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%)