CN113717272B - Freeze-drying protective agent for improving stability of recombinant human collagen - Google Patents
Freeze-drying protective agent for improving stability of recombinant human collagen Download PDFInfo
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- CN113717272B CN113717272B CN202110954486.2A CN202110954486A CN113717272B CN 113717272 B CN113717272 B CN 113717272B CN 202110954486 A CN202110954486 A CN 202110954486A CN 113717272 B CN113717272 B CN 113717272B
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 58
- 108010035532 Collagen Proteins 0.000 title claims abstract description 58
- 229920001436 collagen Polymers 0.000 title claims abstract description 58
- 238000004108 freeze drying Methods 0.000 title claims abstract description 27
- 239000003223 protective agent Substances 0.000 title claims abstract description 17
- 239000000843 powder Substances 0.000 claims abstract description 25
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims abstract description 13
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims abstract description 13
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims abstract description 13
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 12
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 12
- 229930195725 Mannitol Natural products 0.000 claims abstract description 12
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 12
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 12
- 239000000594 mannitol Substances 0.000 claims abstract description 12
- 235000010355 mannitol Nutrition 0.000 claims abstract description 12
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229920003081 Povidone K 30 Polymers 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000005303 weighing Methods 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- 229930182555 Penicillin Natural products 0.000 claims description 4
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 4
- 229940049954 penicillin Drugs 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 241000235058 Komagataella pastoris Species 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 19
- 230000000052 comparative effect Effects 0.000 description 14
- 239000000706 filtrate Substances 0.000 description 10
- 239000008215 water for injection Substances 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 7
- 230000007774 longterm Effects 0.000 description 7
- 238000002372 labelling Methods 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 238000002156 mixing Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 3
- 239000008176 lyophilized powder Substances 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000009477 glass transition Effects 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
Classifications
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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
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a freeze-drying protective agent for improving stability of recombinant human collagen. The protective agent is prepared from 100 parts by weight of recombinant human collagen, 500-2000 parts by weight of trehalose, 100-500 parts by weight of polyvinylpyrrolidone (PVP-K30) and 100-200 parts by weight of mannitol. In the freeze-drying process of the recombinant human collagen, the freeze-drying protective agent is added to obviously 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, and the prepared freeze-dried powder can be placed for a long time at normal temperature.
Description
Technical Field
The invention belongs to the technical field of preservation of biological products, and relates to a freeze-drying protective agent for improving stability of recombinant human collagen.
Background
The recombinant human collagen is based on the original gene sequence of human skin collagen, and the part with strong water solubility and high biological activity is 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 biological activity, 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 adopting a freeze-drying method, and is convenient to store and use. However, the lyophilization process causes denaturation of the natural structure of the recombinant protein because the recombinant collagen forms a hydrated layer on the surface of the aqueous solution, and the removal of water from this hydrated layer during the drying process damages the hydrogen bonding structure on the surface of the protein. In addition, the freeze-dried recombinant human collagen needs to be frozen or refrigerated for preservation, and is easy to degrade when preserved at normal temperature (25 ℃), so that the purity is reduced, and 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-dried 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 above purpose, the technical scheme of the invention is as follows:
the freeze-drying protective agent for improving the stability of the recombinant human collagen is prepared from the following components in parts by weight by taking the recombinant human collagen as 100 parts: 500-2000 parts of trehalose, 100-500 parts of polyvinylpyrrolidone (PVP-K30) and 100-200 parts 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 use 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, sterilizing and filtering the mixture by adopting a filter membrane with the diameter of 0.22 mu m, subpackaging the mixture in penicillin bottles, and then freeze-drying the penicillin bottles in a freeze dryer 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 agents of trehalose, polyvinylpyrrolidone and excipient mannitol are added. Trehalose can raise the glass transition temperature, can serve as a low-temperature protective agent in the freezing process, can also serve as a dehydration protective agent in the drying and dehydration processes, and can protect the hydrogen bond structure on the surface of protein. The polyvinylpyrrolidone can form a heat-resistant framework, so that the influence of heat radiation on recombinant human collagen is reduced, and the degradation of the protein at normal temperature is slowed down. The two protective agents have different protection mechanisms for protecting proteins at different stages of freeze drying, and have synergistic protection effect on recombinant human collagen when used in combination. The freeze-drying protective agent can effectively enhance the resistance of the recombinant human collagen to dry and normal-temperature adverse environments, reduce the degradation range of the purity of the product, prolong the shelf life of the freeze-dried powder at normal temperature (25 ℃) for one year, improve the stability of the product, solve the difficult problem that the freeze-dried powder must be stored and transported at low temperature, and be beneficial to the popularization and application of the product.
Detailed Description
The invention will be further described in detail with reference to specific examples.
In the examples below, the concentration of the recombinant human collagen solution used was 1wt.% and the purity was 100%.
Example 1
The preparation method of the recombinant human collagen freeze-dried powder comprises the following steps:
1. accurately weighing 50g of trehalose, 10g of polyvinylpyrrolidone (PVP-K30) and 10g of mannitol, and adding 300g of water for injection to dissolve.
2. Accurately weighing 500g of recombinant human collagen solution, wherein the solution contains 5g of recombinant human collagen, uniformly mixing with the solution obtained in the step 1, and then adding water for injection, wherein the total weight is 1000g.
3. The solution of step 2 was subjected to aseptic filtration with a 0.22 μm filter membrane to obtain a filtrate.
4. And (3) subpackaging the filtrate obtained in the step (3) in tube bottles, half adding plugs, and freeze-drying in a freeze dryer.
5. After freeze-drying, vacuum tamponading, capping and labeling to obtain recombinant human collagen freeze-dried powder, and placing the powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% for long-term stability experiments.
6. Purity was measured every 3 months and the change was observed.
Examples 2 to 5
The examples are essentially the same as example 1, except that the lyoprotectant used differs in the content of the components, as detailed in table 1.
Table 1 amounts of raw materials added in each example
Comparative example 1
This comparative example is substantially the same as example 1 except that no lyoprotectant is 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, then 500g of water for injection is added, and the total weight is 1000g.
2. The solution of step 1 was subjected to aseptic filtration with a 0.22 μm filter membrane to obtain a filtrate.
3. And (3) subpackaging the filtrate obtained in the step (2) in tube bottles, half adding plugs, and freeze-drying in a freeze dryer.
4. After freeze-drying, vacuum tamponading, capping and labeling to obtain recombinant human collagen freeze-dried powder, and placing the powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% for long-term stability experiments.
5. Purity was measured every 3 months and the change was 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. accurately weighing 125g of trehalose, 30g of polyvinylpyrrolidone (PVP-K30) and 10g of mannitol, and adding 300g of water for injection to dissolve.
2. Accurately weighing 500g of recombinant human collagen solution, wherein the solution contains 5g of recombinant human collagen, uniformly mixing with the solution obtained in the step 1, and then adding water for injection, wherein the total weight is 1000g.
3. The solution of step 2 was subjected to aseptic filtration with a 0.22 μm filter membrane to obtain a filtrate.
4. And (3) subpackaging the filtrate obtained in the step (3) in tube bottles, half adding plugs, and freeze-drying in a freeze dryer.
5. After freeze-drying, vacuum tamponading, capping and labeling to obtain recombinant human collagen freeze-dried powder, and placing the powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% for long-term stability experiments.
6. Purity was measured every 3 months and the change was observed.
Comparative example 3
This comparative example is substantially the same as example 1, except for the kind and content of each component in the lyoprotectant.
The preparation method of the recombinant human collagen freeze-dried powder comprises the following steps:
1. accurately weighing 50g of trehalose, 10g of mannitol and 300g of water for injection for dissolution.
2. Accurately weighing 500g of recombinant human collagen solution, wherein the solution contains 5g of recombinant human collagen, uniformly mixing with the solution obtained in the step 1, and then adding water for injection, wherein the total weight is 1000g.
3. The solution of step 2 was subjected to aseptic filtration with a 0.22 μm filter membrane to obtain a filtrate.
4. And (3) subpackaging the filtrate obtained in the step (3) in tube bottles, half adding plugs, and freeze-drying in a freeze dryer.
5. After freeze-drying, vacuum tamponading, capping and labeling to obtain recombinant human collagen freeze-dried powder, and placing the powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% for long-term stability experiments.
6. Purity was measured every 3 months and the change was observed.
Comparative example 4
This comparative example is substantially the same as example 1, except for the kind and content of each component in the lyoprotectant.
The preparation method of the recombinant human collagen freeze-dried powder comprises the following steps:
1. accurately weighing 10g of polyvinylpyrrolidone (PVP-K30), 10g of mannitol, and adding 300g of water for injection to dissolve.
2. Accurately weighing 500g of recombinant human collagen solution, wherein the solution contains 5g of recombinant human collagen, uniformly mixing with the solution obtained in the step 1, and then adding water for injection, wherein the total weight is 1000g.
3. The solution of step 2 was subjected to aseptic filtration with a 0.22 μm filter membrane to obtain a filtrate.
4. And (3) subpackaging the filtrate obtained in the step (3) in tube bottles, half adding plugs, and freeze-drying in a freeze dryer.
5. After freeze-drying, vacuum tamponading, capping and labeling to obtain recombinant human collagen freeze-dried powder, and placing the powder in a stability incubator with the temperature of 25 ℃ and the humidity of 60% for long-term stability experiments.
6. Purity was measured every 3 months and the change was observed.
The results of the long-term stability experiment purity (%) of the recombinant human collagen lyophilized powder prepared in each comparative example and example are shown in Table 2, and the detection method is shown in Chinese patent application CN109481335A.
TABLE 2 results of purity detection for long-term stability experiments
| Time of placement | 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 months of | 97.1％ | 99.2％ | 99.1％ | 99.3％ | 100％ | 99.8％ | 99.6％ | 100％ | 100％ |
| 6 months of | 95.8％ | 98.3％ | 98.0％ | 98.4％ | 99.4％ | 99.3％ | 99.8％ | 99.6％ | 99.5％ |
| 9 months of | 93.9％ | 97.5％ | 97.2％ | 97.8％ | 99.6％ | 99.4％ | 99.4％ | 99.6％ | 99.2％ |
| 12 months of | 92.8％ | 96.8％ | 96.2％ | 96.5％ | 99.6％ | 98.3％ | 99％ | 99.2％ | 98.7％ |
As can be seen from Table 2, the purity of the recombinant human collagen lyophilized powder prepared in 5 examples after being stored for 12 months at room temperature is obviously higher than that of comparative examples 1-4, which shows that the purity of the recombinant human collagen lyophilized powder protected by the freeze-drying protective agent of the invention is not obviously reduced within 12 months under the condition of normal temperature (25 ℃), and the quality is stable.
Claims (3)
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, wherein the recombinant human collagen is taken as 100 parts: 500-2000 parts of trehalose, 100-500 parts of polyvinylpyrrolidone and 100-200 parts of mannitol, wherein the recombinant human collagen is produced by fermenting pichia pastoris with the preservation number of CGMCC No.5021, and the polyvinylpyrrolidone is PVP-K30.
2. The lyoprotectant of claim 1, wherein the lyoprotectant consists of: 1000 parts of trehalose, 200 parts of polyvinylpyrrolidone and 200 parts of mannitol.
3. The lyoprotectant of claim 1, wherein the lyoprotectant is used in a method comprising: weighing recombinant human collagen, trehalose, polyvinylpyrrolidone and mannitol according to a proportion, adding injection water until the mixture is completely dissolved, sterilizing and filtering the mixture by adopting a filter membrane with the diameter of 0.22 mu m, subpackaging the mixture into penicillin bottles, and then freeze-drying the penicillin bottles in a freeze dryer to obtain the recombinant human collagen freeze-dried powder.
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| CN114010528B (en) * | 2021-12-20 | 2023-09-15 | 广州栋方生物科技股份有限公司 | Recombinant human collagen freeze-dried powder and preparation method thereof |
| CN115252899B (en) * | 2022-09-13 | 2024-03-26 | 济南之羽医疗科技有限公司 | Freeze-dried powder containing recombinant human collagen and preparation method thereof |
| CN115944571B (en) * | 2023-01-10 | 2024-04-02 | 河北纳科生物科技有限公司 | Essence spray for promoting hair growth and repairing hair roots and preparation method thereof |
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