Day 1 :
Keynote: Downstream process development for purification of a novel anti-MRSA antibody: A case study for antibody aggregation removal
Time : 10:35-11:20
Yanfeng Zhang completed his PhD from Michigan State University and Post-doctoral studies from Pacific Northwest National Laboratory and University of Texas HealthrnScience Center at San Antonio. He is Senior Scientist of Process Development of XBiotech, USA, the world’s leading developer of next-generation True Human therapeuticrnantibodies. He has published more than 20 papers in reputed journals and has been serving as an editorial board member of international journals.
Staphylococcus aureus is gram-positive bacteria that commonly causes human health problem such as skin and respiratoryrninfections, and food poisoning. S. aureus has multiple virulence factors including henolysins, toxins and superantigens, andrnthe strains have high genetic variability. Some strains of S. aureus are also resistant to antibiotics (MRSA and VRSA), resultingrnin an infection that is difficult to control. XBiotech has screened the blood of hundreds of human individuals to find antibodiesrnthat directly and specifically target S. aureus. One of these true-human antibodies, 514G3, has shown positive results in preclinicalrnstudies against S. aureus and is currently being evaluated in a Phase I/II study. During the purification of 514G3 antibody,rnan unusually high level of protein aggregation (up to 20%) was observed. Therefore, the protein aggregation problem was firstrnanalyzed by a series of analytical methods. Then, downstream processes were developed to remove the aggregated antibodies. Thisrnpresentation will describe our strategies of resin screen and optimization for the removal of antibody aggregates.
GE Healthcare Life Sciences Cell Culture, USA
Time : 11:35-12:20
W G Whitford is Sr. Manager, Cell Culture, GE Healthcare in Logan, UT with over 20 years’ experience in biotechnology product and process development. He joined therncompany 13 years ago as a Team Leader in R&D developing products supporting biomass expansion, protein expression and virus secretion in mammalian and invertebraterncell lines. Products he has commercialized include defined and animal product-free hybridoma media, fed-batch supplements, and aqueous lipid dispersions. An invitedrnLecturer at international conferences, he has published over 250 articles, book chapters and patents in a number of fields in the bioproduction arena. He now enjoys suchrnindustry activities as serving on the editorial advisory board for BioProcess International.
New approaches in upstream bioproduction include applications of intensified biomanufacturing in both batch and continuousrnculture. Such high cell-density perfusion-supported formats can significantly change basic culture parameters resulting inrnaltered culture media circuits as well as production-cell demands and performance. And, there are a number of distinct highrncell-density, perfusion-based process and instrumentation styles available. Brand new manufacturing approaches including 3Drnbioprinting are creating entirely new demands for their inks, media and matrices. The advanced production platforms of multiplyrnrecombinant null CHO cells and new avian lines are requiring their own specialized SFM formulations. These, as well as such newrntherapeutic products as stem and CAR T-cell therapies all contribute to growing biologic, business and physicochemical demandsrnupon culture media and buffers. Media development strategies must consider such new product and culture mode-unique demandsrnupon primary metabolites and growth factors; media volumes, schedules and storage; materials cost and even definitions of qualityrnattributes. Beyond this, heightened standards for raw and ancillary materials (including single-use product-contact plastics) inrnbiomanufacturing are rising from a number of factors. These include improved assays and testing equipment; model risk-basedrnapproaches adapted from other fields and developing specifications from consortiums and standards-setting bodies.
- Track 3: Downstream processing
Track 12: Fermentation Technology, Bioprocess and cell culture
Biberach University of Applied Science, Germany
Biberach University of Applied Science, Germany
Title: A fermentation strategy for industrial application of purple bacteria, based on computational modeling
Time : 12:20-12:50
Hartmut Grammel, male, microbiologist, PhD about antibiotics production with bacteria at the Institute for System Dynamics and Control, University Stuttgart, Germany, He was a founding member of the Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany in 1996. From 2007-2012, he headed a Forsys (research units in systems bioloy) research group funded by the German Federal Ministry of Education and Research (BMBF), Germany. In March 2012, he was appointed as Professor for Industrial Microbiology at Biberach University of Applied Science, Biberach, Germany. His current research focuses in carbon dioxide fixing enzymes from phototrophic bacteria funded by BMBF.
The high potential of anoxygenic photosynthetic bacteria to produce a range of important compounds such as photosynthetic pigments (porphyrines, carotenoids), coenzymes (Q10), biohydrogen and recombinant membrane proteins is well known since many years. Most of these products are associated with intracytoplasmic photosynthetic membranes (PM) which are induced to maximal levels under anaerobic conditions and low light intensities. However, the industrial large-scale application is so far limited by the requirement for light, which inevitably becomes a limiting factor when cell densities become very high. For the purple non-sulfur bacterium Rhodospirillum rubrum, we developed a process for large scale production of PM and associated products completely separated from the availability of light at microaerobic dark conditions in common bioreactors. The cultivation system was successfully employed in an industrial environment for producing phototoxic pigments for photodynamic tumor therapy. A major challenge for process control is that the term microaerobic is not well defined and cannot be reached by conventional oxygen control using amperometric dissolved-oxygen probes. It is therefore particularly useful that R. rubrum is exceptionally well accessible by optical spectroscopy for determination of various cellular redox components. By coupling fibre optic UV/Vis/NIR and fluorescence spectrometers to a bioreactor, in situ online spectroscopy was established for monitoring of the oxygen response of pyridine nucleotides, cytochrome C, photosynthetic complexes, membrane potential as well as cell growth. Different levels of computational modeling such as metabolic network analysis, and kinetic process modeling contributed to the development of a robust fermentation process for semi-aerobic high-level expression of photosynthetic genes and associated products in the dark. To meet a major requirement for industrial fermentation processes, kinetic process modeling was applied for fed-batch high cell density cultivation yielding 60 g/L cell dry weight. To our knowledge this value represents the highest cell concentration achieved with a phototrophic bacterium so far.
Obafemi Awolowo University, Nigeria
Time : 12:50-13:20
Consumer demand has increased for processed products that keep more of their original characteristics. Downstream processing refers to the separation or purification of biological products, generally in marketable quantities especially in the manufacture of pharmaceuticals such as antibiotics, hormones, antibodies, vaccines, industrial enzymes, natural fragrance, flavor compound, etc. There have been various advances in the drying of foods with respect to quality, rehydration, and energy minimization. Freeze-drying is considered a suitable dehydration process for bacteria, with the ultimate goal of achieving a solid and stable final formulation. Spray drying allows preparation of stable and functional powder products and can be applied to stabilize heat sensitive ingredients, such as enzymes and probiotic bacteria. The choice of an appropriate drying medium is very important in the case of LAB, so as to increase their survival rates during dehydration itself and subsequent storage. This paper is a review of drying as a unit operation in downstream processing and discusses the innovative drying technologies, quality of dried foods and deteriorative reactions during drying, drying methods, stages in downstream processing, separation technology in downstream processing and drying of cell materials. The freeze-drying process, pretreatment prior to freeze drying, freeze drying of microorganisms, optimal spray drying of enzymes, eggs and probiotics, effect of droplet size in spray drying as well as single droplet drying methods were considered. The paper concluded that as drying technologies become more diverse and complex, dryer selection has become an increasingly difficult and demanding task as changes in operating conditions of the same dryer can affect the quality of the product.
GE Healthcare, USA
Time : 14:20-14:50
Bill Whitford is Sr. Manager, Cell Culture, GE Healthcare in Logan, UT with over 20 years’ experience in biotechnology product and process development. He joined the company 13 years ago as a team leader in R&D developing products supporting biomass expansion, protein expression and virus secretion in mammalian and invertebrate cell lines. Products he has commercialized include defined and animal product-free hybridoma media, fed-batch supplements, and aqueous lipid dispersions. An invited lecturer at international conferences, Bill has published over 250 articles, book chapters and patents in a number of fields in the bioproduction arena. He now enjoys such industry activities as serving on the editorial advisory board for BioProcess International.
Such new initiatives as integrated continuous bioprocessing are inspiring new intensified manufacturing approaches including high-cell density perfusion culture. These culture formats can change basic culture parameters resulting in altered cycle rates and metabolic demands by the cultured cells. This can place unique compositional, temporal or physicochemical demands upon the culture media. Furthermore, there are a number of distinct high cell density (or concentrated) perfusion-based processes in development or current use. Modern development strategies must consider perfusion-unique phenomena as providing distinct process-specific demands for primary metabolites/growth factors, volume/schedule/storage, materials costs and definitions of performance. Tools to accomplish this include such modern equipment as mini-bioreactors, techniques as metabolic flux analysis and development algorithms (such as DoE). Finally, it is becoming clear that these new approaches to bio-manufacturing require a new look at not only process control strategies, but the implications of such historical culture quality-surrogate monitoring values as cell division and glucose or oxygen uptake rates.
Federal University of São João del-Rei, Brazil
Time : 14:50-15:20
Daniel Bonoto Gonçalves did a BS in Biochemistry from Federal University of Viçosa, Master in Agricultural Microbiology from the same university and PhD in Microbiology by Federal University of Minas Gerais. He has experience in Industrial Microbiology and Fermentation with emphasis on Optimization of Fermentation Processes and Production of Industrial Enzymes; in Genetics, with emphasis on Molecular Genetics of Filamentous Fungi. He is currently Professor at the Federal University of Sao Joao del Rei, Campus Midwest Dona Lindu, and Deputy-Coordinator of the Graduate Program in Biotechnology.
Brazilian technology of ethanol production from sugarcane is one of the most efficient and well-established of the world and the use of lower cost substrates, such as lignocellulosic byproducts, could result in a more competitive renewable fuel. In this context, the palm press cake generated by biodiesel production process may be a new feedstock for second-generation ethanol production. Our group has been study the bioconversion process of D-xylose to ethanol and xylitol by yeast isolated from the macaúba (Acrocomia aculeata) fruit. Macaúba press cake provided from biodiesel production process was used for hemicellulosic hydrolysates obtaining using acid, hydrothermal and hydrothermal followed by acid pretreatments, which were evaluated and analyzed for sugars, acids and inhibitory compounds. The hydrolysate obtained by acid treatment revealed higher sugar and lower furfural, 5-hydroxymethylfurfural and acetic acid contents. This material was used to study the effect of hydrolysate supplementation and concentration in the Candida boidinii UFMG14 fermentation, while the solid biomass was used to obtain a cellulosic hydrolysate and ferment (SSF) in a mixture experiment. The supplemented hydrolysate containing D-xylose 25 g/L showed the highest ethanol production (12 g/L), YE/S (0.40 g/g) and QE (0.33 g/Lh) compared to hemicellulose hydrolysates supplemented with 10 g/L. The mixture experiment indicated a higher ethanol production when 100% cellulosic hydrolysate was used for fermentation, compared with hydrolysates combination. This research represents the first step in integrating production technologies of the two most important Brazilian renewable energy matrices, ethanol and biodiesel.
Merck & Co, USA
Title: Optimization of high-cell density cultivation to produce monoclonal antibody in glycoengineered Pichia pastoris by real-time monitoring of glycerol and methanol
Time : 15:40-16:10
Sehoon Kim had completed his Ph.D from Chemical Engineering Department, Texas A&M University and postdoctoral studies from University of Texas, MD Anderson Cancer Center. He is an Associate Principal Scientist, Merck & Co., Inc. He has published more than 25 articles combined in both peer-reviewed journals and patents.
Monoclonal antibodies with humanized N-glycosylation have been successfully and highly expressed in glycoengineered Pichia pastoris using high-cell density fed-batch fermentations. High titer and production consistency require tight control of glycerol and methanol as primary carbon source and inducer, respectively, in fed-batch culture of P. pastoris. In this study, glycerol, methanol, and cell density were monitored in real-time for multiple fermentations using a new on-line NIR (near infrared) monitoring system. The real-time measurements provided accurate concentration profiles and kinetic data for process characterization and optimization throughout the entire 52-week study, with no recalibration or user expertise required. Critical operation variables available in real-time include the time of methanol dose, peak methanol concentration, time of methanol depletion, and volumetric methanol consumption rate. Under oxygen-limited fed-batch conditions, the methanol dosage level for these high cell density cultures was optimized at 10 gL-1, with a 30 mmolL-1h-1 oxygen uptake rate (OUR) controlled by adjustment of agitation speed (rpm) and aeration rate (vvm). The real-time kinetic data, including volumetric consumption rates (mmolL-1h-1) of glycerol and methanol, allowed monitoring and control of cell metabolism at various phases of cell growth and induction, thereby improving quality control and culture consistency of industrial bioprocesses for both developmental and scale-up purposes.
Dr.Osama Ibrahim is a highly-experienced Principal Research Scientist with particular expertise in the field of microbiology, molecular biology, food safety, and bioprocessing for both pharmaceutical and food ingredients. He is knowledgeable in microbial screening /culture improvement; molecular biology and fermentation research for antibiotics, enzymes, therapeutic proteins, organic acids and food flavors; Biochemistry for metabolic pathways and enzymes kinetics, enzymes immobilization, bioconversion, and Analytical Biochemistry. Dr. Ibrahim was external research liaison for Kraft Foods with Universities for research projects related to molecular biology and microbial screening and holds three bioprocessing patents. In January 2005, he accepted an early retirement offer from Kraft Foods and in the same year he formed his own biotechnology company providing technical and marketing consultation for new startup biotechnology and food companies. Dr. Ibrahim received his B.S. in Biochemistry with honor and two M.S. degrees in Microbial physiology/ Fermentation and in Applied Microbiology. He received his Ph.D in Basic Medical Science (Microbiology, Immunology and Molecular biology) from New York Medical College. His research dissertation was on the construction of plasmid for the expression of a fusion protein of VEGF121/ Shiga-like toxin as a therapeutic protein for targeting angiogenesis (cancer treatment). Since 1979 he is a member of American Chemical Society, American Society of Microbiology, and Society of Industrial Microbiology.
The original definition of fermentation is the anaerobic conversion of sugars to ethanol and carbon dioxide by yeast. This original definition has been expanded over time to the conversion of organic materials by multiple diversities of organisms (bacteria, yeasts, molds, animal cells, or plant cells) under anaerobic or aerobic conditions into wide ranges of molecules types different In general, fermentation can be divided into four types that are not necessary disjoint from each other: • Production of biomass (viable cellular materials). • Production of extracellular metabolites ( chemical compounds) • Production of intracellular components (proteins). • Transformation of substrates into bio products The key elements of fermentation industry are strain microbial/cells selection, media composition, and conditions optimization Microbial fermentation will be highlighted in this presentation that includes microbial enzymes, metabolic pathways and fermentation process.
Technical University of Denmark, Denmark
Title: Synthetic-natural polyblend nano-micro structured scaffolds for bladder tissue engineering applications
Time : 16:40-17:10
Fatemeh Ajalloueian is a Post-Doc Researcher at Technical University of Denmark (DTU). Her background is science and engineering of polymeric fibers, where she started with fabrication and characterization of nano-micro structured scaffolds and is continuing her research in tissue engineering and regenerative medicine. She has been awarded grants from Danish Council for Independent Research (DFF) and Research Talent (Sapere Aude) to develop nanodelivery systems for urine bladder tissue engineering in a collaboration between DTU, Uppsala University and Karolinska Institute (Sweden). She has published papers in high-ranked journals, and is Reviewer and serving as an Editorial Board Member in reputed journals.
Tissue engineering as a means of functional tissue fabrication or repair requires three-dimensional (3D) scaff olds serving as structural base (matrix) for cell attachment and proliferation. Th us far, both synthetic biodegradable materials such as PLA (polylactic acid), PLGA (Poly(Lactic-Co-Glycolic Acid)), and PCL (Polycaprolactone) and naturally derived polymers such as collagen, chitosan, fi brin and gelatin have been well used in tissue engineering and regeneration. Combination of synthetic and natural biomaterials for experimental investigations involving cells has attracted great deal of attention recently. Such a blend is expected to include the increased strength and durability of the synthetic polymer and the specifi c cell affi nity of the natural one. We fabricated two diff erent polyblend constructs (PCL-collagen and PLGA-collagen) to study their potential for bladder regeneration applications. Both constructs had fi brous stuructures similar to native ECM and the appliled fabrication methods were combination of knitted or electrospun sheet with Plastic Compression of collagen hydrogel (PC-coll). Th e constructs were evaluated by seeding of minced bladder mucosa, followed by in vitro proliferation. It was observed that cells migrated from the minced tissue particles and reorganized on the scaff olds. Th e attachment, viability and proliferation of migrated cells were studied using techniques of microscopy, MTT assay and histological analyses. It was observed that hybrid constructs were more hydrophylic and supported higher rates of cell proliferation compared to the scaff olds made from the syntethic polymer only. Moreover, they demonstrated better dimensional stability and mechanical strength compared to the PC-coll scaff old.