Gibco ExpressionWorld Webcasts

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.

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.

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.

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.

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.

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.

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.

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。

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。

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.

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.

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.

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.

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.

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.

Abstract

Quickly coming from a gene sequence to expressed and purified protein is important for many aspects of basic research and drug development. The usage of expression optimized synthetic genes became a routine for high level protein expression. The basis of gene synthesis and optimization will be explained and how the combination of expression optimized genes and best-in-class expression systems results in increased overall expression reliability and protein yields.

Abstract

In vitro translation (IVT) using mammalian cell extracts is a quick and convenient alternative to in vivo mammalian protein expression. We have developed IVT systems from two mammalian cell lines, HeLa and CHO, that enable rapid expression of genes from mammals, bacteria or protozoa. The Thermo Scientific 1-Step Human Coupled kits,  1-Step Human High-Yield kits, and  1-Step CHO High-Yield IVT kits allow rapid protein expression through the addition of a DNA of interest to a mixture of HeLa or CHO cell lysate and reaction mix followed by incubation for 1-6 hours at 30°C. We have developed a series of IVT vectors (pT7CFE1) for optimal expression in both the HeLa and CHO IVT systems with a variety of purification tags. Using the High-Yield dialysis method, > 250µg/mL of protein of interest was obtained with both IVT expression systems. During this seminar we will present application data demonstrating expression, purification, and activity of several different proteins using both the HeLa and CHO IVT kits. Our results indicate that highly functional proteins can be produced in milligram quantities for a variety of downstream applications. To facilitate construction of IVT templates for high throughput protein expression applications, we have developed Gateway™-compatible pT7CFE1 vectors.  Further, a new protein service option encompassing gene synthesis, IVT expression, and purification will be discussed with turnaround time less than 10 business days.  

Abstract

The talk will focus on the recent advances in transient protein production using high cell density culture e.g., ExpiCHO, Expi293 for supporting pre-clinical and clinical studies including robust generation of critical reagents for assay support. Comparative studies for several classes of protein including optimization for the expression and quality of difficult-to-express proteins will be highlighted.

Abstract

CHO cells are the predominant host for biotherapeutic protein expression, with roughly 70% of licensed biologics manufactured in CHO. Multiple attributes make CHO cells desirable for bioproduction including the ability to adapt to high-density suspension culture in serum-free and chemically-defined media and the incorporation of post-translational modifications that are biologically-active in humans. For these reasons, the ability to produce transient CHO-derived proteins early on during drug development is highly advantageous to minimize, as much as possible, changes in protein quality/function observed when moving from R&D to bioproduction. Unfortunately, CHO cells express lower levels of protein than HEK293 cells in existing transient systems, in some instances 50-100 times less than the best 293-based systems, and only modest titer improvements are obtained through the optimization of individual components of existing transient CHO workflows. To address the significant unmet need for higher transient CHO protein titers, systems-based approaches were employed whereby the latest advances in cell culture media, feeds, transfection reagents and expression enhancers were optimized in conjunction with a new high-expressing CHO cell clone to generate the ExpiCHO transient expression system, a system capable of generating gram per liter protein titers in 10-14 days. These advances allow for unprecedented access to CHO-derived proteins early on during candidate selection and may serve to revolutionize the use of CHO cells for transient protein expression during the drug development process.

Abstract

Complex recombinant protein biologics, such as monoclonal antibodies and coagulation factors, are key components of today’s biopharmaceutical industry. There are many effective paths for generating recombinant proteins in mammalian cells. Stable cell lines made in Chinese Hamster Ovary (CHO) cells are the workhorse for production of biopharmaceuticals due to their relative ease of use and long history of regulatory acceptance. However, in development and laboratory settings, where large numbers of proteins are generated for pre-clinical studies, transient gene expression using the FS293F, Expi293F and ExpiCHO systems is an efficient, rapid and cost-effective alternative to developing stable CHO cell lines. Transient gene expression technology has allowed us to rapidly screen, identify and characterise multiple novel protein-based human therapeutic drug candidates. Notably, we have observed that the choice of expression system has a great influence on not only the quantity but also the quality of the produced recombinant protein. The Freestyle 293, Expi293 and ExpiCHO cell lines are excellent hosts for robust secretion of mammalian proteins, however the cellular machinery for appropriate post-translational modifications for particular proteins is not always optimal. We are currently developing tools that are expected to enable the generation of proteins with appropriate post-translational modifications and hence the desired biological activity and pharmacokinetics.

Abstract

Self-assembling protein microarrays arrays can be used to study protein-protein interactions, protein-drug interactions, search for enzyme substrates, and as tools to search for disease biomarkers. In particular, recent experiments have focused on using these protein microarrays to search for autoantibody responses in cancer patients. These experiments show promise in finding antibody responses that appear in only cancer patients. New methods using click chemistry-based reagents also allow the application of these arrays for discovering new substrates of post translational modification.

Abstract

The ExpiCHO transient expression system allows for the high titer production of a broad range of recombinant proteins. Due to the high titers observed for many proteins in the ExpiCHO system, and differences between CHO culture conditions, purification conditions may vary. In this presentation, protocols are presented for high efficiency, scalable, supernatant purification using Protein A chromatography resins. Differences in purification conditions for each culture condition are noted and product quality is discussed.

Abstract

LRRK2 is a large (2,527 amino acids) multi-domain protein consisting of 7 putative domains, including a Ras-like GTPase domain called ‘Ras of complex proteins’ (Roc) followed by a domain called C-terminal of Roc (COR), which is then followed by a kinase domain (Kin). It remains unclear as to how perturbations of these activities results in disease; however, the most common mutation in LRRK2-associated PD, G2019S in the kinase domain, shows higher kinase activity than wild-type; therefore, its over-activation might be associated with disease pathogenesis. We use Expi293 expression system to produce full-length LRRK2 for biochemical characterization and structural studies.