A process for extracting insulin

Abstract

The present invention relates to the field of biopharmaceuticals, and specifically, to an insulin extraction process, comprising the following steps: raw material pretreatment, extraction, protein removal, purification, and drying; wherein, in the extraction step, an extractant is polyethylene glycol, sodium chloride, glacial acetic acid, and water in a mass ratio of 5-8:6-9:0.1-0.5:80-89. Through the optimization of the extractant, the extraction efficiency is significantly improved, the number of extractions is reduced, and at the same time, after a subsequent purification step, 10g of insulin can be obtained per 1kg of pancreas, and the yield is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to an insulin extraction process. Background Art

[0002] Diabetes mellitus is a syndrome of metabolic disorders such as protein, fat, water and electrolytes caused by absolute or relative insufficient secretion of insulin and reduced sensitivity of target tissue cells to insulin, with hyperglycemia as the main sign. The main clinical manifestations of diabetes are polydipsia, polyuria, polyphagia and weight loss ("three mores and one less"), as well as high blood sugar and glucose in urine (normal urine should not contain glucose). If diabetes is not effectively treated, it can cause damage to multiple systems of the body, such as diabetic retinopathy, diabetic nephropathy, diabetic neuropathy, etc. According to the latest data released by the International Diabetes Federation in 2011, the number of people with diabetes worldwide has reached 366 million, seriously endangering human health.

[0003] Generally, the treatment of diabetes includes oral hypoglycemic drugs and insulin therapy. Insulin is a protein hormone secreted by pancreatic β cells in the pancreas in response to endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc. Insulin participates in regulating sugar metabolism, reducing gluconeogenesis, promoting glycogen synthesis, inhibiting glycogenolysis, accelerating anaerobic glycolysis and aerobic oxidation of glucose, promoting the utilization of glucose by tissues, and promoting the conversion of glucose into fat. Therefore, it can be used to treat diabetes.

[0004] At present, there are roughly three generations of insulin on the market. The first generation of animal insulin is extracted directly from animals, generally from pigs and cows, and has high immunogenicity. The second generation of human insulin uses genetic engineering to transfer human insulin genes to bacteria or yeast bacteria, and uses fermentation technology for large-scale production. The third generation is human insulin analogs, which modify the amino acid sequence of human insulin. The production method is basically the same as the second generation of human insulin. The second and third generation insulin processes are complex. Currently, only a very small number of companies in the world have the production capacity. The products are expensive, which is not conducive to market promotion. For my country's complex domestic market, animal insulin still has a certain share in clinical practice due to its low cost and wide source.

[0005] However, the traditional production process extracts insulin from the pancreas of domestic pigs and sheep, uses acid-alcohol solution to extract and separate it, and then precipitates it through salting out and separates and purifies it. The purity and yield of the product are difficult to meet the quality standards for clinical application. Therefore, there is an urgent need to develop an insulin extraction method with higher product purity and yield. Summary of the invention

[0006] Based on the above-mentioned defects of the prior art, the present invention provides an insulin extraction process with higher product purity and yield.

[0007] To this end, the present invention provides an insulin extraction process, comprising the following steps: raw material pretreatment, extraction, protein removal, purification, and drying;

[0008] Wherein, in the extraction step, the extractant used is polyethylene glycol, sodium chloride, glacial acetic acid and water in a mass ratio of 5-8:6-9:0.1-0.5:80-89.

[0009] Preferably, the raw material pretreatment step comprises: freezing the pancreas at -20 to -40°C for 2 to 4 hours and then storing it at -15 to -18°C.

[0010] Preferably, in the extraction step, the ratio of the mass of the raw material to the volume of the extractant is 1:3-5, and the ratio of the mass to the volume is kg / L.

[0011] Preferably, the extraction temperature is 4-8°C; the extraction time is 24-36h.

[0012] Preferably, the protein removal step comprises adding NaOH solution to the crude protein after extraction, adjusting the pH to 7.8-8.5, separating the solid and the liquid, and retaining the liquid.

[0013] Preferably, the molar concentration of the NaOH solution is 0.5-2 mol / L.

[0014] Preferably, the purification step comprises ultrafiltration and ion exchange chromatography separation.

[0015] Preferably, the ultrafiltration membrane has a molecular weight cut-off of 100,000 Daltons.

[0016] Preferably, the conditions for the ion exchange chromatography separation are: the chromatographic column filler is one or more of SK1BH, WK40L or PK216, the particle size is 5-20 μm, the chromatographic column diameter is 5-10 cm, and the chromatographic column length is 50-100 cm; the balancing liquid is 0.05-0.1 mol / L sodium acetate solution, and the eluent is 0.2-1.0 mol / L sodium acetate solution.

[0017] Preferably, the pH of the balanced solution is 4.0-5.0.

[0018] Preferably, the pH of the eluent is 2.5-3.5.

[0019] The present invention further provides insulin prepared by the above extraction process.

[0020] The beneficial effects of the present invention are:

[0021] The invention provides an insulin extraction method, which significantly improves the extraction efficiency and reduces the number of extractions through the optimization of the extractant. Meanwhile, through the subsequent purification step, 10g of insulin can be obtained per 1kg of pancreas, and the yield is greatly improved. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] This embodiment provides an insulin extraction process, comprising the following steps:

[0025] (1) Raw material pretreatment: The pancreas was frozen at -30°C for 3 h, crushed, and stored at -17°C;

[0026] (2) Extraction: 1 kg of pancreatic powder was added with 4 L of extractant (PEG6000, sodium chloride, glacial acetic acid and water in a mass ratio of 6:8:0.3:85), stirred for 10 min, and then allowed to stand at 6 °C for 30 h. The supernatant and precipitate were removed, and the lower solution was retained.

[0027] (3) Protein removal: The lower layer solution was adjusted to pH 8.0 with 1 mol / L NaOH solution, allowed to stand for 30 min, centrifuged at 4000 rpm for 10 min, and the supernatant was retained;

[0028] (4) Ultrafiltration: Use a 100,000 Dalton ultrafiltration membrane to ultrafilter and then concentrate the insulin extract to 1 / 100 of its original volume to obtain insulin concentrate.

[0029] (5) Ion exchange: A chromatographic column with a diameter of 10 cm and a length of 85 cm was selected, filled with SK1BH filler with a particle size of 10 μm, and equilibrated with 0.1 mol / L sodium acetate solution (pH adjusted to 4.5 with acetic acid) for 2 h. The insulin concentrate was loaded onto the chromatographic column and eluted with 0.2, 0.5, 0.8, and 1.0 mol / L sodium acetate solution (pH adjusted to 3.0 with acetic acid) in sequence, and the target protein was collected (measured using an insulin ELISA kit, and the eluate with a content greater than 1 mg was collected);

[0030] (6) Desalting and drying: The eluate was placed in a dialysis bag, placed in distilled water, and dialyzed at 6°C for 24 h, during which the distilled water was replaced four times. The eluate was then taken out and concentrated to 1 / 10 of the original volume, and freeze-dried to obtain the insulin (11.1 g).

[0031] This embodiment further provides insulin prepared by the above extraction process.

[0032] Example 2

[0033] This embodiment provides an insulin extraction process, comprising the following steps:

[0034] (1) Raw material pretreatment: The pancreas was frozen at -40°C for 2 h, crushed, and stored at -18°C;

[0035] (2) Extraction: 1 kg of pancreatic powder was added with 5 L of extractant (PEG6000, sodium chloride, glacial acetic acid and water in a mass ratio of 6:9:0.1:80), stirred for 10 min, and then extracted at 4 °C for 36 h. The supernatant and precipitate were removed, and the lower solution was retained.

[0036] (3) Protein removal: The lower layer solution was adjusted to pH 7.8 with 0.5 mol / L NaOH solution, allowed to stand for 30 min, centrifuged at 4000 r / min for 10 min, and the supernatant was retained;

[0037] (4) Ultrafiltration: Use a 100,000 Dalton ultrafiltration membrane to ultrafilter and then concentrate the insulin extract to 1 / 100 of its original volume to obtain insulin concentrate.

[0038] (5) Ion exchange: A chromatographic column with a diameter of 5 cm and a length of 100 cm was selected, filled with WK40L filler with a particle size of 20 μm, and equilibrated with 0.05 mol / L sodium acetate solution (the pH was adjusted to 5.0 with acetic acid) for 2 h. The insulin concentrate was loaded onto the chromatographic column and eluted with 0.2, 0.5, 0.8, and 1.0 mol / L sodium acetate solutions (the pH was adjusted to 2.5 with acetic acid) in sequence, and the target protein was collected (the insulin ELISA kit was used for determination, and the eluate with a content greater than 1 mg was collected);

[0039] (6) Desalting and drying: The eluate was placed in a dialysis bag, placed in distilled water, and dialyzed at 6° C. for 24 h, during which the distilled water was replaced 5 times. The eluate was then taken out and concentrated to 1 / 10 of the original volume, and freeze-dried to obtain the insulin (10.6 g).

[0040] This embodiment further provides insulin prepared by the above extraction process.

[0041] Example 3

[0042] This embodiment provides an insulin extraction process, comprising the following steps:

[0043] (1) Raw material pretreatment: The pancreas was frozen at -20°C for 4 h, crushed, and stored at -15°C;

[0044] (2) Extraction: 1 kg of pancreatic powder was added with 3 L of extractant (PEG6000, sodium chloride, glacial acetic acid and water in a mass ratio of 8:6:0.5:89), stirred for 10 min, and then extracted at 8 °C for 24 h. The supernatant and precipitate were removed, and the lower solution was retained.

[0045] (3) Protein removal: The lower layer solution was adjusted to pH 8.5 with 2 mol / L NaOH solution, allowed to stand for 30 min, centrifuged at 4000 rpm for 10 min, and the supernatant was retained;

[0046] (4) Ultrafiltration: Use a 100,000 Dalton ultrafiltration membrane to ultrafilter and then concentrate the insulin extract to 1 / 100 of its original volume to obtain insulin concentrate.

[0047] (5) Ion exchange: A chromatographic column with a diameter of 10 cm and a length of 50 cm was selected, filled with PK216 filler with a particle size of 5 μm, and equilibrated with 0.05 mol / L sodium acetate solution (pH adjusted to 4.0 with acetic acid) for 2 h. The insulin concentrate was loaded onto the chromatographic column and eluted with 0.2, 0.5, 0.8, and 1.0 mol / L sodium acetate solution (pH adjusted to 3.5 with acetic acid) in sequence, and the target protein was collected (measured using an insulin ELISA kit, and the eluate with a content greater than 1 mg was collected);

[0048] (6) Desalting and drying: The eluate was placed in a dialysis bag, placed in distilled water, and dialyzed at 6°C for 24 h, during which the distilled water was replaced three times. The eluate was then taken out and concentrated to 1 / 10 of the original volume, and freeze-dried to obtain the insulin (10.3 g).

[0049] This embodiment further provides insulin prepared by the above extraction process.

[0050] Comparative Example 1

[0051] This comparative example provides an insulin extraction process, which is different from Example 1 only in that PEG6000, ammonium sulfate, glacial acetic acid and water in a mass ratio of 6:8:0.3:85 are used as extractants, and finally 8.4 g of insulin is obtained.

[0052] Comparative Example 2

[0053] This comparative example provides an insulin extraction process, which is different 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 extractants, and finally 3.7 g of insulin is obtained.

[0054] Comparative Example 3

[0055] This comparative example provides an insulin extraction process, which is different 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 extractants, and finally 6.1 g of insulin is obtained.

[0056] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An insulin extraction process, characterized in that: The following steps are involved: Raw material pretreatment, extraction, protein removal, purification, and drying; Wherein, the extraction step uses an extractant of polyethylene glycol 6000, sodium chloride, glacial acetic acid and water in a mass ratio of 5-8:6-9:0.1-0.5:80-89; The raw material pretreatment step includes: freezing the pancreas at -20 to -40°C for 2 to 4 hours and then transferring it to -15 to -18°C for storage; In the extraction step, the ratio of the mass of the raw material to the volume of the extractant is 1:3-5, and the ratio of the mass to the volume is kg / L; The protein removal step includes adding NaOH solution to the crude protein after extraction, adjusting the pH to 7.8-8.5, separating the solid from the liquid, and retaining the liquid; The purification step includes ultrafiltration and ion exchange chromatography separation, and the ultrafiltration membrane has a molecular weight cutoff of 100,000 Daltons; The drying step comprises placing the eluate into a dialysis bag, placing it in distilled water, dialyzing it at 6° C. for 24 hours, replacing the distilled water during the process, then taking it out, concentrating it to 1 / 10 of its original volume, and freeze-drying it.

2. The extraction process according to claim 1, characterized in that The extraction temperature is 4-8°C; the extraction time is 24-36h.

3. The extraction process according to claim 1, characterized in that The molar concentration of the NaOH solution is 0.5-2 mol / L.

4. The extraction process according to claim 1, characterized in that The conditions for the ion exchange chromatography separation are: the chromatographic column filler is one or more of SK1BH, WK40L or PK216, the particle size is 5-20 μm, the chromatographic column diameter is 5-10 cm, and the chromatographic column length is 50-100 cm; the equilibrium liquid is 0.05-0.1 mol / L sodium acetate solution, and the eluent is 0.2-1.0 mol / L sodium acetate solution.