Cambridge Healthtech Institute’s Ninth Annual
Optimising Expression Platforms

Meeting the Demand for Recombinant Antibodies

2-3 November 2016 | EPIC SANA Lisboa Hotel | Lisboa PORTUGAL

The utilisation of recombinant antibodies 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. To meet these demands, an increasing variety of recombinant production platforms are being developed. Unfortunately, there is no “universal” production system which can guarantee high yields of recombinant antibody, particularly as every antibody-based molecule itself causes its own issues in terms of expression.

The Optimising Expression Platforms conference offers comparisons, evaluations and solutions that enable researchers to efficiently express the recombinant antibody of their choice.

Final Agenda

Day 1 | Day 2 | Speaker Biographies | Download Brochure

WEDNESDAY 2 NOVEMBER

07:45 Registration and Morning Coffee

Engineering Expression Platforms to Meet the Demand

08:30 Chairperson’s Remarks

Bjørn Voldborg, MSc, Director, CHO Cell Line Development, Novo Nordisk Foundation Center for Biosustainability (CFB), DTU Biosustain, Technical University of Denmark

09:20 The Effect of Vector Design on the Expression of Bispecific Antibodies

Jason Saunders, MSc, Senior Scientist, Biologics Expression and Technology, Merck & Co., Inc.

09:50 Complementary Approaches for Protein Production in Insect and Mammalian Cell Lines

Konrad Büssow, Ph.D., Research Scientist, Structure and Function of Proteins, Helmholtz Centre for Infection Research

The benefits of the baculovirus system for the production of intracellular and secreted proteins are well known. The human HEK293 cell line represents a valuable alternative, especially for secreted proteins, and allows for virus-free transient transfection with plasmids. Recently, virus-free, plasmid-based transfection of insect cells has been optimised considerably. This system represents a useful alternative that combines advantages of the baculovirus system and transient transfection of HEK293 cells. Examples of proteins produced by transient transfection of HiFive insect cells will be presented, including intracellular and secreted production.

10:20 Strep-Tactin XT - A Superior Next-Generation System for Purification of Proteins, Isolation of Cells & Assay Development

Dennis Niermeier, M.Sc., Scientist, IBA GmbH

The new third-generation Strep-tag® system is based on recently engineered Strep-Tactin®XT and Twin-Strep-tag®. Due to the affinity improved but still reversible binding of Strep-Tactin®XT to Twin-Strep-tag® in the low pM range, the system is superior to other affinity purification systems and now also suitable for assay development.

10:35 Presentation to be Announced

10:50 Coffee Break in the Exhibit Hall with Poster Viewing

ENGINEERING GENES

11:30 Engineering the Baculovirus Genome to Improve Protein Production

Dominic Esposito, Ph.D., Director, Protein Expression Laboratory, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.

The use of insect cell expression systems for the production of pharmaceutically relevant protein targets has dramatically increased over the last few years, with several products already approved by major regulatory agencies. In our laboratory, this system has been vital for production of post-translationally modified RAS proteins essential for cancer drug discovery. In using this system, we have developed a number of process and technology improvements which permit increased protein yield, protein quality, and virus stability. We discuss in detail the enhancements to the system and how they have been applied to high-level production of clinically relevant proteins, and examine ways in which synthetic biology and genome engineering can further enhance the utility of this system.

12:00 SINEUPs: A New Class of Antisense Long Non-Coding RNAs that Specifically Activate Translation of Targeted Proteins

Silvia Zucchelli, Ph.D., Assistant Professor, Health Sciences, University of Eastern Piedmont; CSO, TransSINE Technologies Inc.

SINEUPs represent a new functional class of natural and synthetic antisense long non-coding RNAs that upregulate translation of partially overlapping sense mRNAs through the activity of an inverted SINEB2 element. Given their modular structure, SINEUPs can be designed to increase protein synthesis of potentially any gene of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

12:30 Engineering of CHO Cell Lines for Enhanced Process Robustness

Pierre-Alain Girod, Ph.D., CSO, Selexis

High-quality production cell lines secreting maximal levels of recombinant proteins require stable integration of the recombinant DNA, elevated gene transcription, optimized secretion machinery to handle increased protein secretion and folding loads and, ideally, being easily tracked during manufacturing. Using the data from our CHO-K1 genome and transcriptome, we have engineered our CHO-K1 to address these issues, particularly for difficult-to-express proteins, as well as to provide detailed genomic analysis packages for manufacturing cell lines.

13:00 Luncheon Presentation: A Stable Episomal Expression System to Streamline Mammalian Protein Production

Meelis Kadaja, Ph.D., MBA, Director, Business Development, Icosagen Cell Factory OÜ

We have found the way to stably maintain expression vectors in dividing mammalian cells as extrachromosomal units. This stable episomal expression system is scalable, and requires only 1ug of DNA to produce up to gram quantities of recombinant proteins with low endotoxin levels in few weeks. Our system enables to generate production cell banks in 10 days, and is also used in antibody discovery to express and screen antibodies.

13:30 Session Break

ENGINEERING HOSTS: CHO CELLS

14:00 Chairperson’s Remarks

Nicola Burgess-Brown, Ph.D., Principal Investigator, Biotechnology, Structural Genomics Consortium (SGC), University of Oxford

14:05 CHO Cell Energetics: Understanding the Powerhouse of the Cell through Mitochondrial Deep Sequencing

Paul S. Kelly, Ph.D., Senior Postdoctoral Researcher, School of Biotechnology, National Institute for Cellular Biotechnology, Dublin City University

The predominant metabolic pathways, glycolysis and oxidative phosphorylation, have been linked to critical bioprocess relevant CHO cell phenotypes, growth and productivity. We have shown SEAP producing CHO cells engineered to be depleted of microRNA-23b exhibited a 3-fold increase in product yield associated with elevated mitochondrial activity. Given CHO cells’ genetic instability, we sought to compare the mitochondrial genomic sequence of 22 CHO cell lines and provide a mitochondrial genome reference sequence from the hamster.

14:35 Precision Control of Recombinant Gene Expression for CHO Cell Synthetic Biology

Adam Brown, Ph.D., Research Fellow, Department of Chemical and Biological Engineering, University of Sheffield

To successfully apply the core concepts underpinning synthetic biology to CHO cell engineering, we must develop practical and robust enabling technologies. Fundamentally, we will require the ability to precisely control the relative stoichiometry of numerous functional components that are simultaneously introduced into the host cell factory. In order to enable this, we have developed a suite of complementary technologies that enable precise control of recombinant gene expression in CHO cells. Using synthetic promoters as an exemplar, the function of these tools in the context of CHO cell engineering will be described.

15:05 Inactivation of GDP-Fucose Transporter in CHO Cells: A New Strategy for Producing Fucose-Free Biobetter Antibody Therapeutics

Zhiwei Song, Ph.D., Principal Scientist, Expression Engineering Group, Lead PI for GlycoSing Programme, Bioprocessing Technology Institute, A*STAR

Removal of core fucose from IgG1 has been shown to significantly enhance its affinity to FcgRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity (ADCC). To address these industry-specific needs, we have inactivated the GDP-fucose transporter in CHO cells. The DHFR and GS genes have also been inactivated in these cells as selection markers. Using these cells, stable lines have been developed to produce fucose-free rituximab and fucose-free obinutuzumab (Gazyva).

15:35 Refreshment Break in the Exhibit Hall with Poster Viewing

ENGINEERING HOSTS: CHO CELLS (CONT.)

16:15 A Big Step Forward for Next-Generation CHO Clone Characterization and Selection

Oliver Popp, Dr. rer. nat., Senior Scientist, Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Roche Diagnostics GmbH

In-depth characterization of high-producer cell lines and bioprocesses is vital to ensure robust and consistent production of recombinant therapeutic proteins in high quantity and quality for clinical applications. For that, we established a novel hybrid approach for supporting comprehensive characterization of metabolic CHO clone performance. The proposed approach also provides a mechanistic link between observed clone phenotype, process setup, and feeding regimes, and thereby offers concrete starting points for subsequent process optimization.

16:45 FEATURED PRESENTATION: Engineering the CHO Cell

Bjørn Voldborg, MSc, Director, CHO Cell Line Development, Novo Nordisk Foundation Center for Biosustainability (CFB), DTU Biosustain, 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. The HT cell line engineering pipeline as well as examples of the engineered improved cell lines will be described.

17:15 Cmax: A Heterotrophic Cell Platform for Improved Biologics Production

Nicky C. Caiazza, Ph.D., Principal Scientist, Metabolic Engineering and Synthetic Biology, Heterotrophic Cell Systems, Synthetic Genomics Inc.

We have developed a robustly fermentative heterotrophic system for efficient end-to-end biologics discovery, development and manufacturing. This platform has intrinsic advantages for biologics production and a suite of synthetic biology tools that has enabled high productivity (>1 g/l/d) mAb production with beneficial attributes for downstream processing. Here we show that these organisms can be engineered to secrete genuine, functional monoclonal antibody with favorable characteristics related to protein production and quality.

17:45 Problem-Solving Breakout Discussions

Common Issues with Transient Protein Production

Moderators:
Richard Altman, MS, Scientist, Protein Technologies, Amgen, Inc.
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, Leidos Biomedical Research, Inc.
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

Engineering Cell Factories for Protein Production

Moderator:
Bjørn Voldborg, MSc, Director, CHO Cell Line Development, Novo Nordisk Foundation Center for Biosustainability (CFB), DTU Biosustain, 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?
• Which proteins do we produce: antibodies, hard-to-produce proteins, other?

18:15 Networking Reception in the Exhibit Hall with Poster Viewing

19:15 End of Day

Day 1 | Day 2 | Speaker Biographies | Download Brochure

THURSDAY 3 NOVEMBER

08:00 Registration and Morning Coffee

CASE STUDIES: PROTEIN TO PRODUCT

08:30 Chairperson’s Remarks

Henry C. Chiou, Ph.D., Associate Director, Cell Biology, Life Science Solutions, Thermo Fisher Scientific

08:35 Expression of IgG in Genetically Engineered E. coli

Na Ke, Ph.D., Research Scientist, Protein Expression and Modification Division, New England Biolabs

We have expressed for the first time in the cytoplasm of E. coli full-length human, rabbit and mouse antibodies, along with chimeric versions, including the commercial blockbuster Humira. The expression is further improved by the co-expression of a series of protein-folding helpers. Microbial expression of IgG offers an expanded potential for rapid screening, engineering and expression antibodies.

09:05 Meeting Demand: Optimisation of Our Transient Expression Platforms to Increase Throughput and Titre

Christina Gordon, Scientist, New Meds, UCB Pharma

Development of antibody therapeutics, from early stage research to preclinical and clinical development, requires ever-increasing amounts of reagents. To meet the challenge of furnishing a diverse and full pipeline, we utilise several different transient platforms. Through continuous optimisation, streamlining and automation of component parts of our panel of platforms (utilising both HEK293 and CHO host cells with a variety of transfection methods), we now have the capability to produce microgram-to-gram quantities of panels of purified antibodies and antibody fragments in as few as four weeks from receipt of plasmid DNA.

09:35 Server of Many Masters: The Challenges of a Slim Protein Expression Core Facility

Tsafi Danieli, Ph.D., Director, BioGiv Excubator & Head, Protein Expression Facility, Wolfson Centre for Applied Structural Biology, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem

Running a small core facility that serves multiple academic groups with a wide range of disciplines is extremely challenging. Trying to do it with one technician, two postdocs and no automated platforms sounds like mission impossible. However, it is all about the surrounding! An academic environment and supporting ecosystem allowed our facility to turn the challenge into a new model that successfully accommodates not only academic groups but also early stage biotech startups.

10:05 Application of Streamlined Processes to Improve Process Productivity and Throughput

Naz Dadehbeigi, Pharm.D., Ph.D., Principal Scientist, Fujifilm Diosynth Biotechnologies

10:35 Coffee Break in the Exhibit Hall with Poster Viewing

CASE STUDIES: PROTEIN TO PRODUCT (CONT.)

11:15 Efficient Production of Recombinant Human Monoclonal Antibodies from Single B Cells

Hugo Mouquet, Ph.D., Head, Laboratory of Humoral Response to Pathogens, Immunology, Institut Pasteur

The molecular dissection of anti-pathogen B-cell responses using modern technologies to efficiently generate and characterize antigen-specific human monoclonal antibodies has allowed breakthrough discoveries in antiviral responses to viruses such as Influenza virus and HIV-1. These recombinant antibodies represent unique “fingerprints” for each B-cell clone and when characterized at a molecular and functional level, provide crucial information to help developing therapeutic and vaccine strategies against a given pathogen.

11:45 FEATURED PRESENTATION: Expressing Challenging Proteins at the SGC

Nicola Burgess-Brown, Ph.D., Principal Investigator, Biotechnology, Structural Genomics Consortium (SGC), University of Oxford

The SGC has solved >1700 human protein structures and 6 novel integral membrane proteins. Although we have significantly contributed to structural biology and protein production for functional studies, there are many highly desired targets including protein-protein complexes and known drug targets that remain a challenge to obtain. We present our established expression systems using E. coli and BEVS, and new processes (BacMam and mutagenesis) to generate the most difficult-to-produce proteins.

12:15 Enjoy Lunch on Your Own

13:00 Dessert Break in the Exhibit Hall with Poster Viewing

13:30 End of Optimising Expression Platforms

Day 1 | Day 2 | Speaker Biographies | Download Brochure

Conference Programs

• Display of Biologics
• Engineering Antibodies
• Machine Learning - Part 2

• Antibody-Based Therapies
• Emerging Targets & Approaches
• Membrane Protein Targets

• Safety & Efficacy
• Advancing Bispecifics
• Engineering Bispecifics

• Modulating the TME
• Innovative CAR T Therapy
• Next-Gen Immunotherapies

• Optimisation & Developability
• Analytical Characterisation
• Protein Stability & Formulation

• Data Science & Engineering
• Optimising Expression
• Process Development

• Intro to Machine Learning
• Machine Learning - Part 1
• Machine Learning - Part 2

• Antibody-based Therapies
• Engineering Conjugates
• Next-Gen Immunotherapies