Humulin and Human Growith Hormone

Humulin
Humulin is synthetic human insulin prepared by using genetic engineering. Humulin is

manufactured from DNA sources in laboratory, using recombinant DNA technology.
Synthetic insulin is also called genetically engineered insulin. The synthetic insulin
(humulin) is as effective as hormone insulin secreted by human pancreas.
Primary Structure of Insulin:

Insulin is a proteinaceous hormone secreted by beta-cells of islets of langerhans of
pancreas. Insulin controls, blood sugar level and when there is less secretion of insulin,
it results in diabetes (high blood- sugar level). In 1954, Frederick Sanger determined
primary structure of Insulin. Insulin is a protein formed by two polypeptide chains: A-
chain and B-chain, interlinked by two sulphide bonds (see fig). A-chain is formed of 21
amino acid residue, while B – chain is formed of 30 amino acid residue.
Development of Humulin:
Prior to the approval of Humulin, insulin was manufactured out of pig or cow
pancreases. The non-human insulin has a slightly different chemical composition than
human insulin. In 1978, a US-based small biotech startup called Genentech and City
of Hope National Medical Center, jointly produced human insulin in the
laboratory using recombinant DNA (rDNA) technology. City of Hope scientists had
synthesized the genes for the protein's two chains before inserting them into
Escherichia coli. With its expertise in purifying and handling insulin and the desire to
remain a leader in the field, Eli-Lilly, a US-based pharmaceutical company,
immediately licensed recombinant insulin from Genentech and set about developing it
where it was named "Humulin" or Recombinant Human Insulin. Fortunately,
Lilly also had experience isolating antibiotics from fermentation processes. Because
rDNA guidelines at the time allowed the expression of only inactive protein products,
Eli-Lilly had to use the two-chain method.
In 1982, Eli-Lilly's Humulin became the first genetically engineered drug
approved by the US Food and Drug Administration (US-FDA). The US
approval came just one month after British regulatory authorities allowed its
introduction in the UK. Although the development of Humulin took just four years, it
was a major undertaking for both the developers and regulators, breaking new ground.
Humulin was the first drug produced by genetic engineering techniques to
gain the FDA approval for human use. It was made by inserting human genes
responsible for insulin production into E. coli bacteria, thus stimulating the bacteria to
synthesize insulin. Because this technique could produce large quantities of insulin, the
artificial insulin was expected to help alleviate a shortage of animal insulins predicted to
occur within the next decade. Humulin had proved to be safe and effective in FDA
clinical trials involving more than 400 patients. Today, Humulin is made right here in
Indianapolis in gigantic fermentation vats, 4 stories high and filled with bacteria!!!
These fermentation vats operate 24 hours a day, year round. The human insulin protein
made by the E. coli bacteria is collected from the vats, purified, and packaged for use by
patients with diabetes. It is used by more than 4 million people with diabetes around the
world everyday. Today Humulin is available in both a vial and a disposable prefilled pen
and this helps in choosing a convenient way of taking Humulin.
Advantages of Humulin over Insulin
Earlier insulin required for diabetes was extracted from pancreas of slaughtered cattle,
pigs or salmon. The process was quite tedious and difficult and yields of insulin would
be low. This extracted insulin in some patients, developed allergy or other side effects
due to foreign protein. Due to disadvantages of animal insulin and advantages of
humulin, humulin is regarded superior to animal insulin Humulin is considered better
than animal insulin because:
 Humulin is absorbed more rapidly and show its effectiveness in short duration.
 Humulin causes fewer allergic and autoimmune reactions as compared to animal
insulin.
 Humulin is less expensive than animal insulin
It is due to above advantages, now almost all insulin marketed is human insulin.
Human Growth Hormone (hGH)
Growth hormone (GH or somatotropin) is a 191 amino acid and has a molecular weight
of about 22,000 daltons, and thus is more than three times as large as insulin, single
chain polypeptide hormone which is synthesised, stored and secreted by the
stomatotraph cells within the lateral wings of the anterior pituitary gland, which
stimulates growth and cell reproduction in humans and other vertebrate animals.
Produced by the pituitary gland, human growth hormone (hGH) is essential for proper
growth in children. Some children, however, have disorders that cause reduced HGH
levels. If children go without treatment, they mature as unusually short adults. This
condition is treated by administering HGH, which today is produced by using
recombinant DNA (rDNA) technology. Before the invention of rDNA technology, HGH
could only be produced laboriously by isolating it from pituitary gland tissue taken from
human cadavers.
This process was inefficient, expensive and sometimes unsafe. Production of the drug in
this way continued for more than 20 years. That all came to a halt, however, with the
horrifying discovery that some of the drug was contaminated, having been extracted
from a cadaver infected with Creutzfeldt-Jakob disease (CJD). CJD is similar in effects
to mad cow disease or accelerated Alzheimer's, causing rapid brain degeneration leading
to death within a year of the first symptoms.Infection is caused by proteins called
prions. By eliminating the need for human tissue, rDNA technology avoids these and
other potential contamination problems.
Following the discovery of the contamination in 1985, the U.S. Food and Drug
Administration halted all distribution of cadaver-derived HGH. As luck would have it,
however, a new source for hGH was in the hopper. Biotechnology company Genentech
and drug company Eli Lilly had both independently been working on a new way to
manufacture hGH using recombinant bacteria. The companies genetically modified
bacteria by inserting a gene coding for the production of hGH. This genetic
transformation turned the bacteria into little factories to pump out hGH, leading to a
limitless source of pure hGH with little risk of contamination. Six months after the ban
on the natural source of hGH, the FDA approved Genentech's recombinant
HGH, a drug called Protropin, making it only the second recombinant
pharmaceutical to be sold in the U.S. Biosynthetic human growth hormone, also
referred to as recombinant human growth hormone, is also called somatropin and
abbreviated as rhGH.
Production of human growth hormone (hGH) by recombinant
DNA technology
 The gene for human growth hormone (hGH) is isolated from human pituitary
gland.
 Insertion of whole hGH gene into plasmid vector and cloning into E.coli results
into production of biologically inactive hormone because bacteria can translate the
region of gene that are not translated in human thereby producing a prehormone
containing an extra 26 aminoacids which might be difficult to remove.
 Hence the segment of gene that codes for the first 24 aminoacids of hormone is
constructed chemically from blocks of nucleotide.
Step I: Chemical synthesis of gene for first 24 aminoacids:

 From the known aminoacids sequence of hGH, gene for first 24 aminoacids are
constructed chemically. These genes are constructed in three small fragments
and then they are joined by T4 DNA ligase to get whole gene for first 24
aminoacids.
Step II: Isolation of mRNA for hGH

 In this step mRNA for hGH is isolated from human pituitary gland tissue.
Step III: Reverse transcription

 Using reverse transcriptase enzyme complimentary DNA (cDNA) is synthesized
from mRNA.
 The cDNA obtained by reverse transcription process, is the gene for hGH.
 The full gene is cut with restriction endonuclease enzyme to remove first 24
aminoacids.
Step IV: Joining of synthetic gene and cDNA

 In this step synthetic gene (gene for first 24 aminoacids) and cDNA are joined in
order to obtain full gene with its own initiation codon (AUG). T4 DNA ligase join
these genes.
Step V: selection of suitable vector and recombination:

 Expression vector phGH407 derived from plasmid vector PBR322 is used as
carrier vector.
 HGH gene is ligated into a restriction site just downstream of Lac;
promotor/operator region of the expression vector.
Step VI: selection and recombination into suitable host cell

 E. coli is used as suitable host cell.
 The recombinant expression vector is then transformed into E.coli.
 The recombinant E. coli then starts producing hGH.
 The recombinant E. coli are isolated from the culture and mass production by
fermentation technology to obtain hGH.
Use of recombinant human growth hormone

 Treatment of children suffering from growth deficiency
 Treat the patient with Turner’s syndrome and chronic renal insufficiency
 To treat patient with renal carcinoma
 Bovine somatotropin hormone is used to increases milk production in lactating
cows and also to increase body mass of cattles.

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Humulin

Humulin is synthetic human insulin prepared by using genetic engineering. Humulin is

Primary Structure of Insulin:

Step I: Chemical synthesis of gene for first 24 aminoacids:

Step II: Isolation of mRNA for hGH

Step III: Reverse transcription

Step IV: Joining of synthetic gene and cDNA

Step V: selection of suitable vector and recombination:

Step VI: selection and recombination into suitable host cell

Use of recombinant human growth hormone

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