This talk will focus on coordinated whole system engineering solutions to enhance both i) critical cellular capacities that underpin CHO cell bioproduction phenotypes (e.g. lipid biosynthesis and mitochondrial capacities) and ii) vector-encoded utilisation of the cell factory’s product biosynthesis capacity (i.e. product transcription, translation, translocation, and folding capacities). An engineering toolkit comprising programmable genetic control nodes and synthetic component assemblies of promoters, signal peptides, UTRs, and CDSs will be presented.

As newer therapeutic proteins tend to have much greater complexity than traditional mAbs, an expression host toolbox to target production of ~1-10mg of purified material for 100s of molecules is highly desired to accelerate the drug discovery process. Here, we selected three cell lines (ExpiCHO-S, Expi293, and CHO-K1) and performed combinatorial DOE to systematically vary multiple parameters. We collected multidimensional data for over 2000 samples at medium expression scale (~40ml) after performing 36 DOE iterations to evaluate the robustness of these expression systems on our newly designed end-to-end robotic platform.

Transient transfection is a critical tool for recombinant protein production, as it allows rapid screening for expression without stable integration of genetic material into the target cell genome. Current gold-standard reagents for transient gene transfer are limited by toxicity of the polymer. We developed a panel of cationic polymers, poly(beta-amino ester)s (PBAEs), as reagents for transient transfection and observed enhanced expression of both cytosolic and secreted proteins.

SARS-CoV-2 spike (S) protein purification can be challenging, with engineered and natural variations often resulting in lower yields. Here, we present methods for producing SARS-CoV-2 S ectodomain, its Receptor Binding Domain (RBD), and N terminal Domain (NTD) by transient transfection in mammalian cells. These methods reproducibly yield high-quality preparations of these S protein domains for use in structural, biochemical, and biophysical studies. 

An integral component in early drug discovery efforts is the rapid expression and purification of recombinant proteins. We have optimized automated HEK and CHO transient expression workflows with non-proprietary reagents to reduce costs, enabling the screening of thousands of biological candidates. We also leverage automated magnetic bead-based technologies for higher-throughput, clog-free purifications which further accelerates fit-for-purpose biologics production on our custom platform: Rapid Antibody and Protein Therapeutic Omni Robot (RAPTOR).

Cell lines are an important tool in biologics drug discovery. With most antibody targets being membrane proteins, cell lines provide a great platform to study the activity of the target in its 'native' conformation as well as generate functional antibodies against it. In my talk, I will be sharing the use of cell lines for antibody drug discovery process across all stages from early exploratory to candidate nomination and selection. I will introduce how we use different technologies to generate stable cell lines for difficult targets and the uses thereof in the antibody drug discovery process.

Many next-generation therapeutics remain intrinsically challenging to produce in CHO cells. We exploited a cumate-inducible CHO platform allowing reduced expression of various classes of r-proteins during selection of stable pools. Fed-batch productions showed that pools generated without cumate (OFF-pools) were significantly more productive. Cell viability was lower and pool recovery was delayed during selection of ON-pools (mimicking constitutive expression), suggesting that high producers were likely lost or overgrown by faster-growing, low-producing cells. Using an inducible system to minimize r-protein expression during pool selection can contribute to reduce cellular stresses, including ER stress and metabolic burden, leading to improved productivity.

The use of biotin labeling to immobilize a recombinant protein on a surface is quite useful in the study of binding kinetics (SPR) as well as an orthogonal tag (to commonly used His-tag) when screening DNA-encoded libraries (DEL) to identify small molecule binders. Here, we discuss the use of the baculovirus system to produce avi-tagged recombinant proteins and the use of both in vitro and in vivo methods of labeling with biotin. Case studies of labeling secreted and non-secreted protein in insect cells will be presented along with examples of how tag placement (N- or C-term) can affect assay quality.

To maximize E. coli’s capacity for producing recombinant proteins, production strains have to be customized. Therefore, we have developed a platform enabling to rapidly -i- identify the optimal production rate for a protein and -ii- identify and combine genomic modifications promoting protein’s stability. Compared to mainstream E. coli protein production setups, up to 10-fold increases in production yields were achieved for a variety of targets using customized protein production strains.

Vibrio natriegens has attracted much attention as an additional expression host for recombinant protein expression, largely due to the allure of shorter fermentations as a result of faster growth rate.  While this is a unique and useful feature, there are important considerations in adopting the system that are not well described in the literature. Complications (and our solutions) will be discussed along with examples of proteins that are preferentially produced in V. natriegens rather than E. coli.