Biomedical Engineering and Bioengineering Commons^™

Re-Programming Cho Cell Metabolism Using Mir-23 Tips The Balance Towards A Highly Productive Phenotype, Niali Barron

Cell Culture Engineering XV

microRNA-mediated engineering of CHO cells is an attractive method for enhancing various industrially relevant phenotypes, ultimately boosting recombinant protein titres. A single miRNAs ability to interact with multiple mRNA targets allows their regulatory capacity to extend to processes such as cellular metabolism. Various metabolic states have previously been associated with particular CHO cell phenotypes such as glycolytic or oxidative metabolism accommodating growth and productivity, respectively. miR-23 has previously been demonstrated to play a role in glutamate metabolism resulting in enhanced oxidative phosphorylation through the TCA cycle. Re-programming cellular bioenergetics through miR-23 could tip the balance, forcing mammalian production cells to …

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 …

Building Quality Novel Formats And Development Processes, Steven Lang

Cell Culture Engineering XV

There has been an explosion of interest in bispecific or multi-specific bio therapeutic formats. Monospecific antibodies remain the foundation for most bispecific formats. However, monospecific strategies cannot be directly translated into the discovery and development of bispecific antibodies. Bispecifics present unique challenges especially for efficient and consistent assembly/production processes and analytical methods to ensure high quality drug products. Improvements in selection of bispecific combinations/architecture, yield and efficiency and common analytical issues and resolutions will be discussed. Applying improved methods learned from Fab-arm exchange bispecifics and alternative scaffold-antibody fusions, we have established a foundation of knowledge that enables accelerated development

Predictive Engineering Of Cho Cells Using Systems Biology Models, Nathan Lewis

Cell Culture Engineering XV

Decades of bioprocess optimization have resulted in substantial improvements in recombinant protein production. However, some proteins remain difficult to express, and there is an increasing awareness of the need for improved control of critical quality attributes of recombinant protein drugs. To enable cell engineering efforts to enhance protein production and control product quality, we have enumerated the CHO cell parts through genome sequencing efforts,^1,2 and are now providing context to these parts by reconstructing genome-scale networks of the secretory pathway, glycosylation, and metabolism CHO (Figure 1). Using these models, which account for the activities of more than 2000 genes, …

Accelerated Homology-Directed Targeted Integration Of Transgenes In Cho Cells Via Crispr/Cas9 And Fluorescent Enrichment, Jae Seong Lee, Lise Marie Grav, Gyun Min Lee, Helene Kildegaard

Cell Culture Engineering XV

Development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. With draft genome of several CHO cell lines and targeted genome editing technologies, rCHO cell line development based on site-specific integration has the potential to overcome the limitations of clonal heterogeneity. In a previous study, we demonstrated efficient and precise targeted gene integration in CHO cells using CRISPR/Cas9 technology and homology-directed repair (HDR) pathway¹⁾. However, it requires a selection process, which limits targeted integration of multiple transgenes at multiple sites due to a limited number of selection markers and …

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 …

Expression Of Glycoproteins With Excellent Pharmacokinetic Properties On The Novel Cap-Go Expression Platform, Silke Wissing

Cell Culture Engineering XV

Due to their clinical importance, the development of therapeutic proteins has accelerated immensely over the past years. However, the expression of highly glycosylated recombinant therapeutic proteins like for example blood coagulation factors or serum proteins, has remained a challenging task.

C1-Inhibitor (C1-Inh) holds 7 N- and 8 O-glycosylation sites. Plasma derived C1-Inh (Berinert) as well as recombinant C1-Inh from transgenic rabbits (Ruconest) are approved for the treatment of acute attacks in patients with hereditary angioedema. However, the recombinant product shows a dramatically reduced serum half-life in pharmacokinetic studies in comparison to the plasma derived counterparts.

We have developed the …

Flow Cytometry Screening Strategy For The Enrichment Of High-Producing Chinese Hamster Ovary Cells For Monoclonal Antibody Manufacturing, Takeshi Okumura

Cell Culture Engineering XV

Establishment of Chinese hamster ovary (CHO) cell lines for the production of monoclonal antibodies (mAbs) is generally conducted through transfection and pooling procedures with a laborious and time-consuming screening. It could be one of the most rate-limiting steps in the process development of mAb manufacturing. Therefore, an improvement to screening efficiency will give a positive impact to shortening the period of process development. We here propose an enrichment strategy for screening for high-producing cells using flow cytometry (FCM).

A stable pool prepared by mAb-expression vector transfection was stained with a fluorescent-labeled antibody that binds to an mAb presented on the …

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 …

Identifying Low-Level Sequence Variants Via Next Generation Sequencing To Aid Stable Cho Cell Line Screening, Sheng Zhang, Dean Regier, David Oullette, Ivan Correia, Jerry Carson

Cell Culture Engineering XV

Developing stable Chinese hamster ovary (CHO) cell lines for biotherapeutics is an irreversible process and therefore, key quality attributes, such as sequence variants, must be closely monitored during cell line development (CLD) to avoid delay in the developmental timeline, and more importantly, to assure product safety and efficacy. Sequence variants, defined as unintended amino acid substitution in recombinant protein primary structure, result from alteration at either the DNA or the protein level. Here we report the application of transcriptome sequencing (RNAseq) in an IgG1 monoclonal antibody (mAb) CLD campaign to detect, identify and eliminate cell lines containing low-level point mutations …

Genome-Wide Rnai Screen For Improved Functional Expression Of Recombinant Proteins From Hek 293 Cells, Joseph Shiloach

Cell Culture Engineering XV

For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. 21,585 human genes were individually silenced with three different siRNAs for each gene. 56 genes whose silencing caused the greatest improvement in the luciferase expression were found to be part of several different pathways that are associated with spliceosome formation/mRNA processing, transcription, metabolic process, transport and protein folding. 10 genes whose downregulation significantly enhanced the protein expression were validated by their silencing effect on four different recombinant proteins. Among …

Automated, High Throughput Imaging During Cell Line Development To Increase The Assurance Of Clonality, David Shaw

Cell Culture Engineering XV

Current methodologies to create monoclonal cell lines include limiting dilution or single-cell sorting at conditions that offer statistical assurance of monoclonality. We have implemented an automated, high-throughput imaging workflow that acquires brightfield and fluorescent images of every well during the single-cell cloning process. These images can provide direct evidence on whether the cell line originated from one cell during the cloning step. Characterization of the workflow was completed, including the effect of fluorescent dye on cell culture, effect of exposure to high intensity light on cell culture and rate of random mutagenesis in the presence of fluorescent dye and high …

Karyotype-Based Analysis Of Cell Line Instability And Clonality In Cho Cells, Jong Youn Baik, Kelvin Lee

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cell line instability that can result in unexpected changes in phenotypes such as cell growth, productivity, or product quality is challenging for the biomanufacturing of therapeutic proteins. In addition, understanding cell line instability and its relationship to clonality is critical. We hypothesize that chromosomal rearrangements resulting from genomic instability are associated with cell line instability.

We developed cell line instability models using two cell lines: secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) and their host cells (CHO-DUK). We also developed a karyotype-based framework to quantify chromosomal rearrangements. In the absence of methotrexate (MTX), long-term cultured CHO-SEAP …

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 …

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 …

Pilot Study To Assess Breathing During Sight-Read Stringed Instrument Performance, Collin T. Erickson, Clark Potter Mm, Mfa, Gregory Bashford Ph.D

UCARE Research Products

For many musicians, one common occurrence during a performance is the presence of stage fright. Stage fright, though not always expressed can influence the confidence and self-esteem of a performer, and thus can affect the quality of the performance. Every day musicians are affected by stage fright, and there has been no exact solution as to how to lessen the feeling of anxiety musicians feel before performing. No data has been collected to find the correlation between the regularity of breathing and the level of stage fright that a person feels during their performance. A way to quantify regularity of …

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.