The pursuit for biotherapeutic proteins in basic research, clinical diagnostics and therapy continues at an exponential pace. Consequently, the demands for efficient expression and production of these valuable biomolecules face challenges to improve quantity
and quality while minimising time and cost. Thus, an increasing variety of recombinant production platforms, called “cell factories,” are being developed. Unfortunately, there is no universal production system which can guarantee high
yields, since each protein can vary in terms of expression and production.
Final Agenda
WEDNESDAY 20 NOVEMBER
07:45 Registration (Foyer A) and Morning Coffee (Foyer D)
08:30 Chairperson’s Opening Remarks
Peter Schmidt, PhD, Director, Recombinant Technologies, CSL Behring
08:35 KEYNOTE PRESENTATION: Tag-on-Demand: Exploiting Amber Codon Suppression Technology for the Enrichment of High-Expressing Membrane Protein Cell Lines
Trevor Wilkinson, PhD, Associate Director, Antibody Discovery & Protein Engineering, AstraZeneca Biopharmaceuticals Unit
Multi-spanning membrane proteins such as G-protein-coupled receptors and Ion Channels are of significant therapeutic interest. Targeting these proteins with Monoclonal Antibodies has proven particularly challenging, in part due to the difficulties in
obtaining high-level expression of these membrane protein antigens. In this presentation we will explore case studies illustrating how these challenges may be overcome, including how a Tag-on-Demand strategy can facilitate enrichment of high-expressing
membrane protein cell lines.
09:05 Technologies for High-Level (Membrane) Protein Production in Mammalian Cells
Jonathan Elegheert, PhD, Team Leader, Interdisciplinary Institute for NeuroScience
(IINS), CNRS, University of Bordeaux
Structural, biochemical, and biophysical studies of soluble and membrane proteins typically require their production in milligram quantities. Difficult-to-produce eukaryotic proteins are generally best expressed from close-to-native mammalian cell types.
I will compare different approaches for the production of soluble and membrane proteins from mammalian cells and discuss their strengths and weaknesses in function of the protein target and application, as well as their practical implementation.
09:35 High-Yield Production of “Difficult-to-Express” Proteins in an Improved Cell-Free System
Takanori Kigawa, DSci, Team Leader, Center for Biosystems Dynamics Research, RIKEN
We have developed a new method of E. coli cell extract-based cell-free protein synthesis optimal at lower temperatures (20-25 ºC) achieving high-yield production comparable to the conventional method (30-37 ºC).
This method is suitable for expressing proteins that tend to aggregate and/or be insoluble at optimal temperatures for the conventional method (30-37 ºC). Therefore, our new method is particularly useful for expressing “difficult-to-express”
proteins.
10:05 Accelerating Discovery and Development of Next-Generation Therapeutic Antibodies using the Beacon Platform
John Proctor, PhD, Senior Vice President, Berkeley Lights, Inc.
The Beacon platform dramatically accelerates the discovery and development of next-generation antibody therapeutics. The presentation will demonstrate how Berkeley Lights’ customers are using Plasma B Cell Antibody Discovery to
perform target-to-lead candidate selection in just 1 day. Once lead candidates have been selected, Beacon Cell-Line Development enables the selection of production cell lines with >99% monoclonality assurance in less than 1 week.
10:35 Coffee Break in the Exhibit Hall with Poster Viewing (Rio Pavilion)
11:15 Production of Hard-to-Produce Proteins Using Genome Engineered CHO Cells
Bjørn Voldborg, MSc, Director, CHO Cell Line Development, The Novo Nordisk
Foundation Center for Biosustainability, Technical University of Denmark
Using our in-house developed high throughput CHO cell line engineering platform, we have engineered the glycosylation machinery to make a panel of CHO cell lines for the expression of recombinant proteins with tailored N-glycans. Using these
cells, we have produced a therapeutic protein that until now has only been available from natural human sources. The produced protein resembles the human derived proteins with respect to N-glycan profile and activity.
11:45 The IC-Tagging Platform and Its Use for the Expression of Difficult Proteins
Jose M. Martinez-Costas,
PhD, Profezsor Titular, Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS); Departamento de Bioquímica e Bioloxía Molecular, Universidade de Santiago de Compostela
We have developed a platform that programs cells to construct nano/microspheres that integrate any protein of interest and that are easily purified. Between the multiple applications of this technology, we have recently shown its potential
in the expression of difficult/toxic proteins by expressing, between others, the highly demanded diabetogenic auto-antigen protein IGRP opening the possibility of further studies on type 1 diabetes.
12:15 HEK293 Cell Lines Allow Rescue of Proteins that are Difficult to Produce in CHO – Learning Lessons from Endogenous Secretory Pathway Expression
Magdalena Malm, PhD, MSc, Researcher & Lab Manager, Wallenberg Center for
Protein Research, KTH Royal Institute of Technology
Evaluation of the recombinant expression of 24 secreted human difficult-to-express proteins showed generally improved expression in HEK293 compared to CHO cells. Transcriptomic analysis was used to identify key differences between the secretory
pathways of the two cell lines and to study genes differentially activated upon transgene expression. The findings suggest lessons to be learnt from each cell line based on endogenous secretory pathway gene expression.
12:45 Optimization of E. Coli Solupro® Using Synthetic Biology to Generate a Scalable, High-Yield Chassis Microbe
Melissa Patterson, Purification Group Leader, Downstream Process Development, AbSci
13:00 ALiCE - Makes Production of Difficult-To-Express
Proteins Easy
Ricarda
Finnern, PhD, Research & Development, LenioBio GmbH
ALiCE is the first eukaryotic cell-free platform, scalable according to customer´s needs. Combining the best properties of cell-based, cell-free, eukaryotic and prokaryotic systems, ALiCE is the most versatile protein production system.
It allows single step production of proteins that have been difficult to make using other technologies in unpresented quantities.
13:15 Luncheon Presentation I: Utilising Directed Evolution and Droplet Microfluidics to Increase Titres and Accelerate Cell Line Development Timelines
Devika Kalsi, Principal Scientist, MCC Upstream Process Development, FUJIFILM DIosynth Biotechnologies
Cell line development (CLD) is traditionally a lengthy process, limited by cellular heterogeneity and requirements of high probability and assurance of monoclonality. We employed directed evolution, yielding host lines with improved biomanufacturing
phenotypes. Combining single cell deposition, imaging and screening of Sphere Fluidics Cyto-Mine® with plate imaging created novel workflows for generating clones with high probability and assurance of monoclonality in one cloning
round. This intensified our CHO CLD process from 25 to 10 weeks.
13:45 Luncheon Presentation II: ExpiSf™ Expression system: A Chemically Defined Baculovirus-Based Expression System for Enhanced Protein and Virus Production in Sf9 Cells
Kenneth Thompson, Research & Development Manager, Cell Biology, Thermo Fisher Scientific
The Baculovirus Expression Vector System is a versatile platform for expression of individual recombinant proteins, membrane proteins, virus-like particles and Adeno-associated virus (AAV). Here, we describe the ExpiSf™ Expression
System, the first chemically defined insect system that enables high-yield production of proteins and viral particles at small and large scales. The system includes an improved transfection reagent and updated protocol for
faster baculovirus generation and a chemically defined enhancer to boost protein and virus production.
14:15 Session Break
14:30 Chairperson’s Remarks
Richard Altman, MS, Staff Scientist, Life Science Solutions,
Thermo Fisher Scientific
14:35 The Use of Design of Experiments in Recombinant Protein Production: Concepts and Case Studies
Barry Ryan, BSc (Hons), PGDip, MSc, MA, PhD, Lecturer, Food Science and Environmental Health,
College of Health and Science, Technological University Dublin
Many factors, both intrinsic and extrinsic, can influence recombinant protein yield; however, identifying the most important factors, individually or synergistically, for optimum yield can be time consuming and expensive. Statistical models,
such as Design of Experiments (DoE), can be used as efficient approaches to recombinant protein production optimisation. Fundamental concepts of DoE, with supporting case studies, will underpin an overview of the potential of this method
for enhanced recombinant protein production.
15:05 Playing a Round with Recombinant Protein Production: What is PAR for the Course
Douglas Browning, PhD, Senior Research Fellow, Institute of Microbiology and Infection, School of Biosciences, University of Birmingham
Many promoters used for recombinant protein production (RPP) in Escherichia coli are capable of high-level protein expression but are often too strong for expressing proteins that are difficult to fold or targeted to the membrane
or periplasm. To circumvent this, we have engineered a suite of bacterial promoters (the PAR promoters) and strains that can be used to tailor the expression of target proteins to maximize RPP and product yield.
15:35 Refreshment Break in the Exhibit Hall with Poster Viewing (Rio Pavilion)
16:15 Overview of a High-Throughput Pipeline for Streamlining the Production of Recombinant Proteins for Structural Biology
Raymond J. Owens, PhD, Professor, Research Complex at Hartwell & Rosalind Franklin Institute, University of Oxford
There has been a transformation in the power and throughput of structural methods delivered by large-scale facilities, such as synchrotrons. This has placed an increasing demand on the supply of high-quality samples (purified proteins and
protein crystals) for structural studies. To meet this requirement, protein production has been streamlined to improve efficiency and throughput. Our experience will be reviewed and new trends discussed.
16:45 Improved Production of Recombinant Proteins from Insect Cells through Promoter, Virus, and Strain Enhancements
Dominic Esposito, PhD, Director, Protein Expression
Laboratory, Frederick National Laboratory for Cancer Research
Baculovirus-based insect cell expression platforms are often successfully used for production of pharmaceutically relevant proteins. However, the insect cell system remains suboptimal in terms of technology development related to controlling
the level of protein production, stability of baculoviruses for large-scale production, and modification of host insect cell lines for improved performance. We have begun to address some of these deficiencies and demonstrate the use of
these improved systems for production of clinically relevant drug targets.
17:15 Choosing Right between Transient and Stable Protein Expression Systems While Supporting Fast-Paced Biologics Discovery
Kinjal Mehta, PhD, Principal Scientist, Protein Sciences, Jounce Therapeutics
Within the fast-paced realm of biotechnology, research groups are often tasked with generating large amounts of proteins with the highest quality possible while continuously shortening turnaround times. This becomes a challenge with therapeutic
candidates because factors like glycosylation patterns and consistency with biophysical/chemical properties become critical. Choosing between transient and stable protein production becomes an important step at each stage. We have developed
a platform where we use a CHO-GS KO cell line, typically used under stable conditions, and have adapted it to a transient system with a robust protocol that consistently results in high titers.
17:45 Networking Reception in the Exhibit Hall with Poster Viewing (Rio Pavilion)
18:45 Problem-Solving Breakout Discussions
TABLE 22: Common Issues with Transient Protein Production
Moderators: Richard Altman, MS, Field Application Scientist, Protein Expression, Biosciences Division, Life Sciences Solutions Group, Thermo Fisher Scientific
Henry C. Chiou, PhD, Director, Cell Biology, Life Science Solutions, Thermo Fisher Scientific
Dominic Esposito, PhD, 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 as well as an important tool to generate recombinant proteins for a variety of
other applications. 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?
- How can we maintain volumetric yields while scaling transient expression up or down?
- What cell line(s) should we use and when?
- What parameters can impact the quality or physical attributes of transiently produced proteins?
TABLE 23: High-Throughput Purification, Stability Screening
Moderator: Peter Schmidt, PhD, Director, Recombinant Technologies, CSL Behring
- High-throughput purification approaches
- Conformational stability screening methods
- Colloidal stability screening methods
- Early research formulation strategies
TABLE 24: Cell-Free Protein Synthesis: Pros and Cons
Moderator: Takanori Kigawa, DSci, Team Leader, Center for Biosystems Dynamics Research, RIKEN
- Cell-free expression vs. cellular expression
- Prokaryotic system vs. Eukaryotic system
- Good practices
19:45 End of Day
THURSDAY 21 NOVEMBER
08:00 Registration (Foyer A) and Morning Coffee (Foyer D)
08:30 Chairperson’s Remarks
Renate Kunert, PhD, Professor, Department of Biotechnology, University
of Natural Resources and Life Sciences (BOKU)
08:35 Producing High-Quality Bispecific Material from Transients for Developability Assessment
Claire Pearce, PhD, Senior Research Scientist, CHO Expression Team Leader, Biopharmaceutical Development, Kymab Ltd.
Antibody developability continues to be a hot topic, with the ability to develop and manufacture a molecule having equal importance to its research properties. This presentation will detail the supply and developability assessment
of a panel of bispecific molecules expressed from a rapid transient platform, and the comparability of this material to that expressed from a stable process.
09:05 Novel Highly Productive Production System for Biotherapeutics: Filamentous Fungus Myceliophpthora thermophila C1
Markku Saloheimo, PhD, Senior Principal Scientist, Industrial Biotechnology and Food Research, VTT Technical Research Centre of Finland
We are developing the industrial enzyme production host C1 into an efficient and versatile therapeutic protein production platform. A major topic in our research has been reduction of the host protease level. This has enabled superb
production levels of Mabs, Fc-fusion proteins, bispecifics and vaccine proteins in a 6- to 7-day process in C1. Glycoengineering is also under way to humanize the glycosylation pathway of this production host.
09:35 Rapid Selection of CHO Clones Secreting Chimeric Antibody-Antigen Fusion Constructs Based on 2A-Peptide Cleavage and GFP
Bert Devriendt, PhD, Postdoctoral Scientist, Department of Virology,
Parasitology, Immunology, Physiology, Ghent University
To enable large-scale recombinant antibody production, a high producer cell line is essential. Selecting such a cell line is however time consuming and labor intensive. By combining the design of a tri-cistronic vector expressing GFP
and both antibody chains, separated by a GT2A sequence, with single cell sorting and automated image analysis, a CHO cell line was rapidly selected producing high amounts of recombinant antibodies, which showed minimal degradation.
10:05 Scaling Up and Scaling Out: Pushing the Boundaries of Transient Protein Production
Ian Wilkinson, CSO, Absolute Antibody Ltd.
Whilst transient yields have improved drastically in the last decade, scalable systems are time-consuming and costly to implement. Absolute Antibody has developed systems which scale up and scale out protein expression and purification,
enabling the rapid and cost-effective production of milligram-to-gram quantities of large panels of proteins.
10:20 High Density (HD) Expression Platform: The One-Stop-Solution for Recombinant Antibody Production
Bowu Luan, PhD, Product Manager, GenScript USA, Inc.
GenScript has developed a novel reagent “Cocktail” compatible with HD expression system, which improves antibody yield by increasing cell viability and facilitating protein folding. This HD system works well with all species
and low expressers, readily to scale down and up. Automatic workflow from transfection to purification ensures the quality.
10:35 Coffee Break in the Exhibit Hall with Poster Viewing (Rio Pavilion)
11:15 Influence of Somatic Mutations on mAb Expression and Thermal Stability Properties
Renate Kunert, PhD, Professor, Department of Biotechnology, University
of Natural Resources and Life Sciences (BOKU)
The production potential of monoclonal antibody (mAb) expressing cell lines depends on the intrinsic antibody structure and its interaction with cellular compartments especially the folding and secretion machinery. To get a better
understanding of such relations we expressed different mAbs under isogenic conditions in recombinant CHO cells and studied cellular biology and physicochemical properties of mAbs.
11:45 High Throughput Antibody Production and Purification: Day to Day Challenges and How to Overcome Them
Peter Schmidt, PhD, Director, Recombinant Technologies, CSL Behring
Monoclonal antibodies are the fastest growing segment in the drug market. The development of mAbs requires purification of large numbers of variants with sufficient yield. However, established high-throughput purification strategies
have been limited by the binding capacity of established affinity matrices. The presentation will address some of the known and less known issues and suggest ways to overcome them.
12:15 Luncheon Presentation I: Establishment of a
CHO Transient Expression Platform for Bispecific Fusion Protein Developability Assessment
Thibaut Angevin, PhD,
Pieris Pharmaceuticals GmbH
Pieris Pharmaceuticals’ proprietary molecules, the Anticalin® proteins, are small therapeutics derived from human lipocalins. They can be genetically linked to antibodies to generate different bispecific fusion proteins.
Those proteins display different developability profiles. Pieris’ developability assessment platform has been established in order to screen bispecific constructs at an early stage, with an aim of identifying candidates
that are best suited for advancement to stable cell line development or further optimization of such constructs.
12:45 Luncheon Presentation II: GlycoExpress® - An Alternative Host for Difficult to Express Proteins
Lars Stöckl, PhD, Senior Director BD and Technology, Glycotope GmbH
The era of classical IgG molecules in bio-pharma development is shifting rapidly to more challenging complex biopharmaceuticals. With CHO being a good production cell line for IgG molecules, they might fail to produce more challenging
candidates. The GlycoExpress® (GEX®) system represents an ideal alternative for the production these difficult to express protein molecules and will provide case studies which demonstrate the superiority in productivity
and product quality vs CHO cell expression.
13:15 Dessert Break in the Exhibit Hall with Poster Viewing (Rio Pavilion)
14:00 End of Optimising Expression Platforms
17:00 Dinner Short Course Registration*
17:30 – 20:30 Dinner Short Courses