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.
 

Join us in the Presentation Hall to chat with our Expert on the topic of Mammalian Transient Expression. 

• Do you have questions on the differences/similarities between the Expi293 and ExpiCHO transient expression systems and how they fit together in a protein expression workflow?
• Do you have questions related to protein purification from Expi cell culture supernatants?
• Do you need advice on troubleshooting or adapting the Expi system protocols for your particular needs?

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.
 

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.

Objectives

•        Learn different approaches to produce proteins for display on microarray platforms.
•        Describe different uses of high-throughput protein display for identifying protein interactors or biomarkers.

 

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
 

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.

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.

Learning objectives:

• ​Understanding recent advances in mammalian transient protein expression and how they fit into existing workflows
• Differences and similarities between various cell types used for transient protein expression

Reliability from gene sequence to the expressed and purified protein is essential for many aspects of basic research and drug development. Thermo Fisher Scientific’s complete portfolio from GeneArt™ gene synthesis to advanced expression systems provides researchers the unique ability to optimize their gene expression results. Our proprietary sequence optimization GeneOptimizer™ improve the sequence via a parallel multi-parameter approach. To further maximize the protein yield, you can chose between our best-in-class serum free expression host systems FreeStyle™ or Expi293™ and ExpiCHO™. Offering reagents for in-house work or a convenient and total service solution, this is the most efficient way to streamline your gene-to-protein workflow for maximal performance. The basis of gene synthesis and optimization will be explained and how the combination with best expression system generates expression reliability and protein yields.

Learning Objectives:

• Understand that the combination of gene optimization and best-in-class mammalian expression systems results in increased protein expression & reliability
• Understand Thermo Fisher / Geneart is the best partner to outsource the complete workflow from expression plasmid generation to purified protein