Day 2 :
University of Freiburg Germany
Time : 09:30-10:00
Sandeep K. Vashist is the Head of Immunodiagnostics at Hahn-Schickard-Gesellschaft für angewandte Forschung e.V. (HSG-IMIT) and Laboratory for MEMS Applications, Department of Microsystems Engineering (IMTEK), University of Freiburg, Germany. He was the Bioanalytical Scientist at Bristol-Myers Squibb Company, Dublin, Ireland (2006-2009) and the Team Leader at NUS Nanoscience and Nanotechnology Institute, Singapore (2009-2012) after completing his Ph.D. from Central Scientific Instruments Organisation, India (2006). His research outputs include many successful technology transfers, several in vitro diagnostic and point-of-care technologies, over 80 publications in high impact factor journals and numerous patents, book chapters, and publications/talks in international conferences. He has constantly received highly prestigious fellowships and awards for scientific research excellence and innovation. Besides, he is acting as the Editorial Board and Executive Editor for several reputed journals (such as NPG’s Scientific Reports, Elsevier’s NPBR and MDPI’s Diagnostics). His research interests include in vitro diagnostics, biosensors, point-of-care testing, nanotechnology, and smartphone-based personalized healthcare monitoring and management.
Several proprietary Smartphone-based colorimetric readers (SBCRs) for in vitro diagnostics (IVD) have been developed using a standard Smart phone (Samsung Galaxy SIII mini), a gadget (iPAD mini, iPAD4, iPhone 5S or any similar tablet/Smartphone), a custom-made dark hood and a base holder assembly. A 96 or 24-well microtiter plate (MTP) was positioned on the g adget’s screensaver, enabling white light-based bottom illumination only at the well bottom. The set-up was demonstrated and compared with a commercial MTP reader (MTPR) for human C-reactive protein immunoassay and bicinchoninic acid (BCA) protein estimation assay. Colorimetric images of the reaction solutions were captured by the Smartphone’s back camera with 5 megapixels resolution. The pixel int ensity of the captured images was determined by an image processing algorithm. SBCR has same precision, dynamic range and sensitivity as MTPR. SBCRs would have a considerably commercial opportunity as immunoassays using the clinical IVD kits are based on color detection. Being 20-fold cheaper than MTPR, SBCR could be an ideal point-of-care (POC) colorimetric detection device for the next-generation of cost-effective IVD and versatile tools in bio ana lytical settings. Moreover, they could be employed for colorimetric assays in the visible range using a transparent and flat bio analytical platform such as lab-on-a-chip, lateral flow microfluidics and centrifugal microfluidics-based LabDisk platforms. Considering their widespread use (~7 billion users, ~96% world population), Smartphone’s are under on-going improvement to become smarter, smaller and cheaper. As more and more objects are linked to with a Smartphone, SBCR will become a powerful assistant of analysts to play an important role in IVD, POC, personalized healthcare monitoring and management, telemedicine and remote bio sensing applications.
Merck Millipore Germany
Keynote: Chemically modified cysteine in fed-batch processes and impact on CHO specific productivity
Time : 10:00-10:30
Based in Darmstadt, Germany, Aline is currently responsible for the development of mammalian cell culture media at Merck Millipore. Aline holds an engineering degree in biochemistry and biotechnology from the National Institute of Applied Sciences (INSA) in Lyon, France, a master in cellular physiopatho logy from the University of Lyon and a PhD degree in immunology from the University of Paris. She has worked 3 years in immunotherapy biomarker research at Stallergenes before joining Merck KGaA in 2011. From 2011 to 2012, Aline was responsible for the development of cellular assays to monitor upstream processes at Merck Millipore. From 2013 to 2014, Aline was heading the R&D upstream media performance laboratory responsible mainly for the development of new chemically modified amino acids and their integration in fed-batch platforms. Since April 2014, Aline is heading the mammalian cell culture media development activities at Merck Millipore.
Industrial fed-batch cultivation of mammalian cells is used for the production of therapeutic proteins such as monoclonal antibodies. Beside medium ensuring initial growth, feeding is necessary to improve growth, viability and antibody production. Established commercial systems include a slight acidic concentrated main feed and a separate alkaline amino acid feed containing L-tyrosine and L-cysteine. Since L-cysteine is not s table due to its dimerization to cystine in the presence of air and metal catalysts, a stable L-cysteine derivative is needed to include all amino acids in neutral pH feeds. Those single feed systems are favored to simplify feeding schemes and improve the overall process robustness through stabiliz ation of both pH and DO signals. Here, we suggest the use of a chemically modified cysteine in combination with phosphor tyrosine disodium salt in an industrial single feed fed-batch process applicable at small scale as well as in bioreactor s. Cell culture experiments were carried out either in spin tubes or bioreactors with a CHO suspension cell line expressing a human monoclonal antibody. Viable cell density and viability were measured using an automatic cell counting device. Spent media analysis of supernatants was carried out for amino acids after pre-column derivatization and UPLC analysis and for vitamins using LC-MS/MS. Metabolite measurements were performed with Cedex Bio HT relying on photometric and turbidometric methods. Characterizati on of the monoclonal antibody was performed using 2-AB labeling for glycan analyses, cIEF for charge variants analyses and LC-MS/MS for peptide mapping experiments. Stability studies of the feed containing the modified cysteine derivative showed that the molecule was stable and that no L-cysteine or L-cystine was released over three months when stored at room temperature or 4 ° C. Moreover, no change in the color of the feed was observed over time. Small scale batch experiments where L-cysteine was replaced by the same amount of chemically modified cysteine indicated no change in growth or viability profiles. Use of the modified cysteine derivative in small scale fed-batch processes indicated comparable maximum viable cell density, prolonged viability and increased titer compared to the established two feed system. Bioreactor experiments confirmed the increase in specific productivity described at small scale when the single feed strategy was compared to the two feed strategy. In depth characterization of the monoclonal antibody indicated no change in the glycosylation or charge variant pattern whereas peptide mapping experiments were not able to detect any integration of the modified amino acid in the sequence of the monoclonal antibody.
Professor & Chair of Mechanical Engineering, York University Canada
Time : 10:30-11:00
Sushanta Mitra is a Kaneff Professor in Micro & Nanotechnology for Social Innovation at the York University, Toronto. His research interests are in the fundamental understanding of fluid flow in narrow confinements and its applications in the fields of energy, environment and bio-systems. For his contribution in science and engineering, he has been an elected fellow of the American Society of Mechanical Engineers, the Canadian Society for Mechanical Engineering, the Engineering Institute of Canada, the Canadian Academy of Engineering and the Royal Society of Chemistry.
In recent years there is a significant development in micro and nanotechnology that has enabled fabrication of fine micro/nanostructures, which are often used as building-block towards producing sensors which are specific, selective and sensitive towards number of key biomolecules. The talk will focus on how such micro/nanofabrication tools coupled with fundamental developments in physics and chemistry can create next generation sensors that can be used for monitoring deadly virus like Dengue NS1, food borne pathogen like Listeria monocytogenes, water borne pathogens like E. coli and cardiac markers like myoglobin for early diagnostics of heart attack. The talk will end with example of a translational and transformative research which allows the laboratory proto-type to be tested in field and end-user communities.
- Industrial Aspects of Biotechnology
Location: Flemings Conference Hotel Room 7
University of Guelph, Canada
Title: Novel synthetic anti-microbial defensins through confrontational selection and screening of yeast libraries
Time : 11:45-12:10
K Yankulov is a Professor at the University of Guelph, Canada. His lab is using the yeast S. cerevisiae for research in several closely related areas of genetics , including the stability of extra-chromosomal DNA , protein expr ession and the design and screening of expression DNA library screening . This line of research has produced 33 publicatio ns on genes in influential genetics journals. He is an Editor in PLoS One, Frontiers in Genetics and Genes and Gene Therapy. Recentl y, he has pursued his interest on a group of novel fungal defense molecules (called defensins) and their commercial development as anti-microbial agents
Antibiotics are frequently used in animal feed to boost its efficacy. However, recent policies in EU and USA have restricted s uch use of antibiotics and Canada is expected to follow suit. Consequently, the need for novel anti-microbial agents is a most urgent issue. Defensins form a large group of secreted animal, plant or fungal peptides that kill a broad spectrum of microorganisms includin g food borne pathogens. They have a low probability of developing microbial resistance and are viewed as viable alternatives to antibiotics for both the food and pharmaceutical industries. The industrial development of vertebrate defensins is hindered by concerns of cytotoxicity. In contrast, the known fungal defensins show little side effects in animals and work at doses comparable to these of many antibiotics. We are developing a platform that will use DNA libraries for a huge variety of synthetic defensin-like peptides. We are expressing these libraries in yeast and screen them against model microorganisms. Our aim is to identify novel synthetic anti-microbial peptides that can be used as alternatives to antibiotics. These agents can be improved to incorporate a trypsin-target site to ensure their normal degradation in the stomach and to abolish any side effects on the normal microbiome of the animals. We also aim at the affordable production of such agents by the yeast K. lactis in milk whey. Our progress in these screens will be reported and discussed.
University of Haifa, Israel
Time : 12:10-12:35
Fuad Fares has completed his DSc studies at the Faculty of Medicine, Technion-Israel Institute of Technology and Postdoctoral studies at the Department of Molecular Biology and Pharmacology, School of Medicine, Washington University, St. Louis Missouri. He is the Director of the Department of Molecular Genetics at Carmel Medical Center and Associated Professor at the Department of Human Biology, University of Haifa. He has published more than 75 papers in reputed journals and serving as a member of the Israel Council for Higher Education. He is the inventor of designing long-acting recombinant proteins and the initiator of PROLOR Biotech Company.
One major issue regarding the clinical use of many peptides is their short half-life due to the rapid clearance from the circulation. To overcome this problem, we succeeded to ligate the signal sequence of O-linked oligosaccharides to the coding sequence of the hormones. The cassette gene that has been used contains the sequence of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG) subunit. The CTP contains 28 amino acids with four O-linked oligosaccharide recognition sites. It was postulated that O-linked oligosaccharides add flexibility, hydrophilicity and stability to the protein. On the other hand it was suggested that the four O-linked oligosaccharides play an important role in preventing plasma clearance and thus increasing the half-life of the protein in circulation. Using this strategy we succeeded to ligate the CTP to the coding sequence of follitropin (FSH), thyrotropin (TSH), erythropoietin (EPO) growth hormone (GH) and thus to increase the longevity and bioactivity of these proteins in vivo. Interestingly, the new analogs of FSH and GH were found not immunogenic in human and it is already passed successfully clinical trials phase III and phase II respectively. Moreover, FSH long acting was approved by the European Commission (EC) for treatment of fertility. In addition, our results indicated that long acting GH is not toxic in monkeys and the results from clinical trials phase I and phase II seem to be promising. Designing long acting peptides will diminish the cost of these drugs and perhaps reduce the number of injections in the clinical protocols.
Tanta University, Egypt
Time : 12:35-13:00
Tarek had born in Egypt, Cairo on 1966. Tarek is a Professor of biochemistry in Biochemistry Division, Chemistry Department, Tanta University, Tanta, Egypt started from June 2008 till now. He is associated professor In Tanta University fromm 2003-2008. He worked in National Research Centre from 1992 to 2003 in Molecular Biology Department as a researcher. Tarek is a reviewer for many journals. He shared in 12 national projects. He Supervised more than 20 MSc a Ph.D. thesis. He has 42 publications. Tarek interested in Purification and characterization of enzymes , enzyme inhibitors . Study related enzyme engineering , biochemistry.
Ulocladium atrum inulinase was immobilized on different composite membranes composed of chitosan/nonwoven fabrics. Km values of free and immobilized U. atrum inulinase on different composite membranes were calculated. Free and immobilized U. atrum inulinase had pH optima at 5.6 on polyester nonwoven fabric coated with 3% chitosan solution (PPNWF3) but immobilized U. atrum inulinase on polyester and polypropylene nonwoven fabrics coated with 1% chitosan solution had pH optimum 5. The enzyme had optimum temperature at 40° C for immobilized enzyme on each of polyester and polypropylene composite membranes coated with 1% chitosan meanwhile it was 50° C for free and immobilized enzyme on polypropylene nonwoven fabric coated with 3% chitosan solution. Inulinase was stable at 40° C for free U. atrum inulinase but thermal stability of immobilized enzyme was detected up to 60° C. Reusability of immobilized enzyme was from 38 to 42 cycles of reuse; the immobilized enzyme lost its activity completely. In conclusion, immobilized U. atrum inulinase was considerably more stable than the free enzyme and could be stored for extended periods.
Jiangnan University, China
Title: Systems metabolic engineering of Bacillus subtilis for efficient N-acetyl glucosamine production
Time : 13:45-14:10
Long Liu is currently a life-time professor at School of Biotechnology , Jiangnan University, Wuxi, China. He has been working in the area of bioprocess engineering and metabolic engineering with special reference to bioprocess optimi zation and control. He has authored 2 books, 3 book chapters, 7 review papers, 20 patents, 37 research papers in SCI journals, and 10 conference papers. He has been a recipient of First pri ze of Science and Technology progress, Jiangsu, China (2010), First prize of Science and Technology Progress, China Petroleum and Chemical Industry Federation (2011), the Technological Invention Award of China National Light Industry Council (2013), and Excellent Young Teacher of Jiangsu, China (2014).
N-acetyl glucosamine (GlcNAc) is a pharmaceutically and nutraceutically important compound with wide applications and now is mainly produced by hydrolysis from crab and shrimp shells which can cause severe environmental pollution and has potential risk of allergic reactions. In this work, we achieved the over-production of GlcNAc by systems metabolic engineering of Bacillus subtilis, a generally regarded as safe strain. Specifically, GlcNAc synthesis pathway was strengthened by co-over expression of Glucosamine-6-phosphate (GlmS) synthase and GlcNAc-6-phosphate N-acetyl transferase (GNA1) which realized GlcNAc production (240 mg/L). Next, GlcNAc uptake pathway and intracellular degradation pathway were entirely blocked by knockout of all the encoding genes in GlcNAc catabolic pathway to facilitate GlcNAc accumulation. Then, to balance and strengthen GlcNAc synthetic pathway, DNA-guided scaffold system was introduced and increased GlcNAc titer from 1.83 g/L to 4.55 g/L. Synthetic small regulatory RNAs were then employed to optimize expression level of key enzymes in the nodes of GlcNAc-related network including 6-phosphofructokinase (Pfk) and phospho glucosamine mutase (GlmM). GlcNAc titer was improved to 8.30 g/L by modular regulation of the activities of GlcNAc-related modules. In fed-batch fermentation, the GlcNAc titer was further increased to 31.65 g/L which was 3.8-fold that in the shake flask. Finally, to understand kinetics of metabolite changes in GlcNAc synthesis pathway and glycolysis, targeted metabolomics and dynamic labeling were implemented. Inefficient GlcNAc6P dephosphorylation and undesired GlcNAc phosphorylation were identified as rate-limiting step for GlcNAc synthesis which pinpointed future direction for further pathway optimization. The used systems metabolic engineering strategies may be useful for the construction of versatile B. subtilis cell factories for the production of the other industrially important chemicals.
Jiangnan University China
Title: Enhancing flavonoid production by systematically tuning the central metabolic pathways based on a CRISPR interference system in Escherichia coli
Time : 14:10-14:35
Prof. Jingwen Zhou obtained his Ph.D degree in Fermentation engineering in 2009. After that, he became assistant professor (20 09), associate professor (2011) and full professor (2014) in School of Biotechnology, Jiangnan University. He finished his postdoc training in Department o f Chemistry and Chemical Biology in Harvard from 2012 to 2013. His current research works mainly focused on the metabolic engineering of microorganisms to produce organic acids and plant natural products, especially L-ascorbic acid and flavonoids. He published 52 peer reviewed papers on journals such as Metaboli c Engineering, Applied and Environment Microbiology, and also several invited reviews on Current Opinion in Biotechnology and Biotechnology Advances. Several of the typical products he had been working on were now produced by several manufactures on industrial scale. His achievements were awarded for several times inside China. He is now the Editorial Board of Scientific Reports (Nature Press) and Electronic Journal of Biotechnology (Elsevier Press).
The limited supply of intracellular malonyl-CoA in Escherichia coli impedes the biological synthesis of polyketides , flavonoids and biofuels. Here, a clustered regularly interspaced short palindromic repeats (CRISPR) interference system was constructed fo r fine-tuning the central metabolic pathways to efficiently channel carbon flux toward malonyl-CoA. Using synthetic sgRNA to silence candidate genes, genes that could increase the intracellular malonyl-CoA level by over 223% were used as target genes. The efficiencies of repression of these target genes were tuned to achieve appropriate levels so that the intracellular malonyl-CoA level was enhanced without significantly altering final biomass accumulation (the final OD600 decreased by less than 10%). Based on the results, multiple gene silencing was successful in approaching the limit of the amount of malonyl-CoA needed to produce the plant-specific secondary metabolite (2S)-naringenin. By coupling the genetic modifications to cell growth, the combined effects of these genetic perturbations increased the final (2S)-naringenin titer to 421.6 mg/L which was 7.4-fold higher than the control strain (50.5 mg/L). The strategy described here could be used to characterize genes that are essential for cell growth and to develop E. coli as a well-organized cell factory for the production of other important products that require malonyl-CoA as a precursor such as flavonoids, polyketides and fatty acids.
Jiangnan University, China
Title: One-step biosynthesis of α-keto acids by the L-amino acid deaminase: Biocatalyst construction and process optimization
Time : 14:35-15:00
Guocheng Du is the Dean of School of Biotechnology , Jiangnan University, China. He is the Distinguished Professor of Changjiang Scholars, Ministry of Educat ion, China. His current main research focus is on bioprocess engineering and metabolic engineering. He has about 300 publi cations/communications, which include 68 patents, 10 books, 176 original and review papers, etc. He won two Second Prizes of State Science and Technology Awards in 2006 and 2012, respectively. He has been conferred Honorary Doctorate degree from Jiangnan University, China. He is an Associate Editor of Journal of the Science of Food and Agriculture, and Microbial Cell Factories, and Board Member of Bioresource Technology
L-Amino Acid Oxidases ( LAAOs ) which catalyze the stereo-specific oxidative deam ination of L-amino acids to α-ketoacids and ammonia are flavinadeni ne dinucleotide containing homodimeric proteins. L-Amino acid oxidases are widely distributed in diverse organisms and have a range of properties. In this work, we systematically engineered the LAAO from Proteus vulgaris as a robust biocatalyst for the efficient one-step biosynthesis of a-keto acids including α-ketoglutaric acid, α-keto-γ-methyl thiobutyric acid and phenyl pyruvic acid which are currently produced by multi-step chemical synthesis with heavy environmental pollution. First, the directed evolution of LAAO was done by error-prone Polymerase Chain Reaction (PCR) and then the site-saturation mutagensis was conducted at the mutation sites of the positive mutants yielded by error-prone PCR. The biotransformaton conditions included biocatalyst concentration, the substrates concentration, temperature and pH and so on. The maximum titers of α-ketoglutaric acid, α-keto-γ-methyl thiobutyric acid and phenyl pyruvic acid are 12.3 g/L, 63.6 g/L and 11.8 g/L, respectively under the specific optimal conditions. Compared with the traditional multi-step chemical synthesis, our one-step biocatalytic production of a-keto acids has an advantage in terms of less environmental pollution and thus has great potential for industrial production of a-keto acids.
Reichelt W N is a Project Assistant in the Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses at the Vienna University of Technology (VUT). He studied Biochemistry at the Karl Franzens University in Graz before doing his Master’s thesis in the field of programmed cell death and aging. After a year of PhD research in the field of neurodegeneration (Parkinson), he transitioned to the field of bioprocess technology at VUT to acquire in-depth understanding of pharmaceutical bioprocess production process approaches. Within his PhD work, he is spanning applied to basic science by developing and employing physiological bioprocess control strategies to tweak industrial bioprocesses to maximum productivity. Hereby, the project unifying goal is to gain mechanistic insight into bioprocess physiology to ultimately achieve comprehensive bioprocess understanding and transferable platform knowledge.
Progressively expiring patent protection brings forward generic drug industry and heats up competition for the most efficient and robust bioprocesses. While competition is intensifying, the FDA quality by design (QbD) initiative for increased process understanding is increasing demands concerning bioprocess development. These circumstances cumulate to the necessity of comprehensive process understanding and consequently highly efficient bioprocess development routines. For bioprocess development, literature has comprehensively proven the feeding strategy an especially promising target. Having mastered the feedback control of physiological rates by the use of soft sensors, the subsequent step of biotechnological development could lead towards controlled transient changes of physiological parameters. Whereas the majority of the scientific community has chosen the specific growth rate (µ) as target control parameter, we have invested extensive work in the control of the specific substrate uptake rate (qs). Hereby, the qs control renders physiology accessible, substrate availability being upstream of physiological variables example µ. We want to test the hypothesis that dynamic process control as qs profiles in terms of ramps and oscillations constitute tools for efficient tweaking of physiology. By alternating the substrate availability in the reactor the physiologic state of E.coli shall be modulated between high metabolic load and recreational phases during induction. Focusing on product physiology interrelations, modeling and investigation of scale up effects is explicitly not in scope of our investigation. Hereby, we want to address specific questions as the impact frequency and amplitude for qs oscillation of on physiology as well slope and orientation of qs ramps. The amplitude of oscillation is of great interest can physiological limits be temporarily exceeded by granting periods of recovery? Whereas a high frequency of oscillation of the specific substrate uptake rate could be limited by technical constraints (example sampling capacities, error of measurement) a too long duration could fail to have any impact besides acetate formation.
State University of Campinas, Brazil
Title: Surface activity of surfactin under extreme conditions (pH, temperature and ionic strength): A prospection for microbial enhanced oil recovery
Time : 15:25-15:50
C J Andrade is a PhD student on Biotechnological Processes at State University of Ca mpinas, and has been trying to use industrial wastes as a culture medium . Among the compounds of his interest are biosurfactants, galacto-oligosaccharides, enzyme, flavours, etc. He is also interested on the integration between fermentation and purification, in particular, ultrafiltration.
Petroleum has been playing the most important role in energy sectors. Worldwide oil production has been declining. The primary and secondary techniques of oil recovery, reach ≈35% and ≈20%, respectively. Thus, the aim of enhanced oil recovery technologies is the residual oil, which is ≈45% of the total oil reserves. Microbial-enhanced oil recovery (MEOR) is one of the most important tertiary recovery processes, which rise up the oil recovery ≈3%. However laboratory scale experiments predict up to 10%. The aim of this work was to evaluate the surface activity of surfactin under extreme conditions (pH, temperature and ionic strength) - individually and their interactions – which represents similar environmental of wells. The surface activity of surfactin under extreme condition was already reported. Although, for the best of our knowledge this is the first study that focused on the biosurfactants surface activity under extreme condition at the same time, which is fundamental for MEOR. Surfactin was produced at bioreactor scale (7.5L) using cassava wastewater as substrate. Then, surfactin was collected from the top of bioreactor centrifuged, precipitated (pH 2) and purified by solvent extraction and ultrafiltration. The final product was named produced surfactin and used in the experiments at 74 mg.L-1 of surfactin. The ionic strength was tested using synthesized brine. The pH, temperature and ionic strength were first investigated separately, in which the pH was evaluated from 2 to 12, whereas 3 temperatures were tested during 1h, 79, 100 and 121oC and finally the ionic strength 2.5, 5, 10 and 20 g.L-1 of synthesized brine. Then, were measure surface tension (ST) and its critical micelle dilutions (CMD). Thereafter, central composite design (CCD) experiments evaluated the interactions of temperature, pH and ionic strength. The ionic strength affected the surface activity property of surfactin, in which the higher ionic strength, the higher was the surface tension measurements (ST and CMD). The intermediate pH (4-8) showed highest (best) surface activity. No significant differences were observed for the thermal treatments. Therefore, temperature, pH and ionic strength experiments were aligned with the maintenance of surface activity properties of produced surfactin, which suggest their potential application at extreme systems, in particular MEOR. Regarding to CCD of surfactin, the ANOVA of CMD-1 indicated that temperature, pH and ionic strength were statistically different (95%) with r2 of 0.76. Thus was generated the equation 1: [Y = 67.28 + 8.5 x (ionic strength) – 3.02 x (ionic strength)2]. The ionic strength was the most significant parameters on the surface activity, in which threshold of surface activity of surfactin was at 18.58 g.L-1. The validation (72.17 mN.m-1) was well-aligned to the predicted value (73.26 mN.m-1). Therefore, it seems feasible consider surfactin in MEOR, in which ionic strength is the most important parameter.