When expressing protein, there are a variety of expression systems to choose from. CHO is seen as the ‘king’ in the industry, but does it meet all needs of rapid production and budget constraints? This meeting will explore “Optimizing Protein Expression” through understanding and enhancing expression systems, and will especially focus on CHO cells, and other mammalian systems, E.coli and yeasts. Other host systems, such as baculovirus and algae, will be touched on as well.
Along with case studies, the meeting will feature experts who reveal the underlying mechanisms and insights into the varying systems in order to enhance protein expression. Comparing and contrasting systems will also be featured to provide greater understanding of how these systems function within the context of the results achieved.
Learn the latest protocols and see how protein science leaders are enhancing expression systems to reach greater productivity.
WEDNESDAY, APRIL 27
7:00 am Registration and Morning Coffee
8:00 Chairperson’s Remarks
Donald L. Jarvis, Ph.D., Professor, Molecular Biology, University of Wyoming
8:10 KEYNOTE PRESENTATION:
Expression and Purification of Proteins for Drug Discovery – Challenges and Trends
Ian Hunt, Ph.D., Group Leader & Head, Protein Sciences, Center for Proteomic Chemistry, Novartis Institute for Biomedical Research, Inc.
A key ingredient to the successful identification and optimization of small-molecule compounds in drug discovery is the production of high-quality recombinant proteins to help drive biochemical assays, biophysics, and structural biology studies. As drug discovery continues to evolve and develop, so too do the challenges and demands placed on protein science. This review will describe some of the key strategies developed to meet these challenges. Topics covered will include the discussion of multi-parallel expression and purification strategies, use of HT instrumentation, and the potential utility of a number of new and enabling technologies for dealing with increasingly difficult-to-produce proteins. These include large protein complexes and membrane proteins. The presentation will also discuss some of the projected trends and challenges within the field and beyond.
8:40 Multiple Approaches to Optimize Protein Production for Shortening the Discovery Process of Non-Antibody Protein Therapeutics
Liang Tang, Ph.D., Senior Scientist and Group Head, Molecular Biology and Protein Expression, Global Biologic Research, Global Drug Discovery, Bayer HealthCare
During therapeutic drug discovery, we need to produce non-antibody proteins. Expression of these proteins with proper post translation modifications within a limited time is challenging. At Bayer, we established systems of multiple approaches to optimize the process of non-antibody protein expression starting from expression vector design, evaluation and selection of cell types for protein expression, purification and efficient QC process for shortening the discovery process of new protein therapeutics.
9:10 POSTER SPOTLIGHT
Engineering a Plug-and-Play Mammalian Cell Line Platform for Antibody Expression
Cristina Parola, Ph.D. Student, Systems and Synthetic Immunotechnology, Biosystems Science and Engineering (D-BSSE), ETH Zurich
9:40 Enhanced Synthesis of Recombinant Phosphoproteins in E. coli is Enabled by a New Genetic Code
Jesse Rinehart, Ph.D., Assistant Professor, Cellular & Molecular Physiology, Systems Biology Institute, Yale University School of Medicine
We have recently created a technology that enables site-specific incorporation of phosphoserine into proteins by expanding the genetic code of Escherichia coli. We are now applying this technology to understand the properties of phosphoserine in human kinases. Our current research aims have benefited tremendously from our recent improvements to our phosphoserine technology. Advances on all fronts will be discussed with attention to the broad application of recombinant phosphoprotein production.
10:10 Coffee Break in the Exhibit Hall with Poster Viewing
10:55 Stable Glycoengineering of CHO Cells for Production of Diverse, Homogeneous Glycoproteins
Malene Bech Vester-Christensen, Ph.D., Senior Scientist, Recombinant Expression Technologies, Novo Nordisk A/S
Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells’ generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferase genes controlling N-glycosylation in CHO cells and constructed a design matrix that facilitates the generation of glycoproteins with a desired homogeneous N-glycosylation.
11:25 Direct PCR Methods for Quantification of Residual Host DNA in Monoclonal Antibody Drugs Manufactured in CHO Cells
Musaddeq Hussain, Ph.D., Principal Scientist, BioProcess Development, Biologics and Vaccines Research, Merck Research Laboratories
Chinese hamster ovary (CHO) cells are the host of choice for manufacturing monoclonal antibody (mAb) drugs. Host cell DNA is an impurity of such manufacturing process and must be monitored. Conventional methods require extraction of DNA from the mAb drug before quantification by PCR. Since femtogram amount DNA extraction is typically inefficient, we have developed ‘direct PCR’ methods eliminating extraction step. The method is usable for both qPCR and dPCR.
View the Interview
11:55 To Fucosylate or Not: Utilizing FX Knockout CHO Lines to Express WT or Afycosylated Antibodies on Command
Shahram Misaghi, Ph.D., Scientist, Early Stage Cell Culture, Genentech, a member of the Roche Group
Here we introduce generation and use of a FX-KO CHO host cell line that is capable of expressing antibody molecules with either afucosylated or WT glycan profiles. This host not only obviates the need for undertaking two separate CLD efforts, but it can also be used to generate WT or afucosylated antibody molecules with similar product quality attributes, since both versions of the antibody are made by the same cell-line.
12:25 pm A 2L-12L Orbital-Shaken SUB as an Alternative to Stirred
and Wave Reactors for Protein Expression Experiments
David Laidlaw, CEO, Kuhner Shaker, Inc
Ina Dittler, Ph.D., Zurich University of Applied Sciences
A technically conserved, low-shear and low foaming 2L-12L orbital shaken SUB, the Kühner SB10-X, is presented here as an alternative to stirred and wave reactors for batch protein production. This presentation will describe the bioreactor technical specifications and provide Sf9 and CHOK1 cultivation data for the SB10-X compared to flasks and stirred reactors.
12:40 Automated Cell Culture and Cell Line Development to
Optimize Expression
Russ Mcsweeney, Automation Sales Specialist, Beckman Coulter Life Sciences
12:55 Luncheon Presentation: Shortening the Biotherapeutic Development Timeline: Multi-Gram Yield Antibody Production via Scalable Electroporation
Joan Foster, Field Application Scientist, R&D, MaxCyte
Data developed with MaxCyte’s flow electroporation will demonstrate the production of multiple grams of antibodies, bispecifics, and non-antibody like recombinant proteins following a single transient transfection. Its scalability with CHO, HEK293, insect cells, and other commonly used cells in bioproduction will be demonstrated as well as the rapid generation of high-yield stable cell lines(titer of 6 g/L) within 6-8 weeks of transfection. The glycosylation of stable vs. transiently produced protein will be examined.
1:25 Luncheon Presentation II (Sponsorship Opportunity Available)
1:55 Session Break
2:10 Chairperson’s Remarks
Robert Roth, Ph.D., Associate Principle Scientist, Reagents and Assay Development, Discovery Sciences iMed, AstraZeneca
2:15 miRNA Engineering of CHO Cells
Vaibhav Jadhav, Ph.D., Scientist, Austrian Centre of Industrial Biotechnology, ACIB
In this presentation, I will give an overview of miRNAs and a detailed study of miR-17 overexpression, which resulted in a two-fold increase in specific productivity without loss in growth rate. Dissection of the effects of miR-17 overexpression on the transcriptome and proteome of CHO cells contributes to understanding the molecular mechanisms that control the interaction of growth and productivity, the two most important process-relevant parameters in CHO.
2:45 Designed to be Scaled Up: Pichia pastoris, E. coli, S. cerevisiae and CHO
Anton Glieder, Ph.D., Professor, Molecular Biotechnology, Graz University of Technology
Employing Pichia pastoris as a host strain, we emphasized vector design, development of new chassis strains, and alternative biosynthetic pathway assembly strategies in order to obtain robust microbial strains with stable maintenance of multiple gene copies, opportunities for stepwise increase in bioreactors, and enhanced reliability in larger volumes. The methodology is generic and can be transferred to other hosts, as demonstrated by examples using E. coli, S. cerevisiae and CHO.
3:15 Automated Transient Transfection for High-Throughput Protein Production
Chris Suh, Ph.D., Business Development Manager, PhyNexus, Inc.
Transient transfection of mammalian cell lines is being implemented by the pharmaceutical industry to produce the therapeutic protein candidates very rapidly compared to previous technology thus allowing large numbers of drug candidates to be screened and studied. However, high throughput automated transient transfection is required for increased sample load. Here we describe the integration, implementation and validation of different robotic platforms for automated transient transfection of mammalian cells.
3:45 Refreshment Break in the Exhibit Hall with Poster Viewing
4:45 Problem-Solving Breakout Discussions
Optimizing the Process of High-Quality Non-Antibody Protein Production for Meeting Tight Timelines in Biotherapeutic Drug Discovery
Moderator: Liang Tang, Ph.D., Senior Scientist and Group Head, Molecular Biology and Protein Expression, Global Biologic Research, Global Drug Discovery, Bayer HealthCare
- What are the most difficult problems in your protein production process, and suggestions how to solve these problems?
- What are the routine expression systems you are using in your protein expression and why?
- What are the critical attributes for your expressed proteins and why?
- What kinds of assays are you using in monitoring your protein production, and do they meet your needs?
- What are the most important factors from DNA to protein process for meeting timelines?
Troubleshooting Baculovirus-Insect Cell Technology
Moderator: Donald L. Jarvis, Ph.D., Professor, Molecular Biology, University of Wyoming
- Producing and characterizing baculovirus vectors
- Why can’t I produce my recombinant protein anymore?
- Protein expression and processing in insect cells
- Adventitious agents
Expression of Complex Proteins in Mammalian Cells
Moderator: Gregory T. Bleck, Ph.D., Global Head, Research & Development, Biologics, Catalent Pharma Solutions
- The number of complex proteins in development continues to increase
- These include virus-like particle vaccines, proteins that require additional post-translational modification and various different multi-specific antibody platforms
- Producing these multi-subunit molecules or processing proteins in the correct proportions to obtain maximum titer and biopharmaceutical quality can be difficult
- Techniques and successes in producing cell lines secreting these proteins will be discussed
Transient Expression in Relevant Cell Lines
Moderator: Joan Foster, Field Application Scientist, R&D, MaxCyte
- Using primary cells and human-derived cells
- Conducting R&D in the manufacturing cell
- Working with the right cell for the right application
5:45 Networking Reception in the Exhibit Hall with Poster Viewing
7:00 End of Day
THURSDAY, APRIL 28
8:00 am Morning Coffee
8:30 Chairperson’s Remarks
Vaibhav Jadhav, Ph.D., Scientist, Austrian Centre of Industrial Biotechnology, ACIB
8:35 Improving Insect Cells as Hosts for the Baculovirus System
Donald L. Jarvis, Ph.D., Professor, Molecular Biology, University of Wyoming
Insect cell lines have several limitations as hosts for recombinant protein production. These limitations can and should be addressed to optimize the baculovirus system as a recombinant protein manufacturing platform. For the past 20 years, we have focused on optimizing protein glycosylation in the baculovirus system. This presentation will focus on those efforts, on the new systems emerging from these efforts, and on their major biomedical applications.
9:05 Next-Generation Biotherapeutic Production System: The Filamentous Fungus Trichoderma Reesei
Christopher Landowski, Ph.D., Senior Research Scientist, Industrial Biotechnology, VTT Technical Research Centre of Finland, Ltd.
The filamentous fungus Trichoderma reesei is an important production organism used by industrial enzyme companies worldwide. It secretes its native enzymes at levels exceeding 100 g/L of culture medium and is amenable to large-scale fermentation processes. We have adapted the fungus to become more suitable for biotherapeutic production by reducing secreted protease activity and in some cases altering glycosylation pathways needed for adding mammalian glycoforms.
9:35 Strep-Tactin XT– A Superior Next Generation System for
Purification of Proteins, Isolation of Cells and Assay Development
Uwe Carl, Ph.D., Head, Protein Production, Strep-tag Products and Proteins, IBA GmbH
The new third generation Strep-tag® system is based on recently engineered Strep-Tactin®XT and Twin-Strep-tag®. Due to the affinity improved but still reversible binding of Strep-Tactin®XT to Twin-Strep-tag® in the low pM range, the system is superior to other affinity purification systems and now also suitable for assay development.
9:50 Sponsored Presentation (Opportunity Available)
10:05 Coffee Break in the Exhibit Hall with Poster Viewing
11:05 Multiplexed and Programmable Regulation of Gene Networks with an Integrated RNA and CRISPR-Cas Toolkit in Human Cells
Samuel D. Perli, Ph.D., Post-Doctoral Researcher, EECS and Biological Engineering, Massachusetts Institute of Technology (MIT)
A major limitation for the scalability and integration of the CRISPR-Cas family towards applications in human cells is that the small guide RNAs (sgRNAs) have only been expressed using RNAP III promoters in human cells. We present new strategies to express sgRNAs in human cells by employing Csy4, RNA-triple-helix structures, introns, ribozymes and microRNAs. We thereby demonstrate multiplexed gene regulation, multi-stage cascades and RNA dependent circuits that can be rewired using Csy4 in human cells.
11:35 Expression and Characterization of the Human Cytomegalovirus (HCMV) Pentamer Complex for Vaccine Use
Andrea Carfi, Ph.D., U.S. Head, Protein Biochemistry, GlaxoSmithKline Vaccines
Human cytomegalovirus (HCMV) causes significant disease worldwide. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of neutralizing antibodies (Nabs) in HCMV seropositive individuals and raises high titers of Nabs in small animals and non-human primates . We describe here the development of a mammalian expression system for large scale Pentamer production. We also present initial biochemical, antigenic and structural characterization of this promising vaccine candidate.
12:05 pm Identification of Novel Regulators of Recombinant Protein Expression Using Phenotypic Screening Followed by Target Validation using CRISPR/Cas9
Robert Roth, Ph.D., Associate Principle Scientist, Reagents and Assay Development, Discovery Sciences iMed, AstraZeneca
By creating isogenic cell lines with a defined copy number of the gene encoding the expressed recombinant protein of interest, we minimized genetic locus effects. Using phenotypic screening, we identified positive and negative regulators of recombinant protein secretion, which was then confirmed using precise genome editing. Our findings show that utilising model isogenic reporter cell lines in orthogonal screening assays is a powerful method to rapidly identify novel regulators critical to enhanced protein expression.
12:35 End of Optimizing Protein Expression
5:15 Registration for Dinner Short Courses