Modified amino acid sequences can be produced
through Recombinant DNA technology; aiming to create analogues of insulin with
altered pharmacokinetic properties such as fast or slow onset of actions. High
affinity insulin to receptors can be considered as a potential economic benefit
, by utilizing smaller amount of insulin per therapeutic dose. It has been
identified which amino acid residues are interacting with the insulin receptor  (A1,A5, A19,
A21, B10, B16, B23-25), and a number of analogues containing amino acid
substitutions

at several of these points have been manufactured. Replacement of
histidine by glutamate at the B10 position, for example, leads to the formation
of an analogue displaying five times more activity in vitro. But that does not reflect the same situation in vivo. Disabling dimerization or
stopping the formation of higher polymers at therapeutic doses were attempts to
allow the formation of faster acting insulins.  Amino acids at positions B8, B9, B12–13, B16
and B23–28 form the contact point between insulin dimers/oligomers.  Thus, analogues with various substitutions at
these positions were produced . Promoting steric hindrance and charge repulsion
through the insertion of a bulky or charged amino acid respectively was an
approach adopted to prevent the formation of dimers/oligomers. Therefore
engineered fast acting insulins; are directly absorbed into the bloodstream and
can be taken  at mealtimes rather than 1
or 3 hours before meal.   ‘Insulin
lispro’ (tradename ‘Humalog’) was the first such engineered short-acting
insulin to come to market.

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‘Insulin Aspart’ is a second fast-acting engineered human insulin
analogue now approved for

general medical use. In this insulin; proline at position B28, was
substituted by aspartic acid  It differs
from native human insulin in that the prolineB28 residue has been replaced by
aspartic acid. This inhibits the potential of self -association between the
molecules and thus allowing it  to
directly enter the blood stream from the site of injection immediately upon
administration.

While interests were focused on developing fast-acting insulin; other
people worked on generating longer-acting insulin. Long acting insulin include
Zn-insulin and protamine-insulin suspensions which shows a plasma half -life of
20-25 h. But longer acting insulin were developed through selected amino acids
substitutions and were shown to have a plasma half-life of 35 h . insulin
glargine is one of these insulin and it differs from the native insulin in that
the aspargine residue in the C-terminal of the A chain has been substituted
by  a glycine residue , and the B chain
has been elongated again by two arginine residues from the C-terminus also. The
tradenames of insulin glargine available in the market are lantus and
Optisulin. The ultimate goal is to increase the isoelectric point PI ( the pH
at which the molecule displays a zero charge with minimal solubility) from 5.4
to 7. The engineered insulin is expressed in a recombinant E. coli K12
host strain and is produced via the ‘proinsulin.. The purified product is
formulated at pH 4.0, a pH value at which it is fully soluble and charged. Once
it is injected subcutaneously, the pH rises from 4 to 7, thus precipitating in
the subcutaneous tissue since its 
solubility decreases upon reaching the PI.

Upon s.c. injection, the insulin experiences an increase in pH towards
more neutral values

and, consequently, appears to precipitate in the subcutaneous tissue.
Hence a greatly prolonged release duration since the resolubilization process
is happening slowly. Therefore, it is enough to inject once daily and maintain
of an acceptable basal blood insulin levels.

Levemir (tradename) is an alternative engineered long-acting insulin
product that gained approval for general medical use in 2004  it is different from the native insulin in the
absence of the B30 threonine residue and therefore altering the pharmacokinetic
properties. In addition to that, a 14-Carbon 
fatty acid residue is covalently bonded to the side chain of lysine
residue B29. Subsequently, insulin can bind reversibly to albumin, both in
plasma and site of injection. Prolonged release of free insulin will occur as a
result of this binding ; and the duration of action is up to 24 h.

Product manufacture entails initial expression of insulin in engineered Saccharomyces
cerevisiae, purification and acylation (attachment of the fatty acid
group).

The generation of engineered insulin analogues raises several important
issues relating to product safety and efficacy. Alteration of a native
protein’s amino acid sequence could render the engineered product immunogenic.
Such an effect would be particularly significant in the case of insulin, as the
product is generally administered daily for life. In addition, alteration in
structure could have unintended influences upon pharmacokinetic and/or
pharmacodynamic characteristics of the drug. Preclinical and, in particular,
clinical evaluations undertaken upon the analogues thus are approved, however,
have confirmed their safety and efficacy. The sequence changes introduced are
relatively minor and do not seem to elicit an immunological response.
Fortuitously, neither have the alterations made affected the ability of the
insulin molecule to interact with the insulin receptor, and trigger the
resultant characteristic biological responses.