CN116874586A - Insulin extraction process - Google Patents
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- CN116874586A CN116874586A CN202310908165.8A CN202310908165A CN116874586A CN 116874586 A CN116874586 A CN 116874586A CN 202310908165 A CN202310908165 A CN 202310908165A CN 116874586 A CN116874586 A CN 116874586A
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 title claims abstract description 102
- 102000004877 Insulin Human genes 0.000 title claims abstract description 51
- 108090001061 Insulin Proteins 0.000 title claims abstract description 51
- 229940125396 insulin Drugs 0.000 title claims abstract description 50
- 238000000605 extraction Methods 0.000 title claims abstract description 49
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 32
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 16
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- 239000002994 raw material Substances 0.000 claims abstract description 10
- 210000000496 pancreas Anatomy 0.000 claims abstract description 9
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 7
- 238000001035 drying Methods 0.000 claims abstract description 6
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 3
- 238000000746 purification Methods 0.000 claims abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 10
- 239000001632 sodium acetate Substances 0.000 claims description 10
- 235000017281 sodium acetate Nutrition 0.000 claims description 10
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- 241001465754 Metazoa Species 0.000 description 4
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 description 4
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- 241000894006 Bacteria Species 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
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- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 201000001421 hyperglycemia Diseases 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 1
- 208000032131 Diabetic Neuropathies Diseases 0.000 description 1
- 206010012689 Diabetic retinopathy Diseases 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 206010020710 Hyperphagia Diseases 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920000604 Polyethylene Glycol 200 Polymers 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 208000033679 diabetic kidney disease Diseases 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000004116 glycogenolysis Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003538 oral antidiabetic agent Substances 0.000 description 1
- 229940127209 oral hypoglycaemic agent Drugs 0.000 description 1
- 229940125395 oral insulin Drugs 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 206010036067 polydipsia Diseases 0.000 description 1
- 208000022530 polyphagia Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
- C07K14/625—Extraction from natural sources
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to the field of biological pharmacy, in particular to an extraction process of insulin, which comprises the following steps: pretreating, extracting, deproteinizing, purifying and drying raw materials; wherein, the extraction step adopts the extractant with the mass ratio of 5-8:6-9:0.1-0.5:80-89 parts of polyethylene glycol, sodium chloride, glacial acetic acid and water. Through the optimization of the extractant, the extraction efficiency is obviously improved, the extraction times are reduced, and meanwhile, 10g of insulin can be obtained per 1kg of pancreas through the subsequent purification steps, so that the yield is greatly improved.
Description
Technical Field
The invention relates to the field of biological pharmacy, in particular to an extraction process of insulin.
Background
Diabetes (diabetes mellitus) is a series of metabolic disorders syndrome of proteins, fat water and electrolytes, etc. caused by absolute or relative insufficient secretion of insulin and reduced sensitivity of target tissue cells to insulin, and is mainly marked by hyperglycemia. The main clinical manifestations of diabetes are polydipsia, diuresis, polyphagia and weight loss ("three more and one less"), hyperglycemia, glucose in urine (normal urine should not contain glucose), etc. Diabetes mellitus, if not treated effectively, can cause damage to multiple systems of the body, e.g., diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, etc. According to the latest data published in 2011 of the international diabetes alliance, the number of people suffering from diabetes worldwide reaches 3.66 hundred million, and the people are seriously harmed to human health.
In general, the treatment of diabetes includes oral hypoglycemic agents and insulin treatment. Insulin is a protein hormone secreted by islet beta cells within the pancreas by stimulation with endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, and the like. Insulin participates in regulating glucose metabolism, reducing gluconeogenesis, promoting glycogen synthesis, inhibiting glycogenolysis, accelerating anaerobic glycolysis and aerobic oxidation of glucose, promoting utilization of glucose by tissues, and promoting conversion of glucose into fat, so that the insulin can be used for treating diabetes.
Currently, insulin is generally classified into three generations, and the first generation of animal insulin is directly extracted from animals, generally from two animals, namely pigs and cattle, and has high immunogenicity. The second generation of human insulin is produced in large scale by transferring human insulin gene into bacteria or yeast bacteria by using genetic engineering means and utilizing fermentation process. The third generation is human insulin analogues, the amino acid sequence of human insulin is modified, and the production method is basically the same as that of the second generation human insulin. The second generation and the third generation of insulin have complex processes, and only few international companies have production capacity at present, so that the product has high price and is not beneficial to market popularization. For complex markets in China, animal insulin still has a certain proportion in clinic due to low cost and wide sources.
However, the conventional production process extracts insulin from the pancreas of domestic pigs and sheep, extracts and separates the insulin by acid-alcohol solution, separates the insulin by salting out and then separates and purifies the insulin, and the purity and the yield of the product are difficult to reach the quality standard of clinical application, so that development of an insulin extraction method with higher purity and yield of the product is needed.
Disclosure of Invention
Based on the defects in the prior art, the invention provides an insulin extraction process with higher product purity and yield.
For this purpose, the invention provides an extraction process of insulin, comprising the following steps: pretreating, extracting, deproteinizing, purifying and drying raw materials;
wherein, the extraction step adopts the extractant with the mass ratio of 5-8:6-9:0.1-0.5:80-89 parts of polyethylene glycol, sodium chloride, glacial acetic acid and water.
Preferably, the raw material pretreatment step includes: freezing pancreas at-20 to-40 deg.C for 2-4h, and storing at-15 to-18 deg.C.
Preferably, in the extraction step, the volume ratio of the raw materials to the extractant is 1:3-5, and the ratio of the mass to the volume is kg/L.
Preferably, the extraction temperature is 4-8deg.C; the extraction time is 24-36h.
Preferably, the protein removal step comprises adding NaOH solution into the crude protein after extraction, adjusting pH to 7.8-8.5, carrying out solid-liquid separation, and retaining liquid.
Preferably, the molar concentration of the NaOH solution is 0.5-2mol/L.
Preferably, the step of purifying comprises ultrafiltration and ion exchange chromatography.
Preferably, the ultrafiltration membrane has a molecular weight cut-off of 100000 daltons.
Preferably, the conditions for ion exchange chromatography separation are: the chromatographic column filler is one or more of SK1BH, WK40L or PK216, the particle diameter is 5-20 μm, the diameter of the chromatographic column is 5-10cm, and the length of the chromatographic column is 50-100cm; the balance liquid is sodium acetate solution with the concentration of 0.05-0.1mol/L, and the eluent is sodium acetate solution with the concentration of 0.2-1.0 mol/L.
Preferably, the pH of the balancing solution is 4.0-5.0.
Preferably, the pH of the eluate is 2.5-3.5.
The invention further provides insulin prepared by the extraction process.
The beneficial effects of the invention are as follows:
the invention provides an insulin extraction method, which remarkably improves extraction efficiency and reduces extraction times through optimizing an extracting agent, and simultaneously can obtain 10g of insulin per 1kg of pancreas through subsequent purification steps, thereby greatly improving the yield.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides an extraction process of insulin, comprising the following steps:
(1) Pretreatment of raw materials: freezing pancreas at-30deg.C for 3 hr, pulverizing, and preserving at-17deg.C;
(2) Extraction: taking 1kg of pancreas powder, adding 4L of extractant (PEG 6000, sodium chloride, glacial acetic acid and water in a mass ratio of 6:8:0.3:85), stirring for 10min, standing at 6 ℃ for extraction for 30h, removing supernatant and precipitate, and retaining a lower layer solution;
(3) Removing protein: regulating the pH of the lower layer solution to 8.0 with 1mol/LNaOH solution, standing for 30min, centrifuging at 4000r/min for 10min, and retaining the supernatant;
(4) Ultrafiltration: ultrafiltering with 100000 dalton ultrafilter membrane, and concentrating insulin extractive solution to 1/100 of original volume to obtain insulin concentrate.
(5) Ion exchange: selecting chromatographic columns with the diameter of 10cm and the length of 85cm, filling SK1BH filler with the particle size of 10 mu m, balancing for 2 hours by using 0.1mol/L sodium acetate solution (the pH is adjusted to 4.5 by acetic acid), loading insulin concentrate into the chromatographic columns, eluting by using 0.2, 0.5, 0.8 and 1.0mol/L sodium acetate solution (the pH is adjusted to 3.0 by acetic acid) in sequence, and collecting target proteins (the measurement is carried out by adopting an insulin ELISA kit, and collecting eluent with the content of more than 1 mg);
(6) Desalting and drying: placing the eluate into dialysis bag, placing into distilled water, dialyzing at 6deg.C for 24 hr, changing distilled water for 4 times, taking out, concentrating to 1/10 of original volume, and freeze drying to obtain insulin (11.1 g).
The embodiment further provides the insulin prepared by the extraction process.
Example 2
The embodiment provides an extraction process of insulin, comprising the following steps:
(1) Pretreatment of raw materials: freezing pancreas at-40deg.C for 2 hr, pulverizing, and preserving at-18deg.C;
(2) Extraction: taking 1kg of pancreas powder, adding 5L of extractant (PEG 6000, sodium chloride, glacial acetic acid and water in a mass ratio of 6:9:0.1:80), stirring for 10min, standing at 4 ℃ for extraction for 36h, removing supernatant and precipitate, and retaining a lower layer solution;
(3) Removing protein: regulating the pH of the lower layer solution to 7.8 with 0.5mol/LNaOH solution, standing for 30min, centrifuging at 4000r/min for 10min, and retaining the supernatant;
(4) Ultrafiltration: ultrafiltering with 100000 dalton ultrafilter membrane, and concentrating insulin extractive solution to 1/100 of original volume to obtain insulin concentrate.
(5) Ion exchange: selecting a chromatographic column with the diameter of 5cm and the length of 100cm, filling WK40L filling with the particle size of 20 mu m, balancing for 2 hours by using 0.05mol/L sodium acetate solution (the pH is adjusted to 5.0 by acetic acid), loading insulin concentrate into the chromatographic column, eluting by using 0.2, 0.5, 0.8 and 1.0mol/L sodium acetate solution (the pH is adjusted to 2.5 by acetic acid) in sequence, and collecting target proteins (the measurement is carried out by adopting an insulin ELISA kit, and collecting eluent with the content of more than 1 mg);
(6) Desalting and drying: placing the eluent into a dialysis bag, placing into distilled water, dialyzing at 6deg.C for 24h, changing distilled water for 5 times, taking out, concentrating to 1/10 of the original volume, and freeze drying to obtain insulin (10.6 g).
The embodiment further provides the insulin prepared by the extraction process.
Example 3
The embodiment provides an extraction process of insulin, comprising the following steps:
(1) Pretreatment of raw materials: freezing pancreas at-20deg.C for 4 hr, pulverizing, and preserving at-15deg.C;
(2) Extraction: taking 1kg of pancreas powder, adding 3L of extractant (PEG 6000, sodium chloride, glacial acetic acid and water in a mass ratio of 8:6:0.5:89), stirring for 10min, standing at 8 ℃ for extraction for 24h, removing supernatant and precipitate, and retaining a lower layer solution;
(3) Removing protein: regulating the pH of the lower layer solution to 8.5 with 2mol/LNaOH solution, standing for 30min, centrifuging at 4000r/min for 10min, and retaining the supernatant;
(4) Ultrafiltration: ultrafiltering with 100000 dalton ultrafilter membrane, and concentrating insulin extractive solution to 1/100 of original volume to obtain insulin concentrate.
(5) Ion exchange: selecting a chromatographic column with the diameter of 10cm and the length of 50cm, filling PK216 filling with the particle size of 5 mu m, balancing for 2 hours by using 0.05mol/L sodium acetate solution (the pH is regulated to 4.0 by acetic acid), loading insulin concentrate into the chromatographic column, eluting by using 0.2, 0.5, 0.8 and 1.0mol/L sodium acetate solution (the pH is regulated to 3.5 by acetic acid) in sequence, and collecting target proteins (the measurement is carried out by adopting an insulin ELISA kit, and collecting eluent with the content of more than 1 mg);
(6) Desalting and drying: placing the eluent into a dialysis bag, placing into distilled water, dialyzing at 6deg.C for 24h, changing distilled water for 3 times, taking out, concentrating to 1/10 of the original volume, and freeze drying to obtain insulin (10.3 g).
The embodiment further provides the insulin prepared by the extraction process.
Comparative example 1
This comparative example provides an insulin extraction process which differs from example 1 only in that PEG6000, ammonium sulphate, glacial acetic acid and water in a mass ratio of 6:8:0.3:85 are used as extractant, with 8.4g of insulin finally being obtained.
Comparative example 2
This comparative example provides an insulin extraction process which differs from example 1 only in that PEG6000, sodium chloride, hydrochloric acid and water in a mass ratio of 6:8:0.3:85 are used as extractant, resulting in 3.7g of insulin.
Comparative example 3
This comparative example provides an insulin extraction process which differs from example 1 only in that PEG200, sodium chloride, hydrochloric acid and water in a mass ratio of 6:8:0.3:85 are used as extractant, resulting in 6.1g of insulin.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. An extraction process of insulin is characterized by comprising the following steps: pretreating, extracting, deproteinizing, purifying and drying raw materials;
wherein, the extraction step adopts the extractant with the mass ratio of 5-8:6-9:0.1-0.5:80-89 parts of polyethylene glycol, sodium chloride, glacial acetic acid and water.
2. The extraction process according to claim 1, characterized in that the raw material pretreatment step comprises: freezing pancreas at-20 to-40 deg.C for 2-4h, and storing at-15 to-18 deg.C.
3. The extraction process according to claim 1, wherein in the extraction step, the volume ratio of the mass of the raw material to the extractant is 1:3-5, and the ratio of the mass to the volume is kg/L.
4. The extraction process according to claim 1, wherein the temperature of the extraction is 4-8 ℃; the extraction time is 24-36h.
5. The extraction process according to claim 1, wherein the deproteinizing step comprises adding NaOH solution to the crude protein after extraction, adjusting pH to 7.8-8.5, solid-liquid separation, and retaining the liquid.
6. The extraction process according to claim 5, wherein the molar concentration of the NaOH solution is between 0.5 and 2mol/L.
7. The extraction process according to claim 1, wherein the purification step comprises ultrafiltration and ion exchange chromatography.
8. The extraction process of claim 1, wherein the ultrafiltration membrane has a molecular weight cut-off of 100000 daltons.
9. The extraction process according to claim 1, wherein the conditions of ion exchange chromatography separation are: the chromatographic column filler is one or more of SK1BH, WK40L or PK216, the particle diameter is 5-20 μm, the diameter of the chromatographic column is 5-10cm, and the length of the chromatographic column is 50-100cm; the balance liquid is sodium acetate solution with the concentration of 0.05-0.1mol/L, and the eluent is sodium acetate solution with the concentration of 0.2-1.0 mol/L.
10. An insulin prepared by the extraction process of any one of claims 1-9.
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