How insulin is made - material, manufacture, history, used, parts, components, structure, steps, product
Insulin
Background
Insulin is a hormone that regulates the amount of glucose (sugar) in the       blood and is required for the body to function normally. Insulin is       produced by cells in the pancreas, called the islets of Langerhans. These       cells continuously release a small amount of insulin into the body, but       they release surges of the hormone in response to a rise in the blood       glucose level.
Certain cells in the body change the food ingested into energy, or blood       glucose, that cells can use. Every time a person eats, the blood glucose       rises. Raised blood glucose triggers the cells in the islets of Langerhans       to release the necessary amount of insulin. Insulin allows the blood       glucose to be transported from the blood into the cells. Cells have an       outer wall, called a membrane, that controls what enters and exits the       cell. Researchers do not yet know exactly how insulin works, but they do       know insulin binds to receptors on the cell's membrane. This       activates a set of transport molecules so that glucose and proteins can       enter the cell. The cells can then use the glucose as energy to carry out       its functions. Once transported into the cell, the blood glucose level is       returned to normal within hours.
Without insulin, the blood glucose builds up in the blood and the cells       are starved of their energy source. Some of the symptoms that may occur       include fatigue, constant infections, blurred eye sight, numbness,       tingling in the hands or legs, increased thirst, and slowed healing of       bruises or cuts. The cells will begin to use fat, the energy source stored       for emergencies. When this happens for too long a time the body produces       ketones, chemicals produced by the liver. Ketones can poison and kill       cells if they build up in the body over an extended period of time. This       can lead to serious illness and coma.
People who do not produce the necessary amount of insulin have diabetes.       There are two general types of diabetes. The most severe type, known as       Type I or juvenile-onset diabetes, is when the body does not produce any       insulin. Type I diabetics usually inject themselves with different types       of insulin three to four times daily. Dosage is taken based on the       person's blood glucose reading, taken from a glucose meter. Type II       diabetics produce some insulin, but it is either not enough or their cells       do not respond normally to insulin. This usually occurs in obese or middle       aged and older people. Type II diabetics do not necessarily need to take       insulin, but they may inject insulin once or twice a day.
There are four main types of insulin manufactured based upon how soon the       insulin starts working, when it peaks, and how long it lasts in the body.       According to the American Diabetes Association, rapid-acting insulin       reaches the blood within 15 minutes, peaks at 30-90 minutes, and may last       five hours. Short-acting insulin reaches the blood within 30 minutes, it       peaks about two to four hours later and stays in the blood for four to       eight hours. Intermediate-acting insulin reaches the blood two to six       hours after injection, peaks four to 14 hours later, and can last in the       blood for 14-20 hours. And long-acting insulin takes six to 14 hours to       start working, it has a small peak soon after, and stays in the blood for       20-24 hours. Diabetics each have different responses to and needs for       insulin so there is no one type that works best for everyone. Some insulin       is         sold with two of the types mixed together in one bottle.
History
If the body does not produce any or enough insulin, people need to take a       manufactured version of it. The major use of producing insulin is for       diabetics who do not make enough or any insulin naturally.
Before researchers discovered how to produce insulin, people who suffered       from Type I diabetes had no chance for a healthy life. Then in 1921,       Canadian scientists Frederick G. Banting and Charles H. Best successfully       purified insulin from a dog's pancreas. Over the years scientists       made continual improvements in producing insulin. In 1936, researchers       found a way to make insulin with a slower release in the blood. They added       a protein found in fish sperm, protamine, which the body breaks down       slowly. One injection lasted 36 hours. Another breakthrough came in 1950       when researchers produced a type of insulin that acted slightly faster and       does not remain in the bloodstream as long. In the 1970s, researchers       began to try and produce an insulin that more mimicked how the body's natural insulin worked: releasing a small amount of insulin       all day with surges occurring at mealtimes.
Researchers continued to improve insulin but the basic production method       remained the same for decades. Insulin was extracted from the pancreas of       cattle and pigs and purified. The chemical structure of insulin in these       animals is only slightly different than human insulin, which is why it       functions so well in the human body. (Although some people had negative       immune system or allergic reactions.) Then in the early 1980s       biotechnology revolutionized insulin synthesis. Researchers had already       decoded the chemical structure of insulin in the mid1950s. They soon       determined the exact location of the insulin gene at the top of chromosome       11. By 1977, a research team had spliced a rat insulin gene into a       bacterium that then produced insulin.
Frederick Bonting.
In 1891, Frederick Banting was born in Alliston, Ontario. He graduated         in 1916 from the University of Toronto medical school. After Medical         Corps service in World War I, Banting became interested in diabetes and         studied the disease at the University of Western Ontario.
In 1919, Moses Barron, a researcher at the University of Minnesota,         showed blockage of the duct connecting the two major parts of the         pancreas caused shriveling of a second cell type, the acinar. Banting         believed that by tying off the pancreatic duct to destroy the acinar         cells, he could preserve the hormone and extract it from islet cells.         Banting proposed this to the head of the University of Toronto's Physiology Department, John Macleod. Macleod rejected Banting's         proposal, but supplied laboratory space, 10 dogs, and a medical student,         Charles Best
Begining in May 1921, Banting and Best tied off pancreatic ducts in dogs         so the acinar cells would atrophy, then removed the pancreases to         extract fluid from islet cells. Meanwhile, they removed pancreases from         other dogs to cause diabetes, then injected the islet cell fluid. In         January 1922, 14 year-old Leonard Thompson became the first human to be         successfully treat-ed for diabetes using insulin.
Best received his medical degree in 1925. Banting insisted Best also be         credited, and almost turned down his Nobel Prize because Best was not         included. Best became head of the University of Toronto's         physiology department in 1929 and director of the university's         Banting and Best Department of Medical Research after Banting's         death in 1941.
In the 1980s, researchers used genetic engineering to manufacture a human       insulin. In 1982, the Eli Lilly Corporation produced a human insulin that       became the first approved         genetically engineered pharmaceutical product. Without needing to depend       on animals, researchers could produce genetically engineered insulin in       unlimited supplies. It also did not contain any of the animal       contaminants. Using human insulin also took away any concerns about       transferring any potential animal diseases into the insulin. While       companies still sell a small amount of insulin produced from       animals—mostly porcine—from the 1980s onwards, insulin users       increasingly moved to a form of human insulin created through recombinant       DNA technology. According to the Eli Lilly Corporation, in 2001 95% of       insulin users in most parts of the world take some form of human insulin.       Some companies have stopped producing animal insulin completely. Companies       are focusing on synthesizing human insulin and insulin analogs, a       modification of the insulin molecule in some way.
Raw Materials
Human insulin is grown in the lab inside common bacteria. Escherichia coli is by far the most widely used type of bacterium, but yeast is also used.
Researchers need the human protein that produces insulin. Manufacturers       get this through an amino-acid sequencing machine that synthesizes the       DNA. Manufacturers know the exact order of insulin's amino acids       (the nitrogen-based molecules that line up to make up proteins). There are       20 common amino acids. Manufacturers input insulin's amino acids,       and the sequencing machine connects the amino acids together. Also       necessary to synthesize insulin are large tanks to grow the bacteria, and       nutrients are needed for the bacteria to grow. Several instruments are       necessary to separate and purify the DNA such as a centrifuge, along with       various chromatography and x-ray crystallography instruments.
The Manufacturing
Process
Synthesizing human insulin is a multi-step biochemical process that       depends on basic recombinant DNA techniques and an understanding of the       insulin gene. DNA carries the instructions for how the body works and one       small segment of the DNA, the insulin gene, codes for the protein insulin.       Manufacturers manipulate the biological precursor to insulin so that it       grows inside simple bacteria. While manufacturers each have their own       variations, there are two basic methods to manufacture human insulin.
Working with human insulin
1 The insulin gene is a protein consisting of two separate chains of         amino acids, an A above a B chain, that are held together with bonds.         Amino acids are the basic units that build all proteins. The insulin A         chain consists of 21 amino acids and the B chain has 30.
2 Before becoming an active insulin protein, insulin is first produced         as preproinsulin. This is one single long protein chain with the A and B         chains not yet separated, a section in the middle linking the chains         together and a signal sequence at one end telling the protein when to         start secreting outside the cell. After preproinsulin, the chain evolves         into proinsulin, still a single chain but without the signaling         sequence. Then comes the active protein insulin, the protein without the         section linking the A and B chains. At each step, the protein needs         specific enzymes (proteins that carry out chemical reactions) to produce         the next form of insulin.
STARTING WITH A AND B
3 One method of manufacturing insulin is to grow the two insulin chains         separately. This will avoid manufacturing each of the specific enzymes         needed. Manufacturers need the two mini-genes: one that produces the A         chain and one for the B chain. Since the exact DNA sequence of each         chain is known, they synthesize each mini-gene's DNA in an amino         acid sequencing machine.
4 These two DNA molecules are then inserted into plasmids, small         circular pieces of DNA that are more readily taken up by the         host's DNA.
5 Manufacturers first insert the plasmids into a non-harmful type of the         bacterium E. coli. They insert it next to the lacZ gene. LacZ encodes for 8-galactosidase, a gene widely used in recombinant DNA procedures because it is easy to find and cut, allowing         the insulin to be readily removed so that it           does not get lost in the bacterium's DNA. Next to this gene is         the amino acid methionine, which starts the protein formation.
6 The recombinant, newly formed, plasmids are mixed up with the         bacterial cells. Plasmids enter the bacteria in a process called         transfection. Manufacturers can add to the cells DNA ligase, an enzyme         that acts like glue to help the plasmid stick to the bacterium's         DNA.
7 The bacteria synthesizing the insulin then undergo a fermentation         process. They are grown at optimal temperatures in large tanks in         manufacturing plants. The millions of bacteria replicate roughly every         20 minutes through cell mitosis, and each expresses the insulin gene.
8 After multiplying, the cells are taken out of the tanks and broken         open to extract the DNA. One common way this is done is by first adding         a mixture of lysozome that digest the outer layer of the cell wall, then         adding a detergent mixture that separates the fatty cell wall membrane.         The bacterium's DNA is then treated with cyanogen bromide, a         reagent that splits protein chains at the methionine residues. This         separates the insulin chains from the rest of the DNA.
9 The two chains are then mixed together and joined by disulfide bonds         through the reduction-reoxidation reaction. An oxidizing agent (a         material that causes oxidization or the transfer of an electron) is         added. The batch is then placed in a centrifuge, a mechanical device         that spins quickly to separate cell components by size and density.
10 The DNA mixture is then purified so that only the insulin chains         remain. Manufacturers can purify the mixture through several         chromatography, or separation, techniques that exploit differences in         the molecule's charge, size, and affinity to water. Procedures         used include an ion-exchange column, reverse-phase high performance         liquid chromatography, and a gel filtration chromatography column.         Manufacturers can test insulin batches to ensure none of the         bacteria's E. coli proteins are mixed in with the insulin. They use a marker protein that         lets them detect E. coli DNA. They can then determine that the purification process removes the E. coli bacteria.
PROINSULIN PROCESS
11 Starting in 1986, manufacturers began to use another method to         synthesize human insulin. They started with the direct precursor to the         insulin gene, proinsulin. Many of the steps are the same as when         producing insulin with the A and B chains, except in this method the         amino acid machine synthesizes the proinsulin gene.
12 The sequence that codes for proinsulin is inserted into the         non-pathogenic E. coli bacteria. The bacteria go through the fermentation process where it         reproduces and produces proinsulin. Then the connecting sequence between         the A and B chains is spliced away with an enzyme and the resulting         insulin is purified.
13 At the end of the manufacturing process ingredients are added to         insulin to prevent bacteria and help maintain a neutral balance between         acids and bases. Ingredients are also added to intermediate and         long-acting insulin to produce the desired duration type of insulin.         This is the traditional method of producing longer-acting insulin.         Manufacturers add ingredients to the purified insulin that prolong their         actions, such as zinc oxide. These additives delay absorption in the         body. Additives vary among different brands of the same type of insulin.
Analog insulin
In the mid 1990s, researchers began to improve the way human insulin works       in the body by changing its amino acid sequence and creating an analog, a       chemical substance that mimics another substance well enough that it fools       the cell. Analog insulin clumps less and disperses more readily into the       blood, allowing the insulin to start working in the body minutes after an       injection. There are several different analog insulin. Humulin insulin       does not have strong bonds with other insulin and thus, is absorbed       quickly. Another insulin analog, called Glargine, changes the chemical       structure of the protein to make it have a relatively constant release       over 24 hours with no pronounced peaks.
Instead of synthesizing the exact DNA sequence for insulin, manufacturers       synthesize an insulin gene where the sequence is slightly altered. The       change causes the resulting
A diagram of the manufacturing steps for insulin.
proteins to repel each other, which causes less clumping. Using this       changed DNA sequence, the manufacturing process is similar to the       recombinant DNA process described.
Quality Control
After synthesizing the human insulin, the structure and purity of the       insulin batches are tested through several different methods. High       performance liquid chromatography is used to determine if there are any       impurities in the insulin. Other separation techniques, such as X-ray       crystallography, gel filtration, and amino acid sequencing, are also       performed. Manufacturers also test the vial's packaging to ensure       it is sealed properly.
Manufacturing for human insulin must comply with National Institutes of       Health procedures for large-scale operations. The United States Food and       Drug Administration must approve all manufactured insulin.
The Future
The future of insulin holds many possibilities. Since insulin was first       synthesized, diabetics needed to regularly inject the liquid insulin with       a syringe directly into their bloodstream. This allows the insulin to       enter the blood immediately. For many years it was the only way known to       move the intact insulin protein into the body. In the 1990s, researchers       began to make inroads in synthesizing various devices and forms of insulin               that diabetics can use in an alternate drug delivery system.
Manufacturers are currently producing several relatively new drug delivery       devices. Insulin pens look like a writing pen. A cartridge holds the       insulin and the tip is the needle. The user set a dose, inserts the needle       into the skin, and presses a button to inject the insulin. With pens there       is no need to use a vial of insulin. However, pens require inserting       separate tips before each injection. Another downside is that the pen does       not allow users to mix insulin types, and not all insulin is available.
For people who hate needles an alternate to the pen is the jet-injector.       Looking similar to the pens, jet injectors use pressure to propel a tiny       stream of insulin through the skin. These devices are not as widely used       as the pen, and they can cause bruising at the input point.
The insulin pump allows a controlled release in the body. This is a       computerized pump, about the size of a beeper, that diabetics can wear on       their belt or in their pocket. The pump has a small flexible tube that is       inserted just under the surface of the diabetic's skin. The       diabetic sets the pump to deliver a steady, measured dose of insulin       throughout the day, increasing the amount right before eating. This mimics       the body's normal release of insulin. Manufacturers have produced       insulin pumps since the 1980s but advances in the late 1990s and early       twenty-first century have made them increasingly easier to use and more       popular. Researchers are exploring the possibility of implantable insulin       pumps. Diabetics would control these devices through an external remote       control.
Researchers are exploring other drug-delivery options. Ingesting insulin       through pills is one possibility. The challenge with edible insulin is       that the stomach's high acidic environment destroys the protein       before it can move into the blood. Researchers are working on coating       insulin with plastic the width of a few human hairs. The coverings would       protect the drugs from the stomach's acid.
In 2001 promising tests are occurring on inhaled insulin devices and       manufacturers could begin producing the products within the next few       years. Since insulin is a relatively large protein, it does not permeate       into the lungs. Researchers of inhaled insulin are working to create       insulin particles that are small enough to reach the deep lung. The       particles can then pass into the bloodstream. Researchers are testing       several inhalation devices much like that of an asthma inhaler.
Another form of aerosol device undergoing tests will administer insulin to       the inner cheek. Known as buccal (cheek) insulin, diabetics will spray the       insulin onto the inside of their cheek. It is then absorbed through the       inner cheek wall.
Insulin patches are another drug delivery system in development. Patches       would release insulin continuously into the bloodstream. Users would pull       a tab on the patch to release more insulin before meals. The challenge is       finding a way to have insulin pass through the skin. Ultrasound is one       method researchers are investigating. These low frequency sound waves       could change the skin's permeability and allow insulin to pass.
Other research has the potential to discontinue the need for manufacturers       to synthesize insulin. Researchers are working on creating the cells that       produce insulin in the laboratory. The thought is that physicians can       someday replace the non-working pancreas cells with insulin-producing       cells. Another hope for diabetics is gene therapy. Scientists are working       on correcting the insulin gene's mutation so that diabetics would       be able to produce insulin on their own.
Where to Learn More
Books
Clark, David P, and Lonnie D. Russell. Molecular Biology Made Simple and Fun. 2nd ed. Vienna, IL: Cache River Press, 2000.
Considine, Douglas M., ed. Van Nostrand's Scientific Encyclopedia. 8th ed. New York: International Thomson Publishing Inc., 1995.
Periodicals