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^TM 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.

Lentiviral vectors have become the center of attention for its use as gene transfer vectors in gene therapy. Here, we have developed a new lentiviral production system for the clinical grade production of Lentiviral vectors on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. With new culture media and media supplement, high density cell growth is optimized for maximum LVVs production. Customized lentivirus transfection reagents ensure highly efficient plasmids delivery. LVVs production is further elevated by the boosting enhancers. Taken together, our innovative system is able to deliver >2-3.0E+08 (TU/mL) functional titer with un-concentrated LVVs. We demonstrate that this system can be linearly adapted to bioreactors and wavebags system for large scale LVVs production without compromising the virus yield, which is ideal for industrial needs of gene therapy.

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.

The development of antibody drug conjugates requires milligrams of different mAbs. During the discovery phase of this process, sequences may need to be altered or isotypes changed. Recombinant DNA technology allows for easy manipulation of IgG expression vectors and is not considered a bottle neck. The generation of stable CHO cell lines however is very time and labor consuming. In this brief presentation, I will share my experience using the Expicho system for transient expression and purification of milligram amounts of several mAbs in an academic environment.

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

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.

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.

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 few as four weeks from receipt of plasmid DNA.

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.

The ExpiCHO™ transient expression system offers a turnkey solution for generating high-titer recombinant proteins for therapeutic drug development, reagent generation, as well as an alternative platform for proteins that express poorly 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 with researchers the latest data on the ExpiCHO expression system, tips to help optimize the system for maximal performance and protocols to allow for scalable protein expression from the micro to the macro scale.

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 often hampered by low expression yields limiting subsequent structural and functional analyses.

Thermo Fisher Scientific’s complete portfolio from Invitrogen™ GeneArt™ gene synthesis to advanced expression systems provides researchers the unique ability to optimize their gene expression results. Our proprietary sequence optimization GeneOptimizer™ algorithm improves the sequence via a parallel multi-parameter approach. To further maximize the protein yield, you can choose between 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 as 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 on during drug development to minimize changes in protein quality/function when moving from R&D to bioproduction.

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.

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.

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

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。

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。