Insulin Purification Process
Recombinant human insulin from E. coli — inclusion body recovery, refolding, ion exchange, reverse phase chromatography, and crystallization formulation
Process Overview
Recombinant insulin production in E. coli yields insulin as insoluble inclusion bodies. The downstream process recovers these bodies, solubilizes and refolds the proinsulin to native insulin, then purifies through chromatography. Final product is crystallized as zinc insulin for formulation. Overall yield: 60–75% from inclusion bodies to purified insulin.
Process Steps
1
Cell Disruption
Inclusion Body Isolation
Harvest E. coli cells by centrifugation. Resuspend in buffer, disrupt by high-pressure homogenization (15,000–20,000 psi). Wash inclusion bodies repeatedly with Triton X-100 and urea to remove membranes, endotoxin, and contaminants. Recover pure insulin inclusion bodies.
Yield: >95%
Purity: 70–85%
2
Solubilization & Refolding
Proinsulin Refolding
Solubilize inclusion bodies in 6–8 M urea or guanidine HCl with reducing agent (DTT, β-mercaptoethanol). Dilute slowly into refolding buffer (pH 10.5, redox conditions) to allow correct disulfide bond formation. Refolding yield: 50–80%. Target: correctly folded proinsulin with native disulfide bonds.
Yield: 60–80%
Purity: 30–50%
3
Enzymatic Processing
Enzymatic Cleavage
Add trypsin and carboxypeptidase B to convert proinsulin to mature insulin by removing the C-peptide. Control temperature (2–8°C) and reaction time (30–120 min) to minimize degradation. Stop reaction by acidification to pH 2–3.
Yield: >90%
Product: Mature insulin + C-peptide
4
Ion Exchange Chromatography
Primary Capture (CEX)
Adjust pH to 6.5–7.0. Insulin (pI 5.3) is slightly negative and binds to cation exchange resin (SP Sepharose). Impurities (C-peptide, proinsulin variants, protease) are less retained. Elute with NaCl gradient. This is the primary capture step achieving 60–80% purity.
Yield: 85–92%
Purity: 80–90%
5
Reverse Phase Chromatography
RPC Polishing
Load CEX eluate onto C8 or C18 silica column in acidic ACN/water. Insulin and related impurities (desamido, dipeptides) separate based on hydrophobicity. Gradient elution with ACN (30–40%). RPC achieves >99% purity in a single step. Dry and reconstitute insulin peak.
Yield: 80–90%
Purity: >99%
6
Crystallization & Formulation
Insulin Crystallization
Reconstitute purified insulin in citrate buffer (pH 6.5–7.0) with zinc acetate. Crystallize by adjusting pH to isoelectric point (pI 5.3). Yield: crystalline zinc insulin suspension. Filter, wash, and resuspend in formulation buffer. Final drug substance for fill-finish.
Yield: >95%
Form: Crystalline
Target Molecule: Insulin
| Molecular Weight | 5,808 Da (51 amino acids) |
| Isoelectric Point (pI) | 5.3 (slightly acidic) |
| Charge at pH 7 | −0.5 (slightly negative) |
| Solubility | pH-dependent, min at pI 5.3 |
| Structure | Two polypeptide chains (A: 21 aa, B: 30 aa) linked by 2 disulfide bonds |
View full Insulin molecule page →
Cost Considerations
| Step | Key Cost Driver | Relative Cost |
|---|
| Inclusion Body Isolation | Centrifuge + homogenizer energy | Medium |
| Refolding | Urea, redox reagents, tank volume | High |
| Enzymatic Cleavage | Trypsin, carboxypeptidase B | Medium |
| Cation Exchange | Resin, buffer salts | Medium |
| Reverse Phase HPLC | Silica resin, ACN solvent | High |
| Crystallization | Zinc acetate, buffers | Low |
Refolding and RPC are the highest-cost steps. Refolding requires large tanks and consumes urea and redox reagents. RPC consumes expensive ACN solvent. Continuous refolding and preparative RPC reduce costs. Use
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Frequently Asked Questions
Why is insulin expressed as inclusion bodies in E. coli?
Inclusion bodies form when recombinant proteins aggregate in the bacterial cytoplasm due to high expression levels and improper folding. While this complicates downstream processing, it provides a crude enrichment of the target protein (70–85% purity in the isolated bodies) and protects it from proteolytic degradation.
What is the critical step in insulin purification?
The refolding step is the most critical and lowest-yielding. Insulin requires correct formation of two disulfide bonds (A7-B7 and A20-B19) while avoiding mispaired bonds. Redox buffer conditions (oxidized and reduced glutathione or cysteine/cystine) promote correct disulfide formation. Yield: 50–80% depending on conditions.
Why is reverse phase chromatography used for insulin?
RPC achieves extremely high resolution separation of insulin variants (desamido, deamidated, incorrectly processed). The hydrophobic surface of C18 silica separates these variants that differ by only a few hydrophobic residues. ACN gradient elution followed by acidification yields purified insulin ready for crystallization.
Can insulin be purified without RPC?
Yes, but with lower purity. Alternative approaches include multimodal chromatography (mixed-mode resins), which combines ion exchange and hydrophobic interactions. However, RPC remains the industry standard for achieving >99% purity. The newer insulin glargine (Lantus) uses a different purification strategy due to its modified structure.
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