- Email: [email protected]
Intracellular ribonucleotide pools as a tool for monitoring the physiological state of plant cell suspension culture of Vitis vinifera in response to temperature change
S480
Abstracts / Journal of Biotechnology 136S (2008) S460–S495
V5-P-050
V5-P-052
Characterization of gas–liquid interface in an ultra-scale-down device and its implications for protein formulation
Intracellular ribonucleotide pools as a tool for monitoring the physiological state of plant cell suspension culture of Vitis vinifera in response to temperature change
Hu Zhang 1,2,∗ , Mike Hoare 1 1
The Advanced Center for Biochemical Engineering, University College London, Torrington Plaxe, London WC1E 7JE, United Kingdom 2 College of Life Science, Dalian Nationality University, Dalian 116600, China E-mail address: [email protected] (H. Zhang). It has been well known that gas–liquid interface has a great impact on the stability of protein formulations. The effect of shear forces on biomolecules such as insulin, rhGH, rFXIII, albutropin, ␣-lactoglobulin, and HSA and BSA in the presence of gas–liquid interface have been widely studied and the general findings from them are that damaging effects of shear stress on a protein may take the form of changes in secondary, tertiary, and quaternary structure, altered activity, and aggregation due to exposure of hydrophobic amino acid residues (Virkar et al., 1981). Qualitative studies on these damaging effects have been carried out in stirred tanks, spray driers, and other biological processors (Levy et al., 1999; Zhang et al., 2007). However, quantitative determination of shear or other factors contributing to protein denaturisation at the interface has not been achieved yet due to complicated nature of gas–liquid interface. Recently, we have developed an ultra-scaledown device for studying on the shear and interfacial effects on biological molecules and cells using a small amount of materials but within a well-defined geometries and shear stress. To apply this device into protein formulation, computational fluid dynamics (CFD) was employed to characterize the shape of gas–liquid interface, and the predictions (as shown in Fig. 1) were comparable to those acquired by a high-speed camera. Quantitative values of shear stress were interpreted from the simulation results. The shear forces on the protein can then be determined. Through the use of shear device, the percentage of denaturized protein will be determined experimentally. This percentage of shear damage will be correlated with gas–liquid interface information, such as shear force and interfacial area. This correlation will be independent on the device geometry, thus, it can be used for predicting the protein shear damage in a large-scale biological processors in presence of gas–liquid interface. References Levy, M.S., Collins, I.J., Yim, S.S., Ward, J.M., Titchener-Hooker, N.J., Ayazi Shamlou, P., Dunnill, P., 1999. Effect of shear on plasmid DNA in solution. Bioprocess Engineering 20, 7–13. Virkar, P.D., Narendranathan, T.J., Hoare, M., Dunnill, P., 1981. Studies of the effects of shear on globular proteins: extension to high shear fields and to pumps. Biotechnology and Bioengineering 23, 425–429. Zhang, H., Kong, S., Booth, A., Boushaba, R., Levy, M.S., Hoare, M., 2007. Prediction of shear damage of plasmid DNA in pump and centrifuge operation using an ultra scale-down device. Biotechnology Progress 23, 858–865.
doi:10.1016/j.jbiotec.2008.07.1118
Hao Jiang ∗ , Wei Zhang, Chris Franco Department of Medical Biotechnology, Flinders University of South Australia, Adelaide, Australia E-mail address: jian0020@flinders.edu.au (H. Jiang). Plant cell cultures are sensitive to factors such as mechanical shear stress, surface tension, chemical stress and temperature variations that occur in bioreactor vessels during cultivation and scale up for the production of bioactive compounds. In order to achieve high productivity it is necessary to maintain the plants cells in an optimal physiological state during the production process. An efficient analytical method is required to characterize the physiological state of the plant cells. The method which analyses intracellular ribonucleotide pools as target provides a rapid and reliable assessment of cell state and growth potential and been successfully applied to mammalian cell culture processes (Grammatikos et al., 1999). In this project a more suitable extraction method for intracellular ribonucleotide pool from plant cells was developed. According to our previous study, temperature is one of the fundamental parameters that significantly influences plant cell viability and can be detected by the changes in biomass concentration, water content and pigment content in plant cells. To test our new methodology, intracellular nucleotide pools were measured in plant cell cultures subjected to growth at 24, 26, 28, 30 ◦ C, respectively. The levels of the intracellular ribonucleotide pools were analyzed over the culture period and specific ratios of nucleotide components serve as rapid indicators or predictors of cell growth and secondary metabolite productivity. Reference Grammatikos, S.I., Tobien, K., Noe, W., Werner, R.G., 1999. Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes. Biotechnology and Bioengineering 64, 357–367.
doi:10.1016/j.jbiotec.2008.07.1119 V5-P-053 Empirical modeling as an experimental approach to optimize the hydrolysis of sugarcane bagasse hemicellulose with dilute H2 SO4 Larissa Canilha 1,2,∗ , Walter Carvalho 1 , João Batista Almeida e Silva 1 , Maria das Grac¸as Almeida Felipe 1 , Marco Giulietti 2 1 2
Engineering College of Lorena, Lorena, Brazil Institute of Technological Research, São Paulo, Brazil
E-mail address: [email protected] (L. Canilha). Dilute-acid hydrolysis has frequently been used to hydrolyze the hemicelluloses of many lignocellulosic materials, leading to xylose-rich hydrolysates that can be used for the production of goods like xylitol and ethanol. In the present study, experiments based in a 23 central composite full factorial design were carried out in 200 mL stainless steel containers in order to improve the yield of xylose recovery from the sugarcane bagasse hemicel-
∗ Corresponding author at: The Advanced Center for Biochemical Engineering, University College London, Torrington Plaxe, London WC1E 7JE, United Kingdom.
∗ Corresponding author at: Engineering College of Lorena, Lorena, Brazil.
Abstracts / Journal of Biotechnology 136S (2008) S460–S495
V5-P-050
V5-P-052
Characterization of gas–liquid interface in an ultra-scale-down device and its implications for protein formulation
Intracellular ribonucleotide pools as a tool for monitoring the physiological state of plant cell suspension culture of Vitis vinifera in response to temperature change
Hu Zhang 1,2,∗ , Mike Hoare 1 1
The Advanced Center for Biochemical Engineering, University College London, Torrington Plaxe, London WC1E 7JE, United Kingdom 2 College of Life Science, Dalian Nationality University, Dalian 116600, China E-mail address: [email protected] (H. Zhang). It has been well known that gas–liquid interface has a great impact on the stability of protein formulations. The effect of shear forces on biomolecules such as insulin, rhGH, rFXIII, albutropin, ␣-lactoglobulin, and HSA and BSA in the presence of gas–liquid interface have been widely studied and the general findings from them are that damaging effects of shear stress on a protein may take the form of changes in secondary, tertiary, and quaternary structure, altered activity, and aggregation due to exposure of hydrophobic amino acid residues (Virkar et al., 1981). Qualitative studies on these damaging effects have been carried out in stirred tanks, spray driers, and other biological processors (Levy et al., 1999; Zhang et al., 2007). However, quantitative determination of shear or other factors contributing to protein denaturisation at the interface has not been achieved yet due to complicated nature of gas–liquid interface. Recently, we have developed an ultra-scaledown device for studying on the shear and interfacial effects on biological molecules and cells using a small amount of materials but within a well-defined geometries and shear stress. To apply this device into protein formulation, computational fluid dynamics (CFD) was employed to characterize the shape of gas–liquid interface, and the predictions (as shown in Fig. 1) were comparable to those acquired by a high-speed camera. Quantitative values of shear stress were interpreted from the simulation results. The shear forces on the protein can then be determined. Through the use of shear device, the percentage of denaturized protein will be determined experimentally. This percentage of shear damage will be correlated with gas–liquid interface information, such as shear force and interfacial area. This correlation will be independent on the device geometry, thus, it can be used for predicting the protein shear damage in a large-scale biological processors in presence of gas–liquid interface. References Levy, M.S., Collins, I.J., Yim, S.S., Ward, J.M., Titchener-Hooker, N.J., Ayazi Shamlou, P., Dunnill, P., 1999. Effect of shear on plasmid DNA in solution. Bioprocess Engineering 20, 7–13. Virkar, P.D., Narendranathan, T.J., Hoare, M., Dunnill, P., 1981. Studies of the effects of shear on globular proteins: extension to high shear fields and to pumps. Biotechnology and Bioengineering 23, 425–429. Zhang, H., Kong, S., Booth, A., Boushaba, R., Levy, M.S., Hoare, M., 2007. Prediction of shear damage of plasmid DNA in pump and centrifuge operation using an ultra scale-down device. Biotechnology Progress 23, 858–865.
doi:10.1016/j.jbiotec.2008.07.1118
Hao Jiang ∗ , Wei Zhang, Chris Franco Department of Medical Biotechnology, Flinders University of South Australia, Adelaide, Australia E-mail address: jian0020@flinders.edu.au (H. Jiang). Plant cell cultures are sensitive to factors such as mechanical shear stress, surface tension, chemical stress and temperature variations that occur in bioreactor vessels during cultivation and scale up for the production of bioactive compounds. In order to achieve high productivity it is necessary to maintain the plants cells in an optimal physiological state during the production process. An efficient analytical method is required to characterize the physiological state of the plant cells. The method which analyses intracellular ribonucleotide pools as target provides a rapid and reliable assessment of cell state and growth potential and been successfully applied to mammalian cell culture processes (Grammatikos et al., 1999). In this project a more suitable extraction method for intracellular ribonucleotide pool from plant cells was developed. According to our previous study, temperature is one of the fundamental parameters that significantly influences plant cell viability and can be detected by the changes in biomass concentration, water content and pigment content in plant cells. To test our new methodology, intracellular nucleotide pools were measured in plant cell cultures subjected to growth at 24, 26, 28, 30 ◦ C, respectively. The levels of the intracellular ribonucleotide pools were analyzed over the culture period and specific ratios of nucleotide components serve as rapid indicators or predictors of cell growth and secondary metabolite productivity. Reference Grammatikos, S.I., Tobien, K., Noe, W., Werner, R.G., 1999. Monitoring of intracellular ribonucleotide pools is a powerful tool in the development and characterization of mammalian cell culture processes. Biotechnology and Bioengineering 64, 357–367.
doi:10.1016/j.jbiotec.2008.07.1119 V5-P-053 Empirical modeling as an experimental approach to optimize the hydrolysis of sugarcane bagasse hemicellulose with dilute H2 SO4 Larissa Canilha 1,2,∗ , Walter Carvalho 1 , João Batista Almeida e Silva 1 , Maria das Grac¸as Almeida Felipe 1 , Marco Giulietti 2 1 2
Engineering College of Lorena, Lorena, Brazil Institute of Technological Research, São Paulo, Brazil
E-mail address: [email protected] (L. Canilha). Dilute-acid hydrolysis has frequently been used to hydrolyze the hemicelluloses of many lignocellulosic materials, leading to xylose-rich hydrolysates that can be used for the production of goods like xylitol and ethanol. In the present study, experiments based in a 23 central composite full factorial design were carried out in 200 mL stainless steel containers in order to improve the yield of xylose recovery from the sugarcane bagasse hemicel-
∗ Corresponding author at: The Advanced Center for Biochemical Engineering, University College London, Torrington Plaxe, London WC1E 7JE, United Kingdom.
∗ Corresponding author at: Engineering College of Lorena, Lorena, Brazil.