CN112816595A - Recombinant keratin liquid preparation and purity detection method thereof - Google Patents
Recombinant keratin liquid preparation and purity detection method thereof Download PDFInfo
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Abstract
The invention relates to a recombinant keratin liquid, which comprises, by mass, 0.1-0.2% of recombinant keratin, 8-15% of a stabilizer, 0.01-0.05% of a preservative bacteriostatic agent and the balance of sterile water. Also discloses a purity detection method of the recombinant keratin liquid preparation, which comprises high performance liquid chromatography, wherein gel suitable for separating protein with molecular weight of 5-1250 KDa is adopted as a filler in a chromatographic column, and 0.08% TFA-acetonitrile and 0.1% TFA-water are adopted as mobile phases; the sample loading amount is not less than 10 mu L, the theoretical plate number calculated by recombinant keratin chromatographic peak is not less than 16000 by detection at the wavelength of 280nm, and finally, the calculation is carried out according to an area normalization method. The recombinant keratin is prepared into a liquid preparation for storage, so that the structure of the recombinant keratin can be kept stable, the condition of degradation or aggregation of the keratin is avoided, the recombinant keratin can keep better activity for a long time, and the purity of the recombinant keratin is effectively ensured to be not changed greatly in the storage process.
Description
Technical Field
The invention relates to the technical field of keratin, in particular to a recombinant keratin liquid preparation and a purity detection method thereof.
Background
Keratin is a fibrous animal protein with association and protection functions, is a structural protein of ectodermal cells, and is widely present in hair, feathers and hooves of animals; the keratin has good biological activity and histocompatibility, can play roles in diminishing inflammation, stopping bleeding, repairing nerves, reducing exudation of wound surface and promoting regeneration, repair and healing of wound tissues, can be biodegraded in vivo, and can be used as a good biomedical material. However, the existing keratin is used after being extracted from animals by a complex chemical process, so that the structure of the keratin is easily damaged in the extraction process, and certain separating agents are remained in the keratin, so that the safety and the effectiveness of the keratin cannot be ensured.
Therefore, in order to avoid various influences on keratin caused by an extraction process in the market at present, patent No. 201810123711.6 provides a recombinant keratin which can be obtained as stable, safe, structurally complete and effective keratin; however, the direct storage of recombinant keratin leads to the occurrence of structural instability, degradation or aggregation, the activity is seriously affected, and the purity of the recombinant keratin is affected, thus seriously affecting the effectiveness and the application range of the recombinant keratin. And the prior art cannot detect the purity of recombinant keratin in the preparation.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a recombinant keratin liquid preparation and a purity detection method thereof, which solve the technical problems that the prior recombinant keratin is unstable in direct storage structure, degraded or aggregated, the activity and purity of the recombinant keratin are seriously influenced, the purity of the recombinant keratin in the preparation cannot be detected, and the like.
In order to achieve the aforementioned object, the present invention provides a recombinant keratin liquid preparation comprising, in mass percent: 0.1 to 0.2 percent of recombinant keratin, 8 to 15 percent of stabilizing agent, 0.01 to 0.05 percent of antiseptic bacteriostatic agent and the balance of deionized water.
Preferably, the stabilizer comprises one or any of glycerol, NaCl and sucrose. According to the invention, glycerol and sucrose are used as stabilizers, so that the stability of the recombinant keratin can be enhanced, and on one hand, the change of the secondary structure of the recombinant keratin in the long-term storage process can be prevented; on the other hand, the recombinant keratin can be prevented from being inactivated in the repeated freezing and thawing process, the phenomenon of degradation or aggregation of the recombinant keratin in the long-term storage process is effectively avoided, and the activity and the purity of the recombinant keratin are ensured.
Preferably, the stabilizer comprises 5-8% of glycerol, 1-2% of NaCl and 2-5% of sucrose.
Preferably, the stabilizer comprises 6-7% of glycerol, 1.5% of NaCl1 and 3-4% of sucrose.
Preferably, the preservative bacteriostatic agent is sodium azide. According to the invention, the sodium azide is adopted to carry out corrosion prevention and bacteriostasis on the preserved recombinant keratin, so that the recombinant keratin still has good quality after being preserved for a long time.
Preferably, the content of the sodium azide is 0.02-0.04%.
Preferably, the recombinant keratin has a molecular weight of 47 KDa.
A purity detection method of a recombinant keratin liquid preparation comprises high performance liquid chromatography, wherein a chromatographic column adopts gel suitable for separating protein with molecular weight of 5-1250 KDa as a filling agent, and a mobile phase adopts 0.08% TFA-acetonitrile (containing 0.4ml TFA and 500ml acetonitrile mixed liquid) and 0.1% TFA-water (containing 0.5ml TFA and 500ml deionized water); the sample loading amount is not less than 10 mu L, the theoretical plate number calculated by recombinant keratin chromatographic peak is not less than 16000 by detection at the wavelength of 280nm, and finally, the calculation is carried out according to an area normalization method.
Preferably, the chromatography column is a SEC300 gel chromatography column.
Compared with the prior art, the invention has the beneficial effects that:
1) in the invention, the recombinant keratin is prepared into a liquid preparation for storage, so that the structure of the recombinant keratin can be ensured to be stable, the condition of degradation or aggregation of the keratin is avoided, the recombinant keratin can keep better activity for a long time, the purity of the recombinant keratin is effectively ensured not to change greatly in the storage process, the recombinant keratin with better activity and higher purity can be applied to medical supplies, and good effect can be exerted in the medical supplies; the present invention provides a convenient and effective way of preserving recombinant keratin.
2) The invention also provides a purity detection method of the recombinant keratin liquid preparation, which can effectively detect the purity of the recombinant keratin liquid preparation, thereby finding out the most appropriate storage method of the recombinant keratin under different storage environments and under the condition of purity change along with the storage time; and the effectiveness of the recombinant keratin is ensured by sampling inspection by this method before the liquid formulation is used.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 is a chromatogram of comparative example 1 in a liquid preparation of recombinant keratin according to the present invention.
FIG. 2 is a chromatogram of example 1 of a liquid recombinant keratin preparation of the present invention.
FIG. 3 is a chromatogram of example 2 in a liquid recombinant keratin preparation of the invention.
FIG. 4 is a chromatogram of example 3 in a liquid recombinant keratin formulation of the invention.
FIG. 5 is a chromatogram of example 4 from a liquid recombinant keratin preparation of the invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment can be included in at least one embodiment of the invention. The appearances of the phrase in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. It is explicitly and implicitly understood by one skilled in the art that the embodiments described herein can be combined with other embodiments.
The invention provides a recombinant keratin liquid preparation, which comprises the following components in percentage by mass: 0.1 to 0.2 percent of recombinant keratin, 8 to 15 percent of stabilizing agent, 0.01 to 0.05 percent of antiseptic bacteriostatic agent and the balance of sterile water.
In specific implementation, the stabilizer comprises one or more of glycerol, NaCl and sucrose.
In specific implementation, the stabilizer comprises 5-8% of glycerol, 1-2% of NaCl and 2-5% of sucrose.
In specific implementation, the stabilizer comprises 6-7% of glycerol, 1.5% of NaCl1 and 3-4% of sucrose.
In specific implementation, the antiseptic bacteriostatic agent is sodium azide.
In specific implementation, the content of the sodium azide is 0.02-0.04%.
In particular implementation, the molecular weight of the recombinant keratin is 47 KDa.
A purity detection method of a recombinant keratin liquid preparation comprises high performance liquid chromatography, wherein a chromatographic column adopts gel suitable for separating protein with molecular weight of 5-1250 KDa as a filling agent, and a mobile phase adopts 0.08% TFA-acetonitrile (containing 0.4ml TFA and 500ml acetonitrile mixed liquid) and 0.1% TFA-water (containing 0.5ml TFA and 500ml deionized water); the sample loading amount is not less than 10 mu L, the theoretical plate number calculated by recombinant keratin chromatographic peak is not less than 16000 by detection at the wavelength of 280nm, and finally, the calculation is carried out according to an area normalization method.
Preferably, the chromatography column is a SEC300 gel chromatography column.
Comparative example 1
Preparing a recombinant keratin stock solution with the molecular weight of 47KDa into a liquid preparation with the initial purity of 95% by using deionized water; storing the recombinant keratin liquid preparation at room temperature of 25-30 ℃ for 12 months, injecting the liquid preparation into a SEC300 gel chromatographic column by adopting high performance liquid chromatography at a certain speed, and adopting 0.08% TFA-acetonitrile (mixed liquid containing 0.4ml TFA and 500ml acetonitrile) and 0.1% TFA-water (containing 0.5ml TFA and 500ml deionized water) as mobile phases; the loading amount is 10 μ L, the detection is carried out at the wavelength of 280nm to obtain figure 1, the theoretical plate number calculated by the chromatographic peak of the recombinant keratin is 16000, and finally the purity of the recombinant keratin is 75 percent calculated by an area normalization method.
Example 1
The invention provides a recombinant keratin liquid preparation, which comprises the following components in percentage by mass: taking 0.1% of recombinant keratin stock solution with the molecular weight of 47KDa, preparing the recombinant keratin stock solution with sterile water, and obtaining the initial purity of 95.5%; then stabilizer including 5% of glycerin, 1% of NaCl, 5% of cane sugar and 0.01% of sodium azide as preservative bacteriostat are added. Storing the recombinant keratin liquid preparation at room temperature of 25-30 ℃ for 12 months, and injecting the liquid preparation into a SEC300 gel chromatographic column by adopting high performance liquid chromatography according to a certain speed according to the purity detection method of the recombinant keratin liquid preparation disclosed by the invention, wherein the mobile phase adopts 0.08% TFA-acetonitrile (mixed liquid containing 0.4ml TFA and 500ml acetonitrile) and 0.1% TFA-water (mixed liquid containing 0.5ml TFA and 500ml deionized water); the sample loading amount is 10 μ L, FIG. 2 is obtained by detection at wavelength of 280nm, the theoretical plate number calculated by recombinant keratin chromatographic peak should be 16000, and the purity of the recombinant keratin is 94.69% by area normalization.
Example 2
The invention provides a recombinant keratin liquid preparation, which comprises the following components in percentage by mass: taking 0.15% of recombinant keratin with the molecular weight of 47KDa, preparing the recombinant keratin with sterile water, and obtaining the initial purity of 95.3%; then stabilizer including 6% glycerol, 1.5% NaCl1, 4% sucrose and antiseptic bacteriostatic agent sodium azide 0.02% is added. According to the purity detection method of the recombinant keratin liquid preparation, high performance liquid chromatography is adopted, the liquid preparation is injected into a SEC300 gel chromatographic column at a certain speed, and mobile phase adopts 0.08% TFA-acetonitrile (mixed liquid containing 0.4ml TFA and 500ml acetonitrile) and 0.1% TFA-water (containing 0.5ml TFA and 500ml deionized water); the sample loading amount is 10 μ L, FIG. 3 is obtained by detecting at the wavelength of 280nm, the theoretical plate number calculated by the chromatographic peak of the recombinant keratin should be 16000, and the purity of the recombinant keratin is 94.82% by calculating according to the area normalization method.
Example 3
The invention provides a recombinant keratin liquid preparation, which comprises the following components in percentage by mass: taking 0.15% of recombinant keratin with the molecular weight of 47KDa, preparing the recombinant keratin with sterile water, and obtaining the initial purity of 95.2%; then stabilizer including glycerin 7%, NaCl1.5%, cane sugar 3%, antiseptic bacteriostatic agent sodium azide 0.03% is added. According to the purity detection method of the recombinant keratin liquid preparation, high performance liquid chromatography is adopted, the liquid preparation is injected into a SEC300 gel chromatographic column at a certain speed, and mobile phase adopts 0.08% TFA-acetonitrile (mixed liquid containing 0.4ml TFA and 500ml acetonitrile) and 0.1% TFA-water (containing 0.5ml TFA and 500ml deionized water); the loading amount is 10 μ L, and the detection is performed at a wavelength of 280nm to obtain FIG. 4, the theoretical plate number calculated by the chromatographic peak of the recombinant keratin should be 16000, and finally the purity of the recombinant keratin is 94.92% calculated by an area normalization method.
Example 4
The invention provides a recombinant keratin liquid preparation, which comprises the following components in percentage by mass: taking 0.2% of recombinant keratin with the molecular weight of 47KDa, preparing the recombinant keratin with sterile water, and obtaining the initial purity of 95.8%; then stabilizer including 8% of glycerol, 2% of NaCl, 2% of cane sugar and 0.05% of sodium azide as preservative bacteriostat are added. According to the purity detection method of the recombinant keratin liquid preparation, high performance liquid chromatography is adopted, the liquid preparation is injected into a SEC300 gel chromatographic column at a certain speed, and mobile phase adopts 0.08% TFA-acetonitrile (mixed liquid containing 0.4ml TFA and 500ml acetonitrile) and 0.1% TFA-water (containing 0.5ml TFA and 500ml deionized water); the sample loading amount is 10 μ L, FIG. 5 is obtained by detection at wavelength of 280nm, the theoretical plate number calculated by recombinant keratin chromatographic peak should be 16000, and the purity of the recombinant keratin is 94.89% by area normalization.
The initial concentrations of recombinant keratin and the purity of the recombinant keratin after 12 months storage of the liquid formulations in each example are shown in table 1.
Table 1 purity table of recombinant keratin in liquid formulation in each example
From table 1 it can be analytically determined that the purity of the recombinant keratin in the liquid formulation after 12 months varies by less than 1% compared to the comparative example, wherein the variation in purity of the recombinant keratin in the liquid formulation in example 3 is minimal; the recombinant keratin liquid preparation provided by the invention can be used for effectively preserving recombinant keratin; the condition of keratin degradation or aggregation is avoided, the recombinant keratin can keep good activity for a long time, and the purity of the recombinant keratin is effectively ensured to be not changed greatly in the preservation process, so that the recombinant keratin with good activity and high purity can be applied to medical supplies, and good effectiveness can be exerted in the medical supplies.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
Claims (9)
1. A recombinant keratin liquid preparation, which is characterized by comprising the following components in percentage by mass: 0.1 to 0.2 percent of recombinant keratin, 8 to 15 percent of stabilizing agent, 0.01 to 0.05 percent of antiseptic bacteriostatic agent and the balance of deionized water.
2. The liquid recombinant keratin formulation according to claim 1, wherein the stabilizer comprises one or any combination of glycerol, NaCl and sucrose.
3. The liquid recombinant keratin formulation according to claim 2, wherein the stabilizer comprises 5-8% glycerol, 1-2% NaCl, 2-5% sucrose.
4. The liquid recombinant keratin formulation according to claim 3, wherein the stabilizer comprises glycerol 6-7%, NaCl1.5%, sucrose 3-4%.
5. The recombinant keratin liquid formulation of any one of claims 1 or 4, wherein the preservative bacteriostatic agent is sodium azide.
6. The liquid recombinant keratin formulation according to claim 5, wherein the sodium azide is present in an amount of 0.02% to 0.04%.
7. The liquid recombinant keratin formulation according to any one of claims 1 or 6, wherein the recombinant keratin has a molecular weight of 47 kDa.
8. The purity detection method of the recombinant keratin liquid preparation is characterized by comprising the steps of carrying out high performance liquid chromatography, wherein gel suitable for separating protein with the molecular weight of 5-1250 KDa is adopted as a filling agent in a chromatographic column, and 0.08% TFA-acetonitrile (mixed liquid containing 0.4ml TFA and 500ml acetonitrile) and 0.1% TFA-water (containing 0.5ml TFA and 500ml deionized water) are adopted as mobile phases; the sample loading amount is not less than 10 mu L, the theoretical plate number calculated by recombinant keratin chromatographic peak is not less than 16000 by detection at the wavelength of 280nm, and finally, the calculation is carried out according to an area normalization method.
9. The method for detecting the purity of a liquid preparation of recombinant keratin according to claim 8, wherein the chromatographic column is a SEC300 gel chromatographic column.
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