Contents 1. Insulin Molecule 2. Effect of Insulin in Body 3. History of Insulin 4. Recent Trends in Insulin Productions and Types 4.1 Animal Insulins 4.2 Long-Acting Insulins 4.3 Human Insulins 4.4 Insulin Analogues 4.5 Biosimilar Insulins 5. Insulin Production (Chain A and Chain B Method) 5.1 Upstream Processing 5.2 Downstream Processing 6. The Proinsulin Process 7. Insulin Available in Market with Different Brand Names 8. References

1. Industrial Production of Insulin
Contents
1. Insulin Molecule
2. Effect of Insulin in Body
3. History of Insulin
4. Recent Trends in Insulin Productions and Types
4.1 Animal Insulins
4.2 Long-Acting Insulins
4.3 Human Insulins
4.4 Insulin Analogues
4.5 Biosimilar Insulins
5. Insulin Production (Chain A and Chain B Method)
5.1 Upstream Processing
5.2 Downstream Processing
6. The Proinsulin Process
7. Insulin Available in Market with Different Brand Names
8. References

2. 1. Insulin Molecule
The term “insulin” was derived from the Latin word insula or “island” to describe its origin from
the pancreatic islets of Langerhans. β cells that lie exclusively within these islets produce insulin,
a peptide hormone, which facilitates the entry of glucose into target organs such muscle, fat, and
the liver for further metabolism. The insulin molecule is composed of two polypeptide chains
linked by disulfide bridges: chain A comprising 21 amino acids and chain B comprising 30
amino acids. After it is released, insulin attaches to a glycoprotein receptor on the surface of the
target cell. The α subunit on the glycoprotein receptor binds the insulin hormone, and the β
subunit (a tyrosinasespecific protein kinase) mediates insulin action on metabolism and growth.
Figure 1: Biochemical structure of insulin
2. Effect of Insulin in Body
Insulin is directly released from the pancreatic β cells in a pulsatile fashion into the portal
circulation. Two phases of insulin secretion have been recognized in response to nutrient
(predominantly carbohydrate) ingestion. The first phase is a sharp burst of insulin occurring
within 5–10 min of carbohydrate ingestion; the second phase is a sustained, slow release of

3. insulin which is directly related to the presence of hyperglycemia. Loss of the insulin pulsatility
factor or loss of the first phase and an attenuated second phase of insulin release contributes to
the development of type 2 diabetes mellitus. Insulin secretion decreases in the presence of
hypoglycemia and increases in response to hyperglycemia, certain amino acids, nonesterified
fatty acids, and sympathetic and parasympathetic stimulation. In brief, insulin facilitates glucose
transport in liver and muscle cells by modulation of GLUT4 glucose receptors, stimulates storage
of glucose in the form of glycogen (glycogenesis), stimulates uptake of fatty acid and
triacylglycerol synthesis in adipose tissue and muscle, inhibits lipolysis resulting in lowering of
plasma fatty acids, stimulates amino acid uptake and protein synthesis in liver, muscle and
adipose tissues, inhibits protein breakdown in muscle.
Figure 2: The role of insulin hormone in human metabolism. GH (growth hormone), FFA (free
fatty acid), T2DM (type 2 diabetes mellitus)

4. 3. History of Insulin
The landmark discovery and development of insulin as a medical therapy can be traced back to
the early nineteenth century. Prior to the discovery of insulin, people with diabetes were
subjected to a starvation diet, with little hope for survival.
 In 1922, a series of experiments by Frederick Banting and Charles Best saw the
production of the first pancreatic extract, which later was called “insulin” and
transformed the lives of people with diabetes. In their landmark experiment, Banting and
Best’s rigorous efforts to isolate a purified form of pancreatic extracts from slaughtered
animals saved the life of a young boy, Leonard Thompson, from impending coma and
death due to diabetes.
 Although pancreatic extracts remained the main source of insulin for a long time, in 1936
Hans Christian Hagedorn discovered that the action of insulin could be prolonged with
the addition of protamine, a basic protein widely available from fish sperm. Following
this discovery, protamine insulin, with an approximate duration of 12 h, was increasingly
used in people with diabetes to good effect.
 The subsequent discovery of adding zinc to protamine insulin by Scott and Fisher paved
the way for the development of neutral protamine Hagedorn (NPH). This longer-acting
and more stable insulin suspension was first marketed by Danish pharmaceutical
company Novo Nordisk in 1946.
 The sequencing of insulin by Frederick Sanger then led to the synthesis of human insulin
using DNA recombinant technology, which became widely available through the 1980s
via Eli Lilly pharmaceutical company.

5.  Recognizing the need to improve the physiological profile of insulin to mimic
endogenous insulin secretion and improved knowledge of amino acid sequencing of the
insulin molecule prompted the emergence of synthetic (or analog) insulin. These are now
used extensively in people with diabetes. A summary of key events leading to the
discovery and adoption of insulin for use in diabetes is shown in Table 1.1
Table 1.1 Discovery of insulin timeline
4. Recent Trends in Insulin Productions and Types
Although human insulin has been used for many years, its use does pose a few challenges. For
example, basal (long-acting) neutral protamine Hagedorn (NPH) insulin is associated with an

6. increased risk of nocturnal hypoglycemia, defensive snacking, and weight gain. Additionally, the
need to carefully time regular human insulin injections with food intake is cumbersome and can
restrict people with busy lifestyles. The need to improve the physiological profile of insulin to
mimic endogenous insulin secretion and improved knowledge of amino acid sequencing of the
insulin molecule has prompted the emergence of bioengineered analog insulin and has heralded
an exciting new era in insulin therapeutics. Analog insulin is similar to human insulin with a
slight variation in amino acid composition and structure but with improved pharmacokinetics.
Table 2: Milestones in the development of insulin
In 1996, analog insulin lispro was first marketed. Subsequently, a host of insulin analogs created
by recombinant DNA technology, including rapid- acting (e.g., aspart), premixed, and long-
acting (e.g., glargine and detemir) analogs, have revolutionized diabetes management. With the
recent advent of second-generation long- acting analogs (e.g., degludec, basal insulin, peglispro)
and oral formulations, the future of insulin therapeutics looks promising. However, long-term
data on their clinical efficacy, safety, and economic impact are still needed. Although efforts to
make new insulin formulations more reproducible and similar to human physiology are ongoing,
in recent years, there has also been avid interest in the role of continuous subcutaneous insulin
administration (or insulin pumps), closed loop systems, and “artificial pancreas” combination

7. devices. While these treatments are associated with increased costs and may not be suitable for
all patients, they may improve quality of life.
4.1. Animal Insulins
Following the successful clinical use of insulin, efforts were intensified to improve the purity and
upscale the production of bovine insulin. Initial attempts led to a higher yield, but the product
remained impure. In early 1922, collaboration with the pharmaceutical company Eli Lilly led to
large quantities of refined and relatively “pure” insulin becoming available for clinical use within
6 months. Insulin derived from pig pancreata subsequently became available, and in the
subsequent decades, the purity of preparations steadily improved with the removal of islet
peptides and other pancreatic constituents.
4.2. Long-Acting Insulins
Initially, insulin was available only in native form (so-called soluble or regular) and so had to be
administered on multiple occasions each day. The next major development was in the production
of delayed-action formulations. Initially these were tried without giving concurrent soluble
insulin, until diabetologists better understood how they should be used effectively in clinical
practice. The first of these was protamine insulate which was introduced by Hans Christian
Hagedorn in Denmark in 1936. Subsequently, protamine zinc insulin, globin insulin, neutral
protamine Hagedorn (NPH), and lente insulins became available.
4.3. Human Insulin
The full characterization of the amino acid sequence of human insulin by Sanger in 1955 and the
subsequent discovery of the three-dimensional structure of the molecule by Hodgkin in 1969 led
to the production of the first synthetic insulin from amino acids in the 1960s. In the late 1970s,
genetically engineered synthetic human insulin was produced using recombinant DNA

8. technology, and the first preparation became commercially available in 1982. Human insulin
today is made using the recombinant DNA techniques first developed in the late 1970s. The first
genetically engineered synthetic human insulin was made by inserting the gene that encodes for
human insulin into the bacterium Escherichia coli. The process is similar today, in that the
human gene is cloned and inserted into bacteria. Huge containers of the genetically modified
bacteria can produce large quantities of human insulin, which is purified to provide
pharmaceutical grade pure human insulin or its analogues.
4.4. Insulin Analogues
Advances in genetic engineering allowed manufacturers to alter the amino acid sequence of the
insulin molecule to alter its pharmacokinetic properties and so create analogues of insulin; the
first insulin “analogue,” insulin lispro, which has a more rapid action than conventional soluble
insulin, became available in the 1990s. Insulin analogues have also been developed which have a
much slower rate of absorption and therefore a prolonged duration of action. Stored insulin
forms a biologically inactive hexameric structure, and when injected subcutaneously, the rate of
onset of its action depends on how quickly it dissociates into active monomers. Human insulin
has a faster onset of action than either of the animal-derived insulins (porcine insulin has a faster
onset of action than bovine insulin). However, the older conventional soluble insulins have a
relatively slow onset of action so that a patient should take these insulins about 30 min before
eating food. The rapid-acting human insulin analogues, insulin lispro, insulin aspart, and insulin
glulisine, have weak bonds between the monomeric components, enabling rapid dissociation of
hexamers to monomers. These can then be absorbed rapidly from a subcutaneous injection site,
and their hypoglycemic effect commences within 5–10 min. Conversely, long-acting insulin
analogues— insulin glargine and insulin detemir—slowly dissociate and are slowly absorbed

9. with only a modest peak of plasma insulin and have a protracted effect for 16–24 h. The plasma
insulin concentrations associated with these long-acting analogues reach a plateau level, which
persists for most of the day and more closely mimics basal secretion of insulin in the nondiabetic
state. They are usually administered once or twice daily. Even longer-acting analogues, such as
insulin degludec, last for up to 42 hours with no peak in activity.
Figure 3: Analogues of human insulin
4.5. Biosimilar Insulins
Biosimilars are generic versions of recombinant DNA technology drugs which are synthesized
by a different manufacturing process. Unlike standard generic pharmaceuticals, drugs with a
protein structure, like insulin, which are made by a different manufacturing process to the
original drug may not be absolutely identical because there could be alterations in, for example,
the quaternary (or folding) structure. Biosimilars cannot therefore be approved for clinical use by
following the standard procedure as used for generic drugs, which requires simply the
demonstration of equivalent bioavailability with the reference drug. Instead they have to pass

10. strict mandates from pharmaceutical regulatory authorities. Biosimilars for human insulins have
been developed and are in use in some parts of the world. Enthusiasm to prescribe such drugs has
to be tempered by potential concerns about safety, quality, and comparable efficacy.
5. Insulin Production (Chain A and Chain B Method)
This method consists of chemically synthesizing two oligonucleotides which encodes the 21
amino acid A chain and 30 amino acid B chain individually in two different Escherichia coli (E.
coli) cells, cultured separately in large-scale fermentation vessels, with subsequent
chromatographic purification of the insulin chains produced. The A and B chains are then
incubated together under appropriate oxidizing conditions in order to promote interchain
disulphide bond formation, forming human insulin.
The diagram below illustrates the major steps under molecular level in the production.
Figure 4: ChainA andChainB method

11. 5.1. Upstream Processing
Step 1: Obtaining of human insulin gene
Two general strategies are commonly used to obtain the human insulin gene. They are:
 Complementary DNA (cDNA) obtaining from messenger RNA (mRNA) of the two
chains using enzyme reverse transcriptase
 Cloning of cDNA of both chains using polymerase chain reactions (PCR). This involves
amplification of the cDNA sequences as not every gene yield measurable amounts of
mRNA
Step 2: Insertion of cDNA of both chains into plasmids
Bacterial plasmids are being cut using specific restriction enzymes for the insertion of the two
DNA molecules into separate plasmids. Each cDNA is extended at its 5' terminus with an ATG
(methionine) initiation codon for start of translation, and a translation termination signal at its 3'
with the sticky ends EcoRI and BamHI (later as restriction sites). Two vector plasmids are made
for both the cDNA. They are inserted in the plasmids at the EcoRI and BamHI sites next to the
lacZ gene which encodes for the enzyme β-galactosidase. In E. coli, β-galactosidase is the
enzyme that controls the transcription of the genes. To make the bacteria produce insulin, the

12. insulin gene needs to be tied to this enzyme. The cut plasmids are re-ligated by specific DNA
ligases.
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.

13. Step 4: Media and equipment preparation
The LB broth is prepared using the LB powder. It is antoclaved and ampicillin and lactose are
added (after the sterilization to prevent denaturation or destruction). Inoculation is done by
adding the transformed bacteria into the media. Preparation of the bioreactor is done too. Parts of
the bioreactors are fixed and checked such as the calibration of the pH electrode, pO2 probe,
exhaust condensers and air inlet. The bioreactor is then sterilized.
Step 5: Fermentation
This stage consists of small scaling (enrichment liquid culture in shake flask) to large scaling
(fermentor). The two chains are grown separately. Small scaling (early stage) uses shake flasks
to do the enrichment culture method for selecting the desired type of E. coli for fermentation.
The fermentation broth contains two unique components - an antibiotic known as ampicillin and
lactose. Bacterial cells that have sucessful transformation will contain the plasmic gene which
contains the ampicillin resistance gene and the lac Z gene which encodes for β-galactosidase in
the presence of lactose. These cells therefore can grow in the ampicillin environment and the
transcription of the lac Z gene will in turn result in the transcription of the human insulin chain
DNA. Bacterial cells that have failed the transformation do not contain the ampicillin resistance
gene and the lac Z gene. As a result, the growth of these cells will be suppressed by ampicillin
and will not replicate during the fermentation process.
Moving on to the large scale, where transfected bacterial cells are transferred from the small
flask and replicated under optimal conditions such as temperature, pH in fermentation tanks.
This step involves process monitoring and control. The bacterial cell processes turn on the gene
for human insulin chains and then insulin chains are produced in the cell.

14. 5.2. Downstream Processing
Step 6: Isolation of crude products
Cells are removed from tanks and are lysed using different methods such as enzyme digestion,
freezing and thawing and sonication. For enzyme digestion, lysosome enzyme is used to digest
the outer layer of the bacterial cells and detergent mixture is subsequently added to separate the
cell wall membrane.
Step 7: Purification of crude product
Centrifugation is conducted to helps separate the cell components from the products. Stringent
purification of the recombinant insulin chains must be taken to remove any impurities. This uses
several chromatographic methods such as gel filtration and ion-exchange, along with additional
steps which exploit differences in hydrophobicity.
Step 8: Obtaining of insulin chains
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 therefore, cyanogen bromide is used to split the protein chains at
methionine residues, allowing the insulin chains to be obtained.

15. Step 9: Synthesis 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 and air oxidation, resulting in Humulin - synthetic human insulin.

16. Step 10: PR-HPLC to obtain highly purified insulin
Reverse-phase high performance liquid chromatography (PR-HPLC) is performed lastly to
remove almost all the impurities, to produce highly purified insulin. The insulin then can be
polished and packaged to be sold in the industires.
6. 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. Insulin is naturally synthesized as
pre-proinsulin in the pancreas. It is converted to proinsulin with the N-terminal signal peptide
enzymatically removed. Proinsulin is composed of the amino acid chains that will form insulin
and a connecting 30 residue peptide, that joins one end of chain A to chain B. Enzymatic
proteolysis removes the peptide chain to produce insulin.

17. Figure 5: Proinsulin to insulin
The proinsulin coding sequence is inserted into the non-pathogenic E. coli bacteria and the
bacteria undergo fermentation where they replicate and produce proinsulin. The connecting
sequence between the A and B chains is then spliced away with an enzyme and the resulting
insulin is purified.
The different downstream process is required for the Proinsulin process as compared to the
Chain A and Chain B process. As you can see, the enzymatic proteolysis is a unique step for the
proinsulin production. At the end of the both manufacturing processes, ingredients are added to
insulin to prevent bacteria growth and maintain a neutral pH balance. Towards the end of the
processes the ingredients to produce the desired duration type of insulin are also added. An

18. example is adding zinc oxide to produce longer acting insulin. These additives delay absorption
in the body. Additives vary among different brands of the same type of insulin.
Figure 6: Proinsulin process
7. Insulin Available in Market with Different Brand Names
Types of Insulin available for people with Diabetes are followings
 Rapid-acting: Usually taken before a meal to cover the blood glucose elevation from
eating. This type of insulin is used with longer-acting insulin.
 Short-acting: Usually taken about 30 minutes before a meal to cover the blood glucose
elevation from eating. This type of insulin is used with longer-acting insulin.
 Intermediate-acting: Covers the blood glucose elevations when rapid-acting insulins stop
working. This type of insulin is often combined with rapid- or short-acting insulin and is
usually taken twice a day.

19.  Long-acting: This type of insulin is often combined, when needed, with rapid- or short-
acting insulin. It lowers blood glucose levels when rapid-acting insulins stop working. It is
taken once or twice a day.
Different brand names of insulin are as fallows
 Actrapid
 Apidra
 Humalog
 Human Fastact
 Human Insulatard
 Human Longact
 Human Mixtard
 Human Monotard
 Human Prodica
 Huminsulin
 Iletin N
 Insugen
 Insulin
 Insuman
 Lantus Levemir
 Lentard
 Novolog
 NPH (N)
 Rapidica
 Recosulin
 Regular (R)
 Wosulin
 Zinulin

20. 8. References
 Begg, A. (2013). Insulin therapy: a pocket guide.
 Born, G. V. R. (1970). Handbook of experimental pharmacology.
 Conner, J., Wuchterl, D., Lopez, M., Minshall, B., Prusti, R., Boclair, D., & Allen, C.
(2014). The biomanufacturing of biotechnology products. Biotechnology
entrepreneurship. Elsevier, Cambridge, MA, 351-385.
 Craft, S. (Ed.). (2010). Diabetes, insulin and Alzheimer's disease. Springer Science &
Business Media.
 Crasto, W., Jarvis, J., & Davies, M. J. (2016). Handbook of Insulin Therapies. Springer
International Publishing.
 Flickinger, M. C. (2013). Upstream industrial biotechnology, 2 volume Set. John Wiley
& Sons.
 Flickinger, M. C. (Ed.). (2013). Downstream industrial biotechnology: recovery and
purification. John Wiley & Sons.
 Gronemeyer, P., Ditz, R., & Strube, J. (2014). Trends in upstream and downstream
process development for antibody manufacturing. Bioengineering, 1(4), 188-212.
 http://alwayseatmoresugar.blogspot.com/2008/01/process-description2.html
 http://www.joslin.org/info/insulin_a_to_z_a_guide_on_different_types_of_insulin.ht
ml
 http://www.medindia.net/drug-price/insulin.htm
 https://www.drugs.com/drug-class/insulin.html
 https://www.slideshare.net/salinig27/human-insulin-production-process-
requirement