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Certain proteins have gone down in scientific legend as those that just won’t express in quantity or quality, no matter how many hoops researchers jump through. They are toxic to the host, they won’t fold properly, the quality is poor, they won’t crystallize, are just some of the problems faced. CHI’s 13th 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. Smart planning, done early in the process to provide correct hosts, appropriate affinities, and easy, effective purification is a must. Difficult to Express Proteins presents solutions and strategies to tame these “finicky” proteins and make them tractable, usable and effective.

Final Agenda

Sunday, April 29

Recommended Short Course(s)*

SC1: Preclinical and Clinical Assessment of Immunogenicity: Multidomain Therapeutics and New Modalities, Including Gene Therapy and CAR T

SC5: In silico Immunogenicity Predictions (Hands-On) Workshop


*Separate registration required.


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

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8:30 Chairperson’s Remarks

Hussain Dahodwala, PhD, Postdoctoral Research, University of Delaware, Delaware Biotechnology Institute

8:40 Toolbox for Productivity Boost and Product Quality Improvements of Difficult-to-Express Proteins (DTE) in CHO Cells

Martin_GamerMartin Gamer, PhD, Early Stage Bioprocess Development, Boehringer Ingelheim Pharma GmbH & Co. KG

The increasing number of engineered, often antibody-derived molecule formats entering into biopharmaceutical development poses significant challenges on the generation of high-yielding CHO cell factories. This talk will highlight the most recent advances at Boehringer Ingelheim to improve cell line development of DTE proteins. Our toolbox comprises in silico methods to assess molecule developability leading to tailored development, a rationally designed novel host cell line ensuring high performances, robustness and scalability as well as innovative genetic elements and screening tools to select for outstanding CHO production cell lines.

9:10 Novel Engineered CHO DG44 Host Cell Line Demonstrates Lowered UPR, Increased Titers & Superior Quality of Recombinant Vaccines

Hussein_DahodwalaHussain Dahodwala, PhD, Postdoctoral Researcher, University of Delaware, Delaware Biotechnology Institute

In our workflow, high-producing clones share a common phenotype of increased viable cell density (VCD) and viability at later days in fed-batch culture. To address the production of viral antigens and therapeutic proteins a novel CHO DG44 host clone was engineered and selected for high VCD and viability in later days of fed-batch culture. Using this host, we achieved a 3 X increase in viral antigen titers and mAbs without changing the existing upstream process.

9:40 Optimization for Heterologous Disulfide Bonded Protein Manufacture:  Production of Antibody Fragments in the Periplasm and Cytoplasm of E. coli

Bhupal Ban, PhD, Research Assistant Professor, Antibody Engineering & Technology, University of Virginia

Our results show that the efficient production of soluble, biologically active ScFv and VHH antibody fragments in the cytoplasm of engineered E. coli strain is cost effective and simplistic method.  Further, free cysteine residues can be a major bottleneck hampering the production of correctly folded, soluble protein in both E. coli and mammalian cells.

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

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10:45 Chairperson’s Remarks

Hussain Dahodwala, PhD, Postdoctoral Researcher, University of Delaware, Delaware Biotechnology Institute

10:50 Identification of Intracellular Production Bottlenecks in Suspension-Adapted CHO Cells Producing Complex Biopharmaceuticals Using Fluorescence Microscopy

Kerstin_OtteKerstin Otte, PhD, Professor, Pharmaceutical Biotechnology, Biberach University

With the advance of complex biological format therapeutics, mammalian expression systems often show low performance possibly due to accumulation or haltering of heterologous proteins within the different cellular compartments disturbing transport or secretion. Since neither transcription nor translation processes satisfactorily explain low production capacity, we established a streamlined confocal microscopy-based methodology for CHO production cells which enables the identification of rate-limiting-steps and can also be used for automated detection of production bottlenecks during industrial cell line development processes.

11:20 Improving Transient Gene Expression in Insect Cells

Joop_van_den_HeuvelJoop van den Heuvel, PhD, Department of Structure and Function of Proteins, Helmholtz Centre for Infection Research

Fast expression of recombinant protein is not limited to the use of E. coli. Plasmid based transient gene expression (TGE) in mammalian cell lines is an attractive method to screen for suitable expression constructs and to produce high quality recombinant mammalian proteins. Here we present which steps have been successful to reach high levels of expression in High Five insect cells. Additional miniaturization using an automated micro fermentation system allowed us to implement a high throughput splitGFP screen for expressible constructs. We show that TGE in High Five insect cells is a fast and cheap alternative to produce substantial amounts of high quality recombinant protein.

11:50 Breaking News in Expression Science:  Suppression of the Immune Response as a Tool to Enhance the Expression of Recombinant Proteins in Plants

Fabio Cupri Rinaldi, PhD, Research Associate, Martin Lab, Cornell University

Many pathogens have evolved effector proteins that manipulate host defense mechanisms, including PCD. The plant pathogen Pseudomonas syringae includes in its immune suppression repertoire the effector AvrPtoB. Once inside the host cells, AvrPtoB binds to cellular kinases and inhibits the plant immune response. Extensive research has revealed that AvrPtoB is a general cell-death suppressor in plants. Here we demonstrate how we harnessed the ability of AvrPtoB to suppress PCD, and significantly enhance DTE protein accumulation up to five-fold. We envision developing this technology with the ultimate goal of boosting the production of commercially relevant proteins in plants.


LONZA tagline12:20 pm Moving Beyond the Central Dogma: New Tools for the Industrial Production of Difficult to Express Proteins (DTEPs)

Colin Jaques, PhD, Senior Principal Scientist, Process Development, Research and Technology, Lonza

In recent years, mAbs were the primary protein therapeutic. Now heavily engineered DTEP next generation biologics dominate early phase pipelines. This shift requires tools developed to achieve economically viable concentrations and clinically relevant critical quality attributes. Tools for protein expression were traditionally based on the Central Dogma. For many DTEPs, expression problems occur outside the Central Dogma in post-translational processing. Two new approaches to increase production of DTEPs by targeting post-translational processing will be discussed.

Berkeley Lights 12:50 Luncheon Presentation I: Screening Challenging Clones at Light Speed

Keith_BreinlingerKeith Breinlinger, MS, PhD, CTO, Berkeley Lights, Inc.

Screening thousands of clones for production and function is critical for success when working with difficult to express proteins. The Beacon platform applies light and semiconductor technology in a nanofludic chip to isolate single cells, culture, assay, and export clones of interest. Now this can be done on thousands of clones in just a few days instead of months.


Fujifilm Diosynth1:20 Luncheon Presentation II: Approaches and HTP to Challenges for Recombinant Proteins Expressed in E. coli

Nigel Shipston, PhD, Director, Program Design, FUJIFILM Diosynth Biotechnologies

From this talk you will learn to Avoid “re-inventing the wheel” for every product and to expedite the initial stages of process invention to enable rapid establishment of a process suitable for GMP manufacturing.

1:50 Session Break

2:20 Problem-Solving Breakout Discussions (Commonwealth Hall)

Characteristics of Poorly-Secreted mAb Clones

Host: Haruki Hasegawa, PhD, Department of Therapeutic Discovery, Amgen

  • Notable “behaviors” during biosynthesis in the secretory organelles
  • Influence on cell health during overexpression
  • Sequence-related properties vs. surface properties
  • Protein engineering vs. cell engineering approach
  • Display technology-derived mAb clones vs. animal-derived mAb clones
  • Easier dropping it than fixing it?

How to solve production challenges of Difficult to Express Proteins?

Host: Kerstin Otte, PhD, Professor, Pharmaceutical Biotechnology, Biberach University

  • New classes of engineered proteins pose challenges for production processes
  • There is a need to improve properties as expression, folding, quality, crystallization etc.
  • Which methods are available to tackle these problems and are they successful?

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

PLENARY KEYNOTE SESSION              Amphitheater and Harborview Ballroom

4:00 Chairperson’s Remarks

Peter Fung, PhD, Senior Manager Product Marketing, NanoTemper Technologies

4:10 Challenges and Opportunities in Engineering Protein Biopharmaceuticals

Dane_WittrupK. Dane Wittrup, PhD, C.P. Dubbs Professor, Chemical Engineering and Biological Engineering; Associate Director, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT)

Arthur C. Clarke’s First Law posits that “When a distinguished but elderly scientist states that something is possible, he is almost certainly right. When he states that something is impossible, he is very probably wrong.” Bearing this in mind, in this talk I will highlight areas of protein drug development that appear poised for breakthroughs in the coming decade or so.

4:55 The Next Generation of Cancer Immunotherapy: Targeting Myeloid Immune Checkpoints

Kipp_WeiskopfKipp Weiskopf, MD, PhD, Resident Physician, Internal Medicine, Brigham and Women’s Hospital

Immune cells of the myeloid lineage hold tremendous potential as effectors of cancer immunotherapy. The CD47/SIRPα axis is a key molecular pathway that governs the interaction between myeloid cells and tumors. Therapies that target the interaction are effective across multiple preclinical models of cancer and are now under investigation in clinical trials. Further studies have revealed additional regulators of myeloid cell activation that can be exploited as myeloid immune checkpoints.

Precision for Medicine5:40 Welcome Reception in the Exhibit Hall with Poster Viewing (Commonwealth Hall)


7:15 End of Day


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

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8:25 Chairperson’s Remarks

Matthew A. Coleman, PhD, Senior Scientist, Lawrence Livermore National Laboratory

8:30 Increasing Expression of an Antibody Fab Fragment in Escherichia coli with Unnatural Amino Acid (UAA) Incorporated by Strain Engineering & Process Development

Harun_RashidHarunur Rashid, PhD, Principal Scientist, Ambrx, Inc.

Expression of a ‘difficult-to-express’ antibody Fab fragment with an UAA inserted at a selected site was systematically optimized by expression vector & strain engineering. Among the various genetic elements on expression vector tested, DNA coding sequence, periplasmic chaperone, Fab heavy chain (HC) carboxy-terminal extension and the presence of plasmid partition locus, parB, were beneficial. All of these four components were then put together into a single expression vector that resulted in several hundred-fold improvement in expression titer over the starting strain.

9:00 New Techniques in Heterologous Expression and Purification of Large Polytopic Integral Membrane Proteins

Paul_RoepePaul Roepe, PhD, Professor, Chemistry, Biochemistry and Mol. Biology, Georgetown University

Optimized heterologous expression (codon optimization and other techniques) along with tandem chromatography and other analytical methods has been used to purify several very large malarial parasite membrane proteins. (All examples recently published in a series of “Biochemistry” papers, one additional currently in preparation for publication.)

9:30 A Novel Bicistronic Gene Design Couples Stable Cell Line Selection with a Fucose Switch in a Designer CHO Host to Produce Native and Afucosylated Glycoform Antibodies

Gargi_RoyGargi Roy, MSc, Scientist I, Antibody Discovery and Protein Engineering/Research, MedImmune LLC

Antibodies (mAbs) are complex glycosylated proteins important as therapeutic molecules. mAbs lacking the core fucose at Asn297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Following GlymaxX technology, a Chinese Hamster Ovary (CHO) host cell line was engineered to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in stable expression of high titer, highly potent afucosylated mAb with enhanced ADCC activity suitable for manufacturing.

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

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10:50 KEYNOTE PRESENTATION: Working through Difficult Proteins with Variants, Intensification and Automation

Randal_BassRandal Bass, PhD, Vice President, Just Biotech

Difficult to express proteins will inevitably enter the biotherapeutics development pipeline. As an industry, the better and faster we can adapt, optimize and streamline our response to these proteins, the greater extent to which we will lower costs and improve the chances these molecules will become safe and effective therapeutics. Here, I will discuss efforts that flow from molecular optimization through high-throughput process design, and even into the design of manufacturing facilities, to maximize the potential for molecules to successfully reach commercialization.

11:20 Cell-Free Production of a Functional Oligomeric Form of a Chlamydia Major Outer Membrane Protein for Vaccine Development

Matthew_ColemanMatthew A. Coleman, PhD, Senior Scientist, Lawrence Livermore National Laboratory

Chlamydia is a prevalent sexually transmitted infection that infects over 100 million people worldwide. Chlamydia strains express a major outer membrane protein (MOMP) that is an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein are limited due to poor solubility, low yield, and misfolding. We will present a cell-free co-translation method to make functional MOMP within a nanolipoprotein particle. Our approach solubilizes membrane-bound proteins for biochemical, biophysical characterization and antigen generation.

11:50 Applications of Modular Expression Toolboxes in High-Throughput Protein Expression

Ernest_WeberErnst Weber, PhD, Senior Scientist, Antibody Lead Discovery, Biologics Research, Bayer AG

The presentation will focus on the setup of a modular expression toolbox, consisting of standardized elements influencing expression levels, which allow the rapid generation of multiple expression constructs and also the generation of complex expression optimization libraries. Advantages and implications of a modular cloning system including implementation into protein expression optimization workflows will be discussed and a number of successful case studies will be presented.


12:20 pm LUNCHEON PRESENTATION I: Scaling Cultivations in Your Shaker Incubators: How Vessel, Volume, Speed and Diameter Impact Your Lab-Scale Experiments

David Laidlaw, CEO, Kuhner Shaker, Inc.

Shaker-incubator conditions are as adjustable as those in bioreactors. Reproducible scale-up/down is achievable when culture-influencing shaker parameters are considered during experiment design. We review here how fill volumes, shaking speeds, shaking diameters and closure types influence scalability between deep well plates, tubes, and flasks. Mixing times, kLa’s, surface velocities and evaporative losses will be presented for different vessel types. Specific examples from the literature and Kuhner Shaker in-house data will be shown.

GenScript CRO Logo12:50 Luncheon Presentation II: New Ways to Boost Recombinant Protein and Antibody Yields in Transient Systems

Brady Wu, Associate Director, Protein Production, GenScript USA, Inc.

Transient expression is a fast and reliable way to generate recombinant antibodies and proteins for all forms of research. However, there are several factors limiting the titer of the products in the system, thus the need for optimization at different steps of the process. At this talk, GenScript will demonstrate that one can improve the titer of transient products by tuning the systems in several ways including better anti-apoptosis effect, better cell growth, and more.

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

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2:00 Chairperson’s Remarks

Haruki Hasegawa, PhD, Department of Therapeutic Discovery, Amgen

2:05 Engineering a Patient-Derived Anti-MRSA rF1 Antibody to Develop an Antibody Antibiotic Conjugate (AAC) Therapeutic

Yi_XiaYi Xia, MD, Senior Scientific Researcher, Antibody Engineering, Genentech, Inc.

We have successfully cloned rF1 CDRs into pRK vector and repaired antibody light chain framework position 105-112 from patient’s EIKR-AAA to germline EIKRTVAA (human Kappa 4), boosting antibody production yield from 0.5 mg/L to 75mg/L and solved more than 70% aggregation issues as well. We successfully molecular engineered and generated a promising anti-MRSA therapeutic antibody for antibody-antibiotic conjugate (AAC) clinic use.

2:35 Improving Yield and Stability of Cannabinoid Receptor

Alexei_YeliseevAlexei Yeliseev, PhD, Staff Scientist, Group Leader, LMBB, NIH, NIAAA

We expressed the recombinant cannabinoid receptor CB2 in E. coli cells as well as in expi CHO and expi HEK293 cells in milligram quantities. The yield of the functional target protein was enhanced by modifications of codon usage, testing multiple expression constructs and media compositions. Stabilization of the protein at high concentration in monomeric form was achieved by optimizing the N- and C-terminally truncated constructs of CB2, careful selection of stabilizing lipids and ligands, and controlling glycosylation. Examples of the 19F NMR and DEER measurements will be presented.


3:05 Artificial Protein Folding System to Enhance the Production of “Difficult-To-Express Proteins”

Akinori Hishiya, PhD, Principal Scientist, Biology, SOLA BioSciences

This presentation will discuss: 1) Protein folding is one of the major issues in protein production. Proteins with protein folding problems are so-called “difficult-to-express proteins” due to poor secretion and instability. 2) We have developed a novel technology exploiting protein folding machinery, in which the technology works only for the protein of interest. 3) The technology has successfully enhanced the production of many therapeutic proteins and the expression of some of GPCR proteins were significantly enhanced.

POLYPLUS_withTagline3:20 Development and Optimization of an Advanced Transient Expression System in CHO Cells

Mathieu_PorteMathieu Porte, Project Leader Research & Development – Bioproduction, Research & Development, Polyplus-transfection

Transient expression in CHO cells is commonly used to rapidly produce antibodies but is often limited by transfection efficiency and inherent productivity. To overcome this issue, we developed an advanced transient expression system consisting in the synergistic association of a novel CHO chemically defined medium and a powerful transfection reagent.

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

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4:25 Effects of One Amino Acid Substitution on IgG Biosynthesis, Physicochemical Property, and Biologic Function

Haruki_HasegawaHaruki Hasegawa, PhD, Department of Therapeutic Discovery, Amgen

We investigated the biosynthetic process of two functional human IgG2ĸ mAbs that differ only by one amino acid in the LC-CDR1. Despite their near-identity, one mAb was secreted ∼20-fold less than the other. We found that the poorly-secreted mAb induced Russell body in the ER during overexpression and triggered eIF2α phosphorylation. The observed poor IgG secretion was due to severe down-regulation of global protein synthesis as opposed to physical obstructions of secretory pathway trafficking.

4:55 PANEL DISCUSSION: Emerging Methods to Improve Expression of Difficult Proteins

Haruki_HasegawaHaruki Hasegawa, PhD, Department of Therapeutic Discovery, Amgen

Ernest_WeberErnst Weber, PhD, Senior Scientist, Antibody Lead Discovery, Biologics Research, Bayer AG

Yi_XiaYi Xia, MD, Senior Scientific Researcher, Antibody Engineering, Genentech, Inc.

Alexei_YeliseevAlexei Yeliseev, PhD, Staff Scientist, Group Leader, LMBB, NIH, NIAAA

Randal_BassRandal Bass, PhD, Vice President, Just Biotech

Matthew_ColemanMatthew A. Coleman, PhD, Senior Scientist, Lawrence Livermore National Laboratory

Gregory BleckGregory Bleck, Global Head, Research & Development, Biologics, Catalent Pharma Solutions

5:25 End of Difficult to Express Proteins Conference

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

Recommended Dinner Short Course(s)*

SC8: Introduction to Biophysical Analysis for Biotherapeutics: Discovery & Development Applications

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

SC13: Sub Visible Protein Particles in Immunogenicity: Measurement, Characterization and Impact

Björn Boll, PhD, Head, Particle Lab and Higher Order Structure Protein Analytics, Physical Chemical Analytics, Novartis Pharma AG

Anacelia Ríos Quiroz, PhD, Scientist, Group Leader Particle Lab, Pharma Technical Development Europe (Biologics) Analytics (PTDE-A), F. Hoffmann-La Roche Ltd.

*Separate registration required.

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