Whether for therapeutics, diagnostics or for characterization, some proteins just don’t cooperate when it comes to expression. CHI’s Eleventh Annual Difficult to Express Proteins conference will present the most up-to-date strategies and technologies for successfully expressing the proteins that keep researchers up at night. Folding problems, toxicity to hosts, purification issues or any of a host of other problems make these proteins resist standard expression and purification methods. Difficult to Express Proteins presents solutions and strategies to tame these “finicky” proteins.


7:00 am Registration and Morning Coffee

New Research that Changes the Field

8:30 Chairperson’s Remarks

Haruki Hasegawa, Ph.D., Principal Scientist, Therapeutic Discovery, Amgen, Inc.


The Human Secretome Project - Making and Functional Testing of All Secreted Human Proteins

Rick_DaviesRick Davies, Ph.D., Associate Director, Reagents & Assay Development, AstraZeneca

Through a collaboration between The Royal Institute of Technology (KTH) in Stockholm and AstraZeneca, a project has been initiated to produce the entire ‘Secretome’ using state-of-the-art recombinant expression technology in mammalian cells. In this presentation, I will explain the concept and describe an initial pilot project in which a subset of the ‘Secretome’ consisting of 500 proteins was produced and tested in several different phenotypic assays

9:10 Discovery and Manipulation of Genes that Regulate ER Export and ER to Golgi Transport: Critical Implications for Protein Expression

Jesse_DayJesse C. Hay, Ph.D., Professor, Division of Biological Sciences, University of Montana

ER to Golgi transport is the first and rate-limiting step in the secretory pathway. We identified a calcium signaling pathway that regulates ER to Golgi protein trafficking that can be modified to increase ER export and ER to Golgi flux. This discovery will allow increased secretion into the medium or transport to the cell surface of soluble and membrane proteins, respectively, during manufacturing and research processes.

9:40 Is Endoplasmic Reticulum a Friend or a Foe to the Biopharmaceutical Industry? Lessons We Learned from 12 Angry mAbs

Haruki_HasegawaHaruki Hasegawa, Ph.D., Principal Scientist, Therapeutic Discovery, Amgen, Inc.

The stringency of protein quality control mechanisms in the endoplasmic reticulum (ER) is known for many years. This is something you experience firsthand when your proteins of interest are synthesized but not secreted. Is the ER just giving us the hard time or does this common phenomenon have larger implications? I will discuss how we can take advantage of what the ER is programed to do during protein expression in a mAb-specific manner and why it offers new approach to identifying high quality biotherapeutic lead candidates, fast.

10:10 Coffee Break

Membrane Proteins and Other “Beastly”
Expression Problems

10:45 Chairperson’s Remarks

Haruki Hasegawa, Ph.D., Principal Scientist, Therapeutic Discovery, Amgen, Inc.

10:50 Integrated Cell and Process Development for a Difficult to Express Protein

Christine_AlvesChristine Alves, Ph.D., Cell Culture Development, Biogen

New approaches in cell line and culture process development were utilized to increase expression of a difficult to express, positively charged protein by greater than 10-fold while maintaining product quality similar to early clinical material. A combination of multiple CHO hosts, optimization of media additives, and use of 15mL automated bioreactors resulted in evaluation of over 100 cell lines.

11:20 Dealing with Difficult Proteins via Molecular Design and Advanced Process Platforms

Randal_BassRandal Bass, Ph.D., Vice President, Process Design, Just Biotherapeutics

Proteins destined for clinical and commercial manufacturing require considerable work to establish robust manufacturing with molecular attributes that lead to a successful therapeutic. Difficult proteins require even larger, sometimes herculean efforts to make it through development. We apply an integrated approach from molecule design, process design, and even the manufacturing plant design to optimize the sequence, structure and production of therapeutic proteins.

11:50 Selected Poster Presentation



12:20 pm Development of an Eukaryotic Expression System for Expression of Complex Difficult-to-Express Proteins, Including Ion Channels

Prabuddha K. Kundu, Ph.D., Executive Director, Premas Biotech Pvt Ltd.

Premas Biotech has developed an eukaryotic expression system that enables high fidelity expression of difficult to express proteins, including ion channels, etc. The yeast, S cerevisae system includes the modified strain, vectors, and expression conditions to express the membrane proteins in their correct confirmation and activity. This has been demonstrated for a number of GPCRs, Ion Channels, and difficult to express proteins. The strain has demonstrated high levels of expression and high yields.

12:50 Luncheon Presentation I: Engineering Genes, Vector Elements and Strain Properties for Optimized mAb Production in Mammalian Cell Lines

Claes Gustafsson, Chief Commercial Officer, DNA2.0, Inc.

1:20 Luncheon Presentation II (Sponsorship Opportunity Available)

1:50 Session Break

2:20 Problem-Solving Breakout Discussions

Right from the Start: Designing Your Problems Away

Host: Randal Bass, Ph.D., Vice President, Process Design, Just Biotherapeutics

  • Computational approaches for protein design.
  • High throughput variant production.
  • Early assessment of molecule acceptability.

Optimizing Cell-Free Co-Translation for Difficult to Express Proteins

Host: Matthew Coleman, Ph.D., Senior Scientist, Physics and Life Sciences, Lawrence Livermore National Laboratory

  • Advantages and disadvantages of different cell-free systems
  • Plasmid testing for optimizing co-translation using cell-free lysates
  • Optimizing reaction scale for downstream applications of cell-free produced proteins
  • Additives that enhance receptor and membrane bound protein production in cell-free lysates

Producing Intrinsically Disordered Regions of Proteins - What, How, and Why?

Host: Peter Hwang, Ph.D., Assistant Professor, Biochemistry, University of Alberta

  • Intrinsically disordered regions (IDRs) are common and critical to biological regulation
  • Recombinant production of IDRs requires unique tools
  • Solution NMR spectroscopy is the dominant method for characterizing IDRs


3:20 Refreshment Break in the Exhibit Hall with Poster Viewing


4:00 Chairperson’s Remarks

4:10 The Promise of Cancer Immunotherapy: An Overview of Recent Advances and Jounce’s Approach to Delivering the Right Therapy to the Right Patient

Deborah_LawDeborah Law, D. Phil. CSO Jounce Therapeutics, Inc.

As immunotherapies become an increasingly important component of cancer treatment the challenge will be to identify ways to provide the best therapy(s) to the individual. This presentation will provide an overview of current cancer immunotherapies as well as highlight some of the challenges ahead including selection of optimal combinations, moving outside of T cell-directed approaches, and will highlight how Jounce Therapeutics is using its Translational Science Platform as an approach to develop and deliver the right therapy to the right patient.

4:50 Antibody as Drugs: Then, Now and Tomorrow

Paul_CarterPaul J. Carter, Ph.D., Senior Director and Staff Scientist, Antibody Engineering, Genentech

Antibodies have grown into a clinically and commercially important drug class with more than >45 antibodies marketed for imaging or therapy in the USA and/or Europe and with ~$63 billion in worldwide sales in 2013.  This presentation will highlight progress in developing antibody drugs and consider opportunities for future innovation.  

5:30 Welcome Reception in the Exhibit Hall with Poster Viewing

6:45 End of Day


8:00am Morning Coffee

Cell-Free and Other Cutting-Edge Strategies for Success

8:25 Chairperson’s Remarks

Matthew Coleman, Ph.D., Senior Scientist, Physics and Life Sciences, Lawrence Livermore National Laboratory

8:30 Harmonization of Transient CHO and Stable CHO Expression Platforms for Early Phase Drug Discovery

Gavin Barnard, Ph.D., Group Leader, Eli Lilly & Co.

We describe the development of a transient CHO system capable of generating high titers, currently scalable to 6L. Additionally, we describe the use of stable CHO bulk pools (instead of master wells or clones) for generation of gram quantities of therapeutic protein. Using the same CHO cell line and media package for both platforms streamlines expression during early phase drug discovery.

9:00 Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature

Li_NiuLi Niu, Ph.D., Chemistry Department, Center for Neuroscience Research, University at Albany, SUNY

Animal cells and cell lines are commonly cultured and used to express recombinant proteins at 37 ºC. We show dropping culture temperature to 33 ºC, but not lower, 24 hours after transient transfection with HEK-293 cells will yield ~1.5-fold higher expression of a recombinant protein, such as green fluorescent protein. A mild hypothermia reduces the HEK-293 cell growth rate, while increasing cellular productivity of a protein without affecting the protein function. We demonstrate that this method may be also useful when a recombinant protein is difficult to express, such as in PC-12 cells, using a chemical-based, transient transfection method.

9:30 BacMam Production of Active Recombinant Lecithin-Cholesterol Acyltransferase: Expression, Purification and Characterization

Bil_RomanowWilliam G. Romanow, Senior Associate Scientist, Protein Technologies, Amgen, Inc.

We recently reported a high-resolution crystal structure for lecithin-cholesterol acyl transferase (LCAT). Here we discuss the methods used to produce recombinant LCAT, and other glycoproteins suitable for X-ray crystallography. This will include the use of BacMam as a transient system for the production of secreted proteins, the use of cell lines and reagents to inhibit glycosylation and the bioprocessing of conditioned media and purification.

10:00 Coffee Break in the Exhibit Hall with Poster Viewing and Poster Awards

10:50 Cell-Free Co-Translation Systems for Biophysical and Biochemical Characterization of Proteins and Protein Complexes

Matthew_ColemanMatthew Coleman, Ph.D., Senior Scientist, Physics and Life Sciences, Lawrence Livermore National Laboratory

Here we discuss how our laboratory has incorporated cell-free technologies to produce labeled proteins, protein complexes and trans-membrane proteins in yields that are more than sufficient for biophysical and biochemical characterization. Because cell-free translation is an open process, we have adapted commercial systems for multiple high-throughput screening techniques dependent on the suspected proteins function. For example, labels and co-factors can easily be added to synthesis reactions, such as fluorescent protein tags, peptides, SNAP-tags, small molecule co-factors, nanolipoproteins, lipids and fluorescently-labeled lipids.

11:20 Combining a PagP Fusion Protein System with Nickel Ion-Catalyzed Cleavage to Produce Intrinsically Disordered Proteins in E. coli

Peter_HwangPeter Hwang, Ph.D., Assistant Professor, Biochemistry, University of Alberta

Intrinsically disordered regions (IDRs) are solvent-exposed and unstructured, making them susceptible to post-translational modifications. They are thus important for regulation, though they can be difficult to produce. Unfolded PagP membrane protein is an effective fusion partner for producing IDRs. Using a nickel cleavage-sensitive linker, SRHW, allows for removal of PagP under denaturing conditions, leaving behind a target protein with its native C-terminus.

11:50 Improved Protein Quality through Moss-Based Manufacturing

Andreas_SchaafAndreas Schaaf, Ph.D., CSO, R&D, Greenovation Biotech GmbH

BryoTechnology is a plant-based cGMP expression platform using the moss Physcomitrella patens. As a plant production system it comes with an excellent safety profile, free of human pathogens, antibiotics and animal derived components. Moss genetic engineering is straightforward, time-effective and results in stable strains. From a technical perspective, the system is comparable to mammalian cell processes. Suspension cultures of stable, fully regenerated moss plantlets produce in well-established, single use fermenters from best-known suppliers in a fully controllable, cell-bank based process.

Integral12:20 pm Luncheon Presentation I: New Approaches for MAb Discovery Against GPCRs, Ion Channels, and Transporters

Ross Chambers, Ph.D., Director, Antibody Discovery, Integral Molecular

Integral Molecular has developed new approaches to elicit, characterize, and engineer MAbs against challenging membrane proteins using its MPS Discovery Engine®. Robust immune responses are generated against native antigens using Lipoparticles (high-concentration membrane proteins) and DNA immunization. Chickens are used because most membrane proteins are highly conserved, while both phage display and B-cell cloning are used for isolation. MAbs are engineered using high-throughput Shotgun Mutagenesis and profiled for specificity using a comprehensive membrane proteome array.

Aragen12:50 Luncheon Presentation II: Challenges to Producing and Purifying Difficult to Express Proteins from a CRO Point of View 

Rene Pagila, Scientist Group Leader, Aragen Bioscience

Maurice van der Heijden, Director of R&D, ProteoNic

Aragen Bioscience has established platforms to purify and formulate diverse client proteins from clarified transient cell culture harvest. These products range from antibodies, Fc-fusion proteins, affinity-tagged proteins (e.g. 6X His) as well as novel and native proteins without an Fc or other affinity tag. The platforms work well when the expression levels of the target protein are reasonably high and the aggregate levels are relatively low in the clarified cell culture harvest. However, when a client’s protein is hard to express and has a propensity to be highly aggregated in the clarified cell culture harvest, the established platforms are challenged. In this talk, we will be presenting cases of how we have handled such challenging situations.


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

Case Studies: Making the Difficult Easy

2:00 Chairperson’s Remarks

Arjan Snijder, Ph.D., Associate Principal Scientist, AstraZeneca Discovery R&D

2:05 Efficient Production of Aggregation-Prone Proteins Inspired by How Spiders Store Silk Proteins

Janne Johansson, Ph.D., Professor, Neurobiology, Care Sciences and Society, Karolinska Institutet Center for Alzheimer Research, Novum

Spiders are able to store silk proteins (spidroins) at huge concentrations by sequestering the spridroins’ aggregation-prone regions into micellar structures, in which the very soluble spidroin N-terminal domain (NT) contributes to the shell. We found that yields of soluble NT-transmembrane protein (TM) fusions after expression in E. coli are two to eight times higher compared to conventional tags like thioredoxin and PGB1. Furthermore, NT enables production of non-TM aggregation prone proteins that have previously been refractory to recombinant production.

2:35 Development of an Alternative Purification Method for Difficult to Express Proteins

Arjan_SnijderArjan Snijder, Ph.D., Associate Principal Scientist, AstraZeneca Discovery R&D

We present a purification method where affinity resin is contained in a porous-walled container, which supports clarification, capture and purification, in a single step thus reducing hands-on and processing time without significant investments in equipment. The process will be illustrated with a number of challenging pharmaceutically relevant protein targets, including secreted proteins, membrane proteins, enzymes and kinases.

Wacker Biotech3:05 Manufacturing of Recombinant Biopharmaceuticals by FOLDTEC®
- A Novel Toolbox of Expression Hosts, Plasmids and Refolding Expertise

Andreas Anton, Ph.D., Director, BioProcess Development, Wacker Biotech GmbH

Wacker Biotech is showcasing its novel refolding technology for bioengineered pharmaceutical proteins. With the new technology biopharmaceuticals that tend to aggregate can be efficiently produced in their soluble-active form in high yields. The proprietary process utilizes optimized bacterial strains and a patented, antibiotic-free expression system. WACKER can now cost-efficiently and reliably produce pharmaceutical proteins that are prone to aggregation, and thus difficult to manufacture, in high yields and utmost purity for its customers.

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

Trans-Splicing And Protein Synthesis

4:25 Assembling Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-Splicing

Michaela Mehler, Doctoral Researcher, Biophysical Chemistry, JW Goethe University

Protein trans-splicing using split inteins is established as a tool for protein engineering. We show that this method can be applied to a membrane protein under native conditions in vivo. Our data show that the ligation product is identical to its non-ligated counterpart demonstrating that a correctly folded and functional protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins and they support the development of novel labeling options.

4:55 Eukaryotic in vitro Translation Systems: Cell-Free Synthesis of Post-Translationally Modified Membrane Proteins

Lena_ThoringLena Thoring, Bioanalytics and Bioprocesses, Fraunhofer Institute for Cell Therapy and Immunology (IZI)

In vivo expression systems currently used for protein production processes have often shown issues during synthesis of so called “difficult-to-express” proteins. To circumvent the disadvantages of in vivo protein expression systems novel cell-free systems for the synthesis of proteins were developed based on translationally active extracts of eukaryotic cells. Endogenous microsomal structures present in these platforms enable the direct production of correctly folded, membrane embedded and post-translationally modified proteins.

5:25 End of Difficult to Express Proteins

5:30 Registration for Dinner Short Courses