An Introduction to

Biotechnology

Pioneering science delivers vital medicines

Amgen is a leading human therapeutics company in the biotechnology industry. Since our founding in 1980, we have focused on accomplishing our mission to serve patients by discovering, developing and delivering innovative medicines to treat grievous illnesses. By pioneering the development of novel products based on advances in cellular and molecular biology, Amgens therapeutics have changed the practice of medicine and helped millions of people around the world to fight cancer, kidney disease, rheumatoid arthritis and other serious illnesses.

Table of Contents
Chapter One: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Chapter Two: The Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Chapter Three: How Biology Drives Biotechnology . . . . . . . . . . 9 Chapter Four: The Technology . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Chapter Five: Drug Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Chapter Six: Drug Development . . . . . . . . . . . . . . . . . . . . . . . . . 22 Chapter Seven: Scale-Up and Manufacturing . . . . . . . . . . . . . . 26 Chapter Eight: Biotechnology Medicines . . . . . . . . . . . . . . . . . . 29 Chapter Nine: Future of Biotechnology in Healthcare . . . . . . . . 31 Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Timeline of Medical Biotechnology . . . . . . . . . . . . . . . . . . . . . . 42

Chapter One:

Introduction

In 1919, Karl Ereky, a Hungarian engineer, coined the term biotechnology* to describe the interaction of biology and human technology. He envisioned a new era of technology based on using biology to turn raw materials into socially useful products. Nearly a century later, Erekys vision is being realized by thousands of companies and research institutions.

Introduction

*Terms in boldface are defined in the glossary .

Modern biotechnology began in the 1970s in Northern California and has since grown into a worldwide industry . Amgen, founded in 1980, was one of the first companies to realize the new fields promise by bringing biotechnology medicines to patients . Today, biotechnology industry sectors include healthcare (biologics, devices, diagnostics), agriculture (genetically modified organisms, food safety), industry and environment (biofuels, biomaterials, pollution) and biodefense (vaccines, biosensors). This booklet focuses on healthcare . Drugs A drug is a therapeutic substance used to prevent, manage or cure disease . In the United States, the U .S . Food and Drug Administration (FDA) must approve all drugs before they are sold to the public . Most countries follow global harmonized guidelines and have a regulatory agency similar to the FDA that evaluates drug research and approves drugs for marketing . The most familiar type of drug is the synthesized drug, such as aspirin . The pharmaceutical industry traditionally manufactures synthesized drugs . The biotechnology revolution brought about a new class of drug: the biologic . Biologics are therapies derived from living organisms and include therapeutic proteins, DNA vaccines, monoclonal antibodies and peptibodies [a modality that combines the active portion of a protein (peptide) with a portion of the core structure of an antibody], as well as new experimental modalities such as gene therapy, stem cell therapy, antisense nucleotides and RNA viruses .

Many biotechnology drugs are proteins. Proteins, which are made from amino acids, are the workhorses of the cell and perform all functions within a cell . Because cells produce proteins naturally, the biotechnology industry utilizes cells, not chemicals, to manufacture biologics .
BIoFaCT To bring a new drug to market (from discovery through clinical trials and FDA approval) costs an estimated $1 billion and can take 10 to 15 years or longer .* Only one in 10 new drugs that makes it into human testing actually makes it to market . Given this high failure rate and the tremendous cost of bringing a new therapy to market, companies depend on successful drugs to produce enough revenue to compensate for both the R&D costs of the successful therapies and the expense of failed ones .

* Innovation .org . (February 2007) . Drug discovery and development: Understanding the R&D process [Brochure] . Washington, DC: Pharmaceutical Research and Manufacturers of America .

Introduction

Chapter Two:

The Science

The biotechnology industry is based on living organisms. The cell is the basic unit of life. All living organisms consist of one or more cells. Some organisms are unicellular, such as bacteria and yeast. Others, such as humans, are multicellular, consisting of trillions of cells. All cells have common processes they perform in order to survive. Biotechnology harnesses these processes to make products to treat illness and improve health.

The Science

Cell Processes Cells Replicate (mitosis): Prior to dividing, a cell makes a copy of its DNA and other cell parts . The cell divides and forms two identical cells from the original single cell . These identical cells are referred to as daughter cells . Cells Grow: After replicating, the daughter cells grow to their intended size .

Cell Membrane Nucleus

Nucleus

Cytoplasm

Cytoplasm

Cell Membrane

keep the DNA safe . This DNA never leaves Cells Metabolize: Cell metabolism is the process by which cells process nutrients and maintain a living state . Cells break down large molecules into smaller molecules to produce energy and molecular building blocks, which are used to create new cell structures and control cell function . Cells Respond to Stimuli: Unicellular and multicellular organisms respond to internal and external stimuli . For example, plants grow toward a light source because light is needed for photosynthesis and the production of energy . Light and the ability to respond to its presence are essential to a plants survival . Cells can respond to a whole range of stimuli . Cells Adapt: Organisms may thrive or die based on their ability to adapt to adverse environmental conditions such as changes in temperature, solute concentration, oxygen supply and the presence of hazardous agents . Parts of an Animal Cell The cell can be divided into three main sections: the cell membrane, the cytoplasm (which includes the organelles) and the nucleus.
BIoFaCT

the nucleus . Cell Membrane: The cell membrane is the border that surrounds a cell . It monitors what goes into and out of the cell . Embedded in the cell membrane are receptors. Receptors cross the membranes and act as docking stations for molecules . When a specific type of molecule attaches to a receptor, a series of chemical reactions (cell signaling) can occur in the cell, creating a cellular response . If a receptor is blocked, cell signaling is stopped and no response occurs . This is how some biologic therapies work: they attach to receptors and interrupt cell signaling . Other biologics work by mimicking the signaling molecule . Organelles: There are many different types of organelles within the cytoplasm of a cell, and each performs a specific function . For example, ribosomes make proteins . Mitochondria make energy . The endoplasmic reticulum folds and transports certain proteins . Golgi bodies modify proteins and are involved in their transportation around the cell . Vacuoles store cellular waste products for disposal .

Nucleus: The nucleus houses most of the DNA and is the control center of the cell . It is surrounded by a membrane that lets certain molecules into and out of the nucleus to

Ribosomes can assemble an average-size protein in one minute .

The Science

Determining the Structure of DNA

In 1962, James Watson, Francis Crick and Maurice Wilkins jointly received the Nobel Prize in Physiology or Medicine for discovering the structure of deoxyribonucleic acid (DNA). Because the Nobel Prize is awarded only to the living, Wilkinss colleague Rosalind Franklin, who died from cancer at the age of 37, could not be honored. But many attribute the success of Watson and Cricks 1953 discovery to Franklin, whose X-ray crystallography images of DNA helped them clarify the structure of DNA.
T A T A

Chromosome Gene
C
T A A A T T

C G
G C
A T

C
A A T T

A T

A T

C C G G

C G

C G

DNA

The molecular structure of DNAthe double helix.

DNA All cells contain deoxyribonucleic acid (DNA). DNA is the blueprint for the construction and operation of the cell . DNA is arranged in large segments called chromosomes. Within a chromosome are specific pieces of DNA called genes. Genes vary in length and contain the information a cell needs to make proteins . Proteins control many aspects of cellular function .
BIoFaCT Mitochondria are the only organelles in animal cells to have their own DNA . Mutations in mitochondrial DNA can lead to illnesses such as Kearns-Sayre syndrome, which causes the loss of heart, eye and muscle movement functions .

Cs . Every organism contains DNA, but the number and arrangement of bases are different for every organism . DNA is called a double helix because it is posited to consist of two strands of nucleotides that bond together in a very specific manner . The As bond with Ts, and the Cs bond with Gs . The resulting A-T and C-G combinations are called base pairs. In each human cell, the length of DNA is equal to 3 billion base pairs . If flattened, a DNA segment would look something like a ladder with two side rails (phosphate and ribose groups) and rungs (base pairs) between them . The structure makes DNA very stable and able to carry vast amounts of information .
BIoFaCT

The information in DNA is stored as a code made up of four basic building blocks called nucleotides . DNA is very long, and the order of the nucleotides determines the information stored . Each nucleotide consists of three components: a deoxyribose sugar, a phosphate group and a base . There are four different types of bases: adenine (A), thymine (T), guanine (G) and cytosine (C) . The order of the As, Ts, Gs and Cs in DNA gives meaning to the cell, just as the order of letters in a word gives meaning to that word and, by extension, to a story in which the word appears . The diversity of organisms is a result of the limitless combinations of basesAs, Ts, Gs and Most cells within an organism contain the exact same DNA, but not all genes within each cell are active, or turned on . When a gene gets turned on, the information encoded by the gene is used to produce, or express, the
Humans have 23 pairs of chromosomes, for a total of 46half inherited from the mother and half from the father . Chromosomal abnormalities can involve numeric abnormalities, where there may be more or less than the normal 46 chromosomes, or structural abnormalities, where there may be duplications, translocations, deletions or even inversions of sections of certain chromosomes .

The Science

The number of base pairs does not correspond to the number of genes; the two are indepenUntwist
A T C G G C T A A T C G G C T A A T C G G C T A A T C G G C T A A T C G G C T A A T G C

The U.S. National Center for Biotechnology Information

Established in 1988, the National Center for Biotechnology Information (NCBI) creates and maintains extensive networks of biomedical databases for storing and analyzing genomes. The databases include information about various genome base pair sequences, genes and proteins. NCBI is a division of the U.S. National Library of Medicine at the National Institutes of Health. See http://www.ncbi.nlm.nih.gov.

dent of each other . For example, the human genome has 3 billion base pairs and approximately 20,000 to 25,000 genes . Only 3 percent of the human genome codes for genes; 97 percent is termed noncoding DNAin other words, DNA that does not contain instructions for creating proteins . Biologists have not yet fully discovered the function of noncoding DNA, but they suspect it may be involved in the evolution of species or may have regulatory functions within the cell .
BIoFaCT

Read

GATATTTCAGGCGCGTCAA CTATAAAGTCCGCGCAGTT

encoded protein . When a gene is not active, or is turned off, it is not used to express proteins . Depending on the cells function and needs, genes are either turned on or off . Many diseases are the results of genes improperly turned on or off . Mutations Any change in the DNA sequence is called a mutation . Environmental factors such as exposure to radiation or chemical toxins can cause mutations . Mutations also can occur during the natural process of DNA replication, when the cell is responsible for copying 3 billion base pairs in 20 hours . Bases can be substituted, deleted or repeated . Changes in the DNA sequence may cause proteins to become dysfunctional or may even, occasionally, improve function . Genetic diversity results from an accumulation of mutations over a long period of time, which causes the differences among species . Genomes The term genome refers to the entire genetic information in an organism . The human genome is the entire DNA content found in a human; the corn genome is the entire DNA content found in corn; and so on . All genomes are made up of the same bases: As, Ts, Gs and Cs . The differences between genomes lie in the number and sequence of base pairs and the number and sequence of genes .

Variations in individual nucleotides occur within DNA at the rate of approximately one in every 1,300 base pairs in most organisms . In humans, the rate is one in every 1,200 base pairs . Most of these mutations, however, do not adversely affect us; only a few are involved in the production of dysfunctional proteins or disease states .

Furthermore, the number of base pairs and genes does not correspond to the intelligence or physical capability of an organism . Compare the human genome with the amoeba genome . An amoeba, a unicellular organism, has the largest-known genomewhich may be as large as 670 billion base pairsas compared to the human genome of 3 billion base pairs . Yet a human is a more complex, intelligent and physically capable organism . Proteins Proteins are made of long chains of amino acids that fold into intricate and complex 3-D shapes . The order of amino acids is determined by the DNA sequence in a gene . There are 20 different amino acids . The sequence of amino acids determines the shape, and therefore the function, of the protein .

The Science

BIoFaCT The highest-known number of genes in an organism is around 60,000for the bacterium that causes trichomoniasis which is almost three times as many as in the human genome .

Proteins called enzymes put molecules together and/or break molecules apart . Proteins called signaling molecules allow cells to relay messages to one another . Protein receptors receive signals through a type of communication known as signal transduction or cell signaling . Some proteins move substances into and out of the cell . Structural proteins give shape to cells and organisms . Proteins are involved in cellular recognition and identify different types of cells . Some proteins, such as antibodies, are involved with defending an organism against disease .

Protein synthesis is a complicated, multistep process . Two of the steps involved with making a protein are transcription and translation. Transcription During transcription, the original DNA code is rewritten onto a molecule called messenger RNA (mRNA). mRNA is also made up of a sequence of nucleotides that differ slightly from DNA nucleotides . RNA molecules are very similar to DNA but have a ribose sugar, are single-stranded and use U, or uracil, instead of T, or thymine, as one of the four possible bases . Translation During translation, the ribosomes assemble individual amino acids into proteins . Ribosomes will bind to mRNA . Every three mRNA nucleotides make up a codonthe code for an amino acid . During translation, transfer RNA (tRNA) reads mRNA and picks up the corresponding amino acids . In this way, the amino acids are linked together in a very specific combination as dictated by the sequence of nucleotides on the mRNA . Short chains of amino acids are called peptides. Long chains of amino acids are called polypeptides . Polypeptides fold into a functional protein . Hundreds of different types of proteins perform specific jobs within and between cells .

The Science

Chapter Three:

How Biology Drives Biotechnology

Biotechnology is based on biology, which is the study of life. The basic unit of life is the cell. Biologists study the structure and functions of cellswhat cells do and how they do it. Biotechnologists use this information to develop products. Biomedical researchers use their understanding of genes, proteins and cell parts to pinpoint the differences between diseased and healthy cells. When researchers know how diseased cells are altered and when they learn how to affect those alterations, they are better able to develop innovative medical diagnostics, devices and therapies.

How Biology Drives Biotechnology

Understanding Disease Mechanisms Early-stage drug research and development (R&D) may begin with understanding the underlying biology of a particular disease . Biotechnology medicines are often created specifically to address a particular disease mechanism . To design and develop new drugs, researchers must understand the disease mechanisms involved . Some initial questions researchers Autoimmune Diseases
Rheumatoid arthritis is a chronic autoimmune disease that causes inflammation and tissue damage in joints and tendons. It can be a disabling and painful condition and can lead to substantial loss of functionality and mobility. Psoriasis is a noncontagious disorder that causes red scaly patches to appear on the skin. These patches are areas of inflammation and excessive skin production. Psoriatic arthritis is a type of inflammatory arthritis that affects up to 30 percent of people suffering from psoriasis. Juvenile idiopathic arthritis is the most common form of chronic inflammatory arthritis in children. Ankylosing spondylitis is a chronic, painful, degenerative inflammatory arthritis primarily affecting the spine and causing eventual fusion of the vertebrae. Lupus is a chronic inflammatory disease that most commonly affects women of childbearing age. Lupus can affect the kidneys, joints, blood and skin, among other organs. Its symptoms can include rash, fever, aches, anemia and hair loss.

factor (TNF) plays a major role in regulating inflammation . Researchers know that too much TNF is produced in autoimmune disorders such as rheumatoid arthritis, psoriasis, psoriatic arthritis, juvenile idiopathic arthritis and ankylosing spondylitis . When too much TNF is produced, excessive inflammation occursand that can be damaging to joints, skin and other parts of the body . Biotechnology companies have worked to develop medicines that inhibit the activity of TNF . Models for Studying Disease Researchers often take several different approaches to creating models for studying a particular disease . One approach is to obtain samples of diseased cells and healthy cells and grow them using a method called cell culture. This calls for cells to be incubated and fed with specialized growth media. In culture, the cells do what cells dodivide and express genes to produce proteins . By studying how cellular processes differ between healthy and diseased cells, researchers hope to come to understand the mechanism of disease . Another approach involves studying shared or similar genes and protein equivalents in other species . Since all organisms are made of cells and all cells perform many similar functions, genes and proteins found in humans are also found in other organisms .
BIoFaCT The process of analyzing and interpreting biologic scientific data is called computational biology and involves computer science, applied mathematics and statistics .

ask to understand the underlying mechanisms of a disease are: How does a person get the disease? Which cells become diseased? Is this disease caused by genetics, and if so, what genes are turned on or turned off in the diseased cells? What proteins are producedor not producedin diseased cells as compared to healthy cells? If the disease is caused by a pathogen, what is the interaction between the pathogen and the person?
BIoFaCT The biotechnology industry is one of the most R&D-intensive industries in the world . The United States is recognized as leading the world in biotechnology R&D .

Studying disease mechanisms provides researchers with information that can lead them to identify targets for the early stages of the drug discovery process . An understanding of fundamental biology may lead to effective therapies for patients . Take treatments available for autoimmune disorders, for example . Autoimmune disorders occur when a persons immune system overreacts and attacks proteins, cells and tissues in the body, often leading to inflammation . Biologists have learned that tumor necrosis

The functions of human genes have been revealed by studying parallel genes in

10

How Biology Drives Biotechnology

nonhumans . This approach has added to our understanding of how specific genes and proteins direct the functioning of human cellsboth healthy and diseased .

disease processes or biologic responses to therapeutic treatment and disease intervention . Historically, biomarkers were physiological indicatorssuch as blood pressure or heart rate . Today, disease may also be detected using molecular biomarkers such as prostatespecific antigen, a protein that if elevated, may indicate prostate cancer .
BIoFaCT Comparative genomics is the study of genome structure and function among different species . Researchers have obtained complete genomic sequences for the bacterium Escherichia coli (E. coli), the yeast Saccharomyces cerevisiae, the roundworm Caenorhabditis elegans, the fruit fly Drosophila melanogaster, the laboratory mouse and many other organisms for use in comparative genomic studies with the human genome .

KRAS: A Biomarker Breakthrough

A biomarker can now help physicians determine which patients will not respond to a certain class of anticancer agents used to treat metastatic colorectal cancer (mCRC). Amgen scientists were the first to show in a phase 3 clinical trial that patients with mCRC carrying specific cancerpromoting mutations in a gene known as KRAS are unlikely to benefit from a drug designed to bind to epidermal growth factor receptor (EGFR). By testing for these mutations, physicians can now eliminate anti-EGFR antibodies as a treatment option for patients with mutated KRAS tumors. KRAS research is regarded internationally as an important step toward personalized medicine for cancer treatment.

Bioinformatics Bioinformatics combines biology, computer science and information technology into one discipline . The goal of bioinformatics is to capture, organize and index scientific information so researchers can better understand biology . The challenge for computer programmers is to design databases that allow researchers to easily access existing data as well as submit new data . The scientific community generates volumes of biological data daily . Biotechnology companies use this information to form a comprehensive picture of normal cell activity so researchers can better study diseased cells . This leads to the development of diagnostic tools, therapies and preventive medicines . Because of technological advances in biotechnology, bioinformatics has evolved to focus on nucleotide sequences, genes and amino acid sequences . Biomarkers Biomarkers are substances that can be measured and evaluated to indicate (or serve as markers of) normal biologic processes,

Once a biomarker is validated (meaning all or most patients with the disease test positive for the specific biomarker), it can be used to diagnose disease risk or presence of disease or to help doctors determine patient treatment . Biomarkers also can be used to predict prognosisfor example, how a disease is likely to progress if left untreated . Identifying biomarkers that indicate specific disease is a crucial step in the R&D process for developing new biotechnology diagnostic tools .

BIoFaCT Genetic biomarkers are used in developing diagnostic tests for detecting certain genetic diseases . These tests look for DNA fragments identified as causing disease or associated with susceptibility to a disease .

How Biology Drives Biotechnology

11

Researchers study modifications and changes that occur during cellular processessuch as protein synthesisin various disease states . This can lead to the identification of biomarkers that signal a change in disease progression and can be used as a key development in drug discovery and testing . Types of Cancer
Cancer is a general term used to describe diseases that occur due to abnormal, uncontrolled and rapid cell growth. There are many types of cancer (carcinoma, lymphoma, leukemia, sarcoma, melanoma, etc.) that affect many types of tissue (prostate, skin, breast, brain, ovaries, lungs, etc.). Carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs. At least 80 percent of all cancers are carcinomas. Lymphoma is a cancer of the lymphatic systema network of thin vessels and nodes throughout the body that helps to fight infection. Lymphoma involves lymphocytes, a type of white blood cell found in the immune system. Leukemia is cancer that starts in blood-forming tissuesuch as bone marrowand that causes large numbers of abnormal blood cells to be produced and to enter the blood. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels or other connective and supportive tissue. Melanoma is a cancerous (malignant) tumor that begins in the cells that produce skin coloring (melanocytes). Melanoma is almost always curable in its early stages but can be lethal in later stages.

Understanding protein structure, function and interaction within and between cells is crucial for drug discovery . Proteins are common drug targets because they are responsible for most normal cell functionsand malfunctions, as in the case of disease . A drug target is the molecule the drug interacts with to bring about the desired change . This is sometimes called hitting the target . Cancer: From Biology to Treatment Cancer research is a field that has been at the forefront of utilizing bioinformatics, biomarkers and protein studies to develop new therapies that target specific cellular processes . Cancer biology is a specific field that explores all aspects of the cancer cell . Researchers follow cancer pathways and determine the molecular basis of cancer as they develop diagnostics and treatments . Cancer starts with changes in one cell or a small group of cells that reproduce uncontrollably . In healthy cells, cell division and growth are tightly regulated . However, cancer cells keep dividingwithout normal checks and balancescreating greater opportunity for mutations . A mutation may result from the environment or could have occurred during DNA replication . As the cancer cells replicate and grow, a mass (tumor) is formed . Cells from the tumor can break away (metastasize) and spread through the bloodstream or lymphatic system to other parts of the body, creating new tumors . Traditionally, cancer has been treated with surgery, radiation and chemotherapy . The biotechnology industry has contributed significant advances in cancer treatment by developing hormone therapies, biologics and targeted therapies such as monoclonal antibodies .

Biomarkers are also used in drug discovery to determine whether a drug is effective in animal models and at what doses effectiveness is reached . Finally, biomarkers can be used in disease management to determine whether a drug is having the desired effect and whether the correct dose is being used . This is very important because response to a drug can vary between patients: some require higher or lower doses for the the drug to be effective . Proteomics The entire set of proteins produced by an organism is called its proteome . Proteomics is the study of protein structure and function . Proteins control all aspects of cellular function . Proteins produced in any specific cell of an organism can vary with a number of factors such as time, hormonal change and stress . Proteomics researchers identify all proteins involved with protein synthesis and protein folding . Proteins need to fold correctly into
BIoFaCT Biotechnology has created more than 200 new biotherapeutics and vaccines, including products to treat cancer, diabetes, HIV/AIDS and autoimmune disorders . The majority of these products are therapeutic proteins .

their 3-D shape in order to correctly function . Even small structural defects during the folding process can lead to a number of protein diseases .

12

How Biology Drives Biotechnology

Chapter Four:

The Technology

Biotechnology scientists depend on a wide variety of constantly evolving laboratory techniques and tools. This section focuses on some of these platform technologies. To understand the industry, its helpful to have some basic knowledge of what goes on in the lab.

The Technology

13

Transformed Cells
Transformation of bacterial cells for the production of recombinant proteins usually involves E. coli. Transformation of animal cells, called transfection, usually involves a cell line derived from Chinese hamster ovary (CHO) cells. CHO cells were introduced in the 1960s and remain the most commonly used mammalian host cells for industrial production of recombinant protein therapeutics.

+
Restriction enzymes are proteins that function as molecular scissors and cut DNA.

Restriction Enzymes Biotechnology employs a process called genetic engineering, which combines DNA sequences in order to produce recombinant proteins as potential therapeutics . The process utilizes restriction enzymes .

The counterpart to cutting is called pasting . DNA ligase is a protein (enzyme) that seals two DNA segments together in a process called ligation . The ability to cut and paste DNA is the basis of genetic engineering .

BIoFaCT BIoFaCT Scientists have identified more than 3,800 restriction enzymes, and more than 600 are commercially available for purchase from scientific supply companies . Daniel Nathans, Werner Arber and Hamilton Smith received the 1978 Nobel Prize in Physiology or Medicine for their discovery of restriction endonucleases . Their discovery led to the development of recombinant DNA technology .

Scientists discovered restriction enzymes (endonucleases) in bacteria in the 1970s . They found that these enzymes cut up viral DNA into small, nonfunctional pieces, thereby protecting the bacterium from an invading virus . There are hundreds of restriction enzymes each of them recognizing a specific sequence of DNA called a restriction site . For example, EcoR1, a restriction enzyme found in E. coli, recognizes and cuts at the six-base sequence GAATTC . HaeIII, a restriction enzyme found in Haemophilus-aegyptius, recognizes and cuts at the four base sequence GGCC . All DNA, regardless of where it comes from, is made up of the same four basesAs, Ts, Gs and Cs . HaeIII reads any DNA segment and cuts the DNA every time it encounters the sequence GGCC . All restriction enzymes are specific and reproducible, which are two key characteristics that allow researchers to utilize restriction enzymes to manipulate DNA .

Recombinant DNA When segments of DNA are cut and pasted together, the new DNA is called recombinant DNA. Recombinant DNA can be inserted into cells to produce cells with new characteristics . This genetic altering can include a single-base (letter) change or multiple gene changes . Recombinant DNA can be introduced into a host cell by a vector, which is used to physically carry DNA into a host cell . A host cell can be bacterial, yeast, plant, insect or mammalian . Common bacterial vectors include plasmids and phages . A plasmid is a circular unit of DNA that can be engineered to carry a gene of interest . A phage is a genetically engineered virus that injects DNA into bacteria . Cells that contain recombinant DNA are referred to as genetically modified, transgenic or transformed cells . The process is called transformation.

14

The Technology

BIoFaCT The FDA approved human insulin in 1982 the first medicine made via recombinant DNA technology .

gies, asthma and many other conditions . The number of recombinant proteins has increased greatly in recent years as the technology used for their production and purification has advanced . Cell Culture Cell culture is the technique of growing cells in the laboratory under controlled conditions . Growing large quantities of transformed cells is a major step in the process of producing recombinant protein products . Both transformed bacterial cells and transformed animal cells are used in this process . Simple proteins can be produced using recombinant DNA technology in bacterial cell cultures . Typically, more-complex proteins that are, for example, glycosylated are made in animal cell cultures . During cell culture, cells are grown in petri dishes or flasks containing liquid media . Culture media provides all the nutrients necessary for cell growth . The cultures are grown in an incubator that maintains the appropriate temperature and environment, often requiring certain gas mixtures of oxygen and carbon dioxide . It is very important to maintain specific conditions for cultures, because variation in these conditions may affect the proteins expressed and, therefore, the product that is produced . In the process of commercial production of proteins, cell cultures must be scaled up to produce enough protein to meet demand . Since only a limited number of cells can be grown in small petri dishes or flasks, the cell culture can be transferred to large containers called bioreactors, which are involved in the manufacturing process .

Recombinant Proteins Recombinant DNA can be used to produce recombinant proteins. The host cells use the new DNA information and their own cellular machinery to produce the protein encoded by the recombinant DNA . When recombinant proteins are produced for use as human therapeutics, host cells must be grown in large quantities so that enough recombinant protein is produced to meet demand . The recombinant protein is isolated, purified and analyzed for activity and quality before it goes to market . Producing a protein with the proper order of amino acids isnt always the whole story . Sometimes further processing is required to yield an active or fully functioning protein . Many human proteins are glycosylated, meaning, they have a particular pattern of sugar molecules linked to them . If a protein is translated but not correctly glycosylated, it may not function properly . Adding a phosphate groupa process known as phosphorylationcan act as an on-switch, allowing the proteins to become active proteins . Other biochemical functional groups may be added to the protein to allow it a larger range of function . Recombinant proteins for therapeutic use include vaccines, hormones, monoclonal antibodies and hematopoietic growth factors for the treatment of cancer, AIDS, aller-

The Technology

15

Research Tools Biotechnology professionals depend on leading-edge laboratory equipment . Here are a few pieces used in genetic engineering: Thermocycler Polymerase chain reaction (PCR) is a technique that replicates DNA in a machine called a thermocycler. PCR is a series of cycles that takes a small amount of original DNA and exponentially copies, or amplifies, it . Each three-step cycle doubles the amount of DNA present . Its like a photocopier for DNA . A single piece of DNA can be turned into millions of copies of the same DNA piece . PCR enables researchers to make enough DNA to work with in the lab . There are many applications for PCR, including producing enough of a DNA sequence or gene for use in creating recombinant proteins .

+
Well Gel Gel electrophoresis.

There are different types of electrophoresis . Each is based on the type of separating material used, gels being one type, and the types of molecules being separated . DNA, RNA and proteins can all be separated using electrophoresis in an appropriate apparatus . Gel electrophoresis has many applications in both clinical and research labs . One common use is for verifying PCR productsthat is, checking to see whether the reaction generated the correct DNA fragment . DNA Microarrays A DNA microarray (also called a gene chip) is a small piece of glass or silicon divided into thousands of sections in a grid pattern . Each section has a single-stranded gene fragment corresponding to either a healthy or diseased gene . DNA from an individual is separated into single strands and tagged with a fluorescent dye . The tagged DNA is washed over the microarray . The individuals DNA binds to any complementary DNA sequences on the slide, if they are present, to become doublestranded DNA . With the aid of a computer, the double-stranded, fluorescently tagged DNA spots can be located and measured . The individuals DNA must match the attached gene fragment exactly to bind, thereby indicating the presence of healthy or diseased DNA .

BIoFaCT Kary Banks Mullis developed the polymerase chain reaction in 1983 and received the 1993 Nobel Prize in chemistry for the invention .

Gel Electrophoresis Gel electrophoresis is a technique commonly used in the laboratory for analyzing DNA fragments . Gel electrophoresis allows DNA fragments to be separated within a gel . A gel electrophoresis apparatus holds the gel and allows electricity to run through it . Each DNA fragment is negatively charged, causing it to migrate toward the positive pole of the gel apparatus . Larger fragments of DNA move more slowly than smaller fragments because they encounter resistance from the gel matrix .

16

The Technology

Microarray.

Microarrays are powerful tools allowing researchers to analyze thousands of genes in one test . Microarrays are used for genetic testing, for comparison of genetic information from different individuals or species, and in the discovery of potential drug targets . Microarrays can also be used for researching and identifying genes of interest to be used with recombinant DNA technologies . Other microarrays include protein, tissue, chemical compound and antibody microarraysall of which allow researchers to analyze thousands of data points at one time .

BIoFaCT Microarrays are capable of generating so much data that special microarray databases are needed for storage, searches, analysis and interpretation . There are both public and private microarray databases .

The Technology

17

Chapter Five:

Drug Discovery

During drug discovery, scientists search for moleculeseither chemical or biological agentsthat could alter a disease pathway. As part of the discovery process, they specifically look for ways to change one or more molecular or cellular processes that occur in the affected cells of a diseased tissue or organ.

18

Drug Discovery

Initiating Drug Discovery Research An early step in the drug discovery process is to identify an unmet medical need . What is known about the disease? What are the current treatment options, if any? Does the company have the expertise, technology and financial resources to solve the problem? Potential competitors and barriers, such as regulatory constraints, are also taken into consideration .
BIoFaCT According to the National Center for Health Statistics, the top five diseases causing death in the United States in 2005 were heart disease, cancer, stroke, chronic lower respiratory disease and diabetes .

ask, What are the differences between healthy and diseased cells? Ultimately, disease processes take place at the molecular level . There are various causes of diseases . In inherited diseases, a difference in the expression or in the sequence of genes results in abnormal functioning of a persons cells . Sometimes this leads to a target being present in excess; other times it could be deficient or missing . So the scientists will need to decide if the goal will be to block the target or to enhance or replace it in order to restore healthy function . For a disease caused by an external pathogen, such as a virus or bacterium, the pathogen produces molecules that can damage the host organisms cells . Moreover, the pathogen will, itself, display molecules in the infected individual that are not present in a healthy person . The goal in target discovery is to identify those different molecules . This can be done using a variety of technologies such as microarray experiments, protein electrophoresis, mass spectrometry (MS), DNA sequencing and computerized imaging .
BIoFaCT

Chemical Libraries
In the 1990s, chemists developed huge libraries of chemical compoundsthousands, even millions, of chemicals with different structures used to screen for new drugs. These libraries are often proprietary and constructed by a company explicitly to support its drug discovery programs. Initial screening of drug candidates is relatively simple. Once potential drug candidates or leads are identified, more-complex assays are used at subsequent levels of screening. The sources of the compounds found in the drug candidates are often natural products (from microbes, plants and simple marine life) or chemical compounds synthesized by an organic chemist. Combinatorial chemistry increases the potential of chemical libraries by synthesizing larger, more complex chemicals or chemically related molecules from common chemical structures. Combinatorial chemistry for small-molecule drugs includes the synthesis of large organic molecules by adding together smaller organic molecules, often with improved product results or lessened product side effects.

Target Discovery After identifying an unmet medical need and deciding whether it fits within the companys portfolio, scientists look very closely at the biology behind the disease . Where can they intervene, and what options do they have for intervention? Since the human body is an extremely complex system, scientists have to carefully choose the target . A target is a molecule that plays a critical role in a disease . Scientists estimate that about 8,000 known therapeutic targets exist today . Targets can be secreted factors, cell surface receptors or signaling pathways within a cell . The goal is to develop a drug that affects a target in a way that interferes with the disease process . Its also very important to ensure that the potential benefits of a drug are appropriately weighed against any risks such as possible side effects . Different targets respond to different therapeutic approaches . To select a target, scientists will

Researching the genetic and molecular basis of a disease is called studying the mechanism of disease .

While this sounds straightforward, target discovery is often difficult and may take years to complete . Why? Cells and cell-to-cell interactions are very complex . There may be one or more mechanisms of the disease and many points in the mechanism at which to intercede . Moreover, the difference between healthy and diseased cells can be too minute to easily detect, or a method able to detect the difference may not yet have been invented .

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Cell Receptors and Ion Channels

The most common drug targets are cell receptorsproteins on or inside a cell to which a specific signaling molecule can attach. These signaling molecules can be hormones, neurotransmitters, pharmaceutical drugs, toxins or even infectious agents. When signaling molecules attach to the receptor, a physical change occurs that initiates a specific cellular response. Other common drug targets are ion channels, proteins that form pores in the membranes that surround cells, and enzymes proteins that increase the rate of specific chemical reactions.

The complexity of the bodys response also means scientists could see a difference in the expression of hundreds of genes without being able to determine which ones were critical to the disease . Target Validation Once scientists identify potential targets, the next step is to validate them . Target validation has two components . The first is to show that the target molecule actually plays a role in the disease . The second is to confirm the target is a candidate for therapeutic intervention: Can a safe and effective drug be made against the target? Scientists complete this second component of target validation before the drug enters human testing . There are a number of ways to validate a target, and the process must take into consideration time, cost and technology . At the simplest level, the concept of target validation is to use the target to create the disease in a sample of healthy tissues and then block the target to restore the healthy condition . This is done in cell culture or animal models . The trick is to select a model that is representative and will work . Sometimes people who are born without certain functional molecules express a specific disease type . Studying biological samples taken from such human subjects provides another means of validating a target . Examples of target molecules include receptors, enzymes, ion channels, growth factors, cytokines and DNA binding proteins . The common thread among these targets is that they are often involved in signal transduction processes in and among cells . Signal transduction pathways control cellular processes

such as division, differentiation, protein synthesis and programmed cell death (apoptosis). Initial studies are often done in cell culture . If cell culture studies are positive, a next step is to use an animal model . Sometimes a suitable animal model has to be created to validate a target . Sometimes the target doesnt exist in an animal model or may not mimic the human disease state . Sometimes the drug candidate is so specific to humans, it wont recognize the animal models target or the animal will mount an immune response that blocks any therapeutic effect . For example, Alzheimers disease occurs only in humans, and only recently have mouse models been developed to mimic the disease . Scientists also look at what other effects the drug candidate may have within preclinical (both cell culture and animal) models . Sometimes the target is expressed on other cells or tissues besides those directly involved in the disease . What happens to those cells and tissues in the presence of a drug candidate? Does a drug candidate adversely affect other cells or tissues? Does it raise an immune response; stimulate other, similar targets; or otherwise present any concerns about toxicity?

BIoFaCT Recently, scientists have begun using computer simulation to model drug-target interactions to guide drug discovery .

Preclinical work helps support later human trials that may occur if the drug candidate continues to show promise . Even if the drug gets marketing approval after successfully completing the necessary phases of human

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trials, safety surveillance will continue once the drug has reached the larger patient population . Scientists will continue to answer safety questions throughout the life of a drug . Screening High-throughput screening is a process that combines robotics and data processing to rapidly identify the compounds, antibodies or genes that modulate a particular biomolecular pathway . Large batches of potential drugs are tested for binding activity or biological activity against target molecules . Once a candidate disease is identified, a companys research lab develops a testing method (assay) to determine or measure the pharmacological activity of hundreds to hundreds of thousands of molecules . The assay measures the estimated potential of a molecule to block or stimulate a target . Whats being measured could be as simple as the ability of the drug candidate to kill cancer cells in culture or as complex as measuring its ability to inhibit an enzyme involved in a disease . Generally, the more complex the assay, the more relevant the information but the higher the cost of the assay and the longer it usually takes to get data . Of the molecules that score a hitthat is, a positive result that appears to have a therapeutic potentialsome are identified as lead molecules due to their more druglike properties (solubility, permeability, stability, etc .) . Once a drug candidate is identified, scientists may attempt to optimize its ability to fight disease by changing its molecular structure through combinatorial chemistry for small molecules or protein engineering for large molecules .

Drug Design The design approach to drug discovery starts with scientists understanding the genetic and molecular base of a disease and using that information to select a specific therapeutic target . Drugs are then designed to interact with the target . Through rational drug design, scientists seek to develop a drug that is highly specific to a particular target in a disease in hopes of achieving a better therapeutic outcome with potentially fewer side effects . Scientists can learn more about the structure of the target by using imaging technology such as X-ray crystallography . 3-D structural information about a target enhances drug design strategies . Considerations in designing a therapeutic agent depend on both the nature of the target and the capabilities of the company . If the target is on the exterior surface of the cell membrane or is secreted, protein therapeutics such as monoclonal antibodies or peptides can be used . If the target is on the interior of the cell, only drugs that can cross the cell membrane, such as small molecules, can be used . When designing a drug candidate, scientists must keep in mind the intended method of drug delivery and determine whether the drug will be a pill swallowed, a liquid injected, a spray inhaled or something else .

Choosing the Right Tool for the Target

Designing a targeting strategy usually comes down to a choice between a small-molecule drug and a biologic (most often a recombinant protein or antibody). Each has its particular advantages and disadvantages. Small molecules can usually cross cell membranes and enter cells, allowing them to be used for targets inside cells. Biologics usually cannot cross cell membranes, restricting their use to targets on the surface of, or outside, cells. Small molecules have good specificity for their targets, but recombinant antibodies generally have extremely high specificity, meaning, fewer adverse reactions for the patient. Small molecules have variable half-lives, which is a measurement of how long a drug stays active in the bloodstream or in its target tissues. Biologics often have much longer half-lives, partly because they are modeled on real biological molecules. This means patients dont have to take as many doses of a biologic, which may result in better patient adherence to therapy. Biologics usually need to be injected, whereas small molecules can be taken orally. Small molecules can often cross the blood-brain barrier, but biologics usually cannot, which to date has limited their usefulness for treating diseases of the brain such as psychiatric disorders and neurodegenerative diseases.

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Chapter Six:

Drug Development

After the lengthy process of drug discovery (identifying a target and validating a drug candidate), the process of drug development is still far from complete. Drug development includes the safety, efficacy, formulation and manufacture of the drug. Typically, safety testing begins with a series of experiments called preclinical studies. If these studies predict the drug candidate to be safe, testing begins in humans in a series of studies called clinical trials.

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Preclinical Studies Preclinical studies are tests that take place in a scientifically controlled setting using cell cultures and animals as models . The goal of preclinical studies is to predict what the body does to the drug candidate (pharmacokinetics), what the drug candidate does to the body (pharmacodynamics), and whether the drug candidate may pose potential health hazards or toxic side effects .

Information from these studies is vital . It allows researchers to estimate a safe dosage level for humans in phase 1 clinical trials . Although drug companies are required to submit animal model data to regulatory agencies as part of the drug approval process, companies are taking steps to reduce the number of animals used in testing because of ethical concerns and the cost associated with facilities . In the United States, institutes that conduct

Pharmacokinetic testing provides data to answer questions such as: How is the drug absorbed and transported? Which cells and organs are affected? What enzymes in the body break down the drug, and how fast does this occur? How is the drug or its metabolites (breakdown products) eliminated from the body? Pharmacodynamic studies examine dose-response effects and often monitor biochemical and physiological changes (such as enzyme activities, heart rate, blood pressure and body temperature) in the test subject . Pharmacodynamic testing, which shows what the body does in response to the drug, is used to answer the question: Is the drug harmful or toxic to cells or organ systems? Toxicology studies address the potential of the drug or its metabolites to kill or damage cells and organs, cause cancer or cause reproductive problems, including birth defects or sterility . Pharmacokinetic and pharmacodynamic studies are used together to reach the goal of preclinical studies, which then answer the question: Is the drug safe? In the United States, preclinical studies must be conducted under FDA guidelines known as current Good Laboratory Practice . Many other countries follow global harmonized regulatory guidelines as well .

research involving animals and that receive federal funding must have an Institutional Animal Care and Use Committee (IACUC) . This committee reviews research protocols and evaluates the care laboratory animals receive . The IACUC is responsible for making sure labs comply with the Animal Welfare Act . Animal models greatly enhance scientists ability to test the effectiveness and safety of new drug candidates . In target validation, researchers may use knock-out mice and/or knock-in mice to validate a target . Knock-out mice are genetically altered to remove mouse versions of human disease genes . Human disease genes can also be knocked in to create mouse models with human diseases like cancer, diabetes, Alzheimers and Parkinsons . Drug candidates are tested on these mice, enabling researchers to check for adverse side effects before giving the candidate drug to humans . Initially, many studies of drug safety and toxicity are done using cell lines. Cell lines are engineered to express genes that are often responsible for adverse reactions . The creation of cell line models has decreased the number of animals needed for testing (reducing cost and time) and helps accelerate the drug development process .*

*For more information on Amgens commitment to ethical use of animals in research, visit www .amgen .com/science/ethical_research .html .

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How Is Dosage Determined?

There are two types of phase 1 dosage studies: SAD studies and MAD studies. SAD: Single-Ascending-Dose Studies A few volunteers are given a small dose of the investigational new drug and observed. If there are no adverse reactions, another group is given a slightly higher dose. This is repeated as many times as needed until volunteers start to exhibit intolerable side effects. At these dosage levels, the investigational new drug is said to have reached maximum tolerated dose (MTD). MAD: Multiple-Ascending-Dose Studies The same volunteers receive higher and higher doses of the investigational new drug until the dosage reaches a certain level. Samples of body fluids are collected with each increase in dosage level to understand how the body processes the investigational new drug. Phase 1 clinical trials are usually conducted in an inpatient clinic where full-time staff can observe the study subjects.

If preclinical trials provide sufficient evidence that a drug candidate is safe, companies submit an Investigational New Drug (IND) application to the FDA . After the FDA approves the IND, companies can begin phased clinical trials in humans . Clinical Trials Clinical trials are tests designed to determine the safety, proper dosage, efficacy, adverse reactions and long-term-use effects of a new drug in human subjects . Clinical trials taking place in humans are conducted under global harmonized guidelines, such as the FDAs current Good Clinical Practice (cGCP), which protects the rights and ensures the safety of human test subjects and follows the U .S . Code of Human Research Ethics . Clinical trials are conducted in three successive phases1, 2 and 3and test progressively larger numbers of humans in each phase . Each phase has a different purpose and helps researchers answer different questions . If a phase is successful, the drug candidate proceeds to the next phase . If unsuccessful, clinical trials are halted, the drug is suspended and the sponsor company returns to the discovery phase to look for another drug candidate . Clinical trials are conducted at different testing sites . It takes several years to complete all three clinical trial phases . Clinical trials are often managed by a contract research organization (CRO), which is an independent organization . The CRO is responsible for all the data management and communication between the sponsor company and physicians overseeing the clinical trials . The CRO also ensures that the study volunteers understand and accept the risks

involved in the clinical trials and that cGCP guidelines are followed .
BIoFaCT A crucial component of initiating a clinical trial is recruiting study subjects who agree to participate and sign a document called informed consent . Potential subjects must be informed about all aspects of the study before they decide to participate . Participants can withdraw their informed consent at any time .

Phase 1 Phase 1 trials represent the first time an investigational new drug is tested on humans . The goal is to evaluate the drugs safety, tolerability and safe dosage range . The testing group is often small, ranging from 20 to 50 volunteers . These are usually healthy volunteers who do not have a disease . However, sometimes patient volunteers will be accepted into a phase 1 clinical trial, particularly when testing oncology therapeutics . Usually these patients have been unsuccessful with available treatments or have few treatment options, or the drugs potential side effects are too risky to involve healthy subjects (such as using some chemotherapeutic agents) . Phase 2 The goal of phase 2 trials is to determine the efficacy and safety of the investigational new drug among a larger group of patient volunteersusually 100 to 300 people . A patient volunteer is someone who has the disease the drug is intended to treat . Some companies divide phase 2 trials into phase 2A (to assess dosage) and phase 2B (to assess efficacy) . Most investigational new drugs fail during this stage because of efficacy and/or safety issues .

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must take place in a facility that meets the countrys strict guidelines, such as the FDAs current Good Manufacturing Practice (cGMP), to ensure safety and purity of the product .

Study Designs
Late-stage trials often include a double-blind randomized controlled test. In this type of study, neither the patient volunteer nor the researcher knows which volunteer belongs to the control group or the experimental group. Each patient volunteer is randomly placed into one of the groups. A third party keeps this documentation and releases it only after the study is over.

BIoFaCT One of the largest challenges associated with clinical trials is the shortage of study subjects .

Phase 4 Phase 4 trials occur after an approved drug is on the market . A goal is to monitor the drugs safety and efficacy when utilized in a normal medical setting in a population of patients Phase 3 The goal of phase 3 trials is to confirm the effectiveness of the investigational new drug and compare it with placebos or therapies already available on the market . To do this, hundreds or thousands of patient volunteers are tested . Phase 3 trials are the most expensive and time-consuming, lasting for a couple of years or longer to establish long-term safety . Once phase 3 is successfully complete, the sponsor company files a new drug or biologics application with the countrys regulatory agency . In the United States, the company would file a New Drug Application (NDA) for a small-molecule drug or a Biologic License Application (BLA) for a large-molecule drug with the FDA . If the governing regulatory authority (the FDA in the United States or the European Medicines Agency, known as the EMEA, in Europe) approves the drug, the sponsor company is permitted to market and sell the product in the country or countries regulated by that authority . The final manufacturing of the drugor large-scale production that could number in the millions . Sometimes adverse reactions, which were not seen in a comparatively small cohort of patients (3,000 patient volunteers as compared to millions), are discovered in larger and more diverse populations . If an adverse reaction is discovered, the drug may be withdrawn from the market . Either the sponsor company can voluntarily withdraw the drug or a regulatory body can pull the drug from the marketplace . After further testing, the drug may or may not be reinstated . The stages in product development, or product pipeline, take, on average, 10 to 15 years to complete . Most investigational drugs do not make it . Out of every 1,000 potential new drugs in discovery, only one will make it to approval .

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Chapter Seven:

Scale-Up and Manufacturing

The manufacturing of biologics is complex, since most are proteinslarge molecules often variable in structure and sensitive to environmental conditions. The manufacturing of biologics has become a science that can be summarized in four key steps: producing the master cell line, growing cells and producing protein, isolating and purifying protein from cells, and preparing the biologic for patients. The whole process from creating the master cell bank to preparing the biologic for patients can take years and cost hundreds of millions of dollars.

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Using R&D Specifications During the R&D phase, researchers develop the initial production methods on a small scale . They also determine the drugs final formulation, or physical form, for clinical trialsfor a biotechnology medicine, usually an injection or infusion . Using all of the R&D data from these production steps, companies devise large-scale production methods to produce enough of the product for the intended market . The scale-up and manufacturing process must adhere to cGMP guidelines to ensure product safety and purity . Common Cell Lines Many biotechnology products are proteins that must be produced by cells grown in culture . Chinese hamster ovary (CHO) cells, nonsecreting (NS0) cells (pronounced NS zero) and E. coli are cell lines used for production of biotherapeutics, especially monoclonal antibodies .
BIoFaCT The American Type Culture Collection (ATCC) is a private, nonprofit resource dedicated to the collection, preservation and distribution of authentic cell lines and other biologic materials . (www .atcc .org)

Other cell lines can be used and may be more suitable . The selection of the cell line depends on the expertise of the company, the properties of the cell and the regulatory requirements . Scale-Up Process The scale-up of a cell culture process can be very difficult and time-consuming, taking as long as several months before researchers can obtain a product . The entire process of producing a biotech product from start to finish is often called a campaign and is usually divided into two main parts: upstream and downstream. Upstream processes involve production of the protein product, most often by using cells (microbial, insect or mammalian) growing in culture . Downstream processes include the recovery, purification, formulation and packaging of the protein product . Upstream Phase Upstream processing begins with the cells that scientists create or engineer to make the protein product . Once the desired cell line is made, it is cryopreserved: scientists freeze a large number of vials of the cells to create a cell bank. To begin a campaign, scientists remove and thaw a vial of cells from the cell bank and initiate a cell culture in a flask containing a small volume of growth media . The initial volume of media can be as little as 5 mL . The media provides the nutrients and the optimum environment for cells to survive . Scale-up is done by gradually transferring the growing cells into successively larger growth vessels containing larger media volumes . The cells are constantly dividing as long as the growth environment remains favorable . Therefore, more and more cells are present

Cell Banks
Cell banks involve a two-tiered frozen cell banking system: a master cell bank (MCB) and a working cell bank (WCB). Scientists use one vial of cells from the MCB to create the WCB. Once established, the WCB is used to produce batches of product in the scale-up process. Working from the same stock of cell line reduces the chance of mutations associated with serial cultures. The MCB is a reserve of cells that scientists use only when absolutely necessary. To protect the integrity of the cell lines, companies store their cell banks in two or more locations within their facilities and in one location off-site.

Growth vessels vary in size:

Flasks hold 5 mL pinner flasks or roller bottles S hold 50 to 200 mL ench top bioreactors hold B 5 to 20 liters ilot scale bioreactors hold P 50 to 200 liters roduction vessels hold P 20,000 liters or more

There are a number of reasons to use these cells . Both CHO and NS0 cells synthesize proteins much like human cells do . Both are immortal cell lines, meaning, they should be able to grow and produce product forever . Researchers are well versed in their optimal culture conditions . Both cell lines have generally regarded as safe (GRAS) status for therapeutic protein production . NS0 cells have the additional advantage of being programmed to produce antibodies, but they do not make or secrete any of their own antibody protein .

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with each step . The greater the number of cells, the more protein product is generated . Scale-Up Monitoring The goal of the scale-up process is to grow cells as quickly as possible and to produce as much protein product as possible . Using the same assays or testing methods used in the initial R&D stages, scientists measure cell viability and concentration, product concentration and product activity at each incremental scale-up stage for monitoring purposes . Lab technicians monitor and control the physical environment in which cell cultures grow . They do this manually in the initial scale-up steps to optimize growth parameters such as temperature, pH, nutrient concentration and oxygen level .

assures product quality and testing during the product development stages well before the product is at the stage of marketing, ensuring that the scale-up and manufacturing processes meet certain standards . The QA department is usually responsible for meeting and reporting quality objectives . Downstream Phase In the downstream phase of manufacturing, the protein product is isolated from the cells that produced it . Proteins found inside the cell (intracellular proteins) require special protocols to extract them for purification . Usually this involves bursting the cells open to release the protein product, which then has to be purified away from the other components that were inside the cell . Proteins found outside of the cell (extracellular proteins) can be easier to isolate . After harvesting the protein product, the

The monitoring process is automated once the cell culture is large enough to be grown in bioreactors . It is crucial during the scale-up, fermentation and manufacturing stages that technicians monitor and test the cultures for contamination by bacteria, yeast or other microorganisms . Any contamination of a culture ruins the entire batch of product and costs a company money and time . Technicians follow very strict protocols for maintaining aseptic conditions at all times during the scale-up and manufacturing stages . Quality Control and Quality Assurance Quality control (QC) and quality assurance (QA) departments are responsible for all of the monitoring that is crucial to the success of the scale-up and manufacturing stages of product development . The QC department

next step is clarification. This is where scientists separate the protein from cellular debris . Then they apply the protein solution to a series of chromatography columns to obtain a pure protein product . Purification of protein mixtures by column chromatography separates proteins based on physical and chemical properties such as size, shape or charge (+ or ) . Additional purification steps remove any residual DNA and deactivate any viral particles that may be present . Researchers verify the isolation and purification of the protein product through confirmed testing protocols . The protein product is then formulated according to the R&D specifications and packaged for use by physicians and patients .

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Chapter Eight:

Biotechnology Medicines

The biotechnology industry uses advanced technologies to apply cellular and molecular biology to create new, beneficial products. Medical biotechnology products are used to treat or prevent diseases. These products include therapeutic proteins, monoclonal antibodies, vaccines, allergy immunotherapy products, blood components and tissues and cells for transplantation.

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Monoclonal Antibodies
Though monoclonal antibody technology was invented in the mid-1970s, it took 20 years before the technology showed its true potential. The first experimental monoclonal antibodies developed in mouse models were ineffective because the human immune system rejected mouse antibodies as foreign. The subsequent development of first humanized, then fully human, antibodies has enabled the successful use of this breakthrough technology in fighting cancer and other serious illnesses.

Therapeutic Proteins Scientists use recombinant DNA technology to make therapeutic proteins, often referred to as biologics . Biologics are used in such fields as oncology, rheumatology, immunology, endocrinology and virology . Approximately 50 recombinant therapeutic proteins are approved for clinical use and are currently marketed, and hundreds more are undergoing clinical trials . Some biologics have been in use for more than 20 years and are considered standard therapy . Doctors have long used therapeutic proteins to replace or supplement patients natural body proteinsespecially when natural protein levels are decreased or lost due to disease .

attacked by a pathogenic virus . Recombinant vaccines are safe and easily grown and stored . Antibodies A major area of biologics is the production of humanized or fully human antibodies . Antibodies can attach to antigens found on a pathogen and flag the pathogen for destruction by the immune system . Antibodies also can attach to proteins on immune cells that are involved in autoimmune responses in diseases like rheumatoid arthritis and multiple sclerosis . Humanized antibodies are engineered to be mostly human to avoid problems with rejection . Fully human antibodies are derived from human cells or human antibody genes . Peptibodies

Some recombinant proteins are versions of natural body proteins, and other versions are not exact versions but produce similar effects in the body . Vaccines Vaccines stimulate the immune system and provide protection against particular diseases . The first vaccines were made with inactivated (killed) or weakened virus unable to reproduce in the body but sufficient to provide immunity upon future exposure to the live virus . Vaccines are also created with recombinant proteins . Scientists use genetic engineering to create recombinant vaccines by inserting genes for desired antigens into a vector . A vaccine vector, or carrier, is a weakened virus or bacterium into which harmless genetic material from another disease-causing organism can be inserted . Typically, the body recognizes antigens as foreign, and white blood cells will attack them . Recombinant vaccines, however, do not cause disease but do have the antigen, thus tricking the body into thinking it is being

Peptibodies are engineered therapeutic fusion proteins with attributes of both peptides and antibodies but are distinct from each, and bind to human targets . Diagnostics In addition to recombinant proteins being used as biologic drugs, scientists use recombinant DNA technology to produce a number of diagnostic tests for diseases, including tests for hepatitis and AIDS . In fact, scientists commonly use recombinant protein antigens as diagnostic reagents in enzyme-linked immunosorbent assays (ELISAs) for the detection of infectious agents such as Severe Acute Respiratory Syndrome (SARS) .

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Chapter Nine:

Future of Biotechnology in Healthcare

Biotechnology can offer patients more and better healthcare choices. New, innovative diagnostics and therapies are changing how some human diseases are prevented and others are treated. This monumental healthcare shift is in its early stages, with novel medicines, diagnostics and technologies in development that hold great potential to improve patients lives.

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Personalized Medicine Personalized medicine is the concept that patients should be treated with therapies and medicines based specifically on each patients unique genetic makeup, for optimal results . Currently, the practice of medicine is based on standards of care that are determined by averaging responses across large groups of people . Personalized medicine is a new paradigm that proposes to manage a patients disease based on the individual patients specific characteristics, including age, gender, height, weight, diet, genetics and environment . Genetic testing is beginning to allow the development of genomic personalized medicinemedical care based on a patients genotype or gene expression profile .
BIoFaCT Molecular diagnostic tests analyze DNA, RNA or protein molecules to identify a disease, determine its course, evaluate responses to therapy or predict individual predisposition to a disease .

Advances in DNA technology are the keys to both pharmacogenomics and personalized medicine . These advances allow for testing and identifying an individuals unique genetic makeup and then comparing those differences with the population at large . Knowledge of the human genome, variations of the genome among individuals and variations of the encoded proteins produced enables researchers to develop medicines that address the individual needs of each patient . Pharmacogenomics and personalized medicine promise to improve clinical trials for new drugs, advance screening technology for diseases and result in moreeffective individualized healthcare and advances in preventive medicine . Genetic Testing The biotechnology industry has brought about vast improvements in testing and diagnosis for genetic diseases . The discovery of singlenucleotide polymorphisms (SNPs)singlenucleotide changes in the DNA sequence was one of the major breakthroughs in genetic testing . SNPs (pronounced snips) represent one of the most common forms of genetic variation among individuals . When a SNP occurs in a gene sequence that encodes for a specific protein, it may change that protein and cause a disease or increase a patients susceptibility to a disease . Utilizing technology to detect SNPs allows for more-accurate diagnosis of genetic diseases and therefore facilitates treatment decisions . Genetic testing provides patients with both an understanding of possible risks for certain diseases and possible opportunities for prevention .
BIoFaCT Approximately 10 million SNPs have been identified in the human genome .

Pharmacogenomics A major movement in healthcare is pharmacogenomics. Pharmacogenomics takes advantage of the fact that individuals have unique genomes representing their genetic makeup . Each genome is likely to react differently to a particular drug and dose amount . The challenge is to identify which drug and which dose will work most optimally for each person or for groups of individuals who share similar genetics . By understanding a patients genetic makeup, a physician can better prescribe a drug and dose level that will optimally work to combat a particular disease .

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Gene Therapy Gene therapy is an emerging area of applied genetics that utilizes recombinant DNA techniques . In this case, the recombinant DNA molecules themselves are used for therapy . Gene therapy involves inserting genes, created by recombinant DNA technology, into the cells and tissues of patients to treat their diseases . Scientists are studying gene therapies for a number of inherited human diseases involving defective genes . The idea is to replace them with new, functional genes .

Cell therapies also could be developed in which undifferentiated stem cells may be implanted along with growth factors to guide their differentiation in the patients body . The aim is to replace the damaged cells with healthy, disease-free cellshence the term regenerative medicine for this approach . The hope is that stem cells, directed to differentiate into specific cell types, could be a renewable source of replacement cells and tissues used to treat a wide range of diseases . Nanotechnology

Since the first clinical trial was initiated in 1990, gene therapy research has expanded greatly, with an increasing number of human trials . The field, still in experimental stages, focuses its efforts on patients with severe and life-threatening diseases who usually have few treatment options or who have failed all available therapies . Stem Cells Stem cells are unspecialized cells that can renew themselves indefinitely to produce more stem cells . They can mature and develop specialized functions or differentiate under specific growth conditions . Stem cells eventually differentiate to form all of the different types of cells that make up the body . The broad potential of an undifferentiated stem cell to make a variety of other cells is the focus of stem cell research . Stem cell therapy, which is still in experimental stages, involves growing stem cells in the lab and guiding them toward a desired cell type by adding different growth factors . The differentiated cells are then surgically implanted . The theory is that stem cells may then integrate into the diseased tissue, replace diseased cells and reverse the effects of the disease .

Nanotechnology deals with the manipulation of molecules and structures on a nanometer (one-billionth of a meter) or atomic scale . Applying nanotechnology for the improvement of human health is called nanomedicine. Biotechnology nanomedicine harnesses living organisms and/or their components on a very small scale . One example of nanomedicine is the experimental use of nanoshells to selectively target and destroy cancer cells at the cellular level . Nanoshells are nanoscopic metallic lenses that are selectively delivered to specific organs or tumors through the bloodstream . Nanoshells have the ability to capture infrared light shown through the skin of a cancer patient and convert it to heat, which kills only the targeted cancer cells . Nanoparticles called buckyballsuniquely shaped and constructed carbon molecules are also showing potential for drug delivery to target molecules or cells . They may make it possible to deliver drugs that do not dissolve in water . Also, because of their small size, they allow more of the drug to be delivered per volume . Scientists are working on nanoparticles to unclog blocked arteries .

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New Drug Delivery Systems Biomedical researchers are studying new ways of delivering drugs within the body that could improve effectiveness . One example is the development of microscopic particles called microspheres that have tiny holes just large enough to carry and deliver drugs to their targets . They are made out of materials that resemble naturally occurring fats in cell membranes and are delivered as a mist sprayed into the nose or mouth . Microsphere therapies are currently available for lung cancer and respiratory illnesses . Current research is investigating the use of microspheres to deliver anticancer drugs to active tumors and for use with anesthetics in pain management .

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Looking Ahead

The practice of medicine has changed dramatically over the years through pioneering advances in biotechnology research and innovation, and millions of patients around the globe continue to benefit from the treatments developed by companies that are discovering, developing and delivering innovative medicines to treat grievous illnesses. As companies continue to develop medicines that address significant unmet needs, future innovations in biotechnology research will bring exciting new advances to help millions more people worldwide.

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Glossary
Amino Acids: The building blocks of proteins . The unique sequence of amino acids in a chain defines the character of a protein molecule . Angiogenesis: The process by which the body forms and develops new blood vessels . Angiogenesis can be both beneficial and harmful; while it can be used to stimulate development in new blood vessels to fight clogged arteries, it also allows malignant tumors to increase in size . Angiogenesis is a key area of cancer research . Antibody: A component of the bodys immune

and statistical techniques, and theory to solve formal and practical problems arising from the management and analysis of biological data .
Biologic: A product derived from a living

organism (from animal products or other biological sources) that is used in the diagnosis, prevention or treatment of disease . Examples of biologics include recombinant proteins, allergy shots, vaccines and hematopoietic growth factors .
Biologic License Application: An application filed

response . A Y-shaped protein, it is secreted in response to an antigenic stimulus . It neutralizes the antigen by binding to it .
Antigen: Any substance, almost always a

with the FDA seeking approval to market a novel biologic in the United States . The application contains a description of the trials and results, formulation, dosage, drug shelf life, manufacturing protocols, packaging information, etc .
Biomarker: A substance used as an indicator of

protein, not normally present in the body that when introduced to the body stimulates a specific immune response and the production of antibodies .
Apoptosis: The process of programmed cell death that may occur in multicellular organisms . Programmed cell death involves a series of biochemical events leading to characteristic cell changes and death . Apoptosis is a key area of cancer research . Aseptic: Describes a product or method free

a biologic state . It is a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes or pharmacologic response to a specific therapy . Biomarker identification and measurement are regarded as key developments for the future of disease treatment . Biomarkers are also used in drug discovery to determine whether a drug is effective in animal models and at what doses effectiveness is reached .
Biopharmaceutical: A synthetic drug produced utilizing certain biotechnology methods . Bioreactor: A device or system for growing cells

of microbiological organisms that would lead to contamination .

Assay: A test procedure whereby a property or

concentration of a substance is measured .

Autoimmune Disorders: Diseases whereby an individuals immune system mounts an attack on a portion of its own tissues . Tissues undergoing such an attack can be destroyed in the process . Base Pairs: Two nucleotides on opposite

or tissues in the context of cell culture . The process of fermentation is performed in a bioreactor to grow large volumes of cells for producing specific proteins .
Biosensor: A device that combines a biological

component with a physicochemical detector component to detect a pathogenic agent .

Biotechnology: Technology based on biology,

complementary DNA or RNA strands that are connected via hydrogen bonds . In DNA, adenine (A) forms a base pair with thymine (T), as does guanine (G) with cytosine (C) . In RNA, thymine is replaced by uracil (U) .
Bioinformatics: The application of information

technology to the field of molecular biology . Bioinformatics entails the creation and advancement of databases, algorithms, computational

especially when used in agriculture, food science and medicine . The United Nations Convention on Biological Diversity defines biotechnology as any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use .

36

Glossary

Blood-Brain Barrier: A physiological mechanism

that alters the permeability of brain capillaries so that some substances, such as certain drugs or toxins, are prevented from entering brain tissue, while other substances are allowed to enter freely .
Cell Bank: A facility where cell lines are kept frozen and stored for later use . Cell banks include master cell banks (MCBs) and working cell banks (WCBs) . MCBs house primary cell strains that are kept stored and not used for production purposes . WCBs house cells used in pharmaceutical production grown from those maintained in an MCB so that their stability and uniformity are well characterized . Cell Culture Technology: The growing of cells

mixtures of different molecules may be separated from each other . This is accomplished by subjecting the mixture to many repeated partitionings between a flowing phase and a stationary phase .
Chromosome: A threadlike linear strand of DNA and proteins in a cell that houses genes . Chromosomes are large enough to be seen under a microscope . In humans, all cells other than germ cells usually contain 46 chromosomes: 22 pairs of autosomes and either a pair of X chromosomes (in females) or an X chromosome and a Y chromosome (in males) . In each pair of chromosomes, one chromosome is inherited from the father and one from the mother . Clarification: A step in the downstream phase

outside of living organisms . With mammalian cell culture, it is sometimes possible to replace animal testing with cell testing when evaluating the safety and efficacy of medicines .
Cell Lines: Generations of cells grown from

original primary cells . Primary cells are cultured directly from a living organism . With the exception of some derived from tumors, most primary cell cultures have limited life spans . After a certain number of population doublings, cells usually stop dividing, though they remain alive . An established or immortalized cell line has acquired the ability to proliferate indefinitely through either random mutation or deliberate modification .
Cell Viability: Determining whether a cell popula-

of manufacturing a biologic . After the protein product is harvested, which may include removing intracellular proteins from cells, clarification steps separate the protein from cellular debris . Individual proteins are then separated using chromatography methods .
Clinical Trial: A type of research study that evaluates the safety and efficacy of new drugs, medical devices and biologics in human subjects . These tests are required by regulatory agencies as a precondition of regulatory clearance to market . Cloning: The replication of a DNA sequence

tion is living or dead . Testing for cell viability usually involves looking at a sample cell population and staining the cells or applying chemicals .
Chemical Library (or Compound Library):

from one organism to create an exact genetic copy; processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning) or organisms .
Codon: A string of exactly three mRNA bases

A collection of stored chemicals that may be used in high-throughput screening for drug development . The larger the chemical library, the better the chance that high-throughput screening will find a hit (a potential drug candidate) .
Chinese Hamster Ovary Cells (CHO cells): A cell line often used in biological and medical research, first introduced in the 1960s . CHO cells are used in studies of genetics, toxicity screening, nutrition and gene expression, particularly expression of recombinant proteins . CHO cells are the most commonly used mammalian hosts for industrial production of protein therapeutics . Chromatography: A process by which complex

that code for a specific amino acid during translation of mRNA into DNA .
Colony Hybridization: The screening of a library

with a labeled probe (radioactive, bioluminescent, etc .) to identify a specific sequence of DNA, RNA, enzyme, protein or antibody .
Column Chromatography: A type of chroma-

tography that uses a column for containing and separating a mixture . It is a commonly used method of purifying proteins .
Combinatorial Chemistry: A discipline in which

a large number of new chemicals are created, compiled into a library and screened for potential therapeutic use .

Glossary

37

Cryopreservation: A process whereby cells or

whole tissues are preserved by cooling to low subzero temperatures . At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped .
DNA (Deoxyribonucleic Acid): DNA is a nucleic

Fusion Proteins (or Chimeric Proteins): Proteins created through the joining of two or more genes that were originally coded for separate proteins . Translation of this fusion gene results in a single new protein with functional properties derived from each of the original proteins . Gel Electrophoresis: A technique used for the

acid that contains the genetic information used in the development and functioning of all organisms . Molecular systems interpret the sequence of these nucleic acids to produce proteins .
DNA Fingerprinting: A technique used to distinguish between individuals of the same species using only samples of their DNA . DNA Ligase: The enzyme that creates a bond between the ends of single-stranded DNA segments . Where restriction enzymes are the scissors of recombinant DNA technology, DNA ligase is the glue . DNA Polymerase: An enzyme that attaches

separation of DNA, RNA or protein molecules by using an electric current applied to a gel matrix . The gel is the medium used to contain, then separate the target molecules . Electrophoresis refers to the use of electricity to move the molecules through the gel matrix . Placing the molecules in wells in the gel and applying an electric current moves the molecules through the matrix at different rates based on their size, charge and/or shape .
Gene: A length of DNA that codes for a particular protein or, in certain cases, a functional or structural RNA molecule . Generally Regarded as Safe (GRAS):

complementary nucleotides to a single stranded human DNA .

DNase (Deoxyribonuclease): Any enzyme that catalyzes the breaking up of linkages in the DNA molecule backbone . Downstream Phase: Involves manufacturing processes including the recovery, purification, formulation and packaging of the protein . Enzyme-Linked Immunosorbent Assay (ELISA):

A designation that a substance is considered safe by experts under the conditions of its intended use . Examples are CHO and NS0 cell lines that have GRAS status for therapeutic protein production .
Genetic Engineering: Alteration of the genetic material of cells or organisms in order, for example, to make them capable of making new substances or performing new functions . Glycosylation: The process by which oligosac-

A biochemical technique to detect the presence of an antibody or an antigen in a sample . It is commonly used to detect infectious agents .
Enzymes: The many proteins produced by

charide units are added to proteins .

Half-life: A measurement of the time it takes for

organisms to act as biochemical catalysts . Enzymes are the mediators of cell metabolism .
Epidermal Growth Factor Receptor (EGFR):

a drug to lose half of its pharmacologic activity or half of its administered amount in the bloodstream or in its target tissues .
Hematopoietic Growth Factors: Protein hormones produced by the body to regulate blood development, affecting the production and maturation of blood-forming cells . High-Throughput Screening: The process of screening a sample of compounds rapidly and in parallel, then analyzing the results and choosing further screening compounds based on this information . Hormones: Substances produced by one tissue and conveyed to another through the bloodstream, usually affecting growth or metabolism .

A cell-surface receptor that is activated when bound by epidermal growth factor . Genetic mutations that lead to EGFR overexpression or overactivity have been associated with a number of cancers .
Extracellular Proteins: Proteins found outside

of a cell .
Fermentation: A process of growing, or culturing, cells by using enzymes to effect chemical changes .

38

Glossary

Humanized Antibodies: Monoclonal antibodies that have been synthesized by using recombinant DNA technology to avoid the clinical problem of an immune response to foreign substances . Humanized antibodies are produced by merging the DNA that encodes the binding portion of a monoclonal mouse antibody with human antibody-producing DNA . Cell cultures are used to express this recombinant DNA and produce these partial-mouse and mostly human antibodies . Hybridization: The process of joining two complementary strands of DNA or one each of DNA and RNA to form a double-stranded molecule . Hybridoma: A cell that has been engineered to

termining the chemical structures of molecules, such as peptides or proteins . MS consists of ionizing chemical compounds to generate charged molecules or molecule fragments, then measuring their mass-to-charge ratios .
Media: Nutrient-rich substances in which cells are grown . Messenger RNA (mRNA): A polynucleotide copy of a DNA gene that communicates the code for building a protein to ribosomes so that new proteins can be built . Microarray: A tool that enables analysis of the

produce a desired antibody in large amounts . Hybridomas are created by fusing immortal tumor cells with antibody-producing B-lymphocyte cells that continuously synthesize identical (or monoclonal) antibodies .
Immortal Cell Line: An established cell line that has acquired the ability to proliferate indefinitely through either random mutation or deliberate modification . Immunotherapy: Modulation of the immune

levels of expression of genes in an organism or comparison of gene-expression levels .

Monoclonal Antibody: An antibody produced

system to achieve a therapeutic goal . Monoclonal antibodies are a type of immunotherapy .

Interferon: A naturally occurring cell-signaling

by cells that are all derived from a single antibody-producing cell . Once a cell capable of generating an antibody with desired therapeutic characteristics is selected, laboratory processes are used to clone (make large numbers of) these cells . Since the cells are all identical and are produced by cloning one specific cell in great numbers, they are called monoclonal and can be used to continuously produce identical antibody molecules with these same therapeutic characteristics .
Nanomedicine: The medical application of nanotechnology . Nanotechnology: The study and creation of

protein produced by the immune system in response to infections such as viral infections or parasites .
Intracellular Proteins: Proteins found inside a cell . Investigational New Drug: A drug that has been approved by the FDA for use in human clinical trials . In vitro: The technique of performing an experi-

systems and devices at the level of molecules and atoms .

Neurotransmitters: Chemicals that are used to

relay, amplify and modulate signals between a neuron and another cell .
Non Secreting (NS0) Cells: Mouse myeloma cells that are used frequently in the production of recombinant antibodies . Nucleotide: The name given to an individual unit

ment outside of a living organism, in a controlled environment such as in a cell culture or in cells grown in a petri dish .
Ion Channels: Pore-forming proteins that help

establish and control the small voltage gradient across the plasma membrane of all living cells . Ion channels are involved in a wide variety of biological processes and are a favorite target in the search for new drugs .
Mass Spectrometry (MS): An analytic technique

of the DNA double helix and RNA . A nucleotide contains one sugar, one phosphate and one base .
Nucleus: The organelle within a living cell that

contains genetic material and controls life functions .

Pathogen: A disease-causing agent such as a

bacterium or virus .
Pegylation: The process of adding polyethylene

for determination of the elemental composition of a sample or molecule . It is also used for de-

glycol to a therapeutic protein, which enables

Glossary

39

the therapeutic protein to stay in the body longer .

Peptibodies: Engineered therapeutic fusion

modified structures by altering the genes that direct their composition .

Proteins: Compounds (chains of amino acids)

proteins with attributes of both peptides and antibodies but that are distinct from each and that can bind to human drug targets .
Peptide Bond: A bond that links together two or more amino acids . A protein is a long chain of amino acids joined together with peptide bonds and therefore is sometimes referred to as a polypeptide . Peptides: Short chains of amino acids . Poly-

constituting the ultimate expression product of a gene . Created through the synthesis performed by ribosomes, proteins are the workhorses of living systems, causing chemical processes and changing as their environment changes .
Proteomics: The study of proteins . Proteomics has three major goals: to identify and quantify all the proteins expressed in an organism, to determine the structure and function of each protein and to study the protein-protein interactions that affects how one protein interacts with other proteins to control cellular processes . Receptor (Cell Receptor): A protein molecule,

peptides, or multiple peptides linked together by peptide bonds, are long chains of amino acids .
Personalized Medicine: Use of the information

contained within a patients genome, genotype or genomic signature to design and tailor the best treatment plan for that individual patient .
Pharmacodynamics: Studies performed to determine what a drug does to the body . Pharmacogenomics: The science of under-

standing the correlation between patients genetic makeup (genotype) and their responses to drug treatment .
Pharmacokinetics: Studies performed to deter-

embedded in either the plasma membrane or the cytoplasm of a cell, to which a mobile signaling (or signal) molecule may attach . A molecule that binds to a receptor is called a ligand, and may be a peptide (such as a neurotransmitter), a hormone, a pharmaceutical drug or a toxin, and when such binding occurs, the receptor goes into a conformational change, which usually initiates a cellular response .
Recombinant DNA: A form of DNA that does not exist naturally and is created by combining DNA sequences that would not normally occur together . Recombinant Proteins: Proteins created by

mine what the body does to a drug .

Phosphorylation: The addition of a phosphate (PO4 ) group to a protein or other organic molecule . Protein phosphorylation plays a significant role in a wide range of cellular processes . Polymerase Chain Reaction (PCR): A method

recombinant DNA technology .

Regenerative Medicine: Research into treatments that restore damaged cells with healthy, diseasefree cells . Restriction Enzymes: Enzymes having the ability

for creating millions of copies of a particular segment of DNA . If a scientist needs to detect the presence of a very small amount of a particular DNA sequence, PCR can be used to amplify the amount of that sequence until there are enough copies available to be detected .
Preclinical Trials (or Studies): Tests that take place in a scientifically controlled setting using cell culture and/or animals as disease models . Product Pipeline: In the biomedical industry,

to cut DNA at a certain location . Scientists use these enzymes to isolate certain types of DNA and place them into new environments . Where DNA ligase is the glue of recombinant DNA technology, restriction enzymes are the scissors .
Reverse Transcriptase: An enzyme used by retroviruses to form a complementary DNA sequence (cDNA) from an RNA templateusually the genome of the retrovirus . The enzyme then performs a complementary template of the cDNA strand such that a double-stranded DNA molecule is formed . This double-stranded DNA molecule is then inserted into the chromosome

the term pipeline refers to the number of unique products or processes reported or in development by a company . Drugs that have entered into clinical trials are said to be in the pipeline .
Protein Engineering: A process for isolating and

studying proteins and generating proteins with

40

Glossary

of the host cell, which has been infected by the retrovirus .

Ribonucleic Acid (RNA): A molecule similar to DNA, which helps in the process of decoding the genetic information carried by DNA . RNA is a nucleic acid transcribed from DNA; mRNA is then translated into proteins . Ribosome: The cell structures within which

Transfer RNA: Molecules that carry amino acids

during the process of protein synthesis during translation .

Transformation: The process of transferring DNA

from a donor to a recipient cell . Scientists use this process to introduce recombinant DNA to bacteria, yeast and mammalian cell lines .
Transgenic: A term describing an organism

protein synthesis occurs .

RNA Interference: A mechanism that inhibits

containing genetic material from a source other than its parents .

Translation: The process that converts an mRNA

gene expression at the stage of translation (see translation) or by hindering the transcription (see transcription) of specific genes . This method has been referred to as posttranscriptional gene silencing and is an important tool for gene-expression research .
Signal Transduction: The movement of signals

sequence into a string of amino acids that form a protein . Translation follows transcription (see transcription).
Upstream Phase: Involves the production of the protein product, most often by using cells (microbial, insect or mammalian) growing in culture . Vaccine: An agent bearing antigens on its

from the outside of a cell to the inside . Scientists are attempting to learn more about this process in cancer cells in order to fight the disease .
Single-Nucleotide Polymorphism (SNP): A DNA

surface that causes activation of the immune system without causing actual disease .
Vector: (1) An organism that serves to transfer a

sequence variation that occurs when a single nucleotideA, T, G or Cin the genome differs between members of a species . Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, drugs, vaccines and other agents .
Southern Blotting: Transfer by absorption of

disease-causing organism (pathogen) from one organism to another . (2) A mechanism whereby foreign genes are moved into an organism and inserted into that organisms genome .
X-ray Crystallography: A method of determin-

DNA fragments separated in electrophoretic gels to membrane filters for detection of specific base sequences by radiolabeled complementary probes .
Stem Cell: Undifferentiated, human cells with the

ing the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and scatters in many different directions . From the angles and intensities of these scattered beams, a crystallographer can produce a three-dimensional picture of the density of electrons within the crystal, and the structure of a substance can be determined .

ability both to multiply and to differentiate into specific cells .

Thermocycler: A laboratory apparatus, used to

amplify segments of DNA via the polymerase chain reaction (PCR) process . The device has a thermal block with holes where tubes holding the PCR mixtures can be inserted . The cycler then raises and lowers the temperature of the block in discrete, preprogrammed steps .
Transcription: The process by which enzymes

use the genetic information on a strand of DNA to create a complementary strand of messenger RNA .

Glossary

41

Timeline of Medical Biotechnology

1950s
1952

it into a tool for observing previously invisible cellular processes .

1963

- Dr . George Gey establishes a continuous cell line taken from a human cervical carcinoma isolated from Henrietta Lacks, who died of the cancer in 1951 . This cell line, containing HeLa cells, is commonly used in medical research .
1953

- Independent groups in the United States, Germany and China synthesize insulin, a pancreatic hormone .
1964

- Dr . James Watson and Dr . Francis Crick reveal the 3-D structure of DNA . - Dr . Joseph Murray performs the first kidney transplant between identical twins .
1955

- The existence of reverse transcriptase is predicted . - Dr . Samuel Katz and Dr . John F . Enders develop the first vaccine for measles .
1967

- An enzyme, DNA polymerase, involved in the synthesis of a nucleic acid, is isolated for the first time . - Dr . Jonas Salk develops the first polio vaccine . The development marks the first use of mammalian cells (monkey kidney cells) and the first application of cell culture technology to generate a commercial product .
1957

- Dr . Maurice Hilleman develops the first American vaccine for mumps .

1969

- The first vaccine for rubella is developed . It is combined with the measles and mumps vaccines to form the measles/mumps/rubella vaccine in 1972 .

1970s
1970

- Scientists prove that sickle-cell anemia occurs due to a change in a single amino acid .
1958

- Dr . Arthur Kornberg of Washington University in St . Louis makes DNA in a test tube for the first time . - The first automatic protein sequencer, the Moore-Stein amino acid analyzer, is developed .

1960s
1960

- Restriction enzymes are discovered . These enzymes cut DNA into pieces and are used for various studies and applications . The restriction enzyme technique becomes a fundamental tool in modern genetic research and opens the way for gene cloning. - Dr . Har Gobind Khorana synthesizes the first complete gene at the University of WisconsinMadison .
1972

- French scientists discover messenger RNA (mRNA) .

1961

- Scientists understand genetic code for the first time .

1962

- DNA ligase, which links DNA fragments together, is used for the first time . - The DNA composition of humans is discovered to be 99 percent similar to that of chimpanzees and gorillas . - The purified enzyme reverse transcriptase is first used to synthesize complementary DNA from purified messenger RNA in a test tube .
1973

- Dr . Osamu Shimomura of Boston University discovers the green fluorescent protein Aequorea victoria in jellyfish . He later develops

- Dr . Stanley Cohen and Dr . Herbert Boyer use bacterial genes to perform the first successful recombinant DNA experiment, which inserted

42

Timeline of Medical Biotechnology

a recombinant DNA molecule into a cell for replication . - Dr . Edwin Southern develops a blotting technique for DNA called the Southern blot . It becomes a seminal technology for studying the structure of DNA .
1974

- The first vaccine for meningococcal meningitis is developed .

1980s
1980

- The U .S . National Institutes of Health (NIH) forms a Recombinant DNA Advisory Committee to oversee recombinant genetic research . - The first vaccine for chicken pox is developed in Japan .
1975

- Colony hybridization and Southern blotting are developed for detecting specific DNA sequences . - The first monoclonal antibodies are produced . Dr . Csar Milstein, Dr . Georges Kohler and Dr . Niels Jeme develop monoclonal antibody technology by fusing immortal tumor cells with antibody-producing B-lymphocyte cells to produce hybridomas that continuously synthesize identical (or monoclonal) antibodies .
1976

- The U .S . Supreme Court rules genetically altered life forms can be patented, opening up enormous possibilities for commercially exploiting genetic engineering . The first patent of this nature was awarded to the Exxon oil company to patent an oil-eating microorganism, which would later be used in the 1989 cleanup of the Exxon oil spill at Prince William Sound, Alaska . - Dr . Stanley Cohen and Dr . Herbert Boyer receive a U .S . patent for gene cloning . - The first automatic gene machine, or genesynthesizing machine, is developed in California . - Founding of Amgen, which will grow to become the worlds largest biotechnology medicines company .
1981

- The NIH releases the first guidelines for recombinant DNA research . - Molecular hybridization is used for the prenatal diagnosis of alpha thalassemia . - Yeast genes are expressed in E. coli bacteria .
1977

- Dr . Baruch Blumberg and Dr . Irving Millman develop the first vaccine for hepatitis B (not recombinant) four years after the virus is discovered . - Scientists in Switzerland clone mice . - The first transgenic animals are produced by transferring genes from other animals into mice .
1982

- Protocols are developed to rapidly sequence long sections of DNA . - Genetically engineered bacteria are used to synthesize the human growth protein somatostatin, marking the first time a synthetic recombinant gene is used to clone a protein . Many consider this to be the advent of the Age of Biotechnology . - Dr . Robert Austrian of the University of Pennsylvania develops the first vaccine for pneumonia .
1978

- The U .S . Food and Drug Administration (FDA) approves the first biologicor recombinant protein: Humulin, Genentechs human insulin drug produced by genetically engineered bacteria for the treatment of diabetes .
1983

- Dr . Herbert Boyer of the University of California, San Francisco, constructs a synthetic version of the human insulin gene and inserts it into the bacterium E. coli, allowing the bacterium to produce human insulin . - The first test-tube baby, Louise Brown, is born in the United Kingdom .

- Dr . Luc Montagnier of the Pasteur Institute in Paris isolates the AIDS virus . - Dr . Kary Banks Mullis invents the polymerase chain reaction (PCR), a technique for multiplying DNA sequences . PCR is recognized as the most revolutionary molecular biology technique of the 1980s . - The FDA approves a monoclonal antibodybased diagnostic test to detect Chlamydia trachomatis. - The first artificial chromosome is synthesized . - The first genetic markers for specific inherited diseases are found .

Timeline of Medical Biotechnology

43

1984

- The DNA fingerprinting technique is developed . When a restrictive enzyme is applied to DNA from different individuals, the resulting sets of fragments sometimes differ markedly from one person to the next . Such variations in DNA are called restriction fragment length polymorphisms and are extremely useful in genetic studies . - The first genetically engineered vaccine is developed for hepatitis B . - The entire genome of the HIV virus is cloned and sequenced .
1985

chromosomes, which are expression vectors for large proteins . - Reverse transcription and the polymerase chain reaction are combined to amplify messenger RNA sequences . - DNA microarray technology, the use of a collection of distinct DNAs in arrays for expression profiling, is first described . The arrayed DNAs are used to identify genes whose expression is modulated by interferon . - The FDA approves a diagnostic serum tumor marker test for ovarian cancer .
1988

- Genetic fingerprinting enters the courtroom . - Genentech becomes the first biotechnology company to launch its own biopharmaceutical product . - Genetically engineered plants resistant to insects, viruses and bacteria are field-tested for the first time . - Cloning of the gene that encodes human lung surfactant protein is accomplished . This is a major step toward reducing premature birth complications . - The NIH approves guidelines for performing experiments in gene therapy on humans .
1986

- Congress funds the Human Genome Project, a massive effort to map and sequence the human genetic code as well as the genomes of other species . - The first agreement between two companies with parallel patents for cross-licensing of biotech products occurs and becomes the prototype .
1989

- The FDA approves Amgens first biologically derived human therapeutic . - Oil-eating bacteria are used to clean up the Exxon Valdez oil spill . - A gene responsible for cystic fibrosis is discovered .

- University of California, Berkeley, chemist Dr . Peter Schultz describes how to combine antibodies and enzymes (abzymes) to create therapeutics . - The automated DNA sequencer is invented in California . - The FDA approves the first monoclonal antibody treatment to fight kidney transplant rejection . - The FDA approves first biotech-derived interferon drugs to treat cancer . In 1988, the drugs are used to treat Kaposis sarcoma, a complication of AIDS . - The FDA approves the first genetically engineered human vaccine to prevent hepatitis B .
1987

1990s
1990

- The FDA approves a genetically engineered tissue plasminogen activator to treat heart attacks . - Dr . Maynard Olson and colleagues at Washington University invent yeast artificial

- The first federally approved gene therapy treatment is performed successfully on a 4-year-old girl suffering from an immune disorder called adenosine deaminase deficiency . - The Human Genome Project, the international effort to map all of the genes in the human body, is launched . Estimated cost: $13 billion . - The FDA licenses the first hepatitis C antibody test, which helps to ensure the purity of blood bank products . - The FDA approves a bioengineered form of the protein interferon gamma to treat chronic granulomatous disease . - The FDA approves a modified enzyme for enzyme replacement therapy to treat severe combined immunodeficiency disease . It is the first successful application of enzyme replacement therapy for an inherited disease .

44

Timeline of Medical Biotechnology

1992

- The U .S . Army collects blood and tissue samples from all new recruits as part of a genetic dog tag program aimed at better identification of soldiers killed in combat . - The FDA approve the first genetically engineered blood-clotting factora recombinant protein used to treat hemophilia A . - The FDA approves a recombinant protein to treat renal cell cancer . - American and British scientists unveil a technique for testing embryos in vitro for genetic abnormalities such as cystic fibrosis and hemophilia .
1993

version of human DNase, which breaks down protein accumulation in the lungs of cystic fibrosis patients . It represents the first new therapeutic drug for managing cystic fibrosis in more than 30 years .
1995

- The FDA approves a recombinant protein to treat multiple sclerosismarking the first new multiple sclerosis treatment in 20 years . - An international research team, led by Dr . Daniel Cohen of the Center for the Study of Human Polymorphisms in Paris, produces a rough map of all 23 pairs of human chromosomes . - Two smaller trade associations merge to form the Biotechnology Industry Organization, an international biotechnology advocacy group .
1994

- The first baboon-to-human bone marrow transplant is performed on an AIDS patient . - The first vaccine for hepatitis A is developed . The NIH, the U .S . Army and the Centers for Disease Control and Prevention are significantly involved in the development and clinical testing of the vaccine . - Scientists at the Institute for Genomic Research complete the first full gene sequence of a living organism (other than a virus) for the bacterium Haemophilus influenzae. - A European research team identifies a genetic defect that appears to be the most common cause of deafness .
1996

- The FDA approves a recombinant protein to treat growth hormone deficiency . - Dr . Mary-Claire King at the University of California, Berkeley, discovers the first breast cancer gene, BRCA1 . - The FDA approves a modified enzyme to treat Gauchers disease . - A multitude of genes, human and otherwise, are identified and their functions described . These include: b, a gene predisposing to obesity O CR, a breast cancer susceptibility gene B CL-2, a gene associated with apoptosis B (programmed cell death) edgehog genes (named because of their H shape) produce proteins that guide cell differentiation in advanced organisms pr, a gene governing reproduction of the V HIV virus - Linkage studies identify genes for a variety of ailments, including bipolar disorder, cerulean cataracts, melanoma, hearing loss, dyslexia, thyroid cancer, sudden infant death syndrome, prostate cancer and dwarfism . - The FDA approves a genetically engineered

- The Department of Biochemistry at Stanford University and Affymetrix develop the GeneChip, a small glass or silica microchip that contains thousands of individual genes that can be analyzed simultaneously . This marks a technological breakthrough in gene expression and DNA-sequencing technology . - A group of scientists sequence the complete genome of a complex organism, Saccharomyces cerevisiae, otherwise known as bakers yeast . The achievement marks the complete sequencing of the largest genome to datemore than 12 million base pairs of DNA . - A new, inexpensive diagnostic biosensor test is developed to allow instantaneous detection of a toxic strain of E. coli, the bacteria responsible for many food-poisoning outbreaks .
1997

- The first human artificial chromosome is created . A combination of natural and synthetic DNA is used to create a genetic cassette that can potentially be customized and used in gene therapy . - The FDA approves a recombinant folliclestimulating hormone to treat infertility . - The FDA approves the first bloodless HIV-antibody test that uses cells from patients gums . - Scientists at the Institute for Genomic Research sequence the complete genome of the Lyme

Timeline of Medical Biotechnology

45

disease pathogen, Borrelia burgdorferi, along with the genome for the organism linked to stomach ulcers, Helicobacter pylori. - Scientists at the University of Wisconsin Madison sequence the E. coli genome . - The FDA approves the first therapeutic antibody to treat cancer in the United States . It is used for patients with non-Hodgkins lymphoma .
1998

2002

- An era of very rapid shotgun sequencing of major genomes is completed . Included are the mouse, chimpanzee, dog and hundreds of other species .
2003

- Celera and NIH complete sequencing of the human genome .

2004

- Human skin is produced for the first time in the lab . - Two research teams culture embryonic stem cells . Embryonic stem cells are used to regenerate tissue and create disorders mimicking diseases . - Scientists at the Sanger Institute of the Washington University School of Medicine in St . Louis sequence the first complete animal genome for the Caenorhabditis elegans worm . - A rough draft of the human genome map is produced, showing the locations of more than 30,000 genes . - The first vaccine for Lyme disease is developed . - The FDA approves a novel monoclonal antibody to treat Crohns disease . - A monoclonal antibody therapy used against breast cancer has favorable results, heralding a new era of treatment based on molecular targeting of tumor cells . - Approval of the Her-2 inhibitor for the treatment of breast cancer patients who have tested positive for the Her-2 mutation brings personalized medicine to oncology .
1999

- The FDA approves the first monoclonal antibody that is an antiangiogenic, inhibiting the growth of blood vesselsor angiogenesis for cancer therapy . - The FDA clears a DNA microarray test system, which aids in selecting medications for a variety of conditions . This is an important step toward personalized medicine .
2006

- The FDA approves a recombinant vaccine against human papillomavirus, which causes genital warts and can cause cervical cancer . - Scientists determine the 3-D structure of the human immunodeficiency virus, which causes AIDS .
2007

- Scientists discover how to use human skin cells to create embryonic stem cells .
2008

- The complete genetic code of the human chromosome is deciphered .

2000s
2000

- Chemists in Japan create the first DNA molecule made almost entirely of artificial parts . The discovery can be used in the fields of gene therapy . - Dr . Craig Venter and his team replicate a bacteriums genetic structure entirely from laboratory chemicals, moving a step closer toward creating the worlds first living artificial organism .

- Scientists at Celera Genomics and the Human Genome Project complete a rough draft of the human genome .
2001

- Science and Nature magazines publish the human genome sequence, making it possible for scientists all over the world to begin researching new treatments for diseases that have genetic origins, such as cancer, heart disease, Parkinsons and Alzheimers .

46

Timeline of Medical Biotechnology

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