Insulin most common bacterium used is Escherichia coli.

Insulin is a
hormone which is produced in the pancreas, it regulates carbohydrate, protein
and fat levels through the absorption of glucose. If a patient has diabetes, where
they have high blood glucose levels; exogenous parenteral insulin injections
can be given if their own pancreas cannot produce sufficient amounts of
endogenous insulin to normalise the blood sugar levels.

Insulin gene is
conserved across species through evolution; thus, animal insulins are often
effective in humans too.  Although
Banting & Best first discovered insulin from the pancreas of dogs, previous
large-scale insulin production was sourced from other animals, mainly unused
pancreas of cows & pigs from abattoirs. 
Pharmaceutical production of insulin in the last couple of decades has
moved from isolating animal insulins to producing human recombinant insulin; genetically
engineered human insulin gene is inserted into bacteria (such as E.coli) to
produce human insulin.  Therefore, this
report will be based on pharmaceutical production of insulin from a genetic
engineering plant.

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Insulin is cultivated in a lab inside
bacterium or yeast; the most common bacterium used is Escherichia coli. Human
protein is required to produce insulin, manufactures obtain this by
synthesising DNA by inputting the exact order of amino acids to create insulin into
an ‘amino-acid sequencing machine’.

Synthesising insulin requires
large tanks and a nutrient source for the bacteria to grow. Different machinery
is necessary to produce insulin both for the purification and separation of DNA
and chromatography and crystallography instruments to verify the purity of the
insulin.

The two major
advantages of inserting an insulin gene into microorganisms are: the insulin
produced is identical to the insulin produced in the pancreas and can be
produced in unlimited quantities.

The purity and
structure of the insulin produced needs to be validated after its synthesis; a
variety of methods may be used such as:

·      
High
performance liquid chromatography to detect impurities

·      
X-ray
crystallography to determine the molecular structure

·      
Gel
filtration to  separate proteins

·      
Amino
acid sequencing to ascertain the protein sequence

Tests must also
be performed on the packaging to certify that the vial is protected.

This process flow diagram shows a method that provides human insulin with
a high yield and high purity from proinsulin, these factors are important for
insulin production as the insulin produced will be injected daily into patients

This process makes the liquid
active pharmaceutical ingredient form of insulin,
the steps of this process are shown below:

1)    Prepare a fermentation media and add
phosphate solutions to the fermenter. Prepare two flasks, each containing a
variety of potassium phosphate monobasic and dibasic compounds, and add to the
fermenter. An IPTG solution is also added to encourage transcription in the
proinsulin gene. Incubate for 4 hours and centrifuge the product to produce a
cell paste. 

2)    Disrupt cells using a Niro Soavi
homogenizer

3)    Removal of contaminant protein by
five washes with 3 different buffers

4)    Solubilise the modified proinsulin in
6-8m (6-8 moles) urea and reducing
agents. Adjust pH to 11.8-12 with NaOH.

5)    Dilute the solubilised protein in a
refolding buffer of Glycine and leave for 48 hours for the refolding process to
take place.

6)    The proinsulin derivative is placed
into an immobilised metal ion affinity chromatography (IMAC) column. A buffer
is then used to remove most of the impurities from the column. The proinsulin
derivative is then washed by a solvent (imidazole) to remove any absorbed
substances.

7)    Add citraconic anhydride to the
proinsulin mixture and stir the sample for 3 hours at a low temperature.

8)    Add Ethylenediaminetetraacetic acid
(EDTA) to the sample and exchange the buffer with a membrane of a maximum
weight of 3000 Da.

9)    The buffer exchanged sample is then digested
by a protein to trypsin.

10)
Warm the
digest solution and leave it to rest for 10 hours so that the citraconic
anhydride can release.

The
resulting solution is purified to remove the by-products gained when the
proinsulin was digested by trypsin. This is done using the following steps:

11)
The warmed
solution is loaded onto a cation exchange column with sodium chloride and
propanol. Reversed-phase High-Performance Liquid Chromatography (RP-HPLC) is
then used to separate the insulin into different fractions of purity.

12)
The insulin
is loaded onto a reverse phase column and is lysed using propanol in the presence
of sodium sulphate and phosphoric acid. The fractions are diluted with
phosphate. The RP-HPLC step is repeated.

13)
Exchange
the sample using a membrane of maximum weight of 3000 Da. The pH should be monitored
and kept at 7.5-8 and at 6-10°C. 

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