In beta-cell of the pancreas and is converted

In 1921, insulin was identified to be an anti-diabetic factor by Banting
and Bestand. It  was clinically used in
the year after.  Manufacturing processes
were developed to extract the insulin from porcine and bovine pancreatic tissue
. From 1921 to 1980, the focus was done on  enhancing  the purity of the insulin and providing
different formulations for altering time-action for improved glucose control (Brange,
1987a,b; Galloway, 1988). Purification was improved by enhancing processing
conditions and extraction and by implementing chromatographic processes (size
exclusion, ion exchange, and reversed-

phase) (Kroeff et al., 1989) to reduce the levels of both insulin
related prteins like proinsulin , and  general protein impurities Formulation
development focused on improving chemical stability by moving from acidic to
neutral formulations and by modifying the time-action

profile through the uses of various levels of zinc and protamine.

Recombinant DNA technology allowed the production the unlimited amount
of insulin. Both  advanced purification
techniques,  and r DNA, allowed the
production of the purest human insulin ever made.


Insulin, a 51-amino acid protein, is a hormone that is synthesized as a
proinsulin precursor in the beta-cell of the pancreas and is converted to
insulin by enzymatic cleavage. The resulting insulin molecule is composed of
two polypeptide chains that are connected by two inter-chain disulfide bonds   The A-chain is composed of 21 amino acids and
the B-chain is composed of 30 amino acids. The interchain disulfide linkages
occur between A7–B7 and A20–B19, respectively. A third intrachain disulfide
bond is located in the A-chain, between residues A6 and A11

The original polypeptide contains 108 amino acid polypeptide, 23 amino
acids on the terminal chain form a signal sequence that enables its transfer to
the endoplasmic reticulum . there, the signal sequence is removed by specific
peptidase. This is Proinsulin. Proinsulin-containing vesicles bud off from the
endoplasmic reticulum and fuse with the golgi apparatus. Subsequently,
proinsulin-containing vesicles (clathrin-coated secretory vesicles), in turn,
bud off from the golgi. Once they move away from the golgi apparatus, they
loose their clathrin coat. These vesicles will be stored in the beta cells of
the pancreas, High glucose level, stimulate these vesicles to fuse with the
plasma membrane and therefore releasing the content into the bloodstream by the
mechanism of exocytosis.   Elevated
levels of blood glucose, or other appropriate signals, cause the vesicles to
fuse with the plasma membrane, thereby releasing their contents into the blood
via the process of exocytosis. Porcine insulin (5777 Da) varies from the human
form (5807 Da) by a single amino acid, whereas bovine insulin (5733 Da) differs
by three residues.


Proinsulin, within the coated secretory granule, undergoes proteolysis ,
leading to the formation of mature insulin . and a 35 amino acid connecting
peptide C. the C peptide is then proteolytically modified by removing 2 amino
acids on each end. At the end , the secretory granules contain low level of
proinsulin, C peptide, and protease, plus the insulin itself. The insulin is
stored in the form of zinc-insulin hexamer: 6 molecules of insulin stabilized
by 2 atoms of zinc



Insulin production


Many disadvantages has lead to cease the animal-derived insulin production.
These disadvantages include: 

Immunogenicity: since
animal derived insulin differ from the human insulin, it formed a high risk to
induce an immunological response in humans therefore decreasing the efficacy of
the insulin.  However,  Porcine insulin, differs from human insulin
by only a single amino acid (residue 30 of the B-chain; threonine in humans,
alanine in pigs) and is essentially non-immunogenic in humans. However, many of
the porcine insulin contaminants (including porcine proinsulin) are immunogenic
in humans   Concerns about the
transmission of spongiform encephalopathies linked to the use of animal-derived
materials is the major reason for the product deletion

Availability. Some 170
million people suffer from diabetes worldwide, a fi`            gure projected to double by 2030. Insulin administration
is essential to the survival of those with type-1 (insulin- dependent)
diabetes, and is required to control the progression of a minority of those with
(the more common) insulin-independent type-2 diabetes. The annual insulin
requirement has surpassed 5000 kg and continues to grow, prompting concern of
an insulin shortfall from slaughterhouse sources.

Such issues and concerns underpinned the development of recombinant
human insulin products, now routinely used in the management of diabetes.









Production of human insulin by recombinant DNA

Human insulin can be produced by recombinant DNA technology , thus
producing the same sequence of amino acids and without any risk of



To begin the process, a nucleotide sequence coding for the insulin A and
B chains are inserted into two different K12 strain E.coli bacterial
cells. These cells were allowed to be cultured and reproduced separately in a
large-scale fermentation vessels, with subsequent chromatographic purification
of the chains of insulin produced. After purification, incubation of the A and
B chains together is done under appropriate oxidizing conditions to allow the
formation of the disulfide interchain bond, thus forming the human insulin crb.




An alternative method (developed in the Eli Lilly research
laboratories), entails inserting a

nucleotide sequence coding for human proinsulin into recombinant E.
coli. This is followed by purification of the expressed proinsulin and
subsequent proteolytic excision of the C peptide in vitro. This approach
requires a single step therefore it is much more popular than the previous
approach. What is produced is human insulin prb. The presence of a
single impurity whether insulin derived or microbial derived, can decrease
efficacy of the insulin and induce immunogenicity. Stringent purification
procedures must be done , including several chromatographic steps: gel
filtration, ion exchange, hydrophobic interaction chromatography and
reverse-phase chromatography. An additional step has been included to ensure a
clean product is formed is the RP-HPLC for the prb.