CN113980093A - Method for promoting protein medicine crystallization by polymer and application - Google Patents
Method for promoting protein medicine crystallization by polymer and application Download PDFInfo
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- CN113980093A CN113980093A CN202111251647.8A CN202111251647A CN113980093A CN 113980093 A CN113980093 A CN 113980093A CN 202111251647 A CN202111251647 A CN 202111251647A CN 113980093 A CN113980093 A CN 113980093A
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- 238000000034 method Methods 0.000 title claims abstract description 36
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- 230000001737 promoting effect Effects 0.000 title claims abstract description 14
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- VNDYJBBGRKZCSX-UHFFFAOYSA-L Zinc bromide Inorganic materials Br[Zn]Br VNDYJBBGRKZCSX-UHFFFAOYSA-L 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 claims description 6
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- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
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- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
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- 108010038807 Oligopeptides Proteins 0.000 claims description 2
- 102000015636 Oligopeptides Human genes 0.000 claims description 2
- 229920002873 Polyethylenimine Polymers 0.000 claims description 2
- 125000003158 alcohol group Chemical group 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 230000008020 evaporation Effects 0.000 claims description 2
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 229920000747 poly(lactic acid) Polymers 0.000 claims description 2
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- 239000002904 solvent Substances 0.000 claims description 2
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- 125000003277 amino group Chemical group 0.000 claims 1
- 229960000074 biopharmaceutical Drugs 0.000 claims 1
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- 238000002360 preparation method Methods 0.000 abstract description 3
- 102000016943 Muramidase Human genes 0.000 description 25
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- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 25
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- 239000004325 lysozyme Substances 0.000 description 25
- 235000010335 lysozyme Nutrition 0.000 description 25
- 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 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 101001011741 Bos taurus Insulin Proteins 0.000 description 6
- 102000004877 Insulin Human genes 0.000 description 6
- 108090001061 Insulin Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin 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)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)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(C)C)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 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 description 6
- 229940125396 insulin Drugs 0.000 description 6
- 238000001914 filtration Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 4
- 229960001763 zinc sulfate Drugs 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
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- 239000012535 impurity Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000012835 hanging drop method Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000006911 nucleation Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000012716 precipitator Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Endocrinology (AREA)
- Diabetes (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a method for promoting protein medicine crystallization by a polymer. The method comprises the following steps: adding a polymer into a protein solution, then adjusting the supersaturation degree of the protein solution to 10-15, and stirring for 0.5-6 hours to obtain a protein crystal; the obtained protein crystal is used for protein structure analysis or composition of targeted drug preparation in biological pharmacy. The method has the advantages of simple process flow and short operation time, can improve the crystallization efficiency of protein medicines, can shorten the process of separating and purifying proteins, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for crystallizing macromolecular protein medicines and application thereof.
Background
Protein drug crystals have a wide application prospect in the fields of protein drug production, purification and the like, but due to the fact that the molecular weight of proteins is large, the crystallization time of many proteins is long, and the efficiency is low, and therefore the application of the protein drug crystals in actual production is limited.
In the field of biotechnology for researching protein crystallization, CN107814831 adopts a hanging drop method, uses deoxyribonucleic acid to promote the crystallization of protein, and can improve the success rate of protein crystallization in the hanging drop; CN105566443 uses a method of cross-linking protein crystals to promote protein crystallization and improve the success rate of protein crystallization condition screening; in CN106188220, the use of cyclodextrin and its derivatives as seed crystal improves the success rate of protein crystallization. However, due to the types of the used additives and the crystallization modes, the methods have the defects of strict limitation on the crystallization temperature, long crystallization time and the like, for example, in order to ensure the activity of DNA, CN107814831 needs to be crystallized in a thermostat at 4 ℃; the crystallization time of CN105566443 is 3-7 days.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for promoting protein drugs to crystallize by using a polymer. The method adds high molecular polymer as additive into protein solution, and accelerates the nucleation and crystallization of protein by utilizing the interaction between protein and polymer. The method has the advantages of simple process flow and short operation time, can improve the crystallization efficiency of protein drugs, overcomes the defects of long protein crystallization time and low efficiency in the traditional technology, can shorten the process of protein separation and purification, and has good application prospect.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for promoting crystallization of protein drugs by polymers comprises the following steps:
adding a polymer into a protein solution, then adjusting the supersaturation degree of the protein solution to 10-15 to obtain a supersaturated solution, and stirring for 0.5-6 hours; obtaining protein crystals;
wherein the mass ratio m of protein to polymerProtein/mPolymer and method of making same5-15 parts of a ═ one; the concentration of protein in the supersaturated solution is 1-20 mg/mL;
the method for adjusting the supersaturation degree of the protein solution comprises the steps of adding a precipitator, cooling, freeze drying or reduced pressure evaporation;
the method for adding the precipitating agent for adjusting the supersaturation degree of the protein solution is preferably to add a salting-out precipitating agent;
the precipitant is NaCl solution and ZnSO4Solution, ZnCl2Solution, ZnAc solution, ZnBr2One or more of solution, acetone, phenol, m-cresol, o-cresol and p-cresol;
the polymer is a polymer with carboxyl, amino or hydroxyl;
the polymer is an alcohol, phenol, amine, or organic acid.
The polymer is preferably polyaspartic acid, polyethyleneimine, polylactic acid, or polyethylene glycol.
The protein is oligopeptide, polypeptide, enzyme, hormone, nucleic acid or monoclonal antibody.
The solvent of the protein solution is a buffer solution, preferably a sodium acetate buffer solution, a sodium citrate buffer solution or hydrochloric acid.
The protein medicine prepared by the method is applied to protein structure analysis or composition of a targeted medicine preparation in biological pharmacy.
The invention has the beneficial effects that:
according to the invention, the polymer is mixed with the solution containing the protein, so that the protein crystallization is promoted, the protein crystallization time is effectively shortened, and the protein crystallization efficiency is improved. By the invention, the improvement of the protein crystallization induction rate can be improved by more than 2 times of that of a control group without adding a polymer in the prior art at the minimum. The invention has wide application prospect in the fields of production, purification, crystallization and the like of protein medicines.
Drawings
FIG. 1 is a crystal diagram showing that crystallization of lysozyme was promoted by polyaspartic acid obtained in example 1.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a method for promoting protein drugs to crystallize by using polymers, which comprises the following steps:
and mixing the polymer with the protein solution, and adjusting the supersaturation degree of the protein solution to obtain protein crystals.
In the present invention, a protein solution refers to a solution system containing a protein in which solutes include, but are not limited to, proteins.
The invention promotes the crystallization of protein by introducing the polymer into the protein solution, effectively shortens the crystallization time of the protein, improves the crystallization efficiency of the protein, and has wide application prospect in the production field of protein medicaments.
[ preparation of precipitant solution ]
In a preferred mode of the present invention, the method for preparing the precipitant solution comprises the steps of:
mixing the precipitant and optional assistant with the buffer solution to prepare precipitant solution.
In one embodiment of the present invention, the precipitating agent is NaCl, ZnSO or the like4Or ZnCl2Or ZnAc or ZnBr2And the like.
In one embodiment of the present invention, the auxiliary agent includes, but is not limited to, acetone, or phenol, or m-cresol, or o-cresol, or p-cresol, etc.
[ crystallization of protein promoted by Polymer ]
In one embodiment of the invention, the polymer is mixed with the protein solution to adjust the supersaturation of the protein solution to obtain protein crystals. And filtering, washing and drying the crystals to obtain protein crystals.
In one embodiment of the present invention, the polymer includes, but is not limited to, alcohols, phenols, amines, organic acids, and the like.
In one embodiment of the present invention, the protein includes, but is not limited to, a polypeptide, or an enzyme, or a hormone, or a nucleic acid, or a monoclonal antibody.
In one embodiment of the invention, the final concentration of the protein solution includes, but is not limited to, 1mg/mL, or 5mg/mL, or 10mg/mL, or 15mg/mL, or 20mg/mL, and the like.
The method for promoting protein crystallization by using the polymer provided by the invention is simple to operate, shortens the crystallization time of the protein, improves the crystallization efficiency of the protein, and has wide application prospects in the production field of protein medicines. In order to facilitate understanding of those skilled in the art, the technical solutions provided by the present invention are further described below with reference to examples.
Example 1
The embodiment provides a method for promoting lysozyme crystallization by polyaspartic acid, which is prepared according to the following steps:
(1) NaCl and lysozyme (300mg) were dissolved in 0.1mol/L sodium acetate buffer (8mL, pH adjusted with acetic acid) at 4.6, the dissolved lysozyme solution was filtered through a filter with a pore size of 0.2 μm into a crystallizer, and then 30mg of polyaspartic acid was filtered into the crystallizer to be mixed well with lysozyme (i.e., mass ratio m of protein to polymer)Protein/mPolymer and method of making same10), the dissolved NaCl solution was finally filtered into a crystallizer (i.e. volume ratio 1:1, at which point the supersaturation value was 13.02) to yield a protein solution containing lysozyme at a final concentration of 18.75 mg/mL. Stirring the solution containing lysozyme at the temperature of 20 ℃ with the linear velocity of the outer edge of the stirring paddle being 0.52m/s, wherein the solution begins to be turbid in about 2 hours, small crystals are separated out, and a large amount of crystals are obtained in 6 hours.
(2) Filtering the lysozyme crystal prepared in the step (1), and washing with distilled water for 4 times to remove impurities attached to the lysozyme crystal, thereby obtaining the lysozyme crystal.
As can be seen from FIG. 1, the lysozyme crystals obtained by the method have consistent shape and uniform particle size (the particle size is 10-20 μm), which shows that the addition of the polymer can effectively promote the crystallization of the protein and improve the crystallization efficiency of the protein. The protein crystal with the quality completely meets the requirements of stable crystal property, uniform granularity and easy storage, and can be applied to protein structure analysis or biological pharmacy as a targeted pharmaceutical preparation.
Example 2
The embodiment provides a method for promoting lysozyme crystallization by using polyethylene glycol, which is prepared according to the following steps:
(1) NaCl and lysozyme (300mg) were dissolved in 0.1mol/L sodium acetate buffer (8mL, pH 4.6 with acetic acid), the dissolved lysozyme solution was filtered through a filter with a pore size of 0.2 μm into a crystallizer, and then 30mg polyethylene glycol was filtered into the crystallizer to mix well with lysozyme (i.e., mass ratio of protein to polymer, m.Protein/mPolymer and method of making same10), the dissolved NaCl solution was finally filtered into a crystallizer (i.e. volume ratio 1:1, at which point the supersaturation value was 13.02) to yield a protein solution containing lysozyme at a final concentration of 18.75 mg/mL. The solution containing lysozyme is stirred at the temperature of 20 ℃ for about 2 hours at the linear velocity of the outer edge of a stirring paddle of 0.52m/s, the solution begins to be turbid, small crystals are separated out, and a large amount of crystals are obtained in 6 hours.
(2) Filtering the lysozyme crystal prepared in the step (1), and washing with distilled water for 4 times to remove impurities attached to the lysozyme crystal, thereby obtaining the lysozyme crystal.
Example 3
The present example provides a method for promoting insulin crystallization by polyaspartic acid, which is prepared according to the following steps:
(1) bovine insulin (40mg) was dissolved in HCl solution (8mL) at pH 2 to obtain a bovine insulin solution with a concentration of 5.00 mg/mL. Acetone and zinc sulfate were then added to a sodium citrate buffer solution (8mL, pH adjusted with NaOH) at a concentration of 0.48mol/L, pH ═ 6.58 to give a precipitant solution with an acetone concentration of 10.0% (v/v) and a zinc sulfate concentration of 0.1% (w/w), the bovine insulin solution and the precipitant solution were mixed (i.e. volume ratio 1:1), and then 4mg of polyaspartic acid was added to the mixed solution (i.e. mass ratio of protein to polymer m/1)Protein/mPolymer and method of making same10), the system was left to crystallize at room temperature for 3.5 hours to obtain a large amount of crystals.
(2) Filtering the insulin crystals prepared in the step (1), and washing the insulin crystals for 4 times by using a sodium citrate buffer solution to remove impurities attached to the insulin crystals to obtain the insulin crystals.
Comparative example 1
NaCl and lysozyme (300mg) were dissolved in sodium acetate buffer (8mL, pH adjusted with acetic acid) at a concentration of 0.1mol/L and pH 4.6, respectively, and the dissolved lysozyme solution and the NaCl solution were filtered through a filter having a pore size of 0.2 μm into a crystallizer to obtain a protein solution containing lysozyme at a final concentration of 18.75 mg/mL. The solution containing lysozyme was stirred at a temperature of 20 ℃ for 15 hours at a linear velocity of the outer edge of the stirring paddle of 0.52m/s to obtain lysozyme crystals.
Comparative example 2
Bovine insulin (40mg) was first dissolved in HCl solution (8mL) at pH 2 to give a bovine insulin solution with a concentration of 5.00 mg/mL. Acetone and zinc sulfate were then added to a sodium citrate buffer solution (8mL, pH adjusted with NaOH) at a concentration of 0.48mol/L, pH ═ 6.58 to give a precipitant solution with an acetone concentration of 10.0% (v/v) and a zinc sulfate concentration of 0.1% (w/w), the bovine insulin solution and the precipitant solution were mixed, and the system was left to crystallize at room temperature for 8 hours to give insulin crystals.
It can be seen from the above examples and comparative examples that the mixing of the polymer with the protein-containing solution can promote the crystallization of the protein, effectively shorten the crystallization time of the protein, and improve the crystallization efficiency of the protein. The protein crystallization induction rate was more than 2 times that of the control group to which no polymer was added.
The invention is not the best known technology.
Claims (9)
1. A method for promoting crystallization of protein medicine by polymer, which is characterized in that the method comprises the following steps:
adding a polymer into a protein solution, then adjusting the supersaturation degree of the protein solution to 10-15 to obtain a supersaturated solution, and stirring for 0.5-6 hours to obtain a protein crystal;
wherein the mass ratio m of protein to polymerProtein/mPolymer and method of making same5-15 parts of a ═ one; oversaturationThe concentration of protein in the solution is 1-20 mg/mL;
the polymer is a polymer having a carboxyl group, an amino group or a hydroxyl group.
2. The method of claim 1, wherein the polymer is an alcohol, phenol, amine or organic acid.
3. The method for promoting crystallization of protein medicine according to claim 1, wherein the polymer is preferably polyaspartic acid, polyethyleneimine, polylactic acid or polyethylene glycol.
4. The method for promoting crystallization of protein-based drugs according to claim 1, wherein the protein is an oligopeptide, a polypeptide, an enzyme, a hormone, a nucleic acid or a monoclonal antibody.
5. The method of claim 1, wherein the supersaturation degree of the protein solution is adjusted by adding a precipitant, cooling, freeze-drying or evaporation under reduced pressure.
6. A method of polymer assisted crystallization of a proteinaceous agent as claimed in claim 1 wherein said means for adjusting the supersaturation of the protein solution is preferably by addition of a precipitating agent.
7. The method of claim 6, wherein the precipitating agent is NaCl solution, ZnSO4Solution, ZnCl2Solution, ZnAc solution, ZnBr2One or more of solution, acetone, phenol, m-cresol, o-cresol and p-cresol.
8. The method for promoting crystallization of protein drugs according to claim 1, wherein the solvent of the protein solution is a buffer solution, preferably sodium acetate buffer solution, sodium citrate buffer solution or hydrochloric acid.
9. Use of a proteinaceous medicament prepared according to the method of claim 1, characterized by its composition as a targeted pharmaceutical formulation for use in protein structure analysis or biopharmaceutical manufacturing.
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