We have developed a modular toolbox for genetic code expansion in mammalian cells. With noncanonical amino acids as synthetic building blocks in protein expression, we have applied these tools for dual color live cell fluorescence labeling. We generate cell lines for efficient expression of noncanonical amino acid labeled proteins, an approach with great potential for protein engineering beyond the standard genetic code.

During biomanufacturing, unwanted host cell protein (HCP) expression can affect both yield and quality of drug substances. Our newly developed artificial intronic miRNA technology allows for targeted gene silencing, significantly reducing HCP contamination. This technology facilitates the creation of complex miRNA clusters, enabling simultaneous knockdown of multiple genes. This versatile approach enables one-step cell-line development and engineering and presents a robust solution to various technical development challenges.

Recombinant protein expression is a highly regulated process consisting of transcription, translation, and protein folding. CHO-based expression often stays challenging for artificial therapeutic proteins, like Bispecific T cell Engagers (BiTE), due to reduced productivity compared to mAbs. Investigation of relevant protein production steps unveiled the transcription rate as a root cause. Therefore, we demonstrate quantitative in vitro transcription as a powerful and evolving method for further exploration of this bottleneck.

Drug discovery faces a rising number of projects that demand the generation of cellular reagents with controlled or lower target protein expression. Here, we have tested and optimised several techniques that can be utilised to tune protein expression: stop codon suppression methods, an addition of IRES elements prior to the target sequence, and a panel of weak promoters and construct modifications. These methods for customising protein expression can be combined and applied widely. This presentation will describe techniques that allow tunable protein expression. The optimisation of these methods and generated data will be demonstrated in case studies covering several GSK targets.

Multispecific antibodies (MsAbs) have therapeutic potential for various conditions. However, during production, incorrect chain assembly and co-production of mispaired species impair biological activity. Omics analyses of CHO clones producing trispecific antibodies revealed that high mispairing clones experience endoplasmic reticulum stress, while low mispairing clones exhibit profiles indicative of activated protein translation, enhanced endocytosis, and target protein degradation. A panel of biomarker genes was tested for detecting high mispairing during early bioprocess development. This screening tool could streamline selection of hosts with improved MsAb quality, reducing MsAb development and manufacturing process time and costs.

The development of multi-specific and asymmetric antibodies often requires the simultaneous expression of two or more polypeptide chains. Moreover, such molecules can be challenging to express and assemble correctly in CHO cells. Genetic screening during cell line development has the potential to guide early clone selection by screening for favourable mRNA ratios, gene expression patterns, or genomic liabilities of cell lines. State-of-the-art molecular biological methods, like digital PCR or Next Generation Sequencing, are the perfect tools for screening as they offer unprecedented power and throughput for cell line characterisation.

Recombinant adeno-associated viruses (rAAVs) are preferred vectors for gene therapy but face production challenges in HEK293 cells due to scalability issues and high costs. Currently, CHO cells do not support rAAV production. Our study investigates the proteomic and transcriptomic responses of CHO cells to the expression of AAV elements, aiming to identify and address bottlenecks to enhance rAAV manufacturing.

The selection of proper bacterial strains is required when producing recombinant proteins, particularly when dealing with complex protein domains. This is the case for the human platelet derived growth factor D (PDGFD), a disulfide-rich domain that has been successfully incorporated in recombinant protein nanoparticles that selectively destroy cancer associated fibroblasts (CAFs) in vitro and in vivo. Exploiting Escherichia coli, a promising tool for targeted drug delivery in the tumour microenvironment has been validated.

Self-assembling ferritin NPs are used as a novel vaccine platform to rescue and produce soluble viral glycoprotein in prokaryotic systems and to enhance vaccine stability and immunogenicity for oral administration and broader protection against viral infection. While the glycoprotein alone was insoluble, by genetic fusion with ferritin as a scaffold, we developed a self-assembling IHNV nanovaccine in the E. coli system that is soluble, in the size range (20 nm) ideal for cellular uptake and B-cell activation, biocompatible with living cells (ZFL cells), stable under the harsh GI conditions of the viral host, and induces antiviral immunity in macrophages.