Pharmaceutical

Biotechnology
Dedicated from Prof. Mohammed Bahey-El-Din
Professor of Microbiology and Immunology
Faculty of Pharmacy, Alexandria University

Dr. Nelly Mostafa

Associate Professor of Microbiology and Immunology
Faculty of Pharmacy, Alexandria University
E-mail: [email protected]
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References for Pharmaceutical Biotechnology

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 Introduction
What is Biotechnology?
• Bios: Life Technos: Tool Logos: Study of
• Biotechnology= Study of Living Tools
• “It is the use of living systems (microbial,
yeast, animal, plant cells) to develop
useful products”
• Biotechnology covers a wide range of
applications such as the production of:
• Essential products like life-saving drugs (as
monoclonal antibodies, hormones, antibiotics &
vaccines)
• Environmental application: Biodegradable plastics,
biofuels, genetically modified crops, and using
microorganisms to clean up contaminated sites
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 Fermentation
• Large-scale production of useful products (of medical or commercial
importance) using microorganisms

• The oldest of all biotechnological processes is Fermentation

• “Fermentation” is a biological process that occurs in the absence of
oxygen BUT
• “Fermentation” in industrial-scale productions usually refers to the
process whether performed under anaerobic (no oxygen) or aerobic
(oxygen) conditions
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Examples of Fermentation
• Production of:
• Alcoholic beverages
• Vinegar
• Cheese
• Bread baking
• Production of acetone, ethanol,
and butanol by fermentation of
plant carbohydrates

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 Characteristics of Industrial Microbial
Strains
Industrial microorganisms act like chemical factories and should ideally
have the following properties:
1. Should be a pure culture = not contaminated with other species or low-
producing strains
2. Should produce large amount of the required product
3. Easily cultivated and maintained
4. Should be genetically stable = low rate of mutation
5. Grow rapidly on inexpensive and readily available media
6. Produce the desired product under achievable conditions (pH,
temperature, O2, …..etc).
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 Industrial Culture Media
• Culture media in industrial fermentations are usually by-
products of other industries:
1. Corn steep liquor: by-product of corn wet milling
2. Whey: by-product of cheese industry
3. Molasses: by-product of sugar industry
4. Starches: obtained from different grains but should be degraded to
monosaccharides and oligosaccharides before use in fermentation
• Culture raw materials should be cheap and available locally
• Culture medium should provide the organism with:
1. Carbon
2. Nitrogen
3. Minerals
4. Growth factors
5. Precursors of the intended product
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 Methods to Improve Industrial Fermentation
1. Upstream manipulations (strain improvement):
• Wild strains usually produce low yields of the desired product:
• Improvements should be performed to get a suitable industrial strain
• This can be done by:
a) Induction of mutation (mutagenesis) & Screening for mutants with higher
characteristics
b) Genetic recombination to create improved strains
c) Cell fusion to generate hybrids with improved productivity

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 2. Downstream manipulation
a) Manipulation of the fermentation conditions to
specify the product and maximize the yield

b) Creating mathematical models for optimization of

fermentation parameters for best yield
c) Use of proper methods for separation and
purification e.g. centrifugation, filtration or
chromatography 9
 Fermenter/Bioreactor
• Small fermenters:
• 20 liters in capacity
• Used in research labs
• Usually made of glass
• Large stainless-steel fermenters:
• 25 – 250 liters in capacity
• Used in industry
• Fermenter and substrate solutions:
• Must be sterilized before addition of microbial strain
• Small fermenters are sterilized in autoclave
• Large fermenters are sterilized by steam from a distant boiler 10
• In aerobic fermenters, compressed filtered air is
pumped through a sparger at the bottom of the
fermenter: dispersion of oxygen in the liquid medium
• Impellers and baffles (in big fermenters): transfer
oxygen and mix the content
• Pumps: supply acids and alkalis, adjust pH during
fermentation
• Antifoaming agent: control foam formation
• Foaming occurs due to the vigorous mechanical stirring
and aeration
• If foaming is not controlled: culture is lost by entrapment
in the exhaust gases
• Instrumentation: measure critical parameters: pH,
temperature, dissolved oxygen, harvesting pipe,
inoculation pipe, sampling point, and the power used
by the electric motor
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 Methods of Fermentation

Batch Fed-Batch Continuous

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 Batch Fermentation
• Closed system:
• The fermenter containing the sterilized culture
medium is inoculated with microorganism and
incubation is allowed under optimal condition
for a specific time
• During the fermentation period, nothing is
added except:
1. air
2. anti-foaming agent
3. acid or alkali to adjust pH
• At the end of the fermentation cycle, the
fermenter is shut off and contents collected
to recover the product
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 Continuous Fermentation
• Sterile nutrients are added continuously to
the fermenter and equivalent amount of
product with microorganism are
simultaneously harvested out of the
fermenter
• A steady state is achieved which can last
from days to months
• Methods of monitoring addition of substrates :
1. Chemostat: continuously adjusting the
concentration of one substrate
2. Turbidostat: cell growth is kept constant by
measuring turbidity
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 Fed-Batch Fermentation
• Fed-batch is the intermediary model of bioreactor operation = semi-batch
operation
• Characterized by controlled addition of nutrients into the bioreactor at
intervals during the fermentation process
• Allows a degree of control on the process
• Frequency and concentrations of feed can be controlled by parameters:
• rate of growth of the microorganisms
• or the concentration of the biomass

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 Scale-up
• It is the transfer of small-scale laboratory
fermentation to an industrial large scale
• Fermentation processes are developed in three
stages:
1. Laboratory scale: flasks, laboratory
fermenters from 0.5-10 L
2. Pilot Plant scale: usually 50-200 L
3. Industrial scale: usually 20000 to 2000000 L
• Time required to transition from lab-scale to
manufacturing is typically 3–10 years and a cost of
US $100 million to $1 billion
• Conditions of small-scale fermentation may not
work or may work poorly during scaling-up
optimization is needed 16
Optimization of Fermentation Conditions
(Highest Yield)
The used microorganism strains

Culture media, substrates, precursors and intermediates

pH

Agitation

Aeration

Temperature

Anti-foaming agent
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 Recovery of Fermentation Product
• Involves the purification of the required product which usually
represents a small fraction of the total fermentation medium
• Procedures:
1. Centrifugation 3. Filtration or
2. Precipitation sedimentation to
to separate cells
separate cells

4. Ultrafiltration 6. Reverse osmosis

5.Chromatography
and dialysis

7. Extraction with 8. Fractional

solvents distillation

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Main Categories of Fermentation Products
• 1. Biomass: product is the cells
themselves e.g. Baker’s yeast
• 2. Enzymes: e.g. amylase
• 3. Metabolites:
• Primary: produced during logarithmic
phase of growth such as citric acid
• Secondary: produced during stationary
phase of growth such as antibiotics,
alkaloids, or glycosides

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Main Categories of Fermentation Products
• 4. Biotransformation product: e.g. steroid
transformation
• Traditionally: cortisone was synthesized from deoxycholic acid in a
multi-step chemical process (31 steps) characterized by low mass yields
(0.16%) and high economic costs (200 $/g)
• The incorporation of a biotransformation step with Rhizopus spp.
markedly reduced the number of required chemical steps (11 steps) and
production costs (1$/g)
• 5. Immunological product:
• Vaccines
• Monoclonal antibodies
• 6. Genetically engineered therapeutic proteins:
• Insulin,
• Growth hormone
• Interferon
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Production of Metabolites By Industrial
Fermentation

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