How insulin is made – material, manufacture, history, used, parts, components, structure, steps, product

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 […]

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.

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

Dinsmoor, Robert S. “Insulin: A Never-ending Evolution.”

Countdown

(Spring 2001).


Other


Diabetes Digest Web Page.

15 November 2001. <

Home

>.


Discovery of Insulin Web Page.

16 November 2001. <

http://web.idirect.com/~discover

>.

Eli Lilly Corporation.

Humulin and Humalog Development.

CD-ROM, 2001.


Eli Lilly Diabetes Web Page.

16 November 2001. <

http://www.lillydiabetes.com

>.


Novo Nordisk Diabetes Web Page.

15 November 2001. <

http://www.novonet.co.nz

>.

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