8-Jun-2011

Production of pharmaceutical compounds through microbial fermentation

The original definition of fermentation is ‘the anaerobic conversion of sugar to carbon dioxide and alcohol by yeast’, and most of us will have had first-hand experience of the fermentation process through its most famous and popular use ¬ the brewing of beer.

This original definition has been expanded over time to ‘the conversion of organic materials into relatively simple substances by micro-organisms – essentially efficient, flexible bio factories.’ During their growth and lifespan micro-organisms build a wide range of different molecules types required for viability and multiplication; adaptation to changing environment; stressful conditions and defence against hostile, competitive microbial threats.

Micro-organisms that are typically used within the pharmaceutical industry include: prokaryotes such as bacteria (e.g. Escherichia coli, Staphylococcus aureus) and Streptomycetes (e.g. Streptomyces spp, Actinomyces spp), eukaryotes such as filamentous fungi (e.g., Nigrospora spp, Aspergillus spp,) and yeast (e.g. Saccharomyces cereviciae,

Pichia pastoris).

The molecules that are of primary interest to the pharmaceutical industry are small molecules such as short peptides and low molecular weight organic molecules, larger molecules including proteins and nucleic acids (DNA, RNA) and macromolecules such as lipids and carbohydrate polymers, plus various combinations of product types, for example lipopolysaccharides, lipopeptides, peptidoglycan. Any of these product types could potentially serve as a drug’s Active Pharmaceutical Ingredient (API).

introduction to microbial fermentation

Microbial fermentation is the basis for the production of a wide range of pharmaceutical products, targeting practically any medical indication. Examples range from anticancer cytotoxic drugs and vaccines, anti-infectious disease antibiotics and vaccines, to hormonal disorder therapy and many other indications.

Natural biosynthesis of endogenous molecules involves specific multi-step complex routes, some of which can be manipulated for the biosynthesis of foreign molecules. Micro-organisms may be genetically modified (recombinant technology) or metabolically engineered by substantial alteration of their endogenous routes.

The key elements of fermentation development are strain selection and optimisation, media and process development, and finally, scale-up to maximise productivity. Downstream processing utilises various technologies for extracting, concentrating and purifying the product from a dilute fermentation broth.

Fermentation derived product diversity – the recovery and selective purification of the specific desired product out of the whole molecular repertoire – makes fermentation technology a multi-disciplinary methodology encompassing microbiology, organic chemistry, biochemistry and molecular biology.

When fermenting volumes larger than 10L, necessary biosafety measures are taken, especially when Risk Group 2 (RG2) pathogens are used. These include Biosafety Level 2 Large Scale (BSL2-LS) containment facility design and special operational procedures. As these products can be toxic and hazardous, their recovery and purification require adequate chemical/biochemical facilities and equipment including isolators for handling High-Potent APIs (HPAPIs).

Under cGMP fermentation procedures, quality is built into the entire process ensuring that regulatory agencies requirements are met in terms of safety, product identity, quality and purity.

Deposited in temperature-controlled bio-storage, strains handled under strict aseptic procedures will be identified and characterised for homogeneity (absence of foreign growth).

Why choose microbial fermentation?

Fermentation is the only route to chemical APIs that relies solely on micro-organisms with no equivalent in other biologic systems (e.g. mammalian cells). Examples include antibiotics/secondary metabolites made in fungi serving as anticancer or anti-infectious agents, or lipid A made in gram negative bacteria serving as adjuvants.

These organic molecules can be obtained through multi-step synthesis from their building blocks. However, organic molecules are very complex in nature, potentially encompassing structures such as chiral centres, large stereospecific rings or unique conjugated double bond systems. Going down the synthetic route not only requires significant development but is time consuming and entails higher costs than the fermentation option.

The semi-synthetic approach draws upon the advantages of fermentation in the generation of new drugs. Natural molecules are produced through fermentation then modified synthetically, reducing toxicity, increasing potency and selectivity, and overcoming bacterial resistance to traditional antibiotics.

Fermentation might also be the sole source for natural therapeutic proteins exclusively expressed in microbial systems. Proteins are complex molecules of mid to high molecular weight. Their functionality and stability largely depend upon their secondary and tertiary structure, as well as various post-translational modifications, mainly glycosilation. The synthetic option is limited to very short peptides.

Recombinant technology enables the expression of foreign gene encoding for therapeutic proteins in microbial systems, including those from human source. Using microbial fermentation is advantageous for expression of proteins that do not require post-translational modifications as microbial systems, such as E. coli, lack post-translational mechanics.

A further approach is to reduce the protein expressed to the minimal effective domain (nanobodies/peptibodies in the case of antibodies). The principal advantages of fermentation over the mammalian system, as illustrated in the table below, are time and yield, which ultimately translate to cost.

Therapeutic proteins requiring modification, for example glycosilation of antibodies, were until recently expressed in mammalian cell cultures. Driven by cost considerations, scientists looked to express glycosilated therapeutic proteins in microbial systems, resulting in a novel approach – glycoengineering – whereby the endogenous glycosilation pathway in high yield expression recombinant yeast was modified. The modified pathway reproduced the human pathway therefore allowing the expression of humanized antibody fragments.

Conclusion

Although not a new technology, microbial fermentation continues to evolve and is now frequently the preferred production method for chemical compounds and therapeutic proteins, offering an optimal economic route that allows pharmaceutical companies to shorten production processes and time to market.

Microbial FermentationMammalian Culture
Generation time20 minutes – hourshours – days
Growth length1 – 4 days10 – 14 days
Product typesProteins; Secondary metabolites; Cell wall components; DNAProteins
Crude protein titer1-15g/L1-5g/L
Media costlowhigh
Growth sensitivitylowhigh
Post translational modificationsSome available in yeastYes

Companies

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