21st Century Manufacturing Episode 2 Part 11 pptx

For example, fermentation takes place in a bioreactor: it is a process of growing incu-bating microorganisms on a substrate containing carbon and nitrogen the "food" tures" algae or pond

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Agar contains Two pure cultures containing tetI1Bcline cloned lab;;:DNA F1gure 9.24Procedures for obtaining pure clones containing rabbit DNA: (8) plasmid DNA with resistance to tetracycline and ampicillin is mixed with rabbit

eDNA; (b) DNA ligase is added to join the DNA fragments; (e) E coli cells act as

host; (d) plasmid-containing cells are selected by growth on agar containing

tetracycline, and cells with plasmids joined to rabbit eDNA are identified by screening on ampicillin-containing agar; (e) and(f)these cells grow only on tetracycline;(g)pure cultures containing cloned genes (from Understanding DNA and Gene Claning by Drlica, Copyright © 1992, Reprinted by permission of John

Wiley & Sons, Inc.}

Rabbit DNA

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The eDNA could then be isolated, made single-stranded (typically by boiling), and radioactively labeled

9.8.7 Step 5: Final Screening by Nucleic Acid Hybridization

to Isolate Genomic Clones (Bottom of Figure 9.231

The final step is to screen the colonies grown from the phages containing the orig-inal fragmented rabbit DNA and to find which ones have the desired hemoglobin genes (recall this is on the right side of Figure 9.23).The DNA in each plaque is tested

to see if it will hybridize with the radioactive hemoglobin eDNA nucleic acid com-plement to the hemoglobin genes being sought The technique relies on the principle

of complementary base pairing between the cDNA probes and the desired hemo-globin genes in those very few plaques described in Step 2

Practical Techniques

After the phages were used to growE colicolonies on an agar plate, a piece of filter paper was placed on the agar and removed The cells were thus transferred to the paper, which was placed in a dilute solution of sodium hydroxide (lye or caustic soda) The first key feature was that the sodium hydroxide caused the hemoglobin DNA to become stranded The second key feature was that when the single-stranded,radioactive eDNA was next added, complementary base pairs formed only

if the filter-paper-bound DNA contained the likewise single-stranded hemoglobin DNA gene being sought The radioactive cDNA bound to the filter paper, identifying the locations of the rabbit hemoglobin gene being sought The filter paper was next washed to remove any radioactive probes that were not base paired X-ray film was then used to find bacterial colonies containing the hemoglobin gene of interest This procedure resulted in a pure culture ofE coliin which each cell contained a phage into which the hemoglobin gene of interest had been cloned

In summary, the cDNA radioactive probes derived from mRNA were used to probe the plaques formed from the bacteria-containing phage, which contained rabbit DNA, and eventually isolate the desired genomic clones of hemoglobin 9.9 BIOPROCESS ENGINEERING

9.9.1 Bloreactors

A bioreactor is a container or vessel in which a biological reaction occurs For example, fermentation takes place in a bioreactor: it is a process of growing (incu-bating) microorganisms on a substrate containing carbon and nitrogen (the "food" tures" algae or pond scum

Bioreactors can range from small bench-top fermentors holding 11iter to larger l-miltion-liter production units In addition to producing various food products, bioreactors are used to generate industrial chemicals, enzymes, and biofuels Using poor countries In Finland, for example, baker's yeast has been used in a bioelectro-chemical device in which the bioelectro-chemical process of substrate oxidation-reduction

The biosynthesis process, as well as the resulting relationship between cell growth and bioproduct formation, differs according to the type of bioreactor Two conventional methods are batch and continuous culture fermentation Bioconver-sions can also take place on moist but solid substrates

A primary task in biotechnology processing is to design, operate, and control bioreactors such that conversion rates and yields are economically feasible Another challenge is keeping cells and catalysts alive as they are put through various mashing, only be conversant with process development, equipment design, and scale-up but also understand what is needed to keep organisms viable and growing at an optimal rate

A common type of bioreactor is a mechanically stirred tank, which utilizes a three-phase (gas-solid-liquid) reaction In this type of device, gas is sparged into the

by a mechanical stirrer (Figure 9.25) There are strict constraints in su.ch a proceS& For example, a steady supply of oxygen gas bubbles for aerobic fermentations is crit-ical Stirring must be rapid enough to disperse gas bubbles, develop a homogenous liquid, and ensure a solid suspension Overstirring tends to shear cells, while under-stirring may asphyxiate them Another challenge is optimizing heat removal rates; to-volume ratios decrease, reducing the rate of heat removal

Sterility is another major challenge Processes must be absolutely aseptic Elim-ination of unwanted organisms is required to ensure product quality and to prevent contaminating organisms from displacing the desired production strain This creates significant design and operation difficulties, particularly in combination with other temperature sensors that can stand up to repeated sterilizations

The molecular reactions inside the bioreactor govern the growth charecteris-tics of cells Cell growth patterns depend on a variety of factors, including oxygen availability, nutrient supply, pH, temperature, and population density In a typical batch fermentation, cell growth follows four distinct phases: lag, exponential, sta-the cell restructures its biosynsta-thetic mechanisms to take account of sta-the environment

In the exponential phase, the cell grows as quickly as possible given these factors (In industrial bioprocessing, this phase is typically measured in terms of the time required to double the concentration of cells, known as a biomass.) Exponential growth ceases for a variety of reasons, including nutrient depletion, physical over-crowding, or buildup of by-products of the metabolic process There follows a sta-ribosomes, are degraded to create other enzymes or supply fuel for cell maintenance When internal energy sources are depleted, the cell is unable to carry out basic func-tions The result may be cell lysis (breakage) or inviability (inability to reproduce)

In the depletion phase, the biomass reduces (Figure 9.26)

Optimizing bioreactor functionality involves a number of discrete process variables, including aeration, agitation, mass and heat transfer, measurement and control, cell metabolism, and product expression and preparation of inoculates

Motor drive

Foam breaker

Cooling coils

Baffle plates

Figure 9.25A mechanically stirred bioreactor (adapted from Shuler, 1992)

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~ssemblies

Gas exit

Flat blade turbines

Sterile air inlet Sparger

Time (hours) Figure 9.26 Batch fermentation growth curve.

fermentation process is one area of research One key goal is to be able to correctly identify process problems and correct them online For a fermentation process, this includes sensor, equipment, and process failure monitoring Control and batch maintenance are also important candidates for automation

9.9.2Postprocessing

The bioreactor phase is the heart of the bioprocess However, the recovery and purification of a fermentation product are essential to any commercial process The degree of difficulty in the recovery and purification process depends heavily on the cessing includes filtration, crystallization, and drying techniques Packaging and ship-ping the product are also important postprocessing activities

9.'0 MANAGEMENT OF TECHNOLOGY

9.10.1 Present Trends

Significant advances have been made in a very short time, and these will continue to foster industrial growth in biotechnology For example, the Human Genome Project and the Department of Energy Also in May 1998, a collaboration was announced between The Institute of Genomic Research (TlGR)-a private, nonprofit genetics laboratory-and Perkin-Elmer-the main manufacturer of DNA sequencing instru-ments These projects are focusing on sequencing the 3 billion base pairs of human DNA and identifying approximately 60,000 to 80,000 human genes Naturally enough, these rival projects and enterprises like the Celera Genomics Group, are creating a highly competitive business environment as they race for completion." The early phases of such research are devoted to mapping each human chro-mosome as a step toward ultimately determining all the genes in the DNA sequence

"ror popular press reviews, see "The Race 10 Cash in on the Genetic Code,"The New York1l'mel; August29, 1999,Section 3.A1so,Sciou:e News,VoL 154, October 10, 1998,p,239;and The New Yorker, June 12,

2000 p 66 The Human Genome Project plans to finish sequencing the human genome by 2003 and have

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phase

In the process, researchers are developing methods to automate and optimize genetic mapping and sequencing

Subsequent phases will focus on the development of "molecular medicine" based

on early detection of disease, effective preventive medicine, efficient drug develop-way to sequence the genomes of bacteria, yeast, plants, farm animals, and other organ-automation and optimization routines, will greatly benefit the biotech industry

9.10.2 Manufacturing

Despite these advances in knowledge, the path from the laboratory to the market is full

of obstacles Biotech research is expensive, time-consuming, and frequently fruitless Thescale-up to economically feasible production levels is also a tremendous challenge Biotechnology is in many ways related to one of its parent disciplines, chemical engineering However, it is far more difficult because its raw materials, catalysts, and products are living organisms, which are inherently more fragile and temperamental than petrochemicals and other substances Stringent product safety requirements, especially for therapeutics, create special problems for commercial production Equipment and facilities must meet strict safety and quality control standards to valve design and function) are still being established, making it difficult to design and build biotechnology equipment and systems Lacking manufacturing expertise, many companies arc sticking to research and licensing their technologies to biotech firms that have already developed production capabilities This is similar to the "fabless-IC" model presented in the management of technology section of Chapter 5 The long approval process and other product development risks make it espe-cially important to shorten the critical path for bringing a new product to market With the inherent challenges associated with scale-up of industrial bioprocesses, it is important to begin developing synthesis at the bench and pilot plant scales well before clinical trials are concluded (Figure 9.27) This is similar to the general push for concurrent engineering described in several of the earlier chapters

In addition, there are significant regulatory barriers New medical compounds must be rigorously tested in numerous clinical trials using strict Food and Drug Administration (FDA) regulations The approval process typically takes from five to seven years, and the likelihood that a product will fail is high Agricultural regulation

is less rigorous; the federal government recently relaxed its regulations of field testing and marketing of genetically engineered crops

9.10.3Investment

Given the potential range and impact of its commercial applications (Figure 9.28), it

is not surprising that biotech attracts much attention on Wall Street and with venture

up Each vied to beat the others to market with a breakthrough product Beginning

in the 19905, the well-known pharmaceutical companies also became involved in

issues

Manufacturing

Issues

F1IUre 9.27 Critical path for biotech design for planning to large-scale production (Adapted from O'Connor, 1995 © 1995 IEEE Reprinted, with

permission, from iEEE EngineeringillMedicine and Biology, vot.14,no.2, p.2rf7,March-ApriI1995.)

interest in, the smaller start-up companies As a result, today's picture is one in which

a wide range of company types exists At one extreme, individuals at universities with

molecular and cell biology departments continue to start private research-oriented

companies The integrity of such practices is discussed in Kenney (1986) At the other extreme, the large pharmaceuticals have set up production lines for well-established with a balance of basic research into new products and the production of well-established products

9.10.4The Future

As can be seen in the popular press, biotechnology research creates more ethical debates than the industries reviewed in Chapters 5 through 8 Part of the controversy stems from popular fascination and fears about the potential dangers of "messing with nature." Michael Crichton and Hollywood have helped fuel such concerns with reconstituted dinosaurs wreaking havoc in tropical islands

However, beyond the fanciful terrors of science fiction, biotech does indeed provoke a host of real ethical as well as practical concerns Selective breeding was once the only method available to develop desirable plant and animal characteristics over several generations By contrast, genetic engineering can clone precisely defined species This may be less threatening for the well-known cloning of sheep same possibilities for humans Does society have the right to intervene so forcefully

in the evolutionary processes? What are the implications for biodiversity? Privacy and equity are also a concern Suppose an insurance company decides

to do a DNA test on all its potential clients, and it finds that one of the clients

inher-Software fm drug design

I~olecular

biology

Generate compounds

Molecular targeting

Bench-scale synthesis

Pilot-scale production

Large-scale manufacturing

F1gure9.28 The future of biotech.

available yet As a result, the company refuses to insure the potential client and informs other insurance companies of the risk factor Is this ethical'?

9.10.5 Summary

This brief chapter on biotechnology has been included because the field will experi-ence rapid growth and provide many future career opportunities for people

inter-••• Biotechnology Chap 9 interesting scientific principles that are fundamental to life itself, to the engineering

of gene splicing, to the operation of bioreactors, and finally to the ethicalissues men-tioned

9.11 GLOSSARY

9.11.1 Amino Acids

The building blocks of proteins There are 20 different amino acids that link together via peptide bonds during the process of protein synthesis on the surface of the ribo-some (see Transcription and Translation) according to the genetic information on mRNA

9.11.2 Biar.actors

Vessels for biological reaction through fermentation or other transformation processes Interferon, for example, is manufactured in a genetically engineered fer-mentation process

9.11.3 Biosensors

Combining biology, Ie-design, and IC-microfabrication technologies, biosensors are devices that use a biological element in a sensor Biosensors work via (a) a biological molecular recognition element and (b) a physical detector such as optical devices, quartz crystals, or electrodes

9.11.4 Cen

The smallest unit of living matter capeblc of self-perpetuation

9.11.5 Central Dogma

The concept in molecular biology in which genetic information passes unidirec-tionally from DNA to RNA to protein during the processes of transcription and translation

9.11.6 Chromosome

A subcellular structure consisting of discrete DNA molecules, plus the proteins that organize and compact the DNA

9.11.7 Codon

A set of three nucleotide bases on the mRNA molecule that code for a specific amino combinations Some amino acids can be specified by more than one codon sequence

9.11.8 DNA (Deoxyribonucleic Acid)

The genetic material in every organism It is a long, chainlike molecule, usually

9.11.9 Enzymes

Enzymes are proteins that catalyze reactions

9.11.10 Fermentation

A process of growing (incubating) microorganisms in a substrate containing carbon and nitrogen that provide "food" for the organisms

9.11.11 Gene

The basic unit of heredity, a gene is a sequence of DNA containing the code to con-struct a protein molecule

9.11.12 GeneCloning

One of the most common techniques of genetic engineering, gene cloning is a way to cloned genes are often used to synthesize proteins The basic technique is to construct recombinant DNA molecules, insert the resulting DNA sequences into a carrier mole-cule or vector, and introduce that vector into a host cell so that it propagates and grows 9.11,13GeneticCode

Figure 9.14 shows the 64 possible codons and the amino acids specifiedbyeach 9.11.14Genetic Engineering

The practice of manipulating genetic information encoded in a DNA fragment to con-duct basic research or to generate a medical or scientific procon-duct Selective breeding

is one of the oldest examples of genetic engineering The much newer gene cloning technology is now fairly common practice Genetic engineering is used in three areas:

to aid basic scientific research into the structure and function of genes, to produce pro-teins for medical and other applications, and to create transgenic plants or animals 9.11.15Genome

The total genetic information of an organism

9.11.16 Host

A cell used to propagate recombinant DNA molecules

9,11,17 Natural Selection

The process by which a species becomes better adapted to its environment-the mechanism behind the evolution of the species The process depends on genetic variations produced through sexual reproduction, mutation, or recombinant DNA Variant furms that are best adapted tend to survive and reproduce, ensuring that their genes will be passed on Over hundreds and thousands of generations, a species may develop a whole set of features that have enhanced its survival in a