Stably transfected CHO clonal cell line is the standard platform for manufacturing recombinant glycoprotein therapeutics. The current pandemic, concurrently with improvements in the performance of stable CHO pool platforms, have contributed to their wider acceptance by the regulatory authorities to accelerate clinical evaluation of potentially life-saving drugs. I will present our recent efforts using stable CHO pools to manufacture the SARS-CoV-2 trimeric spike protein as a vaccine subunit antigen.

SARS-CoV-2 receptor domain binding (RBD) protein is a vaccine candidate against COVID.  The wild type RBD protein expressed in yeast has low-yield and tendency to aggregate. By introducing two genetic modifications, we greatly improved the protein expression and stability without changing its biological function. Fermentation process was optimized, and the reproducibility of the process was established with an average yield of 428 ± 36 mg/L. This production process has been transferred to an industrial manufacturer, who has successfully advanced this vaccine candidate into Phase 3 clinical trial.

Generating a bispecific antibody, which is correctly and stably paired, is challenging. Various platforms have developed Heavy-Light chain (HC-LC) mispairing fixes, but there are many rate limiting steps for efficiently expressing these molecules in a CHO system including adaptation of downstream processes. bYlok technology is a design engineering approach that stabilises the interaction between the HC and LC, essentially removing the mispairing problem whilst retaining a more natural antibody structure.

Recombinant SARS-CoV-2 Spike protein has been proposed as a vaccine candidate for immunization against COVID-19. It is a very large, fragile, and difficult to purify protein. Its large size restricts its access to binding sites present in typical resin media used for chromatographic purification and thereby limits its binding capacity. A membrane chromatography-based rapid and scalable method for purification of recombinant SARS-CoV-2 Spike protein will be discussed.

We will discuss the challenges in high-throughput protein production for small and large molecule drug discovery. We demonstrate the parameters and design space required to generate high-quality proteins for HTS, antibody discovery, in vivo and developability studies. Supported by our industry-leading platforms, the Protein Sciences department at WuXi Biologics provides production services utilizing various expression systems for the generation of monoclonal, bispecific and multispecific antibodies, and other recombinant proteins.

Glycosylation is the biochemical process of the attachment of sugar moieties to proteins during co-translational or post-translational modification and occurs during expression in organisms like Saccharomyces cerevisiae.  For integral membrane proteins, such as G-protein coupled receptors, high-level production is especially challenging due to their extreme hydrophobicity and instability in the absence of a cell membrane. This work aims to uncover factors affecting protein subcellular localization during heterologous expression in S. cerevisiae. We postulate a critical link between subcellular localization during expression and the production of functional proteins with an emphasis on GPCRs.

Multiple antigen formats are often needed for antibody discovery to overcome issues associated with avidity and stability. If molecules cannot be standardized, optimization may be needed for each new antigen. Fusion Antibodies has developed a process for the routine production of multiple antigen formats using a unique application of knob-in-hole technology. The molecules generated give greater diversity compared with approaches that use a small number of standard antigens.

A major bottleneck in human GPCR structural biology is the production of sufficient quantities of folded and functional receptors. Expressing GPCRs for nuclear magnetic resonance experiments is made even more challenging by the requirement to incorporate stable-isotope probes.  We present an optimized workflow for recombinant production of human GPCRs in Pichia pastoris for biophysical experiments, illustrated by the production of a human histamine receptor.

Membrane proteins are major drug targets because of their central roles in regulating cellular communication. Despite this, membrane receptors remain underrepresented in interaction databases because of technical limitations. To overcome these challenges, we developed an extracellular vesicle-based method for membrane protein display that enables purification-free and high-throughput detection of receptor-ligand interactions in membranes. We demonstrate that this platform is broadly applicable and can be used to profile endogenous vesicles. We are continuing to improve our vesicle-based methods for the biochemical characterization of difficult-to-purify membrane proteins, membrane-adjacent proteins as well as of the vesicles themselves.

Membrane proteins are integral proteins of the cell membrane and are directly involved in the regulation of many biological functions and in drug targeting. However, our knowledge of such proteins is limited due to difficulties in producing sufficient quantities in a soluble, functional, stable state. Designed, surfactant-like peptides may be used to extract membrane proteins from the cell membrane and stabilize the protein for further studies.

GPCRs are highly sought-after drug targets, but challenges in producing proteins, driving immune responses, and finding hits have limited the development of antibody therapies against these high-value targets. To address these challenges, we leveraged our fully integrated technology stack to rapidly discover functional antibodies against a GPCR target and identify lead candidates with species cross-reactivity and potencies that rival small molecule clinical-stage benchmarks.

During this talk, Catalent will share the latest data leveraging GPEx Lightning to generate highly stable, highly productive cell pools and discuss how the GPEx suite of technologies can be tailored to the specific needs of each individual program on its path to clinical trials.

Many biochemical and structural studies require extensive mutational analysis of the protein of interest. This task can be time-consuming and expensive when using eukaryotic expression systems. Therefore, the use of bacteria as expression system can be very advantageous. Thus, we decided to test the expression of a mitochondrial human ABC transporter, which does not require post-translation modifications, in bacteria. We can successfully use bacteria to produce milligrams of the purified, fully functional human transporter. Our lab is now extending this approach to produce other human mitochondrial ABC transporters.

This talk will cover protein expression, purification, and biochemical/biophysical characterization of the human full-length MOZ 4-protein complex in insect cells. The talk will highlight the challenges of overexpressing a multimeric complex recombinantly, as well as the enablement of various biochemical and biophysical assays that resulted from it.

Binary and ternary complex formation assays are crucial for biotherapeutic research and producing pure, high-quality proteins for SPR analysis may be considered an art. How do we engineer tags and linkers, optimize buffers, and develop workflows for protein complexes, specifically to conserve native folding, promote access to binding pockets, and reduce SPR artifacts? Through thoughtful design and effective strategy, one can accomplish the binding-competence of their SPR dreams.

The Pelican Expression Technology platform (formerly the Pfenex Expression Technology) is a robust, cost-effective, commercially validated platform based on Pseudomonas fluorescens for recombinant protein production with four approved products utilizing the technology. Example expression studies are presented demonstrating the extensive Pelican toolbox of genetic elements, host strains, and automated strain screening that enabled rapid the expression for two complex and challenging antibody formats: a Fab fragment and picobodies.