Thesis (doctoral)--Universität Hamburg, 1979.

Thesis (M.S.)--University of Wisconsin--Madison, 1936. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison, 1936. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Master of Science / Department of Chemical Engineering / James Edgar / The bio-pharmaceutical industry began over 30 years ago with the production of human insulin and has shown incredible growth ever since. With forecasted annual worldwide sales of over $450B in 2025 for biopharmaceuticals, they are expected to be at least 25% of the entire pharmaceutical market. Therapeutics based on monoclonal antibodies (mAbs) make up roughly a third of all biopharmaceutical sales with indications from asthma, to cancer, to Parkinson’s disease. The recent approval of the first biosimilar mAb products in the US and Europe has exposed up to 20 of the top grossing biologic products to competition for the first time, while 75% of the US market is expected to lose patent exclusivity by 2020. With the increased competition from biosimilars, the costs associated with producing mAb based therapeutics will be a constraint on maintaining market share going forward. The majority of the total manufacturing costs for mAbs resides in the downstream processing where Protein A chromatography is the predominantly employed technology for the primary capture step. With Protein A’s high unit cost of up to $15,000 per liter and susceptibility to deamidification when exposed to high pH cleaning and sanitization chemicals, it is no surprise that many mAb manufacturers are considering alternatives. The objective of this work is to review the production process of mAb therapeutics, with a specific focus on the advantages, disadvantages, and alternatives to Protein A affinity chromatography as the primary capture step in downstream processing.

Orientador: Elenice Deffune / Banca: Rosana Rossi Ferreira / Banca: Ana Paula Costa Nunes da Cunha Cozac / Resumo: A tecnologia de produção de anticorpos monoclonais revolucionou a investigação diagnóstica, especialmente a análise dos grupos sanguíneos em imuno-hematologia. O polimorfismo das hemácias humanas torna a produção de novos insumos extremamente necessária. Dentre os sistemas de grupos sanguíneos descritos, o sistema Rh tem se mostrado um dos mais complexos pela vasta composição de antígenos. Analisando aspectos estruturais do antígeno D associado às técnicas de biologia molecular, identificam-se numerosas variantes deste antígeno por trocas de DNA entre genes similares levando a alteração de epítopos e consequentemente de padrão de expressão fenotípica na superfície das hemácias. Desvendar e identificar estas variantes com insumos biotecnológicos desenvolvidos no país foi o desafio desta pesquisa, onde anticorpos monoclonais contra antígenos eritrocitários foram produzidos através de fusão celular, utilizando-se células de baço de camundongos BALB/c imunizados com hemácias fenótipo D categoria IVa. Nove fusões foram realizadas e os sobrenadantes de cultura foram triados por teste de hemaglutinação em microplacas. Foram congelados e mantidos em nitrogêncio líquido 41 híbridos. Estes híbridos foram clonados e mantidos em meio de cultura para a obtenção dos anticorpos monoclonais, resultando em 12 clones IgG1, 5 IgG2a e 10 IgM. Os clones foram selecionados para produção de líquido ascítico, através da injeção dos clones em camundongos BALB/c. Os anticorpos monoclonais produzidos, inicialmente não demonstraram a especificidade foco do presente trabalho que foi a produção de anticorpos anti-D, no entanto, os hibridomas podem ter secretado diferentes especificidades de anticorpos monoclonais, de acordo com os antígenos presentes na membrana das hemácias utilizadas para a imunização dos camundongos. Uma segunda fase da pesquisa está sendo ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The blood groups analyses in immunohematology have been highly benefited based on the advance of the monoclonal antibody technology improvement on diagnostics investigation. The human red blood cell polymorphism increases the need for new alternatives. The Rh system blood group is the most complex of all, due to the vast antigenic composition. When analyzing the structural aspects of the D antigen in combination with the molecular techniques, based upon DNA exchange between similar genes it is possible to identify a huge number of partial D variants, this situation leads to a change of epitopes and hence the red blood cell phenotypic expression pattern. Unveil and identify these variables through the use of local biotechnology was the challenge of this research. Monoclonal antibodies against human red blood cell were produced by cell fusion, using spleen cells from immunized BALB/c mice and DIVa red blood cells. Nine fusions had been made and the culture supernatants were selected to the hemaglutination test using microplates. Forty-one hybrids had been frozen and kept in liquid nitrogen. These hybrids had been cloned and kept in the culture to collect monoclonal antibodies, resulting in 12 IgG1, 5 IgG2a e 10 IgM. The clones had been selected for ascitic fluid production, by injection in BALB/c mice. The monoclonal antibodies produced initially had not produced the anti-D as expected by the research. However, the hibrydomas could have created different specificities of monoclonal antibodies, according to the antigen located in the donor red blood cells membrane used in the mice's immunization. The second phase of the research is being done to confirm the monoclonal antibodies specificities produced through the deployment of new tests to immunoglobulin identification / Mestre

The antral hormone gastrin has generally been regarded as a non antigenic molecule necessitating its conjugation with a large "carrier” to be an effective inducer of antibody. Such conjugation has normally taken the form of covalent binding to a highly antigenic molecule or ionic binding to an inert particle. Alternatively, some success has been achieved by the injection of impure antral extracts. This report describes four approaches to the problem of the induction of antibodies specific for gastrin. Initially, natural hog gastrin was obtained after the procedure of Gregory and Tracy (1964). The pure active hormone was modified by alkaline hydrolysis to liberate an N-terminal amino group for covalent conjugation. The modified gastrin, however, was not specifically identified or isolated in quantity. Consequently, pure and impure antral extracts, hemocyanin-bound synthetic human gastrin and latex-bound gastrin were applied to a variety of animals. Passive hemagglutination tests in ducks revealed titres of 400 to 1600 to both the pure and impure extracts but a series of four injections of pure gastrin into an antrectomized rabbit failed to raise detectable antibody. Injections of 3 mg. of SHG bound to hemocyanin was unsuccessful; antibody titre to the carrier molecule was high but no specificity appeared to be directed to the synthetic hapten. The immunization of chickens with latex-bound natural gastrin was more fruitful. Although a number of precipitin tests established the presence of antibodies to gastrin, it was apparent that the assay was inadequate as a sensitive test for the bivalent antigen. Furthermore, the antibody titre was not sufficiently high to be successfully applied to a radioimmunoassay. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

This investigation involved the production of specific antisera against known International Streptomyces project strains of Streptomyces.

Orientador: Lilian Maria de Castilho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T12:16:53Z (GMT). No. of bitstreams: 1 Melo_Laerciode_D.pdf: 15534575 bytes, checksum: 0d830bc1ab58a6f7460b5beacb32557c (MD5) Previous issue date: 2006 / Resumo: O Programa de Controle de Qualidade Externo em Imunohematologia foi introduzido com o objetivo de avaliar a qualidade do diagnóstico em Imunohematologia. Foram realizadas 41 avaliações em 223 instituições no período de 1992 a 2003 que incluíram testes de proficiência para determinação ABO e RhD, fenotipagem Rh e K, teste direto da antiglobulina humana, pesquisa de anticorpos irregulares e identificação de anticorpos. No período de 12 anos o programa incluiu 8014 determinações de grupo sanguíneo ABO, 8000 classificações RhD, 5193 fenotipagens Rh, 5101 fenotipagens K, 7939 pesquisas de anticorpos irregulares, 4533 identificações de anticorpos e 7912 testes diretos da antiglobulina humana. Respostas incorretas foram classificadas como erros clericais, técnicos, ou não determinados. Ocorreu um número elevado de erros clericais na determinação do grupo sanguíneo ABO (76/76 erros), classificação RhD (34/58 erros) e na fenotipagem Rh (50/73 erros). Erros técnicos ocorreram predominantemente na determinação do antígeno D fraco (91/95 erros), na pesquisa de anticorpos irregulares (252/301 erros) e na identificação de anticorpos (321/335 erros) e estavam associados à sensibilidade das técnicas e dos reagentes utilizados. As avaliações teóricas, como incentivo à educação continuada, auxiliaram na diminuição dos erros, estimularam o treinamento dos participantes e o trabalho em equipe. A detecção dos erros foi importante para propor melhoria técnica com reavaliação dos protocolos bem como ações corretivas visando à melhoria da qualidade. A participação em um programa de controle de qualidade externo bem organizado mostrou ser fundamental na melhoria da qualidade dos testes em imunohematologia, reduzindo a freqüência dos erros e no potencial risco de reações hemolíticas garantido uma melhoria na qualidade das transfusões / Abstract: The Brazilian External Quality Assessment Program in Immunohematology (BEQAPI) was introduced with the objective of evaluating the quality of diagnosis in Immunohematology. From 1992 to 2003, proficiency tests for ABO grouping, Rh (D, C, c, E, e), K phenotyping, Direct Antiglobulin Testing (DAT), Antibody Screening (AS) and Antibody Identification (AI) were performed. Forty-one evaluations were carried out in 223 institutions. Over the period of 12 years, the program included 8,014 ABO typing, 8,000 RhD typing, 5,193 Rh (C, c, E, e), 5,101 K phenotyping, 7,939 AS, 4,533 AI and 7,912 DATs. Erroneous responses were classified as clerical, technical or undetermined. A substantial proportion of erroneous responses due to clerical errors occurred in ABO typing (76/76 errors), RhD typing (34/58 errors) and Rh phenotyping (50/73 errors). Technical errors occurred predominantly for weak D (91/95 errors), AS (252/301 errors) and AI (321/335 errors). Based on these results, since 1996, participants have received ¿Questions and Case Studies¿ as an incentive for training and education. The results of the present study show an improvement in the performance of participants in the course of the program. We found that a well-organized external proficiency program can contribute to the improvement of quality of testing in Immunohematology / Doutorado / Clinica Medica / Doutor em Clínica Médica

archives@tulane.edu / During the 2013-2016 West African outbreak, severe gastrointestinal symptoms were common in Ebola patients and associated with poor outcome. The efficient spread of Ebola virus (EBOV) via vomitus and diarrheal fluids, which contain high concentrations of virus, likely contributed to the scale of the outbreak. The delta-peptide is a conserved product of post-translational processing of the abundant EBOV soluble glycoprotein (sGP). Here, the murine ligated ileal loop model, which is well-established for the study of diarrheal disease, was used to demonstrate that delta-peptide is a potent enterotoxin. Dramatic intestinal fluid accumulation peaked at 9-12 hours following injection of biologically relevant amounts of delta-peptide into ileal loops, along with gross destruction of the villous architecture, loss of goblet cell polysaccharides, and secretion of pro-inflammatory cytokines. Transcriptomic analyses showed that delta-peptide triggers immune and cell survival responses. Delta-peptide may contribute greatly to EBOV-induced gastrointestinal pathology in humans. These findings demonstrate that the EBOV delta-peptide is an enterotoxic viroporin and may be categorized as a novel virulence factor. We then hypothesized that the delta-peptide may also be a druggable target to explore a new avenue for novel EBOV therapeutics, which are direly needed. An unconventional coupling strategy was employed to conjugate a modified version of the 23-residue delta-peptide to a carrier protein Keyhole Limpet Hemocyanin (KLH) in order to immunize rabbits so that they may generate high-affinity binding antibodies against the delta-peptide. Antisera was collected from the rabbits after regular immunizations and the IgG fraction of the antisera demonstrated binding and recognition against several delta-peptide variants confirmed by Western blotting and ELISAs. We then used this knowledge to determine therapeutic index in vitro by testing the antibody against the delta-peptide in the context of synthetic PC-PG vesicles and CHO cells. The purified IgG was then tested against the peptide in vivo to determine therapeutic efficacy by returning to the mouse model of diarrheal pathology mentioned previously. In a small pilot experiment, we were able to successfully block the enterotoxic activity of the delta-peptide and did not observe gastrointestinal distress in the mice that were treated with the peptide and antibody together versus just the peptide alone, signifying that the delta-peptide is a druggable target and may reveal a new therapeutic avenue against EBOV pathogenesis. / 1 / Shantanu Guha