The Master Mix for Insulin: Raw Materials

Raw materials are the most basic components to produce a product. In this section, some of the typical crude raw materials that lead to the industrial production of synthetic insulin will be introduced to you.

- Raw materials used for preparation of recombinant plasmid

(1) Desired Gene

The human insulin gene isolated from the human DNA. However, often, mRNA encodes for insulin is used because finding one gene in the human genome that consists of some 70 000 genes can be considered a mammoth task. Because the B cells of the pancreas make insulin, so make lots of mRNA molecules coding for insulin. This mRNA can be isolated from these cells and used to make cDNA of the insulin gene as this is much simpler.

(2) Vector

Plasmid is the most preferred vector if bacterial host cells are used. The general features of plasmid in insulin production include:

1. It usually replicates independently of bacterial chromosome to produce higher yield of products
2. Permits the reproduction of a foreign DNA by using the bacterial replicating system.
3. Usually contains selective markers to eliminate undesired cells
4. Suitable to accommodate the size of the insulin gene

(3) Specific enzymes

Reverse transcriptase - synthesizes cDNA from the mRNA template that is requiredfor the insertion into the vector

Specific restriction enzymes – to cut DNA at specific sites, such as on the vector for the insertion of the desired insulin gene.

Specific ligases – to join DNA fragments together after the insulin gene has been inserted into the vector so that the gene can be expressed in a host cell.

- Raw Materials used for the fermentation process

(1) The host organism

The bacteria Escherichia coli is commonly used as the host organism to produce the synthetic insulin. However in the 1990s, the insulin production method was improved by using the yeast Saccharomyces cerevisiae instead of E. coli.

This is due to the fact that bacterial cells cannot do post-translational modification. After translation, post-translational conversion to insulin was carried out chemically. By contrast, yeast, as a eukaryote, is capable of post-translational modification, so this simplifies the production of human insulin. (E. coli is used for this website.)

E. coli

Saccharomyces cerevisiae

(2) Media

Luria-Bertani (LB) Medium is used for the production. It is used as both seed-culture and the fermentation media, especially for the cultivation of Escherichia coli. The recipe of LB contains: 1) Bacto-tryptone, 2) Yeast extract, 3) Sodium chloride, 4) dH₂O and 5) pH of 7.5.

Other than the LB components, 2 more components – ampicillin and lactose are present in the media. These two unique components convert the medium into an enrichment liquid culture such that only the desired bacteria that contain the human insulin gene can grow in this type of media. On how it works will be further described in the process description section.

Insulin is a 51 amino acid protein that is made up of two chains, the 21-amino acid A chain and the 30-amino acid B chain, linked by two disulfide bonds.


There are two methods of producing the insulin by genetic recombination:

Method A: The 'A' and 'B' Insulin Chains (generating the chains individually and chemically combining them there after)
Method B: The Pro-insulin Method (creating a single-chain precursor, huma proinsulin, and cleave out a 35-amino acid peptide that joins the two chains)

Methods of Insulin Production: Method A

Method A
This method consists of chemically synthesizing two oligonucleotides which encodes the 21 amino acid A chain and 30 amino acid B chain individually in two different Escherichia coli (E. coli) cells, cultured separately in large-scale fermentation vessels, with subsequent chromatographic purification of the insulin chains produced. The A and B chains are then incubated together under appropriate oxidizing conditions in order to promote interchain disulphide bond formation, forming human insulin.

The diagram illustrates the major steps under molecular level in the production. Click on the diagram below for an enlarged overview:

Two general strategies are commonly used to obtain the human insulin gene. They are:

(a) Complementary DNA (cDNA) obtaining from messenger RNA (mRNA) of the two chains using enzyme reverse transcriptase.

(b) Cloning of cDNA of both chains using polymerase chain reactions (PCR). This involves amplification of the cDNA sequences as not every gene yield measurable amounts of mRNA.

Step 2: Insertion of cDNA of both chains into plasmids

Bacterial plasmids are being cut using specific restriction enzymes for the insertion of the two DNA molecules into separate plasmids. Each cDNA is extended at its 5' terminus with an ATG (methionine) initiation codon for start of translation, and a translation termination signal at its 3' with the sticky ends EcoRI and BamHI (later as restriction sites).

Two vector plasmids are made for both the cDNA. They are inserted in the plasmids at the EcoRI and BamHI sites next to the lacZ gene which encodes for the enzyme β-galactosidase. In E. coli, β-galactosidase is the enzyme that controls the transcription of the genes. To make the bacteria produce insulin, the insulin gene needs to be tied to this enzyme. The cut plasmids are re-ligated by specific DNA ligases.

Step 3: Transfection

Recombinant plasmids enter the bacteria in a process known as transfection. Methods such as the use of CaCl2 treatment and electroporation can be used. These cells are later known as transformed cells.


Step 4: Media and equipment preparation

The LB broth is prepared using the LB powder. It is antoclaved and ampicillin and lactose are added (after the sterilization to prevent denaturation or destruction). Inoculation is done by adding the transformed bacteria into the media.

Preparation of the bioreactor is done too. Parts of the bioreactors are fixed and checked such as the calibration of the pH electrode, pO2 probe, exhaust condensers and air inlet. The bioreactor is then sterilized.

(2) In the Bioreactor

Step 5: Fermentation

This stage consists of small scaling (enrichment liquid culture in shake flask) to large scaling (fermentor). The two chains are grown separately. Small scaling (early stage) uses shake flasks to do the enrichment culture method for selecting the desired type of E. coli for fermentation.

The fermentation broth contains two unique components - an antibiotic known as ampicillin and lactose. Bacterial cells that have sucessful transformation will contain the plasmic gene which contains the ampicillin resistance gene and the lac Z gene which encodes for β-galactosidase in the presence of lactose. These cells therefore can grow in the ampicillin environment and the transcription of the lac Z gene will in turn result in the transcription of the human insulin chain DNA. Bacterial cells that have failed the transformation do not contain the ampicillin resistance gene and the lac Z gene. As a result, the growth of these cells will be suppressed by ampicillin and will not replicate during the fermentation process.

Moving on to the large scale, where transfected bacterial cells are transferred from the small flask and replicated under optimal conditions such as temperature, pH in fermentation tanks. This step involves process monitoring and control. The bacterial cell processes turn on the gene for human insulin chains and then insulin chains are produced in the cell.

(3) Downstream Processing

Step 6: Isolation of crude products

Cells are removed from tanks and are lysed using different methods such as enzyme digestion, freezing and thawing and sonication. For enzyme digestion, lysosome enzyme is used to digest the outer layer of the bacterial cells and detergent mixture is subsequently added to separate the cell wall membrane.

Step 7: Purification of crude product

Centrifugation is conducted to helps separate the cell components from the products. Stringent purification of the recombinant insulin chains must be taken to remove any impurities. This uses several chromatographic methods such as gel filtration and ion-exchange, along with additional steps which exploit differences in hydrophobicity.

Step 8: Obtaining of insulin chains

The proteins isolated after lysis consists of the fusion of β-galactosidase and insulin chains due to the fact that there is no termination or disruption to the synthesis of these two proteins as the genes are linked together.



Therefore, cyanogen bromide is used to split the protein chains at methionine residues, allowing the insulin chains to be obtained.

Step 9: Synthesis of active insulin

Two chains (A and B) forms disulfide bonds using sodium dithionate and sodium sulphite, and the chains are joint through a reaction known as reduction-reoxidation under beta-mercaptoethanol and air oxidation, resulting in Humulin - synthetic human insulin.


Step 10: PR-HPLC to obtain highly purified insulin

Reverse-phase high performance liquid chromatography (PR-HPLC) is performed lastly to remove almost all the impurities, to produce highly purified insulin. The insulin then can be polished and packaged to be sold in the industires.

There are 3 parts to Process Description:

>Part I of Process Description - Brief Introduction<

>You are Here: Part II of Process Description - Method A<

>Part III of Process Description - Method B<

>Part IV of Process Description - Types of Insulin<

Methods of Insulin Production: Method B

The Proinsulin Process:

In 1986, another method to synthesize human insulin using the direct precursor to the insulin gene, proinsulin, was popularized. Many steps are the same as when producing insulin with the A and B chains, except for mostly in the downstream process.

What is proinsulin?
Insulin is naturally synthesized as pre-proinsulin in the pancreas. It is converted to proinsulin with the N-terminal signal peptide enzymatically removed. Proinsulin is composed of the amino acid chains that will form insulin and a connecting 30 residue peptide, that joins one end of chain A to chain B. Enzymatic proteolysis removes the peptide chain to produce insulin.

• The proinsulin coding sequence is inserted into the non-pathogenic E. coli bacteria and the bacteria undergo fermentation where they replicate and produce proinsulin. The connecting sequence between the A and B chains is then spliced away with an enzyme and the resulting insulin is purified.

• At the end of the both manufacturing processes, ingredients are added to insulin to prevent bacteria growth and maintain a neutral pH balance.

There are 3 parts to Process Description:

>Part I of Process Description - Brief Introduction<

>Part II of Process Description - Method A<

>You are Here: Part III of Process Description - Method B<

>Part IV of Process Description - Types of Insulin<

Process Description: Types of Insulin

In the mid 1990s, analog insulin is produced. This is obtained by changing the amino acid sequence. An analog insulin mimics the action of a normal insulin. This somehow "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. They are as the following:-

(1) Humulin insulin does not have strong bonds with other insulin and thus, is absorbed quickly. Glargine changes the chemical structure of the protein to make it have a relatively constant release over 24 hours with no pronounced peaks.

(2) Humalog is an insulin analog that is indicated in the treatment of patients with diabetes mellitus for the control of hyperglycemia. It has faster onset and a shorter duration of action than human regular insulin. Patients with type 1 diabetes uses homalog in regimens that include a longer-acting insulin. However, patients with type 2 diabetes uses homalog without a longer-acting insulin when used in combination therapy with sulfonylurea agents. Humalog may be used in an external insulin pump, but should not be diluted or mixed with any other insulin when used in the pump [3].

There are 3 parts to Process Description:

>Part I of Process Description - Brief Introduction<

>Part II of Process Description - Method A<

>Part III of Process Description - Method B<

>You are Here: Part IV of Process Description - Types of Insulin<

Applications of Insulin

Weight regulation
In the early years of insulin availability, it was marketed for both weight loss and weight gain.

Bodybuilding
Insulin has been discovered by bodybuilders. They know it as "the most anabolic hormone", and a few are injecting themselves with about 10 units of insulin, followed by at least 100 gm of carbohydrates, usually after exercise so that insulin combined with growth hormone will enhance muscle growth rather than fat. This is a very dangerous practice, and could lead to death if sufficient carbohydrates are not consumed in time.

Dialysis shock recovery
A kidney dialysis nurse said that in her clinic they had found that a little bit of insulin added to an elecrolyte solution helped patients absorb electrolytes quickly to recover from dialysis shock.

Cell culture
Insulin is used as an essential growth promoter for mammalian cell cultures in the laboratory .

Organ preserving solution
There are several recipes used for making solution to preserve organs for transplantation, and insulin is an important ingredient in at least some and perhaps all of them. Among other things, the insulin may be increasing cell wall permeability, and helping cells under stress absorb nutrients and eliminate toxins.

Cola drinks
They may be seen as drug delivery systems. The sugar content increases insulin secretion in the body, which may boost caffeine transport across the blood-brain barrier and absorption into cells of the nervous system.

Sport drinks (including Gatorade®)
Oral rehydration solution (water, sugar, and electrolytes). Sugars in these drinks increase insulin secretion in the body, which speeds absorption of water and electrolytes into all the cells of the body.

Therapy for poisoning
By severe calcium channel blocker overdose. A mixture of insulin and glucose may greatly increase survival from this type of poisoning. Many other detoxification applications may exist for acute and chronic poisoning.

Insulin coma therapy
From the mid-1930s to the mid-1950s, this was common in psychiatric treatment of schizophrenia and depression. It was discovered in Germany, and spread to the US with emigration of psychiatrists before World War II. A moderate amount of insulin was given, enough to restrict glucose supply to the brain, causing shock, coma, and convulsions. Electroconvulsive therapy mostly replaced it in the 1950s through 80s. Interestingly, the therapy apparently still persists in Germany, although less insulin is now given, coma is avoided, and it is called "modified insulin therapy". There are fairly recent reports of remarkable success in treatment of schizophrenia and depression. Not being aware of IPT results in Mexico and Canada, psychiatrists have apparently missed out on the benefits of adding medication and nutrients during a mild controlled pulse of hypoglycemia, IPT.

Breast augmentation
In the literature there is a report that insulin self-injected in the breasts of a diabetic woman augmented their size. Further research would have to be done before this could be recommended. Would be concerned about possible increased breast cancer risk.

There are 3 parts to Applications:

>You are Here: Part I - Several Applications of Insulin<

>Part II - Annual Insulin Production<

>Part III - Companies Manufacturing Insulin<

History ~ Raw materials ~ Process Description ~ Applications ~ References
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Annual Insulin Production