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Purificatino of Insulin Glargine
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- Time of issue:2014-01-03 00:00
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Purificatino of Insulin Glargine
nsulin glargine is a type of human insulin analogues which has low solubility in neutral PH solution. It dissolves completely in acid (PH 4) injecta. After injecting into subcutaneous tissue, micro-sediments created by neutralization of acid solution could release small amount of insulin glargine constantly, therefore, it produce expectable, long-lasting, stable, and blood concentration with no peak value.
Chemical name: 21A-Gly-30B a-L-Arg-30Bb-L-Arg-human insulin.
Molecular formula: C267H404N72O78S6.
Molecular weight: 6063.
Purification method:
Zymotic fluid (prosomal) |
Remove cells by centrifugal force |
Supernatant fluid |
Phosphoric acid PH 3.8 |
CQK30S |
Loading amount < 50g/L |
Eluate by ammonium acetate |
|
Crystallize |
Add 0.5% of Phenol into eluant, 4% of AnCl2 and NaOH adjust PH to 4.08, stir slowly for 15min, transfer to 2-8℃environment, stay overnight, centrifugal collecting and precipitating |
Enzymatic conversion |
|
GH C8 reverse phase |
GH C8 (10µm, 120Å) column, 10% acetonitrile-0.25M acetic acid equilibrium liquid, enzymatic conversion liquid and water with 1:5 portion dilution then put the sample, gradient elution by acetonitrile, get product with 94.5% purity; dilute the product from previous step, then use GH C8 (the same as before), acetronitrile-0.2M sodium acetate (PH 5.0) equilibrium liquid, get 98.5% purity product; dilute the product from previous step by water with 1:3 portion, use GH C8 (the same as before), ethanol-0.05M acetic acid equilibrium liquid, gradient elution by ethoanol, get 99% pure insulin glargine finished product |
Crystallize |
Put the sample from previous step into citric acid, ZnCl2 and adjust PH to get crystallized sediments, wash the sediment with cold water |
Insulin glargine finished product (purity >99%) |

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