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 GlycoSwitchĀ® 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>recombinant proteins</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 STRĀ® 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.
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 (HeberBiovacā¢ HB) starts with the expression of the HBsAg in Pichia pastoris….
Heterologous expression of membrane proteins remains a bottleneck for structural characterization by x-ray crystallography. Such proteins represent approximately 30% of the proteome and are not sufficiently represented in the Protein Data Bank (PDB). G-protein-coupled receptors (GPCRs) are an area of particular interest as it is estimated that one third of current FDA approved drugs act through this class of receptors. We have been studying rhodopsin with an interest in determining the conformational change that leads to signal transduction in this class of receptors. Although there has been some success in expressing select members of the large GPCR family in bacterial systems, the best characterized expression systems have generally been in mammalian tissue culture…
The recombinant protein alefacept (AmeviveĀ®) is an immunomodulator approved for the Ātreatment of moderate to severe chronic plaque psoriasis. In phase 3 clinical studies, treatment of psoriatic patients with alefacept at a safe and well-tolerated dose caused Āmeaningful reductions in psoriasis area and severity index and in physician global Āassessment scores as well…
Since the first approval for human use of a recombinant protein therapeutic, this sector of the pharmaceutical market has grown rapidly. The first approved protein therapeutics were small, non-glycosylated proteins such as insulin and human growth hormone; they were produced in bacterial systems. With the advent of mammalian cell-based production systems, it became possible to produce more complex, glycosylated proteins for use as recombinant therapeuticsā¦
In order to unravel new protein activities and functions, we have expressed and purified a large number of human proteins. We have chosen to study secreted proteins and the extra-cellular domains of putative single transmembrane domain-containing proteins. In order to retain the natural protein characteristics as far as possible, we have used a mammalian expression system. Human embryonic kidney (HEK293) cells were chosen as they have been shown to possess a high protein-secretory potential. The secreted proteins were expressed with a carboxy-terminal tag and purified by affinity chromatography. Each protein was produced at a routine scale from 500 ml cell cultures, and the secreted protein was purified from the culture supernatant…
With the continued growth of the biopharmaceutical market, the cell culture industry has seen a major shift away from the use of serum and other animal-derived supplements in the manufacture of biopharmaceuticals. Indeed, supporting guidance from the EMEA and FDA for the manufacture of biopharmaceuticals and medical devices encourages the use of “animal-free” components.Ā The key driver for this can be attributed to the increased concern with contamination from adventitious agents such as transmissible encephalopathiesā¦
The analytical characterization of recombinant protein therapeutic drug products has broadened to include the use of more sophisticated technologies. The expansion of technical abilities has translated into increasing the depth and breadth of our knowledge and understanding of the drug product intended for commercialization. With the availability of more precise methods, the regulatory expectations for understanding the characteristics of a protein therapeutic drug product are increasing. A thorough understanding of a therapeutic proteinās biochemical and biophysical characteristics is necessary to support investigational new drug (IND) applications and other drug regulatory filingsā¦
Proteins are widely used in research, medicine and industry, but its extraction from their natural sources can be difficult, tedious and expensive. Therefore, a simple and inexpensive system that allows large-scale production of safe recombinant proteins will always be highly desirable. Traditional production systems that use microbial, insect and mammalian cell cultures have drawbacks, in terms of cost, scalability and product safety. Several studies have shown that molecular farming in plants has many practical, economic, and safety advantages as compared to these conventional methods. Thus, the use of plants for recombinant protein synthesis is gaining wide acceptanceā¦