Difficult-to-Express Proteins

 

Proteins are each unique and bring unique challenges when attempting to tame them into submission. CHI’s 14th annual Difficult-to-Express Proteins conference examines the challenges researchers encounter when striving for high-yield production of “difficult-to-express” proteins (DTEPs) and the strategies and technologies that have proven successful in overcoming those challenges. Some of the difficulties encountered include solubility, proper folding, inability to crystallize and aggregation and formation of inclusion bodies. Researchers employ a range of problem-specific solutions to achieve expression including genetic modifications, manipulating how a target protein is produced and employing protein tags. In addition, the identification of DNA coding sequences along with the use of high-throughput approaches has brought about significant improvements. The Difficult-to-Express Proteins conference provides the latest developments in improving yield for DTEPs through Case Studies and breakthrough data.

Final Agenda

SUNDAY, APRIL 7

Recommended Short Course(s)*

SC3: Selection, Screening and Engineering for Affinity Reagents

Jonas V. Schaefer, PhD, Lab Head/Investigator II, Novartis Institutes for BioMedical Research (NIBR)

Christian Kunz, PhD, Director, MorphoSys AG

 

*Separate registration required.

MONDAY, APRIL 8

7:00 am Registration (Commonwealth Hall) and Morning Coffee (Harbor Level)


INNOVATING PROCESSES FOR DTEPs

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8:30 Chairperson’s Opening Remarks

Nicola Burgess-Brown, PhD, Principal Investigator, Biotechnology, Nuffield Department of Medicine, Structural Genomics Consortium, University of Oxford


8:40 KEYNOTE PRESENTATION: A High-Throughput Platform to Express All Human Cell Surface Proteins

Love_JamesJames Love, PhD, COO, Protein Production, Institute for Protein Innovation

Generating open-source monoclonal antibodies against every extracellular and secreted protein in humans, has required the development of expression platforms capable of generating high quality antigens and antibodies in HT format. Classes of proteins show somewhat uniform characteristics in ‘expressibility’ and even recalcitrant proteins, such as integral membrane proteins can be processed by rescue pathways if necessary. This talk will outline methods that have proven fruitful and present future areas for investigation.

9:10 Use of a Protein Engineering Strategy to Overcome Limitations in the Production of “Difficult to Express” Recombinant Proteins

Dickson_AlanAlan Dickson, PhD, Professor, Biotechnology; Director, Centre of Excellence in Biopharmaceuticals (COEBP), University of Manchester

Domain engineering opens the potential to manufacture novel recombinant products with innovative functions. Intellectual shuffling of protein domains/parts can be frustrated by the quality control processes in current cell factories. This presentation will focus on our recent work that has localised the sites of limitation of production of model ‘difficult to express’ proteins and development of molecular interventions that enhance production of a desired recombinant protein.

9:40 Teaching an Old Dog New Tricks: Making CHO Cell Line Development Ready for the Difficult-to-Express Protein Challenge

Fischer_SimonSimon Fischer, PhD, Head, BPAD Cell Line Development, Bioprocess & Analytical Development, Boehringer Ingelheim Pharma GmbH & Co. KG

The number of DTE therapeutic proteins appearing in drug development pipelines of pharmaceutical companies has increased dramatically. To address challenges in DTE protein expression, novel cell line development strategies need to be implemented. Being an old workhorse within the industry, CHO cells still represent the predominant production host for large-scale manufacturing. In this talk, we will present new technologies in cell line and molecule engineering to enhance CLD for DTE proteins in the future.

10:10 Networking Coffee Break (Harbor & Mezzanine Level)

BACULOVIRUS & INSECT CELLS

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10:45 Chairperson’s Remarks

Gargi Roy, MSc, Scientist, Antibody Discovery and Protein Engineering, MedImmune, LLC

10:50 Production of Human Integral Membrane Proteins Using Baculovirus and BacMam

Burgess-Brown_NicolaNicola Burgess-Brown, PhD, Principal Investigator, Biotechnology, Nuffield Department of Medicine, Structural Genomics Consortium, University of Oxford

The SGC promotes research advancement through our open access policy, and in the absence of IP. Globally, we have solved more than 2000 human protein structures and 10 novel integral membrane proteins (IMPs). We have made a significant contribution to structural biology and protein production for functional studies, however, IMPs and protein-protein complexes still remain a challenge to produce. I will present our established baculovirus/insect cell expression platform and our promising BacMam pipeline.

11:20 13 Years of Baculovirus Protein Expression in a Core Facility: Evolution of an Ultra-Short Protocol

Suppmann_SabineSabine Suppmann, PhD, Head, Recombinant Protein Production, Biochemistry Core, Max Planck Institute of Biochemistry

In the last three decades, the Baculovirus expression vector system (BEVS) has evolved to one of the most widely used eukaryotic systems for heterologous protein expression. Despite the significant improvements introduced during the past years, the BEV system still has major drawbacks, primarily the time required to generate recombinant virus and virus instability for certain target proteins. We have established and validated an ultra-short BEVS protocol that also eliminates the risk of virus decay.

11:50 Tools for Studying the RAS/RAF/MEK Pathway: Using the BEVS to Produce Complexes and Native Proteins

Gillette_WilliamWilliam Gillette, PhD, Principal Scientist, RAS Reagents Core; Deputy Director, Protein Expression Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research (FNL)

This talk will focus on progress in producing proteins involved in the RAF activation pathway that are suitable for in vitro structural and biochemical studies. Recent advances in using the BEVS to overcome stability issues and producing complexes, as well as more biologically accurate KRAS proteins will be highlighted.

12:20 pm 9g/l in 90 Hours: Development of C1 into a Next-Generation Therapeutic Protein Production System

Tchelet_Ronan_DPXRonen Tchelet, Vice President, Research & Development, Dyadic International, Inc.

This presentation will show the results of the development of the filamentous fungus Myceliophthora thermophila C1 into a next-generation therapeutic protein production system.


thermofisherscientific_no_tagline12:35 Expression, Purification and Characterization of Difficult to Express Membrane Proteins

Jawhari_AnassAnass Jawhari, PhD, CSO, Calixar

This talk will focus on strategies for optimizing expression levels of membrane proteins using the Expi293 and ExpiCHO expression systems.  Additionally, an introduction to newly developed Expi293 engineered cell lines combined with CALIXAR patented technology to improve quality and quantity of challenging membrane proteins will be provided. A case study of KCC2 production and characterization will be further described.


Berkeley-Lights 12:50 Luncheon Presentation: The Beacon™ Platform for the Rapid Discovery of Rare Antibodies to Difficult Targets

Singhal_Anupam_DPXAnupam Singhal, PhD, Technology Development, Berkeley Lights, Inc.

The search of biologics to novel therapeutic targets is hampered by current antibody discovery technologies that are laborious, time-consuming, and generate limited diversity. Here, I will demonstrate how Berkeley Lights’ Beacon® platform’s automated plasma cell antibody discovery workflow mines the vast immune repertoire to identify B cells producing rare, functional antibodies to difficult therapeutic targets in under 24 hours. The Beacon platform links phenotype-to-genotype at the single-cell level, simplifying downstream sequencing, cloning, and bioinformatics analysis.

1:20 Session Break

2:20 Problem-Solving Breakout Discussions - Click here for details (Commonwealth Hall)


3:20 Networking Refreshment Break (Harbor & Mezzanine Level)


PLENARY KEYNOTE SESSION

Amphitheater & Harborview Ballroom

4:00 Chairperson’s Remarks

Rakesh Dixit, PhD, DABT, Vice President, R&D, Global Head, Biologics Safety Assessment, Translational Sciences, MedImmune

PLENARY KEYNOTE SPEAKER

4:10 Vision for How Immunotherapy Will Shape Future of Cancer Care

Leena Gandhi, MD, PhD, Vice President, Immuno-Oncology Medical Development, Lilly Oncology

Immunotherapy is considered by many as a pillar of cancer care today, but in many ways we have only scratched the surface. Our knowledge and understanding of the complexities of immunotherapy and its mechanisms continue to evolve. The future of cancer care will be defined by our ability to systematically identify and implement opportunities for combination therapy to improve and standardize patient response.

YOUNG SCIENTIST KEYNOTE

4:55 The Lassa Virus Glycoprotein: Stopping a Moving Target

keynote-headshot-hastie-400x400Kathryn Hastie, PhD, Staff Scientist, Immunology and Microbiology, The Scripps Research Institute

Lassa virus causes ~5000 deaths from viral hemorrhagic fever every year in West Africa. The trimeric surface glycoprotein, termed GPC, is critical for infection, is the target for neutralizing antibodies, and a major component of vaccines. Structural analysis of Lassa GPC bound to antibodies from human survivors reveals a major Achilles heel for the virus and provides the needed template for development of immunotherapeutics and improved vaccines.

5:40 Welcome Reception in the Exhibit Hall with Poster Viewing (Commonwealth Hall)

7:15 End of Day

TUESDAY, APRIL 9

8:00 am Registration (Commonwealth Hall) and Morning Coffee (Harbor Level)

MEMBRANE PROTEINS

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8:25 Chairperson’s Remarks

Shahram Misaghi, PhD, Senior Scientist, Cell Culture, Genentech, Inc.

8:30 Structures Suggest a Mechanism for Energy Coupling by a Family of Organic Anion Transporters

Stroud_RobertRobert M. Stroud, PhD, Professor, Biochemistry and Biophysics, Pharmaceutical Chemistry, Macromolecular Structure Group (MSG), University of California, San Francisco (UCSF)

Members of the solute carrier 17 family use divergent mechanisms to concentrate organic anions.  Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use electroneutral cotransport to drive efflux from the lysosome. To identify the common features of ionic coupling by the SLC17 family, we determined the structure of E. coli D-galactonate/H+ symporter DgoT in two states: one open to the cytoplasmic side, and the other open to the periplasmic side with substrate bound. The structures identify residues of a proton translocation pathway conserved from bacteria to mammals. Functional analysis suggests that a transition in the role of H+ from flux coupling to allostery may underlie the divergence in energy source.    

9:00 Modulation of STEAP2 Conformation by the Cholesterol Content of Cellular Membrane: An In-Depth Study of Conformational Epitope Located in the Second Extracellular Loop

Hasegawa_HarukiHaruki Hasegawa, PhD, Principal Scientist, Biologics – Protein Technology, Amgen, Inc.

Leveraging a newly-identified mAb that recognizes a conformation-sensitive epitope nested in the second extracellular loop of human STEAP2, we demonstrate that the epitope formation is dependent on the cholesterol content of the membrane in which STEAP2 was embedded. Membrane permeabilization step and membrane cholesterol extraction treatment both abrogated cell surface staining of STEAP2 expressing cells. Given the preexisting difference in cholesterol content among different cellular membranes, STEAP2 conformation appears to undergo compartment-specific modulation during secretory and endocytic trafficking.

9:30 Tag-on-Demand – Exploiting ‘Switchable’ Expression Technology for the Enrichment of High-Expressing Membrane Protein Cell Lines

Britton_ZacharyZachary T. Britton, PhD, Scientist, Antibody Discovery and Protein Engineering, AstraZeneca

Poor expression and detection of membrane protein therapeutic targets have hampered drug discovery and screening efforts. To address this, we have developed the “Tag-on-Demand” approach that exploits ‘switchable’ expression of ‘tagged’ and ‘untagged’ membrane proteins in response to non-natural amino acid supplementation. Expression of ‘tagged’ membrane proteins facilitate detection and selection steps, and expression of ‘untagged’ native proteins can be used directly in whole-cell drug discovery efforts. Validation of this approach using model membrane proteins will be presented.

10:00 Coffee Break in the Exhibit Hall with Poster Viewing (Commonwealth Hall)

MEMBRANE PROTEINS – DETERGENTS & SOLUTIONS

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10:45 Chairperson’s Remarks

Shahram Misaghi, PhD, Senior Scientist, Cell Culture, Genentech, Inc.

10:50 A High-Throughput Approach for Kinetics of Membrane Protein-Detergent Interactions

Movileanu_LiviuLiviu Movileanu, PhD, Professor, Physics, Syracuse University

Interfacial interactions of the membrane protein-detergent complex (PDC) have implications in protein function, structure, stability, and dynamics. Current methods for examining kinetics of the PDC require high amounts of protein and are low throughput. I will talk about our recent developments on an approach for acquiring details of these interactions in a scalable fashion. Further improvements of this semi-quantitative method will impact physical and chemical biology of membrane proteins.

11:20 Peptidisc: A Simple Solution for Capturing Membrane Proteins without Detergent

Duong_FranckFranck Duong, PhD, Professor and Principal Investigator, Biochemistry & Molecular Biology, University of British Columbia

The peptidisc is a straightforward “one fits all” method that allows capture of membrane proteins into functional, heat-stable, water-soluble particles. Addition of lipids or engineering of the scaffold is not necessary. The flexibility of the peptidisc is suited for trapping proteins of various fold, size, architecture and the reconstitution process can be embedded directly within the membrane protein purification protocol.

11:50 Exploration of New Methods to Improve and Streamline Expression of Difficult Membrane Proteins to Support Drug Discovery

Byrne_NoelNoel J. Byrne, MSc, Associate Principal Scientist and Lead, Expression Group, Target Protein Design, Merck Research Laboratories

Integral membrane proteins represent more than 60% of current drug targets. Despite the clinical significance, therapeutic agents that target membrane proteins have been difficult to develop. Poor expression in recombinant systems is the most critical challenge to producing functional integral membrane proteins for antibody discovery, structural and functional studies.  The results from the exploration of different technologies for the streamlined, efficient mammalian expression of several GPCRs and Ion Channels through stable cell-line generation and transient expression (DNA and BacMam) will be presented. 

12:20 pm Luncheon Presentation I: Expi293 Transient Protein Expression System:  New Tools for Structural Biology and Difficult to Express Proteinsthermofisherscientific_no_tagline

Jon Zmuda, PhD, Director of Cell Biology, R&D, Thermo Fisher Scientific

In this presentation, we demonstrate the performance of a new range of Expi293 components, including engineered Expi293 cell lines (e.g. GNTI-, Inducible and Inducible/GNTI- Expi293 cell lines) to allow for regulated expression and/or glycosylation of proteins, as well as an Expi293 Methionine-Deficient System for metabolic labeling of proteins for NMR or multiwavelength anomalous dispersion (MAD).  Together, these new tools significantly enhance the ability of researchers to produce difficult to express proteins for structural biology analyses.

Selexis

12:50 Luncheon Presentation II to be Announced

Ghislaine Arib, PhD, Genomic Director, Selexis SA

Biologics development requires stable, high-producing cell clones yielding high-quality product over years. These clones need stable chromosomal integration, elevated transcription plus optimized metabolic & secretion. Selexis CHO cell line generation combines the SUREtechnologyPlatform™ with precise selection of clones with desirable traits and manipulation of specific genes. This approach allows the isolation of desired clones that are rare events in cell populations and has led to remarkable progress in optimized product quality & titers.

1:20 Ice Cream Break in the Exhibit Hall with Poster Viewing (Commonwealth Hall)

BREAKTHROUGH TECHNOLOGIES TO SUPPORT DTEPs

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2:00 Chairperson’s Remarks

William Gillette, PhD, Principal Scientist, RAS Reagents Core; Deputy Director, Protein Expression Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research (FNL)


2:05 FEATURED PRESENTATION: MicroED: Cryo-Electron Diffraction of 3D Microcrystals

Nannenga_BrentBrent Nannenga, PhD, Assistant Professor, Chemical Engineering, Arizona State University

The growth of large well-ordered crystals is often a barrier to high-resolution biomolecular structure determination. The cryo-electron microscopy technique of microelectron diffraction, or MicroED, is capable of determining high-resolution structures from extremely small microcrystals, promising to overcome this obstacle. In this presentation, the MicroED technique and representative structures will be described, as well as new improvements aimed at structure determination of difficult targets.

2:35 Development of a High-Yielding Expression Platform for the Introduction of Non-Natural Amino Acids

Roy_GargiGargi Roy, MSc, Scientist, Antibody Discovery and Protein Engineering, AstraZeneca

We developed an expression technology that enables site-specific incorporation of non-natural amino acids (nnAA) in a protein sequence. Fully functional, high yielding IgG, in a continuous perfusion process, was produced in hosts stably expressing an orthogonal tRNA synthetase/tRNA pair. These host platforms hold promise to overcome the expression challenges that have encumbered the developability of this technology for manufacturing of antibody-drug conjugates and other protein conjugates.

Lonza-tagline-stacked

3:05 GS Xceed® Gene Expression Toolbox: Overview and the Introduction of New Tools

Peter O’Callaghan, PhD, Principal Scientist, Research & Development, Lonza Pharma & Biotech

In this presentation, we will share an overview of the GS Xceed® Gene expression toolbox will be presented with a focus on new tools that have recently been added. Alongside this some new in-house research will be presented showing the benefit of applying control circuits to boost protein expression.

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

ANTIBODY EXPRESSION

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4:25 Utilizing a Regulated Targeted Integration (RTI) Cell Line Development (CLD) Approach to Systematically Investigate What Makes an Antibody Difficult to Express

Misaghi_ShahramShahram Misaghi, PhD, Senior Scientist, Cell Culture, Genentech, Inc.

A regulated target integration (RTI) system was used to analyze causes of low protein expression for a difficult-to-express antibody (mAb-A). Based on our findings, both antibody heavy chain and light chain subunits of mAb-A independently contributed to its low expression. RTI pools, generated by swapping antibody chains or point-mutations, confirmed that LC expression triggered ER stress and accumulation of intracellular BiP, while HC molecules had impaired degradation and clearance.

4:55 Expression of Multivalent Antibodies that Can Cross the BBB and Detect Aggregates in Amyloid Diseases with Expi293 Cells and PEI

NEW:  Sofia Stenler, PhD, Scientist, Pharmaceutical Biosciences, Protein Drug Design, Uppsala University

I will describe a method of expressing antibodies and bispecifics in EXPI293 cells with PEI. The method is cheap and reliable. We have used the method to express bispecific antibodies that can pass the BBB 80 times better than unmodified antibodies. The antibodies bind monovalently despite having two binding domains and this is what facilitated the high uptake. We have shown that the antibodies can be used to treat or diagnose neurodegenerative diseases.

5:25 End of Difficult-to-Express Proteins

5:30 Registration for Dinner Short Courses (Commonwealth Hall)


Recommended Short Course*

SC9: Introduction to Biophysical Analysis for Antibody Discovery & Development

Christine P. Chan, PhD, Principal Scientist, Global Manufacturing Science & Technology, Sanofi

 

*Separate registration required.


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