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.

Wednesday, September 2

PROTEIN EXPRESSION STRATEGIES

9:05 am

Cell and Process Optimization Strategies to Improve Biologics Manufacturing

Stefan R. Schmidt, MBA, PhD, COO & Head, Operations, BioAtrium AG

In the last decades, a tremendous productivity increase of bioprocessing was observed. Three different innovations drive this evolution: molecular design of expression constructs, engineering of host cell lines and optimization of cultivation processes. These different strategies are used either stand alone or in combination to multiply the cellular output. This presentation gives a comprehensive overview on where and how to implement these approaches, based on successful examples and case studies.

 

Biologicals are one of the fastest growing therapeutic classes and focusing on manufacturing topics is important to be able to match the expanding demand.

As the presentation will cover the whole work stream from gene design to cell culture it will be highly useful to a wide range of the audience.

 

The audience will get insights where and how to implement these techniques and what the benefits (and potential risks) will be. Overall, it will give a comprehensive overview on the current state of the art.

9:25 am

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.

Gregory Bleck, Global Head of R&D, Biologics, Catalent
10:10 am

Session Wrap-Up

Panel Moderator:
William Gillette, PhD, Principal Scientist, Protein Expression Laboratory, Leidos Biomedical Research
Panelists:
Saleha Patel, PhD, Senior Research Scientist, Protein Science, Discovery Biology, AstraZeneca
Stefan R. Schmidt, MBA, PhD, COO & Head, Operations, BioAtrium AG
Gregory Bleck, Global Head of R&D, Biologics, Catalent
10:30 am Speed Networking Coffee Break - View our Virtual Exhibit Hall

CHO CELL EXPRESSION

10:50 am

Multi-Omics Investigation of Hyper Productivity in CHO Cells: Gaining New Insights into the Role of Nuclear Proteomics in Enhancing Cellular Productivity

Hussain Nuruddin Dahodwala, PhD, Director, Upstream Process Development, NIIMBL

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.

11:10 am

Physiological Responses to Increased Productivity: A Transcriptomic and Proteomic Study

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

 

Increased productivity in CHO cells due to gene amplification is a well established technology in the biopharmaceutical industry. What is less well understood are the physiological responses that permit this increased productivity. In this study, we compare a parental cell line and its DHFR-amplified progeny using RNA-Seq and proteomic analysis. A wide range of cellular processes are altered in response to increased productivity, including the protein processing machinery and primary metabolism. These changes provide insights into the use of CHO cells for recombinant protein production and strategies for cell engineering for improved productivity.

 

Sean Taylor, Ph.D., Field Application Scientist Manager, Catalog Products, GenScript Inc. USA

With the ever increasing demand for antibody and protein-based therapeutics, a flexible purification platform that can handle low to high sample volumes and expression levels is critical for screening. Protein purification using traditional chromatography is limited by throughput, time-consuming, and involves labor-intensive sample preparation processes. Magnetic beads-based purification permits the incubation of the beads directly into culture soups. The tools/ application to simplify and significantly augment protein purification and screening cost-effectively will be described.

12:00 pm

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

Theodore Peters, PhD, Sr Scientist, Cell Line Dev, Seattle Genetics

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.

12:20 pm Lunch Break - View our Virtual Exhibit Hall

PROTEIN PRODUCTION ADVANCES

12:45 pm

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

Richard B. Cooley, PhD, Assistant Professor, Biochemistry & 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.

1:05 pm

Integrating Vibrio Natriegens into a Protein Production Workflow

William Gillette, PhD, Principal Scientist, Protein Expression Laboratory, Leidos Biomedical 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.

Lars Stöckl, PhD, Doctor, Glycotope Service Division, 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.

1:50 pm LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Hussain Nuruddin Dahodwala, PhD, Director, Upstream Process Development, NIIMBL
Panelists:
Susan Sharfstein, PhD, Professor, Nanobioscience, Nanoscale Science and Engineering, SUNY Polytechnic Institute
Theodore Peters, PhD, Sr Scientist, Cell Line Dev, Seattle Genetics
William Gillette, PhD, Principal Scientist, Protein Expression Laboratory, Leidos Biomedical Research
Richard B. Cooley, PhD, Assistant Professor, Biochemistry & Biophysics, Oregon State University
Lars Stöckl, PhD, Doctor, Glycotope Service Division, Glycotope GmbH
Sean Taylor, Ph.D., Field Application Scientist Manager, Catalog Products, GenScript Inc. USA
2:10 pm Refresh Break to View our Virtual Exhibit Hall

FEATURED SESSION: BACULOVIRUS

Frederick Boyce, MD, PhD, Assistant Professor, Neurology, and Director, MGH Gene Delivery Technology Core, Massachusetts General Hospital and Harvard Medical School

The popular pFastBac vectors for baculovirus expression have remained relatively unchanged since their publication by Luckow in 1993. We will discuss improvements that we and others have made to these workhorse vectors for protein expression for use in either insect (BEVS) or mammalian (BacMam) systems. Modern versions of these vectors allow more facile and efficient production of recombinant viruses, enabling high-throughput applications, and can also lead to increased protein expression.

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

 

The COVID-19 pandemic is a global crisis. The MultiBac BEVS platform proved central to the COVID-19 response at University and Medical School in Bristol, UK, underpinning serology testing, drug discovery and vaccine candidate production. SARS-CoV-2, the virus causing COVID-19, exposes a glycoprotein called Spike,  the predominant COVID-19 antigen. MultiBac production and Cryo-EM revealed an unexpected druggable pocket in the SARS-CoV-2 Spike. Intriguingly, a similar pocket was utilized previously to develop potent antivirals that defeated rhinovirus in the clinic. Avenues to combat COVID-19 by exploiting the pocket we discovered, will be discussed. 


 

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

New expression systems enable high titer production while simultaneously reducing the expression volume and time. Here we compare the efficiency of the purification of Strep-tagged versus His-tagged proteins from different Expi supernatants. Furthermore, Strep-Tactin®XT can be used in combination with IMCSTips for high throughput purification from automated systems.

Steven Lang, VP, Biologics, Aragen Bioscience

We demonstrate a platform-based upstream process development plan of a biosimilar protein that delivers reduced timelines to GMP manufacturing and consistent product quality. This method can be applied to ensure timely success of CHO biomanufacturing. 

3:30 pm LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Frederick Boyce, MD, PhD, Assistant Professor, Neurology, and Director, MGH Gene Delivery Technology Core, Massachusetts General Hospital and Harvard Medical School
Panelists:
Imre Berger, PhD, Director, Max Planck Centre for Minimal Biology, University of Bristol
Dennis Karthaus, MSc, Director Protein Products & Assays, IBA Lifesciences
Steven Lang, VP, Biologics, Aragen Bioscience
3:50 pm Refresh Break - View Our Virtual Exhibit Hall
4:10 pm Problem Solving Breakout Discussions - Part A

This session provides the opportunity to discuss a focused topic with peers from around the world in an open, collegial setting. Select from the list of topics available and join the moderated discussion to share ideas, gain insights, establish collaborations or commiserate about persistent challenges.

TABLE 23: Prepping Endogenous Protein Complexes for Downstream Analysis

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
4:40 pm Refresh Break - View Our Virtual Exhibit Hall
5:00 pm Problem Solving Breakout Discussions - Part B

TABLE 24: Non-Standard Amino Acid Incorporation in Recombinant Proteins

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 25: Challenges in Cell Therapy Manufacturing Development

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?

 

5:30 pm Close of Day

Thursday, September 3

ESCHERICHIA COLI

9:05 am

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.

9:25 am

Continuous Production with E. coli – USP Concepts and Strategies

Gerald Striedner, PhD, Associate Professor, Biotechnology, University of Natural Resources & 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.

9:45 am

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.

10:10 am LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Jesse Rinehart, PhD, Associate Professor, Cellular & Molecular Physiology, Systems Biology Institute, Yale University School of Medicine
Panelists:
Jan-Willem de Gier, PhD, Professor, Biochemistry and Biophysics, Stockholm University
Gerald Striedner, PhD, Associate Professor, Biotechnology, University of Natural Resources & Life Sciences, Vienna (BOKU)
10:30 am Coffee Break - View our Virtual Exhibit Hall

ENGINEERING CHO

John Gill, Director, CDO R&D Team, Cell Line Development, Samsung Biologics

It can be a critical decision by any institution to
select right host cell line; it can impact quality, cost of goods and timeline. The presentation describes development studies of S-CHOice, Samsung’s proprietary cell line, which can help achieve high quality product, adding value to Samsung’s one-stop CDMO capability.

11:15 am

Multi-Omics Analysis of CHO Cell Lines Reveals Differences in Energy Metabolism and ER Stress

Krishnakumar Malu, PhD, Senior Engineer I, Biogen

Chinese hamster ovary cells are widely used host cell lines in biotherapeutic manufacturing of monoclonal antibodies (mAbs). Cellular energy metabolism and endoplasmic reticulum stress are known to greatly impact cell growth, productivity, and structure of the biotherapeutics. However, molecular mechanisms responsible for these changes are not fully understood. Here, we use multi-omics analysis to investigate differences between cell lines and gain an in-depth understanding of the biological differences that can be exploited to improve the bioprocess.

11:35 am LIVE Q&A:

Session Wrap-Up

Panel Moderator:
Jan-Willem de Gier, PhD, Professor, Biochemistry and Biophysics, Stockholm University
Panelists:
Krishnakumar Malu, PhD, Senior Engineer I, Biogen
John Gill, Director, CDO R&D Team, Cell Line Development, Samsung Biologics
11:55 am Close of Optimizing Protein Expression





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