In this study, it was aimed to investigate the effects of different co-carbon sources on therapeutically important glycoprotein, recombinant human erythropoietin (rHuEPO) production by Pichia pastoris by designing feeding strategies which were applied in the production phase of the bioprocess. During the experiments, the cell growth, sorbitol, mannitol, and methanol consumptions, recombinant human EPO production, alcohol oxidase activity, total protease concentrations and the by-products organic acid concentrations were analyzed. In this context, firstly, laboratory scale air filtered shake bioreactor experiments were performed by P. pastoris Mut+ strain to investigate the effects of mannitol and sorbitol. 50 gL-1 initial concentration of co-substrates was found more affordable and appropriate for cell concentration and recombinant protein production. Thereafter, six pilot scale bioreactor operations were designed and performed. In the first designed strategy (named as SSM strategy), batch-wise 50 g L-1 sorbitol was fed at t=0 h of the production phase and then sorbitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Srb0=0.025 h-1 within t=0-15 h of the production phase together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the following bioreactor experiments co-substrate mannitol was fed to the system with different feeding strategies together with fed-batch methanol feeding with a pre-determined specific growth rate of &mu / M0=0.03 h-1. In the second strategy (MM), only 40 g L-1 mannitol was added to the system at t=0 h of the production phase. In the third strategy (MMM), after adding 50 g L-1 mannitol at t=0 h, mannitol concentration was kept constant at 50 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.11 h-1 within t=0-9 h of the production phase when the same cell concentration was attained in SSM strategy. In the fourth one (MLM), limiting amount of mannitol, 3 g L-1, was added at t=0 h and then mannitol concentration was kept constant at 3 g L-1 by fed-batch feeding with a pre-determined specific growth rate of &mu / Man0=0.005 h-1 within t=0-10 h of the production phase. After these strategies, several pulses, batch-wise, mannitol feeding strategies were performed. In the fifth strategy (MPM), besides 50 g L-1 initial mannitol feeding at t=0 h, adding second batch-wise mannitol at t=6 h, and third one at t=12 h were applied. In the last strategy (MPMG), four 50 g L-1 pulse feeding of mannitol were performed at t=0 h, 7 h, 14 h, and 24 h, containing glycerol, with an initial concentration in the fermentation medium being 8 g L-1. The highest extracellular rHuEPO production was achieved in the fifth strategy MPM as CrHuEPO=645 mg L-1 at t=9 h while the highest cell concentration was achieved in the first strategy SSM as Cx=109 gL-1 at t=48 h. The overall cell and product yields on total substrate were calculated as YX/St=0.22 g g-1 and YP/St=2.23 mg g-1 in the highest rHuEPO production case.

In this study, the effects of pH on therapeutically important protein, recombinant human erythropoietin (rhuEPO), production by Pichia pastoris was investigated at pH=4.0, 4.5, 5.0, 5.5 and 6.0. rHuEPO production was started by methanol induction in fed-batch mode. The highest cell concentration was obtained at pH=4.5 as 81.4 g L-1. The co-substrate substrate sorbitol, which was added batch-wise, was consumed at t=15 h of the operations at pH=4.0, 4.5 and 5.0. However as the pH increases above pH=5.0 the sorbitol consumption rate decreases. The highest rHuEPO concentration was achieved at pH=4.5 as 0.158 g L-1 which was 1.43-, 1.24-, 1.95- and 1.23-fold higher than those obtained at pH=4.0, 5.0, 5.5, and 6.0, respectively. Also at pH=4.5 overall cell yield on substrate was 0.51 g g-1 and overall rHuEPO yield on substrate was 1.45 mg g-1. rHuEPO concentration was decreased in the last 3-6 hour of the operation due to proteolysis. Therefore extracellular protease concentrations in the medium were determined. As expected, since the investigated pH range was acidic, the amount of acidic proteases was found to be higher than neutral and basic proteases. Furthermore the total protease concentration increased linearly in the fermentation broth, having close values at different pH values. Thus, pH did not have a significant effect on extracellular protease activity. Alcohol oxidase (AOX) activities showed similar behavior at different pH. The highest specific AOX activity was attained at pH=4.5, at which the highest rHuEPO concentration was achieved, as 110.1 U g-1 CDW. Keywords:

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Erythropoietin (EPO) is a glycoprotein which stimulates the erythropoiesis (production of hemaceas) and is clinically used for the treatment of renal anemia. EPO formulations present usually elevated contamination by aluminum. Aluminum is known to participate in the pathogenesis of osteodystrophy and encephalopathy associated with chronic hemodialysis, and has been proposed to be involved in central nervous system degeneration diseases such as Alzheimer s diseases. In the present work, the presence of Al as contaminant in erythropoietin formulations consumed by chronic renal patients, was investigated by developing and optimizing a chromatographic method for separation and determination of proteins, with subsequent collection of fractions and measured of the Al present by graphite furnace atomic absorption spectrometry (GF AAS). As the commercialization of pharmaceutical forms of EPO are in glass containers with rubber cap (bottle, vial or syringe), it was investigated the ability of the constituents of EPO formulations (salts, amino acids, urea, and mannitol among others) to promote the extraction of aluminum present in both glass and rubber. The tests were performed considering the process of sterilization and the contact of the solutions during storage. The results demonstrated that the interaction of EPO with aluminum is higher than that of albumin (the other proteic constituent). Among the other constituents, citric acid and citrate presented the highest interaction. For each constituent there is probably a different mechanism for the release of aluminum from glass and rubber, but all constituent of EPO formulations interacted with glass and rubber packaging, leading to their contamination by aluminum. As the level of contamination by aluminum in commercial formulations of erythropoietin was determined, and it was observed that the lyophilized powder presented the lowest contamination by aluminum, this form must be the choice for renal patients, due to the susceptibility of these patients to aluminum toxicity. / A eritropoetina (EPO) é uma glicoproteína que estimula a eritropoiese (produção das hemáceas) e é usada clinicamente para o tratamento de anemias associadas à insuficiência renal crônica. Formulações de eritropoetina apresentam geralmente uma elevada contaminação por alumínio, que é um metal extremamente tóxico para pacientes com insuficiência renal. Este metal é relacionado a doenças neurológicas e ao comprometimento da estrutura óssea dos pacientes, podendo causar a morte se administrado cronicamente ou em concentrações elevadas aos pacientes. Alguns autores também relacionam o desenvolvimento de doenças degenerativas como a doença de Alzheimer com intoxicação por alumínio. A presença do alumínio em formulações farmacêuticas destinadas ao tratamento da insuficiência renal e nutrição parenteral é um problema sério devido a toxicidade do alumínio e ao contato direto com o sangue do paciente. Em nosso laboratório, nos dedicamos a estudar a origem do aluminio nestas soluções e de que modo ele interage com os seus constituintes. No presente trabalho, investigou-se a presença de Al como contaminante em formulações farmacêuticas de eritropoetina consumida por pacientes renais crônicos através do desenvolvimento e otimização de um método cromatográfico para separação e determinação de proteínas, com posterior coleta de frações e medidas do Al presente por espectrometria de absorção atômica com forno de grafite (GF AAS). Como as formas farmacêuticas de comercialização da EPO são em recipientes de vidro com tampa de borracha (frasco-ampola ou seringas), investigou-se a capacidade dos constituintes das formulações de EPO (sais, aminoácidos, uréia e manitol entre outros) em promover a extração do alumínio presente tanto no vidro quanto na borracha. Os ensaios foram realizados considerando o processo de esterilização e o contato das soluções durante o período de armazenamento. Os resultados encontrados demonstraram que a EPO apresenta maior interação com o alumínio que a albumina (o outro constituinte proteico das formulações). Entre os outros constituintes, o ácido cítrico e o citrato apresentaram a maior interação. Para cada constituinte há provavelmente um mecanismo diferenciado de liberação de Al do vidro e da borracha, porém todos os constituintes das formulações de EPO interagiram com o vidro e a borracha, levando a contaminação destas por alumínio. Como foi determinado o nível de contaminação por alumínio das formulações de eritropoetina comerciais, observou-se que a forma farmacêutica de pó liofilizado apresentou menor contaminação por alumínio devendo, portanto, ser a forma de escolha para os pacientes renais, tão sucetíveis a toxicidade do alumínio.

Intracellular calcium is a major determinant of a wide variety of cell functions and thus of organ function. In order to get a clear picture of the intracellular calcium status it is preferable to assess the content of the various intracellular calcium pools as well as the characteristics of the transmembrane calcium movements, Le., the magnitude of the transmembrane Ca2+ flux upon stimulation and the rate of the subsequent return to baseline levels. The first aim of this study was to establish and evaluate the methods in the laboratory. The methods investigated include atomic absorption spectrometry, graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry for the determination of the total cell calcium content, fluorescence spectrophotometry for the determinations of intracellular free Ca2+ and transmembrane Ca2+ movements and transmission electron microscopy for the localisation of intracellular calcium. The methods eventually identified as feasible included fluorescence spectrophotometry for the determination of intracellular free Ca2+ and transmembrane Ca2+ movements and transmission electron microscopy for the localisation of intracellular calcium. The newly developed fluorescent calcium indicator, fura-PE3, was presently shown to be the most reliable fluorescent indicator for the intracellular free Ca2+ determinations. The best method for the calcium localisation by transmission electron microscopy was an adaptation of the antimonate precipitation technique. The following objectives were set in order to contribute to the knowledge in chronic renal failure; examination of the intracellular free Ca2+ content in the neutrophils of end stage renal failure patients on maintenance haemodialysis treatment, as the result of renal failure, dialysis treatment and medication combined; examination of the characteristics of the transmembrane Ca2+ movements; investigation of the intracellular calcium distribution in the neutrophils; exploration of a possible link between the alterations in intracellular calcium status and factors known to influence the calcium status, including the lipid composition of the membrane, the oxidative status as reflected by anti-oxidant vitamin levels, as well as the levels of parathyroid hormone, and ionised serum calcium. This study involved 14 chronic renal failure patients on maintenance haemodialysis. An increase in intracellular free Ca2+, the magnitude of the transmembrane Ca2+ flux upon fMLP stimulation and an increase in the rate of the subsequent decrease in intracellular free calcium were found. In separating the patients into those receiving rHuEPO and those not receiving rHuEPO, it was seen that the significance in the increase in intracellular free Ca2+ could be ascribed to the values obtained in those patients receiving rHuEPO - despite the fact that they were the only patients receiving calcium channel blockers. No overt indications of oxidative stress could be detected by anti-oxidant vitamin levels. Nevertheless, a decrease in the content of specific membrane fatty acids occurred, supporting the previous suggestions of the presence of a mild chronic inflammatory condition in the chronic renal failure patient on maintenance haemodialysis treatment. These results suggest that factors other than those associated with uraemia, such as rHuEPO administration, might result in an increase in intracellular free Ca2+ in cells of CRF/MHT patients. The magnitude of the rHuEPD-induced increase in intracellular free Ca2+ and the effects of the various calcium channel blockers need urgent further investigation as ineffective counteraction of the rHuEPO effect, as indicated by the relative ineffectivity of Norvasc, may have serious side-effects. / Dissertation (MSc)--University of Pretoria, 2000. / Physiology / unrestricted

Para produção de biofármacos protéicos, como a eritropoietina recombinante humana (EPOrh), são necessárias alterações pós-traducionais adequadas que garantam a sua especificidade e atividade biológica. Essas características são obtidas apenas em biorretores baseados em células eucarióticas, como as da glândula mamária. Sistemas baseados nesse tipo celular, tanto in vivo quanto in vitro, já são utilizados para produção estratégica e viável de proteínas recombinantes biologicamente ativas. Assim, tanto o estabelecimento de novas linhagens de células mamárias que apresentem boa expressão protéica quanto o desenvolvimento de sistemas in vivo que utilizem a estrutura da glândula mamária para essa produção de proteínas recombinantes são de grande valia. O presente trabalho teve como objetivo comparar dois métodos de estabelecimento de uma cultura de células de glândula mamárias ovinas, enzimático e não enzimático, e verificar sua capacidade de expressão das proteínas do leite β-lactoglobulina, α-caseína, β-caseína e κ-caseína mediante o tratamento com SFB (soro fetal bovino) ou SOL (soro de ovelha lactante), na presença ou não de Matrigel. Para isso, foi realizado um experimento in vitro, no qual foi estabelecido o cultivo celular até a passagem 12 (P12) de duas linhagens celulares: digerida (LD) e não digerida (LND). Para a LD na P12 foi observado apenas um tipo celular, o qual era positivo para a marcação com vimentina. Essa linhagem apresentou expressão gênica de β-caseína e β-lactoglobulina apenas quando tratada com meio de cultivo acrescido de SFB, sendo a expressão inferior (P=0,001) ao grupo da LND submetido ao mesmo tratamento. Já a LND, quando tratada com meio adicionado com SFB expressou κ-caseína além da β-caseína e β-lactoglobulina. A troca do SFB do meio de cultivo por SOL aumentou a expressão gênica de β-lactoglobulina (P=0,001) para ambas linhagens. Foi realizada a curva de crescimento para LD e LND na P12 com o meio de cultivo acrescido com SFB ou SOL. Para a LND observou-se o efeito do meio na velocidade de crescimento celular, sendo que foi maior para o grupo tratado com SFB (P<0,05). Para a LD, não ocorreu o efeito do meio na velocidade de crescimento celular (P>0,05), não sendo observada diferença com a LND tratada com SOL (P>0,05). A LND apresentou marcação positiva para a presença de vimentina e citoqueratina. Este trabalho visou, ainda, estabelecer um sistema de produção da EPOrh no leite de ovelhas não transgênicas pela técnica de infusão intra-mamária in vivo de dois plasmídeos diferentes e verificar a secreção qualitativa desta proteína por Western-blotting. Assim, foi feito um experimento in vivo no qual glândulas mamárias de ovelhas foram transfectadas com dois plasmídeos diferentes: ALAC (n=2), BGL (n=2) e controle negativo (n=2). Após a infusão dos plasmídeos, foi realizada a eletroporação de cada teto (3 choques de 500 volts com a duração de 15ms cada, sendo realizada a inversão da polaridade). Os animais foram ordenhados durante 20 dias após a transfecção, porém não foi possível detectar a presença de EPOrh nas amostras de leite analisadas. O limiar de detecção do teste utilizado foi de 67,5pg de EPOrh (Eritromax®) em leite controle negativo de ovelha. Concluindo, foi possível estabelecer o cultivo in vitro das LD e LND com capacidade de expressar proteínas do leite, sendo a expressão da β-lactoglobulina aumentada pelo tratamento com SOL. Ambas as linhagens apresentaram marcação positiva para vimentina, mas apenas LND para citoqueratina. Ainda, para o experimento in vivo, não foi possível detectar a expressão de EPOrh no leite das ovelhas transfectadas com os plasmídeos ALAC e BGL. / Some post-translational modifications are necessary for the production of biopharmaceutical proteins, such as recombinant human erythropoietin (rhEPO), with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors, in vivo or in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression and the development of systems for production of recombinant proteins by the mammary gland in vivo are essential studies. One of the main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as β-lactoglobulin, α-casein, β-casein and κ-casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel®. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed β-lactoglobulin and β-casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0,001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed β-lactoglobulin, β-casein and κ-casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of β-lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P<0,05). However, the medium did not have effect on growth speed of LD (P>0,05) and no difference was observed at the NDL treated with LOS (P>0,05). The NDL was positive for staining with vimentin and cytokeratin. The second main objective of this study was to establish an in vivo system for the production of rhEPO in milk of non-transgenic ewes by the intra-mammary infusion of two different plasmids and verify the qualitative milk secretion of this protein by western-blotting. In this way, in the in vivo experiment ovine mammary glands were transfected with two different plasmids: ALAC (n=2), BGL (n=2) and negative control (n=2). Each half udder was filled with plasmid solution and three 3 electric pulses of 500 volts were applied for 15ms each, followed by another three pulses with reversed polarity. The three animals were milked for 20 days after transfection, nevertheless it was not possible to identify rhEPO in any milk sample. The test threshold to identify rhEPO (Eritromax®) in milk from a negative control animal was 67,5pg. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of β-lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. In the in vivo experiment, rhEPO secretion was not detected in the milk from ewes transfected with ALAC and BGL plasmids.

Para produção de biofármacos protéicos, como a eritropoietina recombinante humana (EPOrh), são necessárias alterações pós-traducionais adequadas que garantam a sua especificidade e atividade biológica. Essas características são obtidas apenas em biorretores baseados em células eucarióticas, como as da glândula mamária. Sistemas baseados nesse tipo celular, tanto in vivo quanto in vitro, já são utilizados para produção estratégica e viável de proteínas recombinantes biologicamente ativas. Assim, tanto o estabelecimento de novas linhagens de células mamárias que apresentem boa expressão protéica quanto o desenvolvimento de sistemas in vivo que utilizem a estrutura da glândula mamária para essa produção de proteínas recombinantes são de grande valia. O presente trabalho teve como objetivo comparar dois métodos de estabelecimento de uma cultura de células de glândula mamárias ovinas, enzimático e não enzimático, e verificar sua capacidade de expressão das proteínas do leite β-lactoglobulina, α-caseína, β-caseína e κ-caseína mediante o tratamento com SFB (soro fetal bovino) ou SOL (soro de ovelha lactante), na presença ou não de Matrigel. Para isso, foi realizado um experimento in vitro, no qual foi estabelecido o cultivo celular até a passagem 12 (P12) de duas linhagens celulares: digerida (LD) e não digerida (LND). Para a LD na P12 foi observado apenas um tipo celular, o qual era positivo para a marcação com vimentina. Essa linhagem apresentou expressão gênica de β-caseína e β-lactoglobulina apenas quando tratada com meio de cultivo acrescido de SFB, sendo a expressão inferior (P=0,001) ao grupo da LND submetido ao mesmo tratamento. Já a LND, quando tratada com meio adicionado com SFB expressou κ-caseína além da β-caseína e β-lactoglobulina. A troca do SFB do meio de cultivo por SOL aumentou a expressão gênica de β-lactoglobulina (P=0,001) para ambas linhagens. Foi realizada a curva de crescimento para LD e LND na P12 com o meio de cultivo acrescido com SFB ou SOL. Para a LND observou-se o efeito do meio na velocidade de crescimento celular, sendo que foi maior para o grupo tratado com SFB (P<0,05). Para a LD, não ocorreu o efeito do meio na velocidade de crescimento celular (P>0,05), não sendo observada diferença com a LND tratada com SOL (P>0,05). A LND apresentou marcação positiva para a presença de vimentina e citoqueratina. Este trabalho visou, ainda, estabelecer um sistema de produção da EPOrh no leite de ovelhas não transgênicas pela técnica de infusão intra-mamária in vivo de dois plasmídeos diferentes e verificar a secreção qualitativa desta proteína por Western-blotting. Assim, foi feito um experimento in vivo no qual glândulas mamárias de ovelhas foram transfectadas com dois plasmídeos diferentes: ALAC (n=2), BGL (n=2) e controle negativo (n=2). Após a infusão dos plasmídeos, foi realizada a eletroporação de cada teto (3 choques de 500 volts com a duração de 15ms cada, sendo realizada a inversão da polaridade). Os animais foram ordenhados durante 20 dias após a transfecção, porém não foi possível detectar a presença de EPOrh nas amostras de leite analisadas. O limiar de detecção do teste utilizado foi de 67,5pg de EPOrh (Eritromax®) em leite controle negativo de ovelha. Concluindo, foi possível estabelecer o cultivo in vitro das LD e LND com capacidade de expressar proteínas do leite, sendo a expressão da β-lactoglobulina aumentada pelo tratamento com SOL. Ambas as linhagens apresentaram marcação positiva para vimentina, mas apenas LND para citoqueratina. Ainda, para o experimento in vivo, não foi possível detectar a expressão de EPOrh no leite das ovelhas transfectadas com os plasmídeos ALAC e BGL. / Some post-translational modifications are necessary for the production of biopharmaceutical proteins, such as recombinant human erythropoietin (rhEPO), with a good specific action and a high biological activity. These modifications are obtained only by bioreactors based on eukaryotic cell as mammary cells. Bioreactors, in vivo or in vitro, with this kind of cell have been used for a viable and strategic production of biologically active recombinant proteins. For this reason, the establishment of a new line of mammary cells with high milk protein expression and the development of systems for production of recombinant proteins by the mammary gland in vivo are essential studies. One of the main objectives of this study was to compare two methods, enzymatic and non-enzymatic, to establish ovine mammary cells culture and verify their gene expression of milk proteins such as β-lactoglobulin, α-casein, β-casein and κ-casein with different treatments: LOS (lactating ovine serum) or FBS (fetal bovine serum) added to the culture medium, in the presence or absence of Matrigel®. In this manner, an in vitro study was performed and the culture of two lines were established, digested (DL) and non-digested (NDL), of ovine mammary cell until the passage 12 (P12). In DL was observed just one cellular type that was positive for staining with vimentin. This cell line expressed β-lactoglobulin and β-casein genes with the FBS treatment and without Matrigel. The gene expression was lower (P=0,001) when compared to the NDL under the same conditions of culture. Then, the NDL expressed β-lactoglobulin, β-casein and κ-casein genes when treated with FBS without Matrigel. The treatment with LOS in the culture medium increased the gene expression of β-lactoglobulin for both cell lines. The growth curve was determined with both cell lines in P12 with FBS or LOS treatment. For the NDL, the type of medium had effect on the cell growth speed and was highest with the FBS treatment (P<0,05). However, the medium did not have effect on growth speed of LD (P>0,05) and no difference was observed at the NDL treated with LOS (P>0,05). The NDL was positive for staining with vimentin and cytokeratin. The second main objective of this study was to establish an in vivo system for the production of rhEPO in milk of non-transgenic ewes by the intra-mammary infusion of two different plasmids and verify the qualitative milk secretion of this protein by western-blotting. In this way, in the in vivo experiment ovine mammary glands were transfected with two different plasmids: ALAC (n=2), BGL (n=2) and negative control (n=2). Each half udder was filled with plasmid solution and three 3 electric pulses of 500 volts were applied for 15ms each, followed by another three pulses with reversed polarity. The three animals were milked for 20 days after transfection, nevertheless it was not possible to identify rhEPO in any milk sample. The test threshold to identify rhEPO (Eritromax®) in milk from a negative control animal was 67,5pg. In conclusion, the in vitro culture of NDL and DL was established up to the P12 with expression of milk protein and the LOS treatment increased the expression of β-lactoglobulin. The two cell lines culture were positive for staining of vimentina but only NDL was positive for cytokeratin. In the in vivo experiment, rhEPO secretion was not detected in the milk from ewes transfected with ALAC and BGL plasmids.

In this study, it was aimed to develop a bioprocess using the Pichia pastoris expression system as an alternative to the mammalian system used in industry, for production of the therapeutically important glycoprotein, erythropoietin, and to form stoichiometric and kinetic models. Firstly, the human EPO gene, fused with a polyhistidine-tag and factor-Xa protease target site, in which cleavage produces the native termini of EPO, was integrated to AOX1 locus of P. pastoris. The Mut+ strain having the highest rHuEPO production capacity was selected. The glycosylation profile of rHuEPO was characterized by MALDI-ToF MS and Western blotting. The native polypeptide form of human EPO was obtained for the first time in P. pastoris expression system, after affinity-purification, deglycosylation and factor-Xa protease digestion. Thereafter, effects of medium components and pH on rHuEPO production and cell growth were investigated in laboratory-scale bioreactors. Sorbitol was shown to increase production efficiency when added as a co-substrate. Moreover, a cheap alternative nutrient, the byproduct of biodiesel industry, crude-glycerol, was suggested for the first time for P. pastoris fermentations. Furthermore, methanol feeding strategy was investigated in fed-batch pilot-scale bioreactors, producing 70 g L-1 biomass and 130 mg L-1 rHuEPO at t=24h. Moreover, metabolic flux analysis by using the stoichiometric model formed, which consisted of m=102 metabolites and n=141 reactions, proved useful in further understanding the P. pastoris metabolism. Finally, the first structured kinetic model formed for r-protein production with P. pastoris successfully predicted cell growth, substrate consumption and r-product production rates, where rHuEPO production kinetics was associated with AOX production and proteolytic degradation.

Erythropoietin is a glycoprotein which stimulates the erythropoiesis, clinically used for the treatment of renal anaemia. The biological and chromatographic methods for the potency evaluation of recombinant human erythropoietin (rhEPO) in pharmaceutical products were validated in the present work. The normocythaemic mice bioassay was carried out in female BALB/c strain, with multiple injections schedule and reticulocytes counted by automated flow cytometry. The dose-response curve was linear (r2=0.9708) in the concentration range of 1 to 64 IU/mL. A reversed-phase liquid chromatography method (RP-LC) was developed and validated using a Jupiter C4 column (250 mm x 4.6 mm), maintained at ambient temperature (25°C). The mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) and mobile phase B consisted of 0.08% TFA:acetonitrile (30:70, v/v), run in linear gradient: 0.1-60 min, 0.1-100% of B at flow rate of 0.5 mL/min with detection at 280 nm. The chromatographic separation was obtained within 60 min and it was linear (r2=0.9997) in the concentration range of 10-150 μg/mL. The procedures were validated evaluating parameters such as, the specificity, linearity, precision, accuracy, robustness, and limit of quantitation, as well as, the limit of detection evaluated in the validation of the chromatographic method. The methods were applied for the potency evaluation of the alfa and beta rhEPO in pharmaceutical products, which were analysed by the chromatographic method and compared to the bioassay showing mean differences between the estimated potencies of 11.2% higher for the RP-LC. The alternative established represents a contribution towards the replacement of the animals improving the quality control and assuring the safety and efficacy of the biological product. / Eritropoietina é uma glicoproteína que estimula a eritropoiese e é usada clinicamente para o tratamento de anemias associadas à falência renal crônica. No presente trabalho foram validados métodos biológico e cromatográfico para a avaliação de potência de eritropoietina humana recombinante (rhEPO) em produtos farmacêuticos. O ensaio biológico foi validado em camundongos normocitêmicos, fêmeas, da linhagem BALB/c, com protocolo de injeções múltiplas e contagem automatizada dos reticulócitos por citometria de fluxo. A curva doseresposta foi linear (r2=0,9708) na faixa de concentração de 1 a 64 UI/mL. Paralelamente, desenvolveu-se e validou-se método por cromatografia líquida em fase reversa (CL-FR) empregando coluna Júpiter C4 (250 mm x 4,6 mm), mantida a temperatura ambiente (25°C). A fase móvel A foi composta de ácido trifluoracético 0,1% e a fase móvel B composta de acetonitrila/ácido trifluoracético 0,08% (70:30, v/v), eluída em gradiente linear: 0,1 60 min, 0 100% de B, na vazão de 0,5 mL/min e detecção no ultravioleta a 280 nm. A separação cromatográfica foi obtida no tempo de 60 min, sendo linear (r2=0,9997) na faixa de concentração de 10-150 μg/mL. Ambos procedimentos foram validados com base nos parâmetros de especificidade, linearidade, precisão, exatidão, robustez e limite de quantificação, bem como limite de detecção, avaliado na validação do método cromatográfico. Os métodos foram aplicados na avaliação de potência de rhEPO alfa e beta em produtos farmacêuticos e estudou-se a correlação entre os métodos. Demonstrou-se que as análises dos produtos farmacêuticos forneceram diferenças médias de potência 11,2 % superiores para o CL-FR. Desse modo, estabeleceu-se alternativa no contexto da substituição do uso de animais, contribuindo para aprimorar o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biológico.Eritropoietina é uma glicoproteína que estimula a eritropoiese e é usada clinicamente para o tratamento de anemias associadas à falência renal crônica. No presente trabalho foram validados métodos biológico e cromatográfico para a avaliação de potência de eritropoietina humana recombinante (rhEPO) em produtos farmacêuticos. O ensaio biológico foi validado em camundongos normocitêmicos, fêmeas, da linhagem BALB/c, com protocolo de injeções múltiplas e contagem automatizada dos reticulócitos por citometria de fluxo. A curva doseresposta foi linear (r2=0,9708) na faixa de concentração de 1 a 64 UI/mL. Paralelamente, desenvolveu-se e validou-se método por cromatografia líquida em fase reversa (CL-FR) empregando coluna Júpiter C4 (250 mm x 4,6 mm), mantida a temperatura ambiente (25°C). A fase móvel A foi composta de ácido trifluoracético 0,1% e a fase móvel B composta de acetonitrila/ácido trifluoracético 0,08% (70:30, v/v), eluída em gradiente linear: 0,1 60 min, 0 100% de B, na vazão de 0,5 mL/min e detecção no ultravioleta a 280 nm. A separação cromatográfica foi obtida no tempo de 60 min, sendo linear (r2=0,9997) na faixa de concentração de 10-150 μg/mL. Ambos procedimentos foram validados com base nos parâmetros de especificidade, linearidade, precisão, exatidão, robustez e limite de quantificação, bem como limite de detecção, avaliado na validação do método cromatográfico. Os métodos foram aplicados na avaliação de potência de rhEPO alfa e beta em produtos farmacêuticos e estudou-se a correlação entre os métodos. Demonstrou-se que as análises dos produtos farmacêuticos forneceram diferenças médias de potência 11,2 % superiores para o CL-FR. Desse modo, estabeleceu-se alternativa no contexto da substituição do uso de animais, contribuindo para aprimorar o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biológico.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-29T13:14:34Z No. of bitstreams: 1 ana-paula-araujo.pdf: 1867740 bytes, checksum: ab9d7b2866753474ebe7ecb95d8e32d9 (MD5) / Made available in DSpace on 2012-11-29T13:14:34Z (GMT). No. of bitstreams: 1 ana-paula-araujo.pdf: 1867740 bytes, checksum: ab9d7b2866753474ebe7ecb95d8e32d9 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil. / A eritropoetina (EPO) é um hormônio produzido, principalmente, pelos rins, que regula a produção de células vermelhas do sangue. Trata-se de uma glicoproteína com 166 aminoácidos, três sítios de N-glicosilação e um de O-glicosilação. Apresenta massa molecular na faixa de 30-34 kDa, sendo aproximadamente 40% correspondente aos glicídeos. A EPO está disponível como agente terapêutico produzido através da tecnologia do DNA recombinante em cultura de células de mamíferos. A EPO humana recombinante (rHuEPO) é usada para o tratamento de anemia associada à insuficiência renal crônica. Devido a relevância médica da rHuEPO, sua produção está sendo nacionalizada através do processo de transferência de tecnologia do Centro de Inmunología Molecular (CIM), de Cuba, para o Instituto de Tecnologia de Imunobiológicos (Bio-Manguinhos)/FIOCRUZ. O controle de qualidade sobre o processo produtivo da rHuEPO e seu produto final é bastante rigoroso. Neste contexto, o mapeamento peptídico é um dos mais importantes ensaios usados no controle em processo da produção de proteínas recombinantes. Este ensaio assegura a integridade da estrutura primária das proteínas ao final das etapas de suas purificações. Sendo assim, o objetivo desta dissertação de mestrado foi definir uma metodologia de mapeamento peptídico da rHuEPO, visando o controle de processo da produção deste biofármaco em Bio-Manguinhos. Previamente, a preparação da rHuEPO fornecida pelo CIM foi avaliada quanto a sua homogeneidade e caracterizadapor eletroforeses em gel de poliacrilamida, cromatografia de fase reversa, cromatofocalização eespectrometria de massa. Para obter os peptídeos da rHuEPO, a hidrólise enzimática desta glicoproteína com a enzima tripsina foi avaliada em diferentes tempos de incubação e com relações enzima/substrato distintas. O fracionamento dos peptídeos foi feito por cromatografia líquida de fase reversa em coluna C18, empregando-se diferentes colunas cromatográficas e gradientes de eluição. As principais frações peptídicas isoladas da cromatografia de fase reversa foram analisadas por espectrometria de massa a fim de comprovar a identidade dos peptídeostrípticos da rHuEPO. A especificidade da metodologia desenvolvida foi avaliada através da comparação dos perfis peptídicos do hidrolisado tríptico da rHuEPO e do quimotripsinogênio A. A hidrólise tríptica usando-se relação enzima/substrato de 1/50 (p/p), concentração de rHuEPO de 1 mg/mL e incubação de 1 hora a 37o C hidrolisou mais de 99% da glicoproteína. As colunas cromatográficas de fase reversa Hi-Pore RP 318, Vydac 218TP C18 e Delta Pak C18 se mostraram aptas para o fracionamento dos peptídeos trípticos da rHuEPO. Em comparação à metodologia usada no CIM, o tempo de incubação de hidrólise foi reduzido em, pelo menos,2 horas e a duração da corrida cromatográfica para obtenção do mapa peptídico diminuiu em, pelo menos, 1 hora e 42 minutos. Na análise por espectrometria de massa das frações isoladas da cromatografia de fase reversa, seis peptídeos trípticos da rHuEPO foram observados. Tais frações peptídicas foram identificadas como pertencentes à EPO humana pelo programa Mascot Search Results. Dessa forma, ficou estabelecida uma metodologia de mapeamento peptídico especifica, simples e rápida para ser utilizada no controle em processo da produção da rHuEPO em Bio-Manguinhos. / Erythropoietin (EPO) is a hormone produced mainly by the kidney that regulates the production of red blood cells. It is a glycoprotein with 166 amino acids, three N-glycosylation and one O-glycosylation sites. It has a molecular weight in the range of 30-34 kDa, being approximately 40% of its content corresponding to carbohydrates. EPO is available as a therapeutic agent produced by recombinant DNA technology in mammaliancell culture. The recombinant human EPO (rHuEPO) is used to treat anemia associated with chronic renal disease. Due to rHuEPO medical relevance, its production technology is a subject of transference from the Centro de Inmunología Molecular(CIM), Cuba, to the Instituto de Tecnologia em Imunonobiologicos(Bio-Manguinhos)/FIOCRUZ. The quality control over production process of rHuEPO and its final product is quite rigorous. In this context, peptidemapping is one of the most important tests used in process control of recombinant proteins production. This test ensures the primary structure integrity of a protein after its purification processes. Thus, the objective of this dissertation was to define a methodology for peptide mapping of rHuEPO aiming to be used in the process control of this biopharmaceutical production in Bio-Manguinhos. Previously, the preparation of rHuEPO provided by CIM was characterized by polyacrylamidegel electrophoresis, reversed phase chromatography, chromatofocusing and mass spectrometry. In order to obtain rHuEPO peptides, the enzymatic hydrolysis of this glycoprotein with the enzyme trypsin was assessed at differents incubation times and enzyme/substrate ratio. Peptides fractionation was performed by reverse phase liquid chromatography, using distincts C18 columns and elution gradients. The main peptide fractions from reversed phase chromatography were analyzed by mass spectrometry to confirm the identity of rHuEPO tryptic peptides. The specificity of the developed methodology was evaluated by comparing peptides profiles from rHuEPO and Chymotrypsinogen A tryptic hydrolysates. The tryptic digestion using enzyme/substrate ratio of 1/50 (w/w), substrate concentration of 1 mg/mL and incubation for 1 hour at 37°C hidrolysated more than 99% of the glicoprotein. The reversed phase columns Hi-Pore RP318, Vydac 218TP C18 e Delta Pak C18 were able to fractionate the rHuEPO tryptic peptides. Compared to the methodology used in CIM, the hydrolysis incubation time was reduced by at least 2 hours and the chromatographic running time to obtain the peptide map was decreased by at least 1 hour and 42 minutes. The mass spectrometry analysis of the fractions isolated from reversed-phase chromatography showed six tryptic peptides of rHuEPO. These peptide fractions were identified as belonging to human EPO by the program Mascot Search Results. Thus it was established an specific, simple and fast peptide mapping methodology to use in process control of rHuEPO in Bio-Manguinhos.

O estudo teve como objetivo avaliar a razão de custo-efetividade, sob a perspectiva do Sistema Único de Saúde SUS, do tratamento da anemia de pacientes em Terapia Renal Substitutiva. Duas alternativas foram comparadas: um novo medicamento recentemente registrado no Brasil, o Ativador Contínuo de Receptor de Eritropoetina (Continuous Erythropoietin Receptor Activator), CERA, e outro, atualmente disponível no sistema de saúde brasileiro, a Eritropoetina Recombinante Humana - Epo-rHu. Métodos: Um modelo de Markov simulou o curso de uma coorte de pacientes em Terapia Renal Substitutiva tratados com CERA e Epo-rHu por quatro anos. A qualidade de vida associada ao uso dos medicamentos foi estimada de forma indireta, por meio de entrevista qualificada com os profissionais cuidadores, previamente submetida e aprovada pelo Comitê de Ética em Pesquisa local. Foi realizada análise de sensibilidade no modelo proposto através da variação dos parâmetros: dose dos medicamentos, custo das estratégias, taxa de desconto e efetividade utilizados para sua construção. Resultados: A média da qualidade de vida atribuída aos pacientes tratados foi 6,3 para a Epo-rHu, 7,8 para o CERA e 9,3 para os pacientes transplantados. O modelo demonstrou que a estratégia mais custo-efetiva é a terapêutica com a Epo-rHu, com um custo por QALY de R$ 21.052,00. O custo incremental por QALY ganho associado ao CERA foi de R$ 72.974,00. Conclusão: A utilização mensal do medicamento CERA está associada à maior qualidade de vida quando comparada a Epo-rHu. No entanto, a terapia com o novo medicamento não se mostrou mais custo-efetiva frente ao tratamento com Epo-rHu / This study sought to determine the cost-effectiveness of anemia treatment in dialysis patients for Brazilian Public Health System. Two alternatives were compared: a new drug, the Continuous Erythropoietin Receptor Activator, CERA, recently registred in Brazil, and another one, provided nowadays by the National Health System, Epo-rHu (Recombinant Human Eythropoietin). Methods: A Markov cohort of dialysis patients treated with CERA and Epo-rHu for four years was used to perform the base case analysis. The model outputs were QALYs and costs. The quality of life associated with each drug was measured by interviews applied to health care professionals. These interviews were previously submitted and approved by the local ethics committee. A sensitivity analysis was applied to the model to test it, varying the values of drugs dosage, costs, discount rate and effectiveness. Results: The average quality of life assigned by health care professionals to the patients treated with Epo-rHu, CERA and to kidney transplant receptors were respectively 6,3, 7,8 and 9,3. The model showed that Epo-rHu treatment was more cost-effective than CERA treatment. The cost-effectiveness ratio of Epo-rHu therapy was R$ 21.052,00. In addition, the cost per QALY gained of CERA therapy was R$ 72.974,00. Conclusion: Anemia treatment with CERA is associated with improvement in quality of life compared to Epo-rHu therapy. However, the new drug is not more cost-effective than the drug provided by the Brazilian Public Health System