Lecture 2 introduction to bioprocess
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Introduction to Bioprocess 12 th . July 2010 CEPP, UTM Skudai, Johor Prof. Dr. Hesham A. El Enshasy Faculty of Chemical Engineering CEPP, UTM, Skudai, Malaysia
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Transcript of Lecture 2 introduction to bioprocess
Introduction to Bioprocess
12th. July 2010CEPP, UTM Skudai, Johor
Prof. Dr. Hesham A. El EnshasyFaculty of Chemical Engineering
CEPP, UTM, Skudai, Malaysia
The oldest Known Biotech. Protocol for Yeast production
Bread and Beer Manufacturing Process, the 5th. Dynastry (ca 2400 BC) Leiden Egyptian Museum, Holland
Chronology Milestones
6000 BC Brewing (Sumeria, Babylonia)
2400 BC The first bioprocess complete description (ancient Egyptian)
1680 Yeast under the microscope (van Leeuwenhoek)
1835 Alcoholic fermentation associated with yeast
1857 Fermentation correlated with metabolism (Pasteur)
1877 Term “enzyme” (in yeast) introduced (Kühne)
1923 Industrial production of Citric acid
1930s Industrial production of Amino acids
1940s Industrial production of Antibiotics
1979 Monoclonal antibody production by hybridoma cell
1982 Industrial Production of rHuman Insulin in E. coli (Eli Lily)
1984 First commercial production of therapeutic MAb (Anti CD3)
1994 First commercial vaccine from recombinant yeast (Hepatitis B vaccine)
1996 Completion of the yeast genome project for S. cerevisiae.
2000 10 m3 STR for Mammalian cell culture
2002 Disposable bioreactors were used in industrial scale
2007 The Bioprocess products market exceeded 700 Billion US$. Only the Biopharmaceuticals market reached 70 Billion US$. Mammalian cell culture products reached 25 Billions US$
Milestones of Bioprocess Industry Development
Industrial BioprocessingVaccine
Biopharmaceuticals
Solvents
Baker’s yeast
Organic acids
Amino acids
Antibiotics Enzymes
biopolymers
rProteins MAb
SCP Biosurfactants
Probiotics
Plant Bioactivecompounds BioDiesel X
Pre
1940
sPr
e 19
80s
Post
198
0s
Some of Major Industrial Fermentation Products
Product Annual productionMetric Tons
MainApplication
Microorganism
Citric acid 1,200,000 Food A. niger
Ethanol 26,000,000 Fuel S. cerevisiae
Glutamate 1,000,000 Flavoring C. glutamicum
Lactic acid 400,000 Food, plastics Lactobacillus sp.
Lysine 800,000 Feed C. glutamicum
Penicillin 60,000 Pharmaceutical
P. chrysogenum
Xanthan Gum 100,000 Food, oil drilling
X. campestris
Ref. Bioprocessing-from Biotechnology to Biorefinery (S.T. Yang, Ed.), Elsevier Press, 2007.
Stage Main product Vessels Process control Culture method Quality control Pilot Plant facilities Strain selection
Pre 1900 -Alcohol
-Vinegar
Wooden vessels and
copper used later
Use of thermometer ,
hydrometer
Batch Virtually
Nil
Nil Pure yeast and
bacterial cultures
1900-1940 -Backer´s yeast
-Glycerol
-Citric acid
-Lactic acid
Vessels up to 200 m3
Mechanical stirring
in small vessels
Temp control
pH control
Batch
Fed-batch
Virtually
Nil
Virtually
Nil
Pure cultures
1940-date -Antibiotics
-Amino acids
-Transformation
-Enzymes
Stirred tank
bioreactor (true
bioreactor)
Sterilizable pH and
DO electrodes
Batch
Fed-batch
Continuous
Very important Becomes common Mutation and
selection programms
1964-date Single cell protein Pressure cycle and
pressure jet vessels
development
Use of computer
linked control loop
Continuous culture
with medium
recycling
Very important Very important Genetic Engineering
of producer strain
attempted
1979-date Production of
heterologous proteins
by microbial, animal
cells, monoclonal
antibodies
More development
in STR bioreactor
design, Hollow fiber
bioreactor, Animal
cell bioreactor
More development
in sensors and
Control system.
Immobiliezed cells
Animal cell process
Very important Very important Introduction of
foreign genes into
microbial and animal
hosts
1985-date More heterologous
protein production of
growth factors
More development
in bioreactor design
and agitation
systems. Animal cell
bioreactor
On line control,
autoclavable
biosensors
High cell density
cultivation
Very important Very important Utilization of fungal
cells as host for
heterologous protein
production
1995-date Increase in
heterologous protein
production using
different cell factories
More development
in traditional
cultivation vessels
and new vessels
New era in online
control sensors
(replacable during
process), biosensors
High cell density,
perfusion culture
Very important Very important More cell factories
used such as
invertebrate cells
and insect cells
Stages of Industrial Biotechnology from 1900 to date
Bioprocess Development
Industrial Bioprocessing: Microbial cells
Bioprocess Optimization in Lab. scale(Development of cultivation strategy to reach the maximal productivity)
Scaling up of the Process
Down Stream (Separation/Isolation/Purification and increase of product Stability)
Determination of process bottleneck(s)
CELL
Oxygen
Carbon and Energy
Sources
Nitrogen Source
Other requirements(P, S,Na,K,Mg,etc…)
Carbon dioxide
Biomass
Metabolite(s)
Water
Heat
Substrate Input and Output
General Requirements for Medium Composition
It will Produce the maximal yield of product(s) or biomass per gram of substrate used(High Yield Coefficient)
It will produce the maximal concentration of product or biomass(High volumetric production)
It will permit the maximal yield of product formation (Maximal productivity)
There will be the minimal yield of undesirable products
It will be of consistent quality and be readily available throughout the year
It will cause minimal problems during media preparation and sterilization
It will cause minimal problems in other aspects of the production process particularlyAeration and agitation, extraction and waste treatment
Medium component
DefinedComponent
Un-defined Component
Carbon source (Glucose,Fructose,Glycerol,xylose)
(Sucrose, Starch)
Molasses, Meat extract, Peptone, Plant extracts and Materials (Cellulosic, lignoncellulosic and hemicellulosic materials, Starch complex, etc…)
Nitrogen source Ammonium and Nitrate Salts Yeast extract, Amino acid complex, Casein
Phosphate Mono and di-phosphate salts In traces of complex C- and N-sources
Sulphur Ammonium and Magnesium sulphate
In traces of complex C- and N-sources
Magnesium Mainly Magnesium sulphate In traces of complex C- and N-sources
Mn, Mo, Fe, Zn, etc…
In form of Inorganic salts In traces of complex C- and N-sources
Vitamin and Growth factors
Added in pure form of vitamin and growth factors preparation
Yeast extract, and may found also as traces in some C- and N-sources
Cultivation Media
Element % (w/w) of cell dry weight
Bacteria Yeast Filamentous fungi
Carbon 46-52 46-52 45-55
Nitrogen 10-14 6-9 4-7
Phosphorus 2-4 0.8-2.6 0.4-4.5
Sulphur 0.2-1.0 0.01-0.25 0.1-0.5
Magnesium 0.1-0.5 0.1-0.5 0.1-0.3
Sodium 0.5-1.0 0.01-0.1 0.02-0.5
Calcium 0.01-1.1 0.1-0.3 0.1-1.4
Iron 0.02-0.2 0.01-0.5 0.1-0.2
Manganese 0.001-0.01 0.0005-0.007 -
Molybdenum- - 0.0001-0.0005 -
Overviews on the elemental composition of the microorganisms
From Petri-dish to Bioreactor
What can we use Petri dish for
Strain Isolation and Identification work
As first step for inoculum propagation
Sterility testing
Rapid screening for certain microbial metabolites
Antimicrobial sensitivity testing
Short term strain preservation
What can we use Shake flask for
Primary production of certain metabolites
Genetic material preparation (cell mass production for DNA isolation)
Medium optimization (C-, N-, P- and elements-sources and concentrations)
Cultivation conditions optimization (Temperature, pH)
Primary understanding of oxygen and mixing requirements through the change in (Shaking intensity, Shaking eccentricity, working volume and polymer addition)
Primary data for Growth and product formation kinetics
Bacteria
Actinomycetes
Fungi
Algae
Plant cell
Insect cells
Invertebrate cells
Mammalian cells
Color Anthocyanins V. vinifera Betalaines B. vulgaris Crocetins Gardenia jasminoides Anthraquinones Cinchon ledgeriana
Essential oils Mint oil Mentha piperata Chamomile oil Ma. Chamomilla Jasmine oil Jasmine officinale Anissed oil Pim. anisum
Flavors Vanillin Va. Planifolia Garlic Allium sativum Onion Allium cepa Basmati Oryza sativa Citrus Citrus spp. Cocoa flavour Theobromo cacao
Sweeteners Stevioside Stevia rebaudiana Glycyrrhizin Glycyrrhiza glabra Thaumatin Thaumatococcus danielli
Pharmaceuticals Hepato-protective Ligustrum robustum Anti-oxidant Artemisia judaica Anti-inflammatory Harpagophytum procumbens Anti-cancer Catharanthus roseus Anti-bacterial Ficus microcarpa Anti-fungal Backhousia citriodora Anti-ulcer Different plants
Recombinant products Therapeutic proteins hGM-CSF HBsAg Interleukin-12 Ginseng h-lactoferrin vaccine production MAb
New Process for Plant Metabolites Production
It is independent of geographical and seasonal variations and various environmental factors.
It offers a defined production system, which ensure continuous supply of products, uniform quality and yield
It is possible to produce novel compounds that are not normally found in parent plant.
It is independent of political interface
Efficient downstream recovery with low cost and minimum number of steps.
High efficient production rate with significant short production time
Fully Compliance to cGMP requirements for Biopharmaceuticals
Why Plant Cell Bioreactor ?
PhotoBioreactor
- Plant cells
- Algal cells
Cells as an end product Cells-derived product
Artificial skin Growth factors
Artificial organ-Hepatocyte (liver)-Beta-islet cells (pancreas)
Hormones-Human growth hormones-Insulin
Bone Marrow Interferons
Lymphocytes Monoclonal antibodies
Different levels for mammalian cells / insect cells cultivation
Scaling up
Different levels of cells cultivation
Small scale (T-flask, 24 well)
Spinner flask Rolling bottles
Bioreactor Level(STR, Air-Lift, Hollow fiber)
Shea
r Str
ess
Mix
ing
Cell
Prod
uctiv
ity Non-Optimized
Semi-Optimized
Fully-Optimized
Since mid-1950s no majorChange in STR Engineering
Change mainly in:
- Sampling system- Valves- Material Finish
- Sensors (on-line, in-line and off-line)
- Control system
Bioreactor: The heart of Industrial Bioprocessing Facility
Feed pump(s)
Temperature
Aeration
Power consumption
Stirrer Speed
Exhaust gasAnalyzer
pH
DO
Pressure
Weight / volume
Measurement and open or closed loop control
Measurement only
Common measurement and control of bioreactors as generally accepted as routine equipment
Bioprocess controlAll software and control/recording system must be cGMP approved and validated
Manufacturing Cost in Bioprocess Industries
1. Product
2. Biofactory
3. Expression system
4. Bioprocess
1.1 New Product discovery from different cell factories 1.2 Over-expression of certain metabolites (Pathways Engineering)1.3 Production of new recombinant products1.4. MAb revolution will continue
2.1 Discovery of new biofactories 2.2 Progress research in the omics approach for better pathways engineering
3.1 Development of stronger expression systems 3.2 Process debottle-necking by genetic new approaches
4.1 New economic media formulations (especially for higher Eukaryotic cells)4.4 New Bioprocess design4.5 Improved sensors and control system4.6 Improvement in Downstream separation process (more solvent free processes)
Thank You
