ExpressionWorld 2017

Presentations


An ongoing evaluation of our transient expression systems; Can we predict the appropriate expression system for any drug modality?

Abstract

CHO cells are the predominant host for biotherapeutic protein expression with approximately 70% of licensed biologics manufactured in Chinese Hamster Ovary (CHO) cells. The ability to express therapeutic candidates in CHO cells early in the drug development process is advantageous as it minimizes changes in protein quality and function when transitioning to production scale. To address the lack of a robust and reproducible in-house transient CHO expression system, we incorporated the ExpiCHO transient expression system into our transient expression toolbox, joining the well-established HEK293-6E and Expi293 systems. With an ever-expanding array of protein formats, the next challenge is being able to strategically match a construct with an expression system. We have utilized a diverse set of candidate proteins in an ongoing evaluation comparing material transiently produced in parallel in the HEK293-6E, ExpiCHO and Expi293 systems for expression level and product quality.  We report these results and discuss their implications in how we prioritize our selection of expression systems. Successful prioritization of an expression system for a drug modality will reduce parallel efforts and increase our throughput efficiency by reducing expression timelines.

Biography

Rich Altman has 29 years of experience working in the pharmaceutical industry. In 2016, he joined the Protein Technologies Mammalian Expression group at Amgen San Francisco, supporting biologics drug development. Prior to Amgen, he worked for several pharmaceutical companies on the cloning, expression, purification and characterization of recombinant proteins. This work supported both small-molecule high-throughput screening and protein therapeutic efforts. He received his MS degree from the University of Pittsburgh School of Medicine in the Department of Molecular Biology and Biochemistry.

Richard Altman

Scientist, Protein Technologies
Amgen, Inc., South San Francisco, CA 


Modification of Glycans in Bioprocessing

Abstract

Glycosylation is an important product quality attribute for biotherapeutic proteins expressed in CHO cells. Glycoform variability can significantly affect the safety and efficacy of therapeutic proteins, and can be dependent on several factors, including cell line, media/feeds, and process. As a consequence, it has often been challenging to achieve and maintain preferred glycosylation profiles from cell culture development through bioreactor scale-up. To address these challenges, we have developed a new feed technology in conjunction with a unique fed-batch process that together has been shown not only to maximize protein titers but also to modulate glycan profiles.

Biography

Ryan Boniface has been an R&D Scientist with Thermo Fisher Scientific in development of Gibco™ media and supplement products for nine years. He is experienced in high-throughput medium design process development, validation, and technology transfer. Ryan’s formal education includes an MS in Biotechnology from State University of New York, Buffalo.

Ryan Boniface

Scientist III, Cell Biology, Bioproduction Research and Development
Thermo Fisher Scientific


Basic Concepts in Transient Expression Using 293 and CHO Cells

Abstract

HEK293 and CHO cells are the primary cell lines used in rapid, scalable suspension culture systems for transient protein expression. This presentation provides a primer on fundamental concepts and materials used for transient expression and key differences to consider when using 293 or CHO cells.

Biography

Henry Chiou is an associate director in product management, responsible for the Gibco and Invitrogen protein expression product portfolio at Thermo Fisher Scientific. He has directed or guided development of such products as Lipofectamine LTX, Lipofectamine RNAiMAX, and Lipofectamine 3000 transfection reagents, the FreeStyle MAX CHO Expression System, the Expi293 Expression System, and the ExpiCHO Expression System. He previously worked for small biotech companies on gene delivery and gene therapy. He received his PhD from Harvard University and did his postdoctoral training at the University of Pennsylvania.

Henry Chiou, Ph.D

Associate Director, Cell Biology
Thermo Fisher Scientific


Complete Workflow Solution From Gene to Protein Combines Highly Advanced Expression Systems With Expression-Optimized Genes

Abstract

Reliable autologous expression of recombinant human proteins—from gene sequence to the expressed and purified protein—in human or CHO cells is essential for many aspects of biomedical research and drug development, but is often hampered by low expression yield that limits subsequent structural and functional analyses. From Invitrogen GeneArt gene synthesis to advanced expression systems, Thermo Fisher Scientific offers researchers a portfolio that enables a unique ability to optimize gene expression. Our proprietary GeneOptimizer sequence optimization algorithm improves sequence via a parallel multiparameter approach. To further maximize protein yield, researchers can take advantage of our best-in-class serum-free Gibco FreeStyle or Invitrogen Expi293 and ExpiCHO expression host systems. This holistic approach is the most efficient way to streamline your gene-to-protein workflow for maximal performance, either by purchasing reagents for in-house work or by outsourcing the complete workflow to a highly customized service offered by the GeneArt Protein Team.

The talk will explain the basis of gene synthesis and optimization and how the combination with the best expression system generates unmatched expression reliability and protein yields. This approach is highly advantageous early in the drug development process for minimizing changes in protein quality/function when moving from R&D to bioproduction.

Biography

Stephan Fath is a manager for manufacturing in the Biosciences division at Thermo Fisher Scientific (GeneArt) in Germany. He received his PhD in protein biochemistry from the University of Heidelberg, Germany. After his education and research work on ribosomal biogenesis, he joined Memorial Sloan Kettering Cancer Center in New York as a research fellow, studying COP II proteins by protein crystallography. He has a strong background in protein biochemistry, expertise in different protein expression systems, and has published in renowned journals. In 2008 he joined GeneArt and worked as a senior scientist in R&D before switching to Operations. As a manager for manufacturing he is currently responsible for the highly customized protein expression and purification services.

Stephan Fath, Ph.D

Manager, Manufacturing
Thermo Fisher Scientific


Optimisation of UCB's Transient Expression Platforms

Abstract

Development of antibody therapeutics, from early-stage research to preclinical and clinical development, requires ever-increasing amounts of reagents. To meet the challenge of furnishing a diverse and full pipeline, we utilise several different transient platforms. Through continuous optimisation, streamlining. and automation of component parts of our panel of platforms (utilising both HEK293 and CHO host cells with a variety of transfection methods), we now have the capability to produce microgram to gram quantities of panels of purified antibodies and antibody fragments in as little time as 4 weeks from receipt of plasmid DNA.

Biography

After graduation from the Aachen University of Applied Sciences in Germany, Christina Gordon worked for a number of years as a process development scientist at AstraZeneca in Sweden, in cell-line generation and developing scalable fermentation processes for mammalian cell cultures. After this she moved to the UK and joined the New Meds Division of UCB, working within the transient gene expression group. Over her 10 years at UCB she has implemented many improvements to both the CHO and HEK293 transient expression platforms, which have led to significant improvement in titre and throughput. She is currently part of the alternative antibody format group, helping to design and improve expression of difficult-to-express proteins.

Christina Gordon

Scientist, Protein Sciences
UCB Pharma


Use of Protein Expression System for Influenza Mucosal Vaccine Research

Abstract

In the study of a mucosal vaccine for influenza, we need to evaluate the immune responses against the vaccine in serum and nasal washes. Serum IgG antibodies and mucosal secretory IgA antibodies are needed for this evaluation. We use a mammalian protein expression system to produce the strain-specific influenza virus HA proteins to check the antibody titers. Moreover, we take advantage of the expression system to produce the antibodies that were induced by the mucosal vaccine, to characterize the effectiveness of the vaccine.

Biography

Hideki Hasegawa earned an MD from the Hokkaido University School of Medicine in 1993 and then earned a PhD in 1997, also from Hokkaido University. He studied at Rockefeller University from 1995 to 1996, and was a postdoctoral fellow at University College Dublin from 1996 to 1997. After finishing his studies he joined the National Institute of Infectious Diseases (NIID), where he had done research on infectious diseases in the Department of Pathology. In 2003 he was promoted to chief of the Laboratory of Infectious Diseases and Pathology at NIID, where he is extending his research to the development of a mucosal vaccine against influenza viruses. In 2011 he was promoted to director of the Department of Pathology at NIID.

Hideki Hasegawa, Ph.D

Director, Department of Pathology
National Institute of Infectious Diseases


Stabilization of Native & Functional Membrane Proteins for Drug Discovery 

Abstract

CALIXAR has developed an innovative detergent/surfactant based approach consisting on native isolation and stabilization of therapeutic membrane protein targets such as GPCRs, ion channels, transporters. Here we will explain how this approach can stabilize membrane proteins by modifying their chemical environment instead of their native sequence. We will illustrate using case studies on targets of high medical relevance produced in their most native state without any single mutation, truncation or fusion and that were solubilized, affinity purified while maintaining their functional and structural integrities. A recent collaboration was initiated with Thermo Fisher Scientific as the world leader on the expression of difficult to express proteins. This collaborative effort we will help us tackle the production and characterization of very challenging but very promising and highly druggable targets such as GPCRs, ion channels & transporters. This native isolation approach represents a new hope for the development of more accurate drug discovery (SBDD, FBDD, Antibody discovery & vaccine) and provide a serious alternative to classical protein engineering approaches.

Biography

Anass Jawhari holds a PhD in biochemistry and structural biology from Louis Pasteur University in Strasbourg, France. He worked as a research associate at the Scripps Research Institute in La Jolla, California, and at the Gene Center in Munich, Germany, before joining Transgene as a research investigator. He is now Chief Scientific Officer at CALIXAR. He has more than 18 years of experience in R&D projects related to molecular aspects of vaccines, cancer, and infectious diseases. He is also a member of different consortia on membrane proteins (GPCR and ion channels).

Anass Jawhari, Ph.D

Chief Scientific Officer
CALIXAR


Large-Scale Serum-Free Suspension Lentiviral Production for Cell and Gene Therapy Applications

Abstract

Lentiviral vectors (LVVs) have received attention for their use as gene transfer vectors for gene therapy. We have developed a new system for clinical-grade production of LVVs on a large-scale serum-free suspension platform. This technology employs a newly developed, proprietary set of GMP reagents, which includes culture media, suspension cells, transfection reagent, and boosting enhancers. With new culture media and media supplement, high-density cell growth is optimized for maximum LVV production. Customized lentivirus transfection reagents ensure highly efficient plasmid delivery. LVV production is further elevated by the boosting enhancers. Taken together, our innovative system is able to deliver >2–3 x 108 TU/mL of functional titer with unconcentrated LVVs. We demonstrate that this system can be linearly adapted to bioreactors and the WAVE Bag system for large-scale LVV production without compromising the virus yield, which is ideal for industrial-scale production requirements for potential gene therapy applications. 

Biography

Yongchang Ji is a scientist at Thermo Fisher Scientific in Carlsbad, California. He is currently working on development of a high-titer, large-scale mammalian suspension lentiviral vector production system for gene therapy applications. He received his PhD in cell biology and anatomy from SUNY Upstate Medical University in New York.

Yongchang Ji

Scientist II, Cell Biology
Thermo Fisher Scientific


Gibco ExpiCHO哺乳动物瞬时蛋白表达系统的最新研发进展

Abstract

Gibco ExpiCHO是一款全新优化的瞬时重组蛋白表达系统,可以在瞬转中获得极高的蛋白产量,为临床新药和诊断试剂的研发及快速筛选提供了高效便捷的最佳手段。一些难以在传统HEK293细胞中表达的蛋白亦可在ExpiCHO系统中获得高水平表达。正是基于ExpiCHO系统在CHO细胞重组蛋白瞬时表达方面的突破性进展,该系统作为不可或缺的革命性工具,目前己成为世界上众多生物制药公司创制和生产最具天然结构及活性蛋白的首选表达平台。

在今天的报告中我们将与同道们共同分享ExpiCHO系统应用的最新进展,包括系统扩展性,操作技巧及常见问题指南,以期帮助广大用户全面掌握蛋白瞬时表达最优条件,满足不同规模高效表达的生产需求。

Biography

Chao Yan Liu女士现为Thermo Fisher Scientific公司细胞生物学部门的高级研究员,在美国马里兰州弗雷德里克工作。Chao Yan Liu女士拥有河北医科大学的医学学士学位和军事医学科学院的病理学硕士学位,先后在威斯康辛州血液中心的血液研究所以及纽约州立大学水牛城分校从事细胞生物学和免疫学领域的学术研究工作。Chao Yan Liu女士于2005年加入原Life Technologies公司研发部,对Gibco 品牌下十余款细胞培养产品的研发做出了卓越贡献,包括AlgiMatrix 3D培养系统、OpTmizer CTS T细胞扩增无血清培养基、TrypLE Select酶、适用于胚胎干细胞的FBS、MesenPRO RS 细胞培养基、GlutaMAX 培养基添加剂、FreeStyle F17表达培养基和CD FortiCHO 培养基,在哺乳动物细胞培养,尤其是化学成分确定的无血清和无蛋白培养以及重组蛋白表达方面积累了丰富的经验。最近五年,她作为瞬时蛋白表达课题研发的技术负责人致力于两种新型哺乳动物瞬时蛋白表达系统——Expi293和ExpiCHO的研究开发并取得了突破性进展。

Chao Yan Liu

Senior Staff Scientist, Cell Biology
Thermo Fisher Scientific


使用293和CHO细胞进行瞬时表达的基本概念

Abstract

HEK293和CHO细胞是快速、规模可调的悬浮培养系统中常用的细胞系,可用于瞬时蛋白表达。此次演讲介绍了瞬时表达的基本概念和使用材料,以及使用293或CHO细胞时需要考虑的主要差异。

Biography

刘健先生现为Thermo Fisher Scientific公司细胞生物学部门的资深研究员,在美国马里兰州弗雷德里克工作,拥有华东理工大学的生物化学学士以及硕士学位。刘健先生曾在美国南加州大学从事艾滋病疫苗研发工作长达六年。 2000年,加入Invitrogen公司。在他的任职于Invitrogen公司期间,他曾在多个部门从事多种酶的纯化研究,包括MMLV, SuperScript II and III, Taq DNA聚合酶, RNaseOUT, E. coli DNA聚合酶和DNaseI。 2006年,他加入了公司位于马里兰州弗雷德里克的生物生产部门,并开始从事稳定细胞系的开发,期间他参与开发的DG44和CHO-S的稳定细胞株在流加培养中的单克隆抗体表达超过1克/升。 2014年,他加入了细胞培养部门,专注于新型瞬时蛋白表达系统- ExpiCHO表达系统的规模化开发。

Jian Liu

Thermo Fisher Scientific
Scientist III
Life Sciences Solutions Group


Cost effective protein production for R&D and animal trials

Abstract

The CSIRO Tissue Culture Facility (CTCF) employs a range of in-house optimized platform technologies to identify optimum vector-host cell combinations for both transient and stable protein production in traditional or single-use bioreactors. Our seminar will summarize the recommended steps to use these technologies and provide a quick guide to produce proteins in a cost-effective way. Also, a direct comparison of protein production using transient HEK 293 and the latest ExpiCHO cell cultures will be presented in detail.

Biography

George is Adjunct Professor of RMIT and Monash University Protein Production and Fermentation. He works closely with CSIRO, BioManufacturing Parkville and Clayton laboratories, Australia.

George Lovrecz, Ph.D

Adjunct Professor of RMIT and Monash University
Protein Production and Fermentation CSIRO, BioManufacturing
Parkville and Clayton laboratories, Australia


A Semi-Automated High-Throughput Transient Expression and Purification Platform using the Expi293 Expression System

Abstract

We have developed a semi-automated, high-throughput transient expression and purification system that yields milligram quantities of hundreds of proteins weekly. Starting from a glycerol stock with an automated DNA purification system, followed by semi-automated transfection/feed/clarification/filtration and a fully-automated purification and buffer exchange, we deliver high quality-protein supporting dozens of projects with an eight-day turnaround time.

Biography
  • 2000–2001 Genzyme Biosurgery, GMP Culture of autologous chondrocytes for knee replacement surgery
  • 2001–2002 Harvard School of Public Health, Responsible for culturing of Human Airway Smooth Muscle cells for asthma research
  • 2002–2007 ImmunoGen, Responsible for the stable cell line development of DG44CHO cells secreting monoclonal antibodies
  • 2007–2010 Organon/Schering-Plough/Merck, Responsible for the stable and large scale transient development of DG44CHO and Hek293F cells to produce monoclonal antibodies 

Matthew McKenna

Scientist, High-Throughput Transient Transfection
Pfizer, Inc.


Selecting the Right Resin To Achieve Optimal Results, From High-Throughput Batch To Process-Scale Protein Purification

Abstract

The objective of academic, biotech, or pharmaceutical scientists committed to protein purification is to obtain the purest protein needed for their application, regardless of scale. Toward that pursuit, proper chromatography resin selection is essential to achieving a high-efficiency purification strategy, which includes optimal protein separation and minimal processing time. In this presentation, we discuss a full suite of resins that can be utilized for microgram- to kilogram-scale purifications, whether screening at a high-throughput batch scale to select the right target or increasing production of that target protein from batch to process scale. We will highlight the technological advantages and cost savings of our full portfolio of base beads, from magnetic agarose for convenient small-scale processing, to the uniquely designed Thermo Scientific POROS resin bead for simplified scale-up, which allows for enhanced resolution and faster processing.

Biographies

Scott Meier is a research scientist at Thermo Fisher Scientific, focusing primarily on the development of novel tools for protein isolation and enrichment. He has a strong background in protein expression and purification for structural and functional studies. His most recent project involved developing magnetic agarose affinity beads for high-throughput small-scale purification of recombinant proteins and antibodies. Over the past 6 years, Scott has developed products related to bacterial lysis, membrane protein extraction, high-flow affinity chromatography resins, and glycoprotein immobilization chemistry. He has a BS in bioengineering from the University of Illinois at Urbana–Champaign, and an MS in laboratory investigation from Vanderbilt University School of Medicine.

Aaron McBride is an R&D scientist at Thermo Fisher Scientific, where he has played an instrumental role in the development, commercialization, and support of multiple chromatography resins and recombinant proteins introduced over the past 7 years. Aaron has experience in protein expression, purification, and characterization as well as process development and validation. His development work has included products for protein purification, molecular biology, and in vitro translation. He has an MS in cell and molecular biology from Michigan State University.

Scott Meier

Scientist III, Protein and Cell Analysis
Thermo Fisher Scientific

Aaron McBride

Scientist III, Protein and Cell Analysis
Thermo Fisher Scientific


ExpiCHO Expression Of HIV-1 Immunogens for Structure-Guided Vaccine Design

Abstract

The isolation of broadly neutralizing antibodies from HIV-1–infected individuals raises the hope that a vaccine may elicit a similar immune response. These antibodies target the only viral protein on the surface of HIV, a metastable trimer of heterodimers, Env. A popular option for vaccine design is an engineered version of Env (named SOSIP) that is solubilized and stabilized. Production of many antigenically diverse SOSIP trimers for immunization studies is critical for an increased understanding of the development of neutralizing antibodies.

Biography

Gabriel Ozorowski is a protein biochemist and structural biologist in the field of HIV vaccine design. His work include design and expression of novel immunogens in mammalian cells, stringent purification protocols for animal immunization studies, and quality control assessment of antigens alone or in the presence of adjuvants/buffers, using a combination of biolayer interferometry, differential scanning calorimetry, isothermal titration calorimetry, and electron microscopy. Assessment includes samples from GMP-level production. Additional projects involve high-resolution epitope mapping and structural determination of HIV Env trimers using cryoelectron microscopy, with a focus on trimer dynamics. He has a PhD in molecular biology and biochemistry from the University of California, Irvine.

Gabriel Ozorowski, Ph.D

Sr. Research Associate
Integrative Structural and Computational Biology
The Scripps Research Institute


Ensuring Successful Protein Expression Through Proper Plasmid Purification

Abstract

Plasmid purification is the first critical step in ensuring successful transfection and protein expression. As such, it’s important that the right plasmid purification technology and conditions are chosen to ensure optimal performance. Listen in as our R&D expert discusses the basics and commonly overlooked aspects of plasmid purification to help ensure successful protein expression experiments. In addition, he will review the latest endotoxin-free plasmid purification technology developments at Thermo Fisher Scientific, which includes the PureLink Expi Endotoxin-free plasmid purification portfolio. These kits purify high quality, endotoxin-free plasmid DNA in about half the time, enabling peace-of-mind when going from plasmid to protein. 

Biography

Alexander “Sasha” Vlassov is a Senior Manager, Research and Development, at the Austin site of Thermo Fisher Scientific. Dr. Vlassov has developed multiple product lines including reagents for exosome and microbiome research, siRNA, tools for miRNA functional analysis, and transfection reagents. For the last two  years his team is developing advanced technologies for plasmid DNA purification, with primary downstream application being ExpiCHO and Expi293 transient expression systems.

Sasha Vlassov, Ph.D

Senior Staff Scientist, Cell Biology, Life Sciences Solutions Group
Thermo Fisher Scientific


Latest Developments in High-Titer Transient Protein Expression Using the ExpiCHO Transient Expression System

Abstract

The ExpiCHO transient expression system offers a turnkey solution for generating high-titer recombinant proteins for therapeutic drug development and reagent generation, as well as an alternative platform for proteins that are poorly expressed in 293-based systems. Because of the significant unmet need for high-titer transient CHO protein production, the ExpiCHO system has become a key part of the transient expression workflow in many companies around the world. In this presentation, we will share the latest data on the ExpiCHO expression system, tips to help optimize the system for maximal performance, and protocols for scalable protein expression from micro to macro scales. 

Biography

Jon Zmuda is a director for cell biology at Thermo Fisher Scientific in Frederick, Maryland. He leads a team dedicated to discovering and developing new technologies and products useful for cell biology applications, including transient protein expression, advanced cell culture, and rare-cell analysis. He received his PhD in cell biology from the University of Maryland, College Park. 

Jonathan Zmuda, Ph.D

Director, Cell Biology
Thermo Fisher Scientific