Engineering Commons^™

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 …

P2x7r-Panx1 Complex Impairs Bone Mechanosignaling Under High Glucose Levels Associated With Type-1 Diabetes, Zeynep Seref-Ferlengez, Stephanie Maung, Mitchell B. Schaffler, David C. Spray, Sylvia O. Suadicani, Mia M. Thi

Publications and Research

Type 1 diabetes (T1D) causes a range of skeletal problems, including reduced bone density and increased risk for bone fractures. However, mechanisms underlying skeletal complications in diabetes are still not well understood.We hypothesize that high glucose levels in T1D alters expression and function of purinergic receptors (P2Rs) and pannexin 1 (Panx1) channels, and thereby impairs ATP signaling that is essential for proper bone response to mechanical loading and maintenance of skeletal integrity. We first established a key role for P2X7 receptor-Panx1 in osteocyte mechanosignaling by showing that these proteins are co-expressed to provide a major pathway for flow-induced ATP release. …

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 …

Agricultural Field Robotics For Plant Data Acquisition, Jeremy S. Blackford, Jared Werner, Tyler A. Troyer, Ethan Nutter

UCARE: Research Products

As the demand for food increases, we are presented with the challenge of producing food more efficiently. With the help of agricultural robots it will be possible to achieve greater yields by the application of seeds, fertilizers and chemicals in the most efficient way possible. With more advanced robotic systems accurate crop data can be obtained to improve farming products and techniques.

Flex-Row is a medium sized agricultural robotic platform built for autonomously traversing through rough fields during multiple crop growing stages. This platform consisting of a flexible frame with the ability to vary both width and height will initially …

The Role Of Intrinsic Foot Muscles In Three Running Footwear Conditions, Brian T. Groener

Senior Honors Projects, 2010-2019

Running has grown tremendously in popularity and so has running with minimalist shoes. Injuries such as plantar fasciitis (pain and inflammation of a thick band of tissue that runs across the bottom of the foot) are prevalent in runners despite efforts to design footwear to alleviate the impact of running and to reduce the number of injuries. In the past decade, minimalist running shoes have received considerable attention, causing debate amongst runners and scientists as to their utility in injury prevention. While running barefoot or in minimalist shoes reduces initial impact forces, the claim that they lower injury rates remains …

Orthopedic Drill System With Screw Length Measurement And Heat Sink System, Hunter Wilcox Rafoth, John Patrick Metz

Senior Theses

Most orthopedic drills currently on the market are rudimentary tools that serve only one purpose: to drill holes through bone. Biosphinx envisions a more versatile orthopedic drill that could serve multiple purposes in the operating room. This report outlines Biosphinx’s solution to design a new orthopedic drill system that would include several key features to improve bone screw length measurement, increase patient safety, and reduce time spent in the operating room.

Next Generation Electrode, Joseph Daniel Bivona, Samuel Scott Dacus, Mary Harrell, James Frederick Weber Pike

Senior Theses

This senior thesis involves the results of a biomedical engineering senior design project where the group worked with Rhythmlink International LLC. The project involved the improvement of the manufacturing process of the Invisa Electrode, a CT compatible electrode sold and manufactured by Rhythmlink. The design of the electrode pin was altered to have a hollow center, where the lead wire can be inserted and heat welded, eliminating two key components of the original design and reducing production time and cost. The type of plastic used in the electrodes was also altered to be a 40% carbon loaded conductive plastic.

Dr. Hadi Soroosh Research Presentation | Biological Engineering, Usu College Of Engineering

College of Engineering News

Rise of human population causes an increase of freshwater use and wastewater production worldwide. Wastewater contains various pathogens injurious to health and nutrients (mainly N and P) which would support photosynthesis of chlorophytes and terrestrial plants but can be also an ecological threat to the environment. The conventional wastewater treatment plants (WWTP) require a large amount of energy (for mechanical aeration) and causes emission of CO2 and secondary contamination (by use of flocculants). Microalgae-Bacteria (MaB) biocoenosis can improve the capacity of the treatment system by simultaneous removal of the nutrients and carbon from wastewater with minimum carbon footprint and energy …

Stabilizing The Cold Plasma-Stimulated Medium By Regulating Medium’S Composition, Dayun Yan, Niki Nourmohammadi, Ka Bian, Ferid Murad, Jonathan H. Sherman, Michael Keidar

Biochemistry and Molecular Medicine Faculty Publications

Over past several years, the cold plasma-stimulated medium (PSM) has shown its remarkable anti-cancer capacity in par with the direct cold plasma irradiation on cancer cells or tumor tissues. Independent of the cold plasma device, PSM has noticeable advantage of being a flexible platform in cancer treatment. Currently, the largest disadvantage of PSM is its degradation during the storage over a wide temperature range. So far, to stabilize PSM, it must be remained frozen at −80 °C. In this study, we first reveal that the degradation of PSM is mainly due to the reaction between the reactive species and specific …

Interaction Of Fibrinogen With Fibronectin: Purification And Characterization Of A Room Temperature-Stable Fibrinogen-Fibronectin Complex From Normal Human Plasma, Ayman E. Ismail

Department of Chemical and Biomolecular Engineering: Dissertations, Theses, and Student Research

A fibrinogen-fibronectin complex (γγ’pdFI-pdFN) was purified from normal human plasma using a sequence of cryoprecipitation, ammonium sulfate fractionation, and DEAE Sepharose chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition showed both a 1:1 stoichiometric ratio of fibrinogen (FI) to fibronectin (FN) as well as a stoichiometric ratio of 1:1 of γg to gγ’. The γγ’pdFI-pdFN complex was non-covalent in nature as it was disrupted by affinity adsorption to Gelatin Sepharose where pdFN bound strongly and the disrupted γγ’pdFI fell through the chromatographic column. Surprisingly, the purified γγ’pdFI-pdFN complex was more broadly thermally stable than plasma FI (pdFI) …

Localized Immunotherapy Delivery Using Injectable In Situ Forming Chitosan Hydrogel, Seth Washispack

Biomedical Engineering Undergraduate Honors Theses

Cytokine-based cancer immunotherapies stimulate a host’s immune system to fight cancer. In particular, interleukin-12 (IL-12), a potent pro-inflammatory cytokine, has demonstrated the ability to eliminate tumors in a number of preclinical models. Toxicities associated with the systemic delivery of IL-12 have precluded its use in the clinic. We are developing a novel chitosan-based hydrogel to maintain high local concentrations of cytokines, such as IL-12, in the tumor while minimizing its systemic dissemination. This hydrogel was found to form spontaneously within ten seconds of mixing two proprietary components. To increase the usefulness of the hydrogel, an efficient mixing and delivery system …

Chitosan As An Antiviral, Tayler Pauls

Biomedical Engineering Undergraduate Honors Theses

There is no broad-based antiviral medication available today; there are specific antivirals, for example, the antiretroviral for HIV. However, these specific antivirals are not available in each country and can be problematic for specific patients. Chitosan is proposed as a possible broad-based antiviral, which has already demonstrated antibacterial properties, antiviral properties in plants, is used for wound healing and as a hydrogel among other medical applications. The methods used are transfection of NIH-3T3 cells with GFP-adenovirus with 0.1%, 0.5%, and 1% chitosan added to virus prior to transfection. Fluorescence microscopy and flow cytometry data has validated that the use of …

Determining The Effect Of Delivery Rate On Glucose Uptake By Cancer Cells, Luke Felton

Biomedical Engineering Undergraduate Honors Theses

Radiation therapy is one of the most commonly performed cancer treatment therapies today. Radiation therapy can cause can changes to tumor vasculature and affect blood flow rates. It is important to determine how changes in blood flow rates affect tumor glycolytic uptake to better understand tumor response to radiation therapy (1). The goal of this study was to use a fluorescent analog of glucose, 2-NBDG, to study the effect of varying flow rates on glucose uptake. A microfluidic device was constructed to flow 2-NBDG through. A murine metastatic breast cancer cell line was then cultured inside this microfluidic channel, and …

An Actomyosin Driven, Pressure Based Adhesion Model Of Confined Glioblastoma Multiforme Cell Migration, Jamie Wright

Bioengineering Dissertations - Archive

Glioblastoma multiforme (GBM) is the most aggressive and common form of glial tumors arising in the support cells of the brain. Cell migration plays a critical role in undermining the surgical resection of tumors. Peripheral cells freely detach and invade the surrounding tissue, preferentially collecting and migrating within confined, mechanically distinct environments such as along the borders of white matter tracts and vasculature. These tracks have been mimicked experimentally using small, rectangular PDMS micro-channels. GBM cells will readily enter and migrate through these micro-channels in the absence of any chemoattractant, even without specific cell-substrate adhesion (integrins) if channel dimensions decrease …

Interactive Analytical Tool For Quantitative Corneal Confocal Imaging In Vivo, Madhavi Tippani

Bioengineering Theses - Archive

The objective of this study was to develop an interactive software application for image reconstruction and analysis of the corneal data from an in vivo confocal microscope (Heidelberg Retinal Tomograph with Rostock Corneal Module, HRT-RCM). The optical sectioning ability of confocal microscopy gives high resolution optical section images at different depths within a thick tissue, thereby eliminating the need for physical sectioning. This ability of 4-dimensional (x,y,z and t) imaging makes confocal microscopy uniquely suited for imaging the cornea at the cellular level. The new program was developed to overcome the drawbacks of the original application “Confo” developed in the …

Computer Vision Algorithm Development And Biomedical Applications, Vasant Kearney

Bioengineering Dissertations - Archive

Supervising Professor: Liping Tang and Weihua Mao This study focuses on the development and analysis of novel computer vision algorithms for biomedical applications. Chapter 2 focuses on developing a 2D Canny edge-based DIR algorithm to register in vivo white light images taken at various time points. Using a mouse model, the accuracy of the canny edge-based DIR algorithm was tested using evaluation metrics, a ground truth synthetic scenario, and fluorescent gamma analysis. The results indicate that the Canny edge-based DIR algorithm performs better than all other algorithms. Chapter 3 focuses on the development of a novel longitudinal fluorescent signal tracking …

Investigation Of Prefrontal Cognition Responses To Transcranial Laser Stimulation In Ptsd Using Functional Near Infrared Spectroscopy, Dev Rajanikant Patel

Bioengineering Theses - Archive

In recent years low level laser therapy (LLLT) has been of great research interest because of its therapeutic applications in psychiatry, neurology and ophthalmology. In-vivo transcranial delivery of LLLT has shown to induce beneficial metabolic effects and increase in cognitive functions and hence has been found to be useful in treating neurodegenerative disorders. The aim of this study was to evaluate effects of LLLT on prefrontal cognitive functions, namely attention and short term memory, over six sessions on veterans with Post-Traumatic Stress Disorder (PTSD), using near infrared spectroscopy (fNIRS). In this study the subjects were instructed to perform two neurocognitive …

Diffused Correlation Spectroscopy For Measurement Of Blood Perfusion During Low Level Laser Therapy (Lllt), Soni Sagar

Bioengineering Theses - Archive

Near infrared stimulation or Low Level Laser Therapy (LLLT) is an innovative technique shown to effect the microvasculature hemodynamics. The aim of this study is to use Diffused Correlation Spectroscopy (DCS) to evaluate the physiological effects of LLLT on blood perfusion. This study is divided into two parts: the fist part is the development of DCS system and the second part is investigating the effects of LLLT on biological tissue. DCS is an emerging non-invasive technique to probe deep tissue hemodynamics. DCS uses time-averaged intensity autocorrelation function for the fluctuations caused due to the moving scatterers (RBCs) in biological tissue. …