and lateral collection of permeate. CFD simulations and experimental results have shown that the laterally-fed class of chromatography devices can produce sharper elution peaks, have better peak resolution, and consequently purer product fractions than conventional membrane and resin-based chromatographic formats. The enhanced performance by the laterally-fed class of chromatography devices is attributed to improved system fluidics and narrow solute residence time distribution. One other approach to improving efficiency is to address the tradeoff between purity and recovered yield, due to the non-specific binding nature of many commercial resins and membranes. Purification using high-affinity biological ligands selected on specificity to the target molecule could be a feasible solution. A purification scheme for pertactin was developed with final eluate purity of 90% and approximately 100% recovery. / Thesis / Master of Applied Science (MASc)

FKBP12 est une protéine ubiquitaire, principalement cytosolique, qui est au carrefour de plusieurs voies signalétiques. Son abondance naturelle dans les tissus nerveux peut être reliée à son implication dans les maladies neurodégénératives telles que les maladies d'Alzheimer et de Parkinson ainsi que dans les neuropathies périphériques et diabétiques ou dans des blessures des cordons spinaux. De nombreuses études ont montré que des molécules exogènes (ligands) venant se fixer sur cette protéine permettent la régénération d'un grand nombre de connexions neuronales endommagées. Une difficulté provient cependant du fait que, pour un ligand donné, il n'existe aucune relation claire entre sa structure et sa capacité de liaison à FKBP12. Notre étude vise ainsi à rationaliser la relation entre la structure d'un ligand et son affinité pour cette protéine. Deux complexes modèles, formés entre FKBP12 et chacun des deux ligands 8 et 308, ont été utilisés. Ces deux ligands de haute affinité ont des structures différentes. Notre travail s'est appuyé sur des simulations de dynamique moléculaire pour caractériser l'état intermédiaire qui est formé transitoirement lors du processus de complexation entre la protéine et son ligand. Dans cet état particulier, l'identification des interactions naissantes entre les partenaires a permis (i) de comprendre l'implication des différentes parties du ligand dans le mécanisme de reconnaissance avec FKBP12 et (ii) de rationaliser les affinités de certains ligands apparentés. / FKBP12 is an ubiquitous, mostly cytosolic, protein found at the crossroads of several signaling pathways. Its natural abundance in the nervous tissues can be related to its implication in neurodegenerative diseases like Alzheimer's and Parkinson's as well as in peripheral neuropathies and diabetes or in injuries of the spinal cords. Several studies have demonstrated that exogenous molecules (ligands) that can bind to FKBP12 allow the regeneration of many damaged neuron connections. However, there is no clear relationship between the structure of a ligand and its ability to bind to FKBP12. Our study aims at rationalizing the relationship between the structure of a ligand and its affinity to FKBP12. Two model complexes, formed between FKBP12 and each of the two high-affinity ligands 8 and 308, were studied. These two ligands are structurally different. We used molecular dynamics simulations to characterize the intermediate state that is transiently formed during the binding process between the protein and its ligand. In this state, the analysis of the nascent interactions allowed (i) to unravel the role played by the various ligand moieties in the recognition process with FKBP12 and (ii) to rationalize the affinities of related ligands.