Interleukin-3 is a cytokine, which acts on many target cells within the haemopoietic system, often in synergy with the other cytokines. Streptomyces lividans NCIMB 11416/IL3 p002 secreting human interleukin-3 was used as the host organism in this study of improving target protein production. Streptomyces also produces several proteases including extracellular endoprotease that truncate the N-terminus of the recombinant protein. Federal guidelines and regulations banning animal-derived medium components necessitate the refinement or redevelopment of industrial medium formulations. The development of a defined medium without animal products is most desirable for the production of pure and safe biological products. The objective of the proposed research was the development and application of engineering methodology for the development of a defined medium and the analysis and optimization of a bacterial bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. During the first phase of this study the task was to develop a systematic procedure for the design and optimization of a chemically defined medium. The study aimed at replacing casein peptone in conventional medium for S. lividans with essential amino acids and determining the optimum proportion of the amino acids. To accomplish this, starvation trials with growth limiting amino acids were performed to establish the baseline for the nutritional requirement. The starvation trials revealed that essential amino acids for growth and product formation are amongst the following eight amino acids: Arg, Asn, Asp, Glu, Leu, Met, Phe, and Thr. Following these preliminary experiments, a statistically based experimental method called mixture experiments along with distance-based multivariate analysis revealed that Asp, Leu, Met, and Phe were the essential amino acids. Then, another mixture experiment design known as simplex lattice design was performed and artificial neural networks were employed to obtain the optimum proportions of the essential amino acids. The optimal medium was found to be composed of 56% Asp, 5% Met, and 39% Phe. It was found in previous studies that in complex media, several types of protease are produced during fermentation. Using the defined medium no proteolytic activity was detected in the fermentation broth. The second optimization method was based on metabolic flux analysis. A comprehensive metabolic network was developed for S. lividans. The metabolic network included carbohyderate and amino acid metabolism in both anabolic and catabolic reactions. According to the experimental results, the time course of the fermentation was divided into two phases, Phase E1 and Phase E2. In the first phase amino acids were used as a nitrogen source and in the second phase ammonia was the nitrogen source for growth and product formation. The metabolic network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consisted of 62 intracellular metabolites and 91 biochemical reactions. Two different objective functions were considered for optimization: maximizing the specific growth rate and minimizing the redox equivalent. A linear programming approach was used for optimizing the objective functions. The proposed model was able to predict the specific growth rate very accurately with a maximum error of 10%. The oxygen uptake rate and carbon dioxide evolution rate were evaluated with maximum error of 27% and 35%, respectively. Sensitivity analysis revealed that amino acid uptake was the growth limiting flux during the Phase E1 of the fermentation. During Phase E2 the uptake rate of ammonia had a significant effect on the specific growth rate. Sensitivity analysis of the specific growth rate and redox potential with respect to the biomass components showed that any additional supply of biomass building blocks (amino acids, nucleotides) would not significantly affect the specific growth rate and redox potential production as well as the calculated flux pattern.

Interleukin-3 is a cytokine, which acts on many target cells within the haemopoietic system, often in synergy with the other cytokines. Streptomyces lividans NCIMB 11416/IL3 p002 secreting human interleukin-3 was used as the host organism in this study of improving target protein production. Streptomyces also produces several proteases including extracellular endoprotease that truncate the N-terminus of the recombinant protein. Federal guidelines and regulations banning animal-derived medium components necessitate the refinement or redevelopment of industrial medium formulations. The development of a defined medium without animal products is most desirable for the production of pure and safe biological products. The objective of the proposed research was the development and application of engineering methodology for the development of a defined medium and the analysis and optimization of a bacterial bioprocess for recombinant protein production. The underlying hypothesis is that a significant improvement of target protein productivity is achievable by using appropriate optimization techniques. During the first phase of this study the task was to develop a systematic procedure for the design and optimization of a chemically defined medium. The study aimed at replacing casein peptone in conventional medium for S. lividans with essential amino acids and determining the optimum proportion of the amino acids. To accomplish this, starvation trials with growth limiting amino acids were performed to establish the baseline for the nutritional requirement. The starvation trials revealed that essential amino acids for growth and product formation are amongst the following eight amino acids: Arg, Asn, Asp, Glu, Leu, Met, Phe, and Thr. Following these preliminary experiments, a statistically based experimental method called mixture experiments along with distance-based multivariate analysis revealed that Asp, Leu, Met, and Phe were the essential amino acids. Then, another mixture experiment design known as simplex lattice design was performed and artificial neural networks were employed to obtain the optimum proportions of the essential amino acids. The optimal medium was found to be composed of 56% Asp, 5% Met, and 39% Phe. It was found in previous studies that in complex media, several types of protease are produced during fermentation. Using the defined medium no proteolytic activity was detected in the fermentation broth. The second optimization method was based on metabolic flux analysis. A comprehensive metabolic network was developed for S. lividans. The metabolic network included carbohyderate and amino acid metabolism in both anabolic and catabolic reactions. According to the experimental results, the time course of the fermentation was divided into two phases, Phase E1 and Phase E2. In the first phase amino acids were used as a nitrogen source and in the second phase ammonia was the nitrogen source for growth and product formation. The metabolic network was used to form a set of linear algebraic equations based on the stoichiometry of the reactions by assuming pseudo-steady state for intracellular metabolites. The metabolic flux model consisted of 62 intracellular metabolites and 91 biochemical reactions. Two different objective functions were considered for optimization: maximizing the specific growth rate and minimizing the redox equivalent. A linear programming approach was used for optimizing the objective functions. The proposed model was able to predict the specific growth rate very accurately with a maximum error of 10%. The oxygen uptake rate and carbon dioxide evolution rate were evaluated with maximum error of 27% and 35%, respectively. Sensitivity analysis revealed that amino acid uptake was the growth limiting flux during the Phase E1 of the fermentation. During Phase E2 the uptake rate of ammonia had a significant effect on the specific growth rate. Sensitivity analysis of the specific growth rate and redox potential with respect to the biomass components showed that any additional supply of biomass building blocks (amino acids, nucleotides) would not significantly affect the specific growth rate and redox potential production as well as the calculated flux pattern.

<p>Zemlji&scaron;te predstavlja bogat izvor različitih mikroorganizama čiji<br />produkti metabolizma mogu biti od izuzetnog značaja za čoveka.<br />Dosada&scaron;nja ispitivanja mikrobnog diverziteta u zemlji&scaron;tu su<br />otkrila bogati biosintetski potencijal za proizvodnju novih<br />prirodnih proizvoda kod velikog broja mikroorganizama, naročito<br />kada je u pitanju klasa Actinobacteria. Među zemlji&scaron;nim izolatima,<br />rod Streptomyces prednjači po broju identifikovanih bioaktivnih<br />molekula u odnosu na sve ostale bakterije. Stoga je jedan od<br />ciljeva u okviru ove doktorske disertacije izolacija streptomiceta iz<br />rizosfera medicinski značajnih biljaka sakupljenih na teritoriji<br />Republike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urtica<br />dioica) i ispitivanje njihovog antifungalnog potencijala na različite<br />vrste kandida. Morfolo&scaron;ki različiti izolati (ukupno 103) su izolovani<br />iz uzoraka rizosfera i okarakterisani kao streptomicete. Dve<br />različite podloge i dve procedure za ekstrakciju su kori&scaron;ćene da bi<br />se pospe&scaron;ila detekcija antifungalnih jedinjenja. Ispitan je uticaj<br />ukupno 412 ekstrakata na rast Candida albicans disk difuzionim<br />esejem pri čemu je utvrđeno da 42% (43/103) izolata imaju<br />sposobnost proizvodnje antifungalnih jedinjenja pri ispitivanim<br />uslovima. Pojedini ekstrakti su inhibirali rast važnih humanih<br />patogena poput Candida krusei, Candida parapsilosis, i Candida<br />glabrata. Na osnovu stepena i spektra antifungalne aktivnosti<br />devet izolata je odabrano za dalja istraživanja. Ispitana je<br />sposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnoj<br />kulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na već<br />formirane biofilmove kandide u koncentracijama od 8 do 250<br />pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovu<br />metaboličku raznolikost dobijeni su kori&scaron;ćenjem tečne<br />hromatografije visokih performansi. Tri ekstrakta sa specifičnom<br />antifungalnom aktivno&scaron;ću podvrgnuta su hemijskim analizama s<br />ciljem da se detektuju i strukturno okarakteri&scaron;u molekuli koji su<br />nosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno-<br />rezonantne spektroskopije otkriveno je da su aktivni<br />molekuli genistein, daidzein i staurosporin. Genistein i daidzein<br />koji su poznati fitoestrogeni poreklom iz sojinog bra&scaron;na za koje je<br />poznato da inhibiraju ključne enzime u biosintetskom putu<br />steroida. Njihovo prisustvo je u ovom istraživanju detektovano<br />usled kori&scaron;ćenja sojinog bra&scaron;na u hranljivoj podlozi. Kako<br />streptomicete u čijim ekstraktima su detektovani ovi molekuli<br />pokazuju sposobnost oslobađanja ovih važnih jedinjenja iz<br />kompleksne hranljive podloge, mogu se uzeti u razmatranje za<br />biotehnolo&scaron;ku proizvodnju fitoestrogena. Staurosporin je<br />detektovan kao nosilac antifungalne aktivnosti kod ekstrakta soja<br />Streptomyces sp. BV410. Staurosporin je inhibitor protein kinaza i<br />njegovi derivati i analozi se koriste u kao antitumorski agensi.<br />Biosinteza ovog molekula je optimizovana do prinosa od 36,94<br />mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadrži<br />glukozu, skrob, manitol i sojino bra&scaron;no (JS). Dalja optimizacija<br />hranljive podloge za biosintezu staurosporina ukazala je na<br />sledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba,<br />21,46 g/l manitola, 17,32 g/l sojinog bra&scaron;na. Primenom<br />definisanih optimalnih vrednosti i kori&scaron;ćenjem odgovarajućih<br />matematičkih modela, predviđeno je da će se na ovaj način<br />postići prinosi od 46,88 mg/l staurosporina i 12,05 mg/ml<br />biomase. Validnost predviđenih rezultata potvrđena je<br />izvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta).<br />Ispitana je kinetika biosinteze staurosporina i produkcije biomase,<br />kao i potro&scaron;nje izvora ugljenika i razvijeni su odgovarajući<br />procesni modeli. Dodatna optimizacija je podrazumevala dodatak<br />suplemenata koji prema literature stimuli&scaron;u sekundarni<br />metabolizam streptomiceta (joni cinka, gvožđa, fosfati, metil<br />oleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pH<br />vrednosti (6,5, 7,5 i 8,5) a uspe&scaron;nost bioprocesa je procenjivana 7.,<br />10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatka<br />da dodatak soli gvožđa značajno pospe&scaron;uje biosintezu<br />staurosporina sa povećanjem prinosa od 25%. Dobijeni rezultati<br />potvrđuju da su rizosfere medicinski značajnih biljaka značajan<br />izvor streptomiceta koje proizvode komponente sa<br />antifungalnom aktivno&scaron;ću. Izolacija novog proizvođača<br />staurosporina i optimizacija procesa njegove biosinteze<br />omogućiće dalja istraživanja ovog jedinjenja koje može biti<br />osnova za razvoj novih antifungalnih i jedinjenja koja inhibiraju<br />angiogenezu. Rezultati dobijeni u okviru ovih istraživanja<br />predstavljaju početni korak ka potencijalnoj industrijalizaciji<br />proizvodnje staurosporina.</p> / <p>Different soils are still a source of remarkable microbial diversity<br />which also reflects in the unexplored chemical diversity. Recent<br />advances in assessment of microbial diversity from soil have<br />revealed the extraordinarily rich biosynthetic potential for the<br />production of new natural products among different microbial<br />strains, especially within the group of Actinobacteria. Among<br />bacterial soil isolates, representatives of Streptomyces genus are<br />the most prolific producers of bioactive compounds. One of the<br />objectives of the present study was to isolate Streptomyces spp.<br />from the rhizosphere soils of three ethno-medicinal plants<br />collected in Serbia (Papaver rhoeas, Matricaria chamomilla, and<br />Urtica dioica) and to screen their antifungal activity against<br />Candida spp. Morphologically different sporulating isolates (103<br />in total) were collected from rhizosphere soil samples and<br />determined as Streptomyces spp. Two different media and two<br />extraction procedures were used to induce the production and<br />facilitate identification of antifungals. Overall, 412 crude cell<br />extracts were tested against Candida albicans using disk<br />diffusion assays, with 42% (43/103) of the strains showing the<br />ability to produce antifungal agents. Also, extracts inhibited<br />growth of other important human pathogens: Candida krusei,<br />Candida parapsilosis, and Candida glabrata. Based on the<br />established degree and range of antifungal activity, nine isolates<br />were selected for further testing. Their ability to inhibit Candida<br />growth in liquid culture, to inhibit biofilm formation, and to<br />disperse pre-formed biofilms was assessed with active<br />concentrations from 8 to 250 pg/ml. High-performance liquid<br />chromatographic profiles of extracts derived from selected<br />strains were recorded, revealing moderate metabolic diversity.<br />The most potent extracts were subjected to comprehensive<br />identification and structural characterization of antifungal<br />compounds. Applying a bioactivity-guided isolation approach,<br />active compounds of three extracts were separated, and based<br />on NMR structure elucidation it was shown that active<br />compounds were genistein, daidzein and staurosporine.<br />Genistein and daidzein, soy phytoestrogens, are known to inhibit<br />key enzymes in the steroid metabolism pathway and were<br />coming from the fermentation medium containing soy flower.<br />Since isolated Streptomyces spp. showed good ability to extract<br />these molecules from complex medium, they can be further<br />considered for biotechnological production of these<br />phytoestrogens. One of the isolates, Streptomyces sp. BV410,<br />was characterized as an efficient staurosporine producer.<br />Staurosporine is a potent inhibitor of protein kinases and is<br />considered in anticancer therapy. The biotechnological<br />production of staurosporine by strain BV410 was optimized to<br />yield 36.94 mg/l after 14 days of incubation in soy flowerglucose-<br />starch-mannitol based fermentation medium (JS).<br />Further optimization of medium for biosynthesis of<br />staurosporine indicated the following optimal values of the<br />examined factors: the content of glucose of 20 g/l, starch 0.36<br />g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying the<br />defined optimal values and using the appropriate mathematical<br />models, the following responses were predicted: concentration<br />of staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. The<br />validity of the results was confirmed by performing the<br />biosynthesis of the staurosporine in the medium with optimal<br />composition (JSSta). Kinetics of staurosporine and biomass<br />production and carbon source consumption were examined and<br />process models were developed. Additionally, optimization of<br />staurosporine production was performed with different<br />supplements which, according to literature data, had stimulative<br />effect on secondary metabolism (Zn, Fe and P salts, methyl<br />oleate, grape seed oil). In order to improve the production of<br />staurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time<br />(7, 10 and 14 days) were also examined. It was found that<br />addition of FeS04 significantly improved the staurosporine yield<br />in comparison to the starting conditions (increase of 25%). Our<br />results proved that rhizosphere soils of ethno-medicinal plants<br />are a prolific source of streptomycetes, producers of compounds<br />with good antifungal activity. Isolation of the new staurosporine<br />producing strain, allowed for its detailed bioactivity assessment.<br />Staurosporine scaffold might serve as a lead structure for the<br />development of new antifungal and antiangiogenic agents. Also,<br />results obtained within this research represent the basis for the<br />further scale-up and potential industrialization of the proposed<br />production process.</p>

Monoclonal antibodies (MAbs) have an expanding market for use in diagnostic and therapeutic applications. Industrial production of these biopharmaceuticals is usually achieved based on fed-batch cultures of mammalian cells in bioreactors (Chinese hamster ovary (CHO) and Hybridoma cells), which can express different kinds of recombinant proteins. In order to reach high cell densities in these bioreactors, it is necessary to carry out an optimization of their production processes. Hence, macroscopic model equations must be developed to describe cell growth, nutrient consumption and product generation. These models will be very useful for designing the bioprocess, for developing robust controllers and for optimizing its productivity.<p>This thesis presents a new kinetic model of hybridoma cell metabolism in fed batch culture and typical illustration of a systematic methodology for mathematical modelling, parameter estimation and model-based optimization and state estimation of bioprocesses. <p>In the first part, a macroscopic model that takes into account phenomena of overflow metabolism within glycolysis and glutaminolysis is proposed to simulate hybridoma HB-58 cell cultures. The model of central carbon metabolism is reduced to a set of macroscopic reactions. The macroscopic model describes three metabolism states: respiratory metabolism, overflow metabolism and critical metabolism. The model parameters and confidence intervals are obtained via a nonlinear least squares identification. It is validated with experimental data of fed-batch hybridoma cultures and successfully predicts the dynamics of cell growth and death, substrate consumption (glutamine and glucose) and metabolites production (lactate and ammonia). Based on a sensitivity analysis of the model outputs with respect to the parameters, a model reduction is proposed. <p>In the next step, the effort is directed to the maximization of biomass productivity in fed-batch cultures of hybridoma cells based on the overflow metabolism model. Optimal feeding rate, on the one hand, for a single feed stream containing both glucose and glutamine and, on the other hand, for two separate feed streams of glucose and glutamine are determined using a Nelder-Mead simplex optimization algorithm. Two different objective functions (performance criteria) are considered for optimization; the first criterion to be maximized is the biomass productivity obtained at the end of the fed-batch culture, the second criterion to be minimized is the difference between global substrate consumption and the maximum respiratory capacity.<p>The optimal multi exponential feed rate trajectory improves the biomass productivity by 10% as compared to the optimal single exponential feed rate. Moreover, this result is validated by the one obtained with the analytical approach in which glucose and glutamine are fed to the culture so as to control the hybridoma cells at the critical metabolism state, which allows maximizing the biomass productivity. The robustness analysis of optimal feeding profiles obtained with different optimization strategies is considered, first, with respect to parameter uncertainties and, finally, with respect to model structure errors.<p>Finally, the overflow metabolism model is used to develop an extended Kalman filter for online estimation of glucose and glutamine in hybridoma cell fed-batch cultures based on the considered available measurements (biomasses (on-line), lactate and ammonia (on-line or off-line)). The observability conditions are examined, and the performances are analysed with simulations of hybridoma cell fed-batch cultures. Glutamine estimation sensitivity is enforced by minimizing a cost function combining a usual least-squares criterion with a state estimation sensitivity criterion. <p> / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished