No description available.

No description available.

<p> Improved and efficient bioprocessing technology is a key requirement in the manufacture of biopharmaceuticals. The increasing need to reduce biopharmaceutical cost is driven by the business challenges such as the emerging biogenerics. The great advances in upstream technologies make bioseparation the major cost in bioprocessing. Developing efficient bioseparation technologies is therefore strongly desired. Membrane chromatography is a promising bioseparation technology which combines the advantages of membrane technology and chromatography, thus making high-throughput and high-resolution bioprocesses feasible. This thesis focuses on the novel applications and improvements of membrane chromatography: bioprocessing of transgenic tobacco derived monoclonal antibodies and PEGylated proteins, integrated bioprocessing for antibody fragmentation, study of antibody binding on membranes and the development of novel membranes.</p> <p> Membrane chromatography based bioprocesses were developed for purification of monoclonal antibody (mAb) from transgenic tobacco, primarily addressing the challenge of low mAb abundance in the feed material. PEGylated proteins were purified, demonstrating that high-throughput and high-resolution purification of low-binding-propensity proteins was feasible using membrane chromatography.</p> <p> Membrane chromatography based reactant adsorptive membrane bioreactor separator (RAMBS) systems were developed to integrate enzymatic fragmentation of human IgG with the purification of target fragment. This novel system facilitated the process intensification and led to higher IgG digestion than in liquid phase reaction.</p> <p> The mechanism of hydrophobic interaction based IgG binding on synthetic membranes was studied using the RAMBS system. The results showed that the binding took place primarily through a combination of the hinge and CH2 domain of Fc. This study provides a new approach for studying antibody interaction with membranes and surfaces and could help design membrane-based antibody purification, immunoassay and biomaterials.</p> <p> PEG grafted filter paper was developed as an inexpensive alternative to commercial synthetic membranes. These novel membranes possessed high permeability and low fouling tendency and demonstrated good selectivity and reusability in monoclonal antibody purification.</p> / Thesis / Doctor of Philosophy (PhD)

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / The recombinant expression of heterologous proteins in microorganisms, such Escherichia coli, is often improved by producing the protein of interest translationally linked to another, often unrelated, protein giving rise to a "fusion protein" construct. For many applications it is desirable or imperative to separate the extraneous material from the protein of interest. An increasingly popular approach to this task is the use of site-specific endoproteases 10 excise the protein product. A number of commercially available site-specific proteases exist, but many are not capable of generating an authentic N-terminus for the product, display unsatisfactory specificity leading to adventitious cleavage of the product, or they are unsuitable for an industrial process. Mutants of the serine protease a-Lytic protease have been shown to satisfy many of the criteria for an industrially suitable protease and have been applied to the cleavage of some important fusion proteins used in the production of members of the Insulin-like Growth Factor (IGF) family Lacking from these examples, however, is any viable proteolytic solution for the liberation of human IGF-I from fusion proteins. This has been primarily attributed to the Proline bearing N-terminal tripeptide sequence of this protein, which is known to be refractory to the activity of many site-specific proteases. It has been suggested that in the generation of two combinatorial mutant libraries of a-Lytic protease, the preference for amino acids C-terminal to the cleavage site may have been altered. It is the purpose of this work to first determine if such an alteration has been made in any of the mutants so as to allow cleavage immediately before the N-terminus of human IGF-1, and then to task the lead mutant(s) to the cleavage of the full-length fusion protein. All members of the two mutant libraries were cultured and their activity confirmed and quantified against a generic B-casein substrate in a high-throughput assay. A second high-throughput technique was then employed to query the mutant proteases for their ability to catalyse proteolysis at the required sequence in a peptide model. Finding that many mutants appeared successful at this task, the findings were verified on a longer peptide model of the cleavage site. Initially the yields achieved by cleavage of the full-length IGF-1 fusion protein by a lead candidate mutant a-Lytic protease were not sufficient to satisfy the requirements of an industrial process, despite alteration of the reaction conditions. However, the insight gained from these reactions could be applied to the redesign of the protein structure around the intended site of cleavage, significantly improving site-specific proteolysis. The IGF-1 generated by this cleavage has been shown to be bioequivalent to commercial reference standard to cultured mammalian cells and the yield of this process is approximately 5-fold improved over the existing cleavage system. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1325381 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Yes / It is important for the formulators of biopharmaceuticals to predict the folding–unfolding transition of proteins. This enables them to process proteins under predetermined conditions, without denaturation. Depending on the apparent denaturation temperature (Tm) of lysozyme, we have derived an equation describing its folding–unfolding transition diagram. According to the water content and temperature, this diagram was divided into three different areas, namely, the area of the water-folded lysozyme phase, the area of the water-folded lysozyme phase and the bulk water phase, and the area of the denatured lysozyme phase. The water content controlled the appearance and intensity of the Raman band at ∼1787 cm–1 when lysozyme powders were thermally denatured at temperatures higher than Tm. / MAM gratefully acknowledges CARA (Stephen Wordsworth and Ryan Mundy) and University of Bradford for providing an academic fellowship.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21072号 / 工博第4436号 / 新制||工||1689(附属図書館) / 京都大学大学院工学研究科材料化学専攻 / (主査)教授 大塚 浩二, 教授 松原 誠二郎, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Viruses have emerged as a new class of biotherapeutics used as vectors in gene and cell therapies, vaccines, and as oncolytic agents in novel cancer immunotherapies. While these new and potentially curative new therapies bring great promise for patients, the large-scale purification of viruses is hampered by complicated unit operations, poor overall yields, and high costs. Membrane chromatography (MC) is one of the most ideal options for the removal of host-cell impurities in virus manufacturing. Centred on developing and improving MC processes for virus purification, this thesis focuses on different aspects of downstream processes that are directly related to MC. It describes the development of the first hydrophobic interaction MC process for the purification of vesicular stomatitis virus as a scalable method for the removal of host-cell protein and DNA. It also describes the development of MC for adenovirus purification, and how device design and membrane type impact the resolution; here, the novel laterally-fed membrane chromatography (LFMC) was proven to provide higher resolution than conventional MC devices, and allowed for the first direct comparison between the most popularly used membranes in virus manufacturing – Sartobind Q and Mustang Q. Beyond MC, this thesis also addresses how other downstream unit operations contribute to the final purity. Through an integrated study optimizing clarification, DNA digestion, and MC simultaneously, significant improvement in adenovirus purity was obtained. Finally, the collection of experimental results was used to model complete adenovirus production processes using BioSolve Process and determine the cost-of-goods (COG) of manufacturing for clinical applications. Through simulations of multiple scenarios, critical process parameters were identified and can serve as a guide for future process development decisions. It is anticipated that the contributions herein described will help address critically outstanding questions related to virus purification and thus enable the development of the economical processes for various manufacturing scales. / Thesis / Doctor of Philosophy (PhD) / Certain viruses can be used for human benefit and there are now more than a dozen approved therapies worldwide that use a virus as the main therapeutic agent or as the vector to instruct the patient’s cells to fight cancer and other diseases. The area keeps growing as thousands of other clinical trials continue to be conducted. One of the main challenges that can inhibit patient access to these ground-breaking new options is related to difficulties in producing and purifying enough virus. This study tackles the virus purification challenge by applying and improving membrane chromatography (MC), a promising and scalable technique where virus and impurities are separated based on how differently they interact with a membrane. Different experimental and modelling and simulation tools were applied to optimize MC and other directly-related steps of the production process. The findings in this study can contribute to the development of new virus-based therapeutics so they can reach patients in safe, effective, and affordable ways.

Antibodies and antibody fragments are the largest class of biotherapeutics in development with many products already available in the clinic. Antibodies are promising due to their naturally high affinity and specificity for biological targets. A key stumbling block to biopharmaceutical development compared to small molecule drugs is the general requirement for a stable liquid formulation, which is often difficult to obtain due to issues with aggregation, phase separation, particle formation, and chemical instabilities. Aberrant solution behaviour limits the production, storage and delivery of the monoclonal antibody. Biopharmaceutical solution behaviour is determined by weak, transient protein-protein and protein-solvent interactions. An attractive interaction potential between proteins in solution can lead to association. Irreversible association occurs when proteins undergo large scale structural changes and aggregate. Reversible association is less severe, but can lead to undesirable solution properties such as high viscosity, phase separation and opalescence, which can lead to difficulties throughout the downstream processing and formulation steps. These problems can become exacerbated during formulation of antibodies when trying to achieve high protein concentrations often required for effective antibody dosage. Firstly, we studied the domains of the Fab fragment using statistical models and continuum electrostatic calculations and found that the CH1 domain is more soluble than the other domains and has properties of intrinsically disordered like proteins which is supported by observations in the literature. We then investigated the immunoglobulin superfamily and found 11 proteins which may have a similarly disordered nature. We present a new web server for predicting protein solubility from primary sequence using an in-house algorithm that weighs the contribution of various sequence properties for predicting solubility. Lastly, we conducted physical characterisation of an antibody and human serum albumin in pharmaceutically relevant buffers and found that the interaction potential can be modelled using spherical models from low to high protein concentration. We hope that the work outlined in this thesis will contribute to the theoretical understanding and modelling of protein solution behaviour.

A literature study was performed on a new type of cancer medicine: antibody drug conjugates, or ADCs. These consist of a monoclonal antibody, chemically linked to a cytotoxic agent. What makes them unique is their selective toxicity against cancer cells. The first approval of such a pharmaceutical was in the year 2000, with three or four available in different regions of the world today. In the range of 50 registered drugs in clinical development were found, by major and minor corporations. These have been presented in a table in the appendix according to their properties such as type of linker, cytotoxin, development status etc. Furthermore, a detailed study has been done of the chemistry of the linker conjugation as well as an attempt at studying the ADC market. Finally, the mentioned strengths of the drug were compared to its weaknesses, mainly instability and otherwise poor pharmacokinetics. The main conclusion is that these drugs are expected to play a major role in oncology in the future.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-06-14T10:48:13Z No. of bitstreams: 1 marinnagomes.pdf: 1050831 bytes, checksum: 19f0caa7f0448c9ddc9f79c839e5aa28 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-06-14T11:32:30Z (GMT) No. of bitstreams: 1 marinnagomes.pdf: 1050831 bytes, checksum: 19f0caa7f0448c9ddc9f79c839e5aa28 (MD5) / Made available in DSpace on 2018-06-14T11:32:30Z (GMT). No. of bitstreams: 1 marinnagomes.pdf: 1050831 bytes, checksum: 19f0caa7f0448c9ddc9f79c839e5aa28 (MD5) Previous issue date: 2018-02-21 / Acidentes ofídicos representam um grave problema de saúde pública devido a sua alta incidência e a gravidade dos seus efeitos. No Brasil, o gênero Bothrops é responsável por 85% dos casos de acidentes. A principal forma de tratamento para tais acidentes é feita através da soroterapia, utilizando-se anticorpos eqüinos, porém os mesmos não são capazes de neutralizar os efeitos sistêmicos do envenenamento e podem causar reações adversas. Uma nova perspectiva para o tratamento de acidentes ofídicos é a utilização de anticorpos recombinantes, mais precisamente a porção scFv. O fragmento scFvBap1 é oriundo de um anticorpo monoclonal, produzido em Escherichia coli foi capaz de neutralizar os efeitos hemorrágicos do envenenamento. Atualmente as plantas vêm sendo amplamente utilizadas na produção de proteínas recombinantes de interesse farmacêutico. Neste estudo, objetivou-se a expressão do fragmento scFv em Nicotiana benthamiana, a fim de avaliar seu potencial para ser utilizado como soro antiofídico. Foram avaliadas a expressão transiente do fragmento scFvBap1 em folhas de N. benthamiana, bem como a expressão estável na mesma planta. Os fragmentos produzidos foram capazes de reconhecer e neutralizar as toxinas BaP1 de B. asper, BnP1 de B. newidae e ATX de B. atrox, demonstrando seu potencial para ser utilizado na terapia contra envenenamentos ofídicos. O sistema de produção estável apresentou-se maior rendimento de proteínas (270 μg/g) quando comparado ao sistema de produção transiente (43 μg/g). Por fim, o sistema mostrou-se uma ótima alternativa para produção do fragmento de anticorpo quando comparado ao sistema bacteriano. / Ophidian accidents are a global health problem due to its high incidence and gravity of its effects. In Brazil, the genus Bothrops is responsible for 85% of the cases of accidents. The main form of treatment for snake envenoming is serum therapy using equine antibodies, however, they are not able to neutralize the local effects of the toxin and may also cause adverse reactions. A new perspective for the treatment of snakebite envenoming has emerged with the use of recombinant antibodies, particularly the Fv fragment (scFv). The scFvBap1, is an efficient antibody capable of neutralizing the hemorrhagic effects and proteolytic activity caused by metalloproteinases. Currently the plants have been widely used in the production of recombinant proteins of pharmaceutical interest. In this study, we aimed the expression of the scFv fragment in Nicotiana benthamiana, in order to evaluate its potential as an antiophiidic serum. The transient expression of the scFvBap1 fragment in leaves of N. benthamiana, as well as the stable expression in the same plant, were evaluated.The fragments produced were able to recognize and neutralize the toxins BaP1 from B. asper, BnP1 from B. newidae and ATX from B. atrox, demonstrating their potential to be used in therapy against ophidian poisoning. The stable production system presented higher protein yield (270 μg / g) when compared to the transient production system (43 μg / g). Finally, the system proved to be a good alternative for producing the antibody fragment when compared to the bacterial system.