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Shaping the quality of biopharmaceuticals – a multiscale perspective on the utilization of big data for efficient bioprocess development
S40
Abstracts / New Biotechnology 33S (2016) S1–S213
range of techniques at lab scale. By using simple control tests (PHA from MMC with synthetic substrates), alkaline extraction was investigated in a range of NaOH concentrations (0.05–1.0 M), with or without surfactant sodium dodecyl sulphate (SDS) addition (0.1 w/v% SDS), and for a different treatment time (3–24 h). In particular, the use of SDS allowed maintaining short reaction time (3 h), achieving PHA purity of 99%, with more than 90% of recovery. A treatment with supercritical CO2 (S-CO2 ) has been also investigated at 50◦ C and 20 MPa, whose performance depended on the sample humidity (lyophilisation step not necessary) and on the presence of polarity conditioner (methanol 1% v/v). Even though a better recovery was often obtained, compared to alkaline treatment, the PHA purity did not exceed 90% on cell dry weight, highlighting the need of further improvement. These methods are presently being checked on MMC-PHA from OF-MSW.
engineering studies [2]. These cassettes encode individual 4S pathway reactions as well as different combinations of dsz modules. Results show that all the modules are functional, which opens the chance to develop “à la carte” bacterial synthetic consortia for DBT bioconversions. The refactoring of the 4S route has revealed some unanticipated traits and was shown to be a successful strategy, not only to enhance the conversion of DBT into 2HBP and to overcome the inhibition of the Dsz enzymes by the 4S intermediates, but also for the efficient production of unattainable high added value intermediates that are difficult to obtain using the current monocultures. Acknowledgments: This work has been supported by Saudi Aramco and by project DBR-001/08/WCOD.
http://dx.doi.org/10.1016/j.nbt.2016.06.861
References
O12-9
[1] Gray KA, Pogrebinsky OS, Mrachko GT, Xi L, Monticello DJ, Squires CH. Nat Biotechnol 1996;14:1705–9. [2] Martinez I, Mohamed M, Rozas D, Garcia JL, Diaz E. Metab Eng 2016;35: 46–54.
Bioprocess scale-up from small to large pilot scale using eppendorf fermentation systems
http://dx.doi.org/10.1016/j.nbt.2016.06.863
Christof Knocke 2,∗ , Bin Li 1 , Bruno Sommer Ferreira 2 , Ma Sha 3 O12-11
1
Eppendorf, Inc Vaudaux-Eppendorf AG 3 Biotrend 2
The ultimate goal in bioprocess development is the realization of commercial production. Currently, the scale-up of fermentation processes, which is critical to the success of industrial fermentation for bioproduction, is receiving much attention. Eppendorf fermentation systems cover a wide range of working volumes from less than 1 L to as large as 2,400 L. We investigated their scale-up capabilities from small to pilot scale. Within this presentation, we discuss engineering parameters critical for scale-up, such as vessel and impeller geometry, tip speed, mixing time, oxygen transfer rate (OTR), and power number. Furthermore, we present recent customer data on the production of the biopolymer polyhydroxbutyrate (PHB) using Bacillus sacchari. Successful scale-up of the process from 2 L to 200 L exemplifies the scale-up capabilities of Eppendorf fermentation systems. http://dx.doi.org/10.1016/j.nbt.2016.06.862
O12-10 Enhancing biodesulfurization dibenzothiophene pathway
by
refactoring
the
4S-
Igor Martínez 1,∗ , Magdy El-Said Mohamed 2 , José Luis García 1 , Eduardo Díaz 1 1 2
Biological Research Center, Spain Saudi Aramco, Saudi Arabia
The 4S pathway is the most studied bioprocess for the removal of the sulfur of aromatic heterocyclic compounds present in fuels and that are recalcitrant to the conventional chemical hydrodesulfurization processes. It consists of a set of three sequential reactions, encoded by the dszABCD genes, through which the model compound dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) molecule [1]. In this work, we have pioneered a novel approach based in engineering synthetic dsz cassettes that were implanted in Pseudomonas putida KT2440, a model bacterial “chassis” for metabolic
Shaping the quality of biopharmaceuticals – a multiscale perspective on the utilization of big data for efficient bioprocess development Michael Sokolov 1,∗ , Jonathan Ritscher 1 , Jonathan Souquet 2 , Hervé Broly 2 , Massimo Morbidelli 1 , Alessandro Butté 1 1 2
ETH Zurich, Switzerland Merck Serono SA, Switzerland
The progress in the process development for the production of biopharmaceuticals in the last decades was considerably driven by the advancement of analytical technologies for process characteristics and product attributes. Combined with an enhanced interest in the so-called ‘omics’ technologies (aiming at understanding the process on a molecular level) this resulted in a significant increase of generated process data. This trend was accounted for by the health authorities in the Quality by Design (QbD) initiative, which motivates to develop a reliable process understanding from those data to ensure robust product quality. This work presents several applications of multivariate dataand knowledge-driven tools revealing the versatile possibilities to utilize complex bioprocess data for efficient process understanding and development. The underlying experiments investigate on four different scales (microliter, milliliter, lab and pilot scale) the role of cell line candidates as well as various media components and process settings in fed-batch cell culture processes for the production of therapeutic proteins. The presented examples will cover sequential high throughput screening experiments followed by process scale-up for biosimilar process development, the application of Raman spectroscopy for online monitoring of process and quality variables as well as the process prediction combining statistical and mechanistic approaches. The presented, generally applicable methodologies go far beyond the possibilities offered by commercial software and provide a significant basis for the reduction of the experimental effort, the simplification of the analytical scheme as well as the risk reduction for efficient bioprocess design. http://dx.doi.org/10.1016/j.nbt.2016.06.864
Abstracts / New Biotechnology 33S (2016) S1–S213
range of techniques at lab scale. By using simple control tests (PHA from MMC with synthetic substrates), alkaline extraction was investigated in a range of NaOH concentrations (0.05–1.0 M), with or without surfactant sodium dodecyl sulphate (SDS) addition (0.1 w/v% SDS), and for a different treatment time (3–24 h). In particular, the use of SDS allowed maintaining short reaction time (3 h), achieving PHA purity of 99%, with more than 90% of recovery. A treatment with supercritical CO2 (S-CO2 ) has been also investigated at 50◦ C and 20 MPa, whose performance depended on the sample humidity (lyophilisation step not necessary) and on the presence of polarity conditioner (methanol 1% v/v). Even though a better recovery was often obtained, compared to alkaline treatment, the PHA purity did not exceed 90% on cell dry weight, highlighting the need of further improvement. These methods are presently being checked on MMC-PHA from OF-MSW.
engineering studies [2]. These cassettes encode individual 4S pathway reactions as well as different combinations of dsz modules. Results show that all the modules are functional, which opens the chance to develop “à la carte” bacterial synthetic consortia for DBT bioconversions. The refactoring of the 4S route has revealed some unanticipated traits and was shown to be a successful strategy, not only to enhance the conversion of DBT into 2HBP and to overcome the inhibition of the Dsz enzymes by the 4S intermediates, but also for the efficient production of unattainable high added value intermediates that are difficult to obtain using the current monocultures. Acknowledgments: This work has been supported by Saudi Aramco and by project DBR-001/08/WCOD.
http://dx.doi.org/10.1016/j.nbt.2016.06.861
References
O12-9
[1] Gray KA, Pogrebinsky OS, Mrachko GT, Xi L, Monticello DJ, Squires CH. Nat Biotechnol 1996;14:1705–9. [2] Martinez I, Mohamed M, Rozas D, Garcia JL, Diaz E. Metab Eng 2016;35: 46–54.
Bioprocess scale-up from small to large pilot scale using eppendorf fermentation systems
http://dx.doi.org/10.1016/j.nbt.2016.06.863
Christof Knocke 2,∗ , Bin Li 1 , Bruno Sommer Ferreira 2 , Ma Sha 3 O12-11
1
Eppendorf, Inc Vaudaux-Eppendorf AG 3 Biotrend 2
The ultimate goal in bioprocess development is the realization of commercial production. Currently, the scale-up of fermentation processes, which is critical to the success of industrial fermentation for bioproduction, is receiving much attention. Eppendorf fermentation systems cover a wide range of working volumes from less than 1 L to as large as 2,400 L. We investigated their scale-up capabilities from small to pilot scale. Within this presentation, we discuss engineering parameters critical for scale-up, such as vessel and impeller geometry, tip speed, mixing time, oxygen transfer rate (OTR), and power number. Furthermore, we present recent customer data on the production of the biopolymer polyhydroxbutyrate (PHB) using Bacillus sacchari. Successful scale-up of the process from 2 L to 200 L exemplifies the scale-up capabilities of Eppendorf fermentation systems. http://dx.doi.org/10.1016/j.nbt.2016.06.862
O12-10 Enhancing biodesulfurization dibenzothiophene pathway
by
refactoring
the
4S-
Igor Martínez 1,∗ , Magdy El-Said Mohamed 2 , José Luis García 1 , Eduardo Díaz 1 1 2
Biological Research Center, Spain Saudi Aramco, Saudi Arabia
The 4S pathway is the most studied bioprocess for the removal of the sulfur of aromatic heterocyclic compounds present in fuels and that are recalcitrant to the conventional chemical hydrodesulfurization processes. It consists of a set of three sequential reactions, encoded by the dszABCD genes, through which the model compound dibenzothiophene (DBT) is transformed into the sulfur-free 2-hydroxybiphenyl (2HBP) molecule [1]. In this work, we have pioneered a novel approach based in engineering synthetic dsz cassettes that were implanted in Pseudomonas putida KT2440, a model bacterial “chassis” for metabolic
Shaping the quality of biopharmaceuticals – a multiscale perspective on the utilization of big data for efficient bioprocess development Michael Sokolov 1,∗ , Jonathan Ritscher 1 , Jonathan Souquet 2 , Hervé Broly 2 , Massimo Morbidelli 1 , Alessandro Butté 1 1 2
ETH Zurich, Switzerland Merck Serono SA, Switzerland
The progress in the process development for the production of biopharmaceuticals in the last decades was considerably driven by the advancement of analytical technologies for process characteristics and product attributes. Combined with an enhanced interest in the so-called ‘omics’ technologies (aiming at understanding the process on a molecular level) this resulted in a significant increase of generated process data. This trend was accounted for by the health authorities in the Quality by Design (QbD) initiative, which motivates to develop a reliable process understanding from those data to ensure robust product quality. This work presents several applications of multivariate dataand knowledge-driven tools revealing the versatile possibilities to utilize complex bioprocess data for efficient process understanding and development. The underlying experiments investigate on four different scales (microliter, milliliter, lab and pilot scale) the role of cell line candidates as well as various media components and process settings in fed-batch cell culture processes for the production of therapeutic proteins. The presented examples will cover sequential high throughput screening experiments followed by process scale-up for biosimilar process development, the application of Raman spectroscopy for online monitoring of process and quality variables as well as the process prediction combining statistical and mechanistic approaches. The presented, generally applicable methodologies go far beyond the possibilities offered by commercial software and provide a significant basis for the reduction of the experimental effort, the simplification of the analytical scheme as well as the risk reduction for efficient bioprocess design. http://dx.doi.org/10.1016/j.nbt.2016.06.864