This document summarizes the process of producing human insulin through recombinant DNA technology using E. coli bacteria. It involves growing E. coli in a bioreactor to produce proinsulin inclusion bodies, isolating the inclusion bodies through centrifugation, solubilizing and refolding the proinsulin, and purifying it through affinity chromatography, site-specific cleavage, reverse phase chromatography, and polishing. The purified human insulin can then be stored at 4°C for pharmaceutical use in diabetes treatment.

1. Insulin & its industrial production by
using fermentation technique.
Md.Unuse Ali.
MSc 3rd semester
FLSB,SAU
27-09-16

2. What is insulin?
 Source: Beta cells of Islets of Langerhens.
 Chemistry: Peptide-51 AA, A Chain 21 AA, B chain 30 AA.
 MW: 5808.( First high MW hormone to be by the G.Eng)
 Half life: 5-10 Min.
 Basal Level: 3ng/ml or < 10 mlU/ml.
 Water soluble.
 Fate: Endocytosis & Proteolysis (80% in liver & kidney).
 Species Specificity: Bovine & Human.
 Prolong action: Zn & Protamine.
 It exist in Zn crystals as hexamers & monomer.
 When diluted in the circulation , it exist as monomer.

3. Types of Insulin:

4. Insulin & Diabetes:
 Type I- Here no insulin is produced(IDDM or Juvenile)
 Type II- Here insulin is produced in little quantity than
actually needed (INDDM or Maturity onset).

5. History of Insulin Discovery &
Production:
 In 1921, Canadian Scientist Frederic G. Banting & Charles H. Best
purified Insulin from Dogs pancreas at Connaught Laboratories in
University of Toronto .Later it extracted from Cattle & Pig also.
 On May 30, 1922, Eli Lilly signed an agreement to pay royalties to
the University to increase production.
 First bottles contained U-10 Insulin.
 3 to 5 cc were injected at a time .
 Pain & abscesses were common until
purer U-40 became available.
 In 1982, The first licensed drug produced using
recombinant DNA technology was human
insulin, which was developed by Genentech
& licensed as well as marketed by Eli Lily.

6. Cont.

7. Insulin Timeline:

8. Steps of human insulin production:

9. Why E.coli ?
 Cows & pig – immunogenic.
 Yeast cells –costly.
 Plant cells – not fully developed.
 E.coli – Simple, well-understood genetics.
its very easy to manipulate.
culturing cost is minimal.
high level of expression.
fermentation easy to scale up.
inclusion bodies may be easy to purify.

10. Methods of insulin production:
 There two main methods exits for the production of
recombinant human insulin from genetically modified
bacterial cultures:
 Two chain method (both A & B chain are synthesized
by separate E. coli plasmid) .
 Proinsulin method (intracellular or secreted).
 The proinsulin method is currently the most
efficient method because, single isolation & isolation
steps involved.

11. Innoculum preparation:
 According to design process :
 six 200 ml test tubes need to grow for initial culture of
proinsulin producing bacteria
 1L of tryptic soy broth
 .5g of kanamycin monosulfate
 5g of the genetically altered E .coli
 Grow for 24 hrs at 37oc
 Then placed with a in bioreactor to promote further
growth & proinsulin production.

12. Media preparation:
 The E. coli must be placed in a mixture containing the
essential building blocks for growth, including carbon,
nitrogen, phosphorus.
1.Bioreactor with 23L total volume & 16L working volume.
2.The 1L of E.coli & depleted growth medium is mixed with
9L of growth media in bioreactor.( for C- glycerol & yeast
extract, N- A.sulphate & thiamine,)
3.Buffer: pota. dihydrogen phosphate & dipota. phosphate.
4.pH: at 7 .
5.Other nutrients included in the broth are Na. citrate,
Mg. sulphate, a trace element solution, & a vitamin solution.
6.The oxygen tension is also monitored to be kept at a tension
level of 30 %.

14. Fermentation process of insulin
production:
1.Fermentation: The first 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 & kanamycin
monosulphate.
 All of the accomplished by placing the original growth mixture into
a bioreactor in which the parameters can be controlled for
maximum cell growth & insulin production.
 Within a bioreactor the temperature , PH, foam, & feed can be
controlled automatically to yield maximum result.

15.  Equipment: BioNet Reactor.
 Reagents Involved:
1L inoculation solution
25mL NH3
30mL H3PO4
100mL 87% glycerol feed
25g (NH4)2SO4
30G KH2PO4*H2O
20G KHPO4*2H2O
5g Na3-citrate
10g yeast extract
0.7g thiamine
10mL trace element solution
6.5mL vitamin solution
10mL adecanol LG-109 (antifoam)
10mL B-indole acrylic acid
Water up to 10L total
 Parameters:
10L total working volume
31 hour growth phase
pH 7
37 C

16. 2.Centrifugation: Here used for four times throughout the
process .
 Equipment: Avanti J-HC
i) Cell isolation: The first steps in insulin
production is the isolation of the bacterium
containing proinsulin inclusion bodies,
this process also called cell harvesting.
cell harvesting could be used including filtration
& centrifugation.
 Parameter: 7500g for 10 min.
ii) Inclusion body separation: Proinsulin from rest of cell debris by
centrifugation or reverse osmosis.
 Parameter: 15000g for 30 min.
iii) Additional Separation: for removal of reagents.
 Parameter: 17700g for 33 min.
iv) Volume reduction: Prior to downstream purification.
 Parameter: 17700g for 33min.

17. 3. Cell disruption- Homogenization: By high pressure or alkali
treatment.
 Equipment: Nano DeBEE Electric Blade- type Homogenizer.
 Parameter: homogenizer supplies pressure of 45,000 PSI
& Flow rate:150ml/min ( 3L E.coli cell mixture for 20 min)

18. 4. Centrifugation: Same as previous.
5. 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.
 Parameter: Stirring for 6hr at 37 C
8M Urea added.
6 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 .

19.  Parameter: Performed during 6hr solubilizing step
0.8M Na2SO4
0.3M Na2SO4*2H2O

20. 7. Centrifugation: Same as previous.
8. Dialysis: Effectively removes denaturants (urea, beta-
mercaptoethanol or DDT) & dissolved contaminants ( buffer-
tris-HCL, & solubilizing agents).
 Equipment: Spectra/par 1 membrane,MWCO 6-8000.
 Parameters: 4 repetitions
Washing buffer:10mM Tris-HCl(pH 8).
9.Renaturation: To maximize correct bond formation.
 Parameters: Stirring for 20hr at 4 C
1M glycine NaOH (pH 10.5+)
18:1 molal ratio of B-mercaptoethanol to fusion protein

21. 10. Centrifugation: same as previous.
11.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.
 Equipment: IgG-Sepharose column
SDS-PAGE
Superdex 75 PC 3.2/10
 Parameters: 0.3M acetic acid added until pH of 8 is reached
Ammonium acetate
200mM sodium phosphate

22. ii) Site-specific cleavage: two enzymes-
trypsin &
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.
 Parameters: additional of cleavage : Trypsin & carboxypeptidase B,
Acetonitrile, Trifluoroacetic acid (PH 3).
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.

23.  Equipment : Kromasil C-8
iv)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.
 Storage: At 4oC, for not to allow the insulin to completely
come out of solution.