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Optimizing Cell Culture Technology - Day 2


Optimizing Cell Culture Technology 


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Tuesday, August 23

7:30 am Breakfast Presentation (Sponsorship Opportunity Available) or Morning Coffee

 

CULTIVATING CHO CELLS 

8:25 Chairperson's Remarks

Alan Dickson, Ph.D., Professor, Biotechnology, and Director, Centre of Excellence in Biopharmaceuticals, Faculty of Life Sciences, The University of Manchester

8:30 Optimizing CHO Cell-Based Bioprocesses

Anthony Rossomando, Ph.D., Senior Director, Biotherapeutics, Alnylam Pharmaceuticals

RNAi technology is being utilized in bioprocessing development to silence genes involved in key cellular pathways that impact biologic protein quality, particularly around decreasing fucosylated glycans to improve antibody-dependent cellular cytotoxicity (ADCC).  mRNA levels from genes that affect biotherapeutic fucosylation and cell viability were significantly reduced (>80%) over a 10-14 day culture period using small interfering RNAs (siRNAs) added directly to CHO cells grown in 3L bioreactors with significant improvements in cell viable density and biologic quality.

9:00 Proteomic Analysis of Recombinant CHO Cells and the Potential for Biomanufacturing Process Improvement

Paula MeleadyPaula Meleady, Ph.D., Senior Research Scientist and Programme Leader, Proteomics Core Facility, National Institute for Cellular Biotechnology (NICB), Dublin City University - Biography 

The talk will focus on proteomic approaches we have used to profile poor and good performing recombinant CHO cell lines concentrating on phenotypes related to growth (slow/fast) and productivity (both high/low and sustained productivity). Data will be presented showing outcomes from profiling efforts where phenotypic improvements, using growth as an example, have been achieved following engineering of targets of interest back into recombinant CHO cells.
 

9:30 CHO Cell Culture Improvements Using a Controlled Nutrient Limiting Feed Strategy

Matt GagnonMatthew Gagnon, Scientist, Culture Process Development, BioProcess Research and Development, Pfizer, Inc. - Biography 

Maintaining an optimal environment is essential to promote a healthy and productive CHO cell culture. A well characterized culture vessel ensures ideal mixing and gas transfer. A balanced initial medium and nutrient addition during culture prevents depletion of necessary metabolites. Finally, preventing the accumulation of certain metabolites inhibitory to both cell growth and productivity is critical. An elegant approach to maintain lactate concentrations at low levels in culture will be described in this talk.
 

10:00 Networking Coffee Break with Exhibit and Poster Viewing

10:45 Site-Specific Antibody Production of Different Antibody Cell Lines

Sinyoung ParkSinyoung Park, Ph.D., Scientist, Process Development – Upstream, Ambrx, Inc. - Biography 

Ambrx's proprietary technology enables the production of a site-specific variant of an antibody which is created by incorporation of Ambrx amino acid into the specific location(s) of the molecule. The cell lines containing Ambrx orthogonal t-RNA and tRNA synthetase replace a natural amino acid with an Ambrx unnatural amino acid such as para-acetyl phenylalanine (pAF). The Chinese Hamster Ovary (CHO) cell lines were generated for different types of antibody variants. The cell culture process parameters such as pAF addition time, temperature shift, feed addition, and media selection were examined for different cell lines.
 

11:15 Fluorinert, An Oxygen Carrier, Improves Cell Culture Performance in Deep Square 96 Well Plates by Facilitating Oxygen Transfer – A Scale-Down Model for Multi-Parallel High Throughput Production Clone Selection

Yung TsaoYung-Shyeng Tsao, Ph.D., Senior Principal Scientist, Cell Culture, Merck

Suspension mammalian cell cultures in 96-well plates often fail to reach high cell density under normal agitation presumably due to limitations in oxygen transfer. Although more vigorous agitation can improve gas transfer in 96-well format, it can also harm mammalian cells. We employed Fluorinert, an artificial blood candidate, to overcome oxygen limitation in 96-well plate and enable a recombinant antibody-producing Chinese Hamster Ovary (CHO) cell line to reach high cell density and antibody titer comparable to that of shake flask culture.
 

11:45 pm Perfusion of an IgG producing CHO Cell Line by ATF or by TFF in WAVE Bioreactor

Veronique ChotteauVéronique Chotteau, Ph.D., Animal Cell Technology Group, School of Biotechnology, Division of Bioproduction, KTH - Royal Institute of Technology

Authors: Marie-Françoise Clincke, Carin Mölleryd, Puneeth K Samani, Ye Zhang, Eva Lindskog, Kieron Walsh and Véronique Chotteau

Perfusion of an IgG producing CHO cell line was performed in a WAVE Bioreactor™ using either Alternating Tangential Flow or Tangential Flow Filtration. The properties and performances obtained with both filtration systems were compared. Very high cell densities were achieved and could be stably maintained. Then the cell density could be significantly further increased showing the capacity of the system set-up.
 



Sponsored by
Pall_LifeSciences 
12:15 Luncheon Presentation
Redefining Cell Line Selection and Process Development using Microbioreactors and Design of Experiments (DoE)
Tiffany D. Rau, Ph.D., Global Technology & Technical Manager, Pall CorporationHigh-throughput bioreactors allow one to efficiently conduct Design of Experiments (DoE) which embodies the principles of Quality by Design. Data will be presented showing the Micro-24 successfully being used in cell line selection and process optimization activities and its scalability to larger bioreactors, demonstrating the advantages of a controlled "high-throughput" bioreactor system that allows rapid, very early stage process development which can contribute to shorter development timelines and lower development costs. 
 

 

3D CELL CULTURE 

1:55 Chairperson's Remarks

Thomas C. Killian, Ph.D., Professor, Physics and Astronomy, Rice University

2:00 High-Throughput 3D and Micropatterned Cell Culture

Shuichi TakayamaShuichi Takayama, Ph.D., Professor, Biomedical Engineering, Macromolecular Science and Engineering, College of Engineering, University of Michigan - Biography 

Our laboratory specializes in developing microfluidic tools to control cellular microenvironments. Microfluidic approaches to cell culture, however, are typically cumbersome and challenging to perform in high-throughput formats using conventional liquid handling tools. In this presentation, I will describe recent high throughput cell culture technology developed in our laboratory that incorporates microfluidic concepts but without use of microchannels and in formats compatible with standard 96, 384, and 1536 well technologies. One technology is a 384-well format hanging drop cell culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward as conventional 2D cultures. We show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anti-cancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. I will also describe the use of aqueous two phase system to perform micropatterned gene expression or knockdown, and to micropattern embryonic stem cell niches to modulate their differentiation towards neuronal cells.

2:30 Cells in Gels in Paper: High-Throughput Platform for Investigation of Cell Function in 3D Tissues

Ratmir DerdaRatmir Derda, Ph.D., Assistant Professor, Department of Chemistry, University of Alberta - Biography 

In vitro 3D culture is an important model for tissues in vivo. Cells in different locations of 3D tissues are physiologically different, because they are exposed to different concentrations of oxygen, nutrients, and signaling molecules. The majority of cell-based assays based on 3D cultures, however, can only detect the average behavior of cells in 3D construct. Isolation of cells from specific regions of 3D cultures is possible, but relies on low-throughput techniques such as tissue sectioning. We developed a simple method for generation and analysis of complex 3D tissue structures. Spotting of cells suspended in extracellular matrix (ECM) gel onto the ordinary filter paper creates 200 micron-thick slabs of ECM gel containing cells. Stacking the sheets assembles 3D multilayer constructs. Peeling apart the sheets of paper "sections" the cultures into 200-micron-thick cell-containing slabs. The number of cells plated initially in each layer determines the spatial distribution of cells in the stacked 3D cultures. Multilayer culture, thus, generate 3D tumor models with well-defined gradients of oxygen and nutrients. This capability made it possible to examine the 3D migration/invasion of cells and their susceptibility to therapeutic agents.

3:00 Three-Dimensional Cell Culturing Through Magnetic Levitation

Thomas KillianThomas C. Killian, Ph.D., Professor, Physics and Astronomy, Rice University - Biography 

3D cell culturing through magnetic levitation, developed at Rice University and the University of Texas MD Anderson Cancer Center and marketed by Nano3D Biosciences (n3D) [www.n3dbio.com], is a new paradigm in cell culturing that provides the advantages of 3D cell culturing in a much simpler platform that can easily be incorporated into existing protocols and diagnostics. It is based on magnetization of cells using magnetic-nanoparticles and levitation of the cells in spatially varying magnetic fields. I will describe the general technique and results with a variety of cell lines and primary cells, with an emphasis on primary human lung cells cultured as monoculture and co-culture at the air-media interface, kidney cells used in a wound healing test for toxicology studies, and controlled manipulation and shaping of human gliobalstoma and normal human astrocytes co-culture for invasion assays.

3:30 Networking Refreshment Break with Exhibit and Poster Viewing

 

FUTURE TRENDS FOR CELL CULTURE 

4:15 INTERACTIVE PANEL:    How will Mammalian Cell Culture be Further Innovated?As mammalian cell culture moves forward meeting increased demands for ever higher titer, how will the task of culturing cells be innovated?  What are the emerging technologies and approaches that will bring cell culture to new heights?

Please join this interactive panel discussion as cell culture experts share their insights on how cell culture will be innovated to provide adequate capacity for the biologics industry.

Moderator:  Tiffany D. Rau, Ph.D., Global Technology & Technical Manager, Pall Corporation

Panelists:

 

Paula Meleady, Ph.D., Senior Research Scientist and Programme Leader, Proteomics Core Facility, National Institute for Cellular Biotechnology (NICB), Dublin City University

Yung-Shyeng Tsao, Ph.D., Senior Principal Scientist, Cell Culture, Merck

James C. Warren, Ph.D., Principal Development Engineer, Vaccine Manufacturing Sciences and Commercialization, Merck & Co., Inc.

Jianguo Yang, Ph.D., Principal Scientist, Commercial Cell Culture Development, Commercial Process Development, Biologic R&D, Genzyme

5:15 End of Conference

6:00 – 9:00pm SC 4 - Dinner Short CourseE.coli Innovations 
(Separate registration required)



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