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<