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Protoplast fusion technique in Tolypocladium inflatum for increasing Cyclosporine production
Abstracts / Journal of Biotechnology 131S (2007) S133–S187
of the combination of the three main heterogeneities, dO2 , glucose and pH, that occur simultaneously at the large-scale but not at all at the small-scale. Very similar results were found in the STR-PFR scale down model. However, these studies were carried out using the untransformed wild type bacterial strain, E. coli W3110. Further work considered the additional physiological stress imposed on the cell by having to synthesise a model foreign protein (AP50) when studying process performance on scale-up. It became clear that, even under well-mixed conditions, the expression of AP50 as insoluble inclusion bodies exerts a profound physiological stress on the host cell depending on the time of induction; and, by using the scale-down model, that that there was a further effect of the three major spatial/temporal heterogeneities. Overall, in general, early induction led to lower biomass and lower viability than late induction and the heterogeneities further reduced biomass but increased viability. In this paper, we will briefly discuss the main engineering considerations involved in fermentation scale-up and then critically review those mechanisms thought to be responsible for any detrimental change in bioprocessing at the larger-scale. Though it addresses mainly E. coli fed-batch fermentations, much of the discussion also applies to other single celled aerobic microbial batch and fed-batch fermentations too. References Hewitt, C.J., Lewis, G., Onyeaka, H., Taylor, I.W., Nienow, A.W., 2007. Biotechnol. Bioeng. 96, 495–505. Hewitt, C.J., Nebe-von-Caron, G., Axelsson, B., McFarlane, C.M., Nienow, A.W., 2000. Biotechnol. Bioeng. 70 (4), 381–390. Lewis, G., Taylor, I.W., Nienow, A.W., Hewitt, C.J., 2004. J. Ind. Microbiol. Biotechnol. 31, 311–322. Onyeaka, H., Nienow, A.W., Hewitt, C.J., 2003. Biotechnol. Bioeng. 84 (4), 474–484.
doi:10.1016/j.jbiotec.2007.07.833 4 Accelerating the development cycle of antibody based drugs through the use of multiplexed in situ near infrared spectroscopy and multi-scale models Linda Harvey 1 , Payal Roychoudhury 1 , Ronan O’Kennedy 2 , Timothy Calvert 1,∗ , Brian McNeil 1 1 Strathclyde
Fermentation Centre, University of Strathclyde Glasgow, Scotland, United Kingdom 2 GSK Centre of Excellence in Drug Discovery Beckenham, Kent, United Kingdom The use of mammalian cell culture systems to manufacture antibody based drugs is now routine with levels of protein expressed ranging up to 3 gL−1 . However, the actual process of manufacture typically involves the cultivation of the selected cell line at several scales, and may also involve one or more geographical transfers of the process. These cultures are characterised by slow growth rates making the culture cycle especially lengthy by comparison to microbial expression systems, typically 1–2 weeks for CHO culture, compared with 1–2 days for E. coli. Likewise, the transfer between scales and location can be chal-
S135
lenging. Here we report upon the results of a study into the use of multiplexed in situ near infrared spectroscopic techniques to monitor industrial CHO cultures used for antibody manufacturing by GSK. Specially designed steam sterilisable probes were used in a range of fermenter vessels ranging from small lab scale (7 l) to pilot scale to monitor the progress of the production process, and models were constructed for key analytes based upon data from (A) single probes; (B) multiple probes at the same process scale; (C) multiple probes at differing process scales. All models had good predictive abilities and were robust, proving that the technique was capable of implementation in a manufacturing plant across multiple scales, and could be of practical use throughout the development cycle for antibody based drugs doi:10.1016/j.jbiotec.2007.07.834 5. Protoplast fusion technique in Tolypocladium inflatum for increasing Cyclosporine production Mohammad Reza Bakhtiari ∗ , Masoud Fallahpour, Pupak Foruzanfakhr, Nasrin Moazami Iranian Research Organization for Science & Technology (IROST) Tehran, Islamic Republic of Iran The purpose of our study was the assessment of protoplast fusion technique in order to improve cyclosporine production by the mould, Tolypocladium inflatum. Cyclosporins are valuable natural secondary metabolites with vast biological effects and have several applications, especially in preventing the rejection of transplanted organs and treatment of antoimmune disorders. Hence, increasing the production of this drug is of great importance in pharmaceutical industries. Three different strains of T. inflatum, i.e. PTCC 5252, PTCC 5253, and PTCC 5254 were evaluated in order to select two proper parents with cross differentiating characteristics. Identification of innate and natural traits was studied as the first step. It was found that T. inflatum PTCC 5252 was resistant to 187.5 g/ml of crystal violet and sensitive to 10 g/ml of cyclohexamide, while T.inflatum PTCC 5253 showed reciprocal results. Based on these findings, the two strains were selected as parents. Considering lack of references about protoplast fusion in T. inflatum and the great dependency of the technique on various factors such as, culture medium composition, mycelial age, concentration and contact time of the lytic enzymes, centrifugal velocities, polyethyleneglycol molecular weight and concentration, and composition of regeneration media, the above-mentioned factors were optimized in our study. The technique resulted to 21% of regeneration and 38% of recombination frequencies. In order to identify the high Cyclosporine producing fusants, a primary screening test was designed and performed with 257 colonies. Subsequently, 46
S136
Abstracts / Journal of Biotechnology 131S (2007) S133–S187
selected colonies were transferred to submerged cultures in production flasks. Totally 52% of these fusants showed higher production in comparison to their parents. One of the recombinants produced Cyclosporine 2.8 times more than the parent strain, T. inflatum PTCC 5252, and 2.3 times more than T. inflatum PTCC 5253. doi:10.1016/j.jbiotec.2007.07.835 6. Mid-infrared spectroscopy as a tool for pH prediction and control in bioprocesses Jonas Schenk 1,∗ , Ian W. Marison 2 , Urs von Stockar 1 1 Ecole
Polytechnique F´ed´erale de Lausanne, Lausanne, Switzerland 2 Dublin City University, Dublin, Ireland Calculating the pH of complex aqueous solutions from spectral measurements involves both spectroscopic and thermodynamic modelling, because it implies determining the concentration of species in solution, as well as estimating their activity coefficient. Even though this approach is, therefore, far for being straightforward, it can turn out to be extremely useful when conventional electrochemical pH probes cannot be used and/or space is limited, as in miniaturized systems. An online pH monitoring method based on mid-infrared spectroscopy relevant to bioprocesses will be presented. This approach is non-invasive and does not require the addition of indicators or dies, since it relies on the analysis of species of common buffers used in culture media, such as a phosphate buffer. Starting with titrations of phosphoric and acetic solutions over almost the entire pH range (2–12), it was shown that the infrared spectra of all samples can be expressed as a linear combination of the molar absorbance of the acids and their deprotonated forms. In other words, the pH had no direct influence on the molar infrared spectra themselves, but only on deprotonation equilibria. Accurate prediction (standard error of prediction for pH < 0.15 pH units) was achieved by taking into account the nonideal behavior of the solutions, using the Debye-H¨uckel theory to estimate the activity coefficients. Batch cultures of E. coli were chosen as a case study to show how this approach can be applied to bioprocess monitoring. The discrepancy between the spectroscopic prediction and the conventional electrochemical probe never exceeded 0.12 pH units, and the technique was fast enough to implement a feedback controller to maintain the pH constant during cultivation. doi:10.1016/j.jbiotec.2007.07.836
7. Biosurfactants from grape marc: Stability study Oscar Manuel Portilla Rivera ∗ , Ana Bel´en Moldes Mendui˜na, Ana Mar´ıa Torrado Agrasar, Jos´e Manuel Dom´ınguez Gonz´alez Departamento de Ingenier´ıa Qu´ımica, Facultad de Ciencias, Universidad de Vigo (Campus Ourense) Ourense, Spain The interest in biosurfactants has increased considerably in recent years, as they are potential candidates for many commercial applications. Biosurfactants have several advantages over chemical surfactants including lower toxicity and higher biodegradability and effectiveness at extreme temperatures or pH values (Kosaric, 1998; Cameotra and Makkar, 1998). But in spite of the several advantages of biosurfactants, these must be cost competitive with the chemical synthesis and it is interesting the utilization of cheaper carbon source like agricultural residues for their production. Distilled grape marc, a lignocellulosic material, is one of the most abundant and useless winery wastes, which can be employed as renewable carbon source to obtain biosurfactants. In this work grape marc was submitted to acid hydrolysis in autoclave at 130 ◦ C with 3% sulphuric acid for 30 min using a liquid/wood ratio = 8 g/g. Following hydrolysates selected to ferment were neutralised with CaCO3 to a final pH of 6.5. The clarified liquors were supplemented with nutrients and used directly as fermentation media in order to obtain intracellular biosurfactants employing Lactobacillus pentosus. The maximum biosurfactant concentration after washing L. pentosus cells with buffered saline (PBS) was 5.9 g/L with a critical micelle concentration (CMC) of 2.9 g/L. The effectiveness of biosurfactants from grape marc was compared with the effectiveness of two commercial surfactants: a microbial surfactant (Surfactine) and a synthetic surfactant, sodium dodecyl sulphate (SDS), based on the relative emulsion volume (REV) (Batista et al., 2006), and the percentage of emulsified organic compound using kerosene and commercial gasoline. The biosurfactant from grape marc and surfactine showed a similar behavior, reaching percentages of the emulsified compounds around 60 and 40% towards kerosene and gasoline, respectively. On the contrary, SDS showed a different behavior depending on the organic compound, being the best towards kerosene (reaching 100%), but the worst towards gasoline, with only a 10% of emulsion. The above results indicate that biosurfactants from grape marc can be considered an attractive alternative for the bioremediation of gasoline contaminated soil or water compared with SDS. References Kosaric, N., 1998. Biosurfactants in industry. J. Am. Oil Chem. Soc. 64, 1731–1737. Cameotra, R., Makkar, R., 1998. Synthesis of biosurfactants in extreme conditions. Appl. Microbiol. Biotechnol. 50, 520–529. Batista, S.B., Mounteer, A.H., Amorim, F.R., T´otola, M.R., 2006. Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites. Bioresour. Technol. 97, 868–875.
doi:10.1016/j.jbiotec.2007.07.837
of the combination of the three main heterogeneities, dO2 , glucose and pH, that occur simultaneously at the large-scale but not at all at the small-scale. Very similar results were found in the STR-PFR scale down model. However, these studies were carried out using the untransformed wild type bacterial strain, E. coli W3110. Further work considered the additional physiological stress imposed on the cell by having to synthesise a model foreign protein (AP50) when studying process performance on scale-up. It became clear that, even under well-mixed conditions, the expression of AP50 as insoluble inclusion bodies exerts a profound physiological stress on the host cell depending on the time of induction; and, by using the scale-down model, that that there was a further effect of the three major spatial/temporal heterogeneities. Overall, in general, early induction led to lower biomass and lower viability than late induction and the heterogeneities further reduced biomass but increased viability. In this paper, we will briefly discuss the main engineering considerations involved in fermentation scale-up and then critically review those mechanisms thought to be responsible for any detrimental change in bioprocessing at the larger-scale. Though it addresses mainly E. coli fed-batch fermentations, much of the discussion also applies to other single celled aerobic microbial batch and fed-batch fermentations too. References Hewitt, C.J., Lewis, G., Onyeaka, H., Taylor, I.W., Nienow, A.W., 2007. Biotechnol. Bioeng. 96, 495–505. Hewitt, C.J., Nebe-von-Caron, G., Axelsson, B., McFarlane, C.M., Nienow, A.W., 2000. Biotechnol. Bioeng. 70 (4), 381–390. Lewis, G., Taylor, I.W., Nienow, A.W., Hewitt, C.J., 2004. J. Ind. Microbiol. Biotechnol. 31, 311–322. Onyeaka, H., Nienow, A.W., Hewitt, C.J., 2003. Biotechnol. Bioeng. 84 (4), 474–484.
doi:10.1016/j.jbiotec.2007.07.833 4 Accelerating the development cycle of antibody based drugs through the use of multiplexed in situ near infrared spectroscopy and multi-scale models Linda Harvey 1 , Payal Roychoudhury 1 , Ronan O’Kennedy 2 , Timothy Calvert 1,∗ , Brian McNeil 1 1 Strathclyde
Fermentation Centre, University of Strathclyde Glasgow, Scotland, United Kingdom 2 GSK Centre of Excellence in Drug Discovery Beckenham, Kent, United Kingdom The use of mammalian cell culture systems to manufacture antibody based drugs is now routine with levels of protein expressed ranging up to 3 gL−1 . However, the actual process of manufacture typically involves the cultivation of the selected cell line at several scales, and may also involve one or more geographical transfers of the process. These cultures are characterised by slow growth rates making the culture cycle especially lengthy by comparison to microbial expression systems, typically 1–2 weeks for CHO culture, compared with 1–2 days for E. coli. Likewise, the transfer between scales and location can be chal-
S135
lenging. Here we report upon the results of a study into the use of multiplexed in situ near infrared spectroscopic techniques to monitor industrial CHO cultures used for antibody manufacturing by GSK. Specially designed steam sterilisable probes were used in a range of fermenter vessels ranging from small lab scale (7 l) to pilot scale to monitor the progress of the production process, and models were constructed for key analytes based upon data from (A) single probes; (B) multiple probes at the same process scale; (C) multiple probes at differing process scales. All models had good predictive abilities and were robust, proving that the technique was capable of implementation in a manufacturing plant across multiple scales, and could be of practical use throughout the development cycle for antibody based drugs doi:10.1016/j.jbiotec.2007.07.834 5. Protoplast fusion technique in Tolypocladium inflatum for increasing Cyclosporine production Mohammad Reza Bakhtiari ∗ , Masoud Fallahpour, Pupak Foruzanfakhr, Nasrin Moazami Iranian Research Organization for Science & Technology (IROST) Tehran, Islamic Republic of Iran The purpose of our study was the assessment of protoplast fusion technique in order to improve cyclosporine production by the mould, Tolypocladium inflatum. Cyclosporins are valuable natural secondary metabolites with vast biological effects and have several applications, especially in preventing the rejection of transplanted organs and treatment of antoimmune disorders. Hence, increasing the production of this drug is of great importance in pharmaceutical industries. Three different strains of T. inflatum, i.e. PTCC 5252, PTCC 5253, and PTCC 5254 were evaluated in order to select two proper parents with cross differentiating characteristics. Identification of innate and natural traits was studied as the first step. It was found that T. inflatum PTCC 5252 was resistant to 187.5 g/ml of crystal violet and sensitive to 10 g/ml of cyclohexamide, while T.inflatum PTCC 5253 showed reciprocal results. Based on these findings, the two strains were selected as parents. Considering lack of references about protoplast fusion in T. inflatum and the great dependency of the technique on various factors such as, culture medium composition, mycelial age, concentration and contact time of the lytic enzymes, centrifugal velocities, polyethyleneglycol molecular weight and concentration, and composition of regeneration media, the above-mentioned factors were optimized in our study. The technique resulted to 21% of regeneration and 38% of recombination frequencies. In order to identify the high Cyclosporine producing fusants, a primary screening test was designed and performed with 257 colonies. Subsequently, 46
S136
Abstracts / Journal of Biotechnology 131S (2007) S133–S187
selected colonies were transferred to submerged cultures in production flasks. Totally 52% of these fusants showed higher production in comparison to their parents. One of the recombinants produced Cyclosporine 2.8 times more than the parent strain, T. inflatum PTCC 5252, and 2.3 times more than T. inflatum PTCC 5253. doi:10.1016/j.jbiotec.2007.07.835 6. Mid-infrared spectroscopy as a tool for pH prediction and control in bioprocesses Jonas Schenk 1,∗ , Ian W. Marison 2 , Urs von Stockar 1 1 Ecole
Polytechnique F´ed´erale de Lausanne, Lausanne, Switzerland 2 Dublin City University, Dublin, Ireland Calculating the pH of complex aqueous solutions from spectral measurements involves both spectroscopic and thermodynamic modelling, because it implies determining the concentration of species in solution, as well as estimating their activity coefficient. Even though this approach is, therefore, far for being straightforward, it can turn out to be extremely useful when conventional electrochemical pH probes cannot be used and/or space is limited, as in miniaturized systems. An online pH monitoring method based on mid-infrared spectroscopy relevant to bioprocesses will be presented. This approach is non-invasive and does not require the addition of indicators or dies, since it relies on the analysis of species of common buffers used in culture media, such as a phosphate buffer. Starting with titrations of phosphoric and acetic solutions over almost the entire pH range (2–12), it was shown that the infrared spectra of all samples can be expressed as a linear combination of the molar absorbance of the acids and their deprotonated forms. In other words, the pH had no direct influence on the molar infrared spectra themselves, but only on deprotonation equilibria. Accurate prediction (standard error of prediction for pH < 0.15 pH units) was achieved by taking into account the nonideal behavior of the solutions, using the Debye-H¨uckel theory to estimate the activity coefficients. Batch cultures of E. coli were chosen as a case study to show how this approach can be applied to bioprocess monitoring. The discrepancy between the spectroscopic prediction and the conventional electrochemical probe never exceeded 0.12 pH units, and the technique was fast enough to implement a feedback controller to maintain the pH constant during cultivation. doi:10.1016/j.jbiotec.2007.07.836
7. Biosurfactants from grape marc: Stability study Oscar Manuel Portilla Rivera ∗ , Ana Bel´en Moldes Mendui˜na, Ana Mar´ıa Torrado Agrasar, Jos´e Manuel Dom´ınguez Gonz´alez Departamento de Ingenier´ıa Qu´ımica, Facultad de Ciencias, Universidad de Vigo (Campus Ourense) Ourense, Spain The interest in biosurfactants has increased considerably in recent years, as they are potential candidates for many commercial applications. Biosurfactants have several advantages over chemical surfactants including lower toxicity and higher biodegradability and effectiveness at extreme temperatures or pH values (Kosaric, 1998; Cameotra and Makkar, 1998). But in spite of the several advantages of biosurfactants, these must be cost competitive with the chemical synthesis and it is interesting the utilization of cheaper carbon source like agricultural residues for their production. Distilled grape marc, a lignocellulosic material, is one of the most abundant and useless winery wastes, which can be employed as renewable carbon source to obtain biosurfactants. In this work grape marc was submitted to acid hydrolysis in autoclave at 130 ◦ C with 3% sulphuric acid for 30 min using a liquid/wood ratio = 8 g/g. Following hydrolysates selected to ferment were neutralised with CaCO3 to a final pH of 6.5. The clarified liquors were supplemented with nutrients and used directly as fermentation media in order to obtain intracellular biosurfactants employing Lactobacillus pentosus. The maximum biosurfactant concentration after washing L. pentosus cells with buffered saline (PBS) was 5.9 g/L with a critical micelle concentration (CMC) of 2.9 g/L. The effectiveness of biosurfactants from grape marc was compared with the effectiveness of two commercial surfactants: a microbial surfactant (Surfactine) and a synthetic surfactant, sodium dodecyl sulphate (SDS), based on the relative emulsion volume (REV) (Batista et al., 2006), and the percentage of emulsified organic compound using kerosene and commercial gasoline. The biosurfactant from grape marc and surfactine showed a similar behavior, reaching percentages of the emulsified compounds around 60 and 40% towards kerosene and gasoline, respectively. On the contrary, SDS showed a different behavior depending on the organic compound, being the best towards kerosene (reaching 100%), but the worst towards gasoline, with only a 10% of emulsion. The above results indicate that biosurfactants from grape marc can be considered an attractive alternative for the bioremediation of gasoline contaminated soil or water compared with SDS. References Kosaric, N., 1998. Biosurfactants in industry. J. Am. Oil Chem. Soc. 64, 1731–1737. Cameotra, R., Makkar, R., 1998. Synthesis of biosurfactants in extreme conditions. Appl. Microbiol. Biotechnol. 50, 520–529. Batista, S.B., Mounteer, A.H., Amorim, F.R., T´otola, M.R., 2006. Isolation and characterization of biosurfactant/bioemulsifier-producing bacteria from petroleum contaminated sites. Bioresour. Technol. 97, 868–875.
doi:10.1016/j.jbiotec.2007.07.837