Nowadays, therapeutic monoclonal antibodies (mAbs) are predominantly produced with mammalian cell culture systems such as those using Chinese hamster ovary (CHO) cells. Efforts are underway to reduce the costs of this process to meet the increasing global demand in biopharmaceuticals; meanwhile, cheaper and faster expression systems are being investigated as alternatives. The yeast, Pichia pastoris, has become a substantial workhorse for recombinant protein production. However, the N-linked glycosylation in P. pastoris, namely high mannose glycosylation, is significantly different from that in CHO or other mammalian cells, including human cells. In this study, a SuperMan5 strain of P. pastoris was constructed using Pichia GlycoSwitch® technology to successfully produce a more mammalian-like immunoglobulin G (IgG) fragment crystallizable (Fc), which showcases the potential of P. pastoris as a next-generation mAb production platform. Importantly, in this study, a strong methanol-independent promoter, PUPP, was applied, which only requires glycerol feeding for protein production. Most P. pastoris promoters used for protein expression are derived from genes in the methanol metabolism pathway, creating safety concerns due to the flammable nature of methanol, especially at large scale. Here, a fed-batch SuperMan5 P. pastoris fermentation was carried out in which methanol induction, as well as its affiliated safety risks, were eliminated. Overall, this study provides insights into the development of safe and cost-effective industrial mAb production approaches independent of mammalian cell culture.
Tag: <span>fermentation</span>
Stirred tank single-use bioreactors (SUBs) have been widely adopted for production of biopharmaceuticals such as monoclonal antibodies in mammalian cell culture. However, they are seldom used for commercial production of biologics with microbial fermentation. SUBs offer time-saving advantages because they do not require significant downtime for cleaning and sterilization, so finding a SUB that can perform well with high cell density microbial fermentation processes has the potential to increase the number of production runs. Therefore, for this study, a His-tagged protease inhibitor was chosen as a model protein to demonstrate that the Sartorius Biostat STR® MO, a SUB recently developed for microbial fermentation, is suited for recombinant protein production by high cell density Escherichia coli fermentation processes.
At 50 L scale, the SUB achieved good process control and allowed an oxygen uptake rate (OUR) of up to 240 mmoles/L/h. The fermentation runs produced up to 5.8 g/L of the soluble recombinant protein and a dry cell weight of >60 g/L at the end of fermentation. Additionally, the SUB showed a similar fermentation profile when compared with data from parallel runs in 15 L sterilise-in-place (SIP) vessels using identical media and process parameters. This study indicates that with a minimum investment of capital resources, stirred tank SUBs could be used in pilot-scale manufacturing with high cell density microbial fermentations to potentially shorten the timelines and costs of advancing therapeutic proteins to clinic.
Filamentous fungi are an important class of organisms with significant commercial importance in the biotechnology sector. Those that dominate the markets and are deemed the most important include: Aspergillus, Penicillium, and Trichoderma. They have become indispensable for the production of enzymes due to their exceptionally high capacity to express and secrete proteins. In addition, recent advancements in genomics have allowed for the sequencing or partial sequencing of many of these species. The significance of these fungi and their associated enzymes has accelerated interest in understanding the mechanisms involved in the regulation of gene expression, primarily through the characterisation of the transcriptional elements involved…
Fermentation utilizes organic substrates and organic electron acceptors to produce reduced organic substances as end-products. Bioethanol has a number of advantages over conventional fuels because it is produced from renewable resources. Ethanol is a high octane fuel that can replace lead as an octane enhancer in petrol by helping to oxygenate the fuel mixture so it burns more completely and reduces polluting emissions. The burning of ethanol more closely represents a natural carbon dioxide cycle since the released CO2 is recycled back into plants where the carbon dioxide becomes the carbon source for new sugars, starches and cellulose…
In general, yeasts offer advantages for recombinant protein expression because their intracellular environment is suitable for the correct folding of recombinant proteins and grow very high cell densities in defined fermentation media. Within the yeast kingdom, Pichia pastoris has been successfully used for expressing several recombinant proteins. The genome of this yeast contains two copies of the alcohol oxidase (AOX) gene, where the AOX1 promoter regulates 85% of the alcohol oxidase activity and drives the recombinant protein expression into the cell. One of the most successfully recombinant proteins expressed in Pichia pastoris is the hepatitis B surface antigen (HBsAg). The current manufacturing process of the active pharmaceutical ingredient (HBsAg) of the Cuban hepatitis B vaccine (HeberBiovac™ HB) starts with the expression of the HBsAg in Pichia pastoris….
The manufacture of biological materials by fermentation is based intrinsically on established and well understood processes developed over a number of years. The fundamental basics of supplying a characterised cell line with sufficient nutrients over a period of time, with the intention of harvesting a selected protein for further processing, are similar throughout the industry. However, as a result of economic pressures and the need to control costs of an already expensive, high quality, and high compliance material, all manufacturing companies endeavour to maintain the highest level of productivity…