CN105497925B - A kind of method that functional protein is protected before sterilization by ionizing radiation and its special-purpose protecting agent - Google Patents
A kind of method that functional protein is protected before sterilization by ionizing radiation and its special-purpose protecting agent Download PDFInfo
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- CN105497925B CN105497925B CN201510998411.9A CN201510998411A CN105497925B CN 105497925 B CN105497925 B CN 105497925B CN 201510998411 A CN201510998411 A CN 201510998411A CN 105497925 B CN105497925 B CN 105497925B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0029—Radiation
Abstract
The composite material to functional protein or containing the functional protein is protected before sterilization by ionizing radiation method that the invention discloses a kind of.It includes the following steps: that in liquid-phase system, by described in " functional protein or the composite material containing the functional protein " is mixed with components I and compositionⅱ；The components I is carbohydrate；The compositionⅱ is amino acid and/or amino acid hydrochloride salt.It is demonstrated experimentally that method provided by the present invention has the function of anti-radiation, anti-oxidant and defencive function protein active, has the advantages that 1, is highly-safe: being all pharmaceutic adjuvant, toxic residue problem is not present；2, stability is high: carbohydrate and amino acids physical property are stablized, and are not easy to be oxidized；3, easy to operate, it can be used for laboratory sample production, be also applied for large-scale production.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to one kind protects functional protein before sterilization by ionizing radiation
Method and its special-purpose protecting agent, it is contemplated that reduce sterilization by ionizing radiation to the active damage of functional protein the technical issues of solution
Wound.
Background technique
Bone morphogenetic protein (BMP) is a kind of hydrophobic protein secreted by bone matrix, belongs to transforming growth factor
(TGF-β) superfamily member is put forward for the first time by Urist et al. in nineteen sixty-five, in nineteen eighty-two for the first time from decalcified bone matrix extract
In a kind of isolated functional protein.BMP family includes BMP-1, BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, BMP-
12, BMP-14 etc., wherein BMP-2 has the function of that the undifferentiated fibroblast directed differentiation of induction is osteoblast, and can adjust
Save the differentiation of osteoblast and chondroblast, induced ectopic bon e formation and the function of promoting union.Currently, BMP has become
It is used to function treat fresh fracture, bone nonunion and bone defect and spinal fusion etc..
Simple BMP can be diluted in vivo by tissue fluid and proteases for decomposing, non-in the time for being locally able to maintain effective concentration
It is often limited, therefore BMP is often by compound with suitable carrier material, for treating.Common material has demineralized bone matrix, natural height
Molecular compound (such as absorbable collagen sponge, hyaluronic acid derivatives), calcium containing compound (such as hydroxyapatite, absorbable phosphorus
Sour calcium cement), artificial synthesized high-molecular compound (such as polylactic acid, two polyglycolic acid of polylactic acid, polylactic acid poly ethylene glycol
Deng), BMP and titanium dioxide nano material can also be used in combination.
Sterilizing be simple BMP and BMP with it is essential before Material cladding clinical use.Conventional moist heat sterilization, epoxy second
Alkane sterilizing etc. can all cause the activity of BMP to reduce.Sterilization by ionizing radiation is a kind of good cold sterilization method, sterilization process medium temperature
Degree will not acute variation, can be used for sterilizing and the inactivation of virus of BMP and its composite material.Sterilization by ionizing radiation is mainly penetrated using X
Line, gamma-rays or energetic particle beam sterilizing, strong, wide sterilization spectrum, even action, substantially harmless etc. advantages with penetrability.Cobalt
60 gamma-radiation irradiation sterilizations are the sterilization by ionizing radiation methods of most practical value, and sterilization effect is related with irradiation dose, spoke
Bigger according to dosage, inactivating efficacy is better.Under normal circumstances, the Co 60 gamma-radiation of 20-50kGy dosage can almost inactivate all
Virus, but while inactivating pathogens, he also has an impact to the bioactive molecule of some protein ingredients.
It is now recognized that quality size is related with itself for sensibility of the large biological molecule to ionising radiation, compared with albumen,
Nucleic acid has higher sensibility to sterilization by ionizing radiation.Therefore, it while carrying out sterilization by ionizing radiation, selects reasonable living
Property molecule protected mode, protects functional protein, retains its activity and be possibly realized.Studies in China person's Cheng Wen qin etc. is to Co 60
Under the conditions of gamma-radiation irradiation sterilization, ascorbic acid, sodium ascorbate and tea polyphenols to the protective effect of immune globulin activity into
Research is gone, three kinds of substances have a degree of protective effect to immune globulin activity, but such powerful antioxidant is normal
It is unstable in air under temperature, it is easily oxidized and loses protective effect.QiHong Zhao etc. discloses a kind of Co 60 gamma-radiation radiation sterilization
Under the conditions of BMP-2 active protection method: the sealing centrifuge tube equipped with BMP solution is put into the vacuum flask for fill water and protects
Shield, this method have protective effect to BMP-2 activity under the conditions of Co 60 gamma-radiation radiation sterilization is crossed.But the method for QiHong Zhao etc.
Not only reduce the injury to microorganism and virus simultaneously, while can not reduce and radiate to BMP-2 activity bring consequential damages,
And cumbersome, large-scale production.How the bone morphogenetic protein during sterilization by ionizing radiation is protected, is become
One urgent problem to be solved.
Summary of the invention
The problem to be solved by the invention is to provide one kind before sterilization by ionizing radiation to functional protein or contains the function
Can albumen the method protected of composite material and its special-purpose protecting agent, it is contemplated that reduce ionising radiation the technical issues of solution
Sterilizing is to the active damage of functional protein.
Present invention firstly provides a kind of to answer to functional protein before sterilization by ionizing radiation or containing the functional protein
The method that condensation material is protected, includes the following steps: in liquid-phase system, and " functional protein contains the function by described in
The composite material of albumen " is mixed with components I and compositionⅱ；The components I can be carbohydrate；The compositionⅱ can be amino
Acid and/or amino acid hydrochloride salt.
In the above method, it may also include after the functional protein, components I and compositionⅱ mixing and adjust pH value to 3.5-5.5
The step of.
In the above method, the liquid-phase system can be water or salting liquid.The salting liquid concretely phosphoric acid sodium dihydrogen
The aqueous solution of 2.4mg/mL and sodium chloride 8.8mg/mL.
In the above method, in the liquid-phase system, the quality proportioning of the components I and the compositionⅱ can be as follows
(a1), (a2), (a3), (a4), (a5) or (a6):
(a1) 30:0.1-1800；
(a2) 30:16-90；
(a3) 30:16；
(a4) 30:70；
(a5) 30:17.5；
(a6) 30:90.
In the above method, in the liquid-phase system, the quality of the functional protein, the components I and the compositionⅱ is matched
Than that can be following (b1), (b2), (b3), (b4), (b5) or (b6):
(b1) 1.2:15-30:0.1-1800；
(b2) 1.2:15-30:16-45；
(b3) 1.2:30:16；
(b4) 1.2:15:35；
(b5) 1.2:30:17.5；
(b6) 1.2:15:45.
In the above method, in the liquid-phase system, the concentration of the components I can be 0.1mg/mL-30mg/mL, preferably
15mg/mL-30mg/mL；The concentration of the compositionⅱ can be 0.1mg/mL-45mg/mL, preferably 16mg/mL-45mg/mL.
In the above method, the addition form of the carbohydrate can be sucrose or trehalose；The addition of the amino acid
Form can be methionine and/or tryptophan；The addition form of the amino acid hydrochloride salt can be arginine monohydrochloride, cysteine
Hydrochloride and/or histidine hydrochloride.
In the above method, the method concretely following (c1), (c2), (c3), (c4), (c5), (c6), (c7),
(c8), (c9) or (c10):
(c1) in the method, the components I is sucrose, and the compositionⅱ is arginine monohydrochloride and cysteine hydrochloric acid
Salt；The concentration of sucrose is 30mg/mL；The concentration of arginine monohydrochloride is 15mg/mL；The concentration of cysteine hydrochloride is 1mg/
mL；
(c2) in the method, the components I is trehalose, and the compositionⅱ is histidine hydrochloride and cysteine salt
Hydrochlorate；The concentration of trehalose is 30mg/mL；The concentration of histidine hydrochloride is 15mg/mL；The concentration of cysteine hydrochloride is
1mg/mL；
(c3) in the method, the components I is sucrose, and the compositionⅱ is histidine hydrochloride and methionine；Sucrose
Concentration be 15mg/mL；The concentration of histidine hydrochloride is 30mg/mL；The concentration of methionine is 5mg/mL；
(c4) in the method, the components I is trehalose, and the compositionⅱ is arginine monohydrochloride and tryptophan；Sea
The concentration of algae sugar is 30mg/mL；The concentration of arginine monohydrochloride is 15mg/mL；The concentration of tryptophan is 2.5mg/mL；
(c5) in the method, the components I is sucrose, and the compositionⅱ is histidine hydrochloride and arginine hydrochloric acid
Salt；The concentration of sucrose is 15mg/mL；The concentration of histidine hydrochloride is 15mg/mL；The concentration of arginine monohydrochloride is 30mg/
mL；
(c6) in the method, the components I is sucrose, and the compositionⅱ is arginine monohydrochloride and cysteine hydrochloric acid
Salt；The concentration of functional protein is 1.2mg/mL, and the concentration of sucrose is 30mg/mL；The concentration of arginine monohydrochloride is 15mg/mL；
The concentration of cysteine hydrochloride is 1mg/mL；
(c7) in the method, the components I is trehalose, and the compositionⅱ is histidine hydrochloride and cysteine salt
Hydrochlorate；The concentration of functional protein is 1.2mg/mL, and the concentration of trehalose is 30mg/mL；The concentration of histidine hydrochloride is 15mg/
mL；The concentration of cysteine hydrochloride is 1mg/mL；
(c8) in the method, concretely sucrose, the compositionⅱ are histidine hydrochloride and egg ammonia to the components I
Acid；The concentration of functional protein is 1.2mg/mL, and the concentration of sucrose is 15mg/mL；The concentration of histidine hydrochloride is 30mg/mL；
The concentration of methionine is 5mg/mL；
(c9) in the method, concretely trehalose, the compositionⅱ are arginine monohydrochloride and color ammonia to the components I
Acid；The concentration of functional protein is 1.2mg/mL, and the concentration of trehalose is 30mg/mL；The concentration of arginine monohydrochloride is 15mg/
mL；The concentration of tryptophan is 2.5mg/mL；
(c10) in the method, the components I is sucrose, and the compositionⅱ is histidine hydrochloride and arginine hydrochloric acid
Salt；The concentration of functional protein is 1.2mg/mL, and the concentration of sucrose is 15mg/mL；The concentration of histidine hydrochloride is concretely
15mg/mL；The concentration of arginine monohydrochloride is 30mg/mL.
In the above method, the functional protein can be following (d1), (d2), (d3) or (d4):
(d1) bone morphogenetic protein；
(d2) human bone morphogenesis protein；
(d3) human bone morphogenesis protein-2；
(d4) human bone morphogenesis protein-2 shown in the sequence 2 of sequence table.
In the above method, described in sterilization by ionizing radiation can be following (e1), (e2), (e3) or (e4):
(e1) γ ray sterilization；
It (e2) is the γ ray sterilization of radiation source with radionuclide cobalt -60；
It (e3) is the γ ray sterilization of radiation source, dose of radiation 15kGy-35kGy with radionuclide cobalt -60；
It (e4) is the γ ray sterilization of radiation source, dose of radiation 25kGy with radionuclide cobalt -60.
The present invention also provides a kind of pair of functional protein or the composite material containing the functional protein carries out ionising radiation
The method of sterilizing.
A kind of pair of functional protein provided by the present invention or composite material containing the functional protein carry out ionising radiation
The method of sterilizing, it may include following steps:
(1) described to functional protein or contain the compound of the functional protein before sterilization by ionizing radiation using any of the above-described
The method that material is protected carries out pre-treatment to " functional protein or composite material " containing the functional protein；
(2) it is freeze-dried；
(3) sterilization by ionizing radiation.
The product that the present invention also provides a kind of to protect functional protein before sterilization by ionizing radiation.
A kind of product that functional protein is protected before sterilization by ionizing radiation provided by the invention, including it is any of the above-described
The components I and compositionⅱ.
The product is in use, the quality proportioning of the functional protein to be protected and the product is concretely as follows
(f1), (f2), (f3), (f4) or (f5):
(f1) 1.2:46-60；
(f2) 1.2:46；
(f3) 1.2:47.5；
(f4) 1.2:50；
(f5) 1.2:60.
The product concretely protective agent.
Any of the above-described sterilization by ionizing radiation can be X-ray sterilizing, γ ray sterilization or high-power electron beam sterilizing.
The γ ray sterilization is concretely the γ of radiation source with radionuclide cobalt -60 or radionuclide caesium -137
Ray.
Any of the above-described functional protein can be bone morphogenetic protein, the bone morphogenetic protein concretely people's bone
Morphogenetic proteins.
Any of the above-described human bone morphogenesis protein, concretely recombinant human bone morphogenesis protein BMP-2, amino
Acid sequence is concretely shown in the sequence 2 of sequence table.
It is demonstrated experimentally that one kind provided by the present invention to functional protein or contains the function egg before sterilization by ionizing radiation
The method that white composite material is protected has the function of anti-radiation, anti-oxidant and defencive function protein active.The present invention
The protective agent of offer has the following advantages: 1, highly-safe: to be all pharmaceutic adjuvant, toxic residue problem is not present；2, stability is high:
Carbohydrate and amino acids physical property are stablized, and are not easy to be oxidized；3, easy to operate, it can be used for laboratory sample production,
Suitable for large-scale production.
Detailed description of the invention
Fig. 1 is the liquid chromatogram of blank sample before irradiation sterilization, and peak 1 is rhBMP-2 dimer.
Fig. 2 is the liquid chromatogram of blank sample after irradiation sterilization, and peak 4 is rhBMP-2 dimer.
Fig. 3 is the liquid chromatogram of sample 1 after irradiation sterilization, and peak 2 is rhBMP-2 dimer.
Fig. 4 is the liquid chromatogram of sample 2 after irradiation sterilization, and peak 2 is rhBMP-2 dimer.
Fig. 5 is the liquid chromatogram of sample 3 after irradiation sterilization, and peak 1 is rhBMP-2 dimer.
Fig. 6 is the liquid chromatogram of sample 4 after irradiation sterilization, and peak 3 is rhBMP-2 dimer.
Fig. 7 is the liquid chromatogram of sample 5 after irradiation sterilization, and peak 2 is rhBMP-2 dimer.
Fig. 8 is the liquid chromatogram of sample 6 after irradiation sterilization, and peak 2 is rhBMP-2 dimer.
Fig. 9 is the liquid chromatogram of sample 7 after irradiation sterilization, and peak 1 is rhBMP-2 dimer.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.Experiment weight
It is 3 times multiple, it is averaged.
Cysteine hydrochloride mother liquor in following embodiments: solute and its concentration are as follows: cysteine hydrochloride 100mg/
Ml, sodium dihydrogen phosphate 2.4mg/ml and sodium chloride 8.8mg/ml；Solvent is water；pH 5.5.
C2C12 cell in following embodiments is purchased from Shanghai Inst. of Life Science, CAS cellular resources
The heart.
DMEM high glucose medium is U.S. Hyclone Products.
Glycine-NaOH buffer: glycine-NaOH containing 0.1M (pH9.6), 1mM MgCl2With 1mM ZnCl2Water
Solution.
Arginine monohydrochloride, cysteine hydrochloride and histidine hydrochloride in following embodiments are specially L-arginine
Hydrochloride, L-cysteine hydrochloride and L-Histidine hydrochloride, chemical formula are followed successively by formula I, formula II and formula III:
The preparation of embodiment 1, rhBMP-2 stoste
One, the building of recombinant bacterium
1, by the sequence of sequence table 1, the double chain DNA molecule shown in the nucleotide of 5 ' end 64-477 is inserted into carrier
Between I restriction enzyme site of Nde I and Xho of pET-28a (+), recombinant plasmid is obtained.In recombinant plasmid, the double chain DNA molecule of insertion
It is merged with the part DNA of plasmid itself, fusion shown in the sequence 1 of formation sequence table, shown in the sequence 2 of expressed sequence table
Fusion protein.
2, the recombinant plasmid for obtaining step 1 imports e. coli bl21 (DE3), obtains recombinant bacterium.
Two, the preparation of rhBMP-2 stoste
It takes step 1 to obtain recombinant bacterium, then passes through according to production method for purifying disclosed in patent CN201310432794.4
Purified after purifying, protein renaturation and renaturation before the dissolution of inclusion body, renaturation and etc., obtain rhBMP-2 stoste.RhBMP-2 is
Recombinant human bone morphogenesis protein-2.
The concentration of rhBMP-2 is 1.2mg/mL in the rhBMP-2 stoste.
The composition of the rhBMP-2 stoste: solvent is water, and solute and its solubility are as follows: sodium dihydrogen phosphate 2.4mg/mL, chlorination
The concentration of sodium 8.8mg/mL, rhBMP-2 are 1.2mg/mL.
Embodiment 2, different function protein protective agent are to the protecting effect of rhBMP-2
BMP-2 dimer has the bioactivity of induced synthesis osteoblast, and the polymer of BMP-2 and monomer do not have
The standby bioactivity.BMP-2 usually requires to carry out irradiation sterilization, but can generate BMP-2 poly through cobalt-60γray radiation
Body, to lose bioactivity.The present embodiment is using BMP-2 dimer in high performance liquid chromatography detection sterilizing front and back BMP-2
Purity, while using C2C12 cell method detection sterilizing front and back BMP-2 biological activity, to evaluate provided by the invention
The function and effect of pre-treating method.
One, the production of sample
1, blank sample
(solvent is water to rhBMP-2 stoste prepared by Example 1, and solute and its concentration are as follows: rhBMP-2 albumen
1.2mg/mL, sodium dihydrogen phosphate 2.4mg/mL and sodium chloride 8.8mg/mL) 1mL, it is freeze-dried, is prepared into freeze-dried powder.By the jelly
Dry powder is named as blank sample.
2, sample 1
Sucrose, half Guang ammonia of arginine monohydrochloride and 1mL is added in the rhBMP-2 stoste for taking 99mL embodiment 1 to prepare thereto
Then acid hydrochloride mother liquor adjusts pH to 5.5, obtains mixed liquor 1.In mixed liquor 1, solvent is water, and solute and its concentration are as follows:
RhBMP-2 albumen 1.2mg/mL, sucrose 30mg/mL, arginine monohydrochloride 15mg/mL, cysteine hydrochloride 1mg/mL, phosphoric acid
Sodium dihydrogen 2.4mg/mL and sodium chloride 8.8mg/mL.
Mixed liquor 11mL is taken, is freeze-dried, is prepared into freeze-dried powder.The freeze-dried powder is named as sample 1.
In sample 1, the quality proportioning of rhBMP-2 albumen, sucrose, arginine monohydrochloride and cysteine hydrochloride are as follows:
1.2mg:30mg:15mg:1mg。
In sample 1, the quality proportioning of components I (sucrose) and compositionⅱ (arginine monohydrochloride and cysteine hydrochloride)
Are as follows: 30mg:16mg.
In sample 1, rhBMP-2 albumen and functional protein protective agent (sucrose, arginine monohydrochloride and cysteine hydrochloric acid
Salt) quality proportioning are as follows: 1.2mg:46mg.
3, sample 2
Trehalose, half Guang of histidine hydrochloride and 1mL is added in the rhBMP-2 stoste for taking 99mL embodiment 1 to prepare thereto
Propylhomoserin hydrochloride mother liquor adjusts pH to 5.5, obtains mixed liquor 2.In mixed liquor 2, solvent is water, and solute and its concentration are as follows:
RhBMP-2 albumen 1.2mg/mL, trehalose 30mg/mL, histidine hydrochloride 15mg/mL, cysteine hydrochloride 1mg/mL, phosphorus
Acid dihydride sodium 2.4mg/mL and sodium chloride 8.8mg/mL.
Mixed liquor 21mL is taken, is freeze-dried, is prepared into freeze-dried powder.The freeze-dried powder is named as sample 2.
In sample 2, the quality proportioning of rhBMP-2 albumen, trehalose, histidine hydrochloride and cysteine hydrochloride are as follows:
1.2mg:30mg:15mg:1mg。
In sample 2, the quality proportioning of components I (trehalose) and compositionⅱ (histidine hydrochloride and cysteine hydrochloride)
Are as follows: 30mg:16mg.
In sample 2, rhBMP-2 albumen and functional protein protective agent (trehalose, histidine hydrochloride and cysteine hydrochloric acid
Salt) quality proportioning are as follows: 1.2mg:46mg.
4, sample 3
Sucrose, histidine hydrochloride and methionine is added in the rhBMP-2 stoste for taking 100mL embodiment 1 to prepare thereto,
PH to 5.0 is adjusted, mixed liquor 3 is obtained.In mixed liquor 3, solvent is water, and solute and its concentration are as follows: rhBMP-2 albumen 1.2mg/
ML, sucrose 15mg/mL, histidine hydrochloride 30mg/mL, methionine 5mg/mL, sodium dihydrogen phosphate 2.4mg/mL and sodium chloride
8.8mg/mL。
Mixed liquor 31mL is taken, is freeze-dried, is prepared into freeze-dried powder.The freeze-dried powder is named as sample 3.
In sample 3, the quality proportioning of rhBMP-2 albumen, sucrose, histidine hydrochloride and methionine are as follows: 1.2mg:15mg:
30mg:5mg。
In sample 3, the quality proportioning of components I (sucrose) and compositionⅱ (histidine hydrochloride and methionine) are as follows: 30mg:
70mg。
In sample 3, the quality of rhBMP-2 albumen and functional protein protective agent (sucrose, histidine hydrochloride and methionine)
Proportion are as follows: 1.2mg:50mg.
5, sample 4
Trehalose, arginine monohydrochloride and color ammonia is added in the rhBMP-2 stoste for taking 100mL embodiment 1 to prepare thereto
Acid obtains mixed liquor 4.In mixed liquor 4, solvent is water, and solute and its concentration are as follows: rhBMP-2 albumen 1.2mg/mL, trehalose
30mg/mL, arginine monohydrochloride 15mg/mL, tryptophan 2.5mg/mL, sodium dihydrogen phosphate 2.4mg/mL and sodium chloride 8.8mg/
mL。
Mixed liquor 41mL is taken, is freeze-dried, is prepared into freeze-dried powder.The freeze-dried powder is named as sample 4.
In sample 4, the quality proportioning of rhBMP-2 albumen, trehalose, arginine monohydrochloride and tryptophan are as follows: 1.2mg:
30mg:15mg:2.5mg。
In sample 4, the quality proportioning of components I (sucrose) and compositionⅱ (arginine monohydrochloride and tryptophan) are as follows: 30mg:
17.5mg。
In sample 4, the quality of rhBMP-2 albumen and functional protein protective agent (sucrose, arginine monohydrochloride and tryptophan)
Proportion are as follows: 1.2mg:47.5mg.
6, sample 5
Sucrose, histidine hydrochloride and arginine salt is added in the rhBMP-2 stoste for taking 100mL embodiment 1 to prepare thereto
Hydrochlorate obtains mixed liquor 5.In mixed liquor 5, solvent is water, and solute and its concentration are as follows: rhBMP-2 albumen 1.2mg/mL, sucrose
15mg/mL, histidine hydrochloride 15mg/mL, arginine monohydrochloride 30mg/mL, sodium dihydrogen phosphate 2.4mg/mL and sodium chloride
8.8mg/mL。
Mixed liquor 51mL is taken, is freeze-dried, is prepared into freeze-dried powder.The freeze-dried powder is named as sample 5.
In sample 5, the quality proportioning of rhBMP-2 albumen, sucrose, histidine hydrochloride and arginine hydrochloric acid are as follows: 1.2mg:
15mg:15mg:30mg。
In sample 5, the quality proportioning of components I (sucrose) and compositionⅱ (histidine hydrochloride and arginine hydrochloric acid) are as follows:
30mg:90mg.
In sample 5, rhBMP-2 albumen and functional protein protective agent (sucrose, histidine hydrochloride and arginine hydrochloric acid)
Quality proportioning are as follows: 1.2mg:60mg.
7, sample 6
The rhBMP-2 stoste for taking 100mL embodiment 1 to prepare, sucrose is added thereto, obtains mixed liquor 6.In mixed liquor 6,
Solvent is water, and solute and its concentration are as follows: rhBMP-2 albumen 1.2mg/mL, sucrose 30mg/mL, sodium dihydrogen phosphate 2.4mg/mL
With sodium chloride 8.8mg/mL.
Mixed liquor 61mL is taken, is freeze-dried, is prepared into freeze-dried powder.The freeze-dried powder is named as sample 6.
In sample 6, the quality proportioning of rhBMP-2 albumen and functional protein protective agent (sucrose) are as follows: 1.2mg:30mg.
8, sample 7
Histidine hydrochloride and arginine monohydrochloride is added in the rhBMP-2 stoste for taking 100mL embodiment 1 to prepare thereto,
Obtain mixed liquor 7.In the mixed liquor 7, solvent is water, and solute and its concentration are as follows: rhBMP-2 albumen 1.2mg/mL, group ammonia
Acid hydrochloride 15mg/mL, arginine monohydrochloride 30mg/mL, sodium dihydrogen phosphate 2.4mg/mL and sodium chloride 8.8mg/mL.
Mixed liquor 71mL is taken, is freeze-dried, is prepared into freeze-dried powder.The freeze-dried powder is named as sample 7.
In sample 7, the quality proportioning of rhBMP-2 albumen, histidine hydrochloride and arginine hydrochloric acid are as follows: 1.2mg:15mg:
30mg。
In sample 7, the quality of rhBMP-2 albumen and functional protein protective agent (histidine hydrochloride and arginine monohydrochloride)
Proportion are as follows: 1.2mg:45mg.
Two, sterilization by ionizing radiation
Each sample (blank sample, sample 1, sample 2, sample 3, sample 4, the sample 5, sample respectively prepared by step 1
Product 6 or sample 7) it proceeds as follows: the sample for taking step 1 to prepare carries out the γ that radionuclide cobalt -60 is radiation source and penetrates
Line sterilizes (dose of radiation 25kGy).
Three, protecting effect of the different function protein protective agent to rhBMP-2
Each sample that the blank sample of step 1 preparation and step 2 obtain is proceeded as follows (in order to state respectively
It is convenient, sample prepared by step 1 is known as sample before irradiation sterilization, each sample that step 2 is obtained is known as irradiation sterilization
Sample afterwards):
1, the rhBMP-2 dimer purity in each sample is detected using high performance liquid chromatography
The design parameter of high performance liquid chromatography: the model DIONEX U-3000 of high performance liquid chromatograph；It is arranged using volume
It hinders chromatographic column (Sai Fen Science and Technology Ltd., SRT SEC-300,7.8 × 300mm, 5 μm)；Flowing phase composition is 0.1% trifluoro second
The acetonitrile solution of acid: aqueous solution=300:700 (volume ratio) of 0.1% trifluoroacetic acid, flow velocity 0.5mL/min.Detection wavelength
For 280nm.RhBMP-2 dimer retention time is 19.2-19.5min.
It is calculated according to area normalization method, total peak area ratio, as sample shared by the peak area of rhBMP-2 dimer
The purity of rhBMP-2 dimer.Experimental result is shown in Table 1.
The purity of each sample before and after 1. irradiation sterilization of table
| Sample | Dimer purity (%) |
| Blank sample before irradiation sterilization | 98.64 |
| Blank sample after irradiation sterilization | 31.05 |
| Sample 1 after irradiation sterilization | 95.84 |
| Sample 2 after irradiation sterilization | 96.05 |
| Sample 3 after irradiation sterilization | 92.31 |
| Sample 4 after irradiation sterilization | 93.01 |
| Sample 5 after irradiation sterilization | 85.37 |
| Sample 6 after irradiation sterilization | 49.08 |
| Sample 7 after irradiation sterilization | 51.90 |
The result shows that the purity of rhBMP-2 dimer is 31.05% (Fig. 2) in blank sample after irradiation sterilization, irradiation is gone out
The purity of rhBMP-2 dimer is 95.84% (Fig. 3) in sample 1 after bacterium, rhBMP-2 dimer in sample 2 after irradiation sterilization
Purity is 96.05% (Fig. 4), and the purity of rhBMP-2 dimer is 92.31% (Fig. 5) in sample 3 after irradiation sterilization, and irradiation is gone out
The purity of rhBMP-2 dimer is 93.01% (Fig. 6) in sample 4 after bacterium, rhBMP-2 dimer in sample 5 after irradiation sterilization
Purity is 85.37% (Fig. 7), and the purity of rhBMP-2 dimer is 49.08% (Fig. 8) in sample 6 after irradiation sterilization, and irradiation is gone out
The purity of rhBMP-2 dimer is 51.90% (Fig. 9) in sample 6 after bacterium.
2、C2C12Cell method test sample bioactivity
C2C12Cell is divided into osteoblast under the action of bone morphogenetic protein (BMP-2).And alkaline phosphatase
(ALP) activity is the function and differentiation degree index of osteoblast, therefore can be with by the content of detection detection of alkaline phosphatase
Reflect C2C12The bioactivity for the BMP-2 being added in cell cultivation process.
The method of the retentive activity percentage of sample is specific as follows after detection sterilizing:
1) solution prepares
A, serum: fetal calf serum or newborn bovine serum.
B, complete medium: the DMEM high glucose medium containing 10% serum.
C, basal medium: the DMEM high glucose medium containing 2% serum.
D, digestive juice: accurately weighing pancreatin 0.05g, and EDTA 0.02g adds PBS solution 100ml, stirs and evenly mixs, and solution is adopted
With syringe needle filter (aperture≤0.22 μm) through aseptic filtration method degerming.Packing is managed to EP, is saved backup in -20 DEG C.
E, cell pyrolysis liquid: the glycine-NaOH buffer containing 1%NP40.
F, developing solution: the glycine-NaOH buffer of the pNPP containing 4mg/ml.
G, terminate liquid: 0.2M NaOH aqueous solution.
2) preparation of samples
Sample to be tested is standard items (sample before irradiation sterilization) or test sample (sample after irradiation sterilization).
3) cell culture
With complete medium culture C2C12The degrees of fusion of cell to 70~80%, then by C2C12Cell dissociation passage, connects
(every hole is inoculated with 5000-10000 C to kind in 96 orifice plates2C12Cell) and cultivate for 24 hours.Condition of culture is 5%CO2、37℃。
4) rhBMP-2 acts on cell
After completing step 3), 96 orifice plate is taken, discards culture medium, basis training of the 200 μ l containing sample to be tested is added in every hole
Base is supported, continues to cultivate 72h.Condition of culture is 5%CO2、37℃。
5) alkaline phosphatase assay
After completing step 4), 96 orifice plate is taken, discards culture medium, the PBS buffer solution of 200 μ l is added (by NaCl in every hole
8.0g, KCl 0.2g, Na2HPO41.44g and KH2PO40.2g is dissolved in 900ml water for injection, adjusts pH to 7.4, is settled to
1000mL, sterilizing) twice of washing, blot raffinate；Then 100 μ l cell pyrolysis liquids are added in every hole, and room temperature acts on 1h；Last every hole
It is added 50 μ l developing solutions, after 37 DEG C of effect 40-60min, is terminated and reacted with 50 μ l terminate liquids.Measure the absorbance value of reaction solution
(detection spectrum is 405nm, reference spectrum 465nm, absorbance value=405nm absorbance value -465nm absorbance value).
6) sample retentive activity percentage after sterilizing
The experimental data of step 5) is handled using computer program or four parametric regression calculating methods, and is calculated as follows
As a result:
Ds is test sample pre-dilution multiple；
Dr is standard items pre-dilution multiple；
Es is the extension rate that test article is equivalent to standard items median effective dose；
Er is the extension rate of standard items median effective dose.
Experimental result is shown in Table 2.The result shows that functional protein protective agent provided by the invention has protection sterilization by ionizing radiation
The active effect of rhBMP-2 in the process.
Each sample retentive activity percentage after 2. irradiation sterilization of table
| Sample | Sample retentive activity percentage (%) after irradiation sterilization |
| Blank sample | 30 |
| Sample 1 | 92 |
| Sample 2 | 90 |
| Sample 3 | 95 |
| Sample 4 | 93 |
| Sample 5 | 94 |
| Sample 6 | 53 |
| Sample 7 | 58 |
Claims (19)
1. a kind of side that the composite material to functional protein or containing the functional protein before sterilization by ionizing radiation is protected
Method includes the following steps: in liquid-phase system, will described in " functional protein or the composite material containing the functional protein " and group
Divide I and compositionⅱ mixing；The components I is carbohydrate；The compositionⅱ is amino acid and/or amino acid hydrochloride salt；
The addition form of the carbohydrate is sucrose or trehalose；
The addition form of the amino acid is methionine and/or tryptophan；
The addition form of the amino acid hydrochloride salt is arginine monohydrochloride, cysteine hydrochloride and/or histidine hydrochloride；
The functional protein is bone morphogenetic protein.
2. the method as described in claim 1, it is characterised in that: the bone morphogenetic protein is human bone morphogenesis protein.
3. method according to claim 2, it is characterised in that: the human bone morphogenesis protein is human bone morphogenetic protein
White -2.
4. method a method according to any one of claims 1-3, it is characterised in that: in the liquid-phase system, the components I and described group
The quality proportioning for dividing II is 30:0.1-1800.
5. method as claimed in claim 4, it is characterised in that: in the liquid-phase system, the components I and the compositionⅱ
Quality proportioning is 30:16-90.
6. method as claimed in claim 5, it is characterised in that: in the liquid-phase system, the components I and the compositionⅱ
Quality proportioning is following (a1), (a2), (a3) or (a4):
(a1) 30:16；
(a2) 30:70；
(a3) 30:17.5；
(a4) 30:90.
7. method a method according to any one of claims 1-3, it is characterised in that: the functional protein, described in the liquid-phase system
The quality proportioning of components I and the compositionⅱ is 1.2:15-30:0.1-1800.
8. the method for claim 7, it is characterised in that: in the liquid-phase system, the functional protein, the components I
Quality proportioning with the compositionⅱ is 1.2:15-30:16-45.
9. method according to claim 8, it is characterised in that: in the liquid-phase system, the functional protein, the components I
Quality proportioning with the compositionⅱ is following (b1), (b2), (b3) or (b4):
(b1) 1.2:30:16；
(b2) 1.2:15:35；
(b3) 1.2:30:17.5；
(b4) 1.2:15:45.
10. method a method according to any one of claims 1-3, it is characterised in that: in the liquid-phase system, the concentration of the components I
For 0.1mg/mL-30mg/mL；The concentration of the compositionⅱ is 0.1mg/mL-45mg/mL.
11. according to the method described in claim 10, it is characterized by: in the liquid-phase system, the concentration of the components I is
15mg/mL-30mg/mL；The concentration of the compositionⅱ is 16mg/mL-45mg/mL.
12. method a method according to any one of claims 1-3, it is characterised in that: the method is following (c1), (c2), (c3),
(c4) or (c5):
(c1) in the method, the components I is sucrose, and the compositionⅱ is arginine monohydrochloride and cysteine hydrochloride；
The concentration of sucrose is 30mg/mL；The concentration of arginine monohydrochloride is 15mg/mL；The concentration of cysteine hydrochloride is 1mg/mL；
(c2) in the method, the components I is trehalose, and the compositionⅱ is histidine hydrochloride and cysteine hydrochloric acid
Salt；The concentration of trehalose is 30mg/mL；The concentration of histidine hydrochloride is 15mg/mL；The concentration of cysteine hydrochloride is
1mg/mL；
(c3) in the method, the components I is sucrose, and the compositionⅱ is histidine hydrochloride and methionine；Sucrose it is dense
Degree is 15mg/mL；The concentration of histidine hydrochloride is 30mg/mL；The concentration of methionine is 5mg/mL；
(c4) in the method, the components I is trehalose, and the compositionⅱ is arginine monohydrochloride and tryptophan；Trehalose
Concentration be 30mg/mL；The concentration of arginine monohydrochloride is 15mg/mL；The concentration of tryptophan is 2.5mg/mL；
(c5) in the method, the components I is sucrose, and the compositionⅱ is histidine hydrochloride and arginine monohydrochloride；Sugarcane
The concentration of sugar is 15mg/mL；The concentration of histidine hydrochloride is 15mg/mL；The concentration of arginine monohydrochloride is 30mg/mL.
13. method a method according to any one of claims 1-3, it is characterised in that: the method is following (c6), (c7), (c8),
(c9) or (c10):
(c6) in the method, the components I is sucrose, and the compositionⅱ is arginine monohydrochloride and cysteine hydrochloride；
The concentration of functional protein is 1.2mg/mL, and the concentration of sucrose is 30mg/mL；The concentration of arginine monohydrochloride is 15mg/mL；Half Guang
The concentration of propylhomoserin hydrochloride is 1mg/mL；
(c7) in the method, the components I is trehalose, and the compositionⅱ is histidine hydrochloride and cysteine hydrochloric acid
Salt；The concentration of functional protein is 1.2mg/mL, and the concentration of trehalose is 30mg/mL；The concentration of histidine hydrochloride is 15mg/
mL；The concentration of cysteine hydrochloride is 1mg/mL；
(c8) in the method, the components I is sucrose, and the compositionⅱ is histidine hydrochloride and methionine；Functional protein
Concentration be 1.2mg/mL, the concentration of sucrose is 15mg/mL；The concentration of histidine hydrochloride is 30mg/mL；The concentration of methionine
For 5mg/mL；
(c9) in the method, the components I is trehalose, and the compositionⅱ is arginine monohydrochloride and tryptophan；Function egg
White concentration is 1.2mg/mL, and the concentration of trehalose is 30mg/mL；The concentration of arginine monohydrochloride is 15mg/mL；Tryptophan
Concentration is 2.5mg/mL；
(c10) in the method, the components I is sucrose, and the compositionⅱ is histidine hydrochloride and arginine monohydrochloride；Function
The concentration of energy albumen is 1.2mg/mL, and the concentration of sucrose is 15mg/mL；The concentration of histidine hydrochloride is 15mg/mL；Arginine
The concentration of hydrochloride is 30mg/mL.
14. the method as described in claims 1 to 3 is any, it is characterised in that: described to go out in sterilization by ionizing radiation for gamma-rays
Bacterium.
15. method as claimed in claim 14, it is characterised in that: the γ ray sterilization is to be with radionuclide cobalt -60
The γ ray sterilization of radiation source.
16. method as claimed in claim 15, it is characterised in that: the dose of radiation of the γ ray sterilization is 15kGy-
35kGy。
17. the method described in claim 16, it is characterised in that: the dose of radiation of the γ ray sterilization is 25kGy.
18. the method that a kind of pair of functional protein or the composite material containing the functional protein carry out sterilization by ionizing radiation, including
Following steps:
(1) using the method any in claim 1 to 17, " functional protein is answered containing the functional protein to described
Condensation material " carries out pre-treatment；
(2) it is freeze-dried；
(3) sterilization by ionizing radiation.
19. a kind of product protected before sterilization by ionizing radiation to functional protein, including in claim 1-4 it is any described
Components I and compositionⅱ.
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| CN1427729A (en) * | 2000-03-23 | 2003-07-02 | 科利昂特有限公司 | Method for sterilizing biological materials |
| CN1606457A (en) * | 2001-08-31 | 2005-04-13 | 克里兰特公司 | Methods for sterilizing preparations containing albumin |
| CN102449162A (en) * | 2009-03-31 | 2012-05-09 | 白血球保健股份有限公司 | Means and methods of sterilization of biofunctional compositions |
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| CN1427729A (en) * | 2000-03-23 | 2003-07-02 | 科利昂特有限公司 | Method for sterilizing biological materials |
| CN1606457A (en) * | 2001-08-31 | 2005-04-13 | 克里兰特公司 | Methods for sterilizing preparations containing albumin |
| CN102449162A (en) * | 2009-03-31 | 2012-05-09 | 白血球保健股份有限公司 | Means and methods of sterilization of biofunctional compositions |
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