CN114645040A - Stable creatine kinase MB isoenzyme freeze-dried product and preparation method thereof - Google Patents
Stable creatine kinase MB isoenzyme freeze-dried product and preparation method thereof Download PDFInfo
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- CN114645040A CN114645040A CN202011508450.3A CN202011508450A CN114645040A CN 114645040 A CN114645040 A CN 114645040A CN 202011508450 A CN202011508450 A CN 202011508450A CN 114645040 A CN114645040 A CN 114645040A
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- 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
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- 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
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
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- C—CHEMISTRY; METALLURGY
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- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
- C12Y207/03002—Creatine kinase (2.7.3.2)
Abstract
The invention discloses a stable creatine kinase MB isoenzyme freeze-dried product and a preparation method thereof, wherein 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), trehalose, bovine serum albumin, mannitol, dithiothreitol, isotridecanol polyglycol ether and sodium azide are respectively weighed according to the preparation proportion, are sequentially added into a mixing container of purified water accounting for about 80% of the total amount required by preparation and are stirred to be dissolved, then, the pH value in the mixing container is measured, and 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution is utilized to adjust the pH value to a set range; then, weighing CK-MB enzyme, and adding the CK-MB enzyme into the mixing container after pH adjustment to be uniformly stirred; and finally, adding the purified water into the mixing container to a configured amount, stirring and standing for 15 minutes, sub-packaging by using various brown glass bottles, and freeze-drying according to freeze-drying process parameters, so that the stability is good.
Description
Technical Field
The invention relates to the technical field of medical detection, in particular to a stable creatine kinase MB isoenzyme freeze-dried product and a preparation method thereof.
Background
Creatine Kinase (CK), also known as Creatine phosphokinase, is found mainly in skeletal muscle, cardiac muscle, brain tissue and smooth muscle, and is clinically used mainly for diagnosis of skeletal muscle diseases, cardiac muscle diseases and brain diseases. At present, when the creatine kinase MB isoenzyme determination kit for clinical diagnosis is used for testing, most of calibration products and quality control products are freeze-dried products, and the freeze-dried products have the advantages of convenience in transportation, easiness in storage, long validity period and the like. However, after redissolution, the creatine kinase MB isozyme is still affected by the environment and is easy to be oxidized, so that the test result is inaccurate. Therefore, on the premise of keeping the long-term stability of the creatine kinase MB isoenzyme freeze-dried product, the problem of instability after redissolution is solved, and the accuracy and reliability of creatine kinase MB isoenzyme index determination in the clinical test and diagnosis process are greatly improved.
At present, the preparation principle of a creatine kinase MB isoenzyme freeze-dried product is as follows: by adopting a freeze-drying technology, the frozen matrix liquid of the creatine kinase MB isoenzyme freeze-dried product is directly sublimated from a frozen state under the conditions of low temperature and low pressure, water is removed, drying is completed, and the long-term stability of the physicochemical property and the physiological activity of the creatine kinase MB isoenzyme can be kept unchanged. On the other hand, the stabilizer is added into the freeze-dried matrix solution, so that the activity of the creatine kinase MB isozyme after redissolution can be effectively protected, and the creatine kinase MB isozyme can be used and stored more stably for a long time in the clinical application process. At present, a plurality of composite quality control products containing CK-MB projects exist in the market, but as the plurality of composite quality control products, compared with single CK-MB quality control products, the cost is higher, and the requirements of more professions cannot be met necessarily; the existence of CK enzyme in the composite quality control product can influence the test result of CK-MB enzyme; on the other hand, there are also liquid CK-MB quality control products or CK-MB diluents, and the liquid dosage forms are not suitable for long-term storage.
Disclosure of Invention
The invention aims to provide a stable creatine kinase MB isoenzyme freeze-dried product and a preparation method thereof, and the freeze-dried product has good stability.
In order to achieve the above object, the present invention provides a stable freeze-dried creatine kinase MB isozyme product, which comprises 0.10% -1.00% of HEPES, 1.54% -3.02% of trehalose, 3.55% -6.55% of bovine serum albumin, 1.23% -3.86% of mannitol, 0.00% -0.50% of dithiothreitol, 0.00% -0.50% of isotridecanol polyglycol ether, 0.00% -0.50% of sodium azide, and 7.0-7.5% of CK-MB enzyme 100U/L-250U/L, pH.
Wherein the stable creatine kinase MB isoenzyme freeze-dried product comprises 2g/L of HEPES, 18g/L of trehalose, 40g/L of bovine serum albumin, 15g/L of mannitol, 0.5g/L of dithiothreitol, 0.5mL/L of isotridecanol polyglycol ether and 7.10 of sodium azide 0.5g/L, CK-MB enzyme 120U/L, pH.
Wherein the stable creatine kinase MB isozyme freeze-dried product comprises 3.5g/L of HEPES, 25g/L of trehalose, 50g/L of bovine serum albumin, 25g/L of mannitol, 1g/L of dithiothreitol, 1mL/L of isotridecanol polyethylene glycol ether and 7.30 of sodium azide 1g/L, CK-MB enzyme 160U/L, pH.
In a second aspect, the present invention provides a method for preparing a stable freeze-dried creatine kinase MB isoenzyme product, where the stable freeze-dried creatine kinase MB isoenzyme product according to the first aspect is suitable for preparing a stable freeze-dried creatine kinase MB isoenzyme product, and includes the following steps:
weighing a plurality of preparation raw materials according to the preparation ratio, and sequentially adding the preparation raw materials into a mixing container containing purified water to be stirred until the preparation raw materials are dissolved, wherein the preparation raw materials comprise HEPES, trehalose, bovine serum albumin, mannitol, dithiothreitol, isotridecanol polyglycol ether and sodium azide;
measuring the pH value in the mixing container, and adjusting the pH value to a set range by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution;
weighing CK-MB enzyme, and adding the CK-MB enzyme into the mixing container after pH adjustment to be uniformly stirred;
and adding the purified water into the mixing container to a configured amount, stirring and standing for 15 minutes, subpackaging by using various brown glass bottles, and freeze-drying according to freeze-drying process parameters.
Firstly, respectively weighing HEPES, trehalose, bovine serum albumin, mannitol, dithiothreitol, isotridecanol polyglycol ether and sodium azide according to preparation proportion, sequentially adding the materials into a mixing container of purified water accounting for about 80% of the total amount required by preparation, stirring the materials until the materials are dissolved, then measuring the pH value in the mixing container, and adjusting the pH value to a set range by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution; then, weighing CK-MB enzyme, and adding the CK-MB enzyme into the mixing container after pH adjustment to be uniformly stirred; and finally, adding the purified water into the mixing container to a configured amount, stirring and standing for 15 minutes, subpackaging by using various brown glass bottles, and freeze-drying according to freeze-drying process parameters, so that the stability is good.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a schematic step diagram of a preparation method of a stable creatine kinase MB isozyme freeze-dried product provided by the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
The invention provides a stable creatine kinase MB isozyme freeze-dried product which comprises 0.10-1.00 percent of HEPES (4-hydroxyethyl piperazine ethanesulfonic acid), 1.54-3.02 percent of trehalose, 3.55-6.55 percent of bovine serum albumin, 1.23-3.86 percent of mannitol, 0.00-0.50 percent of dithiothreitol, 0.00-0.50 percent of isotridecyl polyethylene glycol ether, 0.00-0.50 percent of sodium azide and 7.0-7.5 percent of CK-MB enzyme 100U/L-250U/L, pH.
In the present embodiment, the first example is: the stable creatine kinase MB isoenzyme freeze-dried product comprises HEPES 2g/L, trehalose 18g/L, bovine serum albumin 40g/L, mannitol 15g/L, dithiothreitol 0.5g/L, isotridecanol polyglycol ether 0.5mL/L, and sodium azide 0.5g/L, CK-MB enzyme 120U/L, pH of 7.10.
The second embodiment is: the stable creatine kinase MB isoenzyme freeze-dried product comprises 3.5g/L of HEPES, 25g/L of trehalose, 50g/L of bovine serum albumin, 25g/L of mannitol, 1g/L of dithiothreitol, 1mL/L of isotridecanol polyglycol ether and 7.30 of sodium azide 1g/L, CK-MB enzyme 160U/L, pH.
The third embodiment is: the stable creatine kinase MB isoenzyme freeze-dried product comprises 5g/L of HEPES, 30g/L of trehalose, 60g/L of bovine serum albumin, 30g/L of mannitol, 1.5g/L of dithiothreitol, 1.5mL/L of isotridecanol polyglycol ether and 7.45 of sodium azide 1.5g/L, CK-MB enzyme 200U/L, pH.
In the invention, a HEPES buffer system is selected, so that the constant pH range can be controlled for a long time, and no toxic effect is caused to cells. The stabilizer mainly comprises trehalose, bovine serum albumin, dithiothreitol and isotridecanol polyethylene glycol ether, and related components do not participate in the reaction of creatine kinase MB isoenzyme, but can effectively keep the activity of the creatine kinase MB isoenzyme. Mannitol as the excipient of the invention has no hygroscopicity, good chemical stability and can increase storage time. The pH range of the whole system is 7.0-7.5.
Referring to fig. 1, the present invention provides a method for preparing a stable freeze-dried creatine kinase MB isoenzyme product, which is suitable for preparing a stable freeze-dried creatine kinase MB isoenzyme product, comprising the following steps:
s101, weighing a plurality of preparation raw materials according to the preparation ratio, and sequentially adding the preparation raw materials into a mixing container containing purified water to be stirred until the preparation raw materials are dissolved.
Specifically, purified water accounting for about 80% of the total amount required for preparation is added into a mixing container, full batches of HEPES, trehalose, bovine serum albumin, isotridecyl alcohol polyglycol ether, mannitol, sodium azide and dithiothreitol are accurately weighed respectively, and are sequentially added into the mixing container and are stirred one by one until the HEPES, the trehalose, the bovine serum albumin, the isotridecyl alcohol polyglycol ether, the mannitol, the sodium azide and the dithiothreitol are completely dissolved.
And S102, measuring the pH value in the mixing container, and adjusting the pH value to be within a set range by utilizing a 6mol/L hydrochloric acid solution or a 10mol/L sodium hydroxide solution.
Specifically, whether the pH value is in the range of 7.0-7.5 is tested, otherwise, the pH value is adjusted to the range by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution.
S103, weighing CK-MB enzyme, adding the CK-MB enzyme into the mixing container after pH adjustment, and uniformly stirring.
Specifically, the CK-MB enzyme is accurately weighed in full batch, added into the solution with the well adjusted pH value, and stirred uniformly.
S104, adding the purified water into the mixing container to a configured amount, stirring and standing for 15 minutes, sub-packaging by using various brown glass bottles, and freeze-drying according to freeze-drying process parameters.
Specifically, purified water is used for quantifying to a preparation amount, the mixture is uniformly stirred, the solution is kept stand and balanced for 15min, brown glass bottles with different specifications are used for subpackaging according to requirements, and freeze-drying is carried out according to freeze-drying process parameters in the table 1.
The preparation method of the first embodiment is as follows:
adding purified water accounting for about 80% of the total amount required by preparation into a mixing container, accurately weighing a full batch of HEPES 2g/L, trehalose 18g/L, bovine serum albumin 40g/L, isotridecanol polyglycol ether 0.5mL/L, mannitol 15g/L, sodium azide 0.5g/L and dithiothreitol 0.5g/L respectively, sequentially adding into the mixing container, and stirring one by one until the mixture is completely dissolved; then, whether the pH value is in the range of 7.10 +/-0.05 is tested, otherwise, 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution is used for adjusting the pH value to the range; then, accurately weighing 120U/L of the CK-MB enzyme in full batch, adding the weighed CK-MB enzyme into the solution with the well adjusted pH value, and uniformly stirring; and finally, quantifying to a preparation amount by using purified water, uniformly stirring, standing and balancing the solution for 15min, subpackaging by using brown glass bottles with different specifications according to requirements, and freeze-drying according to the freeze-drying process parameters in the table 1.
The preparation method of the second embodiment is as follows:
adding purified water accounting for about 80% of the total amount required by preparation into a mixing container, accurately weighing full-batch HEPES 3.5g/L, trehalose 25g/L, bovine serum albumin 50g/L, isotridecyl alcohol polyglycol ether 1mL/L, mannitol 25g/L, sodium azide 1g/L and dithiothreitol 1g/L respectively, sequentially adding into the mixing container, and stirring one by one until complete dissolution; then, whether the pH value is in the range of 7.30 +/-0.05 is tested, otherwise, the pH value is adjusted to be in the range by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution; then, accurately weighing 160U/L of the CK-MB enzyme in full batch, adding the CK-MB enzyme into the solution with the well adjusted pH value, and uniformly stirring; and finally, quantifying to a preparation amount by using purified water, uniformly stirring, standing and balancing the solution for 15min, subpackaging by using brown glass bottles with different specifications according to requirements, and freeze-drying according to the freeze-drying process parameters in the table 1.
The preparation method of the third embodiment is as follows:
adding purified water accounting for about 80% of the total amount required by preparation into a mixing container, accurately weighing full-batch HEPES 5g/L, trehalose 30g/L, bovine serum albumin 60g/L, isotridecyl alcohol polyglycol ether 1.5mL/L, mannitol 30g/L, sodium azide 1.5g/L and dithiothreitol 1.5g/L respectively, sequentially adding into the mixing container, and stirring one by one until the materials are completely dissolved; then testing whether the pH value is in the range of 7.45 +/-0.05, otherwise adjusting the pH value to the range by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution; then, accurately weighing a whole batch of CK-MB enzyme 200U/L, adding the CK-MB enzyme into the solution with the adjusted pH value, and uniformly stirring; and finally, quantifying to a preparation amount by using purified water, uniformly stirring, standing and balancing the solution for 15min, subpackaging by using brown glass bottles with different specifications according to requirements, and freeze-drying according to the freeze-drying process parameters in the table 1.
TABLE 1 Freeze drying Process parameters
The applicant prepares creatine kinase MB isozyme freeze-dried products according to the 3 embodiments, can be stably stored for 10 days at 37 ℃, and shows good stability after being re-dissolved and respectively placed for 30 days, 7 days and 24 hours at-20 ℃, 2-8 ℃ and 25 ℃, and the specific data are shown in Table 2.
Table 2 stability test data of lyophilized creatine kinase MB isozyme
Firstly, respectively weighing HEPES, trehalose, bovine serum albumin, mannitol, dithiothreitol, isotridecanol polyglycol ether and sodium azide according to preparation proportion, sequentially adding the materials into a mixing container of purified water accounting for about 80% of the total amount required by preparation, stirring the materials until the materials are dissolved, then measuring the pH value in the mixing container, and adjusting the pH value to a set range by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution; then, weighing CK-MB enzyme, and adding the CK-MB enzyme into the mixing container after pH adjustment to be uniformly stirred; and finally, adding the purified water into the mixing container to a configured amount, stirring and standing for 15 minutes, subpackaging by using various brown glass bottles, and freeze-drying according to freeze-drying process parameters, so that the stability is good.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (4)
1. A stable creatine kinase MB isoenzyme freeze-dried product is characterized in that,
the stable creatine kinase MB isozyme freeze-dried product comprises 0.10-1.00 percent of HEPES, 1.54-3.02 percent of trehalose, 3.55-6.55 percent of bovine serum albumin, 1.23-3.86 percent of mannitol, 0.00-0.50 percent of dithiothreitol, 0.00-0.50 percent of isotridecanol polyglycol ether, 0.00-0.50 percent of sodium azide and 7.0-7.5 percent of CK-MB enzyme 100U/L-250U/L, pH.
2. The stable lyophilized creatine kinase MB isoenzyme product of claim 1, wherein the creatine kinase MB isoenzyme is selected from the group consisting of creatine kinase MB isoenzyme,
the stable creatine kinase MB isoenzyme freeze-dried product comprises HEPES 2g/L, trehalose 18g/L, bovine serum albumin 40g/L, mannitol 15g/L, dithiothreitol 0.5g/L, isotridecanol polyglycol ether 0.5mL/L, and sodium azide 0.5g/L, CK-MB enzyme 120U/L, pH of 7.10.
3. The stable lyophilized creatine kinase MB isoenzyme product of claim 1, wherein the creatine kinase MB isoenzyme is selected from the group consisting of creatine kinase MB isoenzyme,
the stable creatine kinase MB isoenzyme freeze-dried product comprises 3.5g/L of HEPES, 25g/L of trehalose, 50g/L of bovine serum albumin, 25g/L of mannitol, 1g/L of dithiothreitol, 1mL/L of isotridecanol polyglycol ether and 7.30 of sodium azide 1g/L, CK-MB enzyme 160U/L, pH.
4. A method for preparing a stable freeze-dried creatine kinase MB isoenzyme product, wherein the method for preparing a stable freeze-dried creatine kinase MB isoenzyme product according to any one of claims 1-3 is suitable for preparing a stable freeze-dried creatine kinase MB isoenzyme product, and comprises the following steps:
weighing a plurality of preparation raw materials according to the preparation ratio, and sequentially adding the preparation raw materials into a mixing container containing purified water to be stirred until the preparation raw materials are dissolved, wherein the preparation raw materials comprise HEPES, trehalose, bovine serum albumin, mannitol, dithiothreitol, isotridecanol polyglycol ether and sodium azide;
measuring the pH value in the mixing container, and adjusting the pH value to a set range by using 6mol/L hydrochloric acid solution or 10mol/L sodium hydroxide solution;
weighing CK-MB enzyme, and adding the CK-MB enzyme into the mixing container after pH adjustment to be uniformly stirred;
and adding the purified water into the mixing container to a configured amount, stirring and standing for 15 minutes, subpackaging by using various brown glass bottles, and freeze-drying according to freeze-drying process parameters.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102128917A (en) * | 2010-12-07 | 2011-07-20 | 江西特康科技有限公司 | Clinical biochemical quality control products and preparation process thereof |
| CN107843469A (en) * | 2017-09-15 | 2018-03-27 | 中生北控生物科技股份有限公司 | A kind of biochemical class compound calibration object of stabilization and preparation method thereof |
| CN109298176A (en) * | 2018-10-29 | 2019-02-01 | 深圳天深医疗器械有限公司 | Myocarditis quality-control product and preparation method thereof, myocarditis detection kit and myocarditis detection device |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102128917A (en) * | 2010-12-07 | 2011-07-20 | 江西特康科技有限公司 | Clinical biochemical quality control products and preparation process thereof |
| CN107843469A (en) * | 2017-09-15 | 2018-03-27 | 中生北控生物科技股份有限公司 | A kind of biochemical class compound calibration object of stabilization and preparation method thereof |
| CN109298176A (en) * | 2018-10-29 | 2019-02-01 | 深圳天深医疗器械有限公司 | Myocarditis quality-control product and preparation method thereof, myocarditis detection kit and myocarditis detection device |
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