2017 Archived Content
![Optimising Expression Platforms Track Banner Optimising Expression Platforms Track Banner](jpg/optimising-expression-platforms-track-banner.jpg)
The utilisation of engineered therapeutic proteins for basic research, clinical diagnostics and therapy continues to expand. Consequently, the efficient expression and production of these valuable biomolecules face challenges in improving their quantity
and quality while minimising time and cost. An increasing variety of recombinant production platforms are being developed, but there is no “universal” production system which can guarantee high yields.
The Optimising Expression Platforms conference offers comparisons, evaluations and solutions that enable protein engineers to efficiently express the therapeutic protein of their choice.
Final Agenda
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Recommended Short Course*
SC4: Transient Protein Expression: A Key Tool to Enable Rapid Protein Engineering
*Separate registration required
WEDNESDAY 15 NOVEMBER
07:45 Registration and Morning Coffee
08:30 Chairperson’s Remarks
Tsafi Danieli, Ph.D., Director, BioGivExcubator & Head, Protein Expression Facility, Wolfson Centre for Applied Structural Biology, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem
08:35 Bottom-Up Strategies for Reconstitution of Multi-Protein Complexes Using the Baculovirus Expression System
Arnaud Poterszman, Ph.D., Research Director (CNRS), Integrated Structural Biology, Institut de
Génétique et de Biologie Moléculaire et Cellulaire (CNRS/INSERM/UdS)
Many eukaryotic proteins function as macromolecular complexes whose subunits act in concert to catalyze specific activities. We illustrate how genome editing technologies allow purified native assemblies from cells and how the baculovirus expression
system emerged as a powerful tool for reconstitution of multi-subunit complexes, focusing on strategies to facilitate manipulation of the baculoviral genome and assemble multi-gene constructs. As model systems, we will use transcription regulators
such as pTefb, nuclear receptors or the 10 subunits transcription factor TFIIH.
09:05 Nature as a Blueprint to Convert Yeast into an Antibody Factory
Alexander Frey, Ph.D., Associate Professor, Molecular Biotechnology, Department of Bioproducts and Biosystems,
School of Chemical Engineering, Aalto University
We have previously created glycoengineered Saccharomyces cerevisiae strains producing human-like comlex N-glycans. Considering the production of antibodies, a major hindrance lies in the yeast endoplasmic
reticulum, as it is not equipped for efficient folding. By rational strain design and high-throughput screening applications we were able to increase the specific secreted antibody yields of S. cerevisiae,
providing a promising strain for further process optimization and platform development for antibody production.
09:35 Combining Synthetic Biology and Molecular Evolution for Optimizing and Understanding Gene Expression
Hadas Zur, Ph.D., Researcher, Department of Biomedical Engineering, Tel Aviv University
Deducing generic causal relations between features of the genetic material and various aspects related to the gene expression dynamic is a fundamental scientific objective with ramifications for disciplines such as biotechnology, human health, and
functional genomics. In this talk I show how, on the one hand, the analysis of endogenous gene expression data with molecular evolution and biophysical modeling tools contributes towards model-based gene expression engineering. On the other hand,
I demonstrate how the design and tailoring of synthetic biology experiments can be used for accurately estimating the direct effect of genomic features on gene expression.
July 2017 Speaker Interview with Colleague Tamir Tuller
10:05 The Strep-Tag® Technology - The Superior Tag System for the Entire Protein Production Workflow
Dennis Niermeier, MSc, IBA Lifesciences
IBA is focused on a comprehensive product portfolio around its proprietary Strep-tag® technology that covers the entire protein production workflow from cloning, expression and purification as well as protein immobilization for assays. Especially
our Strep-Tactin®XT is superior to other systems due to its extremely high affinity while maintaining reversibility.
10:20 Difficult to Express Proteins: Novel Plasmid Technology to Significantly Increase Product Yield in CHO Cells
Marco Cacciuttolo, Ph.D., Director, Operations, Batavia Biosciences
Yield is still an area that requires significant improvement for many promising recombinant proteins and antibodies. Novel vector technology enables rapid generation of stable, CHO cell lines able to provide at least 10-fold more product per cell.
10:35 Coffee Break in the Exhibit Hall with Poster Viewing
11:15 How to Tune Recombinant Protein Expression in E. coli for Enhanced Production of Biopharmaceuticals
David Johannes Wurm, Ph.D., Postdoctoral Researcher, Institute of Chemical, Environmental and Biological Engineering,
Integrated Bioprocess Development, TU Vienna
Strong induction of recombinant protein expression in E. coli can lead to agglomeration of inactive product. We developed a feeding strategy to tune recombinant protein expression on a cellular level
which leads to higher yields of soluble and active product and applied this system to the production of the model protein GFP and an industrially relevant antibody fragment. Furthermore, we developed a fast and effective method for strain
characterization, which is a prerequisite for this feeding strategy.
11:45 An Ongoing Evaluation of Our Transient Expression Systems: Can We Predict the Appropriate Expression System for Any Drug Modality?
Richard Altman, MS, Scientist V, Protein Technologies, Amgen
A robust, flexible transient protein production facility provides critical support to drug discovery efforts. We review the ongoing evolution of our protein production endeavors focusing on two critical components. The first is the strategic assembly
of mammalian expression “tools” that gives us a toolbox capable of expressing diverse and challenging candidate proteins. The second is the harmonization of the entire protein production process, thereby reducing turnaround times
and increasing throughput.
12:15 In-Depth Process and Systems Characterization via PAT/QbD Generates Understanding the Base for New Host Design Concepts
Gerald Striedner, Ph.D., Associate Professor, Department of Biotechnology, University of Natural
Resources and Life Sciences
Implementation of PAT/QbD concepts provides the basis for advanced bioprocess understanding. This fundamental knowledge based on systems behavior under production conditions allows for innovative process-relevant host and process engineering concepts.
The talk focuses on such host design strategies for different systems (CHO, insect cells/baculo system) and process types.
12:45 Approaches for the Rapid Development of Recombinant E.coli Strains for Large Scale GMP Production of Biotherapeutics
Christopher Lennon, Ph.D., Staff Scientist Research & Development, FUJIFILM Diosynth Biotechnologies
Platform processes are key in controlling the approach to Biopharmaceutical process development. Large numbers of variations on strains need to be screened quickly and efficiently in a manner that directly models large scale commercial manufacturing.
Fujifilm Diosynth Biotechnology use the Ambr250(tm) in concert with the pAVEway expression system to rapidly establish a fermentation process.
13:15 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own
13:45 Session Break
14:00 Chairperson’s Remarks
Henry Chiou, Ph.D., Associate Director, Cell Biology, Thermo Fisher Scientific
14:05 KEYNOTE PRESENTATION: Engineering CHO Cells
Bjørn Voldborg, MSc, Director, CHO Cell Line Development, Novo Nordisk Foundation Center for
Biosustainability, Technical University of Denmark
Using high-throughput (HT) technologies, the CHO Cell Line Engineering project at the Center for Biosustainability is genetically modifying CHO cells based on experimental and in silico generated data
to engineer CHO cell lines optimised for the production of therapeutic proteins. We have engineered cells resulting in tailormade glycoprofiles and non-lactate producing cell lines, thereby improving the bioprocess.
14:35 Generation of Superior Host Cell Lines for Biomanufacturing
Holger Laux, Ph.D., Fellow, Cell Line Development, Novartis
CHO cells are the most widely used host for large-scale production of recombinant therapeutic proteins. Using transcriptomic approaches we have identified target genes involved in productivity and product quality. Subsequently a variety of novel
parental CHO cell lines were generated applying cell line engineering techniques. These novel knockout CHO cell lines are superior in respect to productivity and/or product quality.
15:05 Eukaryotic Cell-Free Systems: A Novel Source for Functional Antibodies
Marlitt Stech, Ph.D., Research Scientist, Cell-Free and Cell-Based Bioproduction, Fraunhofer Institute
for Cell Therapy and Immunology
Since antibodies are indispensable tools for diagnostic and therapeutic applications, the development of alternative manufacturing strategies that circumvent the hurdles connected to conventional antibody production technologies is of enormous
interest. To address this issue, we have developed a novel antibody production technology based on a microsome-containing CHO cell-free system that combines the advantages of the eukaryotic expression host with the benefits of cell-free systems
in general.
15:35 Refreshment Break in the Exhibit Hall with Poster Viewing
16:15 Automation and High-Density Mammalian Cultures to Maximize Protein Productivity
Kinjal Mehta, Ph.D., Principal Scientist, Protein Sciences, Jounce Therapeutics
While CHO has proven to be a promising cell line for desirable post-translational modification patterns, HEK293 maintains its role as a conventional mammalian host with regards to desired specifications on product quality and faster turnaround
times in isolated cases. In this presentation, we describe optimized protocols for ExpiCHO and Expi293 cell lines, to demonstrate automated procedures from HTP transfections through purifications enabling generation of several milligrams of
antibody or reagent proteins from smaller cultures with shorter turnaround times.
16:45 Mitochondrial-Derived Small RNAs as Powerful Tools to Boost CHO Cell Productivity
Lisa Alexandra Pieper, Postdoctoral Researcher, Early Stage Bioprocess Development, Boehringer Ingelheim Pharma
GmbH & Co. KG
In this study we show that ectopic expression of a human mitochondrial-derived small RNA (mitosRNA-1978) in IgG expressing CHO cells strongly improved specific productivity by functioning in a microRNA-like fashion. By next-generation sequencing
we identified Ceramide Synthase 2 (CerS2) and Tbc1 domain family member 20 (Tbc1D20) as target genes of mitosRNA-1978. Knockdown of CerS2 and Tbc1D20 in CHO-IgG cells resulted in increased antibody production, thus recapitulating the mitosRNA-1978
phenotype.
17:15 Problem-Solving Breakout Discussions (View All Breakout Discussions)
Common Issues with Transient Protein Production
Moderators: Richard Altman, MS, Scientist V, Protein Technologies, Amgen
Henry C. Chiou, Ph.D., Associate Director, Cell Biology, Life Science Solutions, Thermo Fisher Scientific
Dominic Esposito, Ph.D., Director, Protein Expression Laboratory, Frederick National Laboratory
for Cancer Research
Scalable and rapid transient protein production in mammalian cells continues its evolution as an integral part of the biotherapeutic drug discovery process. We discuss the common issues facing researchers as they try to meet an expanding demand
for transiently produced recombinant protein.
- What are the current challenges to transient protein production?
- What are the keys to optimizing expression?
- How do we optimize the whole protein expression process?
- What scale of expression and level of throughput are commonly being used?
- What cell line should we use and when?
- Characterization of transiently produced proteins
Optimal Expression and Purification of VLP
Moderator: Isabelle Legastelois, Ph.D., Research Unit Head, Virology and Expression
Systems, Sanofi Pasteur
- What is the best expression system?
- Is industrialization feasible, rapidly and at low cost?
- Discussion around the optimal purification process
Engineering Cell Factories for Protein Production
Moderator: Bjørn Voldborg, MSc, Director, CHO Cell Line Development, Novo Nordisk Foundation
Center for Biosustainability, Technical University of Denmark
- Which production hosts do we engineer: microbial, insect and/or mammalian?
- How do we identify engineering targets: modeling, omics and/or literature?
- Which traits are we improving: product yield, product quality, host phenotype, process, other?
- Which proteins do we want to improve the production of: antibodies, hard-to-produce proteins, other?
18:15 Networking Reception in the Exhibit Hall with Poster Viewing
19:15 End of Day
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THURSDAY 16 NOVEMBER
08:00 Registration and Morning Coffee
08:30 Chairperson’s Remarks
Richard Altman, MS, Scientist V, Protein Technologies, Amgen
08:35 Bioprocess Engineering of Insect Cells for Accelerating Vaccines Development
Antonio Roldao, Ph.D., Senior Scientist, Animal Cell Technology Unit, Instituto de Biologia Experimental
e Tecnológica (iBET)
Technological breakthroughs and/or new producer hosts allied to classical systems/molecular biology tools and engineering methodologies are urgently needed to accelerate biologics manufacturing process. Two case studies will be presented where
bioprocess engineering assisted/accelerated vaccines development. In the first case study, the production of multivalent Influenza VLPs could be achieved/optimized by (i) combining stable and baculovirus-mediated expression, (ii) identifying
best infection strategy through DoE, (iii) evolutionary engineering of insect cells phenotype, and (iv) designing a scalable, “universal” and “All-Filtration” purification platform. In the second case study, the
production of Gag-VLP could be achieved/optimized by (i) generating stable insect cell lines using site-specific gene integration based on flipase-mediated cassette exchange technology, and (ii) adaptive laboratory evolution of insect
cells to hypothermic culture conditions and supplementation with productivity enhancers. Overall, the insect cell platforms and bioprocess engineering strategies herein assembled have the potential to assist/accelerate vaccines development.
August 2017 Speaker Interview
09:05 Expression of Influenza Antigens in Conventional and Non-Conventional Eukaryotic Expression Systems
Isabelle Legastelois, Ph.D., Research Unit Head, Virology and Expression Systems,
Sanofi Pasteur
The trimeric transmembrane haemagglutinin, the tetrameric transmembrane neuraminidase of influenza virus or Virus Like Particles (VLPs) are very good candidates to assess expression systems as they are recognized as very challenging to express,
particularly if high yields are targeted. It is the reason why we assessed several conventional but also non-conventional expression systems to obtain the best expression of these candidates in the perspective to develop reagents but also
new vaccines.
09:35 Reaching the High-Hanging Fruit: Expression and Purification Optimization Tools for Production of High-Value Drug Discovery Protein Targets
Dominic Esposito, Ph.D., Director, Protein Expression Laboratory, Frederick National Laboratory
for Cancer Research
Pharmaceutically relevant proteins are often among the most challenging targets for protein production. As part of the NCI RAS Initiative, we are working on a series of complicated targets involved in the MAP kinase activation pathway, and
have had to develop a number of process and technology improvements to permit increased protein yield, protein quality, and proper PTMs. We discuss in detail the enhancements to insect and mammalian expression systems we have developed
and how they can be applied to high-level production of other clinically relevant proteins, using the RAF kinase protein as a particularly challenging example.
10:05 Genome-Wide Signals Detection for Enhanced Cell Line Productivity and Stability
Pierre-Alain Girod, CSO, Selexis
Stable, high-quality production cell lines secreting optimal levels of recombinant protein require stable integration of the recombinant DNA, elevated gene transcription, optimized secretion and metabolic machinery to handle the increased
protein loads along with cellular phenotypic stability. Using the extensive genomic (SNPs) and FISH-RNA/DNA data we have generated for our host (CHO-M) and manufacturing cell lines, we will describe how we are significantly boosting production
capabilities and cell line stability of our CHO-M cell line.
10:35 Coffee Break in the Exhibit Hall with Poster Viewing
11:15 A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting
Abdelmajid Noubhani, Ph.D., Associate Professor, Microbiology, Institute of Chemistry and Biology of Membrane & Nano-Objects (ENSTBB), Institut Polytechnique de Bordeaux
We describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant scFv derived from a human anti-αIIbβ3 antibody selected to target atheromatous lesions for the presence of platelets considered
as relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable
for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The improved conditions allowed for the recovery of highly purified and biologically active scFv
fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques.
11:45 Tuning the Co-Expression of Multiple Polypeptides to Optimize Assembly of Bispecific Antibodies and Other Formats
Giovanni Magistrelli, Head, Protein Engineering, Novimmune SA
The inadequate expression of one or several polypeptide components can significantly affect the assembly and secretion of multi-protein complexes. We found that codon de-optimization - instead of optimization - of an over expressed chain can
lead to significant increase in the assembly of bispecific antibodies and overall yield in CHO cells. This approach to tune the ratio of different polypeptides can be applied to optimize different multispecific antibody formats.
12:15 Luncheon Presentation (Sponsorship Opportunity Available) or Enjoy Lunch on Your Own
13:00 Dessert Break in the Exhibit Hall with Poster Viewing
13:30 End of Optimising Expression Platforms
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