- Email: [email protected]
A new multinozzle encapsulation system for the large-scale production of uniform alginate beads
Abstracts / Journal of Biotechnology 136S (2008) S140–S143
S141
on fluctuation of these parameters, especially at high cell density. The optimization of a shaking flask model focused on the following aspects. First, the effect of the buffer system on pH profile was studied. It was found that the initial pH played an important role in keeping pH value above the lower limit. The relationship between dissolved CO2 and pH was also explored. Secondly, the mass transfer of shaking flasks was evaluated. It was found that DO dropped to a considerably low level when cell density approached a critical value. At the same time lactate accumulation promoted a drop of pH value. It was found that an increase in shaking speed can elevate DO level and help maintain a higher pH level. As a result, the cell growth and final titer from shaking flasks are comparable to bioreactor system by using a proper buffer system and shaking strategy. Finally, the cell aggregation problem of a particular cell line in shaking flasks was investigated. In the second part, an efficient screening process for medium additives and their concentrations was developed. First, a quick assay was established for titer and productivity assessment. In comparison to HPLC, this method applies immuno-turbidometrics technology for measuring IgG or IgG-similar molecules, which is fast, cheap and easy to handle with reasonable accuracy. Secondly, a short-term (a couple of days) screening protocol was developed to identify the compounds which can booster productivity without damaging the cells. Data was carefully analyzed based on the normal/positive controls as well as the sensitivity of the instrument. Potential candidates identified in screening experiments were re-tested in an established scale-down model, and then bench-top bioreactors. Finally, a potent chemically defined medium was developed which has a comparable or even better performance than hydrolysate-containing medium.
selected suitable BAC clones for recognizing 20 chromosomes of CHO DG44. To accurately discriminate among the CHO DG44 chromosomes, it is necessary to determine the chromosome length and centromere position in each CHO DG44 chromosome. Average length and centromere position of 20 chromosomes was determined based on 4 ,6-diamidino-2-phenylindole (DAPI) staining images of CHO DG44 metaphase chromosome. The chromosomal loci of all BAC clones and centromere position of each chromosome were expressed as FLpter (the relative distance from the short arm telomere to the signal fractional length p-terminal) values (Lichter et al., 1990). Finally, we created the diagrammatic summary of the mapping data and FLpter values for 20 chromosomes of CHO DG44. Our BAC-FISH chromosome markers are powerful tools for the genome-wide analysis in CHO cell line. It is expected that these tools contribute to further investigate the cytogenic instability of CHO cell line.
doi:10.1016/j.jbiotec.2008.07.299
A new multinozzle encapsulation system for the large-scale production of uniform alginate beads
II4-O-010
Jing Zhu 1,∗ , Weiyang Xie 1 , Xiudong 1 1 WeitingYu , Wei Wang , Xiaojun Ma 1,∗
Chinese hamster ovary cells BAC library: Application to the characterization of CHO chromosomes Omasa ∗ ,
Takeshi Yihua Cao, Shuiichi Kimura, S.M.A. Haghparast, Yasuhiro Takagi, Kohsuke Honda, Hisao Ohtake Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan E-mail address: [email protected] (T. Omasa). Chinese hamster ovary (CHO) cells were established from ovary of an adult Chinese hamster (Cricetulus griseus) in 1958. The proline-requiring CHO-K1 and the dihydrofolate reductase (DHFR)deficient CHO-DG44 are the most widely used industrial host cells for production of biopharmaceuticals (Griffin et al., 2007). Despite their industrial values, efforts to characterize the CHO genome has lagged behind the human and mouse genome projects. For further improvement of CHO cell lines, genome-wide analysis of CHO cells, including CHO genome sequencing, transcriptomics, proteomics, metabolomics and glycomics are essential. In previous study, we reported the construction of CHO genomic bacterial artificial chromosome (BAC) library from CHO DR1000L-4N cells (Yoshikawa et al., 2000) covering entire CHO genome. CHO chromosomes are ordinarily characterized based on the number of chromosomes and band pattern of chromosome staining. The use of genomic DNA cloned in BACs in combination with fluorescent in situ hybridization (FISH), called BAC-FISH, has been an effective approach for physically mapping specific DNA sequences and identifying individual chromosomes. This study is the first report on CHO chromosome mapping by BAC-FISH using CHO genomic BAC clones. Among more than 200 BAC clones, we
References Griffin, T.J., Seth, G., Xie, H., Bandhakavi, S., Hu, W.S., 2007. Advancing mammalian cell culture engineering using genome-scale technologies. 25, 401–408. Lichter, P., Tang, C.J., Call, K., Hermanson, G., Evans, G.A., Housman, D., Ward, D.C., 1990. High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 64–69. Yoshikawa, T., Nakanishi, F., Ogura, Y., Oi, D., Omasa, T., Katakura, Y., Kishimoto, M., Suga, K., 2000. Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes. Biotechnol. Prog. 16, 710–715.
doi:10.1016/j.jbiotec.2008.07.300 II4-P-004
Liu 2 , Ying
Zhang 1 ,
1
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China 2 College of Environment & Chemical Engineering, Dalian University, Dalian 116622, China E-mail address: [email protected] (X. Ma). Over the past 20 years, cell microencapsulation technology has made a great progress in cell culture, artificial cell therapy, artificial organs in vitro, and bioconversion (Wang et al., 2006). But the time of preparation was an important effect factor of encapsulation, the cells viability decreased with the increase of encapsulation preparation time. To acquire biological microcapsule with high cell viability, it is necessary to prepare the microcapsules with high efficiency on the large-scale in order to bring this technology into the clinic. In this paper, calcium alginate gel beads were prepared through microcapsule preparation system under forced high-voltage electrostatic field (Taylor, 1964) developed in our lab designed with a 19-nozzle system for the encapsulation and immobilization of microorganisms, enzymes or cells. The effect of operation parameters on the large-scale preparation of gel beads was analyzed (Qu et al., 2005). The parameters included the applied potential (U), the distribution of electrostatic field, the frequency and the flow rate of solution through the capillary. The results show that the air pressure of feed is the decisive factor on the size and yield of gel beads, and the size distribution is dependent on several factors including the distribution of electric field, pulse potential, air pressure of feed, pulse frequency, pulse width and threshold potential, etc. Therefore, the monodisperse calcium alginate beads in the range of 0.2–4 mm were produced under sterile and repro-
S142
Abstracts / Journal of Biotechnology 136S (2008) S140–S143
ducible conditions. The gel beads with good sphericity, smooth surface and low dispersivity (CV < 20%) can be prepared by adjusting and matching above parameters. It is more important that the yield of gel beads by microcapsule preparation system is 10–60-fold higher than the traditional electrostatic droplet generator. References Qu, B.B., Yu, W.Y., Liu, X.D., Bao, D.C., Song, X.Y., Qi, W.T., Ma, X.J., 2005. Preparation of monodi sper sed calcium alginategel beads in high voltage elect rostatic field. J. Chem. Ind. Eng. (China) 56 (8), 1547. Taylor, G.I., 1964. Disintegration of water drops in an electric field. Proc. R. Soc. A 280, 383. Wang, W., Liu, X.D., Xie, Y.B., Zhang, H.A., Yu, W.T., Xiong, Y., Xie, W.Y., Ma, X.J., 2006. Microencapsulation using natural polysaccharides for drug delivery and cell implantation. J. Mater. Chem. 16 (32), 3252.
doi:10.1016/j.jbiotec.2008.07.301 II4-P-005 Investigation of the reduction performance of sulfate reducing bacteria enhanced by nano-meter/submicron tourmaline Li Wei 1,2 , Fang Ma 2,∗ , Qiang Wang 1,2 , Hong-yu Wang 3 , Xiaoqi Zhang 1,2 1 School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China 2 State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China 3 Department of Municipal Engineering, Wuhan University, Wuhan 430072, China
E-mail address: [email protected] (F. Ma). The characteristics of nano-meter/submicron tourmaline was studied by chemical analysis, scanning electron microscopy (SEM) and XRD. And the nano-meter/submicron tourmaline was used to investigate the enhanced reduction effects of sulfate reducing bacteria. The results showed that nano-meter/submicron tourmaline can promote the growth of sulfate reducing bacteria. After pure cultivation for 48 h, the amount and enzyme activity of sulfate reducing bacteria increased 38.46% and 19.8%, respectively. The sulfate and COD removal rate were 7.12% and 13.89% higher with nano-meter/submicron than that without nanometer/submicron, respectively (Ellaiah et al., 2003). The pH value of bacterial suspensions can be slightly adjusted and the oxidationreduction potential of bacterial suspensions decreased by 7% with nano-meter/submicron tourmaline, which can improve anaerobic environment (Jin et al., 2003), increase enzyme activity and enhance the reduction effects of sulfate (Xia et al., 2006). Keywords: Nano-meter/submicron tourmaline; Sulfate reducing bacteria; Oxidation-reduction potential; Sulfate reduction performance
doi:10.1016/j.jbiotec.2008.07.302 II4-P-006 Glycolipids production in minireactors: Studies on nutritional requirements of Rhodotorula bogoriensis and Pseudozyma aphidis Isabel A. Ribeiro, Matilde F. Castro, Maria H. Ribeiro ∗ Faculdade de Farmácia, Institute for Medicines and Pharmaceutical Sciences (i-Med) University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal E-mail address: [email protected] (M.H. Ribeiro). Glycolipids are biosurfactants (BS) produced by different microorganisms, show unique properties, like biological activities, as antimicrobial and antiviral, in addition, growth inhibition and differentiation-inducing activities against human leukemia cells have been reported (Kitamoto et al., 2002). Another important application of glycolipids is in gene therapy of cancer, as gene delivery carriers with increased gene transfection in mammalian cells. Due to this properties, and also the mild production conditions, lower toxicity, higher biodegradatibility and environmental compatibility, make the glycolipids among biosurfactants, the most promising for commercial production and utilization. The biological activity is dependent on the carbohydrate structure of the glycolipid (Kitamoto et al., 2002). In this work Rhodotorula bogoriensis and Pseudozyma aphidis were used for the production of different glycolipids type, respectively, sophorolipids (SL) and mannosylerythritol (MEL). The aim of this work was the optimization of the nutrients and process conditions, in minireactors, of glycolipids production with R. bogoriensis and P. aphidis, as well as to provide the feasibility of the mini-scale approach. In order to attain these goals the growth of R. bogoriensis and P. aphidis was carried out in shake flasks of 1000 mL and in microplates of 3 mL, at 25 ◦ C and 150 rpm. In these conditions different parameters were evaluated such as specific growth rate, glucose consumption rate, and glycolipids formation. The glycolipids composition, among others, is dependent on carbon source. In fact, besides glucose, another sources were tested such as, fructose, xilose, lactose, mannose and galactose in the sophorolipids (SL) production and glycerol in the mannosylerythritol (MEL) production. After 53 h a maximum cellular growth of P. aphidis in the media was attained in shake flasks and in microplates, similar results were obtained with R. bogoriensis. An exponential glucose decrease in fermentation media was observed with both microorganisms. Reference Kitamoto, D., Isoda, H., Nakahara, T., 2002. J. Biosci. Bioeng. 94, 187–201.
doi:10.1016/j.jbiotec.2008.07.303 Acknowledgement This research was supported by National Natural Science Funds item (No. 50778052). References Ellaiah, P., Saisha, V., Srinivasulu, B., 2003. Effect of low frequency AC electric fields on urokinase production by MPGN kidney cells. Process Biochem. 39, 1–4. Jin, J.J., Ji, Z.Z., Liang, J.S., 2003. Observation of spontaneous polarization of tourmaline. Chin. Phys. 12, 222–225. Xia, M.S., Hu, C.H., Zhang, H.M., 2006. Effects of tourmaline on the dehydrogenase activity of Rhodopseudomonas palustris. J. Process Biochem. 41, 221–225.
S141
on fluctuation of these parameters, especially at high cell density. The optimization of a shaking flask model focused on the following aspects. First, the effect of the buffer system on pH profile was studied. It was found that the initial pH played an important role in keeping pH value above the lower limit. The relationship between dissolved CO2 and pH was also explored. Secondly, the mass transfer of shaking flasks was evaluated. It was found that DO dropped to a considerably low level when cell density approached a critical value. At the same time lactate accumulation promoted a drop of pH value. It was found that an increase in shaking speed can elevate DO level and help maintain a higher pH level. As a result, the cell growth and final titer from shaking flasks are comparable to bioreactor system by using a proper buffer system and shaking strategy. Finally, the cell aggregation problem of a particular cell line in shaking flasks was investigated. In the second part, an efficient screening process for medium additives and their concentrations was developed. First, a quick assay was established for titer and productivity assessment. In comparison to HPLC, this method applies immuno-turbidometrics technology for measuring IgG or IgG-similar molecules, which is fast, cheap and easy to handle with reasonable accuracy. Secondly, a short-term (a couple of days) screening protocol was developed to identify the compounds which can booster productivity without damaging the cells. Data was carefully analyzed based on the normal/positive controls as well as the sensitivity of the instrument. Potential candidates identified in screening experiments were re-tested in an established scale-down model, and then bench-top bioreactors. Finally, a potent chemically defined medium was developed which has a comparable or even better performance than hydrolysate-containing medium.
selected suitable BAC clones for recognizing 20 chromosomes of CHO DG44. To accurately discriminate among the CHO DG44 chromosomes, it is necessary to determine the chromosome length and centromere position in each CHO DG44 chromosome. Average length and centromere position of 20 chromosomes was determined based on 4 ,6-diamidino-2-phenylindole (DAPI) staining images of CHO DG44 metaphase chromosome. The chromosomal loci of all BAC clones and centromere position of each chromosome were expressed as FLpter (the relative distance from the short arm telomere to the signal fractional length p-terminal) values (Lichter et al., 1990). Finally, we created the diagrammatic summary of the mapping data and FLpter values for 20 chromosomes of CHO DG44. Our BAC-FISH chromosome markers are powerful tools for the genome-wide analysis in CHO cell line. It is expected that these tools contribute to further investigate the cytogenic instability of CHO cell line.
doi:10.1016/j.jbiotec.2008.07.299
A new multinozzle encapsulation system for the large-scale production of uniform alginate beads
II4-O-010
Jing Zhu 1,∗ , Weiyang Xie 1 , Xiudong 1 1 WeitingYu , Wei Wang , Xiaojun Ma 1,∗
Chinese hamster ovary cells BAC library: Application to the characterization of CHO chromosomes Omasa ∗ ,
Takeshi Yihua Cao, Shuiichi Kimura, S.M.A. Haghparast, Yasuhiro Takagi, Kohsuke Honda, Hisao Ohtake Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan E-mail address: [email protected] (T. Omasa). Chinese hamster ovary (CHO) cells were established from ovary of an adult Chinese hamster (Cricetulus griseus) in 1958. The proline-requiring CHO-K1 and the dihydrofolate reductase (DHFR)deficient CHO-DG44 are the most widely used industrial host cells for production of biopharmaceuticals (Griffin et al., 2007). Despite their industrial values, efforts to characterize the CHO genome has lagged behind the human and mouse genome projects. For further improvement of CHO cell lines, genome-wide analysis of CHO cells, including CHO genome sequencing, transcriptomics, proteomics, metabolomics and glycomics are essential. In previous study, we reported the construction of CHO genomic bacterial artificial chromosome (BAC) library from CHO DR1000L-4N cells (Yoshikawa et al., 2000) covering entire CHO genome. CHO chromosomes are ordinarily characterized based on the number of chromosomes and band pattern of chromosome staining. The use of genomic DNA cloned in BACs in combination with fluorescent in situ hybridization (FISH), called BAC-FISH, has been an effective approach for physically mapping specific DNA sequences and identifying individual chromosomes. This study is the first report on CHO chromosome mapping by BAC-FISH using CHO genomic BAC clones. Among more than 200 BAC clones, we
References Griffin, T.J., Seth, G., Xie, H., Bandhakavi, S., Hu, W.S., 2007. Advancing mammalian cell culture engineering using genome-scale technologies. 25, 401–408. Lichter, P., Tang, C.J., Call, K., Hermanson, G., Evans, G.A., Housman, D., Ward, D.C., 1990. High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247, 64–69. Yoshikawa, T., Nakanishi, F., Ogura, Y., Oi, D., Omasa, T., Katakura, Y., Kishimoto, M., Suga, K., 2000. Amplified gene location in chromosomal DNA affected recombinant protein production and stability of amplified genes. Biotechnol. Prog. 16, 710–715.
doi:10.1016/j.jbiotec.2008.07.300 II4-P-004
Liu 2 , Ying
Zhang 1 ,
1
Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China 2 College of Environment & Chemical Engineering, Dalian University, Dalian 116622, China E-mail address: [email protected] (X. Ma). Over the past 20 years, cell microencapsulation technology has made a great progress in cell culture, artificial cell therapy, artificial organs in vitro, and bioconversion (Wang et al., 2006). But the time of preparation was an important effect factor of encapsulation, the cells viability decreased with the increase of encapsulation preparation time. To acquire biological microcapsule with high cell viability, it is necessary to prepare the microcapsules with high efficiency on the large-scale in order to bring this technology into the clinic. In this paper, calcium alginate gel beads were prepared through microcapsule preparation system under forced high-voltage electrostatic field (Taylor, 1964) developed in our lab designed with a 19-nozzle system for the encapsulation and immobilization of microorganisms, enzymes or cells. The effect of operation parameters on the large-scale preparation of gel beads was analyzed (Qu et al., 2005). The parameters included the applied potential (U), the distribution of electrostatic field, the frequency and the flow rate of solution through the capillary. The results show that the air pressure of feed is the decisive factor on the size and yield of gel beads, and the size distribution is dependent on several factors including the distribution of electric field, pulse potential, air pressure of feed, pulse frequency, pulse width and threshold potential, etc. Therefore, the monodisperse calcium alginate beads in the range of 0.2–4 mm were produced under sterile and repro-
S142
Abstracts / Journal of Biotechnology 136S (2008) S140–S143
ducible conditions. The gel beads with good sphericity, smooth surface and low dispersivity (CV < 20%) can be prepared by adjusting and matching above parameters. It is more important that the yield of gel beads by microcapsule preparation system is 10–60-fold higher than the traditional electrostatic droplet generator. References Qu, B.B., Yu, W.Y., Liu, X.D., Bao, D.C., Song, X.Y., Qi, W.T., Ma, X.J., 2005. Preparation of monodi sper sed calcium alginategel beads in high voltage elect rostatic field. J. Chem. Ind. Eng. (China) 56 (8), 1547. Taylor, G.I., 1964. Disintegration of water drops in an electric field. Proc. R. Soc. A 280, 383. Wang, W., Liu, X.D., Xie, Y.B., Zhang, H.A., Yu, W.T., Xiong, Y., Xie, W.Y., Ma, X.J., 2006. Microencapsulation using natural polysaccharides for drug delivery and cell implantation. J. Mater. Chem. 16 (32), 3252.
doi:10.1016/j.jbiotec.2008.07.301 II4-P-005 Investigation of the reduction performance of sulfate reducing bacteria enhanced by nano-meter/submicron tourmaline Li Wei 1,2 , Fang Ma 2,∗ , Qiang Wang 1,2 , Hong-yu Wang 3 , Xiaoqi Zhang 1,2 1 School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China 2 State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China 3 Department of Municipal Engineering, Wuhan University, Wuhan 430072, China
E-mail address: [email protected] (F. Ma). The characteristics of nano-meter/submicron tourmaline was studied by chemical analysis, scanning electron microscopy (SEM) and XRD. And the nano-meter/submicron tourmaline was used to investigate the enhanced reduction effects of sulfate reducing bacteria. The results showed that nano-meter/submicron tourmaline can promote the growth of sulfate reducing bacteria. After pure cultivation for 48 h, the amount and enzyme activity of sulfate reducing bacteria increased 38.46% and 19.8%, respectively. The sulfate and COD removal rate were 7.12% and 13.89% higher with nano-meter/submicron than that without nanometer/submicron, respectively (Ellaiah et al., 2003). The pH value of bacterial suspensions can be slightly adjusted and the oxidationreduction potential of bacterial suspensions decreased by 7% with nano-meter/submicron tourmaline, which can improve anaerobic environment (Jin et al., 2003), increase enzyme activity and enhance the reduction effects of sulfate (Xia et al., 2006). Keywords: Nano-meter/submicron tourmaline; Sulfate reducing bacteria; Oxidation-reduction potential; Sulfate reduction performance
doi:10.1016/j.jbiotec.2008.07.302 II4-P-006 Glycolipids production in minireactors: Studies on nutritional requirements of Rhodotorula bogoriensis and Pseudozyma aphidis Isabel A. Ribeiro, Matilde F. Castro, Maria H. Ribeiro ∗ Faculdade de Farmácia, Institute for Medicines and Pharmaceutical Sciences (i-Med) University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal E-mail address: [email protected] (M.H. Ribeiro). Glycolipids are biosurfactants (BS) produced by different microorganisms, show unique properties, like biological activities, as antimicrobial and antiviral, in addition, growth inhibition and differentiation-inducing activities against human leukemia cells have been reported (Kitamoto et al., 2002). Another important application of glycolipids is in gene therapy of cancer, as gene delivery carriers with increased gene transfection in mammalian cells. Due to this properties, and also the mild production conditions, lower toxicity, higher biodegradatibility and environmental compatibility, make the glycolipids among biosurfactants, the most promising for commercial production and utilization. The biological activity is dependent on the carbohydrate structure of the glycolipid (Kitamoto et al., 2002). In this work Rhodotorula bogoriensis and Pseudozyma aphidis were used for the production of different glycolipids type, respectively, sophorolipids (SL) and mannosylerythritol (MEL). The aim of this work was the optimization of the nutrients and process conditions, in minireactors, of glycolipids production with R. bogoriensis and P. aphidis, as well as to provide the feasibility of the mini-scale approach. In order to attain these goals the growth of R. bogoriensis and P. aphidis was carried out in shake flasks of 1000 mL and in microplates of 3 mL, at 25 ◦ C and 150 rpm. In these conditions different parameters were evaluated such as specific growth rate, glucose consumption rate, and glycolipids formation. The glycolipids composition, among others, is dependent on carbon source. In fact, besides glucose, another sources were tested such as, fructose, xilose, lactose, mannose and galactose in the sophorolipids (SL) production and glycerol in the mannosylerythritol (MEL) production. After 53 h a maximum cellular growth of P. aphidis in the media was attained in shake flasks and in microplates, similar results were obtained with R. bogoriensis. An exponential glucose decrease in fermentation media was observed with both microorganisms. Reference Kitamoto, D., Isoda, H., Nakahara, T., 2002. J. Biosci. Bioeng. 94, 187–201.
doi:10.1016/j.jbiotec.2008.07.303 Acknowledgement This research was supported by National Natural Science Funds item (No. 50778052). References Ellaiah, P., Saisha, V., Srinivasulu, B., 2003. Effect of low frequency AC electric fields on urokinase production by MPGN kidney cells. Process Biochem. 39, 1–4. Jin, J.J., Ji, Z.Z., Liang, J.S., 2003. Observation of spontaneous polarization of tourmaline. Chin. Phys. 12, 222–225. Xia, M.S., Hu, C.H., Zhang, H.M., 2006. Effects of tourmaline on the dehydrogenase activity of Rhodopseudomonas palustris. J. Process Biochem. 41, 221–225.