WO2018232745A1 - 生产可溶性重组人碱性成纤维细胞生长因子(rh-bFGF)的方法 - Google Patents
生产可溶性重组人碱性成纤维细胞生长因子(rh-bFGF)的方法 Download PDFInfo
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- WO2018232745A1 WO2018232745A1 PCT/CN2017/089810 CN2017089810W WO2018232745A1 WO 2018232745 A1 WO2018232745 A1 WO 2018232745A1 CN 2017089810 W CN2017089810 W CN 2017089810W WO 2018232745 A1 WO2018232745 A1 WO 2018232745A1
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P21/00—Preparation of peptides or proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to the field of DNA recombination and biopharmaceuticals. More specifically, the present invention relates to a method for producing soluble recombinant human-basic fibroblast growth factor (rh-bFGF), recombinant human basic fibroblast growth factor obtained by the method (rh-bFGF), and a mutant nucleic acid molecule encoding the recombinant human basic fibroblast growth factor (rh-bFGF).
- Fibroblast growth factor is a kind of heparin-binding protein with similar structure and similar biological functions. At present, 23 kinds of FGF have been found, and the research of FGF-1 (aFGF), FGF-2 (bFGF) and FGF-7 (KGF) is more in-depth.
- bFGF is a very small amount of active substances in mammals and humans, and is highly valued for its wide physiological functions and important clinical application value. It not only stimulates the formation of new blood vessels, but also participates in wound healing and tissue regeneration, and promotes the development and differentiation of embryonic tissues. In recent years, recombinant bFGF has been used in the clinical treatment of diseases such as trauma, ulcers and nervous system.
- bFGF has two pairs of cysteines, which easily form intermolecular disulfide bonds. If stored improperly, dimers and trimers will account for a certain proportion, which will affect the purity of the protein.
- the invention provides a method of producing soluble recombinant human basic fibroblast growth factor (rh-bFGF) comprising: culturing a host cell comprising a mutated nucleic acid molecule; in conditions suitable for expression of rh-bFGF Expression of rh-bFGF in host cells; The rh-bFGF was recovered by purification.
- rh-bFGF human basic fibroblast growth factor
- the invention provides recombinant human basic fibroblast growth factor (rh-bFGF) obtained by the method.
- rh-bFGF human basic fibroblast growth factor
- the invention provides a mutant nucleic acid molecule encoding the recombinant human basic fibroblast growth factor (rh-bFGF).
- the invention provides a pharmaceutical composition comprising the recombinant human basic fibroblast growth (rh-bFGF) and at least one pharmaceutically acceptable excipient.
- rh-bFGF recombinant human basic fibroblast growth
- the invention provides a method of treating dry eye, the method comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition.
- Figure 1 is a diagram showing the structure of the pET-30a(+) plasmid.
- Figure 2 shows the results of Western Blot of rh-bFGF.
- Figure 3 shows the purity of rh-bFGF detected by high performance liquid chromatography.
- Figure 4 shows the molecular mass spectrum of the rh-bFGF intact protein.
- Figure 5 shows the cDNA sequence, amino acid sequence of the native human bFGF and the mutated human bFGF amino acid sequence.
- Figure 6 shows a four-parameter fit curve for rh-bFGF in vitro activity assay, where C is the EC50 value.
- Figure 7 shows the effect of different amino acids, including histidine, glycine and arginine, and Tween on the purity of rh-bFGF under hot pressure conditions, 95% of which refers to the purity standard of rh-bFGF stock solution; A purity of less than 95% means that the purity is unacceptable.
- Figure 8 shows the effect of HSA on the purity change of rh-bFGF under hot pressure conditions.
- Figure 9 shows the effect of different concentrations of rh-bFGF eye drops on tear secretion in an alkali burned New Zealand rabbit dry eye model.
- the symbol "*” or "**” indicates a significant difference (p ⁇ 0.05 or p ⁇ 0.01).
- Figure 10 shows the effect of different concentrations of rh-bFGF eye drops on tear film rupture time in an alkali burned New Zealand rabbit dry eye model.
- the symbol "*" or "**” indicates a significant difference (p ⁇ 0.05 or p ⁇ 0.01).
- the inventors of the present application have developed a novel method for producing soluble recombinant human basic fibroblast growth factor (rh-bFGF) by mutating nucleic acid molecules.
- rh-bFGF human basic fibroblast growth factor
- the present invention unexpectedly achieves efficient and uniform expression of bFGF in soluble form and high expression levels. This method enhances the recovery of recombinant human bFGF protein by highly expressing human bFGF, and retains the biological activity of native human bFGF.
- the invention provides a method of producing soluble recombinant human basic fibroblast growth factor (rh-bFGF) comprising: culturing a host cell comprising a mutated nucleic acid molecule; in conditions suitable for expression of rh-bFGF The rh-bFGF was expressed in a host cell; rh-bFGF was recovered by purification.
- rh-bFGF human basic fibroblast growth factor
- the mutated nucleic acid molecule comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or comprises SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 has a sequence of at least 90%, at least 95% identity, such as a sequence of at least 96%, 97%, 98 or 99% identity.
- sequence of the mutated nucleic acid molecule is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
- the invention provides a vector comprising the nucleic acid molecule.
- Preferred vectors are prokaryotic expression vectors, particularly expression vectors suitable for inducing expression in E. coli, including but not limited to, for example, pET-30a(+), pET-28a(+), pET-23c(+), pET- 15b. Particularly preferred is pET-30a(+).
- the invention provides a host cell comprising the expression vector.
- a preferred host cell is Escherichia coli, and E. coli BL21 (DE3) is particularly preferred.
- the prokaryotic expression vector containing the mutated nucleic acid molecule is transformed into E. coli BL21 (DE3), and the expression is induced to be soluble by adjusting the cell culture temperature, the induction temperature, the pH range, the glucose concentration and the inducer concentration.
- Human bFGF Human bFGF.
- the cells are collected by hollow fiber washing and filtering; the fermenting cells are crushed by high-pressure homogenization and kept at a low temperature; after adding an appropriate amount of nonionic surfactant, the supernatant is collected by low-temperature high-speed centrifugation, and the precipitate is discarded.
- the specific parameters used to induce expression of soluble human bFGF are as follows:
- OD600 before canning about 0.5-4.5
- Bacterial culture temperature about 25-45 ° C
- Bacterial induction temperature about 20-50 ° C
- Glucose concentration about 0.06-0.46%
- Inducer concentration about 0.0125-0.0925g / L
- Induction time about 2-9h.
- purification is carried out as follows:
- the fermentation broth was intercepted with hollow fibers and washed, and the washing buffer was a pH of about 7.0, and a PB solution containing NaCl.
- the high-pressure homogenate was used to maintain the low-temperature crushing of the fermenting cells, and an appropriate amount of Triton was added, and the supernatant was collected by low-temperature high-speed centrifugation, and the precipitate was discarded.
- the invention provides recombinant human basic fibroblast growth factor (rh-bFGF) obtained by the method.
- rh-bFGF recombinant human basic fibroblast growth factor
- the amino acid sequence of the recombinant human basic fibroblast growth factor is SEQ ID NO:5.
- the invention provides a mutant nucleic acid molecule encoding recombinant human basic fibroblast growth factor comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or comprises A sequence having at least 95%, 96%, 97%, 98 or 99% identity to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
- sequence of the mutant nucleic acid molecule is SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
- the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising the recombinant human basic fibroblast growth (rh-bFGF) and at least one pharmaceutically acceptable excipient.
- the excipients include, but are not limited to, buffer systems, thickeners, stabilizers, neutralizing agents, humectants, and the like.
- the present invention provides a pharmaceutical composition
- a pharmaceutical composition comprising the recombinant human basic fibroblast growth (rh-bFGF) and at least one selected from the group consisting of glycine, histidine, arginine, heparin sodium or human A stabilizer for blood albumin (HSA).
- rh-bFGF recombinant human basic fibroblast growth
- HSA human A stabilizer for blood albumin
- the composition is buffered to a pH of between about 6.0 and 8.0, such as 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6. a pH of 7.7, 7.8, 7.9 or 8.0 or any range defined between them.
- the invention provides a method of treating dry eye, the method comprising administering to a patient in need thereof a therapeutically effective amount of the pharmaceutical composition.
- the invention provides the pharmaceutical composition for treating dry eye.
- the invention also provides the use of the composition in the manufacture of a medicament for the treatment of dry eye.
- the pharmaceutical composition of the invention is an ophthalmic pharmaceutical composition.
- the pharmaceutical composition of the invention is in the form of an eye drop or a gel.
- An advantage of the method of the invention is that efficient and uniform expression of bFGF is achieved in soluble form and high expression levels.
- the method enhances the recovery of recombinant human bFGF protein by highly expressing human bFGF, and retains the biological activity of native human bFGF.
- the cDNA sequence encoding the native human bFGF (SEQ ID NO: 4) was mutated without changing the amino acid sequence of the protein, and the three mutated cDNA sequences were designed and synthesized (SEQ ID NO: 1, SEQ ID, respectively).
- the expression vector used was pET-30a (+) (see Figure 1), purchased from Merk KgsA (Cat. No. 69909-3), which carries the T7 promoter, T7 transcription initiation site, His Tag, Coding sequence, S Tag coding sequence, multiple cloning sites (MCS), T7 terminator, lactose coding sequence, kan resistance coding sequence and pBR322 replicon and f1 replicon.
- the MCS comprises the cleavage sites Xho I, Not I, Eag I, HindIII, Sal I, Sac I, EcoR I, BamH I, EcoR V, Nco I, Kpn I, Bgl II, Nsp V, Nde I.
- a Nde I restriction site was designed upstream of the start codon of SEQ ID NO: 1 and a HindIII cleavage site was designed near the terminator.
- the 480bps sequence was completely synthesized by chemical method, and the sequence was digested with Nde I and HindIII and inserted into the same double-digested expression vector pET-30a(+) to obtain 5724bps.
- the recombinant plasmid was heat-transformed into DH5 ⁇ (TaKaRa, 9057) and cultured in LB medium containing 50 ⁇ g/mL kanamycin. Monoclones were selected and the correct transformants were screened by Nde I and HindIII double digestion. The correctness of the transformants was verified again by sequencing.
- pET-28a (+), pET-23c (+) or pET-15b or the like can be used in place of the above pET-30a (+) vector.
- the prokaryotic expression vector containing the sequence 1 was transformed into E. coli BL21 (DE3), and the soluble human bFGF was induced to be expressed by culture, and the specific parameters were as follows:
- Bacterial culture temperature 25-45 ° C
- the cells were collected by hollow fiber washing: the fermentation broth was intercepted with 500 KD hollow fibers, and washed, and the washing buffer was pH 7.0, and a 25 mM PB solution containing NaCl.
- the high-pressure homogenate was used to maintain the low-temperature crushing of the fermenting cells, and an appropriate amount of Triton was added, and the supernatant was collected by low-temperature high-speed centrifugation, and the precipitate was discarded.
- CM-Sepharose Fast Flow ion exchange column was first equilibrated with 25 mM phosphate buffer (pH 7.0) containing sodium chloride, and then the supernatant was passed through the column to 25 mM containing 0.05 M, 0.12 M, 0.4 M sodium chloride. Phosphate buffer (pH 7.0) was subjected to gradient (three-step) elution, and a third elution peak (eluting with 25 mM phosphate buffer containing 0.4 M sodium chloride) was collected.
- Heparin affinity chromatography was used to add an appropriate amount of DTT during the purification.
- the Heparin-Sepharose CL-6B affinity chromatography column was pre-equilibrated with 25 mM phosphate buffer (pH 7.0) containing sodium chloride, and the third elution peak solution of the ion exchange chromatography was passed through the column, containing 0.4 M, Gradient (three steps) of 1.0 M, 1.8 M sodium chloride in 25 mM phosphate buffer (pH 7.0) Elution was performed and a third eluting peak (eluting with 25 mM phosphate buffer containing 1.8 M sodium chloride) was collected.
- the recombinantly prepared human bFGF protein was analyzed by amino acid sequence, and it was confirmed that the amino acid sequence thereof was identical to the amino acid sequence of native human bFGF (see Fig. 5).
- the buffer system may be the above sodium dihydrogen phosphate and disodium hydrogen phosphate, or a boric acid-borax buffer system, a citric acid-disodium hydrogen phosphate buffer system or the like.
- the pharmaceutical composition has a pH of from 6.5 to 7.5.
- the thickener is polyvinyl alcohol, sodium hyaluronate, hypromellose, poloxamer, etc., preferably polyvinyl alcohol.
- step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting with sterile water for injection, that is, obtaining;
- the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
- the carbomer is a series of carbomer 940, carbomer 934, carbomer 974, carbomer 980, etc., preferably carbomer 980 series;
- the neutralizing reagent is sodium hydroxide, potassium hydroxide, potassium hydrogencarbonate, borax and triethanolamine, preferably triethanolamine.
- step (3) Take appropriate amount of room temperature water for injection, add glycerin, human serum albumin, heparin sodium, recombinant human basic fibroblast growth factor, stir evenly, pass the 0.22 ⁇ m filter under sterile conditions, and step (2) The medium gel matrix is mixed, then quantified and stirred evenly.
- Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
- the buffer salt system may be the above sodium dihydrogen phosphate and disodium hydrogen phosphate, or
- the boronic acid-borax buffer system, the citric acid-disodium hydrogen phosphate buffer system, etc. preferably have a pH of 6.5-7.5.
- the thickener is polyvinyl alcohol, sodium hyaluronate, hypromellose, poloxamer, etc., preferably polyvinyl alcohol.
- the humectant is glycerin, propylene glycol or a mixture thereof.
- step (2) solution is filtered through a 0.22 ⁇ m microporous membrane and mixed with the solution obtained in the step (1), and made up to 1 mL with water for injection;
- the volume of the container is in the range of 0.4 g/piece, that is, the finished product is obtained.
- compositions were also prepared (see Table 2). Among them, the preparation of the eye drops was 100 ml, and the preparation of the external gel was 100 g.
- step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
- the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
- step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
- the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
- step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
- the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
- step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
- the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
- step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
- the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
- step (3) mixing the solution obtained in the step (1) and the step (2) uniformly under aseptic conditions, and diluting to 100 mL with sterile water for injection;
- the aseptic solution is filled by the three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4mL, that is, the finished product is obtained.
- step (3) Take appropriate amount of room temperature water for injection, add 2.50g glycerol, 30.0mg human albumin, 7.0mg heparin sodium, recombinant human basic fibroblast growth factor 450,000IU, stir evenly, and pass 0.22 ⁇ m filtration under sterile conditions.
- the membrane is mixed with the gel matrix in step (2), quantified, and stirred uniformly.
- Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
- step (3) Take appropriate amount of room temperature water for injection, add 2.50g glycerin, 30.0mg human albumin, 7.0mg heparin sodium, recombinant human basic fibroblast growth factor 420000IU, stir evenly, and pass 0.22 ⁇ m filtration under sterile conditions.
- the membrane is mixed with the gel matrix in step (2), quantified, and stirred uniformly.
- Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
- step (3) Take appropriate amount of room temperature water for injection, add 2.50g glycerol, 30.0mg human albumin, 7.0mg heparin sodium, recombinant human basic fibroblast growth factor 450,000IU, stir evenly, and pass 0.22 ⁇ m filtration under sterile conditions.
- the membrane is mixed with the gel matrix in step (2), quantified, and stirred uniformly.
- Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
- Aseptic gel is filled by using a three-in-one filling machine of blowing, filling and sealing, and the filling amount is 0.4g, that is, the finished product is obtained.
- rh-bFGF stock solution itself is unstable in nature, polymerization precipitation tends to occur at room temperature. Therefore, different stabilizers are used to initially screen the stability of the stock solution. 5% and 2% mannitol, 5% and 2% glycine, and 2% dextran were used and allowed to stand at 25 ° C for 17 days. Protein concentrations were measured on days 0, 7, and 17, respectively, and the results are shown in Table 3.
- the stability of the stock solution was further selected by using different amino acids (glycine, histidine, arginine) and Tween 20, and placed at 25 ° C for 18 h. The results are shown in Fig. 7.
- New Zealand rabbit dry eye model was selected to observe the clinical indicators of the model (tea secretion, tear film rupture time), and to evaluate the clinical effect of dry eye treatment.
- New Zealand rabbits were divided into negative control group (Negative control, untreated), model control group (Model, treated with alkali burn but no eye drops were added), sodium hyaluronate eye drops treatment group (HA group), and The group D (4.0 ⁇ g/mL), E (8.0 ⁇ g/mL), F (16.0 ⁇ g/mL), and G (32.0 ⁇ g/mL) which were used to prepare the rh-bFGF eye drop concentration of Example 3 were used.
- the negative control group consisted of 72 rats in each group, and the remaining groups were 8 rats in each group at a dose of 300 ⁇ l/eye/day.
- the secretion of tears from New Zealand rabbits was measured using the Schirmer I test. That is, the phenol red cotton thread was clamped with ophthalmology and placed in the outer crotch of the New Zealand rabbit. After 60 s, the wet length of the phenol red cotton thread was taken out. The phenol red cotton thread turns red after being wet, and the eye wetness is determined according to the wet length.
- the experimental results are shown in Figure 9.
- the wet length of the phenol red cotton thread of the model control group was significantly decreased as compared with the negative control eye.
- the wet length of the phenol red cotton thread in the rh-bFGF eye drops D, E, F, and G groups was significantly longer, with statistically significant differences.
- the tear burst time of the model versus group was significantly shorter than the negative control eyes on day 10 of administration compared to the negative control eyes.
- the tear film rupture time of the rh-bFGF eye drops group D, E, F, and G groups was significantly prolonged on the 10th day after administration, with significant statistical difference.
- the phenol red cotton tear secretion test results show that the eye drops of the present invention can improve the tear secretion of the model animals (see Figure 9); the tear film break time test shows that the tear film rupture time of the dry eye model is obvious Improve the effect (see Figure 10).
- the increase of dose rh-bFGF eye drops have a significant improvement on the dry burn model of Alkali-burned New Zealand rabbits.
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Abstract
Description
样品 | 0天 | 7天 | 17天 |
无稳定剂 | 100.00% | 29.96% | 13.63% |
5%甘露醇 | 100.00% | 49.80% | 17.31% |
2%甘露醇 | 100.00% | 53.29% | 15.66% |
5%甘氨酸 | 100.00% | 95.67% | 86.26% |
2%甘氨酸 | 100.00% | 94.74% | 82.73% |
2%葡聚糖 | 100.00% | 51.65% | 16.20% |
Claims (11)
- 一种生产可溶性的重组人碱性成纤维细胞生长因子(rh-bFGF)的方法,包括:培养包含突变的核酸分子的宿主细胞;在适合于rh-bFGF表达的条件下在宿主细胞中表达rh-bFGF;通过纯化回收rh-bFGF。
- 权利要求1的方法,其中所述突变的核酸分子包含选自SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3的序列,或包含与SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3具有至少95%、96%、97%、98或99%同一性的序列。
- 权利要求1或2的方法,其中所述突变的核酸分子的序列为SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3。
- 权利要求1-3中任一项的方法,其中所述宿主细胞为大肠杆菌。
- 通过权利要求1-4中任一项的方法获得的重组人碱性成纤维细胞生长因子(rh-bFGF)。
- 一种编码重组人碱性成纤维细胞生长因子的突变核酸分子,其包含选自SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3的序列,或包含与SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3具有至少95%、96%、97%、98或99%同一性的序列。
- 权利要求6的突变核酸分子,其序列为SEQ ID NO:1、SEQ ID NO:2或SEQ ID NO:3。
- 包含权利要求6或7的突变核酸分子的载体。
- 权利要求8的载体,其中所述载体为pET-30a(+)。
- 一种药物组合物,其包含权利要求5的人碱性成纤维细胞生长因子和至少一种药学上可接受的赋形剂。
- 一种治疗干眼症的方法,所述方法包括向有需要的患者施用治疗有效量的权利要求10的药物组合物。
Priority Applications (7)
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US16/625,191 US11248032B2 (en) | 2017-06-23 | 2017-06-23 | Method for producing soluble recombinant human-basic fibroblast growth factor (rh-bFGF) |
CN201780092424.7A CN110945123B (zh) | 2017-06-23 | 2017-06-23 | 生产可溶性重组人碱性成纤维细胞生长因子(rh-bFGF)的方法 |
JP2020520690A JP7098724B2 (ja) | 2017-06-23 | 2017-06-23 | 可溶性組み換えヒト塩基性線維芽細胞増殖因子(rh-bFGF)を生産する方法 |
PCT/CN2017/089810 WO2018232745A1 (zh) | 2017-06-23 | 2017-06-23 | 生产可溶性重组人碱性成纤维细胞生长因子(rh-bFGF)的方法 |
SG11201912790WA SG11201912790WA (en) | 2017-06-23 | 2017-06-23 | Method for producing soluble recombinant human-basic fibroblast growth factor (rh-bfgf) |
KR1020207002244A KR102428209B1 (ko) | 2017-06-23 | 2017-06-23 | 가용성 재조합 인간-염기성 섬유아세포 성장 인자 (rh-bFGF)를 제조하는 방법 |
EP17915171.7A EP3643781A4 (en) | 2017-06-23 | 2017-06-23 | METHOD FOR PRODUCING A SOLUBLE RECOMBINANT HUMAN BASIC FIBROBLAST GROWTH FACTOR (RH-BFGF) |
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CN115975002A (zh) * | 2022-08-16 | 2023-04-18 | 广东普罗凯融生物医药科技有限公司 | 一种重组人碱性成纤维细胞生长因子及其制备方法与应用 |
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US20200140509A1 (en) | 2020-05-07 |
EP3643781A1 (en) | 2020-04-29 |
SG11201912790WA (en) | 2020-01-30 |
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US11248032B2 (en) | 2022-02-15 |
CN110945123B (zh) | 2023-06-23 |
JP2020533014A (ja) | 2020-11-19 |
JP7098724B2 (ja) | 2022-07-11 |
CN110945123A (zh) | 2020-03-31 |
KR102428209B1 (ko) | 2022-08-02 |
KR20200020900A (ko) | 2020-02-26 |
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