Biomedical Engineering and Bioengineering Commons^™

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 …

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 …

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, …

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 …

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 …

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. …

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

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 …

Cell Express 100tm - A Robust, Simple And Cost Effective Alternative To Highthroughput Automated Platforms For Cell Line Development, Raj Kumar Kunaparaju

Cell Culture Engineering XV

The unrivalled growth in monoclonal antibody (mAb) therapeutics is demanding for a rapid, simple and cost effective expression system. Conventional expression system makes use of Gene Amplification Strategies, Chromatin Modifying Elements and High-Throughput Automated Platforms for cell line development. All these process are complex and costly. CELL EXPRESS 100^TM with its simple selection procedure and being able to generate 100% stable high expressing pool simplifies the isolation of high producing cell line without the need for Amplification, Chromatin Modifying Elements and High Throughput Automated Platforms. CELL EXPRESS 100^TM expression system consists of an expression vector (pUB-CE-100) and a …

Purification Of A Hepatitis C Vaccine Candidate: Comparison Between Multi- Column Chromatographic Processes Operated In Positive And Negative Mode, Ricardo Silva, Alex Xenopoulos, Paula Alves, Manuel Carrondo, Cristina Peixoto

Cell Culture Engineering XV

Given the increasing efficiencies in bioreaction and growing interest on complex biopharmaceutical products such as virus-like particles (VLPs), downstream processing (DSP) is becoming ever more relevant. Therefore, the biopharmaceutical industry is looking for alternative downstream strategies capable of improving purification yields whilst improving product quality and lowering costs. One of most promising improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography.

We report on the development and comparison of two types of multi-column chromatographic systems aimed at the purification of Hepatitis C VLPs, produced using insect cell-based expression with recombinant baculovirus. The first process …

Omics Approach For Generating A High-Yield Cho Cell Line Producing Monoclonal Antibodies, Wei Chi, Hsuan-Pu Chen, Dalton Chen, Hsin-Lin Lu, Bor-Shiun Chen, Chao Yi Teng, Chien-I Lin, Hsueh-Lin Lu, Chi-Chen Hsu, Sheng Jie Huang

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells are extensively used for the industrial manufacture of therapeutic antibodies. Generating high producing cell lines for secretory protein production requires knowing the bottleneck in the cellular machinery for protein expression. Integration site of gene of interest (GOI) is one of the important factors that influence the protein productivity. Even though screening of cells randomly integrated GOI can select high producing cells, the selected cell might not stable due to the chromosome instability. Here, we would like to look for host integration sites where GOI is high yield and stable by screening a single copy integration …

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 …

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, …

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 …

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 …

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 …