Non-Mammalian Eukaryotic Expression Systems Yeast and Fungi in the Production of Biologics

Abstract

Biologics have become an important area of medical research generating therapeutics essential for the treatment of many disease states. Biologics are defined as biologically active compounds manufactured by living cells or through biological processes termed bioprocessing. Compared to small molecules which are chemically synthesised they are relatively complex and therapeutically specific molecules. Biologics include hormones, vaccines, blood products, monoclonal antibodies, recombinant therapeutic proteins, enzymes, gene and cellular therapies amongst others. For biologic production prokaryotic and eukaryotic cells (mammalian and non-mammalian) are used as expression systems. Eukaryotic expression systems offer many advantages over prokaryotic based systems. The manufacture of high-quality proteins for human clinical use via recombinant technologies has been achieved in yeast and filamentous fungal systems. Advances in bioprocessing such as genetic engineering, bioreactor design, continuous processing, and quality by design has allowed for increased productivity and higher yield in in these non-mammalian eukaryotic systems with protein translation similar to mammalian systems. The application of eukaryotic expressions systems for the manufacture of biologics of therapeutic importance are described herein.

Bioprocessing and the Production of Antiviral Biologics in the Prevention and Treatment of Viral Infectious Disease

“…PTMs important for vaccine development include glycosylation, acetylation, sulfation, methylation, amidation, SUMOylation, ubiquitylation, lipidation, formylation and phosphorylation [ 35 ]. PTMs occurs in eukaryotic cell organelles including the nucleus, cytoplasm, endoplasmic reticulum (ER) and Golgi apparatus, which are absent in prokaryotic cells [ 36 ]. Therefore, biologics expressed by bacterial expression systems need in vitro processing and the addition of PTM steps during synthesis, increasing costs and reducing yield [ 37 ].…”

“…Considerations such as the plasmid used, promoter usage, control of proteolytic degradation, expression rate and location (extracellular, intracellular) of proteins affects the quality and quantity of biologics produced in expression systems, with purification strategies also impacting on the vaccine’s antigenic and immunogenic properties [ 38 ]. Bioreactors are operated as batch, fed-batch or continuous systems and with operation considerations including cell type, media composition, substrate concentration, cell density of the biocatalyst, product inhibition, pH, oxygen requirements and temperature, where protein production is typically higher in continuous mode than in batch systems [ 36 ]. Most commercial virus production processes utilise Vero, Madin–Darby canine kidney (MDCK), human foetal lung fibroblast cells (MRC-5) and human lung fibroblast (WI-38) continuous cell lines, which maintain an anchorage-dependent growth [ 39 ].…”

“…Recent advances in vaccine production have generated protein-based subunit vaccines, virus-like particle (VLP) vaccines, viral vector- and nucleic acid-based (RNA and DNA) vaccine modalities [ 40 ]. The production formats of such vaccines aim to lessen the time constraints, issues with reversion to pathogenicity, immunogenicity and biocompatibility issues observed with traditional vaccine types [ 36 ]. The use of RDNA technology along with mammalian and non-mammalian expression systems in bioreactors has allowed for cheaper, faster production of more diverse vaccine types including subunit-based and viral-like particle-based vaccines ( Table 1 ) [ 41 ].…”

“…Yeast are known for their excellent ability to express heterologous proteins and have proven useful in vaccine development. Yeast have many advantages as culture organisms for biologics, including fast growth rate, low-cost media and some PTM ability, and they are considered generally recognized as safe (GRAS) by the FDA [ 36 ]. Yeast are amenable to large-scale production in bioreactors and are amenable to genetic manipulation and the incorporation of plasmids containing the gene of interest coding the desired biologic.…”

“…The cell wall components of yeast chitin, glucan and mannan have adjuvant activity and can act as natural immune stimulators boosting the vaccination [ 87 ]. As a non-pathogenic strain, S. cerevisiae is easily genetically modified, possess strong adjuvant properties and long-term stability, while allowing for oral delivery due to its resistance to gastric degradation [ 36 ]. To date, several antigens including antigens from HBV and porcine epidemic diarrhoea virus were successfully expressed on the yeast surface for the development of oral vaccines [ 88 ].…”