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
TUESDAY, JANUARY 9
1:00 pm Registration
1:30 Refreshment Break in the Exhibit Hall with Poster Viewing
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 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 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 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 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
WEDNESDAY, JANUARY 10
8:00 am Registration and Morning Coffee
8:30 Chairperson’s Remarks
Feras Hatahet, Ph.D., Scientist, Protein Technologies, Amgen
8:35 Protein Production Platform for Rational Design Engineering
Takanori 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 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.
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
10:50 SPEAKER CANCELLATION: Rapid Screening of Cyclotide-Based Libraries against Intracellular Protein-Protein Interactions
Julio 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 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 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
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 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 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 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