Nanobodies are defined as recombinant protein that corresponds to the variable region of a heavy-chain antibody. Due to its size and structure, nanobodies have conferred characteristic properties such as stability, inexpensive mass production, deep tissue penetration and low immunogenicity maintaining the excellent properties from conventional antibodies such as detectability, selectivity and easy to implement in different assays. Some examples will be showed for both diagnostics and therapeutics applications.

There is a growing interest in antibodies with enhanced functional attributes for cancer immunotherapy, such as Fc-engineered and glyco-engineered antibodies and different isotypes to the commonly used IgG1 (IgG4, IgE). I will present our flexible platforms for engineering and glyco-engineering antibodies of different isotypes and specificities, and expression in transient and stable systems. I will illustrate how these can also be used for the generation of scFv-Fc and antibody-drug conjugates.

Glycosylation of therapeutic proteins can have severe implications on activity, half-life, and response. We have engineered a large panel of CHO cell lines for the production of therapeutic proteins with tailor-made glycosylation. Using this panel, we are able to quickly produce defined glycovariants of protein-based drug candidates to screen for the optimal variant to bring into development, thereby improving the likelihood of bringing the optimal candidates into clinical trials.

Recombinant protein production is a widely used technique, yet half of these experiments fail at the expression phase and only a quarter of target proteins are successfully purified. We have discovered that the energetics of RNA structure ensembles, that model the 'accessibility' of translation initiation sites, accurately predicts the expression outcomes of 11,430 recombinant protein production experiments in Escherichia coli. We have further discovered that normalised B-factors, that model the 'flexibility' of amino acid residues, accurately predicts the solubility of 12,158 recombinant proteins expressed in Escherichia coli. We have optimised these B-factors, and derived a new set of values for solubility scoring that further improves prediction accuracy. We call this new predictor the ‘Solubility-Weighted Index’ (SWI). We have developed TIsigner (Translation Initiation coding region designer) and SoDoPE (Soluble Domain for Protein Expression) that allows users to choose a protein region of interest for optimising expression and solubility, respectively. The final results will suggest synonymous codon changes within the first few codons of the DNA fragments of interest, meaning that gene optimisation can be done using standard PCR cloning.

Ipsen Bioinnovation produces engineered protein neurotoxins to support the discovery and development of novel pharmaceutical botulinum toxins. These have traditionally been produced in batch cultures followed by chromatographic purification. We have increased the throughput of neurotoxin production through expression screening. This was achieved using microbioreactors and magnet-based affinity extraction of target proteins. Selected constructs are then produced in a containment laboratory by batch-extracting up to six toxins from lysate in parallel. Under the constraints of working in a contained setting, we can currently produce 24 novel neurotoxins per month.

To support research groups for development of serological tests, generation of high-affinity antibodies and new vaccine candidates, trenzyme has developed a platform for rapid production of high-quality recombinant SARS-CoV-2 proteins by transient transfection, high-producer cell lines and even GMP compliant production cell lines for development of the intranasal vaccine XPOVAX.

Production platforms capable of manufacturing high amounts of vaccines in short timeframes are lacking today. The work herein developed aims at solving this bottleneck by combining evolutionary engineering (i.e. adaptive laboratory evolution of stable insect cells to hypothermic culture conditions) and process intensification (i.e. high cell-density cultures using perfusion). Adapted cells producing influenza HA-Gag-VLPs were cultured to 100x106 cell/ml in perfusion, with specific Gag and HA production rates similar to batch.

Insect cells are an established platform for production of adeno-associated virus (AAV) vectors for gene therapy, but improved understanding of these cells is still necessary to push towards higher productivities and product quality. Based on one-time additions of supplements known to modulate cell metabolism and on statistical design of experiments, we explored the coordinated effect of baculovirus infection and supplementation on insect cell metabolism, cell cycle distribution, and AAV production.

Plasmid-based transient gene expression (TGE) in mammalian cell lines is an attractive method to screen for expression constructs and produce high-quality recombinant mammalian proteins. However, this technology largely depends on licensing EBNA 1 expressing cell lines and using expensive cultivation media. In this presentation, we show which steps have been successful to reach high levels of expression in High Five insect cells. Upon thorough selection of the optimal plasmid, promoter, transfection agent, cell line and cultivation media, an improved procedure was established. Additional miniaturization using an automated micro-fermentation system allowed us to implement a high-throughput splitGFP screen for expressible constructs. We show that the transient gene expression method in the insect cell line High Five is a fast and cheap alternative for production of substantial amounts of challenging recombinant proteins for functional and structural analysis. The transient gene expression in High Five showed to be very efficient for the production of ample amounts of high-quality proteins for structural and functional studies of SARS 2019 CoV-2. The application of the purified recombinant CoV-2 proteins in intervention strategies of host-pathogen interactions will be presented.

The presentation will highlight approaches and technologies applied for the generation of mammalian cell lines that support biologics projects from target identification and candidate generation to manufacturing.

CLD involves labour and resource intensive cloning out a genetically diverse pool of cells engineered to produce the protein of interest. We sought to analyse a new single-cell analysis microfluidic methodology against an industrial ClonePix™ 2 CLD process . We found that there were no statistically significant differences between cell groups generated from the microfluidic or ClonePix™ 2 processes. Using the microfluidic system, it was possible to predict 3 out of the top 5 producing clones for both Etanercept and Blosozumab. Within the standard ClonePix™ 2 CLD group of cell lines, predictions were most accurate from 24-well plate fed-batch and TubeSpin® batch culture ranks. Further, using the microfluidic system, the time from recovery from transfection to cultures that were ambr® ready was reduced from 65 days to 42 days. Based on the findings of this research it is proposed that the new CLD system is an attractive and powerful new tool for industrial cell line development efforts. 

The biopharmaceutical industry is challenged to produce sufficient amounts of representative material for in-depth analysis, while maintaining a large panel of candidate molecules to increase the chance of success within short timelines. We introduced a developability assessment platform to address this challenge through a funnel-like approach with discrete screening steps.