Humulin is the first commercially available biosynthetic human insulin, produced using genetically engineered E. coli bacteria starting in 1978. Insulin is a crucial hormone that regulates blood glucose levels, and its production involves the separate cultivation of insulin chains followed by complex purification processes. Various systems, including different bacterial and yeast strains, are employed for effective insulin production, with a focus on maximizing yield and efficiency.

1. HUMULIN AND IT’S
PRODUCTION

2. HISTORY
Insulin from cattle and pigs was used for many
years to treat diabetes and saved millions of lives,
but it wasn’t perfect, as it caused allergic reactions in
many patients. The first genetically engineered,
synthetic “human” insulin was produced in 1978
using E. coli bacteria to produce the insulin. Eli Lilly
went on in 1982 to sell the first commercially
available biosynthetic human insulin under the brand
name Humulin.

3. WHAT IS INSULIN ?
Insulin is a peptide hormone made and secreted by the pancreas to cause excess
glucose to be taken up from the blood, thereby lowering blood-glucose levels that
could otherwise be toxic . Insulin is made in beta cells, which cluster into islets. The
insulin gene encodes 51 amino acids, a very small protein, composed of two
polypeptides linked by two disulfide bonds . The A chain consists of 21 amino acids
and the B chain consists of 30 amino acids

4. EXPRESSION SYSTEMS USED IN HUMULIN PRODUCTION
Recombinant human insulin is mainly produced either in E. coli or Saccharomyces cerevisiae.
1. Escherichia coli has always been preferred for production of recombinant proteins as it offered
several advantages including high growth rate, simple media requirement, easy to handle, high yield and
very cost effective.
2. Saccharomyces cerevisiae is the most preferred and predominant yeast for large scale commercial
production of insulin, however several other alternate yeast strains have been explored for insulin
production . Among yeast strains, Saccharomyces cerevisiae, Hansenulla polymorpha and Pichia
pastoris are very commonly used for production of recombinant proteins . Like E. coli, they grow
rapidly and are very easy to handle and amenable to various genetic manipulations. Besides, E.coli and
yeast, mammalian cells, transgenic animals and plant expression systems are also employed as a host for
large-scale production of recombinant insulin

5. s
Source E. coli E. coli S. cerevisiae P. pastoris
Destination
of product
Cytoplasm Secreted Secreted Secreted
Biomass cell
dry weight
(g/l)
80, in bioreactor
with fed-batch
culture
1.2, in shake flask
with batch culture
5, in shake flask
with batch culture
59, in bioreactor
with fed-batch
culture
Typical spec.
growth rate
(1/h)
0.08– 0.12 not specified < 0.33 <0.03
Typical spec.
production
rate (mg/gh)
14.2 3.4 0.21 0.375
Product
concentratio
n (g/L)
4.34 0.009 0.075 3.075
Productivity
(mg/l h)
1,085 4.01 1.04 17

6. STEPS INVOLVED IN THE PRODUCTION OF INSULIN

7. STEPS INVOLVED IN THE PRODUCTION OF INSULIN
1. 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.
2. 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.
3. 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. Next to this
gene is the amino acid methionine, which starts the protein formation.
4. 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.

8. 1. 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.
2. After multiplying, the cells are taken out of the tanks and broken open to extract the DNA. 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.
3. The two chains are then mixed together and joined by disulfide bonds through the reduction-reoxidation
reaction. An oxidizing agent is added. The batch is then placed in a centrifuge, a mechanical device that
spins quickly to separate cell components by size and density.
4. The DNA mixture is then purified so that only the insulin chains remain. 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.

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