Chemical process Industries

Hemant Kumara Balsora

Chemical engineering department
hemant.balsora@srict.in
Insulin
 The bloodstream carries glucose-a type of sugar produced from
the digestion of carbohydrates and other foods-to provide
energy to cells throughout the body. Unused glucose is stored
mainly in the liver as glycogen
Normally, blood glucose levels increase after you eat a meal.
When blood sugar rises, cells in the pancreas release insulin,
Liver causing the body to absorb glucose from the blood and lowering
the blood sugar level to normal. When blood sugar drops too
low, the level of insulin declines and other cells in the pancreas
release glucagon, which causes the liver to turn stored glycogen
Pancreas back into glucose and release it into the blood. This brings
blood sugar levels back up to normal.

Intestines Breakdown food in to glucose

Muscles
Pancreas senses the blood sugar& release Insulin

Glycogen
Store molecules of glucose
 What is insulin ?
 Insulin is a chemical messenger that allows cells to absorb glucose, a sugar, from the
blood.
 The pancreas is an organ behind the stomach that is the main source of insulin in the
body.
 Clusters of cells in the pancreas called islets produce the hormone and determine the
amount based on blood glucose levels in the body.
 The higher the level of glucose, the more insulin goes into production to balance sugar
levels in the blood.
 Insulin also assists in breaking down fats or proteins for energy.
 A delicate balance of insulin regulates blood sugar and many processes in the body.
 If insulin levels are too low or high, excessively high or low blood sugar can start to
cause symptoms.
 If a state of low or high blood sugar continues, serious health problems might start to
develop.
 Insulin problems
 In some people, the immune system attacks the islets, and they cease to produce
insulin or do not produce enough.
 When this occurs, blood glucose stays in the blood and cells cannot absorb them to
convert the sugars into energy.
 This is the onset of type 1 diabetes, and a person with this version of diabetes will need
regular shots of insulin to survive.
 In some people, especially those who are overweight, obese, or inactive, insulin is not
effective in transporting glucose into the cells and unable to fulfill its actions.
 Type 2 diabetes will develop when the islets cannot produce enough insulin
Insulin - Production Process

Proinsulin method
 Insulin - Production Process
Proinsulin method

 The first step of the process is to grow enough of the proinsulin producing E. coli bacteria so
as to acquire a sufficient amount of insulin per process.
 In order to do this an original amount of E. coli cells containing the plasmid for proinsulin
production will be grown in test tubes containing tryptic soy broth and kanamycin
monosulfate.
 Within this mixture the tryptic soy broth provides nutrients for the E. coli while the
kanamycin monosulfate acts to kill any bacteria within the mixture which was not given
kanamycin resistance;
Once the growth mixture contains only the growth media and E. coli carrying the plasmid
for proinsulin production,
It is desired that the E. coli cell count be increased and to initiate production of the
proinsulin inclusion bodies.
All of this is accomplished by placing the original growth mixture into a bioreactor in
which the parameters can be controlled for maximum cell growth and insulin production.
Within a bioreactor the temperature, pH, foam, and feed can be controlled automatically
to yield maximum results.
For E. coli the best growth conditions are that of a pH of 7 and a temperature of 37°C.
 2. Cell Isolation

The first step in insulin production is the isolation of the bacterium containing the proinsulin
inclusion bodies, a process also called cell harvesting.
In order to harvest the cells two methods could be used including filtration and centrifugation.
In centrifugation E. coli cells containing proinsulin fusion proteins in the form of inclusion
bodies are separated from the rest of the culture medium. 
As the centrifuge spins, a force greater than gravity acts upon the centrifuged medium, pushing
the more dense particles to the bottom of the mixture.  
In this mixture E. coli cells end up on the bottom.  
Once centrifugation is completed, the supernatant is discarded leaving a mixture with a higher
concentration of bacterial cells.
3. Inclusion Body Separation
After the E. coli bacteria have been disrupted and the proinsulin inclusion bodies have been
released, proinsulin must then be isolated from the rest of the cell debris.  
This may be done by either using centrifugation or reverse osmosis.
Once again the density of the desired product plays a role in separation.
 Proinsulin, having a higher density than most of the other cellular components, will drop to
the bottom of the centrifugation container upon the introduction of forces greater than gravity.
However, due to the low density in comparison to the bacterial cells, the proinsulin will have to
be centrifuged at a greater rpm and centrifugal force to get it to settle to the bottom in an
equivalent amount of time.
Once centrifuged the supernatant will be discarded leaving proinsulin and some impurities.
4. Solubilize inclusion bodies:
Solubilization of inclusion bodies is carried out by addition of a denaturing agent such as
urea or guaminidinium-HCL (GdmHCL).
These agent denature the fusion proteins composing the inclusion bodies.

5. Sulfitolysis:
Sulfitolysis involves addition of –SO3 groups to the reduced sulfur residues on cysteines of
proinsulin polypeptides, preventing the formation of potentially incorrect disulfide bonds
during the solubilization and early purification steps prior to correct refolding of the proteins.
Incorrect disulfide bond formation during the solubilization and renaturation processes of
proinsulin production accounts for a significant decrease in percentage yield
6. Dialysis:
Effectively removes denaturants (urea, betamercaptoethanol or DDT) & dissolved
contaminants ( buffertris-HCL, & solubilizing agents) While retaining intact and chemically-
unmodified fusion protein product.  
7. Renaturation 
In the presence of redox reagents the S-sulfonated proinsulin fusion protein allows quick
reformation of disulfide bonds and shuffling of the bonds to maximize correct bond formation.
During renaturation, the protein must be refolded to its native state as it would occur in the human
body.
The renaturation of proinsulin is “limited by the formation of correct disulfide bonds
 Purification:
i).Affinity chromatography: isolation of proinsulin peptide from fusion protein, further
purification is required to produce an insulin product pure enough for patient use.

ii) Site-specific cleavage: two enzymestrypsin & carboxypeptidase B- its used to cleave the
proinsulin at specific site & convert the proinsulin to native insulin &
C-peptide. Its was also the only method for cleaving the proinsulin.

iii ) Reverse Phase Chromatography: Very effective for purification of C-peptide & human
insulin than Reverse Phase liquid Chromatography, due to high pressure , which increase the
speed & purity of C- peptide & human insulin.

Polishing: Zn+3 complexing and saline dilution: To keep insulin useful for longer periods
of time is by blocking its immediate use by cells , blocking the liver from removing it , &
stabilizing the protein while in the blood stream.