Friday, 28 September
08:00 Breakfast BuzZ Sessions
BuzZ Sessions are one-hour long problem-solving roundtables in which small groups of 5-20 researchers get an opportunity to brainstorm solutions for current problems in the area of proteins. They are facilitated by a researcher knowledgeable in the topic area being discussed and often lead to continuing relationships, collaborations, and correspondence between participants.
Parallel Approaches Continued
09:00 Comments by Session Chairperson
François Arcand, MBA, ERA Biotech
09:10 Expression of a Library of Mammalian Cell-Surface Receptors
Michael Dyson, Ph.D., Atlas of Protein Expression, Department of Biochemistry, University of Cambridge
A non-redundant set of 526 sequence confirmed mammalian cell-surface receptor ecto-domains have been cloned into HEK293E GATEWAY expression vectors. A multiplexed small-scale transient transfection expression screen was developed to rapidly identify expression passes and perform optimizations including tag comparison studies. The screen correlated well for predicting the success of larger scale transient transfections and affinity purifications. Expression failures were rescued by rational or combinatorial construct design methods. The receptor library has been used for both antibody generation and functional assays.
09:40 Development of Highly Efficient Human Cell-Based Cell-Free Protein Expression Systems
Hiroaki Imataka, Ph.D., Senior Research Scientist, Protein Research Group, RIKEN GSC
Because genome projects now focus on the structure and function of human proteins, efficient mammalian cell-derived cell-free systems are needed. The systems in current demands should:
1. express “difficult proteins”. 2. glycosylate proteins efficiently, since a large number of human proteins are glycoproteins. 3. be readily available to researchers. We have developed highly efficient human cell-based cell-free systems. These systems synthesize proteins as large as 200 kD to a high level (200 ?g /ml) and proteins difficult to express in vivo such as gcn2. In addition, we have established a hybridoma-based cell-free system that produces biologically active glycoprotein complexes. Because these systems are derived from established cell lines, researchers can easily prepare them in their laboratories. Furthermore, as an important application, we succeeded in enhancing the synthesis of infectious RNA viruses in cell-free. These novel cell-free systems will be very useful for basic research and pharmaceutical application.
10:10 Morning Coffee, Poster and Exhibit Viewing
10:40 A Model for Parallel Protein Production
Ciaran Cronin, Ph.D., Head, Group Parallel Protein Production, Structural Biology, Pfizer, Inc.
Many recent reports of recombinant protein production have demonstrated the value of processing multiple protein variants of a given target simultaneously in order to identify a particular variant that is optimally suited for biochemical or structural analysis. However, there are a myriad of possibilities available to set up a parallel protein production laboratory. We have taken the recent key discoveries in cloning, expression and purification, and combined them with some novel approaches in order to implement a simple and cost-effective platform for parallel protein production. The process has been applied successfully to provide purified protein from multiple protein families.
11:10 High-Throughput Production of Proteins Using Novel Baculovirus Vectors
Linda King, Ph.D., Professor, Life Sciences, Oxford Expression Technologies
This presentation will cover the principles of a new baculovirus technology -flashBAC- as the first baculovirus expression system designed for automation and high throughput. Recent developments in the original system including the deletion of a number of non-essential virus genes such as chitinase, cathepsin and p10 to improve the quality of recombinant proteins will also be discussed. The presentation will conclude with a discussion of the challenges that still present in high throughput protein production in insect cells.
11:40 Lunch on Your Own or (Luncheon Technology Workshop, Sponsorships Available)
Drug Discovery Applications
13:00 Comments by Session Chairperson
13:15 Miniaturization and Automation of Protein Expression for High-Throughput Screening and Structural Biology
Lorenz Mayr, Ph.D., Executive Director, CPC/LFP, Novartis Pharma AG
Current drug discovery relies on massive screening of chemical and antibody-based libraries against various biochemical & cell-based targets for identification of new drug-like compounds. After implementation of highly automated systems among the High-Throughput Screening (HTS) and Structural Biology (SB) departments in Pharma and Biotech, the actual bottleneck for lead discovery has been shifted towards fast and efficient production of appropriate research tools, e.g. proteins and cell lines. In recent years, development and implementation of novel viral and non-viral protein expression technologies has led to substantial changes in the procedures applied for industrial protein production. Combination of these systems with liquid handling stations and laboratory robotics has enabled strong parallelization and automation for protein expression and protein purification. Applying these novel concepts, multi-parallel protein expression in various systems and combination with various tagging and purification strategies has been applied with great success to particular targets of interest. The presentation from the Novartis Lead Discovery Center (LDC) will describe the basic concept behind miniaturization and automation of protein expression for HTS and SB. Several examples from recent protein expression campaigns will highlight the impact of these novel technologies on reduction of protein production timelines, the increase in success rate for protein expression and the future impact towards low-cost and high-value protein production in pharmaceutical R&D.
13:45 Purified Membrane Protein Targets for Drug Discovery
Niek Dekker, Ph.D., Associate Director, Structural Chemistry Department, AstraZeneca
This will be a presentation on the expression and purification of GPCRs and ion channels in functionally active form as enabling tools for drug discovery. Potential areas for applications are structural studies, affinity screening, biophysical mode of action and biopharmaceuticals. Specific examples on purifications and applications will be presented.
14:15 Overcoming Challenges in Supplying Proteins for Structural Biology
Stephen Irving, Ph.D., Senior Principle Scientist, Structural Biology and Biophysics, Pfizer, Inc. – Sandwich
A series of case studies will be presented on a number of different drug targets, illustrating ways of overcoming various challenges in supplying proteins for structure based drug design that we have applied at Pfizer. Topics that will be discussed include experiences with alternative expression systems, construct design, experiences with different affinity tags, buffer optimization and mutagenesis strategies for improving protein properties. This will include an overview of methodologies employed for different protein classes and summary data drawn from structural studies on approximately 100 target proteins to draw general conclusions on relative success rates, lessons learned and potential future advances that may improve attrition rates through the process of gene to structure solution.
14:45 Poster and Exhibit Viewing, Afternoon Refreshment Break
Emerging Technology Focus—Mammalian Cells
15:15 Advances in Transient Recombinant Protein Production in Mammalian Cells
Scott Pattison, Ph.D., Director of Molecular Biology, Kemp Biotechnologies, Inc.
A huge demand exists for the production of high quality recombinant human proteins for drug target studies and biotherapeutics development. A great deal of high quality R&D work has gone into maximizing the yields, and minimizing the timelines and the costs of producing recombinant human proteins in heterologous expression systems, such as bacterial, insect, yeast, and in vitro systems. Despite the impressive advances made in the field of heterologous gene expression, the final quality of proteins produced in such systems is often sub-par. This is typically due to the fact that these products lack key post translational modifications found on the native human protein. In addition, proteins generated in heterologous systems often require more arduous downstream processing, in order to achieve sufficient purity. We have dedicated our R&D efforts to the development of processes and tools for the rapid, flexible, scalable production of authentic recombinant human proteins in HEK293-derived human cell lines. The advances that we have made in developing optimized expression vectors, novel transfection reagents, customized cell culture media, and innovative cryo-preservation processes have dramatically improved the speed, yields, and reliability of producing human recombinant proteins in a human cell system. We routinely produce and purify 20 to 70 mg of secreted protein per liter of serum-free cell culture, in our optimized transient expression system. Starting with a few micrograms of expression vector, we have repeatedly produced and purified in excess of 1 gram of recombinant protein within 8 weeks. Several case studies will be presented that highlight the advances that we have made in the field of transient recombinant protein production in mammalian cells. Co-Authors: Terri Willstaedt, Sandra Allen, Marjorie Young, Stephanie Makel, April Birch, Chris Kemp
15:45 Applications of High Titre, Stable Mammalian Cell Lines in the Biotech Industry
Igor Fisch, Ph.D., President and CEO, Selexis SA
In recent years the field has begun to re-examine cell line development systems in an effort to develop high titer production systems. We will discuss a platform approach that addresses a multitude of factors that affect both the timeline and yield for MAb production in mammalian cell lines. Within weeks of transfection we have identified clones with very high specific activities (>50 pcd) after screening no more than 50 clones. This unique production system allows for the integration of 50 to 100 copies of the transgene into a single chromosomal locus. The unique genetic elements we use to facilitate high copy number integration also assure all copies of the transgene are actively transcribed. The increase in copy number occurs without the need for amplification. Importantly integration occurs preferentially in a locus near the cellular telomeres. Integration into this select region results in cell lines that are extremely stable and since no drug is required for the increase in copy number, our clones show no evidence of chromosomal breakage or rearrangement. Since no amplification is required, mutant cell lines are also not required. This allows us to use parental cell lines based on CHOK1 that grow to high cell density upon scale-up. In addition to rapidly establishing cell lines for GMP production, this cell line development system has also been used to produce humanized antibody variants needed for preclinical testing. Within 6 weeks of transfection we have obtained titers in shake flasks that exceed 2 gm/liter without any media optimization.
16:15 Closing Comments by Session Chairperson
16:30 Conclusion of Protein Expression – Europe Conference