阅读说明：本技术 提高重组人源胶原蛋白稳定性的冻干保护剂 (Freeze-drying protective agent for improving stability of recombinant human collagen ) 是由 杜尔凤 许乔林 黄建民 赵健烽 于 2021-08-19 设计创作，主要内容包括：本发明公开了一种提高重组人源胶原蛋白稳定性的冻干保护剂。所述的保护剂按重量份数计,以重组人源胶原蛋白为100份,由海藻糖500-2000份、聚乙烯吡咯烷酮(PVP-K30)100-500份和甘露醇100-200份组成。在重组人源胶原蛋白冻干过程中,添加冻干保护剂能够明显提高重组人源胶原蛋冻干粉在常温下的稳定性,防止其纯度和活性下降,制得的冻干粉可在常温下长期放置。(The invention discloses a freeze-drying protective agent for improving the stability of recombinant human collagen. The protective agent comprises 100 parts by weight of recombinant human collagen, 500 parts by weight of trehalose, 500 parts by weight of polyvinylpyrrolidone (PVP-K30) and 100 parts by weight of mannitol. In the freeze-drying process of the recombinant human collagen, the freeze-drying protective agent is added, so that the stability of the recombinant human collagen freeze-dried powder at normal temperature can be obviously improved, the purity and activity of the recombinant human collagen freeze-dried powder are prevented from being reduced, and the prepared freeze-dried powder can be placed at normal temperature for a long time.)

1. The freeze-drying protective agent for improving the stability of the recombinant human collagen is characterized by comprising the following components in parts by weight, taking the recombinant human collagen as 100 parts: 500 portions of trehalose, 500 portions of polyvinylpyrrolidone (PVP-K30) and 200 portions of mannitol.

2. The lyoprotectant according to claim 1, wherein the recombinant human collagen is produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021.

3. The lyoprotectant of claim 1, wherein said lyoprotectant comprises the following components: 1000 parts of trehalose, 200 parts of polyvinylpyrrolidone (PVP-K30) and 200 parts of mannitol.

4. The lyoprotectant of claim 1, wherein said lyoprotectant is administered by a method comprising: weighing recombinant human collagen, trehalose, polyvinylpyrrolidone (PVP-K30) and mannitol according to a proportion, adding injection water until the mixture is completely dissolved, adopting a filter membrane of 0.22 mu m for sterilization and filtration, subpackaging in penicillin bottles, and then putting into a freeze dryer for freeze drying to obtain the recombinant human collagen freeze-dried powder.

Technical Field

The invention belongs to the technical field of preservation of biological products, and relates to a freeze-drying protective agent for improving the stability of recombinant human collagen.

Background

The recombinant human collagen is based on the original gene sequence of human skin collagen, and the parts with strong water solubility and high biological activity are optimized and selected for codon optimization and splicing recombination to obtain a brand new recombinant human collagen sequence. The collagen has good water solubility and high bioactivity, has better performance than animal-derived collagen, and has wide application prospect in the field of biomedical materials.

Because the recombinant human collagen aqueous solution has poor stability and is sensitive to heat, the recombinant human collagen aqueous solution is generally prepared into freeze-dried powder by a freeze-drying method, and is convenient to store and use. However, the freeze-drying process causes the natural structure of the recombinant protein to be denatured because the surface of the recombinant collagen in the aqueous solution forms a hydrated layer, and the water removed by the drying process destroys the hydrogen bonding structure of the protein surface. In addition, the freeze-dried recombinant human collagen needs to be frozen or refrigerated for storage, and is easy to degrade and lower in purity when stored at normal temperature (25 ℃), so that the application of the recombinant human collagen is restricted to a certain extent.

Disclosure of Invention

The invention aims to provide a freeze-drying protective agent for improving the stability of recombinant human collagen. The protective freeze-drying agent can improve the stability of the recombinant human collagen freeze-dried powder at normal temperature and prevent the purity and activity of the recombinant human collagen freeze-dried powder from being reduced.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the freeze-drying protective agent for improving the stability of the recombinant human collagen comprises 100 parts of the recombinant human collagen by weight and comprises the following components: 500 portions of trehalose, 500 portions of polyvinylpyrrolidone (PVP-K30) and 200 portions of mannitol.

The recombinant human collagen is produced by fermenting pichia pastoris with the preservation number of CGMCC NO.5021 and is fully disclosed in Chinese patent ZL 201110327865.5.

Preferably, the lyoprotectant consists of the following components: 1000 parts of trehalose, 200 parts of polyvinylpyrrolidone (PVP-K30) and 200 parts of mannitol.

Further, the application method of the freeze-drying protective agent comprises the following steps: weighing recombinant human collagen, trehalose, polyvinylpyrrolidone (PVP-K30) and mannitol according to a proportion, adding injection water until the mixture is completely dissolved, adopting a filter membrane of 0.22 mu m for sterilization and filtration, subpackaging in penicillin bottles, and then putting into a freeze dryer for freeze drying to obtain the recombinant human collagen freeze-dried powder.

Compared with the prior art, the invention has the following remarkable effects:

according to the invention, the formula of the freeze-drying protective agent is reasonably matched, and the protective agent trehalose, polyvinylpyrrolidone and excipient mannitol are added. The trehalose can improve the glass transition temperature, and can be used as a low-temperature protective agent in the freezing process and a dehydration protective agent in the drying and dehydration process to protect the hydrogen bond structure on the surface of the protein. The polyvinylpyrrolidone can form a heat-resistant framework, reduce the influence of heat radiation on the recombinant human collagen, and slow down the degradation of protein at normal temperature. The two protective agents have different protective mechanisms for protecting proteins at different stages of freeze drying, and have synergistic protective effect on recombinant human collagen when used together. The freeze-drying protective agent can effectively enhance the resistance of the recombinant human collagen to the adverse environment of drying and normal temperature, reduces the degradation amplitude of the product purity, ensures that the shelf life of the freeze-dried powder can reach one year at normal temperature (25 ℃), improves the stability of the product, solves the problem that the freeze-dried powder needs to be stored and transported at low temperature, and is beneficial to popularization and application of the product.

Detailed Description

The present invention will be described in more detail with reference to specific examples.

In the following examples, the concentration of the recombinant human collagen solution used was 1 wt.% and the purity was 100%.

Example 1

The preparation method of the recombinant human collagen freeze-dried powder comprises the following steps:

1. 50g of trehalose, 10g of polyvinylpyrrolidone (PVP-K30) and 10g of mannitol are accurately weighed and dissolved by adding 300g of water for injection.

2. Accurately weighing 500g of recombinant human collagen solution containing 5g of recombinant human collagen, uniformly mixing the solution with the solution obtained in the step 1, and then adding water for injection, wherein the total weight is 1000 g.

3. The solution of step 2 was sterile filtered through a 0.22 μm filter to obtain a filtrate.

4. And (4) subpackaging the filtrate obtained in the step (3) in a tube-type bottle, half plugging, and freeze-drying in a freeze-drying machine.

5. After the freeze drying is finished, performing vacuum tamponade, capping and labeling to obtain the recombinant human collagen freeze-dried powder, and placing the recombinant human collagen freeze-dried powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% to perform a long-term stability experiment.

6. Purity was checked every 3 months and changes were observed.

Examples 2 to 5

The examples are essentially the same as example 1, except that the lyoprotectant employed contains different amounts of the components, as detailed in Table 1.

TABLE 1 amount of each raw material added in each example

Comparative example 1

This comparative example is essentially the same as example 1 except that no lyoprotectant was added.

The preparation method of the recombinant human collagen freeze-dried powder comprises the following steps:

1. 500g of recombinant human collagen solution containing 5g of recombinant human collagen is accurately weighed, and then 500g of water for injection is added, wherein the total weight is 1000 g.

2. The solution of step 1 was sterile filtered through a 0.22 μm filter to obtain a filtrate.

3. And (3) subpackaging the filtrate obtained in the step (2) in a tube-type bottle, half plugging, and freeze-drying in a freeze-drying machine.

4. After the freeze drying is finished, performing vacuum tamponade, capping and labeling to obtain the recombinant human collagen freeze-dried powder, and placing the recombinant human collagen freeze-dried powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% to perform a long-term stability experiment.

5. Purity was checked every 3 months and changes were observed.

Comparative example 2

This comparative example is substantially the same as example 1 except for the content of each component in the lyoprotectant.

The preparation method of the recombinant human collagen freeze-dried powder comprises the following steps:

1. 125g of trehalose, 30g of polyvinylpyrrolidone (PVP-K30) and 10g of mannitol are accurately weighed and dissolved by adding 300g of water for injection.

2. Accurately weighing 500g of recombinant human collagen solution containing 5g of recombinant human collagen, uniformly mixing the solution with the solution obtained in the step 1, and then adding water for injection, wherein the total weight is 1000 g.

3. The solution of step 2 was sterile filtered through a 0.22 μm filter to obtain a filtrate.

4. And (4) subpackaging the filtrate obtained in the step (3) in a tube-type bottle, half plugging, and freeze-drying in a freeze-drying machine.

5. After the freeze drying is finished, performing vacuum tamponade, capping and labeling to obtain the recombinant human collagen freeze-dried powder, and placing the recombinant human collagen freeze-dried powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% to perform a long-term stability experiment.

6. Purity was checked every 3 months and changes were observed.

Comparative example 3

This comparative example is substantially the same as example 1 except for the kinds and contents of the components in the lyoprotectant.

The preparation method of the recombinant human collagen freeze-dried powder comprises the following steps:

1. accurately weighing 50g of trehalose and 10g of mannitol, and adding 300g of water for injection to dissolve.

2. Accurately weighing 500g of recombinant human collagen solution containing 5g of recombinant human collagen, uniformly mixing the solution with the solution obtained in the step 1, and then adding water for injection, wherein the total weight is 1000 g.

3. The solution of step 2 was sterile filtered through a 0.22 μm filter to obtain a filtrate.

4. And (4) subpackaging the filtrate obtained in the step (3) in a tube-type bottle, half plugging, and freeze-drying in a freeze-drying machine.

5. After the freeze drying is finished, performing vacuum tamponade, capping and labeling to obtain the recombinant human collagen freeze-dried powder, and placing the recombinant human collagen freeze-dried powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% to perform a long-term stability experiment.

6. Purity was checked every 3 months and changes were observed.

Comparative example 4

This comparative example is substantially the same as example 1 except for the kinds and contents of the components in the lyoprotectant.

The preparation method of the recombinant human collagen freeze-dried powder comprises the following steps:

1. 10g of polyvinylpyrrolidone (PVP-K30) and 10g of mannitol are accurately weighed and dissolved by adding 300g of water for injection.

2. Accurately weighing 500g of recombinant human collagen solution containing 5g of recombinant human collagen, uniformly mixing the solution with the solution obtained in the step 1, and then adding water for injection, wherein the total weight is 1000 g.

3. The solution of step 2 was sterile filtered through a 0.22 μm filter to obtain a filtrate.

4. And (4) subpackaging the filtrate obtained in the step (3) in a tube-type bottle, half plugging, and freeze-drying in a freeze-drying machine.

5. After the freeze drying is finished, performing vacuum tamponade, capping and labeling to obtain the recombinant human collagen freeze-dried powder, and placing the recombinant human collagen freeze-dried powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% to perform a long-term stability experiment.

6. Purity was checked every 3 months and changes were observed.

The purity (%) of the recombinant human collagen lyophilized powder prepared according to various proportions and embodiments is shown in table 2, and the detection method is shown in chinese patent application CN 109481335A.

TABLE 2 Long-term stability test purity test results

Time of standing

Comparative example 1

Comparative example 2

Comparative example 3

Comparative example 4

Example 1

Example 2

Example 3

Example 4

Example 5

0 month

99.3％

100％

100％

99.6％

100％

100％

100％

100％

100％

3 month

97.1％

99.2％

99.1％

99.3％

100％

99.8％

99.6％

100％

100％

6 month

95.8％

98.3％

98.0％

98.4％

99.4％

99.3％

99.8％

99.6％

99.5％

9 month

93.9％

97.5％

97.2％

97.8％

99.6％

99.4％

99.4％

99.6％

99.2％

12 month

92.8％

96.8％

96.2％

96.5％

99.6％

98.3％

99％

99.2％

98.7％

As can be seen from Table 2, through long-term stability observation, the purity of the recombinant human collagen lyophilized powder prepared in 5 examples is obviously higher than that of comparative examples 1-4 after being stored at room temperature for 12 months, which indicates that the purity of the recombinant human collagen lyophilized powder protected by the cryoprotectant of the invention is not obviously reduced within 12 months and the quality is stable under the condition of normal temperature (25 ℃).