Fermentation Technology for Biopharmaceuticals

With the recent boom in the global demand for biopharmaceuticals, the importance of fermentation technology in meeting this growing demand is now more important than ever. In this article, we discuss the applications of fermentation technology in yielding high quality biopharmaceuticals in a manner that is both efficient and economical.

An overview of some of the most important and game changing biopharmaceuticals is also given, including enzymes, hormones, vaccines, monoclonal antibodies and antibiotics. The future of biopharmaceuticals is facilitated by a combination of biotechnology and fermentation technology, and assures that the era of newer, more efficient and viable methods of mass producing biopharmaceutical products is just around the corner.

Biopharmaceuticals are biotechnological drugs that are used for therapeutic purposes, aiming to cure diseases rather than simply treating symptoms. Structurally, they resemble compounds that are already present in the body. The term ‘biopharmaceutical’ was coined in the 1980s [Gar03], and generally describes any medically useful drug, which is manufactured by microorganisms or genetically modified organisms, or involves bioprocessing.

Other than drugs, biopharmaceuticals may also include the products produced by these organisms, such as enzymes. Their main advantage is that they are highly specific, targeting specific cells, causing fewer side effects compared to traditional pharmaceuticals. They have significantly altered the treatment mechanism for diseases like diabetes, and even malignant disorders, as these can be specifically designed according to the requirements. The first biopharmaceutical product was the biosynthetic human insulin produced using recombinant DNA technology, and with advancing technology, more than 150 biotech drugs exist today, including Interferons, Human growth hormones, and Monoclonal antibodies.

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Majority of these proteins are produced in E. coli, S. cerevisiae or in animal cell lines, and they are mainly manufactured for human use. However, certain products were intended for veterinary application. This includes the bovine growth hormone (Somatotrophin), which is used to increase milk yields from dairy cattle.

Using recombinant technology to produce biopharmaceuticals has a number of advantages. Large quantities of the desired substances can be produced through this technology. These desired substances may also contain the effective domains for a specific disorder.

Production of biopharmaceuticals through fermentation is a less time consuming process, hence the process is faster than other methods. Microbial fermentation requires media with lower costs, reducing the overall cost of the process. This increases the efficiency and productivity, resulting in high quality products.

Simple Fermentation is the mass culture of single celled organisms or a colony of yeast that convert nutrients into useful products. The development of Recombinant DNA Technology, however, has taken the traditional technology of fermentation to a completely new level.

Today, not only genetically engineered microbes but also plants and animal cells are mass cultured to produce a wide range of product including vaccines, antibodies and other therapeutic proteins. Moreover, these modern techniques are used by biopharmaceutical industry not only to produce protein drugs that target medical indications but are also used for extended research and drug testing.

The principle that forms the basis of the production of a wide range of Biopharmaceuticals is Microbial Fermentation. In manufacturing of Biopharmaceuticals, microorganisms utilize the sugar source releasing energy and use this energy for biosynthesis of different molecular product of prophylactic and therapeutic roles.

Examples include vaccines and cytotoxic drugs against cancer, infectious diseases and hormonal disorder therapy. The pharmaceuticals industries are interested in products that can serve as potential Active Pharmaceutical Ingredient (API) in a drug. These products may be small molecules (short peptides), large molecules (proteins, DNA, RNA), macromolecules (lipids, carbohydrates), conjugated molecules (lipopolysaccharides) etc.

For efficient manufacturing of biopharmaceutical products, two components which play a key role are choice of the organism and the surrounding environment provided to the microbes for growth and metabolism. The expected yield can only be attained if the organisms are well designed to biosynthesize the desired molecule and they are provided with a tightly controlled favorable environment. Under optimum conditions, organisms will exhibit maximum growth rate and produce the molecule of interest.

As, mentioned earlier, choice of the organism is one of the most important component of fermentation. The Biopharmaceutical Industry uses several different microbes and they are broadly categorized as: Bacterial cells have an edge over other microbial systems in that they contain plasmid DNA in addition to chromosomal DNA. The small size of plasmids makes them an easy target for manipulation and gains bacterial cells the advantage of genetic engineering.

The bacterial species used in Biopharmaceutical industry are Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Streptomyces (e.g. Streptomyces spp, Actinomyces spp). The specie preferred for expression of therapeutic proteins is Escherichia coli (E. coli) owing to the fact that it is the most well studied bacteria with reference to the genetic sequence and structure. Almost 30% of Biopharmaceuticals are produced using E. coli as cell factory.  Examples of antimicrobial compounds obtained through bacteria are Streptomycin, Rifamycin, Erythromycin which are effective against Tuberculosis.

Yeast is the most widely used fungi for biopharmaceutical manufacturing. Examples of yeast species used include Saccharomyces cereviciae, Pichia pastoris but S. cerevisiae is preferred and accounts for 20% of the Biopharmaceuticals produced). The dominant among these products are Insulin, Hepatitis vaccines, Human serum albumin and virus like particles.

The advantage of using S. cerevisiae is that, being a unicellular eukaryotic model, it allows for the easy cultivation and at the same time, the synthesis and folding of complex human proteins. Moreover, yeast having eukaryotic machinery have the advantage over bacteria of performing Post- translational modification of proteins. In addition, the information available on S. cerevisiae through high-throughput studies, databases and sequenced genomes is enough to make it an ideal for further manipulation.

A different approach to obtain Biopharmaceuticals is the use of Mammalian cell culture. Fermenters or Bioreactors are used to provide the Mammalian cell lines that are complex and more sensitive to the environmental conditions, thus require tightly regulated conditions to grow. The desired product are secreted by the cells and in some cases, the cells are the therapeutic agents themselves, for example Apligraf from Novartis Pharmaceuticals, human skin that can be grafted, produced through the research conducted on human stem cell therapies.

Mammalian cells are now becoming dominant cell factories for complex therapeutics because they add post translational modifications to the complex proteins they produce but in contrast to fungi, these modifications are more humanlike. Chinese Hamster Ovarian cell lines (CHO cells) are mainly used as mammalian cell cultures and about 40% of Biopharmaceuticals are currently being produced by them. Mammalian cells derived pharmaceuticals are important for diagnosis and treatment of disease conditions like Adalimumab for rheumatoid arthritis, Ofatumumab for leukemia and Panitumumab to treat colon-rectal cancer.

The organism or the system finally selected for the biopharmaceutical production depends on a number of technical and economic factors. The benefits and limitations to using different systems are assessed which in turn are governed by the product that is required to be produced.

Bacteria and yeast are both unicellular organisms which have simple nutritional needs, can easily grow suspended in a liquid medium. The cells are independent and each cell is responsible for carrying out its own metabolic activity. Furthermore, these microbes are resistant to damage due to the presence of cell wall.

In contrast, multicellular organisms, like mammalian cells, are fragile and most likely to be damaged by the harsh environment. Unlike, unicellular organisms, they are evolved to function as a part of a collective group of cells in organs or tissues. Thus, it is costly to maintain animal or mammalian cell cultures, with their replication being slow and requirement of complex nutrients for growth.

Size of gene of interest. The size of the gene that can be inserted in bacteria and yeast is a limitation to these systems despite their easy culturing. In case a larger clone of DNA needs to be clones, a mammalian cell culture needs to be used.

Same protein produced by bacterial and animal cell can have different effect. This is because a protein after expression goes through post-translational modification and glycosylation (improves molecular stability of protein pharmaceuticals) in eukaryotes. These processes effect the way protein folds into its final form and it has a great effect on the activity of protein in a particular process.

Therefore, if protein needs to be glycosylated or post translational modification is essential for it; such a protein should be made using mammalian cell culture and fungi e.g. Insulin Aspart and Insulin Glargine [Bhu2010] manufactured using yeast, Pichia pastoris and Erythropoietin-alpha having similar biological effects as Erythropoeitin [Bhu2010]. Similarly, non-glycosylated products require bacterial cells e.g. Streptokinase manufactured as a non-glycosylated polypeptide chain using Recombinant E. coli.