CN1390854A

CN1390854A - Process for rourifying insuline

Info

Publication number: CN1390854A
Application number: CN 02113186
Authority: CN
Inventors: 罗桂荣; 彭淑华; 陈慧
Original assignee: Jiangsu Wanbang Biological Pharmaceutical Co Ltd
Current assignee: Jiangsu Wanbang Biological Pharmaceutical Co Ltd
Priority date: 2002-06-15
Filing date: 2002-06-15
Publication date: 2003-01-15
Anticipated expiration: 2022-06-15
Also published as: CN1165549C

Abstract

A process for purifying insuline includes such steps as salting out, sodium salt crystallizing, zinc salt recrystallizing, and collecting dried insuline crystal powder. Its advantages are short period and high output rate.

Description

The purification process of Regular Insulin

Technical field

The invention relates to the purification process of Regular Insulin, the crystallization method of the Regular Insulin of more specifically saying so is used to prepare high purity insulin.

Background technology

At present, known insulin production mostly adopts zinc crystallization technology, and its technical process is: the thing lysate of saltouing → remove acid albumin → filtrations → zinc precipitation → dissolving → and except that basonuclin → filtration → crystallization → collection drying.Its shortcoming is that product yield is low, operation steps is many, production cycle is long, the supplementary product onsumption height, in the technology simultaneously output need again recovery article about 15%, in particular for high purity insulin production, obviously its technology is unreasonable, Chinese patent publication number CN1077203A invention disclosed patent application " a kind of crystallizing process of insulin sodium salt ", its technology is: the thing lysate → filtration → crystallization of saltouing → drying collection → insulin sodium salt crystal powder.This technology is compared with zinc crystallization technology, and operation is few, and is with short production cycle, only improves 3-5% but the shortcoming that exists is the yield of tiring.

Summary of the invention

The objective of the invention is, a kind of purification process of Regular Insulin is provided, this method not only makes Regular Insulin purity height, stable yield, and overcome the CN1077203A patent application yield shortcoming on the low side of tiring.

Technical scheme of the present invention is: its purification process is: Regular Insulin saltout thing → sodium salt crystal → recrystallization → collection dry the insulin crystals powder; The method of sodium salt crystal is:

The Regular Insulin thing of saltouing becomes the lysate (W/V) of 20-30% with the 0.1N diluted hydrochloric acid dissolution; With the 10N sodium hydroxide solution this lysate is transferred about PH7, added 0.5-2/ ₁₀The sodium chloride solution of 30% concentration of amount stirs; Mend with the 5N sodium hydroxide solution again and transfer to PH7.5-8.5, stirring at room crystallization 8-10 hour.Leave standstill again, crystallization is avaled fully after, inhale and to remove supernatant liquor, with centrifugal or filtering method collection crystallization; Cold sodium chloride solution with concentration 2-10% is washed crystallization 1-2 time, gets the wet crystallization of insulin sodium salt.The method of recrystallization is:

The wet crystallization of insulin sodium salt is analysed dried by 26-30%, calculate cumulative volume by the 0.5-2% densitometer; Add solid citric acid 1-1.5 gram by every gram dry product, be dissolved in water into about 1/3 amount of cumulative volume again; Leave standstill 10-15 hour after-filtration and get clear filtrate the cold house; Adding concentration by 1-1.5 gram dry product in filtrate is 20% zinc acetate solution 1-2ml, cold acetone 16-20ml, be diluted to cumulative volume with cold water again after, transfer PH6.0-6.5 with 4N ammoniacal liquor, stir at a slow speed more than 5 hours, change the cold house over to and leave standstill crystallization is avaled fully.The collection drying means is:

Suction goes supernatant liquor to collect crystallization with centrifugal method, and with cold distilled water washing 1-2 time, acetone, ether dewater, do siccative vacuum-drying with Vanadium Pentoxide in FLAKES then, promptly get the insulin crystals powder again.

Term " Regular Insulin saltout thing " is that present technique field professional is known.

Described cold house is meant centigradetemperature 5-10 ℃.

Advantage of the present invention is: overcome the shortcoming of simple zinc crystallization or simple sodium salt crystal, insulin crystals yield and purity are improved, the yield of tiring improves 10-15%.

Embodiment 1

With the 1Kg Regular Insulin thing 0.1N diluted hydrochloric acid dissolution of saltouing, make into volume 5 liters, transfer PH8.2 with 10 Equivalent Hydrogen sodium oxides, add sodium chloride solution 0.5 liter of concentration 30%, stir, mend with 5N sodium hydroxide again and transfer to PH8.2, stirring at room left standstill after 8 hours, the centrifugal collection crystallization in back of avaling fully to be crystallized, with 3% sodium chloride solution wash crystallization 1 time, the sodium salt crystallization of wetting.

Recrystallization

Analyse by 26% dried, by dry weight 0.5/ ₁₀Calculate cumulative volume, add solid citric acid 1 gram by every gram dry product,

Be dissolved in water to 1/3 amount of cumulative volume, after about 10 hours are left standstill in the cold house, filter, in filtrate, press 1.0

1 milliliter of the zinc acetate of gram dry product adding 20%, 20 milliliters of cold acetones are diluted to always with 5-10 ℃ of cold water

Volume is transferred PH6.1-6.3 with 4N ammoniacal liquor, stirs at a slow speed 5 hours, and 10 hours are left standstill to crystallization in the cold house

Avale fully.Collect drying, get the insulin crystals powder.The yield of tiring improves 10-15%.

Embodiment 2

With the 1Kg Regular Insulin thing 0.1N diluted hydrochloric acid dissolution of saltouing, make into volume 3 liters, transfer PH7.8 with 10 Equivalent Hydrogen sodium oxides, add sodium chloride solution 0.15 liter of concentration 30%, stir, mend with 5N sodium hydroxide again and transfer to PH7.8, stirring at room left standstill after 8 hours, the centrifugal collection crystallization in back of avaling fully to be crystallized, with 3% sodium chloride solution wash crystallization 1 time, the insulin sodium salt crystallization of wetting.

Recrystallization

Analyse driedly by 30%, calculate cumulative volume, add solid citric acid 1.2 grams by every gram dry product by dry weight 2/10,

Be dissolved in water to 1/3 amount of cumulative volume, after leaving standstill about 15 hours, filter, in filtrate, add 1.5 milliliters of 20% zinc acetates by 1 gram dry product, 20 milliliters of cold acetones are diluted to cumulative volume with 5-10 ℃ of cold water, transfer PH6.1-6.3 with 4N ammoniacal liquor, stirred at a slow speed 5 hours, the cold house leave standstill 10 hours complete to crystallization.Collect drying, get the insulin crystals powder, the yield of tiring improves 10-15%.

Claims

1, a kind of purification process of Regular Insulin is characterized in that this method is: Regular Insulin saltout thing → sodium salt crystal → recrystallization → collection dry the insulin crystals powder; The method of described sodium salt crystal is: the thing of (a) Regular Insulin being saltoutd becomes the lysate (W/V) of 20-30% with the 0.1N diluted hydrochloric acid dissolution; (b) with sodium hydroxide solution lysate is transferred PH7.5-8.5, stirring at room crystallization 8-10 hour; (c) leave standstill, it is back with centrifugal or filtration collection crystallization that crystallization is avaled fully; (d) wash crystallization 1-2 time with the cold sodium chloride solution of 2-10%, get the wet crystallization of insulin sodium salt.The method of described recrystallization is: the crystallization of (a) insulin sodium salt being wet is analysed dried by 26-30%, press 0.5-2/ ₁₀Densitometer is calculated cumulative volume, adds solid citric acid 1-1.5 gram by every gram dry product, is dissolved in water into 1/3 amount of cumulative volume again; (b) 10-15 hour after-filtration left standstill in the cold house, and filtrate need be clarified; (c) adding concentration by 1 gram dry product in filtrate is 20% zinc acetate solution 1-2ml, cold acetone 16-20ml, be diluted to cumulative volume with cold water again after, transfer PH6.0-6.5 with 4N ammoniacal liquor, stir at a slow speed more than 5 hours and make crystallization complete, leave standstill to crystallization and avale fully the cold house again.Described collection drying means is: inhale and to remove supernatant liquor, collect crystallization with centrifugal method, again with cold distilled water washing 1-2 time, acetone, ether dewater, do siccative vacuum-drying with Vanadium Pentoxide in FLAKES and promptly get the insulin crystals powder then.

2, method according to claim 1, it is characterized in that: (b) step of sodium salt crystal is to use the 10N sodium hydroxide solution, lysate is transferred about PH7, and 30% the sodium chloride solution that by volume adds 0.5-2/10 amount again stirs, and mends with the 5N sodium hydroxide solution and transfers PH7.5-8.5.

3, method according to claim 1 is characterized in that: cold house's temperature is 5-10 Celsius ℃.