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 …

Continuum And Molecular-Dynamics Simulation Of Nanodroplet Collisions, Raunak Bardia, Zhi Liang, Pawel Keblinski, Mario F. Trujillo

Mechanical and Aerospace Engineering Faculty Research & Creative Works

The Extent to Which the Continuum Treatment Holds in Binary Droplet Collisions is Examined in the Present Work by using a Continuum-Based Implicit Surface Capturing Strategy (Volume-Of-Fluid Coupled to Navier-Stokes) and a Molecular Dynamics Methodology. the Droplet Pairs Are Arranged in a Head-On-Collision Configuration with an Initial Separation Distance of 5.3 Nm and a Velocity of 3 Ms-1. the Size of Droplets Ranges from 10-50 Nm. Inspecting the Results, the Collision Process Can Be Described as Consisting of Two Periods: A Preimpact Phase that Ends with the Initial Contact of Both Droplets, and a Postimpact Phase Characterized by the Merging, …

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. …

Wireless Authentication Of Smart Doors Using Rfid, Austin A. Hentrup, Deyi Lu, Peter R. Roldan

Honors Thesis

In an increasingly interconnected world, the traditional metal lock-and-key method of securing homes and businesses is becoming more outdated and inconvenient when compared to modern solutions. Modern systems are smarter, faster, lighter, more secure and more integrated than ever before. Though the costs of Radio Frequency Identification (RFID) are shrinking overall, professional solutions remain prohibitively expensive. The aim of this project is to develop an inexpensive, secure, and internet-enabled RFID door authentication system. This is to transparently investigate the design limitations for these systems and help determine the ultimate feasibility of RFID growing to define the modern door-authentication standard.

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 …

Electric Vehicle And Roadway Conference To Showcase Wireless Charging | College Of Engineering, Usu College Of Engineering

College of Engineering News

NEWS RELEASE – May 8, 2016 – The fourth annual Conference on Electric Roads & Vehicles (CERV) will showcase several exciting technologies including a first-ever demonstration of wireless charging of an electric bus in motion.

The Context Of Tool Control In An Aircraft Assembly Environment Related To Productivity, Taylor Jay Sisson

KSU Journey Honors College Capstones and Theses

The aircraft industry focuses a large portion of its resources on tool control during the assembly of aircraft. Tool control is a strict process that demands time from the assembly process. This time is removed from the value-added time spent actually assembling the aircraft. A study at Lockheed Martin conducted in the Spring of 2016 is discussed that examines the time spent on tool control. Tool control is necessary in aircraft assembly to prevent tools from entering compartments of the aircraft. If such an event occurs, the tool may damage the aircraft. All aircraft assembly processes must be tool controlled, …

Congratulations Class Of 2016 | College Of Engineering, Usu College Of Engineering

College of Engineering News

May 7, 2016 — The College of Engineering congratulates its graduating class of 2016 along with the master's and PhD students who graduated yesterday. Here's the breakdown of our graduates.

• Biological Engineering: 37
• Computer Science: 69
• Computer Engineering: 11
• Civil & Environmental Engineering: 69
• Electrical Engineering: 47
• Environmental Engineering: 9
• Mechanical and Aerospace Engineering: 145

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 …

Transportation Rating Systems And Social Sustainability: A Comprehensive Analysis, Kelsey Lineburg

Senior Honors Projects, 2010-2019

The purpose of this study is to explore the social sector of sustainability in transportation design and engineering. Along with establishing a definition for social sustainability in transportation from existing literature, this document also includes a comprehensive analysis of current sustainability rating systems based on their evaluation of social sustainability metrics. The goal of this thesis is to inform transportation professionals about the existing social sustainability gaps in transportation literature and sustainability rating systems.

Social sustainability in transportation is comprised of two fundamental concepts: social equity and sustainability of community (Bramley and Power, 2009; Dempsey, 2009; McKenzie, 2004; Magis, 2010; …

Understanding Identity And Personality Authenticity Of Engineering Students, Kylie D. Stoup

Senior Honors Projects, 2010-2019

Women are underrepresented in engineering, with 18-20% of engineering students being women¹. We, therefore, used the framework of identity theory and self-concept differentiation to better understand female and male engineering student retention. An exploratory approach is used to measure freshman and senior engineering students’ personality and authenticity of personality across engineering and non-engineering contexts. First, we found personality profiles among engineering freshmen and seniors in engineering settings, and then compared them to their personality in nonacademic settings for authenticity purposes. Big 5 Personality and Authenticity scale were methods used through a survey to determine personality and authenticity in …

A Concrete Water Pasteurization Device For Third World Countries, Andrea R. Murchie

Senior Honors Projects, 2010-2019

In the rural areas of South Africa, as well as many other developing countries, people cook food and sterilize their water by heating over open fires. Many times the fires are inside of homes, which more often than not are very small. Children will often be the ones to tend to the fire, out of necessity while parents and other family members are tending to other work and needs.

The purpose of my Senior Honors Project was to design a concrete vessel that can hold water and heat the water to pasteurization (149°F) when exposed to sunlight for an extended …

A Comparison Of Algorithms And Heuristics For Solving The O 1 I N D 1 Shortest Route Problems, Steven D. Young

Senior Honors Projects, 2010-2019

Shortest-path problems have seen a huge amount of study over the course of the last 50 years. Not surprisingly, this means the original problem of finding the shortest path from some starting location to some destination location has been thoroughly studied. However a large number of slight variations on the original problem still have yet to be thoroughly examined. In this paper we examine one of those variations, namely the problem of determining the optimal route from some source location to some destination location such that at least one location in an intermediate set is included in the route. Three …

Leadership, Culture, And Academic Success, An Internal And External Departmental Analysis And Comparison Of The James Madison University Engineering Program, Sierra Hahn-Ventrell

Senior Honors Projects, 2010-2019

Madison Engineering is a unique young program that does not follow the traditional teachings of engineering education. The following study analyzes the output variable of academic success of the Madison Engineering program in relation to the culture of the program and the leadership styles of those in management positions within the department. Eight comparative schools were selected, analyzed, and surveyed to compare to Madison Engineering and cross-examine how different leadership styles affect certain outputs.

From extensive research and benchmarking, data was collected to compare measures of leadership and culture to output variables of academic success. A survey was further conducted …

Creating A 3d Imaging Device, Bryce Walker

Undergraduate Honors Capstone Projects

This report provides a summary of Bauble, a 3D imaging device designed at Utah State University by Bryce Walker. It is designed to work somewhat like a high tech Mirascope. Unfortunately, it has not yet been successful. Many setbacks occurred in the manufacturing process, detailed below. Simulations of Bauble show both that Bauble is an exciting idea, and that the technology required to manufacture a Bauble is not currently available.

Historical Trends In Mean And Extreme Runoff And Streamflow Based On Observations And Climate Models, Behzad Asadieh, Nir Y. Krakauer, Balázs M. Fekete

Publications and Research

To understand changes in global mean and extreme streamflow volumes over recent decades, we statistically analyzed runoff and streamflow simulated by the WBM-plus hydrological model using either observational-based meteorological inputs from WATCH Forcing Data (WFD), or bias-corrected inputs from five global climate models (GCMs) provided by the Inter-Sectoral Impact Model Intercomparison Project (ISI-MIP). Results show that the bias-corrected GCM inputs yield very good agreement with the observation-based inputs in average magnitude of runoff and streamflow. On global average, the observation-based simulated mean runoff and streamflow both decreased about 1.3% from 1971 to 2001. However, GCM-based simulations yield increasing trends over …

Scalable Three-Dimensional Grasping Mechanism, Ikya Mamidala

Master of Technology Management Plan II Graduate Projects

In this work, we develop a scalable end-effector mechanism for grasping three- dimensional objects with sizes ranging from micrometer to millimeter scale. The design architecture of the gripper comprises an array of identical fingers patented in a circular fashion. Each finger is designed from a novel linkage mechanism whose end effector is manipulated by two independent actuators. In this research, we study three finger gripper device, where each is obtained from a 3 - linkage mechanism. The device is controlled by three independent piezo actuators, and one electro-magnetic solenoid common to each mechanism. The gripping capability depends on how fingers …

Measuring What We Value: Using Performance Measures To Achieve Goals, Chris Rall

PSU Transportation Seminars

Performance measures are commonly used in transportation planning, but how effectively are public agencies using them, and to what ends?

Metro, ODOT and many cities use performance measures to evaluate investment choices and monitor progress. Drawing from Transportation for America’s report Measuring What We Value, and some of the most cutting edge examples of performance-based planning around the nation, Chris' presentation will step back to consider what makes a performance-based planning approach effective at achieving an agency’s goals.