Engineering Commons^™

Proof That Can Travel - Documented Clonality Report For Regulatory Submission, Ian Taylor, Paul Miller

Cell Culture Engineering XV

Clonality is a key element of cell line development and is an important component of a regulatory submission. Indeed for BLA, the clonality of MCB is mandatory. Historically, the regulator has insisted upon 2 rounds of cloning for developing a cell line and assurance of monoclonality based on statistical outgrowth measurements. Recent improvements in high resolution whole well imaging of cells in microplates, enables the creation of indisputable image-based evidence for the growth of a colony from a single cell. This evidence eliminates a round of sub-cloning and results in several months of cell line development time being saved. However, …

An Integrated Cell Line Development Platform For Generation Of High Yielding Cho Stable Cell Lines Expressing A Stabilized Trimeric Pre-Fusion Rsv F Recombinant Viral Glycoprotein, Richard Schwartz, Amritha Menon, Mingzhong Chen, Giulia Fabozzi, Althaf Hussain

Cell Culture Engineering XV

Accelerating timelines to deliver stable cell lines with high productivity is challenging, especially for biologics portfolios which include complex recombinant vaccine constructs. The VRC’s current integrated approach to shorten CHO cell line development timeline utilizes host cells pre-adapted to production medium, optimization of the expression vectors, improved pre and post-transfection methodology, automated ClonePix^TM technology and top clone selection and ranking in micro-scale ambr bioreactor technology. This platform was developed with monoclonal antibodies as model proteins. While this integrated platform enabled generation of high yielding clones expressing monoclonal antibodies, it was initially much less successful with a difficult to express …

Overcoming Process Intensification Challenges To Deliver A Manufacturable And Competitive Integrated Continuous Biomanufacturing Platform, Jason Walther, Neha Shah, Myles Hollenbach, Jonathan Wang, Marcella Yu, Jiuyi Lu, Yang Yang, Konstantin Konstantinov, Chris Hwang

Cell Culture Engineering XV

Groups in both industry and academia have achieved high densities and productivities in perfusion cell culture processes. At Sanofi, we have demonstrated perfusion densities greater than 100 million cells/mL (with associated high productivities) at a cell-specific perfusion rate of only 20 pL/cell/day. This process intensification reduces the footprint of upstream unit operations as well as capital and operating expenses of manufacturing facilities. The continuous nature of perfusion cell culture also creates opportunities for integration of continuous downstream operations, leading to further process intensifications and volume reductions.

In this presentation, we will discuss our work on several upstream challenges that must …

Cre-Loxp-Controlled Cell-Cycle Checkpoint Engineering In Chinese Hamster Ovary Cells, Takeshi Omasa, Rima Matsuyama, Tomomi Tsusui, Noriko Tamano, Masayoshi Onitsuka, Kyoung Lee

Cell Culture Engineering XV

The gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the productive cell line construction of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering¹⁾. Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system²⁾. A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by …

Generation Of Desirable Cho Cell Factories With Predictive Culture Performance Using Crispr/Cas9-Mediated Genome Engineering, Helene Faustrup

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus, genome engineering of CHO cells for improved product quality and yield is of great interest. Here, I will demonstrate our latest advances in improving the efficiency of CRISPR/Cas9-mediated genome engineering to generate attractive knockout and knockin CHO cell lines. Analysis of the dynamics and efficiency of the technology will be demonstrated for genes involved in glycosylation and apoptosis. Combined with multiplexing and fluorescent enrichment, application of CRISPR/Cas9 genome editing facilitated disruption of several genes simultaneously and accelerated …

A Novel Platform For High Throughput Cell Line Screening & Development, Maria Wendt, Christoph Freiberg, Cristopher Smith, Hans Fischer

Cell Culture Engineering XV

The provision of stably expressing, high-yield mammalian cell lines is a key step in bioprocess development. Recent progress in clone screening automation, and the increased use of mini-bioreactors, such as ambr^®, have resulted in higher experimental throughput and a significant increase in data that needs to be handled and interpreted.

Co-developed in close collaboration with leading biopharmaceutical companies, we have implemented a dedicated cell line development platform for fully automating the clone line selection and assessment process to increase process efficiency and quality. The new platform supports the entire cell line development workflow including seeding, selection, incubation, passaging, …

Glycoengineering Of Chinese Hamster Ovary Cell For Modulating Glycoprotein Nlinked Sialylation, Chengyu Chung

Cell Culture Engineering XV

Erythropoietin (EPO), a highly glycosylated protein was widely used in anemia of kidney failure. It has been confirmed that enhancing sialic content on EPO can prolong its circulatory half-life, improving EPO therapeutic efficacy. On the other hand, limiting EPO sialylation can be beneficial in medical and diagnostic application. EPO has been evaluated as a potential radiolabeled diagnostic candidate. Thus, the development of genetic engineering strategies to finetune the sialic acid content on EPO as well other protein therapeutics is needed.

To enhance sialylation, we focused on engineering Chinese Hamster Ovary cell (CHO) to overexpress Beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1). Combinantorial overexpression …

Insight Into Single Cell Cloning In Serum-Free Media, Tsuyoshi Yamaguchi, Yumi Sugita, Mie Fukuda, Keina Yamaguchi, Yuka Mashio, Koichi Yamamoto, Toshiyuki Suzawa

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells have been used as host cells for the manufacturing of therapeutic recombinant proteins over the past decade. It is thought that the development of high performance cell lines, which satisfy both productivity and regulatory expectations, is one of the key success drivers to establish good manufacturing processes. The cell line for the clinical and commercial productions should be derived from a single progenitor or clone, and so the single cell cloning is an essential step during the cell line development. Recently serum-free media have been widely applied for this step. But under such conditions, the …

Sub-Physiological Culture Temperature Boosts Expression Levels Of Membrane Proteins In Cho Cells, Sampath Kumar, Lihui Zhong, Beata Bobrowicz, Juergen Nett

Cell Culture Engineering XV

Membrane proteins especially G-protein coupled receptors (GPCR) are the targets of a large number of small molecule therapeutics. With the advances in biopharmaceutical therapeutic approach, development of antibodies against the membrane proteins are increasingly attempted. Generation of therapeutic antibodies against these targets is a major challenge due to the limited accessibility of these proteins in vivo. Stable clonal cell lines expressing the membrane proteins are used as antigen-presenting cells for antibody development in one of two ways: whole-cell based selection including complexing with B cells, or immunization. They also serve as valuable reagents in characterizing the binding properties of …

Targeted Integration Of Multiple Active Sites In Cho Genome For Rapid Generation Of Stable And High Monoclonal Antibody Producing Cell Lines, Yuansheng Yang

Cell Culture Engineering XV

Mammalian cell lines used for manufacturing recombinant therapeutic proteins need to have high productivity, long term stable production and good product quality. The process of cell line development currently used in industry is based on random integration of the plasmid vectors into genome. Due to clonal variation, hundreds to thousands of clones are screened through multiple stages of evaluation. The whole process takes at least a few months and is extremely tedious. Targeted integration-based cell line development is attractive as the plasmid vectors integrate into predetermined active sites in genomes. All targeted cell lines have same genetic background and thus …

Bridging The Gap Of Screening And Scale Up In Cho, Hybridoma, Hek293 And Other Cell Lines: Single Use Optimum Growth Flasks 30ml-5l Flasks With Transfer Caps, And Ports, Sam Ellis

Cell Culture Engineering XV

Optimum Growth Flasks^TM (patented) are a break through design to shake flasks, and the first in 100+ years. By using Optimum Growth Flasks^TM users are able to grow 15mLs-3L of Cell Culture with the same scalability. The Optimum Growth Flasks^TM have replaced disposable Fernbach Flasks and also small Wave^® Bags (5L & 10L) in the larger sizes as production vessels. The Optimum Growth Flasks ^TM also give high viability cultures with a great use of space as shown by our data in CHO, Hybridoma, HEK293 and other Cell Lines. We have made a series of Transfer …

Cho-K1 Host Cell Engineering Strategy Enabling The Establishment Of Strains Producing Higher Yields Of Recycling Antibodies, Hisahiro Tabuchi, Ikumi Murakami, Masaki Yamaguchi

Cell Culture Engineering XV

As described elsewhere (Biotechnol Bioeng 2010, 2013), our DXB11 (dhfr^–) host cell engineering strategy achieved high cell viability for a prolonged period (more than 1 month) and enhanced mAb productivity (>100 pg/cell/day) by nutritional control. Introduction of taurine transporter (TAUT) into DXB11 parent cells increased glutamine uptake and accelerated glutathione metabolism. By forcing the overexpression of TAUT, we were able to control DXB11 host cell functions and thereby increase the monoclonal antibody (mAb) titer up to 8.1 g/L/31 days under conventional 1-L bioreactor fed-batch conditions. Furthermore, the mAb produced by the DXB11/TAUT cells was comparable in quality …

Challenges Developing Biologics For The Prevention And Treatment Of Infectious Diseases Impacting Global Health, Steven Hadley

Cell Culture Engineering XV

Biologics have enjoyed tremendous success as products in high income countries of the world. However the high cost of these products today prevent their use in low income countries and with the notable exception of palivizumab there has been little commercial interest in developing biologics for infectious diseases that are prevalent in these geographies. Recent advances in the study of HIV, malaria, pertussis and Ebola highlight the potential of monoclonal antibodies (mAb) for prevention or treatment of these diseases. The evolution of the industrialized CHO process over the past 30 years to satisfy market demands for mAbs is remarkable. The …

Varied Productivity According To The Differences Between Targeted Locations Of Antibody Expression Vectors In Chinese Hamster Ovary Cells, Noriko Yamano, Jana Frank, Masayoshi Onitsuka, Kota Yoshitomi

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cell lines are widely used in the pharmaceutical industry to produce therapeutic antibodies. However, trial and error cell selection methods are still used to construct high-producing cell lines. Exogenous genes are predicted to express differently depending on the expression vector integration sites. Chromosomal instability is one of the characteristics in CHO cells. We have previously constructed the CHO genomic bacterial artificial chromosome (BAC) library that is expected to cover entire the CHO-DG44 genome (Omasa et al., Biotechnol. Bioeng., 104, 986-994, 2009). The BAC-based physical map is a powerful tool to identify each chromosome and analyze …

The Renaissance Of Cancer Immunotherapy Is A Revolution For Patients, Ira Mellman

Cell Culture Engineering XV

Over just the past few years, cancer immunotherapy has transformed how we think about cancer care and cancer biology. This transformation is remarkable not only for its rapidity but also because cancer immunology itself had received little serious consideration for decades. At least three factors have been responsible: (i) the advent of actual data from the clinic as opposed to promising ideas from the laboratory; (ii) a more sophisticated understanding of the human immune response to cancer; and (iii) the realization that despite many years of effort targeting the oncogenic drivers of cancer, for the vast majority of patients the …

Design And Testing Of Sorbents For Co2 Separation Of Post-Combustion And Natural Gas Sweetening Applications, Jiajun He, John To, Peter Psarras, Jianguo Mei, Jen Wilcox

CO2 Summit II: Technologies and Opportunities

In post-combustion processes, sorbents with both high capacity and selectivity are required for reducing the cost of carbon capture. Although physisorbents have the advantage of low energy consumption for regeneration, it remains a challenge to obtain both high capacity and sufficient CO₂/N₂ selectivity at the same time. A novel N-doped hierarchical carbon has been developed, which exhibits record-high Henry’s law CO₂/N₂ selectivity among physisorptive carbons while having a high CO₂ adsorption capacity. Specifically, the synthesis involves the rational design of a modified pyrrole molecule that can co-assemble with the soft Pluronic template via …

Techno-Economic Challenges Associated With Biomass Energy Utilization And Ccs, Carl Bozzuto

CO2 Summit II: Technologies and Opportunities

Despite the recent agreement by world leaders regarding the need for reductions in CO2 emissions from the global energy system, there will still be substantial amounts of fossil fuels utilized for the next few decades. According to IEA estimates, there will likely be an “overshoot” of the CO₂ concentration that is estimated to be needed to restrain average global temperatures increases in the range of 1.5–2˚C. As a result, biomass energy has been proposed as one means to reduce net CO₂ emissions and, perhaps, even reduce the level of CO₂ in the atmosphere. One approach has been …

Evaluation Of Physical Adsorbents For Post-Combustion Co2 Capture, Youssef Belmabkhout

CO2 Summit II: Technologies and Opportunities

CO₂ capture using physical adsorbents such as activated carbon, zeolites, and metal-organic frameworks (MOFs) have attracted a lot of attention because of the technical capability of adsorption technology using such separation agents to retrofit the energy demanding liquid amine scrubbing process. Nevertheless, this will be conceivable only if the separation agents (adsorbents) in question fulfil many unambiguous attributes in terms of porosity, CO₂ affinity, kinetics, energetics, stability, throughout the right capture mechanism, in addition to the adsorbents cost.

In this paper we report recent study about the evaluation of physical adsorbents for CO₂ capture from simulated flue …

Metallic Membranes For N2 Separation & Post-Combustion Co2 Capture Improvement, Simona Liguori, Kyoungjin Lee, Jennifer Wilcox

CO2 Summit II: Technologies and Opportunities

Carbon (CO₂) capture represents an important role in the reduction greenhouse gas emissions. Among various CO₂ capture technologies currently investigated, post-combustion capture allows for the retrofitting of existing plants and industrial units. Today, the amine scrubbing is considered the most competitive method for CO₂ removal in the flue gases from power plants in comparison to other technologies. Nevertheless, recent work has shown that the energy requirement for solvent recovery can decrease the overall efficiency of the power plants up to 16%¹. Moreover, additional costs may occur in the solvent absorption technology because of solvent …

Co2 Capture Using Nanoporous Tio(Oh)2/Tetraethylpentamine, Maryam Irani, Maohong Fan, Khaled Gasema

CO2 Summit II: Technologies and Opportunities

In this work, an inorganic-organic CO2 sorbent was prepared by immobilizing tetraethylenepentamine (TEPA) onto nanoporous titanium oxyhydrate (TiO(OH)2). The prepared sorbents were characterized using X-ray diffraction, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and Brunauer-Emmett-Teller (BET) analysis. At the optimal TEPA loading of 60 wt.% on TiO(OH)2, its CO2 sorption capacity reached 3.1 mmol CO2/g-sorbent for 1 vol.% CO2 in N2 along with ~1 vol.% H2O at 60°C. Studies of adsorption kinetics and thermodynamics showed that the activation energies for CO2 adsorption and desorption of TiO(OH)2/TEPA are 38.31 kJ/mol and 44.51 kJ/mol, respectively. Its low …

Nitrogen-Functionalized Porous Carbons For Enhanced Co2 Capture, Peter Psarras, Jennifer Wilcox

CO2 Summit II: Technologies and Opportunities

The global consequences of increased anthropogenic carbon dioxide emissions are well documented. To avoid the catastrophic long-term damages associated with climate change, much attention has been devoted to the development of CO₂ emission mitigation strategies. A present challenge in the mitigation of anthropogenic CO₂ emissions involves the design of less energy- and water-intensive capture technologies. Traditional capture methods, e.g. MEA solvent scrubbing, have prohibitive regeneration costs, and recent concerns in drought-ridden states like California have brought these water-intensive processes under increased scrutiny. Sorbent-based capture represents a promising solution to the aforementioned hurdles, as they do not incur the …

Direct Air Capture Versus Post Combustion Capture For Coal Fired Power Plants: Energy Balance And Life Cycle Environmental Assessment, Christoph Meinrenken

CO2 Summit II: Technologies and Opportunities

Direct air capture (DAC) has been suggested as a possible alternative to post combustion capture (PCC), as a way to lower the climate impact of fossil-based electricity generation. However, various literature suggests that the much lower concentration of CO2 in ambient air (compared to flue gas) will cause DAC to require ~3x higher energy consumption than PCC per ton CO2 captured, in turn leading to impractically high parasitic loads, costs, and potentially other adverse environmental impacts of DAC for purposes of mitigating emissions from e.g. coal-fired power plants. Here we present a comparative techno-economic and life cycle assessment (LCA) of …

Optimal Molecular Design Of Poly (Ionic Liquids) For Co2 Capture From The Atmosphere, Kun Ge, Tao Wang

CO2 Summit II: Technologies and Opportunities

Polymeric ionic liquids (PILs) are promising CO₂ adsorption materials for that they combine the unique properties of ionic liquids with the flexibility and properties of macromolecular architectures. Previous studies have demonstrated that a carbonated-functionalized quaternary ammonium based PIL could adsorb CO₂ from atmosphere when dry and release CO2 when wet, which constituted a moisture swing cycle, which provides an economical approach for air capture. Moreover, the performance of the PIL (such as CO₂ affinity, reaction kinetics, swing size and hydrophobicity), could be improved owing to its fine tunability. To find out its optimal structure, molecular simulation was …

Pathway To Achieve Negative Co2 Emissions - Combining Biomass With Ccs, Antti Arasto, Janne Karki, Kristin Onarheim

CO2 Summit II: Technologies and Opportunities

The urgency to stabilize the global temperature rise at 1.5°C as highlighted in Paris COP21 and the IPCC Fifth Assessment Report calls for solutions that can remove CO2 from the atmosphere. The ability of carbon negative processes to offset historic emissions and emissions across different sectors is also highlighted in recent scenarios by IEA and WEC. Achieving negative CO₂ emissions by removing CO₂ from the atmosphere is possible by applying carbon capture in processes utilizing biomass (bio-CCS). Biomass has the capability of withdrawing and storing atmospheric CO₂. As a result, CO₂ released during biomass thermo-chemical …

Co2 Mitigation Opportunities In China, Wayne Xu

CO2 Summit II: Technologies and Opportunities

Over the past thirty years China has focused on urbanization and economic growth through rapid development and modernization of its manufacturing capability. Much of this growth was achieved though massive adoption of coal based power generation with industry and manufacturing accounting for over 70% of all electricity consumption. Although the use of coal and other hydrocarbons have successfully fueled China into an era of prosperity and global recognition, it has come at a cost to the environment and quality of life. Significant efforts to reduce coal based generation emissions have recently been achieved through the development and adoption of world …

Carboncycle And Other Profitable Strategies For Air Capture Of Co2, Deane Little

CO2 Summit II: Technologies and Opportunities

Development of an effective and economically viable strategy for air capture of CO2 would be a game-changing development in the fight against global warming. If widely adopted such a process would allow “chemostatting” the atmosphere at sustainable CO2 levels and might not require widespread social change in how we use fossil fuels. Unfortunately the concept that air capture of CO2 “doesn’t work” has taken hold in academic and policy circles, based on the assumption that such a process is inherently expensive and results in no useful products. In fact, air capture of CO2 can be profitable and offers fundamental advantages …

Metal Oxides With Ionic-Electronic Conductivity For Thermochemical Energy Storage, Eric Coker, Sean Babiniec, Andrea Ambrosini, James Miller

CO2 Summit II: Technologies and Opportunities

One of the key approaches to developing a supply of renewable energy is Concentrating Solar Power (CSP). However, the diurnal and intermittent nature of the solar resource necessitates efficient energy storage solutions if a reliable energy supply is to be expected. By increasing the temperature and energy density of thermal storage, improvements in power cycle efficiency and energy storage cost are possible and can result in increased economic competitiveness. Thermochemical energy storage (TCES) holds promise for enabling this goal. Metal oxides (MOs), with their elegantly simple reduction/re-oxidation (redox) chemistry, approach an ideal medium for TCES in many respects. For instance, …

Kinetic Enhancement Of Adsorbent For Co2 Capture From Atmosphere By Porous Material, Tao Wang, Jun Liu, Kun Ge, Zhongyang Luo

CO2 Summit II: Technologies and Opportunities

Strategies for stabilizing atmospheric greenhouse gas concentrations will need to consider future CO2 emissions from an enormous resource of worldwide fossil fuel supplies and a diverse range of mitigation technologies. In 2013, global CO2 emissions due to fossil fuel use (and cement production) were 36 gigatonnes (Gt CO2), and are projected to increase by an additional 2.5% in 2014. Even if all emissions from large fixed sources could be captured, the roughly 30-50% of global emissions due to transportation and mobile sources would still be released into the atmosphere. To ensure the concentration of atmospheric CO2 in the scope of …

Factors That Impact The Co2 Mitigation Potential Of Cogeneration, Carl Bozzuto

CO2 Summit II: Technologies and Opportunities

Cogeneration systems that produce both heat and power are being promoted as a means of improving the overall energy efficiency around the world, especially in the industrial and power sectors. Claims are often made regarding dramatic improvements in efficiency for such systems. For example, a cogeneration system is advertised as being 85% efficient, while, for example, the U.S. grid is only 31% efficient. However, comparing such efficiency figures directly can be misleading. An 85% efficient cogeneration system is being evaluated at full load and on a first law of thermodynamics basis (i.e., input = output at steady state). The average …

Zerronox Corporation: Using Pulsed Electron Beams For The Removal Of Carbon Dioxide, Nitrogen Oxides And Other Emissions From Power Plants, Stephen Kennedy

CO2 Summit II: Technologies and Opportunities

Zerronox Corporation is a technology company addressing the market for emission abatement from fossil fuel power plants, specifically coal, natural gas, oil and diesel. Our mission is to commercialize the patented pulsed electron beam (PEB) technology for the cost effective removal of carbon dioxide, nitrogen oxides (NOx), and other exhaust stream pollutants such as sulfur oxides and mercury. The core technology was developed by the Naval Research Laboratory over the past fifteen years with a total investment from the DOE of more than $150M. Consequently, the developed technology is industrial scale in scope and has been tested for durability.

The …