2015 Archived Content
Cambridge Healthtech Institute’s Eighth Annual
Applying Expression Platforms
Meeting the Demand for Recombinant Proteins
4-5 November 2015
Biopharmaceuticals represent the fastest‐growing sector of the pharmaceutical industry, driven by the rapidly expanding manufacture of recombinant protein‐based drugs. Key properties of individual expression systems and characteristics of the targeted
recombinant protein affect the quality and quantity of production. Thus, early steps in the development of a functionally active protein often require multiple iterations to optimise expression, production and purification.
The Applying Expression Platforms conference addresses the strategies required in the transition from producing recombinant proteins for the laboratory to preparing for clinical production.
Final Agenda
Day 1 | Day 2 | Speaker Biographies | Download Brochure
Wednesday, 4 November
07:45 Registration and Morning Coffee
08:30 Chairperson’s Opening Remarks
Zhiwei Song, Ph.D., Principal Scientist, Lead PI for GlycoSing Programme, Bioprocessing Technology Institute, A*STAR
KEYNOTE PRESENTATION
08:35 Alternative Cell Line Development: The Use of Polycistronic Vectors and Alternate-Splicing for Multimeric Protein Expression
Pierre Moretti, Ph.D., Head, Cell Line Development, Glenmark Pharmaceuticals
This talk introduces the alternate splicing-based multicistronic vectors developed at Glenmark for the expression of multimeric protein such as bispecific antibodies. September 2015 Speaker Interview
09:20 Boosting Antibody Cell Line Performance without Compromising the Desired Glycan Composition
Volker Sandig, M.D., Ph.D., CSO, ProBioGen
An optimal and consistent glycan pattern is desired for novel antibodies while reproduction of the original pattern is critical for biosimilar development. When selecting the final clone, posttranslational modifications may get in conflict with process
performance and maximal titer. An advanced flexible vector platform and control of complex N-Glycans (including fucosylation, galactosylation and sialylation) with heterologous enzymes, glycan precursors and microelements support the perfect choice
for each antibody.
09:50 A Quick and Efficient Method to Generate Mammalian Stable Cell Lines Based on a Novel Inducible Alphavirus DNA/RNA Layered System
Alejandro Aranda, Ph.D., Principal Investigator, Vectors Development Lab, END-ICAP Unit, INSERM, University
of Versailles – Saint Quentin en Yvelines
We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. New cell
lines can be generated based on MCL by recombinase mediated cassette exchange, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest.
10:20 Platform Expression Systems for Rapid Development of Microbial & Mammalian Cell Lines for Biopharmaceutical Production
Ian Hodgson, Ph.D., BSc, Head, Molecular Biology, FUJIFILM Diosynth Biotechnologies
The process development of biopharmaceuticals demands concise timelines (to get products into clinic quickly), high product titres (with correct product quality), coupled with the ability to use the same expression system throughout the product life cycle.
We have developed platform approaches to cell line development for both bacterial and mammalian expression, which combine all of these attributes. We will describe the development of these systems, and show data that demonstrates their performance.
10:50 Coffee Break in the Exhibit Hall with Poster Viewing
11:30 Expression and Cell Line Development of Complex and Fc-Modified Therapeutic Antibodies
Johannes Auer, Ph.D., Principal Scientist, Large Molecule Research, Roche Pharma Research & Development,
Roche Innovation Center Penzberg
The process of generating complex therapeutic antibodies from first test expressions until the selection of cell clones for the primary seed bank will be described. Exemplified insights will be presented into how we determine individual characteristics
of a given molecule, how we balance quantity and quality of the product and how we deal with proof of monoclonality of cell clones and their stable productivity for clinical supply.
12:00 Generation of Tagged Mammalian Cell Lines with an Improved Recombinase Exchangeable Cassette
Johannes Spehr, Ph.D., Research Scientist, Recombinant Protein Expression, Helmholtz Centre for Infection
Research
Recombinase mediated cassette exchange (RMCE) is a fast and reliable technique to stably integrate a gene-of-interest into a well-characterized master cell line. By using the trifunctional selective marker Hygromycinephosphotransferase-Thymidinkinase-eGFP
(HTG) we improved and accelerated the generation of new producer cell lines for high expression. The HTG combines positive selection for Hygromycin and eGFP during master cell line generation with negative selection on Ganciclovir during cassette
exchange.
12:30 Rapid Production of Recombinant Proteins and Stable Cell Lines
Peer Heine, Ph.D., Field Application Scientist, MaxCyte, Inc.
Success means getting to market fast. Therefore, fast and efficient protein production for drug candidate development, characterization, and selection is critical. Creating a stable cell line for clinic trial, takes months to more than a year for
complicated proteins. High efficiency, scalable electroporation can reduce stable cell line development timelines by up to 50%, even in difficult-to-transfect cell lines. In this presentation, data will show the rapid production of proteins and
cell lines at different scales.
13:00 Enjoy Lunch on Your Own
13:30 Session Break
14:00 Chairperson’s Remarks
Cecília Calado, Ph.D., Professor, Chemical Engineering Department, ISEL-Instituto Superior de Engenharia de Lisboa, Instituto Politécnico de Lisboa
14:05 Sequencing the CHO DXB11 Genome Reveals Regional Variations in Genomic Stability and Haploidy
Christian
Schrøder Kaas, Research Scientist, Mammalian Cell Technology, Novo Nordisk A/S
The copy number from each gene in the genome was calculated from next-generation sequencing data and revealed an unexpected degree of haploidy in Chinese Hamster Ovary (CHO) cells. The data can further be mined to reveal areas of the genome shown
to be stable, such as chromosomes one and four, which can be hypothesised to host favourable landing platforms for targeted integration of transgenes encoding coagulation factors or antibodies.
14:35 Tunable Gene Expression Control of the RiboTite System
Neil Dixon, Ph.D., MRSC, BBSRC David Phillips Research Fellow, Manchester Institute of Biotechnology, Faculty of Life
Sciences, University of Manchester
Here we report the development and application a novel recombinant E. coli expression system that operates at translational level. The RiboTite system has been benchmarked against classical gene expression control mechanisms for the production
of various reporter and target proteins of clinical importance. The system permits tunable cellular level regulation, tight basal control, high levels of expression upon induction, and predictable specific production rates across a range of growth
temperatures.
15:05 Minimally Invasive Host Cell Design and Growth Decoupled Protein Production - New Concepts to Further Advance E. coli Expression Systems
Gerald Striedner, Ph.D., Assistant Professor, Biotechnology, University of Natural Resources and
Life Sciences
Our E. coli expression system design is strongly focused on making them fit for production conditions. One strategy to improve fitness and performance of production cells is the reduction of the recombinant infrastructure to essential compounds
to minimise metabolic load and cell response to recombinant gene expression. An alternative concept is based on decoupling cell growth and product formation to allocate cells’ full protein synthesis capacity to recombinant protein production.
15:35 Refreshment Break in the Exhibit Hall with Poster Viewing
16:15 Cell Line Development and Early Bioprocessing: An Integrated Data Management Solution to Select the Right Clone
Claudia Götzberger-Schad, Ph.D., Senior Scientist, Global Biologics, Bayer Pharma AG
We present an integrated data management platform supporting the clone selection in the Cell Line Development (CLD) process. The system tracks all clones screened in high-throughput mode, collects all relevant characterisation data, such as productivity
and quality parameters, and streamlines high-throughput workflows by interfacing with automation equipment and bioreactors at different scales. A special focus of the presentation will be on fermentation in small-scale bioreactors to assess
quality and to guide clone selection and upscaling campaigns.
16:45 In situ Near-Infrared versus High-Throughput Mid-Infrared Spectroscopy to Monitor Biopharmaceuticals Production
Cecília Calado, Ph.D., Professor, Chemical Engineering Department, ISEL-Instituto Superior de
Engenharia de Lisboa, Instituto Politécnico de Lisboa
Global regulatory agencies impose stringent quality guidelines concerning biopharmaceutical production that have been changing to meet the Quality by Design (QbD) framework. Highly sensitive analytical techniques, as the ones based on Fourier
Transformed Infra-Red (FTIR) spectroscopy enabling simultaneous characterisation of heterologous product synthesis and physiologic cell behavior, are therefore desirable. We compare in situ near-FTIR versus high-throughput Mid-FTIR
spectroscopy concerning monitoring of critical process variables and general cell physiology.
17:15 PROBLEM SOLVING ROUNDTABLE DISCUSSIONS
Table 19: Opportunities and Challenges of Bacterial Production Strain Engineering Using Synthetic Biology
Moderator: Neil Dixon, Ph.D., MRSC, BBSRC David Phillips Research Fellow, Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester
- Bottom-up vs. top-down, which approach is most realistic for success - Can minimal genomes provide a good foundation on which to build?
- Can technologies such as CRISPR and MAGE enable high-throughput strain engineering for protein production?
- What are the current production strain bottlenecks or upstream challenges that could be targeted with such engineering approaches?
- Can strain engineering of bacterial hosts ever realistically produce complex authentic mammalian glycoproteins?
Table 20: Protein Glycosylation: Challenges and Opportunities for the Biotech Industry
Moderator: Zhiwei Song, Ph.D., Principal Scientist, Lead PI for GlycoSing Programme, Bioprocessing Technology Institute, A*STAR
- Does protein N- and O-glycosylation affect the quality of your recombinant therapeutics?
- Would you like to improve the quality of your product by glycoengineering the host cell?
- What analytical methods do you use to assess the glycosylation profiles?
- What should be added to your analytical toolbox?
Table 21: How to Shorten the Timeline for Drug Development with Large-Scale TGE in CHO Cells
Moderator: Peer Heine, Field Application Scientist, MaxCyte
- Achieve >2 g/L titers in CHO cells
- Stable vs. Transient Transfection
- Scale-up/Scale-down of transient transfection
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, 5 November
08:00 Morning Coffee
08:30 Chairperson’s Remarks
Georg Klima, Executive Director, Process Science Austria, Biopharmaceuticals Division, Boehringer Ingelheim RCV
FEATURED PRESENTATION
08:35 From Bench to Clinic – Fast Track Process Development for Novel Biotherapeutics in Microbial Expression Systems
Georg
Klima, Executive Director, Process Science Austria, Biopharmaceuticals Division, Boehringer Ingelheim RCV
Novel biotherapeutic formats pose unique development challenges as established platform processes are rarely applicable. To achieve rapid assessment of novel constructs’ safety in nonclinical and clinical testing, alternative process development
strategies are required. A versatile toolbox approach utilising E. coli and Pichia pastoris expression systems and automated high-throughput process development will be presented. Further, we highlight a systematic approach
for robust scale-up and transfer from bench to clinical scale manufacturing. September 2015 Speaker Interview
09:05 Eukaryotic Lysates for Cell-Free Bioproduction
Marlitt
Stech, Ph.D., Research Scientist, Cell-Free Bioproduction, Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses Potsdam-Golm (IZI-BB)
We introduce novel systems for the cell-free production of glycoproteins, membrane proteins and functional antibody fragments in an endotoxin-free environment. The eukaryotic translation systems developed are based on translationally active lysates
from cultured Sf21 and CHO cells which contain endogenous microsomal vesicles. The presence of these microsomes enables a co-translational translocation of target proteins, improved conditions for protein folding and enrichment and their subsequent
posttranslational modification.
09:35 Modular Expression Approaches Using Transient and Stable Mammalian Production
Mark Trautwein, Ph.D., Senior Research Scientist, Cell and Protein Sciences, Bayer HealthCare
Successful antibody lead discovery requires high-quality antigens, either as recombinant proteins or displayed on cellular surfaces. However, antigen production has notorious pitfalls in expression and purification. To ensure high probability
of success while minimising resource demands and keeping short timelines, we have implemented a step-wise approach using transient and stable expressions in different mammalian host cell lines taking advantage of a modular expression toolbox
allowing high-throughput expression optimisation.
10:05 cGMP Biologics Production Using Corynex® :
A Highly-Productive Gram-Positive Microbial Protein Secretion System
Yoshimi Kikuchi, Ph.D., Principal Researcher, Ajinomoto Co., Inc.
Corynex® is Ajinomoto’s highly productive protein expression system based on the gram-positive bacteria C. glutamicum. The easy-to-handle platform can secrete correctly-folded proteins directly into media with high purity, free from
cell lysis, refolding, endotoxins, and spores. We recently demonstrated successful 1000L GMP biologics production using Corynex®.
10:35 Coffee Break in the Exhibit Hall with Poster Viewing
11:15 The Incorporation of Non-Natural Amino Acids Enables Empowered Antibodies for Cancer Therapy
Gang
Yin, Ph.D., Principal Scientist, Protein Biochemistry, Sutro Biopharma, Inc.
A cell-free protein synthesis system was engineered for site-specific incorporation of non-natural amino acids (nnAAs). Methods have been developed for conjugation of synthetic molecules to antibodies or antibody to antibody by Azide/DBCO
or Tetrazine/Trans-cyclooctenes reaction at desired sites. Incorporation of non-natural amino acids and development of conjugation chemistries enable empowered antibodies for cancer therapy. Three applications were discussed: antibody
drug conjugates with single warhead and combination warheads, bispecific antibodies, and extended half-life antibodies by pegylation.
11:45
Wacker Biotech Refolding Technology – The Complement to our ESETEC® Secretion Platform
Guido Seidel, Ph.D., Managing Director, Operations, Wacker Biotech GmbH
Wacker Biotech is known as THE MICROBIOAL CMO. We lead the development of innovative, cost-saving E. coli technologies for recombinant manufacturing of biopharmaceuticals. Depending on customers need we can produce difficult to expressed proteins
via E. coli Secretion Technology - ESETEC® or with our proprietary refolding technology.
12:15 Enjoy Lunch on Your Own
13:00 Dessert Break in the Exhibit Hall with Poster Viewing
13:30 End of Applying Expression Platforms
Day 1 | Day 2 | Speaker Biographies | Download Brochure