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.

Oncolytic viruses (OVs) are a class of cancer therapy that is currently undergoing clinical trials on its way to full regulatory approval. At present, the downstream processing of OVs relies on a combination of chromatography and membrane-based processes to remove process-related (e.g. host-cell proteins and nucleic acids) and product-related impurities (e.g. aggregated virus particles). This thesis explores various methods that can potentially be used to address the challenges associated with downstream processing during the production of OVs. To this end, the Rhabdoviral vector, which is currently undergoing clinical trials (phase I/II) for use in treating advanced or metastatic solid tumors, was selected as a promising oncolytic virus. One potential improvement in the downstream process that was investigated was the use of monolithic column chromatography for Rhabdovirus purification. Two monolithic anion-exchange columns (2 and 6 µm pore size) and one hydrophobic interaction column (6 µm pore size) were used to examine how column pore size affects virus recovery and contaminant removal. This investigation ultimately inspired the development of a purification process based on monolithic hydrophobic interaction column chromatography. Furthermore, this work is also the first to investigate how additives, namely glycerol, impact the hydrophobic interaction chromatography of virus particles. The developed process could be readily implemented for the scaled-up purification of the Rhabdoviral vector. Another challenge associated with the downstream processing of OVs is membrane fouling, which is characterized by a dramatic rise in transmembrane pressure (TMP) and low virus recovery. Indeed, membrane fouling poses a significant challenge, as some recent studies have reported that it can result in viral vector titer losses of over 80%. One critical use of membranes in downstream processing is for the sterile filtration of OVs, which is a required final step that is conducted right before vialing and involves passing the virus particles through a validated sterile filter. One of the main objectives of this thesis was to develop a fundamental understanding of the sterile filtration process and to optimize it in order to achieve higher throughput and lower losses, which are both essential to the large-scale production of OVs. To this end, a dead-end sterile filtration setup was designed, and various commercially available filters were evaluated to examine how membrane morphology affects fouling and product recovery. The results of these tests showed that double-layered composite filters enabled higher virus recovery and filtration capacity compared to single-layered sterile filters. Another cause of membrane fouling is the aggregation of virus particles, which is mediated by various interactions in the solution. To study this, the above-described setup was re-designed to create an effective procedure that utilizes minimal volumes of virus solution, while also enabling the rapid assessment of microscale filtration performance and a comprehensive understanding of virus-virus and virus-membrane interactions. This setup was used to study how different additives, including various proteins (bovine serum albumin and α-lactalbumin) and polymers (polyethylene glycol and polyvinylpyrrolidone), affect the microfiltration of the Rhabdoviral vector and, consequently, the TMP profile. Furthermore, the correlation between the membrane fouling rate (via TMP profiles) and virus recovery was also investigated. This investigation revealed that proteins significantly increase virus transmission and that polymers are incapable of mimicking the effects of the proteins. To explain this phenomenon, a theory based on the biophysical structure of proteins, mainly heterogenicity in charge distribution, was proposed. Moreover, membrane surface modification tests were conducted using bovine serum albumin, with the results indicating that this approach has considerable potential for enhancing virus transmission. Due to the similarities between the test setup and actual downstream processing unit operations, the results from this part of the thesis could be easily and accurately applied to process optimization. / Thesis / Candidate in Philosophy / There is considerable interest in the development of oncolytic viruses for cancer immunotherapy. Indeed, at the time of this thesis’ writing, a Canadian team of researchers is conducting the world’s first clinical trial using a combination of two viruses to kill cancer cells and stimulate an immune response. The process of manufacturing oncolytic viruses is generally divided into two major steps: upstream processing and downstream processing. While upstream processing focuses on virus propagation, downstream processing aims at removing process-related and product-related impurities. However, research into downstream process design and optimization has largely been neglected in favour of a focus on upstream processing, aimed at increasing bioreactor yields and achieving high viral titers. Consequently, downstream processing has become the main bottleneck in virus manufacturing processes, accounting for as much as 70% production costs. This thesis aims to identify and develop a fundamental understanding of the main challenges associated with the downstream processing of oncolytic viruses and to investigate methods for addressing them. Specifically, the present work focuses on the purification and final sterile filtration steps in the manufacturing of oncolytic Rhabdoviral vectors.

Orientadores: Telma Teixeira Franco, Reginaldo Guirardello / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-07-28T22:33:20Z (GMT). No. of bitstreams: 1 Santos_EveraldoSilvinodos_D.pdf: 4348712 bytes, checksum: f3506ac5746394a5083752c78e0b99b3 (MD5) Previous issue date: 2001 / Resumo: O presente trabalho refere-se ao uso da técnica de adsorção em lejto expandido para recuperar e purificar enzimas. Aspectos fundamentais da adsorção em leito expandido são abordados, utilizando lisozima e soro albumina bovina como proteínas modelo, e as enzimas, gliceraldeído-3-fosfato desidrogenase de Saccharomyces cerevisae, xilanase (extrato comercial) e quitosanase produzida por Bacil/us cereus. A avaliação da influência do uso de dois distribuidores, poroso e do tipo prato perfurado, mostrou que o distribuidor do tipo prato perfurado favoreceu mais à adsorção em leito expandido e que os adsorventes Streamline@ SP e Streamline@ DEAE possuem uma ampla distribuição de tamanho de partículas favorecendo ao fenômeno de segregação (Capítulo 2 - Artigo publicado no IEX 2000 (Cambridge/UK)). O uso de um processo integrado para recuperar e purificar a enzima intracelular Gliceraldeído 3-fosfato desidrogenase mostrou-se bem sucedido. O desempenho hidrodinâmico e cromatográfico foi estudado usando um adsorvente de estrutura pelicular (Pellicular) e dois adsorventes porosos comerciais (Stream1ine e Macrosorb) . Os resultados mostraram que o adsorvente Pellicular apresentou as melhores propriedades hidrodinâmicas e cromatográficas. (Capítulo 3 - Artigo em parceria e que será submetido à periódico internacional). O estudo da influência do uso de uma altura do leito empacotado, de 0,050 m e de 0,075 m na ALE operando-se em 10% da curva de ruptura, mostrou que uma altura de 0,075 m foi mais eficiente. Para o sistema lisozima - Streamline@ DEAE o rendimento aumentou com o aumento da velocidade linear enquanto que para o sistema BSA - Streamline@ SP esse fato não foi observado. Neste caso, para o sistema BSA - Streamline@ SP, a transferência de massa parece ser limitada pela menor densidade de carga acarretando assim em menores valores de eficiência em 10% de ruptura. (Capítulo 4 - Artigo aceito para publicação no periódico Biosepara_ion). O estudo da influência do conteúdo de células na adsorção em leito expandido mostrou que quando foi utilizado o extrato com o conteúdo de 5% de células (peso úmido), em leito expandido, o desempenho da purificação foi comprometido. (Capítulo 5 - Artigo aceito para publicação no periódico Joumal of Chromatography A). A adsorção de quitosanase de um caldo de fermentação de Baci/lus cereus em leito empacotado permitiu à obtenção de um pico com um fator de purificação de 7,6 e uma recuperação de 67,4% que eluiu com 0,51 M de NaCL Valores muito próximos a estes foram obtidos com o uso da adsorção em leito expandido com ambos os caldos, bruto e clarificado. Eletroforese em gel de poliacrilamida (SDS­P AGE) realizada para o tubo que exibiu a maior atividade, quando o leito foi no modo expandido e com células, mostrou a presença de duas bandas. Este fato sugere três possibilidades, a existência de duas quitosanases, a presença de um proteína contaminante ou a existência de uma quitosanase com duas sub-unidades (dímera). Entretanto, grande parte dos contaminantes foram separados da quitosanase em uma única etapa. (Capítulo 6 - Artigo que será submetido à um periódico internacional) / Abstract: This work deals with the application of Expanded Bed Adsorption (EBA) to recovery and purify enzymes. Model proteins, lisozyme and bovine serum albumin (BSA), as well as some enzymes, glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from baker's yeast, xylanase (from a commercial extract) and chitosanase from Bacil/us cereus were used to study the EBA background and application. The influence of two distributor (porous and perfurated plate) showed that the perfurated plate distributor was more favorable for the EBA. The Strearnline@ SP and the Strearnline@ DEAE adsorbents have a wide size distribution favourable for the segregation phenomena. (Chapter 2 - artic1e published in IEX 2000 (Carnbridge/UK». An integrated process was successful to recover and to purify an intracellular enzyme, Glyceraldehyde 3-Phosphate Dehydrogenase (G3PDH). Performance of the hydrodynamic and chromatographic properties using a pellicular adsorbent (Pellicular) and two porous commercial adsorbents (Stream1ine and Macrosorb) showed that the former presented the best hydrodynarnic and chromatographic properties. (Chapter 3 - paper that wiil be submmited to an international journal). The influence of two settled bed height, 0.050 m and 0.075m, respectively, in the EBA operating at 10% of the breaktrough curve, showed that the former was more efficient. For the lisozyme - Strearnline@ DEAE system yield increased with the increase of the velocity while for the BSA - Strearnline@ SP system this behaviour was not observed. In this case, mass transfer seems to be limited problably due to its lower charge density. (Chapter 4 - paper accepted for publication in the Bioseparation Journal). The influence of cell contents in the EBA using a 5% (wet weight) cell content showed that the purification perfonnance was hampered. (Chapter 5 ­paper accepted for publication in the Journal ofChromatography A). The chitosanase adsorption of fermentation broth from Bacillus cereus, in packed mode, showed a chitosanase peak that eluted with 0.51 M NaCl, in this case a 7.6-fold purification factor with 67.4% of activity recovery were obtained. Similar values were obtained for both c1arified and unc1arified fermentation broth using the bed in the expanded mode. Experiment in expanded mode showed that two bands were present in the peak showing the highest activity, this suggests three possibilities, the existence of two chitosanases, the existence of a contaminant protein or the existence of a chitosanase with two sub-units. However, it was possible to separate the chitosanase from the-main contaminants in just one step. (Chapter 6 - paper that will be submitted to an international journal) / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

The need of purified biomolecules, such as proteins or antibodies, has required the biopharmaceutical industries to look for new recovering solutions to reduce time and costs of bioseparations. In the last decade, the emergent field of membrane chromatography has gained attention as possible substituent of the common used protein A affinity chromatography for bioseparations. In this scenario, gold nanoparticles can be used as means for offering affinity, mainly because of their biocompatible and reversible binding behavior, together with their high surface area-to-volume ratio, which offers a large number of binding sites. This work introduces a new procedure for purification of monoclonal antibodies based on polymeric membranes functionalized with gold nanoparticles. This novel approach shortens the process of purification by promoting selective binding of antibodies, while separating a mixture of biomolecules during a filtration process. The effects of gold nanoparticles and the surrounding ligand on the proteins adsorption and filtration are investigated. The results confirm that the functionalization helps in inducing a selective binding, preventing the non-selective one, and it also improves the selectivity of the separation process.

The biopharmaceutical industry has experienced remarkable progress in the upstream production capacity of life-saving proteins. This is while the downstream processing has failed to keep pace, including unit operations which are working close to their physical limit with no economy of scale. Column chromatography which is an integral unit in different stages of downstream purification is considered as the major bottleneck in this section. The packed-bed resin media is costly and the processes are labor-intensive and extremely time consuming. Membrane chromatography which uses a stack of adsorptive membranes as the chromatographic media is one of the most promising alternatives for conventional chromatography techniques. The performance of membrane adsorbers is consistent over a wide range of flow rates which is owing to the dominance of convective solute transport as opposed to the diffusion-based nature of mass transfer within the pores of the resin beads. This translates to much higher productivity and considerably lower buffer consumption (even as high as 95%), leading to much lower overall processing costs. The other advantages are significantly lower footprints and decreased pressure drops, both contributing to diminished capital costs. Membrane adsorbers are greatly scalable and used in a single-use manner. The latter eliminates the cleaning and validation steps and brings about much shorter processing times and higher flexibility in process development. Due to the performance advantages of membrane chromatography, this technique is now widely used in purification of high volumes of samples in late-stage polishing. Currently available membrane adsorbers have radial-flow spiral-wound configuration with high frontal surface area to bed height ratio according to which dilute impurities are removed in a flow-through format at very high flow rates and low pressure drops. Nevertheless, they fail to give high-resolution for bind-and-elute separations which makes them unsuitable for many unit operations, highly restricting their application. Severe design deficiencies such as large dead volumes and varying membrane area over the bed height result in broad and poorly resolved peaks. Herein, a novel device design was successfully developed which addresses the abovementioned shortcomings. The laterally-fed membrane chromatography (LFMC) devices house a stack of rectangular membrane sheets with two rectangular lateral channels on both sides of the stack as the feed and permeate channels. The design offers balanced pressure over the sides of the stack as well as even solute flow path lengths due to which the solute residence time is very uniform. Also, the small dead volumes minimize the dispersion effects. These features make the LFMC technology highly suitable for bind-and-elute applications, the improvement which is brought about by a simple design. The devices are easy to fabricate and highly scalable. The LFMC devices containing cation-exchange (CEX) membranes with 7 mL bed volume were examined for bind-and-elute separation where they outperformed the equivalent commercially available radial-flow devices. The design was further modified to give even lower dead volumes and more cost-effective fabrication. The latest embodiment of the device gave resolutions which were comparable with the ones obtained with the commercially packed resin columns in 1 mL and 5 mL scale with consistency over wide range of flow rates. The results were all acquired using a three component model protein system. Upon the approval of suitability of the device for bind-and-elute separation, the CEX-LFMC was used for purification of monoclonal antibodies (mAbs), the largest class of biopharmaceuticals. The device showed great performance in separation of mAb charge variants when extensively shallow gradients (60 membrane bed volumes) were required. The devices offered very stable conductivity gradients at high flow rates. LFMC devices in three different preparative scales gave great performance in separation of mAb aggregates which was approved for different mAb samples. The other application studied with the CEX-LFMC devices was the single-step preparative purification of mono-PEGylated proteins which is as well very challenging due to the physicochemical similarities between the target molecules and the impurities. Collectively, the LFMC devices combine the high-resolution with high-productivity which is highly desirable in downstream purification of biological molecules with great potential to expand the application of membrane chromatography. Finally, the LFMC devices were modified to adapt the analytical scale where they were integrated with a stack of hydrophilized PVDF membranes. The device successfully delivered ultra-fast separation of mAb aggregates in less than 1.5 minutes based on hydrophobic interaction membrane chromatography (HIMC). The assay times achieved with the HI-LFMC technique outclassed the currently available ultra-high performance chromatography (UPLC) methods at the same time with being extremely cost-effective. The application of the LFMC technology in analytical scale has great potential to offer cheap and rapid analysis in process development and quality control section of biopharmaceutical manufacturing. / Thesis / Doctor of Philosophy (PhD)

Transgenic plant systems have been proposed as bioreactors in the production of pharmaceutical and industrial proteins. The economic benefits of inexpensive plant production systems could be erased if the downstream processing ends up being expensive. To avoid monoclonal antibody (mAb) modification or fouling of chromatography resins, removal of phenolics from plant extracts is desirable. Removal of major phenolics in Lemna extracts was evaluated by adsorption to PVPP, XAD-4, IRA-402 and Q-Sepharose resins. Analysis of phenolics adsorption to XAD-4, IRA-402 and Q-Sepharose showed superior dynamic binding capacities at pH 4.5 than at 7.5. The economic analysis using SuperPro Designer 7.0 indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The overall mAb processing cost can be reduced by implementing a phenolics removal step. To understand phenolics-resin interactions, adsorption isotherms of phenolic compounds (chlorogenic acid, ferulic acid, rutin, syringic acid and vitexin-2-O-rhamnoside) from different phenolic classes on three resins (IRA-402, PVPP, XAD-4) at pH 4.5 and 7.5 were determined. Differences in adsorption with the type of phenolics were observed, and PVPP was not efficient for phenolics removal. Screening of sugarcane lines for bovine lysozyme (BvLz) accumulation indicated that expression levels are still inadequate for commercial development. To maximize BvLz extraction, pH and ionic strength were evaluated; five conditions resulted in equivalent BvLz/TSP ratio. Membrane filtration process using BvLz extracts attained partial removal of native proteins by the 100 kDa membrane step, but also BvLz loss (21-29%). Regardless of the extraction condition, at least 47% of the starting BvLz was lost during the membrane processing. None of the evaluated extraction conditions caused a substantial recovery of BvLz in the concentrate. Alternative purification options for the IEX+HIC process, which achieved 95% BvLz purity, were tested. Direct loading of sugarcane extract concentrate on HIC and XAD-4 pretreatment of juice did not recovered BvLz as effectively as the IEX chromatography. Pure BvLz was obtained by the XAD+HIC process, but higher purification fold and HIC yield were achieved by the IEX+HIC process, due to the complete separation of BvLz and 18-kDa protein.

Plants are potential sources of valuable recombinant and native proteins that can be purified for pharmaceutical, nutraceutical, and food applications. Transgenic rice and corn germ were evaluated for the production of novel protein products. This dissertation addresses: 1) the extraction and purification of the recombinant protein, human lysozyme (HuLZ), from transgenic rice and 2) the processing of dry-milled corn germ for the production of high protein germ and corn protein concentrate (CPC). The factors affecting the extraction and purification of HuLZ from rice were evaluated. Ionic strength and pH was used to optimize HuLZ extraction and cation exchange purification. The selected conditions, pH 4.5 with 50 mM NaCl, were a compromise between HuLZ extractability and binding capacity, resulting in 90% purity. Process simulation was used to assess the HuLZ purification efficiency and showed that the processing costs were comparable to native lysozyme purification from egg-white, the current predominant lysozyme source. Higher purity HuLZ (95%) could be achieved using pH 4.5 extraction followed by pH 6 adsorption, but the binding capacity was unexpectedly reduced by 80%. The rice impurity, phytic acid, was identified as the potential cause of the unacceptably low capacity. Enzymatic (phytase) treatment prior to adsorption improved purification, implicating phytic acid as the primary culprit. Two processing methods were proposed to reduce this interference: 1) pH 10 extraction followed by pH 4.5 precipitation and pH 6 adsorption and 2) pH 4.5 extraction and pH 6 adsorption in the presence of TRIS counter-ions. Both methods improved the binding capacity from 8.6 mg/mL to >25 mg/mL and maintained HuLZ purity. Processing of dry-milled corn germ to increase protein and oil content was evaluated using germ wet milling. In this novel method, dry-milled germ is soaked and wet processed to produce higher value protein products. Lab-scale and pilot-scale experiments identified soaking conditions that reduced germ starch content, enhanced protein and oil content, and maintained germ PDI (protein dispersibility index). Soaking at neutral pH and 25 degrees C maintained germ PDI and improved CPC yield from defatted germ flour. CPC with greater than 75% protein purity was produced using protein precipitation or membrane filtration.

Orientador: Everson Alves Miranda / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T01:42:54Z (GMT). No. of bitstreams: 1 Robic_Goran_M.pdf: 2208429 bytes, checksum: 860e813410e0f18d21e22d51ca77e727 (MD5) Previous issue date: 2005 / Resumo: Os trabalhos realizados utilizando plantas transgênicas como biorreatores para produção de proteínas recombinantes indicam que a cano Ia, o milho e a soja são candidatos de grande potencial. Apesar da semente de soja e suas proteínas serem sistemas bem estudados, não existem estudos sistemáticos e comparativos sobre da utilização da soja como um biorreator, no tocante à extração e purificação de proteínas recombinantes. Até hoje, segundo a literatura consultada, só existe uma tentativa de usar soja como biorreator (Russel et al., 2005), mas por causa da baixa expressão da proteína recombinante (hormônio de crescimento humano), este estudo aparentemente não teve continuidade. O objetivo deste trabalho foi avaliar, sob o ponto de vista de recuperação (extração) e purificação, sementes de soja como biorreatores para produção de proteínas recombinantes, usando b-glucuronidase recombinante (rGUS) como proteína modelo / Abstract: The work done on the field of using trasgenic plants as bioreactors indicates the soybean, canola and corn as a plants of choice. Although the extraction and purification of soybean seed proteins is well studied and the plant is relatively easy to transform, there is practically no study done with transgenic soybean seeds expressing recombinant proteins in terms of downstream processing. To our knowledge, soybean was used to produce human growth hormone (Russel et al., 2005), but the study of purification was not done due to the low expression level of that recombinant protein. The objective of this work to evaluate the soybean seeds, in terms of recuperation (extraction) and purification, as a bioreactor for production of recombinant proteins using b-glucuronidase (rGUS) as a model protein / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Orientador: Everson Alves Miranda / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-06T23:00:08Z (GMT). No. of bitstreams: 1 Lima_LeonardoHenriqueFrancade_M.pdf: 992316 bytes, checksum: 57f2f7216633dfbabec7d916c3adb88d (MD5) Previous issue date: 2006 / Resumo: A precipitação de proteínas através da adição de sais (por exemplo, o sulfato de amônio e cloreto de sódio) é uma técnica comumente utilizada em recuperação e purificação de proteínas. Contudo, a remoção de sais do precipitado e tratamento da solução remanescente contendo altas concentrações de sal são etapas limitantes, devido ao custo do processo e a regulamentação ambiental. O uso de eletrólitos voláteis é uma alternativa aos sais convencionais neste tipo de processo, visto permitirem um fácil processamento e redução do custo do tratamento de efluentes: os sais voláteis podem ser removidos com a redução de pressão ou elevação da temperatura. Neste trabalho foram determinadas as curvas de solubilidade para a lisozima e insulinas bovina e suína em soluções aquosas de sais voláteis, em sistema vedado contendo carbonato, carbamato e bicarbonato de amônio em equilíbrio com CO2 e NH3. Estas curvas de equilíbrio foram determinadas como função da concentração salina (1,00 a 7,00 mol.kg-1), da temperatura (5,0 a 25,0 °C), e razão nitrogênio por carbono (RN/C) (2,0 e 2,5) das soluções.O aumento do conteúdo de nitrogênio aumentou a solubilidade das proteínas que, de uma forma geral, apresentaram um comportamento de solubilidade retrógrada em relação à temperatura. Espectros de dicroísmo circular sugeriram uma pequena desnaturação causada pela precipitação / Abstract: Protein precipitation induced by salt addition (e.g., ammonium sulfate and sodium chloride) is a commoly used technique in the downstream processing of proteins. However, salt removal from the precipitate and disposal of the salt containing liquid phase are key steps of the process due to cost and environmental concerns. Volatile salts are alternatives to conventional salts in this process since they can allow easy processing and reduce the cost of waste disposal: the volatile salt can be removed by pressure reduction and temperature increase. In this work we reported the solubility curves for lysozyme and swine and bovine insulins in aqueous solutions of the volatile salts of the system comprised of ammonium carbonate, carbamate, and bicarbonate in equilibrium with CO2 and NH3. These equilibrium curves were determined as funciton of the salt concentration (1.0-7.0 mol/kg), temperature (5.0-25.0 ºC), and N/C ratio (2.0 and 2.5) of the solutions. The increase of the N content of the solutions increased the proteins solubility that in general had a retrograde solubility. Circular dichroism spectra suggested some denaturation caused by the procipitation / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Orientador: Telma Teixeira Franco / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-07-28T03:46:25Z (GMT). No. of bitstreams: 1 Sousa_KathiaAssisde_M.pdf: 2724531 bytes, checksum: 3490f460776b8b8cf4564482c815165b (MD5) Previous issue date: 2001 / Resumo: Foram investigadas estabilidade frente a pH e temperatura e condições ótimas da enzima peroxidase do extrato bruto da casca da soja (Glycine max). Foi verificada a afinidade entre a enzima e íons cobre imobilizados no gel "Chelating Sepharose FastFlow" (CSFF) e também foi estudado o efeito do pH sobre a adsorção desta enzima. Foram construídas isotennas de adsorção para a peroxidase do extrato bruto de casca da soja e para as peroxidases padrão de soja e de nabo (horseradish), para verificar a capacidade máxima de adsorção do complexo CSFF-IDA-CU2+ para estas enzimas. Curvas de ruptura para a peroxidase do caldo bruto de casca da soja foram construídas para estudar a eficiência do complexo na adsorção da enzima. A purificação da peroxidase do extrato bruto da casca da soja foi estudada na coluna HR 5/5 empacotada com o complexo CSFF­IDA-CU2+ equilibrado com tampão fosfato de sódio O,IM a pH 6,0. Foi verificado que a peroxidase do extrato bruto da casca da soja apresentou condições ótimas de atividade a pH 4,5, mostrou-se estável por três horas em temperaturas entre 1 e 55°C. Foi observado que a adsorção mais seletiva da peroxidase do extrato bruto de casca da soja se deu a pH 6,0, quando 51% da enzima foi retida após dez minutos de contato entre a peroxidase e o complexo CSFF -IDA-Cu2+ a 25°C em tampão fosfato de sódio O,IM. A adsorção das peroxidases da casca da soja, padrão comercial de nabo e padrão comercial de soja no complexo CSFF-IDA-Cu2+ obedeceu ao modelo proposto por Langmuir. Com a construção das curvas de ruptura foi verificado que a pH 6,0 houve a melhor seletividade na separação da atividade de peroxidase, quando 96,9% de atividade foi recuperada. Na adsorção da peroxidase do extrato bruto da casca da soja na coluna HR 5/5 empacotada com CSFF-IDA-CU2+ a pH 6,0, foi obtido um fator de purificação de 5,9 vezes com um rendimento de 83,4% / Abstract: The conditions for the soybean hull peroxidase activity were investigated for pH and temperature. It was observed that the best pH for maximum activity was at 4.5, however the activity was only 5% reduced at pH values 5.0 and 5.5 and was 20% reduced for pH's between 6.0 and 7.0 and 45% reduced at pH 8.0. It was stable at this pH interval for four hours period, however, it lost 20% activity at pH 4.5 after one hour incubation. Best temperature for the enzyme active was 55 °C and it was stable from 1 to 55 °C for at least three hours. The affinity between the soybean hull peroxidase and copper ions immobilized in a solid matrix was investigated. The maximum capacity ofthe CSFF-IDA-CU2+ to interact with the enzyme was calculated by plotting the concentrations of the proxidase found in the liquid phase in equilibrium with the peroxidase concentrations found in the solid phase (isotherms). Breakthrough curves were built to study the efficiency ofCSFF-IDA-CU2+ bed to adsorb the peroxidase of soybean hulI and also two standards peroxidases commercially available from soybean and ITom horseradish. The effect of pH on the adsorption of the enzyme was also investigated and it was observed that the most selective adsorption of the soybean hulI peroxidase was at pH 6.0. Purification of the soybean hull peroxidase was studied in the column HR 5/5 packed with the complex CSFF-IDA-CU2+ in 100 mM phosphate buffer at pH 6.0. The adsorption of the soybean hull peroxidase, soybean and horseradish peroxidases by the CSFF-IDA-CU2+ was observed to folIow Langmuir model. The final peroxidase purified by the process developed in this work showed that the specific activity of the enzyme was about 5.9 fold higher than that of cru de extract and the yield was about 83.4% / Mestrado / Desenvolvimento de Processos Químicos / Mestre em Engenharia Química