ProClin Preservative ®
for Diagnostic Reagents
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Production of Pharmaceutical Compounds
Through Microbial Fermentation
Silvian Shama, Ph.D., cGMP Fermentation Development Manager
Overview
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 microorganisms- essentially
efficient, flexible bio factories.’ During their growth
and lifespan microorganisms 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.
Microorganisms 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).
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Microbial Fermentation–an Introduction
Microbial fermentation is the basis for the production
of a wide range of pharmaceutical products, targeting
practically any medical indication. Examples range
from anti cancer 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. Microorganisms 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 optimization, media and process
development, and finally, scale-up to maximize
productivity. Downstream processing utilizes 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.
ProClin Preservative ®

for Diagnostic Reagents

SAFC Pharma ®

Packaging
pipeline Information
partners

Why Choose Microbial Fermentation?

Fermentation is the only route to chemical APIs that A further approach is to reduce the protein expressed
relies solely on microorganisms with no equivalent in to the minimal effective domain (Nanobodies/
other processes. Examples of its applications include Peptibodies in the case of antibodies). The principal
mammalian cells, antibiotics/secondary metabolites advantages of fermentation over the mammalian
made in fungi serving as anti cancer or anti infectious system, as illustrated in the table below, are time and
agents, or lipid A made in gram negative bacteria yield which ultimately translate to cost.
serving as adjuvants.
Microbial Mammalian
These organic molecules can be obtained through Fermentation Culture
multi-step synthesis from their building blocks. Generation
20 minutes-hours hours-days
However, organic molecules are very complex in time
nature, potentially encompassing structures such Growth length 1-4 days 10-14 days
as chiral centers, large stereospecific rings or unique Proteins
conjugated double bond systems. Going down Secondary metabolites Proteins
Product types
the synthetic route not only requires significant Cell wall components
development but is time consuming and entails DNA

higher costs than the fermentation option. Crude protein

1-15g/L 1-5g/L
titer
The semi-synthetic approach draws upon the Media cost low high
advantages of fermentation in the generation of new Growth
low high
drugs. Natural molecules are produced through sensitivity
fermentation then modified synthetically, reducing Post
Some available
toxicity, increasing potency and selectivity, and translational Yes
in yeast
overcoming bacterial resistance to traditional modifications
antibiotics.
Therapeutic proteins requiring modification, for
Fermentation might also be the sole source for example glycosilation of antibodies were, until recently,
natural therapeutic proteins exclusively expressed in expressed in mammalian cell cultures. Driven by
microbial systems. Proteins are complex molecules cost considerations, scientists looked to express
of mid to high molecular weight. Their functionality glycosilated therapeutic proteins in microbial systems,
and stability largely depend upon their secondary and resulting in a novel approach – Glycoengineering-
tertiary structure, as well as various post-translational whereby the endogenous glycosilation pathway in high
modifications, mainly glycosilation. The synthetic yield expression recombinant yeast was modified. The
option is limited to very short peptides. modified pathway reproduced the human pathway
therefore allowing the expression of humanized
Recombinant technology enables the expression antibody fragments.
of foreign gene encoding for therapeutic proteins
in microbial systems, including those from human Conclusion
source. Using microbial fermentation is advantageous Although not a new technology, microbial fermentation
for expression of proteins that do not require continues to evolve and is now frequently the preferred
post-translational modifications as microbial systems, production method for chemical compounds and
such as E. coli, lack post-translational mechanics. therapeutic proteins, offering an optimal economic
route, which allows pharmaceutical companies to
shorten production processes and time to market.

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