Optimizing Protein Expression

Part of the Tenth Annual PEGS: the essential protein engineering summit
May 7-8, 2014 | Seaport World Trade Center| Boston, MA


When expressing proteins, there are a variety of expression systems to choose from. CHO is seen as the ‘king’ in the industry, but does it meet all needs of rapid production and budget constraints? This meeting will explore “Optimizing Protein Expression” through understanding and enhancing expression systems, and will especially focus on CHO cells and other mammalian systems, E.coli and baculovirus. Other host systems, such as yeast and algae, will be touched on as well.

Along with case studies, the meeting will feature experts who reveal the underlying mechanisms and insights into the varying systems in order to enhance protein expression. Comparing and contrasting systems will also be featured to provide greater understanding of how these systems function within the context of the results achieved. Learn the latest protocols and see how protein science leaders are enhancing expression systems to reach greater productivity.

Day 1 | Day 2 | Download Brochure | Speaker Bios 


WEDNESDAY, MAY 7

7:00 am Registration and Morning Coffee

 

Protein Expression in CHO Cells 

8:00 Chairperson's Opening Remarks

Alexei Yeliseev, Ph.D., Staff Scientist and Head, Protein Expression Group, LMBB, NIH

8:10 Engineering Recombinant Proteins for Improved Manufacturability in CHO Cells

Sujeewa D Wijesuriya, Ph.D., Senior Scientist, XOMA (US) LLC

Case studies will be presented that identify manufacturability issues with two recombinant proteins. First, we greatly improved expression of CAB-2, a hybrid recombinant protein, by eliminating unproductive mRNA coding sequences. Second, we established that light chain was responsible for poor expression and high aggregate levels in a recombinant antibody. Light chain shuffling yielded an antibody with high binding affinity, and improved expression and product quality.

8:40 Development and Optimization of a Suspension CHO-EBNA Transient Gene Expression System

Robert RothRobert Roth, Ph.D., Associate Principal Scientist, Discovery Sciences, Reagents and Assay Development, AstraZeneca

Recent advances in mammalian expression hosts for protein expression have led to their increased utilization in efforts to meet the challenge of generating large amounts of functional proteins for drug discovery research. We present the engineering of a suspension CHO-EBGS cell line and subsequent optimization of polyethyleneimine-mediated transient gene expression using a factorial experimental design approach. Data from a case study will be presented highlighting why this high-yielding, high-throughput CHO-EBGS expression system has become an integral expression tool within the Reagents and Assay Development (RAD) department at AstraZeneca.  

9:10 Aeration Strategies and the Control of Interfacial Stress in Mammalian Cell (CHO) Culture

Daniel G. BracewellDaniel G. Bracewell, Ph.D., Reader, Biochemical Engineering, University College London

Understanding gas transfer is critical to the design and scale-up of bioreactor conditions with landmark events being the use of stirred tank reactors for penicillin in 1944 and discovery of surfactants for mammalian cell culture in 1968. The advent of biosimilars and the commodification of antibodies challenges the depth of our understanding. We present our recent results on the subject in this context.

9:40 Estimation of Metabolic Rewiring in CHO Cell Culture Using Combined 13 C-Metabolic Flux Analysis

Woo Suk AhnWoo Suk Ahn, Ph.D., Research Associate, Bioinformatics and Metabolic Engineering, Massachusetts Institute of Technology (MIT)

13C-Metabolic flux analysis (13C-MFA) is a powerful methodology to quantify intracellular metabolism using stable isotopic tracers. We introduced multiple tracers in parallel to quantify CHO central metabolism. Here, we revealed metabolic rewiring from growth to non-growth phase. In addition, by design of multiple isotopic tracers, we achieved high accuracy and resolution of flux values. This technology enables us to identify and optimize CHO cellular metabolism.

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

 

Expression Strategies 

» 11:10 KEYNOTE PRESENTATION:
The Production of Complex Proteins in Drug Discovery: Challenges & Solutions

René AssenbergRené Assenberg, Ph.D., Research Investigator, Novartis Pharma AG

Recently there’s been a trend towards greater complexity of proteins targeted for drug discovery. The production challenges of such proteins require a range of different tools, including different expression systems, novel protein engineering and analytical approaches. This talk will outline some of the challenges our group faces in this area, with emphasis on glycoproteins and protein complexes, as well as the solutions we deploy.

11:40 FEATURED PRESENTATION:
Protein Expression Technologies - Evolution or Revolution?

Lorenz MayrLorenz Mayr, Ph.D., Vice President & Global Head, Reagents & Assay Development, AstraZeneca

I will give an overview of protein expression at AstraZeneca Pharmaceuticals, including: An overview of established protein expression technologies; Novel trends and demands for proteins in hit discovery and lead optimization; Novel technologies and trends for protein expression in drug discovery research; Case studies for difficult-to-express proteins and protein complexes; Finding the balance between in-house efforts and outsourcing, and summary and outlook.

iba
 
12:10 pm From Strep-Tag® to Super-StrepTactin®

Schmidt_ThomasThomas Schmidt, Ph.D., COO, IBA GmbH

The widely used Strep-tag®/Strep-Tactin® purification system provides highly pure and functional proteins from crude lysates of any host after one chromatographical step only. We have now developed a new Strep-Tactin®, called Super-StrepTactin®. Super-StrepTactin® has been designed for the very stable binding of Twin-Strep-tag fusion proteins (T1/2=13h) with the provision that binding can still be reversed under physiological conditions. This opens new application fields for Strep-tag users beyond purification, particularly new assay capabilities in drug discovery processes. 


 

POLYPLUS_withTagline12:25 Superior Protein Yields in CHO and HEK-293 Cells Using a Novel Highly Efficient Reagent for Transient Transfection

Guerin_Peyrou_GeraldineGéraldine Guérin-Peyrou, Bioproduction Product Specialist, Polyplus-transfection

Low transfection efficiency of CHO cells is a major bottleneck hampering protein production in Transient Gene Expression (TGE). Polyplus-transfection, with its 10+ year expertise in transfection, will present superior results obtained in CHO and HEK-293 cells with its novel, technically advanced reagent.
  

DNA 2.012:40 Luncheon Presentation I:
Engineering of Genes, Proteins and Pathways for Systematic Optimization of Therapeutic Protein Production

Welch_MarkMark Welch, Ph.D., Director, Gene Design, DNA2.0, Inc.

Protein production yield is dependent on a range of factors, including gene coding sequence, vector elements, protein sequence, host genotype, and fermentation conditions. Gene synthesis allows manipulation of essential factors at all levels from the production gene to the host genome, opening the door to systematic, thorough engineering of production systems. We describe applications of gene synthesis and informatics technologies for the optimization of genes, vectors, and cell lines for maximum protein yield in a variety of host types.

 

Icosagen1:10 Luncheon Presentation II: 
Lessons from DNA Viruses: Customized and Flexible Protein Production in CHO Cells

Kadaja_MeelisMeelis Kadaja, Ph.D., CSO, Icosagen Cell Factory

By combining favorable features of DNA viruses, Icosagen has developed technology for fast and efficient production of recombinant proteins in CHO cells. Our QMCF Technology is flexible and can be tailored for the optimal expression of each protein. It is also suitable for cell bank generation and long-term protein production.  

 

1:40 Session Break


Baculovirus / Insect Cells 

2:00 Chairperson's Remarks

Donald Jarvis, Ph.D., Professor, Molecular Biology, University of Wyoming

2:05 A Novel Baculovirus Vector for Production of Non-Fucosylated Recombinant Glycoproteins

Donald JarvisDonald Jarvis, Ph.D., Professor, Molecular Biology, University of Wyoming

The inability to provide human-type protein glycosylation patterns has precluded the use of baculovirus-insect cell systems for therapeutic glycoprotein production. Recent glycoengineering efforts have advanced the platform through the creation of improved systems that can produce recombinant glycoproteins with human-type, complex, terminally sialylated N-glycans. We produced the first baculovirus-insect cell systems capable of producing non-fucosylated recombinant glycoproteins, including antibodies, and characterized their capabilities.

2:35 Insect Cell Culture for Rapid GMP Manufacturing of Seasonal and Pandemic Influenza Vaccines

Nikolai KhramtsovNikolai Khramtsov, Ph.D., Associate Director, Upstream Development, Product Realization, Protein Sciences Corp.

We developed a universal process for the generation of recombinant baculovirus banks for seasonal and pandemic influenza antigens, as well as for the expression and purification of these antigens at large scale. Less than 30 days is required for the introduction of new antigens to GMP manufacturing. Influenza seasonal vaccine Flublok, containing three different hemagglutinin antigens, can be manufactured during two months.

MaxCyte3:05 The Impact of Scalable Transfection: Maximizing CHO Antibody Production Using Flow Electroporation to Accelerate Development Timelines

Brady_JamesJames Brady, Ph.D., MBA, Director, Technical Applications, MaxCyte

CHO transient gene expression (TGE) greatly accelerates antibody development by eliminating the need to change cell backgrounds during scale up to biomanufacturing. Flow electroporation enables scalable transient transfection of a variety of CHO cell lines producing sufficient quantities of antibodies for early and mid-stage antibody development. The production of antibody titers >1g/L yielding multiple grams of antibody from a single transfection, as well as the rapid generation of stable CHO cell lines will be discussed.

3:35 Refreshment Break in the Exhibit Hall with Poster Viewing

4:20 Problem-Solving Breakout Discussions

42.    Expression Systems for Research Applications

Moderator: Mark Welch, Ph.D., Director, Gene Design, DNA2.0, Inc.

  • Overview of systems used for tools generation
  • Key features of each system (benefits/limitations)
  • Unique selling points for each (differentiating factors)
  • Selection of the most appropriate system
  • Experiences & Learnings
 

43.    Having Problems with Your Baculovirus-Mediated Gene Expression Projects?

Moderator: Donald Jarvis, Ph.D., Professor, Molecular Biology, University of Wyoming

  • What's the best way to produce my baculovirus vector?
  • To plaque purify or not...that is the question.
  • I used to get good expression, but not anymore...what happened?
  • How much virus should I use?
  • Do I really have to use insect cells passaged <100 times in culture?
  • What's all this stuff about insect glycosylation patterns; does it really matter?
  • And now, for the complicated questions...
 

44.    Protein Production Using the Baculovirus-Insect Cell Expression System

Moderator: Nikolai Khramtsov, Ph.D., Associate Director, Upstream Development, Product Realization, Protein Sciences Corp.

  • generation of high titer baculovirus stock
  • short storage of working virus stocks
  • reducing the generation of defective genomes
  • identification of the best conditions for protein
 

45.    Known Unknowns: What are the Remaining Challenges at the Upstream / Downstream Interface?

Moderator: Daniel G. Bracewell, Ph.D., Reader, Biochemical Engineering, University College London

  • Product quality issues associated with host cell enzymes e.g. Proteases and reductases
  • Batch to batch variability in solid-liquid separation.
  • Analytical needs.
  • Improvements in scale-down models of filtration and centrifugation.
  • Potential areas of unknown unknowns.
 

46.    Optimization of Purification Protocols and Fermentation Conditions to Maximize Recovery of Recombinant Membrane Receptors

Moderator: Alexei Yeliseev, Ph.D., Staff Scientist, NIAAA, NIH

  • Use of expression and solubilization tags
  • Affinity tags for efficient recovery of target proteins from dilute solutions
  • Performance of affinity tags in the presence of detergents
  • Specialized fermentation: cultivation in minimal media and stable isotope labeling
 

47.    Achieve High Yield Production in CHO and HEK-293 Cells using Transient Gene Expression

Moderator: Géraldine Guérin-Peyrou, Bioproduction Product Specialist, Polyplus-transfection

  • Towards more transient transfection in bioproduction
  • Choose cells & medium according to your goal
  • Optimization of transient transfection
  • Optimization of cell culture parameters post-transfection
 

48.    Optimization of Protein Expression in Yeast

Moderator: Piotr Bobrowicz, Ph.D., Director, Open Innovation, Adimab, LLC

  • Choosing appropriate host
  • Important elements of the expression vector
  • Expression optimization strategies
  • Strain engineering
 

49.    Strategies for Producing Protein Complexes

Moderator: René Assenberg, Ph.D., Research Investigator, Novartis Pharma AG

  • Expression strategies: 
         Which host?
         What tag(s)?
         Co-infection/transformation vs co-expression? Etc.
  • Strategies for the purification and analysis of (large) complexes

5:20 Networking Reception in the Exhibit Hall with Poster Viewing

6:30 End of Day


Day 1 | Day 2 | Download Brochure | Speaker Bios