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)

Linear covalently closed (LCC) DNA minivectors serve to be superior to conventional circular covalently closed (CCC) plasmid DNA (pDNA) vectors due to enhancements to both transfection efficiency and safety. Specifically, LCC DNA minivectors have a heightened safety profile as insertional mutagenesis is inhibited by covalently closed terminal ends conferring double-strand breaks that cause chromosomal disruption and cell death in the low frequency event of chromosomal integration. The development of a one-step, E. coli based in vivo LCC DNA minivector production system enables facile and efficient production of LCC DNA minivectors referred to as DNA ministrings. This novel in vivo system demonstrates high versatility, generating DNA ministrings catered to numerous potential applications in gene therapy and vaccine development. In the present study, numerous aspects pertaining to the generation of gene therapeutics with LCC DNA ministrings have been explored with relevance to both industry and clinical settings. Through systematic assessment of induction duration, cultivation strategy, and genetic/chemical modifications, the novel in vivo system was optimized to produce high yields of DNA ministrings at ~90% production efficiency. Purification of LCC DNA ministrings using anion exchange membrane chromatography demonstrated rapid, scalable purification of DNA vectors as well as its potential in the separation of different DNA isoforms. The application of a hydrogel-based strong Q-anion exchange membrane, with manipulations to salt gradient, constituted effective separation of parental supercoiled CCC precursor pDNA and LCC DNA. The resulting DNA ministrings were employed for the generation of 16-3-16 gemini surfactant based synthetic vectors and comparative analysis, through physical characterization and in vitro transfection assays, was conducted between DNA ministring derived and CCC pDNA derived lipoplexes. Differences in DNA topology were observed to induce differences in particle size and DNA protection/encapsulation upon lipoplex formation. Lastly, the in vivo DNA minivector production system successfully generated gagV3(BCE) LCC DNA ministrings for downstream development of a HIV DNA-VLP (Virus-like particle) vaccine, thus highlighting the capacity of such system to produce DNA ministrings with numerous potential applications.

Les antibiotiques sont fréquemment utilisés dans l’alimentation de la volaille afin de prévenir certaines maladies, dont l’entérite nécrotique, ce qui occasionne l’émergence de souches bactériennes résistantes aux antibiotiques. Une alternative prometteuse est l’utilisation de peptides antimicrobiens (AMPs) comme suppléments alimentaires, tels les AMPs provenant des produits laitiers. L’objectif du projet était de développer une méthode de production d’extraits peptidiques à partir de coproduits de la transformation alimentaire (babeurre, lactoferrine, isolat de protéines de pois), afin de tester si ces extraits peptidiques possédaient une activité antimicrobienne sur les pathogènes spécifiques aviaires suivants : Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli et Staphylococcus aureus. Les protéines ont été mises en suspension dans l’eau (5% p/p) et hydrolysées par la pepsine, 6 heures, pH de 2.5. Les peptides furent récupérés par ultrafiltration (< 10 kDa), puis fractionnés selon leur charge nette : totaux, cationiques, anioniques et non liés. L’effet antimicrobien a été évalué surmicroplaques, par la survie bactérienne en présence de concentrations croissantes d’extraits peptidiques. Les extraits cationiques de babeurre ont démontré une efficacité à une concentration inférieure ou égale à 5 mg/mL; perte de 3 log pour Escherichia coli O78 :H80. En comparaison, la lactoferrine cationique a été efficace à une concentration inférieure ou égale à 0.6 mg/mL; perte de 6 log pour E. coli O78 :H80. Les extraits peptidiques du pois ont démontré une efficacité faible. Cette méthode s’avère prometteuse pour le développement d’une alternative ou d’un complément pour la réduction de l’utilisation des antibiotiques dans l’alimentation de la volaille. / Antibiotics are frequently used in poultry feed in order to prevent certain diseases, including necrotic enteritis, which causes the emergence of bacterial strains resistant to antibiotics. A promising alternative is the use of antimicrobial peptides (AMPs) as dietary supplements, such as AMPs from dairy products. The objective of this project was to develop a production method for the extraction peptides, from co-produced food processing (buttermilk, lactoferrin, pea protein isolates). These peptides were tested for the detection of antimicrobial activity on the following specific poultry pathogens; Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli and Staphylococcus aureus. Proteins were suspended in water (5% w/w) and pepsin hydrolyzed by pepsin for 6 hours at pH 2.5. Peptides were recovered by ultrafiltration (< 10 kDa) and fractionated based on the basis of their ionic charges: total, cationic, anionic and unbound peptides, to specifically target the fractions with antimicrobial activities. Bacterial survival was measured in contact with different peptides concentrations. Cationic buttermilk extracts were effective at a concentration less or equal to 5 mg / mL; loss of 3 log for Escherichia coli O78: H80, compared with lactoferrin which was effective at a concentration less than or equal to 0.6 mg / mL; loss of 6 log for E. coli O78: H80. The peptide extracts from pea showed low efficiency. The use of antimicrobial peptides, from buttermilk, lactoferrin and peas, is promising for the development of an alternative or a complement to reduce antimicrobial use.

La chromatographie membranaire est une alternative à la chromatographie classique sur résine basée sur le transport convectif des solutés à travers une membrane microporeuse plutôt que par le transport diffusif des solutés dans les particules de résines. Cette technique présente les avantages de diminuer les phénomènes de diffusion, de réduire les temps de séjour et les pertes de charge, et de permettre la purification rapide de quantités importantes de molécules. La chromatographie membranaire connaît un fort succès commercial. Une gamme importante de membranes chromatographiques mettant en jeu différents mécanismes de rétention (échange d’ions, affinité, etc.) et différentes géométries (feuille, spirale, etc.) est actuellement commercialisée. Malgré ce succès, différents aspects relatifs à la chromatographie membranaire restent mal connus. Cette thèse de doctorat se propose de répondre à certaines questions relatives à cette technique / Membrane chromatography (MC) is an alternative to traditional resin packed columns chromatography. The solute mass transport in the membrane occurs in convective through-pores rather than in stagnant fluid inside the pores of the resins particles, which is limited by the slow diffusive transport. MC offers the main advantage of reducing diffusion phenomena, shorter residence time and lowered pressures drops, and thus, facilitates rapid purification of large quantities of molecules. A wide range of chromatographic membranes involving different molecules retention mechanisms (ion exchange, affinity, etc...) is now commercialized. Despite their success, the influence of the geometry of the membrane chromatography devices remains relatively unexplored from a theoretical point of view. This doctoral thesis is aimed to clarify some ambiguous points related to this technique

<p>Monoclonal antibodies (mAbs) as therapeutic proteins have shown great potential in treatment of various human diseases because of their highly specific nature. This has attracted worldwide attention leading to increased demand for such mAb products. To meet this demand large scale manufacturing is carried out using recombinant mammalian cell culture techniques for high yields and faster production. mAb products are worth the investment if produced in their native state. The quantity of mAb present in such cell cultures is very less and therefore special care is needed while handling them. Purifying antibody molecules from heterogeneous cell culture impurities and maintaining their native functional state is a critical task mainly because these antibodies are labile in nature. Care also need to be exercised during processing because mAbs have inherent tendancy to aggregate which is undesirable since such aggregates in antibody formulation produces immunogenic reaction when injected in humans. The other important factor in mAb purification is the processing cost involved since majority of the total production cost is utilized for purification of mAb. Protein-A chromatography is the first choice for purifying antibodies and is widely adopted. However failure in distinguishing between monomer and aggregate antibody molecules along with harsh acidic processing conditions necessitates the use of further purification steps.</p> <p>In this work various techniques for mAb processing are discussed and are outlined below:</p> <p>Removal of impurities from mAbs is a major challenge and this thesis discusses various processing options available to purify these mAbs. Impurities in mAb products are usually the aggregate byproducts formed due to unfolded monomer antibody molecules. These molecules are naturally hydrophobic in nature and display great differences in hydrophobicity on aggregation. Hydrophobic interaction membrane chromatography (HIMC) makes use of this hydrophobicity difference and helps in removal of aggregate impurities from monomer antibody.</p> <p>Heavy chain mAbs (hcmAbs) are promising new developments in the area of biopharmaceuticals because of their unique structural composition. Similar to conventional mAbs these hcmAbs are also rapidly finding their way into therapeutic markets. Purifying hcmAbs will be an important step in their development and for this purpose we use HIMC technique for removing impurities and obtain pure product.</p> <p>Antibody molecules are almost always lost as aggregates which leads to great economic losses and the ability to disaggregate these mAb oligomers would be of significant practical and scientific interest. In this work a novel thermalcycling technique is discussed to disaggregate such mAb oligomers and potentially recover functional monomer mAb molecules.</p> / Master of Applied Science (MASc)