Optimizing Protein Expression

Expression of heterologous proteins presents many challenges, and understanding expression systems is key. The 10th Annual Optimizing Protein Expression conference delves into protein expression by examining and enhancing expression systems, including CHO, and other mammalian systems, E. coli, yeast, and baculovirus. What is the best expression system for expressing your protein of choice? Ease and cost of scale-up must be considered to ensure successful bottom-line results. Experts will share case studies and disclose data, while divulging details of expression systems’ underlying mechanisms. Comparing and contrasting systems will also be featured to increase understanding in the quest for greater productivity.

Final Agenda


Recommended Short Course*

SC9: Introduction to Biophysical Analysis for Biotherapeutics: Development Applications

*Separate registration required.


7:15 am Registration and Morning Coffee

7:25 Women in Science Panel Discussion with Continental Breakfast

Rochard_LucieLucie Rochard, PhD, Liaison, Scientific & Entrepreneurial Initiatives; Director, Innovation Services, Massachusetts Biotechnology Council

Nora MinevaNora Mineva, PhD, CSO, Adecto Pharmaceuticals

Liu_TinaTina Liu, MBA, Co-Founder and CEO, Ally Therapeutics

Chadwich_JenniferJennifer Chadwich, PhD, Vice President, Biologic Development, BioAnalytix, Inc.


8:40 Chairperson’s Opening Remarks

William Gillette, PhD, Principal Scientist, Protein Expression Laboratory, Frederick National Laboratory for Cancer Research

8:50 KEYNOTE PRESENTATION: Optimising Recombinant Protein Expression in Mammalian Cells for Research and Preclinical Discovery, and Development of Novel Human Therapeutics

Catherine Owczarek, PhD, Director, Recombinant Protein Expression Group, CSL Limited

Despite numerous advances, there remain challenges in generating proteins, especially those with increased complexity, multiple functions, difficult to express proteins or where the cellular machinery for specific post-translational modifications may be best suited to only one cellular system. A robust, flexible transient production strategy is necessary to achieve our goal of generating proteins with appropriate post-translational modifications, and hence the desired biological activity and pharmacokinetic profiles for pre-clinical research programs for the discovery, generation and development of novel human therapeutics.

9:20 Begin with the End in Mind: Seamless Transitioning of Novel Biologics from RES to DEV and Beyond

Ingo Gorr, PhD, Director and Head, Early Stage Bioprocess Development (USP&DSP), Boehringer Ingelheim Pharma GmbH & Co. KG

Right formatting of therapeutic proteins is key for its efficacy and formats are getting more complex. Novel formats often present challenges to CMC with respect to performance or product quality. Early involvement of CMC and smart molecule selection prevent later unexpected CMC findings and thus costly trouble shooting.

9:50 Recombinant Protein Production Strategies for Multi-Protein Complexes in Drug Discovery

Saleha Patel, PhD, Senior Research Scientist, Protein Science, Discovery Biology, AstraZeneca

The increasing complexity of drug targets, many of which are multi-protein complexes, provides a challenge for successful reagent generation for drug discovery. Whilst there have been advances in the downstream analytical technologies (such as cryo-EM) the ways in which recombinant proteins are produced remains largely unchanged. However, to ensure the protein reagents are fit for purpose, for these novel technologies, new strategies are required within the protein production workflow.

10:20 Coffee Break in the Exhibit Hall with Poster Viewing

10:30 Women in Science Speed Networking in the Exhibit Hall


11:05 Investigation of Gene Expression Patterns in Stable and Unstable Clonally Derived CHO Cell Lines

Theodore Peters, PhD, Senior Scientist, Cell Line Development Group, Seattle Genetics, Inc.

Development of biologic-producing CHO clones in accelerated timelines is encumbered by demonstrating production stability in candidate cell lines. Screening out unstable lines earlier in development could mitigate the risk of advancing unstable candidates. Our work reveals significant phenotypic heterogeneity in clonal populations by characterizing subclones from stable and unstable clones. This highlights the prevalence of phenotypic drift in clonal cell lines providing a basis for investigating gene expression patterns.

11:35 An Innovative siRNA-Based Technology versus Traditional Antibiotic-Selection in the Creation of Stable CHO Cell Clones

Susan Sharfstein, PhD, Professor, Nanobioscience, Nanoscale Science and Engineering, SUNY Polytechnic Institute

Current DHFR, GS, and antibiotic selection methods to establish stable Chinese hamster ovary cell lines are time-consuming and labor-intensive, often requiring auxotrophic cell lines. A novel siRNA-based technology, PTSelect™ overcomes these hurdles using an siRNA cloned upstream of the gene of interest (GOI). After transfection, cell selection is performed by transfecting CD4-mRNAs, which are regulated by the siRNA. Cells with stably integrated GOI are selected by exploiting the variable expression of CD4 on the cell surface, yielding high-expressing clones with greatly shortened timelines.

12:05 pm Multiomics Investigation of Hyper Productivity in CHO Cells: Gaining New Insights into the Role of Nuclear Proteomics in Enhancing Cellular Productivity

Hussain Nuruddin Dahodwala, PhD, Associate Site Director, AMBIC, Delaware Biotechnology Institute, University of Delaware

IgG-producing cell clones were analyzed using a combination of transcriptomic, proteomic, phosphoproteomic, and chromatin immunoprecipitation (ChIP) techniques. Our data suggested increased in vivo CMV promoter-transcription factor interaction in the higher producing cell line. We show here that the nuclear proteome and phosphoproteome have an important role in regulating final productivity of recombinant proteins from CHO cells.

12:35 Presentation to be Announced

12:50 Case Study on a Cost-Efficient Refolding Process for a Biotherapeutic Protein with FOLDTEC®

Monique Janowski-Egler, PhD, Head of USP, BioProcess Development Halle, BioProcess Development, Wacker Biotech GmbH

Wacker Biotech GmbH will present a new case study with FOLDTEC, a unique technology for the sophisticated manufacturing of biopharmaceuticals by refolding. Our engineered strains, plasmids and know-how lead to formation of high-quality IBs, efficient refolding and downstream processing with optimized yields.

1:05 Session Break

1:10 Luncheon Presentation I to be Announced


Glycotope 1:40 Luncheon Presentation II to be Announced

2:10 Session Break


2:25 Chairperson’s Remarks

Johannes van den Heuvel, PhD, Research Group Leader, Structure and Function of Proteins, RG Recombinant Protein Expression, Helmholtz Centre for Infection Research

2:30 Using Recoded Organisms for the Expression of Post-Translationally Modified Proteins: Leveraging the Benefits while Avoiding the Pitfalls

Richard Cooley, PhD, Research Assistant Professor, Biochemistry and Biophysics, Oregon State University

Genetic Code Expansion (GCE) permits site-specific incorporation of non-canonical amino acids into proteins at UAG amber stop codons. To increase yields and mitigate premature termination, organisms in which UAG codons have no functional assignment are gaining widespread use as GCE expression hosts. However, heterogeneously modified proteins are often obtained with these systems. Here, we develop GCE systems for homogenous, multi-site incorporation of phosphoserine and nitro-tyrosine in recoded organisms, and demonstrate their benefits for accessing biologically relevant post-translationally modified proteins.

3:00 Integrating Vibrio Natriegens into a Protein Production Workflow

William Gillette, PhD, Principal Scientist, Protein Expression Laboratory, Frederick National Laboratory for Cancer Research

Vibrio natriegens offers an alternative host to express recombinant proteins. Although there are some minor differences in basic E. coli protocols, much, including expression constructs, is bifunctional across the two systems. Case studies that cover how the system has improved some aspects of protein production will be presented.

3:30 Overcoming Limitations of Conventional Tag Systems – Strep-Tactin®XT Applications

Karthaus_DennisDennis Karthaus, MSc, Director Protein Products & Assays, IBA Lifesciences

The Strep-TactinXT:Twin-Strep-tag-purification system enables protein purification at high yields and purity under physiological conditions. Providing the highest binding affinity among all affinity tag systems, the technology fulfills the demands of detections and monitoring of biomolecular interactions in real time and is available for applications like SPR and Octet/BLItz.

3:45 Presentation to be Announced

4:00 Refreshment Break in the Exhibit Hall with Poster Viewing

5:00 Problem-Solving Breakout Discussions - View All Breakout Discussion Topics

TABLE: Non-Standard Amino Acid Incorporation in Recombinant Proteins

Moderator: Jesse Rinehart, PhD, Associate Professor, Cellular & Molecular Physiology, Systems Biology Institute, Yale University School of Medicine

  • Non-standard amino acids have been incorporated in a variety of protein expression systems
  • Each non-standard amino acid, the choice of cellular host, and incorporation strategy brings with it a host of unique challenges
  • From basic set up/trouble shooting to mass spectrometry/proteomics of non-standard amino acid containing proteins
  • Bacterial systems using non-standard amino acids and proteomics

TABLE: Challenges in Cell Therapy Manufacturing Development

Moderator: Zhimei Du, PhD, Director, Process Development, Merck and Co., Inc.

  • How to mitigate the variability during cell therapy manufacturing?
  • What are the practical strategies to reduce COGS of cell therapy manufacturing?
  • Is current the list of critical quality attributes sufficient to define product and process control?
  • What is the next generation manufacturing strategy for cell therapy?

TABLE: Prepping Endogenous Protein Complexes for Downstream Analysis

Moderator: John LaCava, PhD, Group Leader, Laboratory of Macromolecules and Interactomes, European Research Institute for the Biology of Aging, University Medical Center Groningen

  • Quality assurance / quality control
  • Affinity capture, best practices
  • Intermediate readout - which tool for the job?
  • End game - routine structural analysis of endogenous complexes

6:00 Taste of New England Networking Reception in the Exhibit Hall with Poster Viewing

7:15 End of Day


8:00 am Registration and Morning Coffee


8:30 Chairperson’s Remarks

Theodore Peters, PhD, Senior Scientist, Cell Line Development Group, Seattle Genetics, Inc.

8:35 FEATURED PRESENTATION: The MultiBac System: A Perspective

Imre Berger, PhD, Director, Max Planck Centre for Minimal Biology, the University of Bristol

MultiBac is a BEVS originally developed for producing multiprotein complexes for structural studies. We continuously optimized MultiBac, providing reagents and protocols to facilitate its use. We generated MultiBac variants tailored to produce glycoproteins, kinases, viral polymerases and synthetic vaccines. We implemented MultiBacMam for genomic engineering applications. Exploiting synthetic biology, we engage in optimizing the properties of our baculoviral genome. Here we provide a perspective of MultiBac, past, present and future.

9:05 Improving the Reproducibility of the Virus-Free Transient Gene Expression in High Five Insect Cell Lines

Johannes van den Heuvel, PhD, Research Group Leader, Structure and Function of Proteins, RG Recombinant Protein Expression, Helmholtz Centre for Infection Research

Virus-free transient gene expression (TGE) in high five insect cells recently evolved to an efficient protein production method. However, up to now, the published TGE protocols were not standardized and remain difficult to establish and reproduce in different labs. In this study, we analyzed and optimized the factors determining the reproducibility and robustness of the method. The type of polyethylenimine, growth phase of the cells, passage number, origin of cell line isolates and cultivation media were considered to create a robust method with high protein yields.

9:35 Unleashing the Power of Transient Transfection to Overcome Expression Challenges

Markus Hildinger, PhD, CEO, evitria AG

Time to market is crucial in biopharmaceutical development. Transient transfection offers one of the fastest ways to express proteins and to overcome challenges for difficult-to-express proteins. evitria has developed an integrated transient transfection platform based on CHO cells to overcome the major hurdles in protein expression with a particular focus on antibodies. Its platform has been successfully applied in more than 50,000 transfections and more than 10,000 antibody purifications.

9:50 Sponsored Presentation (Opportunity Available)

10:05 Coffee Break in the Exhibit Hall with Poster Viewing and Poster Award


11:05 Large-Scale Synthetic Human Phosphoproteomes to Decode Novel Protein-Protein Interaction Networks in E. coIi

Jesse Rinehart, PhD, Associate Professor, Cellular & Molecular Physiology, Systems Biology Institute, Yale University School of Medicine

Protein phosphorylation encompasses a central cellular language that determines every facet of normal cellular biology. We have developed technologies that enable site-specific incorporation of phosphorylated amino acids into proteins by expanding the genetic code of Escherichia coli. I will describe our new capability to synthesize and observe phosphoproteome-scale libraries of human phosphoproteins that enable answers to systems level questions. Our work has also advanced the protein-protein interaction field by enabling the first system for large-scale genetic screening of phosphorylation-dependent protein-protein interactions.

11:35 Continuous Production with E. coli – USP Concepts and Strategies

Gerald Striedner, PhD, Associate Professor, University of Natural Resources and Life Sciences, Vienna (BOKU)

Genetic stability of E. coli expression systems represents the major barrier in context with development of continuous production processes. In this talk, data from successful solutions, comprising host cell engineering (genome integrated plasmid free systems, growth decoupled systems) and USP process design strategies (multistage cultivation) will be presented and aspects for further improvements will be discussed.

12:05 pm Engineering E. coli Strains for Recombinant Protein Production

Jan-Willem de Gier, PhD, Professor, Biochemistry and Biophysics, Stockholm University

My laboratory has been using an engineering approach to create E. coli strains with improved properties for recombinant protein production. This will be illustrated by showing examples of the construction of strains for the production of proteins in the periplasm and in inclusion bodies in the cytoplasm.

12:35 End of Optimizing Protein Expression

Recommended Short Course*

SC16: Assembling an Effective Toolbox of Expression Systems to Support your Research Efforts

*Separate registration required.

* The program is subject to change without notice, due to unforeseen reason.

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