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Optimization of cultivation conditions for mammalian cell lines producing complex biosimilars
Jure Strnad, 2011, dissertation

Abstract: EXTENDED ABSTRACT The possibility to replace standard single use 250 mL shake flasks with single use 50 mL spin tubes was investigated using the design of experiments methodology. Experimental throughput could be tripled to a maximum of 120 spin tubes per shaker-incubator unit if similar process performance could be proven in shake flasks and spin tubes. A D-optimal response surface design was used to model titer values in a seven day batch process producing recombinant erythropoietin. Shaking rate and working volume in spin tubes were adjusted to simulate titers that are produced in shake flasks at the reference setting. Research results indicated that equivalent titers as in shake flasks at the reference setting can be produced in spin tubes; moreover, even higher titers are possible. The maximal titers in spin tubes reached values as observed in 6 L bioreactors. Furthermore, a comparison of process performance and product quality attributes between two spin tube settings, the reference shake flask setting, a standard bioreactor run and a bioreactor run without pH control was made. Process performance and product quality attributes in spin tubes at the equality setting (shaking rate of 180 rpm and 30 mL working volume) were comparable to the ones derived from the reference shake flask setting. Results derived for both bioreactor runs were not fully comparable to the spin tube and shake flask systems. The statistical model for calculating titers on day seven of a batch process in spin tubes was successfully validated and can be used for titer prediction in the proposed design space. The optimized spin tube settings were further used in a repetitive batch process where in the harvest phase of the process medium was daily exchanged to prevent component depletion or build-up of inhibitors. Spin tubes and shake flasks were used to simulate the industrial process of erythropoietin production. The effect of process mode change from seven-day batch to repetitive batch was investigated on process performance and product quality attributes, such as isoform distribution and glycan group distribution. Spin tube performance at the equality setting was comparable to the shake flask performance also in the repetitive batch process. Performance, especially titers at the maximal titer spin tube setting was, however, not fully comparable to the results obtained in previous optimization experiments. The spin tube equality setting was also used for cultivating two cell lines producing different monoclonal antibodies. The goal was to investigate how different cell lines influence process performance and product quality attributes, such as monoclonal antibody charge variant distribution and glycan group distribution. Both cell lines were derived from a Chinese hamster ovary parental cell line, therefore, it was proposed that maybe the optimal spin tube setting derived for the erythropoietin producing cell line, which was also derived from the Chinese hamster ovary parental cell line, could also be used for these subtypes. Cell growth of the monoclonal antibody producing cell lines was extensively better as observed for the erythropoietin producing cell line, which meant that culture demands were more pronounced, such as oxygen transfer or mass transfer. It was observed that the erythropoietin derived equality spin tube setting did not produce similar process responses as shake flask at the reference setting for both monoclonal antibodies. The foremost difference was that the metabolite lactate was being consumed in shake flasks after it reached a maximum value but was not consumed in spin tubes for both monoclonal antibody producing cell lines. In these experiments also amino acid time profiles during a seven day batch process were monitored and subsequently compared. It was seen that several amino acids seemed to be in excess as most of them were only half way consumed. Product quality attributes also differed between the spin tube and the shake flask setting. The conclusion of the experimental work was that some fine tuning of th
Keywords: design of experiments, spin tube, shake flask, optimization, erythropoietin, monoclonal antibodies
Published: 22.12.2011; Views: 2566; Downloads: 133
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Analysis of growth models for batch kefir grain biomass production in RC1 reaction system
Marko Tramšek, Andreja Goršek, 2008, original scientific article

Abstract: This work describes the statistical analysis of three mathematical models, modified for describing the kefir grain biomass growth curve. Experimental data of time-dependent kefir grain mass increase were used. The propagation was performed in RC1 batch reaction system under optimal bioprocess parameters (temperature, rotational frequency of stirrer, glucose mass concentration) using traditional cultivation in fresh, high-temperature, pasteurized whole fat cow's milk. We compared values of biological parameters obtained by applying the nonlinear regression of experimental data in logistic, Gompertz and Richards models. The most statistically appropriate model was determined using the seven statistical indicators. We established that the kefir grain biomass growth curve during batch propagation under optimal bioprocess conditions can be most successfully described using the Gompertz growth model.
Keywords: chemical processing, milk products, kefir grain growth, process parameters, design of experiments, modeling, mathematical models, Gompertz growth model, RC1
Published: 31.05.2012; Views: 1961; Downloads: 97
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Quantitative examination of process parameters during kefir grain biomass production
Andreja Goršek, Marko Tramšek, 2007, short scientific article

Abstract: This article examines the propagation of kefir grains in fresh HTP whole fat cows' milk, with some additions (glucose and bakers yeast). The objective of our work was an experimental determination of the various process parameters relative influence on the propagation and daily kefir grain increase mass, using the Taguchi method for experimental design. The effects of medium temperature, glucose mass concentration, bakers yeast mass concentration and the rotational frequency of the stirrer at four levels were studied. Orthogonal array layout of L16 was selected for the proposed experimental design. All experiments were performed in an automated laboratory reaction calorimeter RC1 (Mettler-Toledo) with the same milk (3.5 % fat). The gravimetric method was used to determine daily kefir grain mass increases. Relative contributions of the proposed influencing process parameters on the daily kefir grains increase mass were estimated by analysis of the variance (ANOVA). The highest increase (51.5 %) was found at the rotational frequency of the stirrer 90 (1/min), at glucose mass concentration 20 g/L, and at mediumtemperature 24 °C. Within the observed range of yeast mass concentrationthis process parameter was found to be insignificant compared to others. The rotational frequency of the stirrer has the highest relative influence on the daily kefir grains increase mass (37.3 %) while glucose mass concentration and medium temperature have lower ones, 18.8 % and 9.9 %, respectively. The remaining fraction represents error influence. The main reason for its relatively high value (34.0 %) is that kefir grains are bulky and awkward to handle. This fact confirms the importance of optimal kefir grains production management.
Keywords: chemical processing, milk products, kefir, kefir grain growth, process parameters, design of experiments, Taguchi method, RC1
Published: 31.05.2012; Views: 1755; Downloads: 36
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Optimization of extrusion process by genetic algorithms and conventional techniques
Zoran Jurković, Miran Brezočnik, Branko Grizelj, Vesna Mandić, 2009, original scientific article

Abstract: The purpose of this research is the determination of the optimal cold forward extrusion parameters with the minimization of tool load as objective. This paper deals with different optimization approaches in order to determine optimal values of logarithmic strain, die angle and friction factor with the purpose to find minimal tool loading obtained by cold forward extrusion process. Two experimental plans based on factorial design of experiment and orthogonal array have been carried out. Classical optimization, according to the response model of extrusion forming force, and the Taguchi approach are presented. The obtained extrusion force model as the fitness function was used to carry out genetic algorithm optimization. Experimental verification of optimal forming parameters with their influences on the forming forces was also performed. The experimental results show an improvement in the minimization of tool loading. The results of optimal forming parameters obtained with different optimization approaches have been compared and based on that the characteristics analysis (features and limitations) of presented techniques.
Keywords: metal forming, forward extrusion force optimization, design of experiments, Taguchi approach, genetic algortihm
Published: 31.05.2012; Views: 1154; Downloads: 66
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Dušica Mirković, 2019, doctoral dissertation

Abstract: The aim of this doctoral research was to develop and optimize parenteral nanoemulsions as well as the total parenteral nutrition (TPN) admixture containing a nanoemulsion obtained in the course of the optimization process (hereinafter referred to as optimal nanoemulsion), and to examine their physicochemical and biological quality as well. In addition, the quality of the prepared nanoemulsions was compared with the quality of the industrial nanoemulsion (Lipofundin® MCT/LCT 20%), and, in the end, the TPN admixture initially prepared was also compared with the admixture into which the industrial emulsion was incorporated. Parenteral nanoemulsions that were considered in this dissertation were prepared by the high-pressure homogenization method. This method is the most widely applied method for the production of nanoemulsions due to the shortest length of homogenization time, the best-obtained homogeneity of the product and the smallest droplet diameter. For the nanoemulsion formulation, preparation and optimization purposes, by using, firstly, the concept of the computer-generated fractional design, and, after that, the full experimental design, the assessment of both direct effects of different formulation and process parameters (the oil phase type, the emulsifier type and concentration, a number of homogenization cycles and the pressure under which homogenization was carried out) as well as the effects of their interactions on the characteristics of prepared nanoemulsions was performed. Monitoring the nanoemulsion physical and chemical stability parameters was carried out immediately after their preparation, and then after 10, 30 and 60 days. It included the visual inspection, the measurement of the droplet diameter (the mean and volume droplet diameter), the polydispersity index, the ζ-potential, the pH value, the electrical conductivity, and the peroxide number. After the preparation and after 60 days, the biological evaluation (the sterility test and the endotoxic test) of the prepared nanoemulsions was carried out. As far as the characterization of the TPN admixture is concerned, it included practically the same parameters. The dynamics of monitoring the characteristics of the TPN admixture was determined on the basis of practical needs of hospitalized patients (0h, 24h and 72h). The scope and comprehensiveness of this issue indicated the need to divide the doctoral dissertation into three basic stages. The first stage was preliminary. Using the 24-1 fractional factorial design, nanoemulsions for the parenteral nutrition were prepared. They contained either a combination of soybean and fish oil, or a combination of medium chain triglycerides and fish oil. In addition, the type and the amount of an emulsifier used, a number of high-pressure homogenization cycles, and the homogenization pressure, were also varied. The measurement of the above-mentioned parameters for the industrial nanoemulsion was parallely carried out (Lipofundin® MCT/LCT 20%). The objective of this part of the research was to identify critical numerical factors having the most significant effect on the characteristics that define the prepared parenteral nanoemulsions. Parameters that were singled out as the result of this stage of the research (the emulsifier concentration and a number of homogenization cycles) were used as independent variables in the second stage of the research.
Keywords: nanoemulsions, total parenteral nutrition admixtures, high pressure homogenization, design of experiments, optimization, analysis of variance, artificial neural networks
Published: 07.06.2019; Views: 501; Downloads: 0
.pdf Full text (2,82 MB)

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