In 1921, insulin was identified to be an anti-diabetic factor by Bantingand Bestand. It was clinically used inthe year after. Manufacturing processeswere developed to extract the insulin from porcine and bovine pancreatic tissue. From 1921 to 1980, the focus was done on enhancing the purity of the insulin and providingdifferent formulations for altering time-action for improved glucose control (Brange,1987a,b; Galloway, 1988). Purification was improved by enhancing processingconditions and extraction and by implementing chromatographic processes (sizeexclusion, ion exchange, and reversed-phase) (Kroeff et al.
, 1989) to reduce the levels of both insulinrelated prteins like proinsulin , and general protein impurities Formulationdevelopment focused on improving chemical stability by moving from acidic toneutral formulations and by modifying the time-actionprofile through the uses of various levels of zinc and protamine.Recombinant DNA technology allowed the production the unlimited amountof insulin. Both advanced purificationtechniques, and r DNA, allowed theproduction of the purest human insulin ever made. Insulin, a 51-amino acid protein, is a hormone that is synthesized as aproinsulin precursor in the beta-cell of the pancreas and is converted toinsulin by enzymatic cleavage. The resulting insulin molecule is composed oftwo polypeptide chains that are connected by two inter-chain disulfide bonds The A-chain is composed of 21 amino acids andthe B-chain is composed of 30 amino acids. The interchain disulfide linkagesoccur between A7–B7 and A20–B19, respectively. A third intrachain disulfidebond is located in the A-chain, between residues A6 and A11The original polypeptide contains 108 amino acid polypeptide, 23 aminoacids on the terminal chain form a signal sequence that enables its transfer tothe endoplasmic reticulum .
there, the signal sequence is removed by specificpeptidase. This is Proinsulin. Proinsulin-containing vesicles bud off from theendoplasmic reticulum and fuse with the golgi apparatus. Subsequently,proinsulin-containing vesicles (clathrin-coated secretory vesicles), in turn,bud off from the golgi. Once they move away from the golgi apparatus, theyloose their clathrin coat. These vesicles will be stored in the beta cells ofthe pancreas, High glucose level, stimulate these vesicles to fuse with theplasma membrane and therefore releasing the content into the bloodstream by themechanism of exocytosis.
Elevatedlevels of blood glucose, or other appropriate signals, cause the vesicles tofuse with the plasma membrane, thereby releasing their contents into the bloodvia the process of exocytosis. Porcine insulin (5777 Da) varies from the humanform (5807 Da) by a single amino acid, whereas bovine insulin (5733 Da) differsby three residues. Proinsulin, within the coated secretory granule, undergoes proteolysis ,leading to the formation of mature insulin . and a 35 amino acid connectingpeptide C. the C peptide is then proteolytically modified by removing 2 aminoacids on each end. At the end , the secretory granules contain low level ofproinsulin, C peptide, and protease, plus the insulin itself. The insulin isstored in the form of zinc-insulin hexamer: 6 molecules of insulin stabilizedby 2 atoms of zinc Insulin production Many disadvantages has lead to cease the animal-derived insulin production.These disadvantages include: Immunogenicity: sinceanimal derived insulin differ from the human insulin, it formed a high risk toinduce an immunological response in humans therefore decreasing the efficacy ofthe insulin.
However, Porcine insulin, differs from human insulinby only a single amino acid (residue 30 of the B-chain; threonine in humans,alanine in pigs) and is essentially non-immunogenic in humans. However, many ofthe porcine insulin contaminants (including porcine proinsulin) are immunogenicin humans Concerns about thetransmission of spongiform encephalopathies linked to the use of animal-derivedmaterials is the major reason for the product deletion Availability. Some 170million people suffer from diabetes worldwide, a fi` gure projected to double by 2030. Insulin administrationis essential to the survival of those with type-1 (insulin- dependent)diabetes, and is required to control the progression of a minority of those with(the more common) insulin-independent type-2 diabetes. The annual insulinrequirement has surpassed 5000 kg and continues to grow, prompting concern ofan insulin shortfall from slaughterhouse sources.Such issues and concerns underpinned the development of recombinanthuman insulin products, now routinely used in the management of diabetes.
Production of human insulin by recombinant DNAtechnologyHuman insulin can be produced by recombinant DNA technology , thusproducing the same sequence of amino acids and without any risk ofimmunogenicity. To begin the process, a nucleotide sequence coding for the insulin A andB chains are inserted into two different K12 strain E.coli bacterialcells. These cells were allowed to be cultured and reproduced separately in alarge-scale fermentation vessels, with subsequent chromatographic purificationof the chains of insulin produced.
After purification, incubation of the A andB chains together is done under appropriate oxidizing conditions to allow theformation of the disulfide interchain bond, thus forming the human insulin crb. . An alternative method (developed in the Eli Lilly researchlaboratories), entails inserting anucleotide sequence coding for human proinsulin into recombinant E.coli. This is followed by purification of the expressed proinsulin andsubsequent proteolytic excision of the C peptide in vitro. This approachrequires a single step therefore it is much more popular than the previousapproach. What is produced is human insulin prb. The presence of asingle impurity whether insulin derived or microbial derived, can decreaseefficacy of the insulin and induce immunogenicity.
Stringent purificationprocedures must be done , including several chromatographic steps: gelfiltration, ion exchange, hydrophobic interaction chromatography andreverse-phase chromatography. An additional step has been included to ensure aclean product is formed is the RP-HPLC for the prb.