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
1953 1970 1973 1975 1978 1978 1980 1981 1983 1984

Early Developments

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
1986

Middle Developments

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
1994 1995 1996 1997

Recent Developments

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 Se e d Fe r m e n t or

St e r ilize Fe r m e n t or & M e d iu m

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

CH 1.8O 0.5 N 0.2

Concept : Growth Reaction

Growth Can be represented by:

Considering primary constituents:

In general:

Concept : Cell Yield

Experimental observation
300

Cell mass is proportional to available substrate

250 200 150 100 50 0 0 10 20 Glucose, mM 30 40 Slope = 7.2 g/ml per mM

Cell Yield Formal Definition

Cell Yield is:

Yx / s

Change in Cell Mass ! Substrate Consumed Yx / s dX ! dS dP ! dS

Similarly, product yield is defined as:

YP / S

Cell Growth in Batch Culture

Cell Growth - Kinetics

During log phase, growth kinetics is expressed as:

X ! X0 e

Qt

ln(2) Doubling Time ! t d ! Q

Typical Growth Rates

Organism E. coli Yeast Hybridoma Insect Cells

Growth Rate [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
0.5 0.4

Q mS Q! Ks  S
How does one experimentally determine cell parameters?

0.3 0.2 0.1 0 0 5 10 15 20 25 S, g/ L

Population Growth Rate?

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, 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 of

CO2/S

+ YNH3/S g of NH 3 = YX/S g of Biomass + Y

CO2 + others

rs rO 2 rx ! !  1 Yx / s  YO 2 / s

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 37

o o

h -1

45o
1

30

Growth Rate

47

21

17 0.5 48

13.5
0.1

3.1

3.15

3.2

3.25

3.3

3.35

3.4

3.45

3.5

3.55

1,000/T(K)

Temperature range of life?

Factors Affecting Growth Rate - pH

1.1

Dimentionless Specific Growth Rate

1 0.9 0.8 0.7 QQm 0.6 0.5 0.4 0.3 0.2 0.1 0 2 4 6 8 10 Without Adaptation With Adaptation

pH

Class discussion on adaptation

Factors Affecting Growth Rate - DO

1

QQm

E-Coli

0.95 0.9 0.85 0.8 0.75 0.7 0 0.1

o o o o o o

o o

Dimensionless Growth Rate

o o

0.2 0.3 D.O (mg/l)

0.4

Fish ?

How does discharge affects DO in rivers?

Growth Associated Product Formation

Product 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

Cell or Product Concentration

CELL, X

PRODUCT, P

Time

Non-Growth Associated Product Formation

Cell or Product Concentration

CELL, X

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

Example: Monoclonal Antibody expression by hybridoma.

Time

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.