How is insulin manufactured

Related Content. How insulin is made using yeast Synthetic human insulin was the first golden molecule of the biotech industry and the direct result of recombinant DNA technology. Genentech Genentech, the first biotechnology company, established in Biography Herb W. Producing rat insulin using recombinant DNA, Walter Gilbert Walter Gilbert talks about his group's early success with isolating the rat insulin gene and making recombinant rat insulin.

To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. After the discovery of insulin in Milestone 1 , the use of porcine or bovine insulin to control blood glucose levels in individuals with diabetes became widespread and saved many lives.

This immunogenicity was thought to be the result of contamination of insulin with other pancreatic substances and small differences in amino acid composition between human and animal insulin. Purified animal insulins were developed and reduced the occurrence of allergic reactions, but further improvement was needed.

In the s, advances in DNA synthesis and recombinant DNA technology raised the possibility that bacteria could be genetically altered to produce human insulin. A major step towards this goal was made in when, for the first time, a functional polypeptide was generated from a chemically synthesized gene. Itakura et al. The plasmid was inserted into Escherichia coli and the resulting strain produced a polypeptide containing the somatostatin amino acid sequence.

Cyanogen bromide was used to cleave somatostatin from this larger protein in vitro. Somatostatin is just 14 amino acids long and was chosen in part because of its size — the time-consuming nature of chemical gene synthesis was considered a barrier to the synthesis of longer polypeptides such as the amino-acid insulin.

Nevertheless, just 2 years later, Goeddel et al. Native human insulin consists of two amino acid chains — the A chain and the B chain — that are linked by two disulphide bonds. Due to this, subcutaneously injected recombinant insulin usually have a slow onset with peak plasma concentration after 2 hours of injection and longer duration of action that last for hours [ 62 ]. Hence, in order to develop a fast- acting insulin analogue, it was required to modify the amino acids residues whose side chains are involved in dimer or oligomer formation.

It has been shown that amino acids residues in insulin B-chain particularly B8, 9,12, 13, 16 and play critical role in oligomerization [ 63 ],[ 64 ]. Lispro, developed by Eli Lilly, was the first fast acting insulin analogue to obtain regulatory approval in , for therapeutic use [ 60 ]. Insulin Lispro is engineered in such a way that it has similar amino acid sequence as the native insulin but has an inversion of proline-lysine sequence at position 28 and 29 of the B-chain, which resulted in reduced hydrophobic interactions and thus prevented dimer formations.

Another rapid-acting insulin analogue, produced in E. Insulin Glulisine have been generated by replacing B3 asparagine by a lysine and B29 lysine replaced by glutamic acid [ 14 ].

To avoid multiple injection, long-acting insulin analogues with prolonged duration of actions have also generated. Insulin Glargine is one of such long-acting insulin analogues, which was developed by Aventis Pharmaceuticals and approved by regulatory authorities of USA and EU in Insulin Glargine was generated by replacing the C-terminal asparagine of the A-chain with a glycine residue and the C-terminal of the B- chain was modified by adding two arginine residues.

These modifications resulted in increase of the isoelectric point pI from 5. Glargine was produced as proinsulin and expressed in E. However, after subcutaneous administration, it precipitated due to neutral pH in the subcutaneous tissue. Resolubilization of insulin occur slowly, resulting in longer duration for its release in the blood [ 14 ]. Yeast is a preferred host for expression of various heterologous proteins that require post-translational modifications for its biological activity.

Yeast cell has the ability to carry out numerous post-translational modifications such as phosphorylation, O-linked glycosylation, N-linked glycosylation, acetylation and acylation. Recombinant proteins are expressed in soluble form in yeast and properly folded in functionally active form.

Production of biopharmaceuticals using yeast expression system is also very cost effective and is amenable to scale up using large bioreactors. However, one major concern for producing therapeutic glycoprotein for human application is that yeast N-glycosylation is of the high-mannose type, which confers a short half-life in vivo and hyper-immunogenicity and thus render the therapeutic glycoprotein less effective. Various attempts have been made to humanize yeast N-glycosylation pathways in order to produce therapeutic glycoproteins with humanized N-glycosylation structure [ 65 ].

The therapeutic proteins produced in yeast are specifically from Saccharomyces cerevisiae and include hormones insulin, insulin analogues, non-glycosylated human growth hormone somatotropin, glucagon , vaccines hepatitis B virus surface antigen , uprate oxidase from Aspergillus flavus , granulocyte-macrophage colony stimulating factor, albumin, hirudin of Hirudo medicinalis and human platelets derived growth factor [ 34 ].

Alternate yeast strains, besides S. Specifically, Pichia pastoris has the ability to attain high cell densities by its robust methanol-inducible alcohol oxidase 1 AOX1 promoter and simple developmental approaches contribute to high quality and quantity of recombinant proteins production.

In comparison to Saccharomyces cerevisiae , Pichia pastoris provides a major advantage in the glycosylation of secreted proteins because it does not hyperglycosylate the heterologous proteins.

Both yeast strains have a majority of N-linked glycosylation of the high-mannose type, but the length of the oligosaccharides chain added to proteins in Pichia around mannose residues per side chain is much shorter than those expressed in Saccharomyces cerevisiae approximately mannose residues per side chain , suggesting that glycoproteins produced in Pichia pastoris may be more suitable for therapeutic use in humans [ 66 ],[ 67 ].

Therefore, Pichia pastoris can be an attractive alternate for large-scale production of recombinant insulin and insulin analogues. Comparing the different insulin production systems where the bacterial expression systems show higher average specific productivity and maximum biomass concentrations are higher in yeast, the overall production space-time yield remains similar as shown in Table 1 [ 70 ].

Saccharomyces cerevisiae has been extensively used to produce recombinant human insulin since early s [ 17 ],[ 18 ] and a large proportion of recombinant commercial insulins are produced by this yeast expression system [ 19 ],[ 74 ]. For efficient expression and secretion of recombinant proinsulin in yeast, insulin construct was engineered to contain the native A-chain and a B-chain lacking the C-terminal B30 threonine, either directly fused or linked via a short synthetic C peptide like AAK.

The single chain proinsulin was purified and converted to active insulin by a trypsin-mediated transpeptidation reaction in presence of threonine ester [ 19 ]. Besides native recombinant insulin, various insulin analogues are also being produced in S. Insulin Aspart is another fast-acting insulin analogue, which was produced in S. Insulin Aspart was generated by replacing proline residue at position 28 with aspartic acid in the B-chain.

This genetic modification resulted in an increase in inter-chain charge repulsion, decrease in self-association and thus causing rapid entry into the blood from the site of subcutaneous injection [ 63 ],[ 75 ]. Insulin Detemir is another recombinant long-acting insulin analogue that was commercially produced in S. Recombinant Detemir have been generated by removing the threonine residue at the 30 position of the B-chain, and a C14 fatty acid chain covalently attached to the lysine residue at the 29 position of the B-chain.

These genetic alterations resulted in the binding of insulin to albumin in plasma, which ensured the slow and constant release of insulin and thus prolonging its duration of action up to 24 hours [ 76 ]-[ 78 ].

Sacharomyces cerevisiae has been reported for the production of more than 40 different recombinant proteins [ 79 ]. A few of which related to diabetes are illustrated in Table 2 , along with different characteristics. Furthermore, a synthetic leader sequence had been developed by Kjeldsen and associates at Novo Nordisk for more efficient protein secretion in yeast [ 79 ],[ 80 ]. Transgenic plants have been utilized to produce recombinant proteins because of their advantage of cost effectiveness, high quality protein processing, absence of human pathogens, ease of production and presence of eukaryotic machinery for posttranslational modifications.

Initially, the human growth hormone was the recombinant protein product extracted from transgenic tobacco plant [ 81 ]. After that, numerous different products have developed from plants such as Hepatitis-B-Virus surface antigen, antibodies, industrial proteins and milk proteins. Recombinant human insulin has been successfully expressed and produced in oilseeds of plant Arabidopsis thaliana [ 27 ]. This technology involved the targeted expression of insulin in subcellular organelles known as oilbodies that allowed very high level of expression with easy recovery of recombinant insulin.

Oilbodies are storage organelles inside the oilseeds, which comprises of hydrophobic triacylglycerol core encapsulated by phospholipid membrane and an outer wall of proteins known as oleosins. Genetically engineered oil seeds have been generated with recombinant protein specifically targeted to oilbodies as oleosin fusion [ 27 ],[ 82 ],[ 83 ].

Then the oilbodies are easily separated from other seed components by liquid-liquid phase separation, which reduced the number of chromatography steps required to obtain purified insulin.

It has been observed that insulin accumulated to high level in transgenic seed 0. Recombinant insulin was cleaved from the oleosin fusion partner and matured with trypsin digestion following oil body purification to yield a biologically active insulin.

This study clearly demonstrated that expression of insulin as oleosin fusion protein in plant allow accumulation of large amount of recombinant insulin within the seed and also provide simple downstream purification by centrifugation i. Subsequent maturation to obtain biologically active insulin can be accomplished using standard enzymatic methods currently used for commercial production of insulin from E. Oilseeds also act as a natural cellular warehouse, where recombinant insulin can be stockpiled until required [ 27 ].

In another approach, transgenic plants have been generated, in which, tobacco and lettuce chloroplasts were transformed with human proinsulin comprised of A, B and C-chains fused with the cholera toxin B subunit [ 28 ]. Oral delivery of unprocessed proinsulin encapsulated in plant cell or by injection into mice revealed lowering of blood glucose levels similar to commercially available insulins.

C-peptide of proinsulin, which is not present in current commercially available insulin and insulin analogues derived from E. Very high level of expression of biologically active proinsulin in tobacco and lettuce leaves and long-term stability in dried leaves offers a reliable low-cost technology for both injectable as well as oral delivery of proinsulin.

Dietary and lifestyle changes are causing dramatic increase in diabetes incidence all over the world. Both Type I and Type II diabetic patients use insulin, however late stage Type II diabetes patients require large doses of insulin as they develop insulin resistance. The dramatic increase in the number of diabetic patients globally and exploration of alternate insulin delivery methods such as inhalation or oral route is bound to escalate the demand for recombinant insulin in near future.

Current manufacturing technologies will not be able to meet the growing demand of insulin due to limitation in production capacity and high production cost. Recombinant human insulin is produced predominantly using E.

However, there is an upmost need to increase the production by several fold of a biologically active insulin and its analogues from E. Another strategy, using a different expression host other than E. Moreover, transgenic seeds can also act as warehouse where recombinant insulin can be stockpiled until required. Nielsen J: Production of biopharmaceutical proteins by yeast. Landes Biosci Bioengineered. The for-profit companies who are supposed to negotiate, PBMs, do so in their own interests and not the interests of patients.

Patients are left powerless, and are shamed publicly for their weakness. In the current rebate system, for some PBMs, the incentive may be to favor branded Humalog. Therefore, the remainder of this article will focus on barriers to insulin affordability and pricing by focusing on the manufacturers, and not the impact of pharmacy benefit managers, payers, or other actors in the healthcare industry.

Diabetes Sci. See Beran et al. Biologics may also be cells or tissues used in transplantation. Biologics as a class include therapeutic proteins, toxins and antitoxins, viruses, blood and blood products, gene therapy products, and whole cells, among others.

As a general matter, they are much more complex than traditional small-molecule drugs. See 21 U. I NDA process. See generally 42 U. Other insulin follow-on products and biosimilars are in the pipeline. Pharmacy Tech. Biocon in cooperation with Mylan Gan and Lee, and Wockhardt have all begun Phase 3 clinical trials for follow-on Lantus products. See id. While some articles use follow-on biologic and biosimilar interchangeably, see, e.

Biosimilars are approved under the BLA pathway while follow-on biologics, for the purposes of this article, are those insulins approved through the FDCA b 2 pathway. Jack T. See also Cefalu et al.

Automatic substitution is largely governed by state law. Some states already have laws allowing prescriptions for a biologic to be filled with biosimilars deemed interchangeable, just like generics. See also Gary M. A high hurdle for what constitutes interchangeability will limit automatic substitution of affordable. If a biosimilar is to be dispensed to a patient, it must be prescribed-selected by a treating physician for that patient. State law will not make the choice automatically.

Preston Atteberry et al. Blog, Apr. This impression has been reinforced by case reports regarding novel side effects, reducing the proclivity of physicians to prescribe biosimilars. One study suggested that brand loyalty may factor into insulin competition. Another patient perspective study agreed that brand loyalty was a factor in comparison to company loyalty , but those patients surveyed indicated that they would be willing to try insulins made by other smaller companies.

Biosimilars , 17 Hous. Health L. Even more so than for small-molecule drugs, the manufacturing complexity and development costs for biologics can serve as a potent barrier to entry, keeping competitors off the market. Dzintars Gotham et al. See also Martin K. See also Michael S. Sinha et al.

Cohen, supra note 30, at 3. Aaron S. These price drops would likely be followed by continued escalating prices after their competitors exit the market, potentially leaving patients worse off than before.

This tactic, often called predatory pricing, could be an antitrust violation under the Robinson-Patman Act, 15 U. The Supreme Court, however, has not generally been receptive to predatory pricing cases, especially when the low price is above cost.

See generally Brooke Group Ltd. If the insulin manufacturers were to drop their prices below cost, which would be difficult and unlikely, there could be a successful claim. Even if the conduct did not constitute an antitrust violation, it would have significant anticompetitive consequences in the insulin market and would have devastating consequences for patients in the short term. Avik S. The study by Gotham et al. Erwin A. Fuhr, Jr. Not only are the fixed costs of manufacturing insulin and other biologic high, but the variable, marginal, and fractional costs are also high in comparison to small molecule drugs.

See Price, Making Do , supra note 99, at — Ravi Gupta et al. Reciprocal approval could also facilitate regulatory responses to mitigate bad public health outcomes when drugs face shortages or dramatic price increases.

Thus, international sources could lead to increased US competition for a meaningful number of drugs and might be worth pursuing in concert with other strategies. These strategies would include continuing the increased resources and capacity at FDA; prioritizing approvals and waiving application fees for drugs with few generic versions; and where medically appropriate, permitting automatic substitution of drugs within treatment 49 classes at the pharmacy level.

Bovine insulin is manufactured by USV product Longact. Human insulins are manufactured by Wockhardt product Wosulin and Biocon product Insugen. Insulin glargine is manufactured by Wockhardt product Glaritus and Biocon product Basalog. Other insulin combination products are available manufactured by Cadila. Gupta et al. Importation is distinct from purchasing insulin manufactured abroad: Novo Nordisk and Sanofi largely manufacture their insulin products abroad.

May 30, Some cities, counties, and schools even have policies of importing drugs from Canada, despite the fact it is illegal and discouraged by the FDA.

These figures are an estimate and would be dependent on several decisions of exporting countries. These figures may underestimate savings: if potential exporting countries are willing to provide unlimited supply for export to the United States and forced-sale provisions can be fully enforced, savings may be higher. These estimates may also overestimate savings: if major export sources permit higher launch prices or experience launch delays, and if the supply of new drugs shifts toward products that are exempt from importation, savings may be lower.

See Atteberry et al. Bach et al. For a more detailed analysis of price capping and pharmaceutical price regulation more generally, see generally Michelle M. Howard et al. Perspectives , ; Cong. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.

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