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>antibody expression</span>
Many laboratories have utilized cell-free systems or prokaryotic systems designed to produce biological molecules with single polypeptide chains, limited folding requirements, and without glycosylation. The yeast systems are used to generate glycoproteins; however, their glycosylation profiles are vastly different from those of mammalian cells. Without significant glycoengineering, the yeast-produced recombinant glycoproteins may be unsuitable as therapeutic molecules. As such, the use of mammalian cells is still the preferred method to produce complex biological molecules…
Directed evolution has profoundly changed the way antibodies or antigen binding fragments (Fab) are produced today. Lead generation for product candidates is oftentimes based on complex combinatorial libraries containing a large collection of variant antibodies. The selection of candidate molecules by screening procedures like phage display is considered the gold standard. One major advantage of these methods is that initial candidate molecules can be further improved in terms of affinity, specificity, etc. by reiterative in vitro maturation processes using the very methods that have been used for lead generation…
Despite the existence of effective vaccines against Hepatitis B virus, the infection with it remains an important problem worldwide due to its association with hepatocellular carcinoma. Several procedures have been used to purify the Hepatitis B surface antigen (HBsAg) for immunization purposes. Immuno-purification using HBsAg-specific murine monoclonal antibodies (MAbs) has been one of the most successful strategies for such a purpose due to the high antigen selectivity (high affinity) of MAbs…
Biopharmaceuticals are predicted to become the main driving force of the pharmaceutical market in the near future. Other than blockbuster products such as erythropoietin, an increasing number of approved recombinant therapeutic proteins are based on antibody technology (e.g., fusion proteins or monoclonal antibodies [MAbs]). In contrast to relatively simple products produced in Escherichia coli bacteria (e.g., insulin), proteins which require complex posttranslational modifications such as glycosylation have to be produced in eukaryotic cells. In this context, production systems have been dominated by mammalian cell culture. Nevertheless, alternative eukaryotic expression technologies based on yeast, insect cells, transgenic animals, or transgenic plants are under development. Plants are a particularly promising alternative to mammalian cell culture because of their excellent safety aspects and estimated cost-efficient upstream/cultivation processes. In addition, plants are well known for their ability to express biologically functional monoclonal antibodies. In comparison to the seed plants most widely used for transgenic protein expression — tobacco, corn, and rice — mosses provide unique, beneficial features…
A number of antibody drugs are currently in clinical development and 22 antibodies (including five diagnostic antibodies) have received FDA market approval in the last decade. A number of different technologies are now being used successfully to isolate potent therapeutic antibodies with minimal immunogenicity and improved safety. These include chimerisation (mouse/human antibodies), humanisation (complementarity-determining region [CDR] grafting), transgenic mice, phage display, ribosome display, and other emerging technologies. The phage and ribosome display technologies used at Cambridge Antibody Technology (CAT) are based on the physical linkage of gene to gene product which enables the recovery and enrichment of genetic material encoding the selected antibody…