It is challenging to create artificial cellular systems that rival the dynamic chemical-biological response and resilience of natural cells. The artificial cellular systems are hybrid material-bacteria systems with superior applications in drug discovery, disease treatment, and basic biological study. Dr. Tan will present his work on controlling the information processing of artificial cellular systems. The work has broad impact on biotechnology applications and disease treatment.

The biological importance of carbohydrates (or glycans) can be better understood and exploited by creating a robust toolkit. Here, we highlight recent advances in glycoengineered microbial strains/plasmids and enzymes development for in vivo conversion of azido sugars to designer glycans (e.g., Human Milk Oligosaccharides). We discuss how such a toolkit can be used for efficient recombinant protein production as well as directed evolution of carbohydrate-active enzymes (e.g., glycosynthases).

Reference: Bandi et al. 2021, ACS Synthetic Biology, 10:4, 682–689. https://doi.org/10.1021/acssynbio.0c00449.

Non-native synthetic DNA sequences have become a common resource in recombinant protein work due to the low cost of DNA synthesis. Many of these DNAs are “optimized” through commercial tools despite there being little guidance for what defines an optimal sequence. We surveyed nine optimization algorithms and found significant variability between their optimized DNAs, leading us to propose a set of best practices when designing synthetic sequences.

Production of high-value protein targets often faces significant challenges due to their poor and variable levels of expression and active folding, as well as the long turnaround times required for their end-to-end manufacturing and testing. This presentation describes strategies for robust production of active proteins that are applicable to a wide range of DNA and mRNA expression formats in the context of cell-free, cell-based and gene therapy platforms.

When it comes to the commercialization of biologics, effective, high-quality cell line development (CLD) is essential for success. The key to minimizing risk and optimizing outcome is having a reliable partner that offers experience, expertise, and a comprehensive portfolio of products and services. This presentation will highlight the key challenges and points to consider during biopharmaceutical CLD and the impact early decision-making can have at later stages of the development process.

Genetic engineering of proteins from the classical secretion pathway (CSP) is an alternative for obtaining CHO cell clones with enhanced productivity. However, few CSP targets have been detected from homogenates in previous differential proteomic studies. Hence, we used subcellular proteomics to intensify the identification of CSP proteins associated with cell productivity. Differentially expressed proteins (DEP) from this study participate in protein synthesis and translocation, autophagy, proteasomal degradation, calcium regulation, vesicular transport, ER stress and UPR. About 80% of DEP had not been associated with productivity and their further modulation could have a positive impact on bioprocesses. Supported by IN210419.

Decades of cell line development and media optimization of CHO cell lines has led to notable improvements in recombinant protein (rProtein) production yield.  However, the expression of some rProteins results in little to no yield and remains a challenge.  Our lab has taken a systems biology approach, including machine learning and network analyses, to elucidate host and protein-specific properties that act as determinants of rProteins yield in CHO.

Process intensification strategies in CHO production cultures can potentially increase productivity, lower cost of goods, and improve facility utilization. However, process intensification often triggers apoptotic cell death in the later phases of intensified production process. Here we show that apoptosis-resistant CHO cell lines counteract this undesired outcome, resulting in not only better viability but also enabling extended productivity that significantly improve volumetric productivity without affecting product quality.

With the aim of increasing protein productivity, we generate a novel CHO host with favorable biomanufacturing phenotypes and improved functionality. Producer pools and clones generated from the new host outperformed the standard host by displaying (1) improvement in productivity, (2) reduced product aggregation, (3) enhanced cell viability, (4) low lactate production and (5) improved cell cloning efficiency. Proteomics and western blot analysis demonstrate that the new host acquired multifaceted protection against mitochondrial dysfunction and ER stress.

The control of nutrient availability is critical to large-scale manufacturing of biotherapeutics. Genome-scale metabolic modeling offers a promising approach for in silico monitoring and predicting the consumption of proteinogenic amino acids, which is critical for bioprocess control. However, the prediction accuracy is challenged by the discrepancy between the model assumption and the biological variances in CHO cultures. We demonstrate such challenge can be addressed by integrating a CHO-specific metabolic network model with machine learning to achieve accurate prediction throughout the fed-batch process.

Industry 4.0 can potentially revolutionize biomanufacturing. Digital twins, virtual copies of a process interacting with the process, will play an important role in this transformation. This talk has focus on understanding the needs and challenges faced by the biomanufacturing industry when dealing with this digitalized paradigm. Two major building blocks of a digital twin, data and models, are highlighted. Data characteristics and collection strategies are examined, and new methods and tools for data processing are highlighted. Furthermore, different modelling approaches are presented in view of their use in a digital twin. Potential roadblocks for digital twin implementation are discussed.

Bispecific antibodies have become increasingly of interest for therapeutic applications. These antibodies can be difficult to express (DTE) with much lower productivity than classical monoclonal antibodies. Besides protein engineering, vector and bioprocess optimization, CHO host engineering shows potential to enhance DTE protein productivity. Here, we analyzed the transcriptome of cells expressing various DTE proteins and identified genes that were differentially transcribed in bispecific antibody expressing cells. The effects of these genes on productivity were subsequently evaluated by gene overexpression or gene knock out/down. Our results indicate that proper host cell engineering is one solution to improve recombinant therapeutic protein production.

Insect cell systems have become a major tool in the toolbox for the production of pharmaceutically relevant protein targets. In our laboratory, this system has been vital for production of multiprotein complexes for structural biology and drug discovery research. We have developed a number of process and technology improvements which permit increased protein yield, protein quality, virus stability, and efficiency of protein complex formation. We will discuss in detail the enhancements to the system and how they can be applied to high-level production of other clinically relevant proteins.

Launched only a few years ago, the Leap-In Transposase platform has rapidly become an industry standard technology for the generation of CHO cells for the manufacturing of antibodies and other biologics.  This presentation will highlight achievements and case studies of the platform including high titer mAb manufacturing, rapid anti-COVID responses, and some novel, next generation, applications.  

Ambrx’s EuCODE^TM platform utilizes engineered CHO cells with an expanded genetic code to produce engineered precision biologics. Quick material delivery with high productivity is critical for rapid project development. MaxCyte’s STX electroporation system enables systemic optimization of transient expression conditions and a facile scale-up to support material delivery need at different stages of projects.