Biotechnology
Raj Mutharasan
Dept of Chemical Engineering
Drexel University
Products of Biotechnology
Applications
Agriculture
Plant breeding to improve resistance to pests, diseases, drought and salt
conditions
Mass propagation of plant clones Bioinsecticide development modification of
plants to improve nutritional and processing characteristics
Chemical Industry
Production of bulk chemicals and solvents such as ethanol, citric acid, acetone
and butanol
Synthesis of fine specialty chemicals such as enzymes, amino acids, alkaloids
and antibiotics
Medicine
Development of novel therapeutic molecules for medical treatments
Diagnostics
Drug delivery systems
Tissue engineering of replacement organs
Gene therapy
Applications II
Food Industry
Production of bakers' yeast, cheese, yogurt and fermented foods such as
vinegar and soy sauce
Brewing and wine making
Production of flavors and coloring agents
Veterinary Practice
Vaccine production
Fertility control
Livestock breeding
Environment
Biological recovery of heavy metals from mine tailings and other industrial
sources
Bioremediation of soil and water polluted with toxic chemicals
Sewage and other organic waste treatment
History – Early Developments
1953
1970
1973
1975
1978
1978
1980
1981
1983
1984
Watson and Crick determined DNA structure
First restriction endonuclease isolated
Boyer and Cohen establish recombinant DNA methodology
Kohler and Milstein describe production of monoclonal
antibodies
Genentech produced human insulin in E. Coli
John Baxter reported cloning the gene for human growth
hormone
Kary Mullis(Cetus Corp.) conducted in vitro, the polymerase
chain reaction (PCR).
Genentech, Inc. cloned interferon gamma. Bill Rutter and Pablo
Valenzuela identify hepatitis B surface antigen. Scientists at
Ohio University produced the first transgenic animal.
Syntex received FDA approval for a monoclonal antibody based
diagnostic test for Chlamydia trachomatis.
Chiron Corp. announced the first cloning and sequencing of the
entire human immunodeficiency virus (HIV) genome
History – Middle Developments
1986
1987
1989
1990
1993
1994
Orthoclone OKT3® (Muromonab CD3) approved for reversal of
acute kidney transplant rejection. Automated DNA fluorescence
sequencer invented. First recombinant vaccine (for hepatitis) to
Chiron Corp approved. First genetically engineered crop, gene
altered tobacco plant approved.
Genentech received FDA approval to market rtPA (genetically
engineered tissue plasminogen activator) to treat heart attacks.
Recombivax HB® (recombinant hepatitis B vaccine) approved.
Epogen® (Epoetin alfa) a genetically engineered protein
introduced.
Approval for human somatic cell gene therapy trial. GenPharm
International, Inc. created the first transgenic dairy cow. The
Human Genome Project launched. Estimated cost: $13 billion.
Chiron's Betaseron is approved for multiple sclerosis.
Researchers clone human embryos and successfully nurture
them in a Petri dish for several days.
The BRCA1 gene reported to cause of non-inherited breast
cancers. Vpr, a gene governing reproduction of the HIV virus.
History – Recent Developments
1994
1995
1996
1997
1998
2000
The enzyme telomerase reported as responsible for the
unchecked growth of cells seen in human cancers.
Gene therapy, immune system modulation and genetically
engineered antibodies enter clinical trials.
Biogen's recombinant interferon drug. Avonex® approved for
multiple sclerosis.
Researchers at Roslin Institute report cloning a sheep named
Dolly from the cell of an adult ewe. Polly the first sheep cloned
by nuclear transfer technology bearing a human gene appears
later. A new DNA technique combines PCR , DNA chips, and
computer programming providing a a new tool in the search for
disease
causing genes.
Favorable results with a new antibody therapy against breast
cancer, HER2neu (Herceptin), herald a new era of treatment
based on molecular targeting of tumor cells.
A near complete draft of the human genome map is produced,
showing the locations of more than 30,000 genes.
An Overview
Market Values
Organic molecules, $5 - $10 / g
Biopolymers, $1 - $5 / g
Food & beverage additives, < $1 / g
Biopharmaceuticals, > $20,000 / g
5 mg hGH = $100
$100 / 0.005 g = $20,000 / g
(bulk diamonds = $10,000 / g)
Early Success
Cloning the human insulin gene (story)
Cloned by Genentech scientists in 1978
Licensed to Eli Lilly (why?)
First recombinant drug marketed, 1982
Product revenues from human insulin
(humulin) reached $839 million in 1996
(will exceed $1 billion for 1999)
Cloning Challenges
Heterologous gene expression
mRNA processing
Codon usage
Protein folding
Post-translational modifications
Inclusion bodies
Regulatory Challenges
Manufacturing reproducibility
Proof of product identity
Proof of product purity
Proof of product activity
Creation of CBER in FDA
Business Challenges
Protection of proprietary technology
(genetic material and manufacturing)
Large-scale cGMP manufacturing
Development timelines (7 - 10 years)
Cost of development ($300 - $500 mill)
Clinical trials
Industry Summary - 1999
No of companies - 1300
Employees – 155,000
Market Capitalization - $ 100 b
Sales = $13 b
Revenue = $18 b
R&D = 10b
Net Loss = 5 b
Human therapeutics = 75%
Medical diagnostics = 18%
Jobs in Biotechnology
Bioinformatics
Combinatorial chemistry
Corporate development
cGMP and validation
Large-scale cell culture
Process engineering and scale-up development
Protein purification and downstream processing
Regulatory affairs
Structure of Bacteria
Live cell pics at : http://www.cellsalive.com/
Bacteria – SEM view
With permission from: http://www.uq.edu.au/nanoworld/
Structure of an Animal Cell
Source: http://www.biosci.uga.edu/almanac/bio_103/notes/may_15.html.
Biochemicals of Life
Pairing of Nucleotides
Central Dogma
DNA makes RNA makes Protein
Fig 1 Single Strand of DNA
Genetic Code
Essential Cloning Steps
Essential Cloning Steps
Insert the DNA into plasmids. Gene of interest is inserted into small DNA molecules
known as plasmids, which are self-replicating, extrachromosomal genetic elements
originally isolated from the bacterium, Escherichia coli. The circular plasmid DNA is opened
using the same endonuclease that was used to cleave the genomic DNA.
Join the ends of DNA with the enzyme, DNA ligase. The inserted DNA is joined to the
plasmid DNA using another enzyme, DNA ligase, to give a recombinant DNA molecule. The
new plasmid vector contains the original genetic information for replication of the plasmid in
a host cell plus the inserted DNA.
Introduce the new vector into host. The new vector is inserted back into a host where
many copies of the genetic sequence are made as the cell grows and divide with the
replicating vector inside.
Isolate the newly-synthesized DNA, or the protein coded for by the inserted gene. The
host may even transcribe and translate the gene and obligingly produce product of the
inserted gene. Alternatively, many copies of the DNA gene itself may be isolated for
sequencing the nucleic acid or for other biochemical studies.
Amino Acids – Back bone of Proteins
Amino Acids – II
Process Overview
St ock Cu lt u r e >>
Sh a k e Fla s k s
St e r ilize
Fe r m e n t or &
M e d iu m
Se e d
Fe r m e n t or
P r od u ct ion
Fe r m e n t or
Ce ll
Se p a r a t ion
P r od u ct
P u r ifica t ion
What are cells made of?
Element
C
O
N
H
P
S
K
Na
Ca
Mg
Cl
Fe
others
% DW
50
20
14
8
3
1
1
1
0.5
0.5
0.5
0.2
0.3
A Good Approximation –
“Formula for Cell”
CH1.8O0.5 N 0.2
Concept : Growth Reaction
Growth Can be represented by:
Considering primary constituents:
In general:
Concept : Cell Yield
Experimental observation –
Cell mass is proportional
to available substrate
300
250
Slope = 7.2 µg/ml per mM
200
150
100
50
0
0
10
20
Glucose, mM
30
40
Cell Yield – Formal Definition
Cell Yield is:
Yx / s
Change in Cell Mass

Substrate Consumed
Yx / s
dX

dS
Similarly, product yield is defined as:
YP / S
dP

dS
Cell Growth in Batch Culture
Cell Growth - Kinetics
During log phase, growth kinetics is expressed as:
X  X0 e
t
ln( 2)
Doubling Time  t d 

Typical Growth Rates
Organism
E. coli
Yeast
Hybridoma
Insect Cells
Growth
Rate
µ [h-1]
2
0.3
0.05
0.06
Doubling
time
[h]
0.35
2.3
13.9
11.6
Nature of Specific Growth
Monod Kinetics
0.4
µ , 1/h
 mS

Ks  S
0.5
0.3
0.2
0.1
0
How does one experimentally
determine cell parameters?
Population Growth Rate?
0
5
10
15
20
25
S, g/ L
Monod Kinetics. Dependence of Growth Rate on
Limiting Substrate. Specific growth rate reaches a
maximum value of 0.5 h-1. Value of KS here is 0.5 g
L-1. Note that when S = 0.5 g L-1, µ is half of its
maximum.
Metabolic Quotients
1 g S + YO2/S g of O 2 + YNH3/S g of NH 3 = YX/S g o
YNH3/S g of NH 3 = YX/S g of Biomass + Y
CO2/S
rs
rO 2
rx


 1 Yx / s  YO 2 / s
CO2 + others
Metabolic Quotients - II
General Definition:
Organism
qglucose
qO2
g/(g h) g/(g h)
E. coli
2.5
0.3
Yeast
0.5
0.2
Hybridoma
0.2
0.02
What is your “food” metabolic quotient?
Factors Affecting Growth Rate - Temperature
3
39 o
2
42
o
37 o
h -1
o
45
30 o
Growth Rate
1
47
21 o

17 o
0.5
48
o
13.5
o
0.1
3.1
3.15
3.2
3.25
3.3
3.35
1,000/T(K)
3.4
3.45
3.5
3.55
Temperature range of life?
Factors Affecting Growth Rate - pH
1.1
0.9
With
Adaptation
0.8
0.7
m
Dimentionless Specific Growth Rate
1
0.6
0.5
Without
Adaptation
0.4
0.3
0.2
0.1
0
2
4
6
8
10
pH
Class discussion on adaptation
Factors Affecting Growth Rate - DO
Dimensionless Growth Rate
m
1
E-Coli
o
0.95
o
o
o
o
0.9
o o
o
0.85
0.8
o
o
o
o
0.75
0.7
0
0.1
0.2
0.3
D.O (mg/l)
0.4
Fish ?
How does discharge affects DO in rivers?
Cell or Product Concentration
Growth Associated Product Formation
Product expression
occurs as a
consequence of growth.
Product formation is
linked to energy
metabolism.
CELL, X
PRODUCT, P
Time
Example: alcohol
fermentation. Ethanol
is produced as cell
metabolizes substrate
sugar to derive energy,
and ethanol is a
byproduct of metbolism
Cell or Product Concentration
Non-Growth Associated Product Formation
Product expression
occurs as a product of
secondary metbolism,
and is not linked to
energy metabolism.
CELL, X
PRODUCT, P
Time
Example: Monoclonal
Antibody expression by
hybridoma.
Oxygen Transfer in Bioreactors
Volumetric oxygen transfer rate in a sparged bioreactor is:
Oxygen balance over reactor:
O2 transfered from Gas Phase - O2 consumed by Cells = Accumulation
Oxygen Transfer in Bioreactors-II
Oxygen Transfer Dynamics
If supply of oxygen is balanced exactly by metabolic oxygen demand, then
How does one measure oxygen transfer coeff.?
Oxygen Transfer Coefficient
When cells are absent:
If supply of oxygen is shut off, then
Solution:
O2 Off
O2 on
CL
time
A Design Example
You are part of a tech service team asked to evaluate if the available 10,000 liter
fermentor is adequate to produce 10 kg/day of a recombinant protein using a strain
of E. coli that expresses the protein as 20 % of cellular protein. In order to enhance
plasmid stability, the nutrients are manipulated to give a low specific growth rate is
0.2 h-1. The oxygen demand is 0.15 g O2/g cell - h. Assume that the recombinant
protein formation is cell growth associated.
Data: The lag phase is 4 hours. Typical clean-up time following a fermentation
batch and preparation for the next batch is 8 hours. The plant runs three shifts.
Cell yield on substrate is 0.55 g cell/g substrate. Available support services can
supply inoculum of a maximum of 6 kg of cells every 24 hour period. Maximum
KLa for the available fermentor is 500 h-1. Fermentor accessories are capable of
handling cell concentrations of 60 g/L. Assume any other parameters you need to
complete the calculation.
Class discussion of solution
Biotechnology – The next steps
This module gives a basic introduction to principles of
biotechnology and biochemical engineering.
With bit more background in biology, you can pursue
further reading in gene therapy, human genomics,
tissue engineering, cancer diagnostic tools, use of
biotechnology in crime detection, and a whole score of
other applications.