RECOMBIN

ANT
INSULIN
 HISTORY
Sharpey-Shafer propsed that patients with diabetes had just one hormone lacking
01 1910 from their pancreas.

02 1921 Frederick Banting discovered how to extract insulin from a dog pancreas.

Leonard Thompson,a 14 year old child became the first person to recieve an
03 1922 injection of insulin.

04 1923 Eli Lilly a pharmaceutical company ,began mass producing insulin shortly after.

05 1936 Novo Nordisk Pharmaceuticals introduced the first slower-acting insulin.

E.Coli bacteria were used to manufacture the first genetically engineered synthetic
06 1978 “human” insulin.

Eli Lilly went to market the first commercially accessible biosynthetic human
07 1982 insulin under the brand name HUMULIN.
 STRUCTURE OF INSULIN

• Insulin is composed of two protein chains, with 21 amino acids

in the A chain and 30 amino acids in the B chain, totalling 51
in number, linked by disulphide bridges.

• The pro-hormone proinsulin, from which insulin is derived,

contains 74 amino acids, has a molecular weight of 5802 Da,
and has an isoelectric point of 5.5.

• Proinsulin secreted by the beta cells is relatively inactive

under biological conditions, but after cleavage in two places
yields the two chains (B and A) of the active hormone insulin, and
the biologically inactive C peptide.
 RECOMBINANT INSULIN PRODUCTION

Prior to genetic engineering, many diabetics cannot make insulin and therefore need an external source.
Since human insulin could not isolated readily, animal insulin such as the pig or cow was used for injection
although the insulin from these species is not identical to the human protein.
Currently, insulin is industrially made by genetic engineering.

Production of insulin by recombinant means display several advantages, including

1) Reliability of supply

2) Elimination of the risk of accidental transmission of disease due to the presence of pathogen in
animals

3) Economically attractive (once the initial capital investment has been made).
 PRODUCTION STEPS IN INSULIN
WORKING
• Single colony of E.coli strain BL21 harboring
either of above mentioned plasmids was grown in
LB medium(10g/L bacto tryptone,5/L yeast
extract and 5g/Nacl).
• The cultures were then diluted with freezing
medium(contains cryo preserving agent to protect
the cells from damage due to the low
temperature)and stored at-80℃ or at -170℃.
INOCULUM
• Sterile LB medium supplemented with 30 microgram/ml of
kanamycin or 50 microgram/ml of ampicilin in a shake flask
was inoculated from working bank. and incubated 15 hours on a
shaker at 37° C and approximately 250 rpm.
• Sterile medium was inoculated with 5-10%flask culture where,
INCUBATION TIME: 5-8 hours
INCUBATION TEMPERATURE: 37℃
PH: 6.9±0.5
AGITATION AND AERATION: To maintain the dissolved
oxygen level above 20% air saturation.
 PRODUCTION

• The production medium was inoculated with 1-10%

inoculum culture and incubated at 37° C.
• Agitation-aeration rates were set to maintain the dissolved
oxygen level above 20% air saturation.
• The pH was maintained at 6.9±0.5.
• NH4OH/ Propinol was used as antifoaming agent
• Sterile solution of 50%glucose was infused to supply
carbon source
• Once cell concentration reached an OD600 of about
30 ,sterile solution of IPTG-Isopropyl beta-D-1-
thiogalactopyranoside(final concentration 1mM)was
infused and growth continued for 6 hours ,cell concentration
reached an approximate OD660 OF 50.
• The culture was then chilled and cells were recovered by
centrifugation

PRODUCTION MEDIUM TRACE ELEMENTS

 Production of insulin

There are two ways of producing insulin

using recombinant DNA technology,

two-chain method Producing proinsulin

 Materials Required:
Desired
Gene
In humans, proinsulin is
encoded by the INS gene.

Vector
Plasmid is the most preferred vector
if bacterial host cells are used.

Specific
enzymes
Reverse transcriptase, Specific
restriction enzymes, Specific ligases

The host organism

Bacteria, yeast etc
 Upstream Processing

01 02 03 04

Insertion of cDNA of
Obtaining of human Transfection Fermentation
both chains into
insulin gene plasmids
 Method A:

Step 1: Obtaining of human insulin gene:

Gene involved in the production

of insulin in human – INS

Chromosome number: 11
Step 2: Insertion of cDNA of both chains into plasmids

Insertion of cDNA of both The cutting pattern of EcoRI

chains A and B into two
different plasmids.
 Step 3: Transfection

Recombinant plasmids enter the bacteria in a process known as transfection.

Methods such as the use of CaCl2 treatment and electroporation can be used.
These cells are later known as transformed cells.
 Method B: The Proinsulin Process

In 1986, another method to synthesize human insulin using the direct

precursor to the insulin gene, proinsulin, was popularized. Many steps
are the same as when producing insulin with the A and B chains,
except for mostly in the downstream process.
What is Proinsulin ?
 DOWNSTREAM PROCESS
Insulin Enzymatic Polishing of
isolation Cleavage humulin
By Centrifugation &
Using cyanogen bromide Using Zinc complexation
Microfiltration

1 3
5

2 4

Purification of
crude product RP-HPLC
 precipitation Last step for purification
 chromatography
 DOWNSTREAM PROCESS
INSULIN
ISOLATION

The fermented broth is subjected to differential centrifugation to obtain the inclusion

bodies.
Then they undergo microfiltration to avoid unnecessary cell contents to enter the
chromatography process for further purification.
In large scale production ,
 cell homogenization is done to lyse the cell.
 Continuous centrifugation and filtration is done simultaneously to remove the
impurities.
 DOWNSTREAM PROCESS
PURIFICATUION
OF CRUDE
PRODUCT
 Precipitaion ,
1. nucleic acids : polycationic agents such as polyethyleneimine (PEI)
2. addition of salts or using acidic pH decreases the load of host cell protein.

chromatographic techniques used :

i. Size exclusion chromatography
ii. Ion exchange chromatography & affinity chromatography
 DOWNSTREAM PROCESS
PURIFICATUION
OF CRUDE
PRODUCT
 For direct capture from crude feedstocks, expanded bed adsorption
(EBA) chromatography has been integrated into a number of
downstream processes with industrial focus.

 EBA may be used for feeds containing intact cells or cell debris,
however, clogging and fouling due to interaction with the resin may
compromise resin performance and cleanability.

 Scale-up of EBA chromatography has so far been limited to 1 m

diameter EB columns; for processing of large volumes several
columns have to be operated in parallel.
 DOWNSTREAM PROCESS

Enzymatic cleavage

The proteins isolated after lysis consists of the

fusion of β-galactosidase and insulin chains due
to the fact that there is no termination or
disruption to the synthesis of these two
proteins as the genes are linked together.

Thus , cyanogen bromide is used to split the

protein chains at methionine residues, allowing
the insulin chains to be obtained.
 DOWNSTREAM PROCESS

Production of active insulin :

Two chains (A and B) forms disulfide bonds using
sodium dithionate and sodium sulphite, and the
chains are joint through a reaction known as
reduction-reoxidation under beta-mercaptoethanol
(REDUCING AGENT) and air oxidation, resulting in
Humulin - synthetic human insulin.
 DOWNSTREAM PROCESS
RP-HPLC
 Reverse-phase high performance liquid chromatography (RP-HPLC) is performed lastly to
remove almost all the impurities, to produce highly purified insulin.
 RP-HPLC is a common method used to analyze insulin products since it can separate insulin
into its different species, and the use of high pressure increases the speed of the process
and purity of the product.
 RP-HPLC involves a non-polar stationary phase and a polar mobile phase. Since insulin is
non-polar and large, it adheres to the stationary phase column whereas the mobile phase
contains methanol or acetonitrile in a buffer solution.
 Conveniently, both the chains of insulin can be separated using this method where for the A
chain, trifluoroacetic acid is used while for the B chain, formic acid is used.
 DOWNSTREAM PROCESS
Polishing of humulin

 zinc ions are used to form crystal complexes with insulin, named 2Zn insulin,
which have a hexagonal configuration while exhibiting axial symmetry, hence
slowing the release of insulin to the body and preventing its immediate use by
cells.
 The formation of crystals is achieved through batch crystallization, and the
solution is stored at 4°C.
 crystallization with zinc can help alleviate zinc deficiency.
 70% of the complexed crystalline insulin is mixed with 30% monomeric insulin,
which allows for immediate use of the monomeric insulin by the body, while the
complex insulin is released slowly over an extended period.
 STORAGE
There are many ways to store insulin depending on whether it has been opened or not:
 Before opening, insulin must be stored between +2°C and +8°C up to the expiry date
indicated on the box.
 After opening, insulin can be stored at room temperature (between +15°C and +25°C)
for up to 1 month, so long as it is kept in its packaging, away from heat and light.
However, even if it has not been fully used, the insulin must be thrown away 1 month
after opening.
 In any case, you must ensure that the insulin does not freeze, in which case it will lose
its efficacy. It must not be stored alongside the walls of the refrigerator. In the same
way, it must never be subjected to intense heat so as not to alter its efficacy.
 THERMODYNAMICS OF THE FORMATION OF
INSULIN HEXAMER
• The thermodynamics of formation of the insulin
hexamer, which is stabilized by two Zn 2+ ions.

• An original method involving EDTA chelation of Zn2+

from the hexamer is done.

• The two metal ions are chelated sequentially,

reflecting stepwise Zn2+ binding and stabilization
of the quaternary structure.

• Two to three H+ bind to the hexamer upon its

formation at pH 7.4, which is both enthalpically and
entropically favored.

• The former is due to Zn2+ coordination to His residues Stability constant of EDTA-zn2+ complex- 7×10^12
from three subunits, and the latter is associated with Enthalpy of formation of EDTA-zn2+ complex- -11.2 kcal/mol
desolvation that accompanies the protonation and the Enthalpy of formation of insulin-zn2+ complex-17 kcal/mol
packing of the subunits in the hexamer.
THERMODYNAMICS OF THE INTERACTION OF
INSULIN WITH ITS RECEPTOR

• Insulin binding to its cellular receptors is markedly dependent on the temperature.

• The thermodynamic parameters for the reaction of insulin with the high affinity state of its receptor have been
evaluated from equilibrium studies at multiple temperatures between 5˚ and 37°C.

• The “hydrophobic reaction” plays an important role in the reaction with its receptor of insulin and other
polypeptide hormones like glucagon.

• The insulin monomer appears to be folded around an inside core of buried hydrophobic residues, but there
are also two patches of clustered hydrophobic residues on the surface of the monomer,

a) The first hydrophobic domain is part of the putative receptor-binding region, constitutes most of the
“cooperative site”, and is involved in the monomer-monomer interactions in the insulin dimer.

b) The second hydrophobic domain is involved in dimer-dimer interactions in the insulin hexamer.
 EQULLIBRIUM CONSTANT AND FREE ENERGY
CHANGE

• The equilibrium constant for the association reaction in the high

affinity state, Ke, varies markedly with temperature.

• A van’t Hoff plot of the data is curvilinear with a maximum around

20°C for the corrected data.

• The standard free energy change is ΔG = ΔG° + RT ln Ke where R is

the gas constant and T the absolute temperature.
 EQULLIBRIUM CONSTANT AND FREE ENERGY
CHANGE
• A plot of ΔG against the temperature also shows a marked
curvilinearity with a maximum at 37°C.

• Therefore, a regression analysis of the free energy change was done

by using Equation,
ΔG = -RTlnKe =A+BT+CT^2

• The best fit analysis is done and the above equation is given as,

• And hence we get,

• The heat capacity change can also be given as,

Variation of free energy change of insulin-receptor
binding with temperature.
 VARIATION OF ΔH°, ΔS° AND ΔCp° WITH
TEMPERATURE
• The changes in both enthalpy and entropy for the insulin-receptor
association decrease continuously and within the limits studied
vary quasi-linearly with temperature.

• The thermodynamic stability of the complex is maximum at 37°C.

• The change in heat capacity, that is the slope of the ΔH° curve, is
negative, indicating that the heat capacity for the separated
insulin and receptor molecules is greater than in the
hormone.receptor complex.

• ΔCp°, also varies with temperature.

• The absolute value of the variation is, however, a second

derivative of the initial data plot and thus relatively imprecise.

• The value of ΔCp°, is traditionally expressed at 25°C and is in

this case -766 cal/(mol deg).
 DRIVING FORCES IN THE INSULIN-RECEPTOR
INTERACTION

Represents a complete analysis of the driving forces

in the formation of the insulin.-receptor complex in
the case where the effect of temperature on
ionization is “eliminated” by appropriate correction.
 INFERENCE
• From the large negative heat capacity change observed in our study, it appears that the insulin-receptor
interaction is critically dependent on the properties of water.

• Since ionic and polar groups interact strongly with water, a large part of the driving force in the insulin-
receptor interaction is probably contributed by the removal of water from the less polar moieties (probably
surface residues of both proteins) and the resultant changes in the cooperative organization of the hydrogen
binding between water molecules.

• We have calculated the theoretical contribution of the non-polar residues in the putative receptor-binding regions of and
it amounts to -460 cal/(mol deg).

• Thus, insulin may contribute more than half of the heat capacity change of the reaction as determined in our study, (-
766 cal/(mol deg)), and burying hydrophobic residues from the receptor or membrane site must contribute the other
half.

• All the datas were obtained based on the assumption that the bindin is a single step reaction H + Re  HRe
with the equillibrium constant Ke.

• But if the reaction involves more than one step, for example H + Re ↔HRe ↔ HR’e, or that there is another coupled
reaction, the resulting steady state may yield curvilinear van’t Hoff plots even in the absence of hydrophobic
effects.
 TYPES OF INSULIN AVAILABLE IN MARKET

1 2 3 4

RAPID- SHORT- INTERMEDIATE-

LONG-ACTING
ACTING ACTING ACTING
• Covers the blood glucose • Often combined when needed
• Usually taken before • Usually taken before elevations when rapid acting with rapid or short acting insulin
meal. 30 min before meal. insulin stop working.
• Lowers blood glucose levels
• Used in combination • Used in combination • This type of insulin is often
when rapid acting insulin stop
with longer acting with longer acting combined with rapid or short working.
insullin . insulin. acting insulin.
• Taken once or twice a day
• Usually taken twice a day.
 Absorption Of Insulin

A B

A KMD
Equilibrium constant between
monomer and dimer form of insulin
KDH
B Equilibrium constant between dimer
and hexamer form of insulin
• The insulin monomers and
dimers are readily absorbed by
blood capillaries. Insulin
hexamers, however, are not
absorbed into the capillaries.
 Factors affecting the absorption of insulin:
1.) Concentration:

• Depot concentration and absorption of

insulin is inversely proportional.
2.) Subcutaneous Blood Flow
Increased SBF results in accelerated insulin absorption.

Serum albumin :
 Serum albumin dissociates hexameric Zn2+-insulin into active monomers.

 Several different variables are implicated in the absorption rate of

subcutaneously injected insulin, such as subcutaneous fat thickness, skin
temperature and, especially, insulin characteristics.

 capillary diffusion capacity depends on the size of the insulin molecule.

 Insulin monomer (approximately 6000 kD) is absorbed rapidly, and the

larger Zn2+-insulin hexamer (approximately 36000 kD) is absorbed at
much lower rates.
Thank you