Cambridge Healthtech Institute’s 7th Annual
Difficult to Express Proteins
Solving Problems with Finicky Proteins
3-4 November 2014
This opening meeting of the protein stream for PEGS Europe will focus on the “finicky” proteins that are extremely challenging to expression. Vaccines, hormones, membrane proteins, ion channels, low-abundance proteins and protein complexes continue to cause issues for bench scientists. This meeting will focus on the novel and imaginative solutions for these problems.
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Monday, 3 November
12:00 Conference Registration
13:45 PEGS Europe Team Welcome
13:50 Chairperson’s Opening Remarks
William Finlay, Ph.D., Director, Global Biotherapeutic Technologies, Pfizer, Inc.
14:00 The Impact of the New Regulatory Guidance Landscape on the Validation of the Manufacturing Process and the Characterisation of Starting Materials, Drug Substance and Drug Product
Steffen Gross, Ph.D., Head, Monoclonal and Polyclonal Antibodies, Paul-Ehrlich-Institut
The regulatory guidance landscape regarding biological products changes rapidly. Documents overseeing process validation is currently facing major overhauls. New guidelines are published defining the requirements for starting materials and excipients. This might have a deep impact not only on the process development of traditional monoclonal antibody type products but also on new antibody formats such as conjugates as well as on new developed formulations.
14:45 Current Progress with Armed Antibody Products
Dario Neri, Ph.D., Professor, Chemistry and Applied Biosciences, ETH Zurich
There is an emerging trend in Pharmaceutical Biotechnology to “arm” antibodies, capable of selective localization at the site of disease, with suitable therapeutic payloads (e.g., drugs, cytokines, radionuclides, a second antibody moiety, hence producing a bispecific product). This strategy aims at concentrating therapeutic agents in diseased part of the body, while sparing normal tissues. In this lecture, I will present a comparative evaluation of different classes of armed antibody products, developed in my laboratory in collaboration with Philogen. In particular, I will present preclinical and clinical data of armed antibodies in the field of cancer, of chronic inflammation and of endometriosis.
15:30 Refreshment Break
16:00 Chairperson’s Opening Remarks
16:05 KEYNOTE PRESENTATION
Production of Protein Tools in Support of R&D Pharma Projects
Jacques Dumas, Ph.D., Head, Protein Production, Global Biotherapeutics, sanofi
The genomic era of the 90’s is culminating in the identification and production of novel protein targets for drug discovery. Increasing numbers of “high value” proteins are used for crystallography and High Throughput Screening (HTS). Proteins are produced by recombinant technology and purified to homogeneity using platform strategies. In the last 10 years, strategies have evolved to adapt to difficult to express proteins, such as kinases. In addition, the revival of biotherapeutic proteins has generated the need for new protein tools.
16:35 High-Yield, Zero-Leakage Expression System in Escherichia coli
Yusuke Kato, Ph.D., Senior Researcher, Genetically Modified Organism Research Centre, National Institute of Agrobiological Sciences
We designed a high-yield, zero-leakage expression system using a transcriptional-translational double-regulation for toxic protein production in E. coli. “Translational switches” were constructed using site-specific unnatural amino acid incorporation at the amber stop codons that were inserted in target genes. Under repression conditions, leakage-expression was completely abolished by a synergistic repression both at the transcriptional and translational levels. In contrast, both transcription and translation were fully activated under induction conditions.
17:05 Addressing Challenges for Production of Human Proteins and Multi-Protein Complexes Using the Baculovirus Expression System (BEVS)
Arnaud Poterzman, Research Director, Integrated Structural Biology, IGBMC/CNRS
We present here recent advances for the production of multi-subunit complexes in the baculovirus expression system using human multi-subunit transcription factors as model systems: Vector development for parallel expression/co-expression screening, use of Lambda red recombination in E. coli for manipulation and improvement of the baculoviral genome and assembly of multi-gene constructs from synthetic biology approaches. We will also discuss use of fluorescent proteins as infection makers and of baculovirus-infected insect cells (BIIC) for storage and standardization.
17:35 Sponsored Presentation (Opportunity Available)
18:05 Welcome Reception in the Exhibit Hall with Poster Viewing
19:05 End of Day One
Tuesday, 4 November
07:45 Breakfast Presentation (Sponsorship Opportunity Available) or Morning Coffee
08:30 Chairperson’s Remarks
08:40 Higher Yield – An Ultimate Goal for Biological Product Development: A Case Study about Use of Process Intensification Technology for a Difficult to Express Glycoprotein
Mallika Singh, Ph.D., Associate Director, Upstream Development & Manufacturing, Teva Biopharmaceuticals USA, Inc.
The case study will show the use of an emerging technology (process intensification technology using Alternating Tangential Flow, ATF) which will perhaps be the direction of cell culture manufacturing process for biologics in near future.
09:10 Exploring Codon Optimisation Strategies for Production of Membrane Proteins
Morten Nørholm, Ph.D., Academic Entrepreneurial Research Group Leader, DTU Biosustain; Technical University of Denmark, Novo Nordisk Foundation Center for Biosustainability
Using a library of GFP-tagged membrane proteins, we have compared different codon optimisation strategies including synonymous mutations in the 5´end, complete re-coding using multiparameter optimisation algorithms and complementing rare codon usage with additional copies of the corresponding low-concentration tRNAs.
09:40 Chaperones Enable Native Folding of a Disulfide-rich Scorpion Toxin in the E.coli Periplasm
Andrias O’Reilley, Ph.D., Researcher, Department of Physiology and Pathophysiology, Friedrich-Alexander Universität Erlangen-Nürnberg
Animal neurotoxin peptides are valuable probes for studying ion channel pharmacology. Misfolding through formation of incorrect disulfide isomers has hindered recombinant toxin expression in E.coli. We report that co-secretion of a suite of protein disulphideisomerases and peptidyl-prolylcis/trans-isomerases into the E.coli periplasm boosts expression and produces correct folding of an insecticidal scorpion toxin, as validated by X-ray crystallography.
10:10 Coffee Break in the Exhibit Hall with Poster Viewing
10:50 A S. cerevisiae-Based High Yield Expression System for Efficient Purification of High Quality Human/Eukaryotic Membrane Proteins for Structural Studies
Per Armstrup Pedersen, Ph.D., Professor, Department of Biology, University of Copenhagen, Copenhagen, Denmark; Center for Microbial Biotechnology, Department of Systems Biology, Technical University of Denmark
We have managed to develop a S. cerevisiae-based platform with the capacity to deposit eukaryotic membrane proteins to a density of up till 8% of total membrane protein content and identified conditions that allow efficient purification of high quality eukaryotic membrane proteins. We believe that our expression platform is of relevance for any membrane protein as we have managed to express and purify, 7TM receptors, P-type ATPases, K-channels, amino acid/hexose transporters/tranceptors.
11:20 Sponsored Presentation (Opportunity Available)
11:50 Problem Solving Roundtable Discussions
Using Codon Optimisation to Facilitate Expression of Membrane Proteins
Moderator(s): Morton Nørholm, Ph.D., Academic Entrepreneurial Research Group Leader, DTU Biosustain; Technical University of Denmark, Novo Nordisk Foundation Center for Biosustainability
- Codon optimization choices
- Host-related decisions
- What to expect
Recombinant Expression of Toxins and Peptides
Moderator: Jacques Dumas, Ph.D., Head, Protein Production Vitry, Biologics SCP, Vitry Research Center, sanofi
- Expression systems
- Peptide size limitations
- Mammalian vs microbial
- Is recombinant expression competitive to synthesis?
Overcoming Challenges in Protein Expression with Baculovirus
Moderator: Arnaud Poterzman, Research Director, Integrated Structural Biology, IGBMC/CNRS
- When is baculovirus better?
- Tools for improving expression
- Tweaking the system
12:50 Luncheon Presentation (Sponsorship Opportunity Available) or Lunch on Your Own
14:00 Dessert Break in the Exhibit Hall with Poster Viewing
14:30 Chairperson’s Remarks
Stefan Schmidt, Ph.D., Vice President, Downstream Processing, Rentschler Biotechnology
14:35 Modular Approaches to Express Complex Therapeutic Proteins
Stefan Schmidt, Ph.D., Vice President, Downstream Processing, Rentschler Biotechnology
Difficult to express proteins represent an increasing amount of therapeutic molecules. This causes bioprocessing challenges such as controlling glycosylation, increasing titers and yields, suppressing aggregation and purification without specific affinity resins. Here we demonstrate how to develop modular quasi-platform processes solving these issues. The case studies with examples from various molecule classes highlight successful process design, optimisation strategies and critical manufacturing parameters. Additionally practical advice will be given what to consider when designing a novel molecule.
15:05 Enhancing Protein Secretion of Clinically-Important Proteins
Tsafi Danieli, Ph.D., Head of Protein Expression Facility, Wolfson Centre for Applied Structural Biology, Alexander Silberman Institute of Life Sciences
The ability to improve recombinant protein secretion has significant biological and commercial benefits; Based on high throughput screening experiments, followed by bioinformatics analysis of protein databases of secreted vs. non-secreted proteins, we have developed an algorithm that can predict whether a protein will be poorly or efficiently secreted. We have tested our algorithm on several poorly secreted proteins, and found they all contained specific motifs that we predicted to impair translocation. We then demonstrated that disruption of these motifs by conserved replacement of a few amino acids resulted in a dramatic (up to 15 fold) enhancement of protein secretion in E.coli, insect cells and mammalian cells.
15:35 Heterologous Expression and Purification of an Active Human TRPV3 Ion Channel
Stefan Kol, Ph.D., Protein Biochemist, Novo Nordisk Foundation Centre for Biosustainability, Danish Technical University
In spite of great progress in the TRP-channel characterization, very little is known about their structure and interactions with other proteins at the atomic level. This is mainly caused by difficulties in obtaining functionally active samples of high homogeneity. I will present here the high-level Escherichia coli expression of the human TRPV3 channel, which retains its current inducing activity.
16:05 Sponsored Presentation (Opportunity Available)
16:35 Refreshment Break in the Exhibit Hall with Poster Viewing
17:15 Expression of Soluble and Active Interferon Consensus in SUMO Fusion Expression System in E. coli
Emmanuelle Laurine, Ph.D., PolyTherics Ltd.; The London Bioscience Innovation Centre
Interferon consensus (IFN-con) is a non-natural recombinant interferon alpha displaying superior activity compared to most clinically used IFNα-2a. However, production of this therapeutically promising protein remains challenging. Herein, we describe the optimisation of the production of IFN-con in E.coli as a soluble IFN-con. The recoded IFN-con gene was cloned into the Champion™ pET SUMO expression vector downstream of the SUMO fusion protein under T7lac promoter and expressed in E.coli SHuffle strain. The fusion protein was efficiently expressed in soluble form and IFN-con remained stable following removal of the SUMO fusion partner. In addition, antiviral activity of the produced IFN-con was proven to be greater than activity of IFN α-2a.
17:45 A Technology for the Consistent Generation of Functional Antibody-Drug Candidates to Membrane Proteins
C. Davis Farmer, Jr., Chairman, MSM Protein Technologies
MSM Protein Technologies has developed proprietary methods for generating cell lines that over-express complex membrane proteins. We also have displays to present these proteins in their native conformation, highly purified for use in antibody discovery. To date we have generated fully human antibodies to several GPCRs that have met very stringent criteria as drug candidates. We will present an overview of the platforms and examples of antibodies that we have generated.
18:15 Expression of USP18 (UBP43) for Enzymatic and Structural Analysis using a Trigger Factor Fusion System in E.Coli and Baculovirus Expression
Klaus Peter Knobeloch, Ph.D., Head, Liebniz Institute for Molecular Pharmacology
Protein modification by ISG15 represents an interferon effector system counteracted by USP18. In mice, we selectively inactivated USP18 protease activity. Enhanced ISGylation increased resistance against influenza-virus infections qualifying USP18 inhibition as a potential antiviral strategy. Using a trigger factor fusion system in E.Coli and baculovirus expression we overcame poor USP18 expression yields, weak enzymatic activity and limited solubility, enabling us to perform inhibitor screens and solve the USP18/ISG15 complex crystal structure.
18:45 End of Difficult to Express Proteins
Speaker Biographies | Download Brochure | Register