We have developed a CHO host with specific productivity similar to the levels observed in professional secretory cells.  This CHO host cell also produces complex molecules such as multi-specific antibodies with higher quality compared to other CHO host cells. The superior performance of this host cell may be explained by its significantly higher ER / Golgi content.

Based on the outcome of our CHO cell line engineering research, we have developed CHO cell based platforms, enabling rapid production of tailored glycoforms of therapeutic proteins, with improved protein quality and predictable cell line development. Through the glycoengineered CHO platform (geCHO), the effect of N-glycans on therapeutic proteins can be screened, and the optimal glycoform can then be manufactured using the geCHO cell lines. 

Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a surface protein co-inhibitory receptor expressed on tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). GPNMB may interact directly with T-cells, impairing T cell activation to down-modulate anti-tumor immune response. Human GPNMB has soluble and membrane-bound forms, and soluble GPNMB may act as a decoy receptor, resulting in an antibody sink. Here we report an approach for developing antibodies binding specifically to membrane-bound GPNMB.

Chinese Hamster Ovary (CHO) cells are one of the major workhorses for Transient Gene Expression (TGE) of recombinant antibodies. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used (including Filler DNA), and post-transfection culture conditions, we arrived at an uniquely optimized TGE process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply.

Mammalian metabolic inefficiencies lead to accumulation of waste by-products. Untargeted and targeted metabolomics for identification of novel metabolites identified six CHO metabolic inhibitors. They negatively impact growth and titer production. Inhibitors were shown to accumulate across different mammalian cell lines. A holistic methodology incorporates metabolomics analysis into cell culture studies.

Although there are many different methods (e.g., temperature, pH, feed) to improve protein production in CHO cells, the role of physiologically relevant hydrostatic pressure in CHO cell culture has not been fully explored. In this study, hydrostatic pressures similar to in vivo interstitial pressure values (0~90 mmHg) were applied to batch CHO cell culture, and their cell growth/metabolism and IgG₁ production were examined. Our results indicate that hydrostatic pressure can increase the maximum cell concentration by up to 50%. These findings are important for the optimization of fed-batch or perfusion culture for directing cell growth and improving antibody production.

Cell-free expression systems allow evaluation of gene expression from DNA constructs without cloning, accelerating characterization timelines. Low reaction volumes and experimental assembly using acoustic liquid handling further increase the throughput possible. We have shown rapid screening of a library of genetic regulatory sequences by varying PCR primers. We further report use of a similar approach to modify the composition of the cell-free reaction to optimize expression of a target gene.

Numerous developments have made the cell-free expression platform more accessible to new users and have expanded its range of applications. However, the workflow for E. coli cell extract preparation has remained a bottleneck. We have developed the Cell-Free AutoInduction (CFAI) workflow that allows users to grow cells, produce extracts, and conduct cell-free protein expression within 24 hrs. Additionally, the CFAI workflow reduces the time and cost of generating cell extracts.

Our unique platform technology, PUREfrex, is a fully reconstituted (or rebuilt) cell-free protein expression system. It's easy to customize the system for various applications, and useful for high throughput screening of various kinds of biologics as well. Plus, we established robust ribosome display with customized PUREfrex and named PUREfrexRD, which has great advantage in screening of highly diversified library to generate new biologics such as antibodies or cyclic peptides.

Production of biotherapeutic protein and antibodies relies on successful selection of high performing clone for manufacturing. To accelerate this process, the right transfection reagent is key to produce milligram to gram quantities of proteins and to overcome productivity challenges of diverse proteins from therapeutic antibodies to difficult-to-express proteins. We demonstrate how FectoPRO transfection reagent is the go-to transfection reagent that combines productivity and flexibility in suspension CHO and HEK-293 cell lines.

Sequencing of genomes has become an essential tool to study human/viral/microbial and cell-line diversity. CHO cell-line development benefits from these multi-modal sequencing advancements. We will present our solutions that assess comprehensively the genotypic stability of clones, the location and architecture of transgene integration and additionally the quality and risk associated with a clone selection. 

An elegant highly active transposase equipped with epigenetic readers carries expression units to most active spots in the host genome providing desired expression levels and stability. With this foundation the focus for screening producer clones is entirely on correct pairing and PTMs. We will discuss how this system is adapted to various formats and removing a critical bottleneck for bi-specifics during clinical development.

The aßT cell receptor (aßTCR) governs the recognition of virally infected or cancerous cells by aßT cells to initiate an immune response. The aßTCR probes cells in its vicinity for ligand, proteolytically-derived peptide bound to major histocompatibility complex molecules (pMHC). The aßTCR-pMHC recognition was recently found to be force-dependent. To probe the origins of mechanosensing, a toolbox was developed for single-molecule (SM) study of the TCR-pMHC interaction. A eukaryotic transient expression system was utilized, leveraging antibody affinity purification. Production of the heterodimeric aßTCR for SM proved to be quite robust, allowing systematic deconstruction of TCR mechanosensing.

The N protein of the SARS-CoV-2 virus constitutes an attractive target to be used in a subunit vaccine. To improve the quality of N protein as antigen, it was fused to the extracellular domain of human CD154 (CD40L). Subsequently, the N-CD gene was cloned into a lentiviral vector. Lentiviral particles bearing the N-CD gene were used to transduce HEK-293 cells. Polyclonal populations were obtained and characterized. Next, cell clones were obtained by limiting dilution cloning and adapted to growth in serum-free medium and suspension culture. Finally, experiments were carried out on animals to verify the immunogenicity of this antigen.

This presentation will focus on key platform technologies to address and overcome timeline challenges in therapeutic drug development. Starting with a high through-put antibody discovery platform (MonoRab) and optimized mammalian expression system (TurboCHO) developed in-house, our goal is to provide key reagents (like anti-ID antibodies and kits to enable PK studies) in an effort to meet faster timelines without compromising quality.

High producing stable mammalian cells are often required for producing recombinant proteins. Traditional methods like antibiotic selection cause high levels of cell stress during the selection or require high initial costs for instruments. Here, we present a new bead based method for generating high producing stable cell pools that avoids substantial negative effects on viability or cell growth after selection and that does not have high acquisition costs for instruments. 

Astex has successfully applied its proprietary FBDD platform to generate multiple new drug candidates that are progressing in clinical development. The platform employs X-ray crystallography and a range of biophysical methods for FBDD. Moreover, Astex has established a state-of-the-art cryo-EM facility. Production of suitable quality proteins is a prerequisite for supporting these platforms. I will share our experience and case studies on expression, purification, and characterisation of challenging drug targets.

Baculovirus expression systems composed of Cap-, Rep-, and trans-gene baculoviruses are routinely employed for the scalable expression of adeno-associated virus (AAV) gene therapy vectors. Here, we describe the characterization and development of a baculovirus system devoid of the Cap-gene for expression of capsid-free closed-ended DNA (ceDNA) for non-viral gene therapy. The expression challenges faced and methods for controlling and maximizing ceDNA expression yields will be discussed.