CN112358541B - Freeze-drying protective agent for recombinant human gamma-interferon - Google Patents
Freeze-drying protective agent for recombinant human gamma-interferon Download PDFInfo
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- CN112358541B CN112358541B CN202011339745.2A CN202011339745A CN112358541B CN 112358541 B CN112358541 B CN 112358541B CN 202011339745 A CN202011339745 A CN 202011339745A CN 112358541 B CN112358541 B CN 112358541B
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- 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
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
- C07K14/57—IFN-gamma
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Abstract
The invention belongs to the technical field of reagent protection, and discloses a freeze-drying protective agent of recombinant human gamma-interferon, which comprises sitosterol; it can ensure that the freeze-drying loss of the recombinant human gamma-interferon is not more than 20 percent, and the freeze-drying loss does not generate any side effect on the performance and the effect of the recombinant human gamma-interferon; the sitosterol is added into a freeze-drying protective reagent of the recombinant human gamma-interferon, so that the stable recombinant human gamma-interferon with lower cost can be obtained, and the method has practical and wide popularization effect.
Description
Technical Field
The invention relates to the technical field of reagent protection, in particular to a freeze-drying protective agent for recombinant human gamma-interferon.
Background
Gamma interferon, also known as IFN-gamma or type ii interferon, is produced by mitogens that stimulate T lymphocytes. Interferon is a highly effective antiviral bioactive substance, and is also a lymphokine with a wide range of immunoregulatory actions. The product is widely applied to interferon release assay (IGRA) kits. The gamma-interferon serving as a key component of the standard substance of the kit has important influence on the quality of the kit.
However, the prior various recombinant human gamma-interferon solutions have the problem of instability, and the effective period is far lower than the requirements of a kit. After the freeze-drying treatment, the freeze-drying loss is large. There is therefore a need to reduce the lyophilization loss of recombinant human interferon-gamma.
Disclosure of Invention
In order to overcome the defects of the prior art, the primary object of the invention is a recombinant human gamma-interferon lyoprotectant.
The second purpose of the invention is to provide a buffer solution containing the lyophilized protectant of the recombinant human gamma-interferon.
The purpose of the invention is realized by the following scheme:
the application of sitosterol as the freeze-drying protective agent of recombinant human gamma-interferon.
According to the invention, the buffer solution containing sitosterol is added into the recombinant human gamma-interferon, so that the protection of the recombinant human gamma-interferon is obviously higher than that of a control group, and therefore, the added sitosterol can be used as an excellent freeze-drying protective agent for the recombinant human gamma-interferon.
Preferably, the addition concentration of sitosterol is 1-5%; in addition, the sitosterol has no obvious difference on the excellent freeze-drying protection effect of the recombinant human gamma-interferon within the concentration range.
The invention also provides a freeze-drying protective agent of the recombinant human gamma-interferon, which contains sitosterol.
Preferably, the addition concentration of the sitosterol in the cryoprotectant is 1-5%.
The invention also provides a buffer solution of the freeze-drying protective agent containing the recombinant human gamma-interferon, wherein the buffer solution also comprises mannitol, BSA and trehalose.
As a preferred technical scheme, the formula of the buffer solution is PBS solution containing 1% sitosterol, 3% BSA, 4% mannitol and 5% trehalose.
As a preferred technical scheme, the formula of the buffer solution is PBS solution containing 2% sitosterol, 3% BSA, 3% mannitol and 5% trehalose.
As a preferred technical scheme, the formula of the buffer solution is PBS solution containing 3% sitosterol, 3% BSA, 2% mannitol and 5% trehalose.
As a preferred technical scheme, the formula of the buffer solution is PBS solution containing 4% sitosterol, 3% BSA, 1% mannitol and 5% trehalose.
As a preferable technical scheme, the formula of the buffer solution is PBS solution containing 5% sitosterol, 3% BSA and 5% trehalose.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a freeze-drying protective agent of recombinant human gamma-interferon, which comprises sitosterol; it can ensure that the freeze-drying loss of the recombinant human gamma-interferon is not more than 20 percent, and the freeze-drying loss does not generate any side effect on the performance and the effect of the recombinant human gamma-interferon; the sitosterol is added into a freeze-drying protective reagent of the recombinant human gamma-interferon, so that the stable recombinant human gamma-interferon with lower cost can be obtained, and the method has practical and wide popularization effect.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
In the following embodiments, methods for determining the content of recombinant human interferon-gamma are known to those skilled in the art, and whether the lyophilization solution contains sitosterol does not have any influence on the method steps for determining the content of recombinant human interferon-gamma. The freeze-drying liquids with different sitosterol contents are all prepared by adjusting the ratio of sitosterol to mannitol so as to ensure that the solid content of the freeze-drying liquid is 13 percent.
Example 1
Respectively preparing PBS buffer solution (lyophilized solution 1) of 3% BSA, 5% mannitol and 5% trehalose and PBS buffer solution (lyophilized solution 2) of 1% sitosterol, 3% BSA, 4% mannitol and 5% trehalose, dissolving a certain amount of gamma-interferon to reach the concentration of 1mg/ml, and subpackaging into penicillin bottles with 1ml per bottle.
Freeze-drying the subpackaged penicillin bottles in a freeze dryer under the primary drying conditions of-30 ℃, 0.2mbar and 10 hours; the secondary drying condition is 25 deg.C, 0.1mbar, 3 h.
And (3) determining the content of gamma-interferon:
the determination of the gamma-interferon content was performed using ELISA. Taking a bottle of gamma-interferon freeze-dried products with different freeze-dried liquid formulas, redissolving the gamma-interferon freeze-dried products by using 1ml of ultrapure water, and determining the concentration of the freeze-dried liquid after redissolution by using a gamma-interferon international standard product preparation standard curve. The results are shown in Table 1.
The data generated in this study were analyzed by comparison using statistical analysis software, and the statistical differences between the different lyophilisates were studied for the values of the gamma-interferon concentration of the two lyophilisates. The data show that there is a significant difference in lyophilization loss using lyophilized liquid 1 versus lyophilized liquid 2 to lyophilize gamma-interferon. The protective agent-containing freeze-drying liquid (freeze-drying liquid 2) has significantly higher protection for gamma-interferon during the freeze-drying process than the freeze-drying liquid (freeze-drying liquid 1) without the protective agent. Therefore, the freeze-drying liquid added with the protective agent has better protection effect on the gamma-interferon in the freeze-drying process.
TABLE 1 determination of the content of gamma-interferon in two lyophilized solutions
Example 2
Respectively preparing a formula I (1% sitosterol freeze-dried solution for short): 1% sitosterol, 3% BSA, 4% mannitol, 5% trehalose in PBS buffer;
formula two (2% sitosterol freeze-dried solution for short): 2% sitosterol, 3% BSA, 3% mannitol, 5% trehalose in PBS buffer;
formula three (3% sitosterol freeze-dried solution for short): 3% sitosterol, 3% BSA, 2% mannitol, 5% trehalose in PBS buffer;
formula four (abbreviated as 4% sitosterol freeze-dried liquid): 4% sitosterol, 3% BSA, 1% mannitol, 5% trehalose in PBS buffer;
formula five (abbreviated as 5% sitosterol freeze-dried liquid): 5% sitosterol, 3% BSA, 5% trehalose in PBS buffer.
Dissolving a certain amount of gamma-interferon in each prepared freeze-drying solution to enable the concentration of the gamma-interferon to reach 1mg/ml, and subpackaging the mixture into penicillin bottles with 1ml per bottle. Freeze-drying the subpackaged penicillin bottles in a freeze dryer under the primary drying conditions of-30 ℃, 0.2mbar and 10 hours; the secondary drying condition is 25 deg.C, 0.1mbar, 3 h.
And (3) determining the content of gamma-interferon:
the determination of the gamma-interferon content was performed using ELISA. Taking a bottle of gamma-interferon freeze-dried products with different freeze-dried liquid formulas, redissolving the gamma-interferon freeze-dried products by using 1ml of ultrapure water, and determining the concentration of the freeze-dried liquid after redissolution by using a gamma-interferon international standard product preparation standard curve. The results are shown in Table 2.
Comparative analysis of the data generated in this study was performed using statistical analysis software and the data showed that there was no significant difference in lyophilization loss using lyophilizates of different concentrations of protectant. Therefore, the freeze-drying liquid with the protective agent concentration in the range of 1-5% has no difference on the protective effect of the gamma-interferon in the freeze-drying process.
TABLE 2 determination of the content of gamma-interferon in different lyophilized solutions
Claims (3)
1. The application of sitosterol as the freeze-drying protective agent of recombinant human gamma-interferon.
2. The use according to claim 1, wherein the sitosterol is added at a concentration of 1% to 5%.
3. A buffer solution of a freeze-drying protective agent of recombinant human gamma-interferon is characterized in that,
the buffer solution is PBS solution containing 1% sitosterol, 3% BSA, 4% mannitol and 5% trehalose, or; the buffer solution is PBS solution containing 2% sitosterol, 3% BSA, 3% mannitol and 5% trehalose, or; the buffer solution is PBS solution containing 3% sitosterol, 3% BSA, 2% mannitol and 5% trehalose, or; the buffer solution is PBS solution containing 4% sitosterol, 3% BSA, 1% mannitol and 5% trehalose, or; the buffer was a PBS solution containing 5% sitosterol, 3% BSA, 5% trehalose.
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| HU228877B1 (en) * | 1998-03-26 | 2013-06-28 | Merck Sharp & Dohme | Formulations for stabilization of peg-interferon alpha conjugates and method for preparation such formulations |
| CN1267152C (en) * | 2003-06-02 | 2006-08-02 | 长春生物制品研究所 | Polythylene glycol-interferon alpha foreeze dried preparation |
| CN102327241A (en) * | 2011-10-20 | 2012-01-25 | 北京凯因科技股份有限公司 | Polyethylene glycol-integrated interferon variant freeze-dried preparation |
| CA2905243C (en) * | 2012-03-14 | 2021-11-09 | Membrane Protective Technologies, Inc. | System and substances for cryopreservation of viable cells |
| CN104474553A (en) * | 2014-11-26 | 2015-04-01 | 嘉应学院医学院 | Drug-releasing agent based on beta-sitosterol and preparation method of drug-releasing agent |
| CN106244653A (en) * | 2016-08-29 | 2016-12-21 | 芜湖英特菲尔生物制品产业研究院有限公司 | A kind of preparation method of recombination chicken interferon gamma standard substance |
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