Co-culture of multiple organisms in the same vessel can be used to facilitate bioproduction or to integrate living cells as analytics in bioprocessing. However, growing organisms together that have different growth rates can lead to one type of cell outcompeting the others.  Here we discuss a materials science solution to enable co-cultures by using cell encapsulation to contain the fast-growing population, allowing cells to grow together in harmony. We have successfully deployed this approach to enable bacterial whole-cell biosensors to be grown alongside mammalian producer cells without detrimental effects on recombinant protein production.

Macromolecular complexes are cornerstones of most, if not all, biological processes in cells and their preparation in quantity and quality is often a bottleneck. After an overview of widely used recombinant expression approaches, we will illustrate the potential of the CrispR/Cas9 editing technology.  By generating knock-in cell lines where the protein of interest is fused to an affinity tag, the CRISPR methodology facilitates the isolation of macromolecular complexes assembled under physiological conditions. Benefits and limitations of this approach for the purification of multi-protein complexes as well as for other applications will be discussed.

Recombinant protein production burdens cell metabolism which may affect titer and quality. Stable HEK293 clones producing either secreted erythropoietin or GFP at different rates were subjected to multi-omics characterization. EPO producers displayed both a shift in oxidative phosphorylation and in ribosomal structure compared to host and GFP. A super producer clone of EPO displayed high expression of negative regulation of ER stress genes which was functionally validated to increase titer.

We built a comprehensive library of human proteins engineered for controlled cell surface expression. Coupled to tetramer-based screening for increased binding avidity, we developed a high throughput cell-based platform that enables systematic interrogation of receptor-ligand interactomes. Using this technology, we characterized the cell surface proteins targeted by the receptor binding domain (RBD) of the SARS-CoV spike protein. Host factors that specifically bind to SARS CoV-2 but not SARS CoV RBD were identified, including proteins that are expressed in the nervous system or olfactory epithelium.

A robust secretory phenotype is fundamental for the high-level production of biopharmaceuticals, particularly for those with complex molecular architecture. However, the current CHO cell platforms present deficient protein secretion rates (when compared to dedicated secretory cells). Here, we will discuss the use of regulatory transcription factors as tools for reshaping cellular phenotype and show their potential for engineering CHO cells towards an increased protein secretion.

The presentation covers recent advances of Novartis Cell Line Development toolbox of plasmid elements and novel engineered CHO cell lines, which resulted in increased clone productivity and genetic stability as well as an improved product quality for complex therapeutic proteins. Combining vector technologies with a robust CHO cell line, an accelerated cell line development process was developed for antibodies with significantly reduced efforts for the generation of high-producing CHO clones.

Selecting potent regulatory sequences with robust transgene expression is critical for CHO cell line engineering. This talk will summarize the development and validation of a dual luciferase reporter system for quantitative interrogation of such sequences in transient and stable transfectants of CHO cells. Functional execution of this toolkit was achieved with several known constitutive promoters. Experimentally, CMV-mIE yielded the highest transcriptional activity in transient transfectants, while CHEF1a was the strongest in stable pools of CHO-DG44 cells. Together, the reporter system is a viable tool for selecting established or identifying novel regulatory sequences in CHO and perhaps other mammalian cell lines.

We built StoCellAtor, a bacterial whole-cell modelling (WCM) framework that entails a detailed, codon-level translation model combined with the stochastic version of an existing WCM. StoCellAtor can be used to link a synthetic construct’s modular design (promoter, ribosome binding site, and codon composition) to protein yield and cellular burden during continuous culture, with a particular focus on the effects of low-efficiency codons and their impact on ribosomal queues.

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

N-linked glycosylation is a critical quality attribute of many biopharmaceutical products that needs to be controlled. A screening of a miRNA library identified 82 miRNA sequences capable of altering galactosylation, sialylation, and fucosylation. Subsequent validation provided insight into the intracellular mode of action of miRNAs regulating cellular glycosylation pathways. Moreover, a multiplex modulation approach and rational design of artificial miRNAs demonstrated the potential of miRNAs to fine tune expressed glycosylation patterns.