An introduction to biotechnology

Pioneering science delivers vital medicines
Since Amgen’s founding in 1980, the company’s focus has been on discovering, developing, and delivering novel medicines for patients with serious illnesses. Amgen’s scientists are pioneers in the field of biotechnology, delivering treatments based on advances in cellular and molecular biology. And Amgen therapies have helped millions of people worldwide to fight cancer, kidney disease, bone disease, rheumatoid arthritis, and other serious illnesses.

What is biotechnology?
In 1919, Hungarian agricultural engineer Karl Ereky foresaw a time when biology could be used for turning raw materials into useful products. He coined the term biotechnology to describe that merging of biology and technology. Ereky’s vision has now been realized by thousands of companies and research institutions. The growing list of biotechnology products includes medicines, medical devices, and diagnostics, as well as more-resilient crops, biofuels, biomaterials, and pollution controls. While the field of biotechnology is diverse, the focus of this guide is on biotechnology medicines. How do biotechnology medicines differ from other medicines? A medicine is a therapeutic substance used for treating, preventing, or curing disease. The most familiar type of medicine is a chemical compound contained in a pill, tablet, or capsule. Examples are aspirin and other pain relievers, antibiotics, antidepressants, and blood pressure drugs. This type of medicine is also known as a small molecule because the active ingredient has a chemical structure and a size that are small compared with large, complex molecules like proteins. A medicine can be made by chemists in a lab. Most medicines of this type can be taken by mouth in solid or liquid form. Biotechnology medicines, often referred to as biotech medicines, are large molecules that are similar or identical to the proteins and other complex substances that the body relies on to stay healthy. They are too large and too intricate to make using chemistry alone. Instead, they are made using living factories—microbes or cell lines—that are genetically modified to produce the desired molecule. A biotech medicine must be injected or infused into the body in order to protect its complex structure from being broken down by digestion if taken by mouth. In general, any medicine made with or derived from living organisms is considered a biotech therapy, or biologic. A few of these therapies, such as insulin and certain vaccines, have been in use for many decades. Most biologics were developed after the advent of genetic engineering, which gave rise to the modern biotechnology industry in the 1970s. Amgen was one of the first companies to realize the new field’s promise and to deliver biologics to patients. Like pharmaceuticals, biologics cannot be prescribed to patients until their use has been approved by regulators. For example, in the United States, the Food and Drug Administration evaluates new medicines. In the European Union, the European Medicines Agency manages that responsibility.
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DNA

The molecular structure of DNA—the double helix
Illustration is copyrighted material of BioTech Primer, Inc., and is reproduced herein with its permission.

The science of biotechnology
Biotechnology has been used in a rudimentary form since ancient brewers began using yeast cultures to make beer. The breakthrough that laid the groundwork for modern biotechnology came when the structure of DNA was discovered in the early 1950s. To understand how this insight eventually led to biotech therapies, it’s helpful to have a basic understanding of DNA’s central role in health and disease. What does DNA do? DNA is a very long and coiled molecule found in the nucleus, or command center, of a cell. It provides the full blueprint for the construction and operation of a life-form, be it a microbe, a bird, or a human. The information in DNA is stored as a code made up of four basic building blocks, called nucleotides. The order in which the nucleotides appear is akin to the order of the letters that spell words and form sentences and stories. In the case of DNA, the order of nucleotides forms different genes. Each gene contains the instructions for a specific protein. With a few exceptions, every cell in an organism holds a complete copy of that organism’s DNA. The genes in the DNA of a particular cell can be either active (turned on) or inactive (turned off) depending on the cell’s function and needs. Once a gene is
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How does the body make a protein?
Protein production is a multistep process that includes transcription and translation. During transcription, the original DNA code for a specific protein is rewritten onto a molecule called messenger RNA (mRNA); mRNA has nucleotides similar to those of DNA. Each successive grouping of three nucleotides forms a codon, or code, for one of 20 different amino acids, which are the building blocks of proteins. During translation, a cell structure called a ribosome binds to a ribbon of mRNA. Other molecules, called transfer RNAs, assemble a chain of amino acids that matches the sequence of codons in the mRNA. Short chains of amino acids are called peptides. Long chains, called polypeptides, form proteins.

activated, the information it holds is used for making, or “expressing,” the protein for which it codes. Many diseases result from genes that are improperly turned on or off. What functions do proteins control? The amino acids that form a protein interact with each other, and those complex interactions give each protein its own specific, three-dimensional structure. That structure in turn determines how a protein functions and what other molecules it impacts. Common types of proteins are: • Enzymes, which put molecules together or break them apart. •  Signaling proteins, which relay messages between cells, and receptors, which receive signals sent via proteins from other cells. •  Immune system proteins, such as antibodies, which defend against disease and external threats. • Structural proteins, which give shape to cells and organs. Given the tremendous variety of functions that proteins perform, they are sometimes referred to as the workhorse molecules of life. However, when key proteins are malfunctioning or missing, the result is often disease of one type or another.

Genetic engineering tools
To manipulate cells and DNA, scientists use tools that are borrowed from nature, including: Restriction enzymes. These naturally occurring enzymes are used as a defense by bacteria to cut up DNA from viruses. There are hundreds of specific restriction enzymes that researchers use like scissors to snip specific genes from DNA. DNA ligase. This enzyme is used in nature to repair broken DNA. It can also be used to paste new genes into DNA. Plasmids. These are circular units of DNA. They can be engineered to carry genes of interest. Bacteriophages (also known as phages). These are viruses that infect bacteria. Bacteriophages can be engineered to carry recombinant DNA.

How does genetic engineering work? Genetic engineering is the cornerstone of modern biotechnology. It is based on scientific tools, developed in recent decades, that enable researchers to: • Identify the gene that produces the protein of interest. •  Cut the DNA sequence that contains the gene from a sample of DNA. •  Place the gene into a vector, such as a plasmid or bacteriophage. •  Use the vector to carry the gene into the DNA of the host cells, such as Escherichia coli (E coli) or mammalian cells grown in culture. •  Induce the cells to activate the gene and produce the desired protein. • Extract and purify the protein for therapeutic use. When segments of DNA are cut and pasted together to form new sequences, the result is known as recombinant DNA.

When recombinant DNA is inserted into cells, the cells use this modified blueprint and their own cellular machinery to make the protein encoded by the recombinant DNA. Cells that have recombinant DNA are known as genetically modified or transgenic cells. • G  enetic engineering allows scientists to manufacture molecules that are too complex to make with chemistry. This has resulted in important new types of therapies, such as therapeutic proteins. Therapeutic proteins include those described below as well as ones that are used to replace or augment a patient’s naturally occurring proteins, especially when levels of the natural protein are low or absent due to disease. They can be used for treating such diseases as cancer, blood disorders, rheumatoid arthritis, metabolic diseases, and diseases of the immune system. • Monoclonal antibodies are a specific class of therapeutic proteins designed to target foreign

invaders—or cancer cells—by the immune system. Therapeutic antibodies can target and inhibit proteins and other molecules in the body that contribute to disease. •  Peptibodies are engineered proteins that have attributes of both peptides and antibodies but that are distinct from each. • Vaccines stimulate the immune system to provide protection, mainly against viruses. Traditional vaccines use weakened or killed viruses to prime the body to attack the real virus. Biotechnology can create recombinant vaccines based on viral genes. These new modes of treatment give drug developers more options in determining the best way to counteract a disease. But biotech research and development (R&D), like pharmaceutical R&D, is a long and demanding process with many hurdles that must be cleared to achieve success.
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How are biotechnology medicines discovered and developed?
The first step in treating any disease is to clarify how the disease is caused. Many questions must be answered to arrive at an understanding of what is needed to pursue new types of treatments. • How does a person get the disease? • Which cells are affected? •  Is the disease caused by genetic factors? If so, what genes are turned on or off in the diseased cells? •  What proteins are present or absent in diseased cells as compared with healthy cells? •  If the disease is caused by an infection, how does the infectious organism interact with the body? In modern labs, sophisticated tools are used for shedding light on these questions. The tools are designed to uncover the molecular roots of disease and pinpoint critical differences between healthy cells and diseased cells. Researchers often use multiple approaches to create a detailed picture of the
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disease process. Once the picture starts to emerge, it can still take years to learn which of the changes linked to a disease are most important. Is the change the result of the disease, or is the disease the result of the change? By determining which molecular defects are really behind a disease, scientists can identify the best targets for new medicines. In some cases, the best target for the disease may already be addressed by an existing medicine, and the aim would be to develop a new drug that offers other advantages. Often, though, drug discovery aims to provide an entirely new type of therapy by pursuing a novel target. Selecting a target The term target refers to the specific molecule in the body that a medicine is designed to affect. For example, antibiotics target specific proteins that are not found in humans but are critical to the survival of bacteria. Many cholesterol drugs target enzymes that the body uses to make cholesterol.

Scientists estimate there are about 8,000 therapeutic targets that might provide a basis for new medicines. Most are proteins of various types, including enzymes, growth factors, cell receptors, and cell-signaling molecules. Some targets are present in excess during disease, so the goal is to block their activity. This can be done by a medicine that binds to the target to prevent it from interacting with other molecules in the body. In other cases, the target protein is deficient or missing, and the goal is to enhance or replace it in order to restore healthy function. Biotechnology has made it possible to create therapies that are similar or identical to the complex molecules the body relies on to remain healthy. The amazing complexity of human biology makes it a challenge to choose good targets. It can take many years of research and clinical trials to learn that a new target won’t provide the desired results. To reduce that risk, scientists try to prove the value of targets through research

Models for studying disease
The following tools help researchers gain insights into how disease develops. Cell cultures. By growing both diseased and healthy cells in cell cultures, researchers can study differences in cellular processes and protein expression. Cross-species studies. Genes and proteins found in humans may also be found in other species. The functions of many human genes have been revealed by studying parallel genes in other organisms. Bioinformatics. The scientific community generates huge volumes of biological data daily. Bioinformatics helps organize that data to form a clearer picture of the activity of normal and diseased cells. Biomarkers. These are substances, often proteins, that can be used for measuring a biological function, identifying a disease process, or determining responses to a therapy. They also can be used for diagnosis, for prognosis, and for guiding treatment. Proteomics. Proteomics is the study of protein activity within a given cell, tissue or organism. Changes in protein activity can shed light on the disease process and the impact of medicines under study.

experiments that show the target’s role in the disease process. The goal is to show that the activity of the target is driving the course of the disease. Selecting a drug Once the target has been set, the next step is to identify a drug that impacts the target in the desired way. If researchers decide to use a chemical compound, a technology called drug screening is typically used. With automated systems, scientists can rapidly test thousands of compounds to see which ones interfere with the target’s activity. Potent compounds can be put through added tests to find a lead compound with the best potential to become a drug. In contrast, biologics are designed using genetic engineering. If the goal is to provide a missing or deficient protein, the gene for that protein is used for making a recombinant version of the protein to give to patients. If the goal is to block the target protein with an antibody, one common approach is to expose

transgenic mice to the target so as to induce their immune systems to make antibodies to that protein. The cells that produce these specific antibodies are then extracted and manipulated to create a new cell line. The mice used in this process are genetically modified to make human antibodies, which reduces the risk of allergic reactions in patients. Developing the drug Once a promising test drug has been identified, it must go through extensive testing before it can be studied in humans. Many drug safety studies are performed using cell lines engineered to express the genes that are often responsible for side effects. Cell line models have decreased the number of animals needed for testing and have helped accelerate the drug development process. Some animal tests are still required to ensure that the drug doesn’t interfere with the complex biological functions that are found only in higher life-forms.

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If a test drug has no serious safety issues in preclinical studies, researchers can ask for regulatory permission to do clinical trials in humans. There are three phases of clinical research, and a drug must meet success criteria at each phase before moving on to the next one. Phase 1. Tests in 20 to 80 healthy volunteers and, sometimes, patients. The main goals are to assess safety and tolerability and explore how the drug behaves in the body (how long it stays in the body, how much of the drug reaches its target, etc.). Phase 2. Studies in about 100 to 300 patients. The goals are to evaluate whether the drug appears effective, to further explore its safety, and to determine the best dose. Phase 3. Large studies involving 500 to 5,000 or more patients, depending on the disease and the study design. Very large trials are often needed to determine whether a drug can prevent bad health outcomes. The goal is to compare the effectiveness, safety, and tolerability of the test drug with another drug or a placebo. If the test drug shows clear benefits and acceptable risks in phase 3, the company can file an application requesting regulatory approval to market the drug. In the United States, the Food and Drug Administration evaluates new medicines. In the European Union, the European Medicines Agency manages that responsibility. Regulators review data from all studies and decide whether the medicine’s benefits outweigh any risks it may have. If the medicine is approved, regulators may still require a plan to reduce any risk to patients. A plan to monitor side effects in patients is also required.

A company can continue doing clinical trials on an approved medicine to see if it works under other specific conditions or in other groups of patients, and additional trials may also be required by regulatory agencies. These are known as phase 4 studies. The whole drug development process takes 10 to 15 years to complete on average. Very few test drugs are able to clear all the hurdles along the way.

The right tool for the target
A key early decision in drug discovery is whether to pursue a target by using a small-molecule chemical compound or a large-molecule biologic. Each has its advantages and disadvantages. Small molecules can be designed to cross cell membranes and enter cells, so they can be used for targets inside cells. Some may also cross the blood-brain barrier to treat psychiatric illness and other brain diseases. Biologics usually cannot cross cell membranes or enter the brain. Their use is largely restricted to targets that sit on the cell surface or circulate outside the cell. Small molecules often have good specificity for their targets, but therapeutic antibodies tend to have extremely high specificity. Most large molecules stay in the body longer, resulting in the need for less frequent dosing.

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How are biotechnology medicines made?
The manufacture of biologics is a highly demanding process. Protein-based therapies have structures that are far larger, more complex, and more variable than the structure of drugs based on chemical compounds. Plus, protein-based drugs are made using intricate living systems that require very precise conditions in order to make consistent products. The manufacturing process consists of the following four main steps: 1.  Producing the master cell line containing the gene that makes the desired protein 2. Growing large numbers of cells that produce the protein 3. Isolating and purifying the protein 4. Preparing the biologic for use by patients Some biologics can be made using common bacteria, such as E coli. Others require cell lines taken from mammals, such as hamsters. This is because many proteins have structural features that only mammalian cells can create. For example, certain proteins have sugar molecules attached to them, and they don’t function properly if those sugar molecules are not present in the correct pattern. Maintaining the right growth environment The manufacturing process begins with cell culture, or cells grown in the laboratory. Cells are initially placed in petri dishes or flasks
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containing a liquid broth with the nutrients that cells require for growth. During the scale-up process, the cells are sequentially transferred to larger and larger vessels, called bioreactors. Some bioreactor tanks used in manufacturing hold 20,000 liters of cells and growth media. At every step of this process, it is crucial to maintain the specific environment that cells need in order to thrive. Even subtle changes can affect the cells and alter the proteins they produce. For that reason, strict controls are needed to ensure the quality and consistency of the final product. Scientists carefully monitor such variables as temperature, pH, nutrient concentration, and oxygen levels. They also run frequent tests to guard against contamination from bacteria, yeast, and other microorganisms. When the growth process is done, the desired protein is isolated from the cells and the growth media. Various filtering technologies are used to isolate and purify the proteins based on their size, molecular weight, and electrical charge. The purified protein is typically mixed with a sterile solution that can be injected or infused. The final steps are to fill vials or syringes with individual doses of the finished drug and to label the vials or syringes, package them, and make them available to physicians and patients.

What does the future of biotechnology therapies look like?
Biotechnology is still a relatively new field with great potential for driving medical progress. Much of that progress is likely to result from advances in personalized medicine. This new treatment paradigm aims to ensure that patients get the therapies best suited to their specific conditions, genetic makeups, and other health characteristics. For example, a new discipline called pharmacogenomics seeks to determine how a patient’s genetic profile affects his/her responses to particular medicines. The goal is to develop tests that will predict which patient genetic profiles are mostly likely to benefit from a given medicine. This model is sometimes called personalized medicine. Pharmacogenomics has already changed the way clinical trials are conducted: Genetic data is
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routinely collected so that researchers can determine whether different responses to a test medicine might be explained by genetic factors. The data is kept anonymous to protect patients’ privacy. Biotechnology is also revolutionizing the diagnosis of diseases caused by genetic factors. New tests can detect changes in the DNA sequence of genes associated with disease risk and can predict the likelihood that a patient will develop a disease. Early diagnosis is often the key to either preventing disease or slowing disease progress through early treatment. Advances in DNA technology are the keys to pharmacogenomics and personalized medicine. These developments promise to result in more effective, individualized healthcare and advances in preventive medicine.

Emerging treatments
Gene therapy involves inserting genes into the cells of patients to replace defective genes with new, functional genes. The field is still in its experimental stages but has grown greatly since the first clinical trial in 1990. Stem cells are unspecialized cells that can mature into different types of functional cells. Stem cells can be grown in a lab and guided toward the desired cell type and then surgically implanted into patients. The goal is to replace diseased tissue with new, healthy tissue. Nanomedicine aims to manipulate molecules and structures on an atomic scale. One example is the experimental use of nanoshells, or metallic lenses, which convert infrared light into heat energy to destroy cancer cells. New drug delivery systems include microscopic particles called microspheres with holes just large enough to dispense drugs to their targets. Microsphere therapies are available and being investigated for the treatment of various cancers and diseases.

Looking ahead
The practice of medicine has changed dramatically over the years through pioneering advances in biotechnology research and innovation; and millions of patients worldwide continue to benefit from therapeutics 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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