Cambridge Healthtech Institute’s Twentieth Annual

Recombinant Protein Expression and Production

Achieving Quality and Quantity

January 9-10, 2018


Biopharmaceuticals currently represent the fastest-growing sector of the pharmaceutical industry, driven by a rapid expansion in the manufacture of recombinant protein-based drugs. Consequently, the efficient expression and production of these valuable biomolecules face challenges in improving their quantity and quality while minimizing time and cost. To meet these demands, an increasing variety of recombinant production platforms are being developed. Unfortunately, there is no “universal” production system which can guarantee high yields of recombinant protein, particularly as every biomolecule itself causes its own issues in terms of expression. To meet the demand, it is crucial to increase the throughput of expression, production and purification processes and systems.

Cambridge Healthtech Institute’s Recombinant Protein Expression and Production conference explores the newest data and innovations relating to the best choices in hosts/systems, as well as ways to “rescue” existing systems and make them work more effectively to produce the quality and quantity of the desired biotherapeutic.

Final Agenda


1:00 pm Registration

1:30 Refreshment Break in the Exhibit Hall with Poster Viewing

Optimizing Production Platforms

2:00 Chairperson’s Opening Remarks

Mark Arbing, Ph.D., Core Facility Director, UCLA-DOE Institute, University of California, Los Angeles


2:05 Combining Biophysical Analytics with NGS-Based Genetic Characterization and Gene Editing Technologies to Improve mAb Producing CHO Cell Lines

Holger_ThieHolger Thie, Ph.D., Associate Director, Molecular Biology, Early Stage Bioprocess Development, Boehringer Ingelheim Pharma GmbH & Co. KG

This study demonstrates how state-of-the-art technologies foster the development of production cell lines by gaining a more holistic understanding of these cells to ensure high performance and product quality. Here, the process is shown from the detection of an unfavorable molecule property (remarkable differences in N-linked glycosylation between two production clones derived from the same CHO cell line), the identification of the genetic background by NGS and how this can be fixed by applying gene editing technologies.

2:45 Hosts, Partners, and Accessories: Keys to Productive Protein Production

Mark Arbing, Ph.D., Core Facility Director, UCLA-DOE Institute, University of California, Los Angeles

Successful production of recombinant proteins requires an appropriate expression host and careful consideration of protein expression conditions. Important factors to consider are the origin of the target protein, the context in which the target protein natively exists, and additional factors (e.g., chaperones) that may be required for proper folding of the target protein.

3:15 A Chemically-Defined Baculovirus-Based Expression System for Enhanced Protein Production in Sf9 Cells

Jonathan_ZmudaJonathan Zmuda, Ph.D., Director, Cell Biology R&D, Thermo Fisher Scientific

The Baculovirus Expression Vector System (BEVS) is one of the major platforms for recombinant protein production. Unlike mammalian systems, insect systems rely on yeastolate-containing media that can exhibit significant variability in cell growth and protein expression. Here, we present the development of a novel Sf9-based Baculovirus expression system based on a high-density, chemically-defined medium, a high-expressing Sf9 cell line and enhancers that allow for consistent protein production with improvements in titers compared to traditional workflows.

3:45 Refreshment Break in the Exhibit Hall with Poster Viewing

4:30 Redirecting Bacterial Microcompartment Systems to Improve Expression of Toxic Proteins

Mimi_YungMimi Cho Yung, Ph.D., Biomedical Staff Scientist, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory

Recombinant expression of toxic peptides/proteins remains a challenging problem. One potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. As a proof-of-concept, we redirected recombinant bacterial microcompartment (BMC) systems in Escherichia coli to shield the toxicity and enhance the expression of lysis protein E from bacteriophage ϕX174. Ultimately, our recombinant system achieved a ~7-fold improvement in protein yield compared to prior reports. Ongoing efforts to express antimicrobial peptides within encapsulin microcompartments to enhance their recombinant expression will also be discussed. General strategies described here can be applied to enhance expression of other toxic proteins in recombinant E. coli systems.

5:00 Development of Diatoms (Algae) as Therapeutic Protein Expression Systems

Mark_HildebrandMark Hildebrand, Ph.D., Research Scientist and Director, Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego

We have developed microalgae called diatoms as protein expression systems, particularly for vaccine production. We use a novel inducible promoter that suppresses expression during growth (enabling expression of toxic proteins), and induces under nutrient-induced growth cessation – increasing yields by enabling channeling of energy and metabolites into protein synthesis. Diatom silica cell walls are an effective adjuvant, and the system is an all-in-one package of adjuvant and slow-release particulate antigen.

5:30 Close of Day

5:30 - 5:45 Short Course Registration

5:45 - 8:45 Dinner Short Courses*

* Separate registration required


8:00 am Registration and Morning Coffee

Cell-Free Systems

8:30 Chairperson’s Remarks

Feras Hatahet, Ph.D., Scientist, Protein Technologies, Amgen

8:35 Protein Production Platform for Rational Design Engineering

Takanori_KigawaTakanori Kigawa, Ph.D., Team Leader, Quantitative Biology Center, RIKEN

We have established a protein production platform based on cell-free technologies, which can produce milligram quantities of a hundred kinds of newly generated mutant proteins within a day totally without recombinant DNA technology. By using this platform, time and labor consuming site-directed mutagenesis and subsequent mutant protein expression/purification are dramatically accelerated. Therefore, this platform is highly useful for protein engineering with rational design approaches.

9:05 Membrane Protein Production and Characterization in Tailored Lipid Environments

Frank_BernhardFrank Bernhard, Ph.D., Lab Leader, Institute of Biophysical Chemistry, Goethe University Frankfurt

Using proprietary cell-free expression platforms, we synthesize membrane proteins directly into supplied preformed nanoparticles containing defined lipid compositions. The process has been optimized for preparative scale production yielding up to 100 µM concentrations of membrane protein containing nanoparticles in the reactions. We show applications of GPCRs, ion channels, transporters as well as of large assemblies and demonstrate the complete detergent-free structural analysis of membrane proteins by crystallization and NMR. The production pipeline is fast, highly successful and ideal for studying lipid effects on folding, stability, enzymatic activity and oligomeric assembly of membrane proteins.

Premas Biotech  9:35Oral Insulin: Significant COGs Reduction via Innovative Process Development and Production for Late Stage Clinical Trials

Prabuddha K Kundu, Ph.D.,Cofounder, Executive Director, Premas Biotech

10:05 Coffee Break in the Exhibit Hall with Poster Viewing

Antibody Production in E. coli

10:50 SPEAKER CANCELLATION: Rapid Screening of Cyclotide-Based Libraries against Intracellular Protein-Protein Interactions

Julio_CamareroJulio A. Camarero, Ph.D., Professor, Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California

We report novel methods for the biosynthesis of natively folded MCoTI-based cyclotides inside live E. coli cells using split protein splicing units. The cyclotide MCoTI-cylotides are potent trypsin inhibitors recently isolated from the seeds of Momordica cochinchinensis, a plant member of the cucurbitaceae family. Biosynthesis of genetically encoded cyclotide-based libraries opens the possibility of using single cells as microfactories where the biosynthesis and screening of a particular inhibitor can take place in a single process within the same cellular cytoplasm. The cyclotide scaffold has tremendous potential for the development of therapeutic leads based on its extraordinary stability and potential for grafting applications. We show an example, where a large cyclotide-based genetically encoded library was used to screen for low nanomolar antagonists for the Hdm2-HdmX RING-mediated E3 ligase activity. We also present different strategies to improve the cellular uptake and pharmacokinetic profiles of bioactive cyclotides.

11:20 Super Secretory Production of Recombinant Antibody Fragments by Precisely Controlled Fed-Batch Culture of E. coli

Yoichi_KumadaYoichi Kumada, Ph.D., Associate Professor, Department of Functional Chemistry, Kyoto Institute of Technology

Large-scale production of single-chain Fv antibodies by recombinant E. coli was investigated. Periplasmic secretion signal, pel B reader was often effective for leaking proteins to supernatant, while most of them were aggregated as inclusion body in flask culture. When the fed-batch culture was performed by Jar fermenter with tightly regulated DO control system, the scFvs expressed were highly secreted to the culture supernatant. Consequently, final concentration of scFv reached at more than 4g/L, and solubility of scFv was approx. 50%. These results potentially suggested that precisely regulated fed-batch fermentation is a promising way for secretory production of target recombinant proteins.

11:50 Soluble Expression of Antibody Fragments in the Cytoplasm of E. coli

Feras_HatahetFeras Hatahet, Ph.D., Scientist, Protein Technologies, Amgen

E. coli is widely used for the production of proteins of pharmaceutical importance. However, the production of soluble functional proteins can be hampered when disulfide bonds are required. We demonstrate the feasibility of producing single chain variable fragments (scFv) of antibodies and multi-chain peptides in the cytoplasm of genetically altered E. coli. This approach is potentially quite useful for assessing the convertibility of antibodies into multi-specific antibody formats containing scFvs.

12:20 pm Enjoy Lunch on Your Own

CHO Cell Development for Efficient Protein Production

2:00 Chairperson’s Remarks

Bjørn Voldborg, Director, CHO Cell Line Development, The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark

2:05 Optimizing Expression of Proteins in CHO through a Systems Biology Approach

Nathan_LewisNathan E. Lewis, Ph.D., Assistant Professor, Department of Pediatrics, University of California, San Diego

In our lab, we are mapping out the cell pathways controlling CHO cell growth, protein synthesis, and protein glycosylation. Here I discuss our work in which we have developed computational models to predict the cell costs for protein synthesis and identify how to improve protein synthesis through media and genetic modifications.

2:35 Optimizing Biologics by Cell-Based Glycan Display

Claus_KristensenClaus Kristensen, Ph.D., CEO, GlycoDisplay Aps

Glycan structures are important for efficacy and distribution of biologics. Optimization of glycans has been hampered by inefficient technologies for glyco-engineering in mammalian cells. Now GlycoDisplay offers technologies allowing development of novel glyco-optimized biologics. GlycoDisplay has applied targeted cell engineering to generate cell lines with different glycosylation capacities. By expressing a drug candidate protein in panels of glycoengineered cell lines, followed by screening novel glyco-optimized leads are identified.

3:05 Refreshment Break in the Exhibit Hall with Poster Viewing

4:00 Overexpression of Ebola Virus Envelope GP1 Protein

Zhongcheng Zou, Ph.D., Staff Scientist, Structural Immunology Section, Lab of Immunogenetics, NIAID/NIH

To elucidate the role of the mucin-like domain of GP1 in Ebola-host attachment and infection and to facilitate vaccine development, we constructed a GP1 expression vector containing the entire attachment region. Cysteine 53 of GP1 was mutated to serine to avoid potential disulfide bond mispairing. Stable expression clones using codon optimized open reading frame were developed in human 293-H cells with yields reaching ~ 25 mg of GP1 protein per liter of spent medium.

4:30 Using GlycoExpress to Overcome Production Limitations for Difficult-to-Express Proteins

Lars_StöcklLars Stöckl, Ph.D., Director, Glycoprotein Development and PTM Analytics, Glycotope GmbH

Even though productivity for CHO systems has remarkedly improved over the last years, some biopharmaceuticals like bispecific constructs or complex glycoproteins remain very challenging. We present case study data from clone and upstream perfusion development for the human GlycoExpress cell line, which overcomes productivity limitations.

5:00 Expression of Recombinant Blood Coagulation Factor VIII: Importance for Human Healthcare and Approaches to Improve the Protein’s Yield and Quality

Andrey G. Sarafanov, Ph.D., Chemist, Principal Investigator, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration (FDA)

Deficiency in factor VIII (FVIII) results in abnormal bleeding (Hemophilia A), which is treated by infusions of FVIII. However, the FVIII production is challenging as the protein is expressed at low levels both in plasma (0.3 nM) and heterologous systems. The presentation overviews approaches to improve FVIII production, in particular, re-design of the protein and its gene, optimization of the protein expression and purification, and selection of test methods.

5:30 - 6:45 Networking Reception in the Exhibit Hall with Poster Viewing

6:45 Close of Recombinant Protein Expression and Production Conference


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