Chapter 33 — Case Study 1: Insulin Synthesis — A Century of Progress
"Insulin's history is a microcosm of the history of biotechnology. From extracted protein (1921), to chemical synthesis (1963), to recombinant DNA (1978), to engineered analogs (1990s+), insulin shows how chemistry and biology together have transformed medicine." — paraphrase from a biotechnology history text
Insulin was the first protein hormone to be isolated, the first to be sequenced, the first protein drug to be made by chemical synthesis, and the first recombinant pharmaceutical. Its century-long history mirrors the development of protein chemistry, peptide synthesis, and biotechnology. This case study traces that history with focus on the chemistry.
What is insulin?
Insulin is a small protein (51 amino acids) that regulates glucose homeostasis in animals. It is produced by pancreatic β-cells (the islet of Langerhans cells in the pancreas) in response to elevated blood glucose. Its job: signal cells to take up glucose, signal the liver to store glucose as glycogen.
Without insulin, blood glucose rises uncontrollably (Type 1 diabetes); cells starve for energy despite high blood glucose; ketogenesis ramps up, leading to acidosis. Untreated Type 1 diabetes was uniformly fatal before insulin was discovered.
Insulin's structure
Mature insulin has two polypeptide chains held together by disulfide bonds: - A chain: 21 amino acids (sequence GIVEQCCASVCSLYQLENYCN). - B chain: 30 amino acids (sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT). - Disulfide bonds: - Cys6 (A) – Cys11 (A): intra-A disulfide. - Cys7 (A) – Cys7 (B): A-B linkage. - Cys20 (A) – Cys19 (B): A-B linkage.
Both chains are derived from a single precursor (proinsulin) which is processed in the β-cell to mature insulin + a "C peptide" (which is excreted).
The 3D structure has the two chains in a compact, helical arrangement held by the disulfides. Both chains are essential — neither alone has activity.
1921: Discovery and isolation
Frederick Banting, Charles Best, and others at the University of Toronto isolated insulin from animal pancreas in 1921 (Banting + Macleod won the 1923 Nobel Prize). The first patients (Leonard Thompson, 14 years old, dying of T1D) were treated in 1922 — and lived.
Initial production: extract from cattle or pig pancreas (animal insulin differs from human by 1-3 amino acids). Eli Lilly (USA) and Novo Nordisk (Denmark) became major insulin manufacturers. Animal insulin was the standard treatment for ~60 years.
1955: Sequencing
Frederick Sanger sequenced insulin (1953, published 1955) — the first protein ever sequenced. Sanger received the 1958 Nobel Prize in Chemistry for this work. The sequencing methodology (Edman degradation + dinitrophenyl labeling) became a standard technique.
The sequence revealed: - Two chains of specific length. - Three disulfide bonds at specific positions. - Conserved across mammalian species (with small variations).
This was the first proof that proteins have specific, defined sequences — not random or mixtures. It opened the door to chemical synthesis.
1963: Chemical total synthesis
Du Vigneaud and Bauer (Cornell, 1963) accomplished the first chemical total synthesis of insulin. They used solution-phase peptide synthesis with classical methods (mixed anhydride coupling, hydrogenolysis of benzyl protecting groups). The two chains were synthesized separately and then joined by oxidative folding.
The synthesis was a tour de force, but: - Yields were extremely low (each step was 30-50% yield over many steps; total yield ~1%). - Each chain alone took weeks of work. - Scale: very limited; not practical for medicine.
Why so low-yielding? Solution-phase peptide synthesis runs into: - Side reactions at each coupling step. - Need to purify intermediates (crystallization, chromatography). - Difficulty re-folding correctly (getting the right disulfide pattern).
Total synthesis gave proof-of-concept but was not commercially viable.
1965-1980s: SPPS and continued challenges
Bruce Merrifield invented Solid-Phase Peptide Synthesis (SPPS) in 1963 (Nobel 1984). The method dramatically simplified peptide synthesis: each amino acid added one at a time on a solid resin support; no need to purify between couplings.
But SPPS for insulin was still difficult: - 51 amino acids is long for SPPS; coupling efficiency drops with chain length. - Disulfide formation must be done carefully to avoid scrambling. - Each chain needs separate synthesis + correct disulfide pairing.
By the late 1970s, SPPS could produce insulin at research scale but not commercial.
1978: The recombinant breakthrough
The decisive change came from biotechnology, not chemistry. Genentech (founded by Bob Swanson and Herb Boyer in 1976) used recombinant DNA to make human insulin in bacteria:
- Synthesize the human insulin gene chemically (or use a cloned cDNA).
- Insert the gene into E. coli plasmids.
- Express the protein in bacterial fermentation (large-scale).
- Refold and process the recombinant proinsulin to mature insulin.
Genentech's product, Humulin (1982, marketed by Eli Lilly), was the first FDA-approved recombinant pharmaceutical. It dramatically scaled insulin production and eliminated the immunogenicity issues of animal insulin.
The chemistry was less of an issue once cells could produce the protein. Humans now had unlimited insulin supply.
1990s+: Engineered insulin analogs
With the gene available, scientists designed insulin analogs — variants of human insulin with modified properties:
Lispro (Humalog, Lilly 1996)
Two amino acids in the B chain are swapped (Pro28-Lys29 in human insulin → Lys28-Pro29 in lispro). This destabilizes the dimer/hexamer that normal insulin forms, making lispro rapid-acting (begins working in 15 minutes, peaks at 1 hour).
Use: pre-meal injection to control post-meal blood glucose spikes.
Glargine (Lantus, Sanofi 2000)
Modified to shift the isoelectric point (pI). At physiological pH 7.4, glargine precipitates and is slowly released, giving long-acting insulin (lasts 24 hours).
Use: basal (overnight) glycemic control.
Detemir (Levemir, Novo 2004)
A fatty acid (myristic acid, C14) is attached to a Lys residue in the B chain. This makes detemir bind to albumin in the bloodstream, slowing absorption and prolonging duration.
Use: basal control with less variability.
Aspart (NovoLog), Glulisine (Apidra)
Other rapid-acting variants with different specific modifications.
These engineered insulins have transformed diabetes care. Patients can now match insulin to their meals and lifestyle, achieving better glycemic control and fewer hypoglycemic episodes.
SPPS today: the GLP-1 revolution
Modern SPPS, with improved coupling reagents (HATU, Oxyma) and better protecting groups, can synthesize peptides up to ~50 amino acids efficiently. While insulin (51 amino acids, 3 disulfides) is still mostly recombinant, GLP-1 agonists are the new SPPS workhorses:
- Semaglutide (Ozempic, Wegovy): 31 amino acids; fatty-acid-modified; SPPS-made.
- Liraglutide (Victoza, Saxenda): 31 amino acids; SPPS-made.
- Tirzepatide (Mounjaro, Zepbound): 39 amino acids; dual GIP/GLP-1; SPPS-made.
Semaglutide alone is a > $20 billion/year drug as of 2024. Its synthesis is pure Chapter 33 chemistry: SPPS, solid-phase peptide synthesis with Fmoc protection and HATU coupling.
The GLP-1 agonist class has revolutionized: - T2D treatment (potent glucose lowering + weight loss). - Obesity treatment (Wegovy approved 2021). - Cardiovascular risk reduction (semaglutide reduces CV events).
Take-home
- Insulin is a 51-amino acid hormone, 2 chains held by 3 disulfide bonds.
- Discovered 1921; sequenced 1955 (Sanger Nobel); first chemical synthesis 1963 (Du Vigneaud); first recombinant pharmaceutical 1982 (Genentech Humulin).
- Engineered insulin analogs (lispro, glargine, detemir, aspart, glulisine) are made by recombinant DNA + small synthetic modifications. Each has specific kinetics for clinical use.
- Modern peptide drugs (GLP-1 agonists like semaglutide) are made by SPPS at industrial scale. SPPS uses Fmoc protection + HATU coupling + protected side chains.
- Insulin's history shows how chemistry, biotechnology, and clinical needs together transform medicine.
- Mastery of Chapter 33 amino acid and protein chemistry is the foundation for understanding peptide drug development.