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

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

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)

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.

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

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

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

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

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

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: Chain A and Chain B method

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

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.

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.

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.

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.

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.

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

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.

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

8. References

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(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

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