A method for large scale preparation of basic fibroblast growth factor from human placenta

Abstract

This invention provides a method for large-scale preparation of basic fibroblast growth factor (bFGF) from human placenta, comprising the following steps: step (1). washing; step (2). homogenization; step (3). acid extraction; step (4). centrifugation; step (5). clarification and filtration; step (6). concentration and buffer replacement; step (7). obtaining human placental basic fibroblast growth factor through cation exchange chromatography and heparin affinity chromatography. This invention can purify approximately 100 μg of high-purity human basic fibroblast growth factor from 1 kg of human placenta (Bradford method). The process has been verified through multiple experiments, showing good repeatability and stability; it improves the yield and reduces costs; and the use of low-speed centrifugation facilitates industrial production, effectively avoiding the disadvantages of the classic ammonium sulfate fractionation precipitation method, which is difficult to operate in industrial production and has significant environmental pollution caused by ammonium sulfate. It has excellent application prospects for large-scale industrial preparation.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical manufacturing technology, specifically a method for large-scale preparation of basic fibroblast growth factor from human placenta. Background Technology

[0002] In recent years, with the increasing application of fibroblast growth factor in pharmaceuticals, wound repair devices, and cosmetics, natural humanized fibroblast growth factor has attracted widespread attention due to its advantages such as low immunogenicity and high activity. Human basic fibroblast growth factor (hbFGF) is a high-purity, fully human hbFGF obtained from healthy human placenta through a combination of homogenization, acid extraction, centrifugation, clarification, filtration, ultrafiltration concentration with buffer replacement, ion exchange, and affinity chromatography. The advantages of hbFGF purified from human placenta are that the raw material is human waste tissue, free of foreign proteins, and has low immunogenicity to humans, while recombinant hbFGF is mostly bovine or partially humanized hbFGF expressed by bacteria. Furthermore, hbFGF extracted from human placenta possesses a natural structure, with its amino acid sequence being 100% derived from humans. Compared to recombinant hbFGF, it has not undergone gene modification, and its secondary and tertiary structures retain their natural spatial conformation. hbFGF is clinically used for the repair of fresh wounds after cosmetic surgery (eyebrow tattooing, lip tinting, rhinoplasty, eyebrow removal, eyeliner cutting); and for the rapid repair of fresh wounds such as burns, scratches, scalds, and falls.

[0003] Current methods for extracting bFGF from animal tissues mostly employ ammonium sulfate fractionation for pretreatment. However, this method uses high concentrations of ammonium sulfate, which can cause environmental pollution and is difficult to implement industrially. US Patent 4902782 uses ammonium sulfate fractionation and high-speed centrifugation to extract bFGF from bovine brain, but this is not easily scaled up industrially. The literature "Isolation and characterization of two different molecular forms of basic fibroblast growth factor extracted from human placental tissue" describes large-scale preparation of bFGF from placenta. This method involves pH adjustment to 3.2, acid extraction for 30 min, followed by precipitation with 15% ethanol and centrifugation. Subsequent preparation steps include concentration, cation exchange chromatography, and heparin affinity chromatography. Ultimately, 15 μg of bFGF can be extracted from 1 kg of placental tissue. The literature "Biochemical, and biological characterization of bFGF extracted from human placenta" reports that 10 μg of bFGF can be prepared from 1 kg of placenta. All these reported methods are difficult to scale up industrially and have low yields. Summary of the Invention

[0004] The technical problem solved by this invention is to provide a method for large-scale preparation of basic fibroblast growth factor from human placenta, so as to solve the problems mentioned in the background art.

[0005] The technical problem solved by this invention is achieved by the following technical solution: a method for large-scale preparation of basic fibroblast growth factor from human placenta, comprising the following steps:

[0006] Step (1). Cleaning: Select suitable placentas, thaw them, and clean them with saline solution;

[0007] Step (2). Homogenization: Add 0.15M ammonium sulfate at a W / V ratio of 1:5, homogenize for 1.5 min / time using a tissue homogenizer, and homogenize 3 times; then homogenize the homogenate again for 20 minutes using a pre-disperser.

[0008] Step (3). Acid extraction;

[0009] Step (4). Centrifugation: Centrifuge using a cloth bag centrifuge with a centrifugal force of 500g to 1000g to remove the residue from the homogenate extraction;

[0010] Step (5). Clarification and filtration: Dilute the liquid after centrifugation in the filter bag until the conductivity is below 10 mS / cm, add chitosan to 0.01% to 0.05%, adjust the pH to 6.5 to 7.5, let stand for 10 to 12 hours, siphon the supernatant, and further filter the supernatant with 0.45 μm hollow fiber tangential flow filter or direct current filter cartridge;

[0011] Step (6). Concentration and buffer replacement: The filtrate is concentrated 5 to 20 times by ultrafiltration using a 10KD ultrafiltration membrane, and washed with pH 6.0 100mM PB buffer to replace the buffer.

[0012] Step (7). The product is obtained by cation exchange chromatography and heparin affinity chromatography.

[0013] In step (3), acid extraction refers to adjusting the pH of the homogenate to 4.5 and stirring for 2 hours for extraction.

[0014] The cation exchange chromatography conditions in step (7) include: Equilibration Buffer: pH 6.0 100 mM PB, Elution Buffer I: pH 6.0 100 mM PB + 0.15 M NaCl, and Elution Buffer II: pH 6.0 100 mM PB + 0.6 M NaCl.

[0015] The heparin affinity chromatography conditions in step (7) include: Equilibration Buffer: pH 7.0-10mM Tris-HCl + 0.6M NaCl, Elution Buffer I: pH 7.0-10mM Tris-HCl + 1.1M NaCl, and Elution Buffer II: pH 7.0-10mM Tris-HCl + 2M NaCl.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention mainly innovates through steps (2), (4), (5), and (6), namely, by using a pre-dispersant to enhance the homogenization strength and improve the release of the target protein; by using low-speed centrifugation (such as a bag centrifuge) to achieve large-scale industrial centrifugation, which has the advantages of low cost, low heat generation, low energy consumption and low mechanical shear force; by combining the conductivity of the diluted feed liquid and chitosan clarification, a large number of impurities are removed by using chitosan clarification, which improves the yield of the target protein; the chitosan clarification method replaces the traditional high-concentration ammonium sulfate precipitation method to reduce the loss of the target protein; the filtration treatment after chitosan clarification is highly operable and can reduce the loss of the target protein more than the commonly used ammonium sulfate precipitation method; finally, the ultrafiltration concentration and buffer replacement are used to connect the chromatography process.

[0017] In summary, this invention can prepare approximately 100 μg of high-purity human basic fibroblast growth factor from 1 kg of human placenta (Bradford method), which is far higher than previously reported. Furthermore, the low-speed centrifugation and clarification process facilitates industrial production, reduces costs, and effectively avoids the disadvantages of the classic ammonium sulfate fractionation precipitation method, which is difficult to operate in industrial production and causes significant environmental pollution from high-concentration ammonium sulfate. It has excellent application prospects for large-scale industrial production. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the preparation process of human placental-derived bFGF according to the present invention.

[0019] Figure 2 This is an SDS-PAGE electrophoresis image of human placental bFGF from the present invention.

[0020] Figure 3 This is an isoelectric focusing (IEF) electrophoresis image of human placental bFGF from the present invention.

[0021] Figure 4 The cell proliferation curve of Bal b / c 3T3 cells with human placental-derived bFGF according to the present invention is shown.

[0022] Figure 5 This is a diagram showing the skin irritation test of albino rabbits with human placental-derived bFGF according to the present invention.

[0023] Figure 6 The images show before-and-after comparisons of the effects of bFGF on tear trough repair in volunteers using this invention. Detailed Implementation

[0024] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific illustrations.

[0025] I. Origin of Human Placenta

[0026] The raw material of this invention, human placenta, is collected from healthy human placentas for use in the production of biological products under strict aseptic conditions. To ensure its quality and safety, the collection includes not only the human placenta but also maternal plasma, placental plasma, a signed informed consent form from the mother, and a completed placental blood and placenta collection registration form. The collecting hospital is a Grade II Class A hospital or above, qualified for initial screening tests for HBV, HCV, TP, and HIV. In accordance with the requirements of the 2020 edition of the Chinese Pharmacopoeia and the "Quality Control Standards for Major Raw and Auxiliary Materials of Chinese Biological Products," the collection subjects of the raw material placentas undergo strict screening. Each placenta is tested for HBsAg, syphilis, HIV-1 / HIV-2 antibodies, and HCV antibodies using reagent kits approved by the national drug administration authority. Only placentas with all negative results are used.

[0027] Table 1 Experimental Instruments and Equipment

[0028]

[0029]

[0030] II. Preparation Method

[0031] like Figure 1 As shown, a method for large-scale preparation of basic fibroblast growth factor from human placenta includes the following steps:

[0032] Step (1). Cleaning: Select 20 placentas (10kg) that meet the requirements, thaw them, and clean them with physiological saline.

[0033] Step (2). Homogenize with 0.15M ammonium sulfate at a W / V ratio of 1:5, homogenize for 1.5 min / time, and repeat 3 times; then homogenize the homogenate again for 20 minutes using a pre-disperser.

[0034] Step (3). Acid extraction;

[0035] Step (4). Centrifugation: Centrifuge using a cloth bag centrifuge with a centrifugal force of 500g to 1000g to remove the residue from the homogenate extraction;

[0036] Step (5). Clarification and filtration: Dilute the liquid after centrifugation in the filter bag until the conductivity is below 10 mS / cm, add chitosan to 0.01% to 0.05%, adjust the pH to 6.5 to 7.5, let stand for 10 to 12 hours, siphon the supernatant, and further filter the supernatant with 0.45 μm hollow fiber tangential flow filter or direct current filter cartridge;

[0037] Step (6). Concentration and buffer replacement: The filtrate is concentrated 5 to 20 times by ultrafiltration using a 10KD ultrafiltration membrane, and washed with pH 6.0 100mM PB buffer to replace the buffer.

[0038] Step (7). The product was obtained by CMFF chromatography and heparin affinity chromatography.

[0039] In step (3), acid extraction refers to adjusting the pH of the homogenate to 4.5 and then stirring for 2 hours for extraction.

[0040] In step (7), the chromatographic medium used for cation exchange chromatography is CMFF, 500 ml, and the chromatographic conditions include equilibration buffer: pH 6.0 100 mM PB, elution buffer I: pH 6.0 100 mM PB + 0.15 M NaCl, and elution buffer II: pH 6.0 100 mM PB + 0.6 M NaCl.

[0041] In step (7), the heparin affinity chromatography conditions refer to the use of 50 ml of affinity resin (Pall) and the operation of an AKTAAVANT150 fully automated intelligent protein purification system. The chromatography conditions include: Equilibration Buffer: pH 7.0 10 mM Tris-HCl + 0.6 M NaCl, Elution Buffer I: pH 7.0 10 mM Tris-HCl + 1.1 M NaCl, and Elution Buffer II: pH 7.0 10 mM Tris-HCl + 2 M NaCl.

[0042] III. Experimental Results

[0043] The heparin target elution peak was collected, and the total protein concentration was determined (Bradford method). The protein passed sterility and endotoxin tests. Simultaneously, the molecular weight of the target protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the isoelectric point (pI) was determined by isoelectric focusing. Subsequently, cell proliferation experiments, animal experiments, and volunteer experience experiments were conducted. The results are as follows:

[0044] like Figure 2 As shown, the purified human placenta bFGF, analyzed by SDS-PAGE electrophoresis, had a molecular weight of 17.355 kD and a purity of 95% as shown by thin-layer chromatography.

[0045] like Figure 3 As shown, the isoelectric point (pI) of the bFGF sample purified from human placenta was determined by isoelectric focusing (IEF). The results showed that there was only one obvious basic band in the pI 9.3-10.0 region, with a pI of approximately 9.7.

[0046] like Figure 4 As shown, the biological activity of human placental bFGF was determined using a cell proliferation assay (BALB / c 3T3 cells). The experimental data were processed using a four-parameter regression method. The results showed that, at the same dilution factor, both bFGF purified from human placenta (dark color) and commercially available recombinant bFGF (light color) promoted fibroblast proliferation and growth, with consistent proliferation curves. Furthermore, the biological activity of human placental bFGF (dark color) was higher than that of the commercially available recombinant bFGF sample (light color). This was further confirmed by EDTA. 50 The estimated biological activity of human placental bFGF is greater than 10,000 U / ml, and the estimated specific activity is greater than 1×10⁻⁶. 6 U / mg.

[0047] Animal safety testing

[0048] (1) In the abnormal toxicity test in mice, according to the method in Appendix 1141 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, 0.5 ml of human placental-derived hbFGF sample was injected intraperitoneally into 5 mice. After 7 days of observation, no mice died and their weight increased normally. The test requirements were met.

[0049] (2) In the guinea pig adverse toxicity test, according to Appendix 1141 of the 2020 edition of the Chinese Pharmacopoeia, two guinea pigs were each injected with 5.0 ml of hbFGF sample. Seven days later, there were no deaths, no abnormal reactions, and the weight gain was normal. The test met the requirements.

[0050] (3) Intradermal reaction test, such as Figure 5 As shown, according to the requirements of Biological Evaluation of Medical Devices Part 10: Testing for Irritation and Delayed-Type Hypersensitivity (GB / T 16886.10-2005 / ISO 10993-10:2002), intradermal injection of purified human placenta bFGF was used to evaluate the potential for irritation in the sample. The test results showed that after injecting albino rabbits with the test sample hbFGF and the blank control (physiological saline), no erythema or edema was observed at the injection sites (sample and control) at 24h, 48h, and 72h. Based on the scoring criteria in the evaluation results, the difference between the average score of the test sample and the control was less than 1.0, indicating that the sample had no potential for irritation.

[0051] like Figure 6 As shown, 10 male and 10 female volunteers were recruited to experience bFGF repair for tear troughs via microneedling. bFGF has a certain repairing effect on tear troughs.

[0052] bFGF prepared from human placenta can be used in multiple fields, including the repair of fresh wounds after cosmetic surgery (eyebrow tattooing, lip tinting, rhinoplasty, eyebrow removal, eyeliner cutting); rapid repair of fresh wounds such as burns, scratches, scalds, and falls, etc.

[0053] In summary, the basic fibroblast growth factor prepared by the method of this invention has a high yield, stable quality, and good effect on promoting fibroblast growth; moreover, the method of this invention is simple and easy to implement, suitable for large-scale production, and has good application prospects.

[0054] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for large-scale preparation of basic fibroblast growth factor from human placenta, characterized in that... Includes the following steps: Step (1). Cleaning: Select suitable placentas, thaw them, and clean them with saline solution; Step (2). Homogenization: Add 0.15M ammonium sulfate at a W / V ratio of 1:5, homogenize for 1.5 min / time using a tissue homogenizer, and homogenize 3 times; then homogenize the homogenate again for 20 minutes using a pre-disperser. Step (3). Acid extraction; Step (4). Centrifugation: refers to low-speed centrifugation using a cloth bag with a centrifugal force of 500g~1000g to remove the residue from the homogenate extraction. Step (5). Clarification and filtration: Dilute the liquid after centrifugation in the filter bag until the conductivity is below 10 mS / cm, add chitosan to 0.01%~0.05%, adjust the pH to 6.5~7.5, let stand for 10~12h, siphon the supernatant, and further filter the supernatant with 0.45μm hollow fiber tangential flow filter or direct current filter cartridge; Step (6). Concentration and buffer replacement: This refers to concentrating the filtrate by ultrafiltration using a 10KD ultrafiltration membrane 5 to 20 times, and then washing and replacing the buffer with pH 6.0 100mMPB buffer. Step (7). Chromatography: cation exchange chromatography and heparin affinity chromatography; The cation exchange chromatography conditions in step (7) include: equilibration buffer: pH 6.0 100mM PB; elution buffer I: pH 6.0 100mM PB + 0.15M NaCl; elution buffer II: pH 6.0 100mM PB + 0.6M NaCl; the heparin affinity chromatography conditions in step (7) include: equilibration buffer: pH 7.0 10mM TrisHCl + 0.6M NaCl; elution buffer I: pH 7.0 10mM TrisHCl + 1.1M NaCl; elution buffer II: pH 7.0 10mM TrisHCl + 2M NaCl. In step (3), acid extraction refers to adjusting the pH of the homogenate to 4.5 and stirring for 2-5 hours.

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