Pharmaceutical Biotechnology
(PHA412) Lecture: 10
Topic: Insulin
Mohammad Abbas Gani
Lecturer, Department of Pharmacy, IUB
Insulin is a naturally occurring hormone made by your pancreas that helps your
body use sugar for energy and lowers the level of glucose (a type of sugar) in
the blood. In diabetes, the pancreas doesn't make enough insulin or the body can't
respond normally to the insulin that is made. This causes the glucose level in the
blood to rise.
Discovery and Development
• Insulin was discovered by Banting and Best in 1921. Soon afterward,
manufacturing processes were developed to extract the insulin from porcine and
bovine pancreas.
• From 1921 to 1980, efforts were directed at increasing the purity of the insulin and
providing different formulations for altering time-action for improved glucose
control.
• Purification was improved by optimizing extraction and processing conditions and
by implementing chromatographic processes (size exclusion, ion exchange, and
reversed-phase) to reduce the levels of both general protein impurities as well as
insulin related proteins such as proinsulin and insulin polymers.
• Formulation development focused on improving chemical stability by moving from
acidic to neutral formulations and by modifying the time action profile through
the uses of various levels of zinc and protamine.
Discovery and Development
• The evolution of recombinant DNA (rDNA) technology led to the unlimited
availability of human insulin, which has eliminated issues with sourcing
constraints while providing the patient with a natural exogenous source of insulin.
• Combining the improved purification methodologies and recombinant DNA
technology, manufacturers of insulin are now able to provide the purest human
insulin ever made available, >98%.
• Bovine and porcine insulin preparations have also been made commercially
available. But difficulties obtaining sufficient supplies of bovine or porcine
pancreas and recent concerns over transmissible spongiform encephalopathies
associated with the use of animal-derived materials are major reasons for the
product deletions.
• Therefore, human insulin and insulin analog products are predominately used
today as the first-line therapies for the treatment of diabetes.
Chemical Description
• Insulin is synthesized by β-cells of the pancreas in the form of a single chain of
three peptides B, C and A in the order: B chain-C peptide-A chain.
• This proinsulin is converted to mature insulin after the removal of the central C-
peptide by the action of proteolytic enzymes.
• The mature insulin (51-amino acid protein) consists of two polypeptide chains
that are connected by two inter-chain disulfide bonds.
• The A-chain is composed of 21 amino acids and the B-chain is composed of 30
amino acids.
• The inter-chain disulfide linkages occur between A7–B7 and A20–B19,
respectively.
• A third intra-chain disulfide bond is located in the A-chain, between residues A6
and A11.
Chemical Description
Figure: Primary sequence of insulin.
Chemical Description
Figure: Insulin
Chemical Description
Figure: Primary sequence of insulin. The colored (yellow) amino
acids represent sites of sequence alterations.
Chemical Description
Chemical Description
• The net charge on the insulin molecule is produced from the ionization potential of
four glutamic acid residues, four tyrosine residues, two histidine residues, a lysine
residue, and an arginine residue, in conjunction with two α-carboxyl and two α-
amino groups.
• Insulin has an isoelectric point (pI) of 5.3 in the denatured state; thus, the insulin
molecule is negatively charged at neutral pH. This net negative charge-state of
insulin has been used in formulation development.
• In addition to the net charge on insulin, another important intrinsic property of the
molecule is its ability to readily associate into dimers and higher order states.
• The driving force for dimerization appears to be the formation of favorable
hydrophobic interactions at the C-terminus of the B-chain.
(The isoelectric point (pI) is the pH of a solution at which the net charge of a protein becomes zero.
At solution pH that is above the pI, the surface of the protein is predominantly negatively charged)
Chemical Description
• Insulin can associate into discrete hexameric complexes in the presence of various
divalent metal ions, such as zinc at 0.33g-atom/monomer, where each zinc ion (a
total of two) is coordinated by a HisB10 residue from three monomers.
• Physiologically, insulin is stored as a zinc containing hexamer in the β-cells of the
pancreas. The ability to form discrete hexamers in the presence of zinc has
been used to develop therapeutically useful formulations of insulin.
• Commercial insulin preparations also contain phenolic excipients (e.g., phenol, m-
cresol, or methylparaben) as antimicrobial agents. These phenolic species also
bind to specific sites on insulin hexamers, causing a conformational change that
increases the chemical stability of insulin in commercial preparations.
• The phenolic ligands are stabilized in a binding pocket between monomers of
adjacent dimers by hydrogen bonds as well as numerous van der Waals contacts.
Figure: Schematic representation of insulin association in the presence and absence of zinc
and phenolic antimicrobial preservatives.
Chemical Description
• In addition to the presence of zinc and phenolic preservatives, modern insulin
formulations may contain an isotonicity agent (glycerol or NaCl) and/or a
physiologic buffer (sodium phosphate).
• The isotonicity agent is used to minimize subcutaneous tissue damage and pain
on injection.
• The physiologic buffer is present to minimize pH drift in some pH-sensitive
formulations.
Insulin Production by rDNA Technology
• The basic step in recombinant DNA technology is similar for insulin production
also.
1. At first suitable vector (plasmid) is isolated from E. coli and then it is cut open by
restriction endonuclease enzyme.
2. The gene of interest (i e., insulin coding gene) is isolated from β-cell and inserted
in opened plasmid.
3. Plasmid and gene of interest are recombined together by DNA ligase enzyme.
4. This recombined plasmid is inserted into suitable host cell (i e., E. coli) and now
this recombined host cell starts producing insulin hormone.
