CN113952310A - Preparation of lyophilized powder - Google Patents

Preparation of lyophilized powder Download PDF

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CN113952310A
CN113952310A CN202111005410.1A CN202111005410A CN113952310A CN 113952310 A CN113952310 A CN 113952310A CN 202111005410 A CN202111005410 A CN 202111005410A CN 113952310 A CN113952310 A CN 113952310A
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maintaining
vacuum
lyophilized powder
heating
sucrose
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郭志栋
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Shandong Duomeikang Biomedical Co ltd
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Abstract

Disclosed are lyophilized powders comprising recombinant type III humanized collagen, sucrose and mannitol, wherein the sucrose comprises from about 1.0% to about 2.0% by weight of the lyophilized powder, and the mannitol comprises from about 1.0% to about 2.0% by weight of the lyophilized powder.

Description

Preparation of lyophilized powder
FIELD
The present disclosure relates generally to the field of formulations, and more particularly, to lyophilized powders.
Background
Collagen is a large group of molecules consisting of various glycoproteins, the major protein component of connective tissue, and accounts for approximately 25% of the total protein in the human body. To date, more than 30 genes encoding collagen chains have been discovered, and these different collagen chains, combined in different ways, can form at least more than 16 collagen molecules. The 16 different types of collagen molecules are mostly formed by twisting 3 alpha chains, and are shaped like rods. Some of them consist of 3 identical alpha chains, e.g. [ alpha ]1]3Constituting a homogeneous trimer, and some of them consisting of 3 different alpha chains, e.g. [ alpha ]1]2α2Or alpha1α2α3A heterogeneous trimer is composed. The size of the alpha chain typically varies from 600 to 1000 amino acids. New collagen chains and collagen types are being discovered continuously. In the molecular structure, each collagen peptide chain is mainly composed of Gly-X-Y (X, Y is any amino acid residue except Gly) triplet repeat, and the unique structure is necessary for forming collagen fiber higher-order structure, which determines collagen proteinHas excellent biocompatibility and low immunogenicity, and is widely applied to the industries of medicines, health care products and cosmetics.
The conventional method for producing collagen is to treat animal-derived tissues by acid, alkali, and enzymatic hydrolysis to extract collagen derivatives. The collagen extracted by the methods loses the original biological activity and cannot be applied to the biomedical field to play a real function. By utilizing the genetic engineering technology and selecting various host cells such as transgenic animals, transgenic plants, insect cells, bacteria, yeast and the like to produce the recombinant human collagen and gelatin, the product has the advantages of good safety, good reproducibility, stable quality and the like, overcomes the defects of hidden danger of mad cow disease virus and the like existing in the traditional extraction method, and simultaneously improves the performances of hydrophilicity, immunological rejection and the like of the collagen. The application of the recombinant collagen product can gradually replace the animal-derived collagen in the future, particularly in the application of high-end fields.
SUMMARY
In one aspect, the present disclosure relates to a lyophilized powder comprising recombinant type III humanized collagen, sucrose and mannitol, wherein the sucrose comprises 1.0% to 2.0% by weight of the lyophilized powder and the mannitol comprises 1.0% to 2.0% by weight of the lyophilized powder.
In another aspect, the present disclosure relates to a lyophilization process for preparing a lyophilized powder comprising:
cooling the solution containing recombinant type III humanized collagen, sucrose and mannitol to-35 deg.C to-45 deg.C under vacuum of 0.20 + -0.05 mbar to obtain solid;
raising the temperature of the solid to-5 ℃ to 5 ℃ under a vacuum of 0.20 ± 0.05mbar, thereby obtaining a powder; and
raising the temperature of the powder to 20 ℃ to 30 ℃ under a vacuum of 0.20 +/-0.05 mbar to obtain the lyophilized powder,
wherein the lyophilized powder comprises recombinant type III humanized collagen, sucrose and mannitol.
In yet another aspect, the present disclosure relates to a lyophilized powder prepared by a lyophilization process comprising the steps of:
cooling the solution containing recombinant type III humanized collagen, sucrose and mannitol to-35 deg.C to-45 deg.C under vacuum of 0.20 + -0.05 mbar to obtain solid;
raising the temperature of the solid to-5 ℃ to 5 ℃ under a vacuum of 0.20 ± 0.05mbar, thereby obtaining a powder; and
raising the temperature of the powder to 20 ℃ to 30 ℃ under a vacuum of 0.20 +/-0.05 mbar to obtain the lyophilized powder,
wherein the lyophilized powder comprises recombinant type III humanized collagen, sucrose and mannitol.
Brief description of the drawings
FIG. 1 shows SDS-PAGE patterns before and after lyophilization of recombinant type III humanized collagen in example 17 of the present disclosure.
Detailed description of the invention
In the following description, certain specific details are included to provide a thorough understanding of various disclosed embodiments. One skilled in the relevant art will recognize, however, that the embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth.
Throughout this specification and the claims which follow, unless the context requires otherwise, the words "comprise", "comprising", and "have" are to be construed in an open, inclusive sense, i.e., "including but not limited to".
As used in this disclosure and the appended claims, a singular reference of an element without a numerical designation includes a plural reference unless the context clearly dictates otherwise.
Reference throughout the specification to "one embodiment," "an embodiment," "in another embodiment," or "in certain embodiments" means that a particular reference element, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
It should be understood that, as used in the specification of the present disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a lyophilized powder comprising "recombinant humanized collagen" includes one recombinant humanized collagen, or two or more recombinant humanized collagens.
Definition of
In the present disclosure, the term "recombinant humanized collagen" refers to a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombination technology, or a combination containing functional fragments of human collagen.
In the present disclosure, the term "type III" refers to recombinant collagen encoded based on the collagen COL3a1 gene or a partial gene.
In the present disclosure, the term "sucrose" refers to a substance which is obtained by condensing and dehydrating a hemiacetal hydroxyl group of one molecule of glucose and a hemiacetal hydroxyl group of one molecule of fructose with each other and has the following structure
Figure BDA0003237000370000041
In the present disclosure, the term "trehalose" refers to a substance which is formed by condensation of two glucose molecules through a hemiacetal hydroxyl group and which has the following structure:
Figure BDA0003237000370000042
in the present disclosure, the term "mannitol" refers to a substance having the structure:
Figure BDA0003237000370000043
in the present disclosure, the term "Phosphate Buffered Saline (PBS)" refers to a Phosphate Buffered Saline (PBS) containing Na as a major component2HPO4、KH2PO4NaCl and KCl.
In certain embodiments, the term "eutectic temperature" refers to the temperature at which water in the material completely freezes into ice crystals.
In the present disclosure, the term "redispersibility" refers to the complete reconstitution time.
In the present disclosure, the term "lyophilization", i.e. vacuum freeze-drying, refers to a technique of freezing a material below a eutectic temperature to change water in the material into solid ice, then directly subliming the ice into water vapor under a proper vacuum degree, and then condensing the water vapor by using a water vapor condenser in a vacuum system to obtain a dried product.
Detailed Description
In one aspect, the present disclosure relates to a lyophilized powder comprising recombinant type III humanized collagen, sucrose and mannitol, wherein the sucrose comprises about 1.0% to about 2.0% by weight of the lyophilized powder and the mannitol comprises about 1.0% to about 2.0% by weight of the lyophilized powder.
In certain embodiments, the sucrose comprises 1.0% to 2.0% by weight of the lyophilized powder.
In certain embodiments, the sucrose comprises about 1.5% by weight of the lyophilized powder.
In certain embodiments, the mannitol comprises 1.0% to 2.0% by weight of the lyophilized powder.
In certain embodiments, the mannitol comprises about 1.5% by weight of the lyophilized powder.
In certain embodiments, the sucrose comprises about 1.5% by weight of the lyophilized powder, and the mannitol comprises about 1.5% by weight of the lyophilized powder.
In certain embodiments, water comprises about 4% to 6% by weight of the lyophilized powder.
In certain embodiments, water comprises 4% to 6% by weight of the lyophilized powder.
In certain embodiments, water comprises 5% by weight of the lyophilized powder.
In certain embodiments, a lyophilized powder consists essentially of recombinant type III humanized collagen, sucrose and mannitol, wherein the sucrose comprises from about 1.0% to about 2.0% by weight of the lyophilized powder, and the mannitol comprises from about 1.0% to about 2.0% by weight of the lyophilized powder.
In certain embodiments, a lyophilized powder consists of recombinant type III humanized collagen, sucrose, mannitol, and other unavoidable impurities, wherein the sucrose comprises from about 1.0% to about 2.0% by weight of the lyophilized powder, and the mannitol comprises from about 1.0% to about 2.0% by weight of the lyophilized powder.
In certain embodiments, the quality criteria after lyophilization of the recombinant type III humanized collagen are consistent with those before lyophilization.
In certain embodiments, the lyophilized powder of the present disclosure has a low moisture content and a high stability.
In certain embodiments, the lyophilized powder of the present disclosure is suitable for storage and transportation.
In another aspect, the present disclosure relates to a lyophilization process for preparing a lyophilized powder comprising:
cooling the solution comprising recombinant type III humanized collagen, sucrose and mannitol to about-35 ℃ to about-45 ℃ under a vacuum of about 0.20 + -0.05 mbar to obtain a solid;
raising the temperature of the solid to about-5 ℃ to about 5 ℃ under a vacuum of about 0.20 ± 0.05mbar, thereby obtaining a powder; and
raising the temperature of the powder to about 20 ℃ to about 30 ℃ under a vacuum of about 0.20 + -0.05 mbar to obtain the lyophilized powder,
wherein the lyophilized powder comprises recombinant type III humanized collagen, sucrose and mannitol.
In certain embodiments, the temperature of the solid is raised to about-15 ℃ to about-25 ℃, then to about-10 ℃ to about-15 ℃, and finally to about-5 ℃ to about 5 ℃ under a vacuum of about 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to-15 ℃ to-25 ℃, then to-10 ℃ to-15 ℃, and finally to-5 ℃ to 5 ℃ under a vacuum of 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to-15 ℃, then to-10 ℃ and finally to 0 ℃ under a vacuum of about 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to-15 ℃ to-25 ℃ over 1 to 2 hours under a vacuum of 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to-15 ℃ over 2 hours under a vacuum of 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to about-15 ℃ to about-25 ℃ in about 1 to about 2 hours under a vacuum of about 0.20 ± 0.05mbar, maintained for about 12 to about 15 hours.
In certain embodiments, the temperature of the solid is raised to-15 ℃ to-25 ℃ over 1 to 2 hours at a vacuum of 0.20 ± 0.05mbar, maintained for 12 to 15 hours.
In certain embodiments, the temperature of the solid is raised to-15 ℃ under a vacuum of 0.20 ± 0.05mbar for 15 hours.
In certain embodiments, the temperature of the solid is raised to-15 ℃ over 2 hours at a vacuum of 0.20 ± 0.05mbar for 15 hours.
In certain embodiments, the temperature of the solid is raised to about-15 ℃ under a vacuum of about 0.20 ± 0.05mbar, and then raised to about-10 ℃ to about-15 ℃ over about 0.5 to about 1 hour.
In certain embodiments, the temperature of the solid is raised to-15 ℃ under a vacuum of 0.20 ± 0.05mbar, and then raised to-10 ℃ to-15 ℃ over 0.5 to 1 hour.
In certain embodiments, the temperature of the solid is raised to-15 ℃ under a vacuum of 0.20 ± 0.05mbar and then to-10 ℃ within 0.5 hours.
In certain embodiments, the temperature of the solid is raised to about-15 ℃ under a vacuum of about 0.20 ± 0.05mbar, and then raised to about-10 ℃ to about-15 ℃ over about 0.5 to about 1 hour, for about 10 to about 12 hours.
In certain embodiments, the temperature of the solid is raised to-15 ℃ under a vacuum of 0.20 ± 0.05mbar, then raised to-10 ℃ to-15 ℃ over 0.5 to 1 hour, for about 10 to about 12 hours.
In certain embodiments, the temperature of the solid is raised to-15 ℃ and then to-10 ℃ for 12 hours under a vacuum of 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to-15 ℃ under a vacuum of 0.20 ± 0.05mbar, and then raised to-10 ℃ over 0.5 hours for 12 hours.
In certain embodiments, the temperature of the solid is raised to about-15 ℃, then to about-10 ℃, and finally to about-5 ℃ to about 5 ℃ over about 1 to about 2 hours under a vacuum of about 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to-15 ℃, then-10 ℃ and finally-5 ℃ to 5 ℃ within 1 to 2 hours under a vacuum of 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to-15 ℃, then-10 ℃ and finally to 0 ℃ over 2 hours under a vacuum of 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the solid is raised to about-15 ℃, then to about-10 ℃, and finally to about-5 ℃ to about 5 ℃ over about 1 to about 2 hours, under a vacuum of about 0.20 ± 0.05mbar, for about 2 to about 5 hours.
In certain embodiments, the temperature of the solid is raised to-15 ℃, then-10 ℃ and finally further to-5 ℃ to 5 ℃ over 1 to 2 hours, under a vacuum of 0.20 ± 0.05mbar, for 2 to 5 hours.
In certain embodiments, the temperature of the solid is raised to-15 ℃, then to-10 ℃ and finally to 0 ℃ again under a vacuum of 0.20 ± 0.05mbar for 2 hours.
In certain embodiments, the temperature of the solid is raised to-15 ℃, then-10 ℃ and finally to 0 ℃ over 2 hours under a vacuum of 0.20 ± 0.05mbar for 2 hours.
In certain embodiments, the temperature of the powder is raised to about 20 ℃ to about 30 ℃ over about 0.5 to about 1.5 hours under a vacuum of about 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the powder is raised to 20 ℃ to 30 ℃ over 0.5 to 1.5 hours under a vacuum of 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the powder is raised to 25 ℃ within 1 hour under a vacuum of 0.20 ± 0.05 mbar.
In certain embodiments, the temperature of the powder is raised to about 20 ℃ to about 30 ℃ for about 0.5 to about 1.5 hours under a vacuum of about 0.20 ± 0.05mbar, maintained for about 5 to about 8 hours.
In certain embodiments, the temperature of the powder is raised to 20 ℃ to 30 ℃ for 0.5 to 1.5 hours under a vacuum of 0.20 ± 0.05mbar for 5 to 8 hours.
In certain embodiments, the temperature of the powder is raised to 25 ℃ under a vacuum of 0.20 ± 0.05mbar for 7 hours.
In certain embodiments, the temperature of the powder is raised to 25 ℃ over 1 hour at a vacuum of 0.20 ± 0.05mbar for 7 hours.
In certain embodiments, the solution comprising recombinant type III humanized collagen, sucrose, and mannitol has a eutectic point temperature of about-15 ℃ to about-8 ℃.
In certain embodiments, the pH of the solution comprising recombinant type III humanized collagen, sucrose, and mannitol is about 6.0 ± 0.1.
In certain embodiments, the solution comprising recombinant type III humanized collagen comprises about 20mmol/L phosphate buffered saline.
In certain embodiments, the amount of recombinant type III humanized collagen in the solution comprising recombinant type III humanized collagen, sucrose and mannitol is about 20 to 50 g/L.
In certain embodiments, the solution comprising recombinant type III humanized collagen, sucrose and mannitol is pre-cooled to 5 ℃ to-5 ℃ under a vacuum of 0.20 ± 0.05mbar, and the solution comprising recombinant type III humanized collagen, sucrose and mannitol is cooled to-35 ℃ to-45 ℃ to obtain a solid.
In certain embodiments, a solution comprising recombinant type III humanized collagen, sucrose, and mannitol is lyophilized in vials filled at a size of 2 ml/vial.
In certain embodiments, the methods of the present disclosure are suitable for large scale production.
In certain embodiments, using the lyophilization methods of the present disclosure, the post-lyophilization quality criteria for recombinant type III humanized collagen are consistent with that before lyophilization.
In yet another aspect, the present disclosure relates to a lyophilized powder prepared by a lyophilization process comprising:
cooling the solution comprising recombinant type III humanized collagen, sucrose and mannitol to about-35 ℃ to about-45 ℃ under a vacuum of about 0.20 + -0.05 mbar to obtain a solid;
raising the temperature of the solid to about-5 ℃ to about 5 ℃ under a vacuum of about 0.20 ± 0.05mbar, thereby obtaining a powder; and
raising the temperature of the powder to about 20 ℃ to about 30 ℃ under a vacuum of about 0.20 + -0.05 mbar to obtain the lyophilized powder,
in certain embodiments, the lyophilized recombinant type III humanized collagen in the lyophilized powder obtained using the methods of the present disclosure is consistent in quality criteria after lyophilization as before lyophilization.
In certain embodiments, the lyophilized powder obtained using the methods of the present disclosure has a low moisture content and a high stability.
In certain embodiments, the lyophilized powder obtained using the methods of the present disclosure is suitable for storage and transport.
Hereinafter, the present disclosure will be explained in detail by the following examples in order to better understand various aspects of the present application and advantages thereof. It should be understood, however, that the following examples are not limiting and are merely illustrative of certain embodiments of the present disclosure.
Examples
The reagents and equipment used in the examples of the present disclosure are conventional and commercially available.
For example:
recombinant type III humanized collagen, biotechnological division of north china pharmaceutical products, inc; 20200302 batches
Sucrose, Jiuxiang pharmaceutical products, Inc., Hunan; CAS number: 57-50-1
Mannitol, Jiu-classic pharmaceutical Co., Ltd, Hunan; CAS number: 887-78-5
Na2HPO4National pharmaceutical chemicals; CAS number: 7558-79-4
NaH2PO4National pharmaceutical chemicals; CAS number: 10049-21-5
NaCl, dengyucheng, refined salt works ltd; CAS number: 7647-14-5
Precision analytical balance (Mettler Toledo AL104-IC, Switzerland), PH meter (Raymagnetic PHS-25), electromagnetic stirrer (ETS-D4, IKA, Germany), freeze dryer (LGJ-S30, Beijing four-ring sailing science and technology Co., Ltd.), electrophoresis apparatus (Bio-rad), ultraviolet spectrophotometer (UV-7540, Shanghai Xinmao apparatus Co., Ltd.)
Preparation examples
Example 1
Weighing 1.0g of sucrose in 100ml of recombinant III type humanized collagen solution (protein content is 20-50g/L, buffer solution is 20mmol/LPBS, pH is 6.0 +/-0.1), stirring and mixing uniformly until the sucrose is completely dissolved, filtering and sterilizing at 0.22 mu m, then subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 0 deg.C under vacuum of 0.15mbar, cooling to-40 deg.C for 1.5 hr, and maintaining for 2 hr;
(2) sublimation drying: keeping the vacuum degree at 0.15mbar, heating to-15 ℃ within 1.5h, and maintaining for 12 h; heating to-10 deg.C for 0.5h, and maintaining for 10 h; the temperature is raised to 5 ℃ for 2h, and the temperature is maintained for 2 h.
(3) And (3) resolving and drying: adjusting the vacuum degree to 0.20mbar, heating to 20 ℃ within 1 hour, and maintaining for 8 hours to obtain the freeze-dried powder.
Example 2
Weighing 1.5g of sucrose in 100ml of recombinant III type humanized collagen solution (protein content is 20-50g/L, buffer solution is 20mmol/LPBS, pH is 6.0 +/-0.1), stirring and mixing uniformly until the sucrose is completely dissolved, filtering and sterilizing at 0.22 mu m, then subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 5 deg.C under vacuum of 0.2mbar for 1 hr, cooling to-45 deg.C, and maintaining for 2 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.15mbar, heating to-15 ℃ within 1.5h, and maintaining for 12 h; heating to-10 deg.C for 0.5h, and maintaining for 10 h; heating to 5 ℃ for 2h, and maintaining for 2 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.15mbar, heating to 25 ℃ within 1.5h, and keeping for 7h to obtain the freeze-dried powder.
Example 3
Weighing 2.5g of sucrose in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the sucrose is completely dissolved, filtering and sterilizing at 0.22 mu m, then subpackaging the sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-5 deg.C under vacuum of 0.25mbar, cooling to-35 deg.C for 0.5 hr, and maintaining for 3 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.15mbar, heating to-20 ℃ within 2h, and maintaining for 14 h; heating to-15 ℃ for 1h, and maintaining for 12 h; heating to 0 ℃ for 1.5h, and maintaining for 3 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.20mbar, heating to 30 ℃ within 1 hour, and maintaining for 5 hours to obtain the freeze-dried powder.
Example 4
Weighing 10g of sucrose in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the sucrose is completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging the sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-10 deg.C under vacuum of 0.20mbar for 1 hr, cooling to-40 deg.C, and maintaining for 2 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.25mbar, heating to-25 ℃ within 1 hour, and maintaining for 12 hours; heating to-15 ℃ for 1h, and maintaining for 10 h; heating to 5 ℃ for 1h, and maintaining for 5 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.20mbar, heating to 25 ℃ within 0.5h, and maintaining for 7h to obtain the freeze-dried powder.
Example 5
Weighing 1.0g of trehalose in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the trehalose is completely dissolved, filtering and sterilizing at 0.22 mu m, then subpackaging the sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 0 deg.C under vacuum of 0.15mbar for 2 hr, cooling to-35 deg.C, and maintaining for 2 hr;
(2) sublimation drying: keeping the vacuum degree at 0.15mbar, heating to-20 ℃ within 1h, and maintaining for 14 h; heating to-10 deg.C for 0.5h, and maintaining for 12 h; heating to 5 ℃ for 2h, and maintaining for 4 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.20mbar, heating to 20 ℃ within 1 hour, and maintaining for 8 hours to obtain the freeze-dried powder.
Example 6
Weighing 1.5g trehalose in 100ml recombinant type III humanized collagen solution (protein content 20-50g/L, buffer solution 20mmol/LPBS, pH6.0 + -0.1), stirring and mixing well until completely dissolving, filtering and sterilizing at 0.22 μm, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain lyophilized powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-10 deg.C under vacuum of 0.25mbar for 1 hr, cooling to-45 deg.C, and maintaining for 3 hr;
(2) sublimation drying: keeping the vacuum degree at 0.25mbar, heating to-15 ℃ within 1h, and maintaining for 13 h; heating to-15 deg.C for 0.5h, and maintaining for 10 h; heating to 0 ℃ for 2h, and maintaining for 2 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.20mbar, heating to 25 ℃ within 0.5h, and maintaining for 7h to obtain the freeze-dried powder.
Example 7
Weighing 2.5g of trehalose in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the trehalose is completely dissolved, filtering and sterilizing at 0.22 mu m, then subpackaging the mixture into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-5 deg.C under vacuum of 0.20mbar, cooling to-35 deg.C for 1.5 hr, and maintaining for 2.5 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.25mbar, heating to-20 ℃ within 0.5h, and maintaining for 15 h; heating to-10 deg.C for 0.5h, and maintaining for 12 h; heating to 5 ℃ for 1h, and maintaining for 5 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.25mbar, heating to 30 ℃ within 1h, and maintaining for 6h to obtain the freeze-dried powder.
Example 8
Weighing 10g of trehalose in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the trehalose is completely dissolved, filtering and sterilizing at 0.22 mu m, then subpackaging the mixture into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 5 deg.C under vacuum of 0.25mbar, cooling to-35 deg.C for 1.5 hr, and maintaining for 3 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.20mbar, heating to-25 ℃ within 2h, and maintaining for 15 h; heating to-15 deg.C for 0.5h, and maintaining for 12 h; heating to 0 ℃ for 1h, and maintaining for 3 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.25mbar, heating to 30 ℃ within 1 hour, and maintaining for 5 hours to obtain the freeze-dried powder.
Example 9
Weighing 1.0g of mannitol into 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the mannitol is completely dissolved, filtering and sterilizing at 0.22 mu m, then subpackaging the filtrate into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 0 deg.C under vacuum of 0.15mbar for 2 hr, cooling to-40 deg.C, and maintaining for 2 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.20mbar, heating to-15 ℃ within 2h, and maintaining for 15 h; heating to-10 ℃ for 1h, and maintaining for 12 h; heating to 0 ℃ for 2h, and maintaining for 5 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.20mbar, heating to 25 ℃ within 0.5h, and maintaining for 8h to obtain the freeze-dried powder.
Example 10
Weighing 1.5g mannitol in 100ml of recombinant III type humanized collagen solution (protein content 20-50g/L, buffer solution 20mmol/LPBS, pH6.0 + -0.1), stirring and mixing well until completely dissolving, filtering and sterilizing at 0.22 μm, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain lyophilized powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-10 deg.C under vacuum of 0.25mbar, cooling to-35 deg.C for 0.5 hr, and maintaining for 3 hr;
(2) sublimation drying: keeping the vacuum degree at 0.25mbar, heating to-20 ℃ for 2h, and maintaining for 13 h; heating to-15 deg.C for 0.5h, and maintaining for 10 h; heating to-5 ℃ for 1h, and maintaining for 3 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.25mbar, heating to 30 ℃ within 0.5h, and maintaining for 7h to obtain the freeze-dried powder.
Example 11
Weighing 2.5g mannitol in 100ml of recombinant III type humanized collagen solution (protein content 20-50g/L, buffer solution 20mmol/LPBS, pH6.0 + -0.1), stirring and mixing well until completely dissolving, filtering and sterilizing at 0.22 μm, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the lyophilized powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 0 deg.C under vacuum of 0.20mbar for 1 hr, cooling to-45 deg.C, and maintaining for 2 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.25mbar, heating to-25 ℃ within 2h, and maintaining for 14 h; heating to-10 deg.C for 0.5h, and maintaining for 12 h; heating to 0 ℃ for 1h, and maintaining for 4 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.25mbar, heating to 20 ℃ within 1h, and maintaining for 6h to obtain the freeze-dried powder.
Example 12
Weighing 10g of mannitol into 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the mannitol is completely dissolved, filtering and sterilizing at 0.22 mu m, then subpackaging the filtrate into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-5 deg.C under vacuum of 0.20mbar, cooling to-35 deg.C for 0.5 hr, and maintaining for 2 hr;
(2) sublimation drying: keeping the vacuum degree at 0.20mbar, heating to-15 ℃ within 1h, and maintaining for 15 h; heating to-10 deg.C for 1h, and maintaining for 11 h; heating to-5 ℃ for 1h, and maintaining for 5 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.15mbar, heating to 30 ℃ within 1.5h, and maintaining for 5h to obtain the freeze-dried powder.
Example 13
Weighing 100ml of recombinant III type humanized collagen solution (protein content 20-50g/L, buffer solution 20mmol/LPBS, pH6.0 +/-0.1), filtering for sterilization at 0.22 μm, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain lyophilized powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 5 deg.C under vacuum of 0.15mbar for 2 hr, cooling to-40 deg.C, and maintaining for 2 hr;
(2) sublimation drying: keeping the vacuum degree at 0.15mbar, heating to-20 ℃ within 1.5h, and maintaining for 12 h; heating to-10 deg.C for 1h, and maintaining for 11 h; heating to 0 ℃ for 1h, and maintaining for 2 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.20mbar, heating to 25 ℃ within 1 hour, and maintaining for 7 hours to obtain the freeze-dried powder.
Example 14
Weighing 1.5g of sucrose, 1.5g of mannitol and 1.5g of trehalose, adding into 100ml of recombinant III type humanized collagen solution (with the protein content of 20-50g/L, the buffer solution of 20mmol/LPBS and the pH of 6.0 +/-0.1), stirring, mixing uniformly until the solution is completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 0 deg.C under vacuum of 0.15mbar for 1 hr, cooling to-35 deg.C, and maintaining for 3 hr;
(2) sublimation drying: keeping the vacuum degree at 0.15mbar, heating to-15 ℃ for 2h, and maintaining for 15 h; heating to-10 deg.C for 0.5h, and maintaining for 12 h; heating to 0 ℃ for 2h, and maintaining for 2 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.20mbar, heating to 30 ℃ within 1h, and maintaining for 8h to obtain the freeze-dried powder.
Example 15
Weighing 1.5g of sucrose and 1.5g of trehalose in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the sucrose and the trehalose are completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 5 deg.C under vacuum of 0.25mbar for 1 hr, cooling to-35 deg.C, and maintaining for 2 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.15mbar, heating to-20 ℃ within 1.5h, and maintaining for 15 h; heating to-15 ℃ for 1h, and maintaining for 10 h; heating to-5 ℃ for 2h, and maintaining for 4 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.15mbar, heating to 20 ℃ within 0.5h, and maintaining for 8h to obtain the freeze-dried powder.
Example 16
Weighing 1.5g of mannitol and 1.5g of trehalose in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the mannitol and the trehalose are completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-10 deg.C under vacuum of 0.20mbar for 2 hr, cooling to-35 deg.C, and maintaining for 2 hr;
(2) sublimation drying: keeping the vacuum degree at 0.2mbar, heating to-20 ℃ for 2h, and maintaining for 13 h; heating to-10 deg.C for 0.5h, and maintaining for 12 h; heating to 5 ℃ for 2h, and maintaining for 5 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.25mbar, heating to 30 ℃ within 1.5h, and maintaining for 5h to obtain the freeze-dried powder.
Example 17
Weighing 1.5g of sucrose and 1.5g of mannitol in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the protein is completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 5 deg.C under vacuum of 0.20mbar for 2 hr, cooling to-40 deg.C, and maintaining for 2 hr;
(2) sublimation drying: keeping the vacuum degree at 0.2mbar, heating to-15 ℃ for 1h, and maintaining for 15 h; heating to-10 deg.C for 0.5h, and maintaining for 12 h; heating to 0 ℃ for 2h, and maintaining for 2 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.2mbar, heating to 25 ℃ for 1h, and maintaining for 7h to obtain the freeze-dried powder.
Example 18
Weighing 1.0g of sucrose and 1.0g of mannitol in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the protein is completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 0 deg.C under vacuum of 0.15mbar for 1 hr, cooling to-35 deg.C, and maintaining for 2 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.2mbar, heating to-25 ℃ within 1 hour, and maintaining for 15 hours; heating to-15 deg.C for 0.5h, and maintaining for 10 h; heating to-5 ℃ for 2h, and maintaining for 5 h; and
(3) and (3) resolving and drying: keeping the vacuum degree at 0.2mbar, heating to 20 ℃ within 0.5h, and maintaining for 6h to obtain the freeze-dried powder.
Example 19
Weighing 2.0g of sucrose and 2.0g of mannitol in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the protein is completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-5 deg.C under vacuum of 0.25mbar, cooling to-35 deg.C for 0.5 hr, and maintaining for 3 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.2mbar, heating to-15 ℃ within 2h, and maintaining for 12 h; heating to-10 ℃ for 1h, and maintaining for 12 h; heating to 5 ℃ for 1h, and maintaining for 2 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.15mbar, heating to 25 ℃ within 1.5h, and maintaining for 8h to obtain the freeze-dried powder.
Example 20
Weighing 1.0g of sucrose and 2.0g of mannitol in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the protein is completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at-10 deg.C under vacuum of 0.20mbar for 1 hr, cooling to-40 deg.C, and maintaining for 2 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.25mbar, heating to-15 ℃ within 1 hour, and maintaining for 12 hours; heating to-10 ℃ for 1h, and maintaining for 12 h; heating to 0 ℃ for 2h, and maintaining for 2 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.15mbar, heating to 20 ℃ within 1.5h, and maintaining for 6h to obtain the freeze-dried powder.
Example 21
Weighing 2.0g of sucrose and 1.0g of mannitol in 100ml of recombinant III type humanized collagen solution (the protein content is 20-50g/L, the buffer solution is 20mmol/LPBS, and the pH is 6.0 +/-0.1), stirring and mixing uniformly until the protein is completely dissolved, filtering and sterilizing at 0.22 mu m, subpackaging into sterilized penicillin bottles according to the specification of 2 ml/bottle, and freeze-drying to obtain the freeze-dried powder. The specific freezing steps are as follows:
(1) pre-freezing: pre-freezing at 0 deg.C under vacuum of 0.15mbar for 2 hr, cooling to-45 deg.C, and maintaining for 2 hr;
(2) sublimation drying: adjusting the vacuum degree to 0.25mbar, heating to-15 ℃ within 2h, and maintaining for 15 h; heating to-10 deg.C for 0.5h, and maintaining for 10 h; heating to 0 ℃ for 1h, and maintaining for 5 h; and
(3) and (3) resolving and drying: adjusting the vacuum degree to 0.20mbar, heating to 25 ℃ within 1 hour, and maintaining for 7 hours to obtain the freeze-dried powder.
Effects of the embodiment
1. Appearance: visual observation shows that the freeze-dried powder has smooth and full appearance and no obvious collapse or atrophy.
2. Color: visual observation shows that the freeze-dried powder has uniform color and no layering and color difference.
3. Redispersibility: taking the freeze-dried powder to redissolve with 2ml of purified water, visually observing, and recording the time of complete redissolution.
TABLE 1 evaluation index of formulation
Figure BDA0003237000370000191
The results of scoring analysis of each example were performed according to the above criteria and are shown in Table 2.
TABLE 2 Scoring results for lyophilized powders of examples 1 to 21
Figure BDA0003237000370000192
Figure BDA0003237000370000201
Redissolving examples
After the freeze-dried product is redissolved, protein content, purity (enzymolysis method) and SDS-PAGE detection are carried out, and the quality of the recombinant III type humanized collagen of each embodiment is changed compared with that before and after freeze-drying.
Selecting the freeze-dried powder of example 17, opening a penicillin bottle cap, adding 2ml of purified water, recording the adding time A, naturally dissolving, visually observing, and recording the complete dissolving time B, wherein the redissolving time is C ═ B-A.
And taking the completely redissolved solution, measuring the absorbance value at 562nm by using an ultraviolet spectrophotometer, preparing 6 tubes of test solution by using a pure collagen standard substance, respectively measuring the absorbance, making a standard curve, and calculating the protein content value of the sample by using the absorbance as a vertical coordinate.
Taking the completely-redissolved solution, determining by an SDS-PAGE method, comparing the area percentage of the main peak with a standard substance, and calculating the purity of the sample.
TABLE 3 results before and after lyophilization
Figure BDA0003237000370000202
The lyophilized powders of examples 18 to 21 have similar properties to example 17.
In the present disclosure, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
From the foregoing it will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the disclosure. Such variations and modifications are intended to fall within the scope of the appended claims of this disclosure.

Claims (10)

1. A lyophilized powder comprising recombinant type III humanized collagen, sucrose and mannitol, wherein the sucrose comprises 1.0% to 2.0% by weight of the lyophilized powder and the mannitol comprises 1.0% to 2.0% by weight of the lyophilized powder.
2. A lyophilized powder of claim 1, wherein the sucrose comprises 1.5% by weight of the lyophilized powder and the mannitol comprises 1.5% by weight of the lyophilized powder.
3. A lyophilized powder according to claim 1 or 2, wherein water comprises 4% to 8% by weight of the lyophilized powder.
4. A lyophilization process for preparing a lyophilized powder comprising:
cooling the solution containing recombinant type III humanized collagen, sucrose and mannitol to-35 deg.C to-45 deg.C under vacuum of 0.20 + -0.05 mbar to obtain solid;
raising the temperature of the solid to-5 ℃ to 5 ℃ under a vacuum of 0.20 ± 0.05mbar, thereby obtaining a powder; and
raising the temperature of the powder to 20 ℃ to 30 ℃ under a vacuum of 0.20 +/-0.05 mbar to obtain the lyophilized powder,
wherein the lyophilized powder comprises recombinant type III humanized collagen, sucrose and mannitol.
5. A lyophilization process according to claim 4 wherein the temperature of the solid is raised to-15 ℃ to-25 ℃, then-10 ℃ to-15 ℃ and finally-5 ℃ to 5 ℃ under a vacuum of 0.20 ± 0.05 mbar.
6. The lyophilization process according to claim 4, wherein the temperature of the solid is raised to-15 ℃ to-25 ℃ within 1 to 2 hours under a vacuum of 0.20 ± 0.05mbar and then maintained for 12 to 15 hours, preferably then raised to-10 ℃ to-15 ℃ within 0.5 to 1 hour, maintained for 10 to 12 hours, more preferably finally raised again to-5 ℃ to 5 ℃ within 1 to 2 hours and maintained for 2 to 5 hours.
7. The lyophilization process according to any one of claims 4 to 6, wherein the temperature of the powder is raised to 20 ℃ to 30 ℃ within 0.5 to 1.5 hours under a vacuum of 0.20 ± 0.05mbar for 5 to 8 hours.
8. The lyophilization process of any one of claims 4 to 7, wherein the solution comprising recombinant type III humanized collagen, sucrose and mannitol has a eutectic point temperature of-15 ℃ to-8 ℃.
9. Lyophilized powder prepared by the lyophilization method of any one of claims 4 to 8.
10. A lyophilized powder of claim 9, wherein the sucrose comprises 1.0% to 2.0% by weight of the lyophilized powder and the mannitol comprises 1.0% to 2.0% by weight of the lyophilized powder.
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