Biotechnology Raj Mutharasan

Dept of Chemical Engineering Drexel University

Products of Biotechnology

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

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

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

) conducted in vitro. the polymerase chain reaction (PCR). Chiron Corp. Scientists at Ohio University produced the first transgenic animal. Inc. announced the first cloning and sequencing of the entire human immunodeficiency virus (HIV) genome .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. Syntex received FDA approval for a monoclonal antibody based diagnostic test for Chlamydia trachomatis. Bill Rutter and Pablo Valenzuela identify hepatitis B surface antigen. cloned interferon gamma. Genentech. Coli John Baxter reported cloning the gene for human growth hormone Kary Mullis(Cetus Corp.

Recombivax HB® (recombinant hepatitis B vaccine) approved. Genentech received FDA approval to market rtPA (genetically engineered tissue plasminogen activator) to treat heart attacks. First genetically engineered crop. The Human Genome Project launched. . created the first transgenic dairy cow. First recombinant vaccine (for hepatitis) to Chiron Corp approved. a gene governing reproduction of the HIV virus. Chiron's Betaseron is approved for multiple sclerosis. Automated DNA fluorescence sequencer invented. Epogen® (Epoetin alfa) a genetically engineered protein introduced. Estimated cost: $13 billion. The BRCA1 gene reported to cause of non-inherited breast cancers. gene altered tobacco plant approved. Researchers clone human embryos and successfully nurture them in a Petri dish for several days. Approval for human somatic cell gene therapy trial. Inc. Vpr.History 1986 Middle Developments 1987 1989 1990 1993 1994 Orthoclone OKT3® (Muromonab CD3) approved for reversal of acute kidney transplant rejection. GenPharm International.

Biogen's recombinant interferon drug. showing the locations of more than 30. Gene therapy. and computer programming providing a a new tool in the search for disease causing genes. immune system modulation and genetically engineered antibodies enter clinical trials. herald a new era of treatment based on molecular targeting of tumor cells. . Avonex® approved for multiple sclerosis. Polly the first sheep cloned by nuclear transfer technology bearing a human gene appears later. A near complete draft of the human genome map is produced. A new DNA technique combines PCR . Favorable results with a new antibody therapy against breast cancer.000 genes. HER2neu (Herceptin).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. DNA chips. Researchers at Roslin Institute report cloning a sheep named Dolly from the cell of an adult ewe.

An Overview .

> $20.005 g = $20.000 / g (bulk diamonds = $10. $1 . < $1 / g Biopharmaceuticals.$10 / g Biopolymers.000 / g) . $5 .000 / g 5 mg hGH = $100 $100 / 0.Market Values Organic molecules.$5 / g Food & beverage additives.

1982 Product revenues from human insulin (humulin) reached $839 million in 1996 (will exceed $1 billion for 1999) .Early Success Cloning the human insulin gene (story) Cloned by Genentech scientists in 1978 Licensed to Eli Lilly (why?) First recombinant drug marketed.

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 .

$500 mill) Clinical trials .Business Challenges Protection of proprietary technology (genetic material and manufacturing) Large-scale cGMP manufacturing Development timelines (7 .10 years) Cost of development ($300 .

1999 No of companies .000 Market Capitalization .Industry Summary .1300 Employees ± 155.$ 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 : .

au/nanoworld/ .uq.Bacteria ± SEM view With permission from:

edu/almanac/bio_103/notes/may_15.uga.biosci.html.Structure of an Animal Cell Source: http://www. .

Biochemicals of Life .

Pairing of Nucleotides .

Central Dogma DNA makes RNA makes Protein Fig 1 Single Strand of DNA .

Genetic Code .

Essential Cloning Steps .

which are self-replicating. The host may even transcribe and translate the gene and obligingly produce product of the inserted gene. 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. Isolate the newly-synthesized DNA. DNA ligase. Alternatively.Essential Cloning Steps Insert the DNA into plasmids. or the protein coded for by the inserted gene. The inserted DNA is joined to the plasmid DNA using another enzyme. . DNA ligase. 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. The new plasmid vector contains the original genetic information for replication of the plasmid in a host cell plus the inserted DNA. Gene of interest is inserted into small DNA molecules known as plasmids. to give a recombinant DNA molecule. extrachromosomal genetic elements originally isolated from the bacterium. Introduce the new vector into host. Escherichia coli. 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 .

2 .5 0.2 0.5 0.8O 0.5 0.5 N 0.3 A Good Approximation Formula for Cell CH 1.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.

Concept : Growth Reaction Growth Can be represented by: Considering primary constituents: In general: .

2 µg/ml per mM .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.

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 .

growth kinetics is expressed as: X ! X0 e Qt ln(2) Doubling Time ! t d ! Q .Cell Growth .Kinetics During log phase.

6 .05 0.3 0.06 Doubling time [h] 0.9 11.3 13.Typical Growth Rates Organism E. coli Yeast Hybridoma Insect Cells Growth Rate µ [h-1] 2 0.35 2.

Nature of Specific Growth Monod Kinetics 0.3 0. µ is half of its maximum. g/ L Population Growth Rate? Monod Kinetics. Dependence of Growth Rate on Limiting Substrate.2 0. .4 Q mS Q! Ks  S How does one experimentally determine cell parameters? 0.5 g L-1. Note that when S = 0. Value of KS here is 0.5 g L-1.1 0 0 5 10 15 20 25 S.5 h-1. Specific growth rate reaches a maximum value of 0.5 0.

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 .

02 What is your food metabolic quotient? .2 0.2 Hybridoma 0.Metabolic Quotients .5 0.5 0. coli 2.II General Definition: Organism qglucose qO2 g/(g h) g/(g h) E.3 Yeast 0.

2 3.35 3.3 3.1 3.1 o 3.5 3.4 3.Factors Affecting Growth Rate .5 48 o o 13.Temperature 3 39 o 2 42 37 o o o h -1 45o 1 30 Growth Rate 47 3 21 o 17 0.55 1.000/T(K) Temperature range of life? .25 3.45 3.5 0.15 3.

Factors Affecting Growth Rate .2 0.8 0.6 0.3 0.1 0 2 4 6 8 10 Without Adaptation With Adaptation pH Class discussion on adaptation .7 QQm 0.5 0.4 0.9 0.1 Dimentionless Specific Growth Rate 1 0.pH 1.

DO 1 QQm E-Coli 0.3 D.8 0.95 0.Factors Affecting Growth Rate .7 0 0.75 0.1 o o o o o o o o o o Dimensionless Growth Rate o o 0.9 0.O (mg/l) 0.4 Fish ? How does discharge affects DO in rivers? .85 0.2 0.

Ethanol is produced as cell metabolizes substrate sugar to derive energy. X PRODUCT. and ethanol is a byproduct of metbolism Cell or Product Concentration CELL. Product formation is linked to energy metabolism.Growth Associated Product Formation Product expression occurs as a consequence of growth. Example: alcohol fermentation. P Time .

and is not linked to energy metabolism.Non-Growth Associated Product Formation Cell or Product Concentration CELL. P Example: Monoclonal Antibody expression by hybridoma. PRODUCT. Time . X Product expression occurs as a product of secondary metbolism.

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.? .

then Solution: O2 Off O2 on CL time .Oxygen Transfer Coefficient When cells are absent: If supply of oxygen is shut off.

the nutrients are manipulated to give a low specific growth rate is 0.55 g cell/g substrate.15 g O2/g cell .h. 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. Data The lag phase is 4 hours. Cell yield on substrate is 0. The plant runs three shifts. Typical clean-up time following a fermentation batch and preparation for the next batch is 8 hours. coli that expresses the protein as 20 % of cellular protein. Class discussion of solution .000 liter fermentor is adequate to produce 10 kg/day of a recombinant protein using a strain of E.A Design Example You are part of a tech service team asked to evaluate if the available 10. In order to enhance plasmid stability. Fermentor accessories are capable of handling cell concentrations of 60 g/L. The oxygen demand is 0. Assume that the recombinant protein formation is cell growth associated. Assume any other parameters you need to complete the calculation.2 h-1.

. use of biotechnology in crime detection. you can pursue further reading in gene therapy. cancer diagnostic tools. With bit more background in biology. human genomics. and a whole score of other applications.Biotechnology ± The next steps This module gives a basic introduction to principles of biotechnology and biochemical engineering. tissue engineering.

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