How is Insulin Made

Millions of people all over the world die due to diabetes every year. It is one of the most dreaded diseases in the underdeveloped countries. The prime cause of diabetes is inadequate production of insulin by the body.

What is Insulin?
Insulin is basically 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 called Islets of Langerhans in the pancreas. These cells keep on releasing small amount of insulin into the body. Whenever a person consumes something, the blood glucose rises. This raised blood glucose triggers the cells in the Islets of Langerhans to release the necessary amount of insulin. Insulin helps to transport the blood glucose from the blood into the cells. Cells have an outer wall, called a membrane, which controls what enters and exits the cell. Though researchers do not know exactly how insulin works, they do know insulin binds to receptors on the cell's membrane. This activates a set of transport molecules, which lets the glucose and proteins enter the cell. The cells can utilize this glucose as a source of energy to carry out its functions. Once transported into the cell, the blood glucose level returns to normal within hours.

In the absence of insulin, the blood glucose builds up in the blood, and the cells are deprived of their energy source, giving rise to several disorders. If the body fails to produce any or adequate insulin, people need to take artificial insulin. Diabetic patients benefit the most, from manufactured insulin, as they are incapable of producing it naturally.

Background
The attempts to produce insulin outside human body, date back to 1921 when Canadian scientists Frederick G. Banting and Charles H. Best successfully purified insulin, from a dog's pancreas. In 1936, researchers came up with a way, to make insulin with a slower release in the blood. They added a protein found in fish sperm, protamine, which the body is able to break down slowly. One injection lasted for 36 hours. In the 1970s, researchers began to try and produce an insulin that had more resemblance to the body's natural insulin. However, a major breakthrough came in 1980's, with revolutionary inventions in the field of biotechnology. Researchers used the principles of genetic engineering to manufacture human insulin. In 1982, the Eli Lilly Corporation successfully produced a human insulin, that became the first approved genetically engineered pharmaceutical product. Without having to depend on animals, researchers could now produce genetically engineered insulin and that too in abundant quantities.

Raw Materials
Human insulin is grown in the lab inside Escherichia coli, a common bacteria. Though it is by far the most widely used type of bacterium, yeast can also be used as a substitute. The human protein that produces insulin, is obtained through an amino-acid sequencing machine that synthesizes the DNA. Manufacturers input insulin's amino acids (the exact number of which is known to the manufacturers), and the sequencing machine connects the amino acids together. Large tanks are needed to grow the bacteria and also to synthesize insulin. Apart from this, several instruments are necessary to separate and purify the DNA , such as a centrifugation machine and various chromatography and X-ray crystallography instruments.

Manufacturing Process
The insulin gene is a protein, which consists of two separate chains of amino acids, A and B, that are held together with bonds. Amino acids are the basic building blocks of all proteins. The insulin A chain consists of 21 amino acids while B chain has 30. Before producing an active insulin protein, preproinsulin is produced. It is one single long protein chain with the A and B chains together. There is a section in the middle, which links the chains together and a signal sequence at one end, stimulating the protein to start secreting outside the cell. After preproinsulin, the chain evolves into proinsulin, which again is a single chain but without the signaling sequence. Finally, the active protein insulin, the protein without the section linking the A and B chains is generated. At every step, the protein requires specific enzymes to generate the next form of insulin. Any of the following methods can be adopted to produce insulin.

Starting with A and B Chains
One method of manufacturing insulin is, to grow the two insulin chains A and B separately, so as to avoid manufacturing each of the specific enzymes needed at every step. Two mini-genes are needed for this process, one that produces the A chain and the other for the B chain. Since the manufacturers know the exact DNA sequence of each chain, they synthesize each mini-gene's DNA in an amino acid sequencing machine. These two DNA molecules are then inserted into plasmids, that are more readily assimilated in the host's DNA.

Manufacturers then insert the plasmids, into a non-harmful type of the bacterium E. coli. The insertion is done next to the lacZ gene, which allows the insulin to be removed, so that it does not get lost in the bacterium's DNA. Adjacent to this gene is the amino acid methionine, which initiates the protein formation.

Plasmids are mixed up with the bacterial cells to enter the bacteria in a process called transfection. The bacteria synthesizing the insulin then undergo a fermentation process in the large tanks and start multiplying rapidly. After multiplying, the cells are extracted from the tanks and broken down to obtain the DNA. The bacterium's DNA is then treated with cyanogen bromide, a reagent that splits protein chains, at the methionine residue, to separate the insulin chains from the rest of the DNA. The two chains are then mixed together and connected by disulfide bonds through the reduction-reoxidation reaction. An oxidizing agent is then added to this. Centrifugation is done to separate cell components by size and density. At the end the DNA mixture is purified so that only the insulin chains remain.

Proinsulin Process
This is another method to synthesize human insulin. The process starts with the direct precursor to the insulin gene, proinsulin. Many of the steps are the same as in the above method, except in this method the amino acid machine synthesizes the proinsulin gene.
The sequence that codes for proinsulin is inserted into the cells of non-pathogenic E. coli bacteria. The bacteria go through the fermentation process to reproduce and produce proinsulin. Then the connecting sequence between the A and B chains is broken with an enzyme and the resulting insulin is purified.

Analog Insulin
This is an improved method and is primarily done by changing its amino acid sequence and creating an analog, a chemical substance that mimics another substance so well, that it fools the cell. Analog insulin clumps less and assimilates more readily into the blood, allowing the insulin to start working in the body minutes after an injection. Humulin is a type of analog insulin that does not have strong bonds with other insulin and thus, is absorbed quickly. Another analog insulin, called Glargine, changes the chemical composition of the protein to make it have a relatively constant release over 24 hours, with no pronounced peaks.

Over the years, researchers have come up with more advanced procedures to produce insulin, which is very similar to its natural form. People all over the world have become aware of the benefits of manufactured insulin and have started consuming it in large numbers.