Evolutionary development of eukaryotic microorganisms—and yeast in particular—has provided an ideal chassis for fast and predictable process development and realizing next-generation intensified manufacturing processes of recombinant proteins. This talk will explore the features of alternative hosts that make them well-suited for addressing both speed and costs, while retaining quality, in manufacturing recombinant proteins. In an era of genomic sequencing and gene editing technologies, simple eukaryotic microorganisms like yeast have become ‘software’-like and can be reprogrammed to make a range of recombinant proteins from cytokines to vaccines to nanobodies to monoclonal antibodies (mAbs). Alternative hosts offer great potential for predictable and fast cycles of development and process intensification and some have proven use in manufacturing FDA-approved products. There remains, however, conceptual and practical barriers to widespread adoption, including considerations of potential quality variations and sufficient productivity for commercial use in some applications, as well as limited shared experience with alternative hosts more broadly in the industry. The AltHost Consortium at MIT has created a venue for sharing the risk of developing new host biology in an innovation-friendly model based on open-source principles akin to software. This talk will highlight examples of this approach to progress a model host, Komagataella phaffii (“Pichia”), to improve volumetric productivity of mAbs, create molecular tools for engineering, and remodel pathways for glycosylation. Some topics of discussion will include genome-scale screening for improved production, refinements of sequences for quality of produced mAbs, and ML-guided process-based improvements for improved production of proteins. Implications for these advancing capabilities for a next-generation platform for biomanufacturing will be presented.

The baculovirus expression system is recognised as one of the main platform technologies for the production of proteins in insect cells. For many years, the Sf9 cell line has been popular for both the amplification of recombinant viruses and the production of proteins. It was produced by clonal selection from Sf21 cells, which were originally derived from the pupal ovarian cells of Spodoptera frugiperda (Fall armyworm). In this study, we aimed to produce a rhabdovirus-free insect cell line for production of proteins for use as human vaccines. We used a frozen vial of early passage Sf21 cells (Oxford, 1981) as our starting material. A number of cell lines were derived by rounds of single-cell cloning and testing for the presence or absence of the insect rhabdovirus by RT-PCR. Putative rhabdovirus-free cell lines were expanded in ES-AF (animal-free) medium and were also tested for the ability to support baculovirus amplification and protein production. One cell line, SfC1B5, was selected for banking and further study. The SfC1B5 cell line has since been adapted to grow in chemically-defined medium and extensive testing has demonstrated the cell line supports recombinant baculovirus production, amplification and protein yields similar to that obtained with Sf9 cells. We report use of the cell line to make Gc and Gn surface glycoproteins of Crimean Congo Hemorrhagic Fever virus as a candidate vaccine. However, for a range of secreted proteins, including viral antigens, higher yields were obtained in the SfC1B5 cell line. Where the secreted protein is to be used as a vaccine for human or animal health, the increase in yield significantly reduces the cost of vaccine production per unit dose.

The utilization of diverse cell expression platforms, including insect cells, HEK (Human Embryonic Kidney) cells, CHO (Chinese Hamster Ovary) cells, and Escherichia coli (E. coli), has become a pivotal strategy in addressing challenges associated with “difficult-to-express" biological targets. Each cell expression platform offers unique advantages tailored to the specific requirements of the target proteins. E. coli systems are favoured for their rapid growth and high yield, though they may struggle with complex post-translational modifications (PTMs). Insect cell systems, utilizing baculovirus vectors, excel in producing proteins with intricate PTMs, crucial for functional studies. HEK cells are widely used for their human-like PTMs and ease of genetic manipulation, making them ideal for producing proteins in their native conformation. CHO cells are indispensable for producing therapeutic proteins with human-like modifications and are extensively used in biopharmaceutical production. HEK and CHO cells also offer the advantage of secreting proteins into the surrounding medium making downstream processing much easier and cost effective. As the Department of Protein Science and Structural Biology within Sygnature Discovery, we are a contract research organization (CRO) that provides custom made, soluble and membrane proteins for use in drug discovery, research applications as well as X-ray crystallography, NMR and Cryo-EM structures of both novel and precedented proteins. Here we show real examples of how we’ve utilized each of our different expression platforms (Insect, HEK, CHO, E. coli) to successfully express membrane proteins (for structural work), intrinsically disordered transcription factors, large multi subunit protein complexes (the largest being a 12 subunit protein complex) and labelled proteins for NMR studies. This was achieved by leveraging the strengths of each individual expression platform to overcome the limitations posed by “difficult-to-express" targets; thereby, facilitating advancements in drug discovery, structural biology and therapeutic development.

The use of non-clonal CHO cell derived materials for preclinical studies has been found to be a valuable approach to accelerate the development of monoclonal antibodies (mAbs) for first-in-human (FIH) studies. In a comprehensive study, we assessed the culture performance, productivity, and product quality of non-clonal cell lines compared with clonal cell lines expressing various biologic modalities to determine if this approach can be applied to complex molecules. We evaluated a multi-specific antibody, a cytokine-Fc fusion protein, and a mAb produced using the stable pool, the pool of top clones, and the lead clone utilizing transposase-mediated integration. The results indicated that the attributes were comparable regardless of the source of cells. Building upon these findings, the study progressed to the preclinical manufacturing of two multi-specific antibodies using both the pool of top clones and the lead clone. Subsequently, clinical manufacturing of these multi-specific antibodies was performed using the lead clone. The batches produced with the pool of clones and the lead clone demonstrated a high level of comparability in culture performance, productivity, and product quality. In conclusion, non-clonal CHO cell derived materials can be effectively utilized for preclinical studies of complex molecules without compromising their quality, allowing for accelerated development for FIH studies.

Hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used effectively in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, GigaGen established a platform for development and manufacturing of recombinant polyclonal antibodies (pAbs), which comprise a mixture of >1,000 individual antibodies produced en masse. Two drugs of this class have now entered clinical development: GIGA-2050, for treatment of SARS-CoV-2, and GIGA-2339, for treatment of chronic Hepatitis B virus. To achieve these milestones GigaGen overcame several key challenges, including development of a single site targeted integration cell line, optimization of the upstream process using that cell line, as well as development of novel methods to monitor the upstream and downstream processes to ensure lot to lot consistency.