CHI’s 5th annual Protein Expression System Engineering conference examines the functioning of the cellular machinery harnessed during protein biosynthesis and how to engineer hosts to efficiently express a protein of
interest. The intricate steps required to achieve properly folded protein will be discussed, including verification and sequence analysis of the gene, codon optimization, vector construction, selecting and optimizing a clone and selecting a host system.
In addition, engineering host cells to sustain expression for longer time periods will be discussed, along with overcoming cellular stress response to produce and secrete functionally active recombinant proteins.
Final Agenda
THURSDAY, APRIL 11
12:00 pm Registration (Commonwealth Hall)
12:35 Luncheon in the Exhibit Hall with Poster Viewing (Commonwealth Hall)
1:40 Chairperson’s Opening Remarks
Susan Sharfstein, PhD, Professor, Nanobioscience, Nanoscale Science and Engineering, SUNY Polytechnic Institute
1:50 KEYNOTE PRESENTATION: Mammalian Synthetic Biology: Foundations and Application to Cell Line Engineering
Ron
Weiss, PhD, Professor, Biological Engineering, Massachusetts Institute of Technology (MIT)
In this research, we appropriate from established engineering fields proven design principles such as abstraction, standardization, modularity, and computer aided design. But we also spend considerable effort towards understanding what makes synthetic
biology different from all other existing engineering disciplines and discovering new design rules that are effective for the biological substrate. Building on this foundation, I will describe our recent application of synthetic biology tools and
principles towards the improvement of cell line engineering and biomanufacturing.
2:20 Communicating with and Controlling Gene Expression via Redox-Linked Bioelectronics
William E. Bentley, PhD, Robert E. Fischell Distinguished Chair, Engineering; Inaugural Director, Robert E. Fischell Institute for Biomedical Devices, Chemical and Biomolecular Engineering, University of Maryland, College Park
We are developing tools of “biofabrication” that enable facile assembly of biological components within devices, including microelectronic devices, that preserve their native biological function. We have created redox-based synthetic biology
to sample, interpret and report on biological information contained in molecular communications circuitry. We have also developed synthetic genetic circuits that enable electronic actuation of gene expression. These tools enable unparalleled means
to control genetic circuits, creating new and exciting means to actuate and control biology.
2:50 Steering N-Glycosylation of Recombinant Proteins Using Systems Engineering
Michael J. Smanski, PhD, Assistant Professor, Biochemistry, Molecular Biology & Biophysics, Biotechnology Institute, University of Minnesota
Chinese Hamster Ovary cells are used for industrial production of protein-based therapeutics (i.e. ‘biologics’), but systems-level genetic engineering of beneficial traits is slow, difficult, and empirically-guided. We exploit systems- and
synthetic-biology approaches to design, build, and screen multi-gene constructs that rationally perturb the post-translational glycosylation of a secreted Immunoglobulin G (IgG) towards high galactose incorporation. Our approach allows for rapid hypothesis
testing and quantification of synergistic behavior from genetic perturbations.
3:20 Fusion Partners for Robust Peptide Production in Pseudomonas Fluorescens
Diane Retallack, PhD, Senior Director, Upstream Processing and Intellectual Property, Pfenex
3:50 Networking Refreshment Break (Commonwealth Hall)
4:20 A Multi-Landing Pad DNA Integration Platform for Mammalian Cell Engineering
Liliana Wroblewska, PhD, Principal Scientist, Biomedicine Design, Pfizer, Inc.
Reliable, large-scale engineering of CHO cells through precise insertion of large amounts of heterologous DNA into well-characterized genomic loci would have broad applications for mammalian synthetic biology, recombinant protein production, and biomanufacturing.
Using multi-gene payload vectors, cell lines with multiple landing pads, and recombinase technology, we demonstrated controlled integration of up to nine copies of a monoclonal antibody (about 100 kb of heterologous DNA), and a corresponding linear
increase in antibody expression.
4:50 Implementing Next-Generation Sequencing for DNA-Based Sequence Variant Analysis of Recombinant Proteins
Ulrich Göpfert, PhD,
Principal Scientist, Cell Line & Molecular Development, Roche Innovation Center Munich
Sequence variants are unintended amino acid substitutions in biopharmaceuticals, which can either be due to the manufacturing process or mutations of the transgene. Transgene mutations are permanent properties of affected cell lines and may give rise
to critical quality attributes. Therefore, mutated cell lines need to be identified and excluded from development. We will share our experience with next-generation sequencing as an efficient and highly sensitive method to detect DNA-based sequence
variants.
5:20 End of Day
5:20 Registration for Dinner Short Courses (Commonwealth Hall)
FRIDAY, APRIL 12
8:00 am Morning Coffee (Harbor Level)
8:30 Chairperson’s Remarks
Christopher H. Gray, PhD, Staff Scientist & Team Leader, Structural Biology, Drug Discovery Program, CRUK Beatson Institute
8:35 FEATURED PRESENTATION: Methylation Analysis of Cell Lines with Varying Productivities
Susan Sharfstein, PhD, Professor, Nanobioscience, Nanoscale Science and Engineering, SUNY Polytechnic Institute
DNA methylation plays a critical role in regulating gene expression, and it is well known that the CMV promoter contains a CpG island that is subject to silencing by methylation. Using a novel next-generation sequencing approach, we have analyzed the
methylation status of the CMV promoter for cell lines with varying productivity to provide insight into the role of methylation in control of transgene expression.
9:05 Improving Cytidine and Adenine Base Editors by Expression Optimization and Ancestral Reconstruction
Luke Koblan, PhD, Scientist, Chemical
Biology, Chemistry & Chemical Biology, Harvard University
Base editors enable targeted single-nucleotide conversions in genomic DNA. The usefulness of base editors for research and therapeutic applications strongly depends on the efficiency with which they modify target nucleotides. Optimizations to improve
editor expression, nuclear localization, and the component deaminase domain enable substantially improved editing by both cytidine and adenine base editors in a variety of mammalian cell types. BE4max, AncBE4max, and ABEmax represent the current state-of-the-art
base editors.
9:35 Development and Production of Biologics Beyond Antibodies
Ulrich Kettling, Vice President, Business Development, CEVEC Pharmaceuticals GmbH
While antibodies have become standard, now more and more complex, glycosylated proteins get into the focus of biopharma research, as cell therapy reagents and therapeutics. While such complex proteins represent a large portion of the human proteome they
are notoriously difficult to express in CHO or microbials. The presentation discusses the CAP cell lines and CAP-Go expression platform which are specifically designed for production of high-end biologics with authentic post-translational modifications
and tailor-made glycosylation patterns.
10:05 Networking Coffee Break (Commonwealth Hall)
10:35 POSTER SPOTLIGHT
A High-Throughput Approach to Construct Generation and Expression Screening for Recombinant Protein Production
Shu Ti, Scientific Researcher, R&D, Genentech, Inc.
11:05 Cell-Free Synthetic Biology for Therapeutics, Sensing, and Remediation
David
Karig, PhD, Associate Professor, Systems and Synthetic Biology, Bioengineering, Clemson University
Cell-free protein expression systems offer a number of advantages for implementing synthetic biology applications. They simplify system composition and tuning, avoid evolution away from the intended function, and alleviate safety concerns associated
with the spread of engineered living cells. Key developments in the preservation and ruggedization of cell-free reagents will enable therapeutics production in the field as well as environmental sensing and remediation.
11:35 Identification of ADP-Ribosylation by Tandem Mass Spectrometry
Guy
Poirier, PhD, Professor, Faculty of Medicine, Scientific Advisor, Proteomics Platform CHU of Quebec, Université Laval
We have developed a new method to identify all the ADP-ribosylation sites independent of the acceptor amino acid. This method uses tandem mass spectrometry amenable to CID or other modes of fragmentation. The mass spectrometry signature is very unique
and is stable in any mode of fragmentation.
12:05 pm Production of Antimicrobial Peptides Using Protein-Cage Carrier Proteins
Mimi Cho Yung, PhD, Staff
Scientist, Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory (LLNL)
The bioproduction of antimicrobial peptides (AMPs) in bacterial expression systems remains a challenging problem due to host toxicity and proteolysis. We will discuss our recent efforts to engineer encapsulin nanocompartment systems to enhance expression
of AMPs in Escherichia coli.
12:35 Luncheon Presentation: Strategies to Increase the Speed of Cell Line Generation for Biomanufacturing Whilst Maintaining Performance
Fay Saunders, PhD, Head, Mammalian Cell Culture, PD, FUJIFILM Diosynth Biotechnologies
Increasing efforts are focused towards reducing the time taken to move from gene to GMP manufacturing. In this study we demonstrate how to leverage (i) host cell line directed evolution strategies to improve bioprocess relevant phenotypes and increase
mAb titres up to 2-fold; and (ii) key technology enablers that allow intensification of cell line development timelines.
1:05 Networking Refreshment Break (Commonwealth Hall)
1:35 Chairperson’s Remarks
David Karig, PhD, Associate Professor, Systems and Synthetic Biology, Bioengineering, Clemson University
1:40 Molecular Approaches that Improve Soluble Protein Yields from Bacterial Expression Systems
Christopher H. Gray,
PhD, Staff Scientist & Team Leader, Structural Biology, Drug Discovery Program, CRUK Beatson Institute
Expression systems targeting well folded products often employ contradictory strategies, pushing production with strong promoters and codon-enhanced cDNAs, while simultaneously slowing the process by titrating back inducing reagents or culture temperature.
Often, elevated total expression levels aren’t matched by a similar increase in the recovery of soluble protein. We compare a series of alterations to key codons and expression-vector sequence elements that attenuate protein production rates
and maximise soluble recovery.
2:10 Recombinant (Membrane) Protein Production in Yeast
Roslyn M. Bill, DPhil, Professor,
Biotechnology; Associate Dean, Research, Aston University
My lecture will focus on methods that are available for protein synthesis in yeasts, which are an important source of recombinant eukaryotic membrane proteins. I will provide an overview of approaches to optimize the expression plasmid, host cell
and culture conditions, as well as the extraction and purification of functional protein for further study.
2:40 Development of the Filamentous Fungus Myceliophthora thermophila C1 into a Next-Generation Therapeutic Protein Production System
Anne Huuskonen, MSc, Research Scientist, VTT Technical Research Centre of Finland, Ltd.
We are utilizing the vast protein production capability of the filamentous fungus Myceliophthora thermophila to construct a highly potent therapeutic protein production platform. Superb productivities of full-length antibodies, up to 2.5
g/l/day, have been reached. We have also successfully produced several difficult-to-express proteins such as bispecific antibodies and vaccine proteins in titers superior to other expression systems. Our work also aims at humanizing the glycosylation
pathway of this fungus, and the first steps in this research line have been successful.
3:10 End of Conference