Biotechnology

Raj MutharasanDept 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

EnvironmentBiological 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 Watson and Crick determined DNA structure1970 First restriction endonuclease isolated1973 Boyer and Cohen establish recombinant DNA methodology1975 Kohler and Milstein describe production of monoclonal

antibodies1978 Genentech produced human insulin in E. Coli1978 John Baxter reported cloning the gene for human growth

hormone1980 Kary Mullis(Cetus Corp.) conducted in vitro, the

polymerase chain reaction (PCR). 1981 Genentech, Inc. cloned interferon gamma. Bill Rutter and

Pablo Valenzuela identify hepatitis B surface antigen. Scientists at Ohio University produced the first transgenic animal.

1983 Syntex received FDA approval for a monoclonal antibody based diagnostic test for Chlamydia trachomatis.

1984 Chiron Corp. announced the first cloning and sequencing of the entire human immunodeficiency virus (HIV) genome

History – Middle Developments

1986 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.

1987 Genentech received FDA approval to market rtPA (genetically engineered tissue plasminogen activator) to treat heart attacks. Recombivax HB® (recombinant hepatitis B vaccine) approved.

1989 Epogen® (Epoetin alfa) a genetically engineered protein introduced.

1990 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.

1993 Chiron's Betaseron is approved for multiple sclerosis. Researchers clone human embryos and successfully nurture them in a Petri dish for several days.

1994 The BRCA1 gene reported to cause of non-inherited breast cancers. Vpr, a gene governing reproduction of the HIV

virus.

History – Recent Developments

1994 The enzyme telomerase reported as responsible for the unchecked growth of cells seen in human cancers.

1995 Gene therapy, immune system modulation and genetically engineered antibodies enter clinical trials.

1996 Biogen's recombinant interferon drug. Avonex® approved for multiple sclerosis.

1997 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.

1998 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.

2000 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

ProductionFermentor

SterilizeFermentor &Medium

SeedFermentor

Stock Culture >>Shake Flasks

CellSeparation

ProductPurification

What are cells made of?

C 50O 20N 14H 8P 3S 1K 1Na 1Ca 0.5Mg 0.5Cl 0.5Fe 0.2

Element % DW

others 0.3

A Good Approximation –“Formula for Cell”

2.05.08.1 NOCH

Concept : Growth ReactionGrowth Can be represented by:

Considering primary constituents:

In general:

Concept : Cell Yield

Glucose, mM

0

50

100

150

200

250

300

0 10 20 30 40

Slope = 7.2 µg/ml per mM

Experimental observation –

Cell mass is proportionalto available substrate

Cell Yield – Formal Definition

Similarly, product yield is defined as:

dS

dXY s/x

Cell Yield is:

ConsumedSubstrate

MassCellinChangeY s/x

dS

dPY S/P

Cell Growth in Batch Culture

Cell Growth - Kinetics

During log phase, growth kinetics is expressed as:

t0 eXX

)2ln(tTimeDoubling d

Typical Growth Rates

Growth Rate

Doubling time

µ [h-1] [h]E. coli 2 0.35Yeast 0.3 2.3Hybridoma 0.05 13.9Insect Cells 0.06 11.6

Organism

Nature of Specific Growth

SK

S

s

m

S, g/ L

µ ,

1/h

0

0.1

0.2

0.3

0.4

0.5

0 5 10 15 20 25

Monod Kinetics

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.

How does one experimentallydetermine cell parameters?

Population Growth Rate?

Metabolic Quotients

1 g S + YO2/S g of O2 + YNH3/S g of NH3 = YX/S g of Biomass + YCO2/S CO2 + others

1 g S + YO2/S g of O2 + YNH3/S g of NH3 = YX/S g of Biomass + YCO2/S CO2 + others

s/2O

2O

s/x

xs

Y

r

Y

r

1

r

Metabolic Quotients - II

General Definition:

Organism qglucose qO2

g/(g h) g/(g h)E. coli 2.5 0.3Yeast 0.5 0.2Hybridoma 0.2 0.02

What is your “food” metabolic quotient?

Factors Affecting Growth Rate - Temperature

0.1

1

Gro

wth

Rat

e

3.1 3.15 3.2 3.25 3.3 3.35 3.4 3.45 3.5 3.55

1,000/T(K)

47

45

42

3937

30

21

17

13.5

oo

o

o o

o

o

o

48o0.5

2

3

h-1

Temperature range of life?

Factors Affecting Growth Rate - pH

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1D

imen

tion

less

Sp

ecif

ic G

row

th R

ate

2 4 6 8 10

pH

WithAdaptation

WithoutAdaptation

m

Class discussion on adaptation

Factors Affecting Growth Rate - DO

How does discharge affects DO in rivers?

0.7

0.75

0.8

0.85

0.9

0.95

1

Dim

ensi

onle

ss G

row

th R

ate

0 0.1 0.2 0.3 0.4D.O (mg/l)

o

o

oo

o

oo

o

oo o o

E-Coli

m

Fish ?

Growth Associated Product Formation

Time

PRODUCT, P

CELL, X

Cel

l or

Pro

duct

Con

cent

rati

onProduct expression occurs as a consequence of growth. Product formation is linked to energy metabolism.

Example: alcohol fermentation. Ethanol is produced as cell metabolizes substrate sugar to derive energy, and ethanol is a byproduct of metbolism

Non-Growth Associated Product Formation

Time

CELL, X

PRODUCT, P

Cel

l or

Pro

duct

Con

cent

rati

on

Product expression occurs as a product of secondary metbolism, and is not linked to energy metabolism.

Example: Monoclonal Antibody expression by hybridoma.

Oxygen Transfer in Bioreactors

Volumetric oxygen transfer rate in a sparged bioreactor is:

O2 transfered from Gas Phase - O2 consumed by Cells = Accumulation

Oxygen balance over reactor:

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

CL

time

O2 on

O2 OffSolution:

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.