CN113956343A - Method for large-scale preparation of basic fibroblast growth factor from human placenta - Google Patents
Method for large-scale preparation of basic fibroblast growth factor from human placenta Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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Abstract
The invention provides a method for preparing basic fibroblast growth factor (bFGF) from human placenta in a large scale, which comprises the following steps: step (1), cleaning; homogenizing; step (3), acid extraction; step (4), centrifuging; step (5), clarifying and filtering; step (6), concentrating, replacing and buffering; and (7) preparing the human placenta basic fibroblast growth factor by cation exchange chromatography and heparin affinity chromatography. According to the invention, 1kg of human placenta can be purified to obtain about 100 mu g of high-purity human basic fibroblast growth factor (Bradford method), and the process is verified by multiple tests, so that the repeatability and the stability are good; the yield is improved, and the cost is reduced; and the low-speed centrifugation is favorable for industrial production and preparation, so that the defects that the conventional ammonium sulfate fractional precipitation method is difficult to operate in industrial preparation and ammonium sulfate has great environmental pollution are effectively avoided, and the industrial large-scale preparation has good application prospect.
Description
Technical Field
The invention relates to the technical field of biological product production, in particular to a method for preparing basic fibroblast growth factor from human placenta in a large scale.
Background
In recent years, with the wider application of fibroblast growth factors in the fields of medicines, wound repair instruments, cosmetics and the like, the advantages of low immunogenicity, high activity and the like of natural humanized fibroblast growth factors are paid extensive attention. Human Basic Fibroblast Growth Factor (hbFGF) is high-purity fully Human hbFGF obtained by purifying healthy Human placenta by methods of homogenizing, acid extraction, centrifugation, clarification, filtration, ultrafiltration concentration, buffer replacement, combination of ion exchange and affinity chromatography and the like. The hbFGF purified from human placenta has the advantages that the raw material is derived from human waste tissue material, has no foreign protein and low immunogenicity to human, and the recombinant hbFGF is mostly bovine or partially humanized hbFGF expressed by bacteria; the hbFGF extracted from human placenta has a natural structure, the amino acid sequence of the hbFGF is 100% from human beings, and compared with the recombinant hbFGF, the hbFGF has no gene modification, and the secondary structure and the tertiary structure keep natural spatial conformation. The hbFGF is clinically used for treating the fresh wound repair after cosmetic plastic surgery (eyebrow tattooing, lip bleaching, nose humping, eyebrow washing and eye line cutting); quick repair of fresh wound surfaces of burns, scratches, scalds, falling injuries and the like.
The existing method for extracting bFGF from animal tissues mostly adopts an ammonium sulfate fractional precipitation method for pretreatment, the ammonium sulfate fractional precipitation method uses ammonium sulfate with high concentration, which is easy to cause environmental pollution, and the ammonium sulfate fractional precipitation method is not easy to operate in industrial preparation. US4902782 uses ammonium sulfate fractional precipitation and high speed centrifugation to extract bFGF from bovine brain, which is not easy to be scaled up industrially. Large-scale preparation of bFGF from placenta is carried out in the literature "Isolation and catalysis of two differential molecular force of basic fiber growth factor extracted from human placenta tissue", wherein pH3.2 is adjusted for acid extraction for 30min, 15% ethanol is used for precipitation and centrifugation, concentration, cation exchange chromatography and heparin affinity chromatography are adopted for subsequent preparation operations, and finally 15 mu g of bFGF can be extracted from 1kg of placenta tissue. The literature, "Biochemical, and biological characterization of bFGF extracted from human placenta", reports that 10. mu.g bFGF can be prepared from 1kg placenta. These reported methods are not easy to scale up for industrial production and have low yield.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a method for preparing basic fibroblast growth factor from human placenta in large scale, so as to solve the problems proposed in the background art.
The technical problem solved by the invention is realized by adopting the following technical scheme: a method for preparing basic fibroblast growth factor from human placenta in large scale comprises the following steps:
step (1), cleaning: selecting a placenta meeting the requirement, unfreezing, and cleaning with normal saline;
step (2), homogenizing: W/V1: 5 adding 0.15M ammonium sulfate, homogenizing for 1.5 min/time and 3 times by a tissue homogenizer; homogenizing the homogenate for 20 minutes by using a pre-dispersing machine;
step (3), acid extraction;
step (4), centrifuging: centrifuging by a cloth bag centrifuge, wherein the centrifugal force is 500 g-1000 g, and removing homogenate extraction residues;
and (5) clarifying and filtering: diluting the feed liquid after the cloth bag centrifugation to the conductivity lower than 10mS/cm, adding chitosan to 0.01-0.05%, adjusting the pH value to 6.5-7.5, standing for 10-12 h, siphoning the supernatant, and further performing tangential flow filtration or filter core direct flow filtration by using hollow fibers of 0.45 mu m;
step (6), concentrating, replacing and buffering: ultrafiltering and concentrating the filtrate by a 10KD ultrafiltration membrane by 5-20 times, and washing and filtering the buffer solution of pH6.0100mM PB for buffer replacement;
and (7) preparing the product through cation exchange chromatography and heparin affinity chromatography.
The acid extraction in the step (3) is to adjust the pH of the homogenate to 4.5 and stir and extract for 2 hours;
the cation exchange chromatography conditions in step (7) include equilibrium Buffer: pH6.0100mM PB, elution Buffer I: pH6.0100mM PB +0.15M NaCl, elution Buffer II: pH6.0100mM PB +0.6M NaCl.
The heparin affinity chromatography conditions in the step (7) comprise balance Buffer: pH7.010mM Tris-HCl +0.6M NaCl, elution Buffer I: pH7.010mM Tris-HCl +1.1M NaCl, elution Buffer II pH7.010mM Tris-HCl +2M NaCl.
Compared with the prior art, the invention has the beneficial effects that: the invention mainly adopts the innovations of the step (2), the step (4), the step (5) and the step (6), namely, the homogenate intensity is enhanced through a pre-dispersing machine, and the release of target protein is improved; the industrial large-scale centrifugal treatment is realized through low-speed centrifugation (such as a bag centrifuge), and the low-speed centrifugation has the advantages of low cost, low heat production, low energy consumption, low mechanical shearing force and the like; by combining the dilute feed liquid conductance and the chitosan clarification, a large amount of impurities are removed by the chitosan clarification, the yield of the target protein is improved, the chitosan clarification method replaces the traditional high-concentration ammonium sulfate precipitation method to reduce the loss of the target protein, the operability of filtration treatment after the chitosan clarification is strong, and the loss of the target protein can be reduced more than that of the common ammonium sulfate precipitation method; finally, the buffer linking chromatography process is replaced by ultrafiltration concentration.
In conclusion, the high-purity human basic fibroblast growth factor prepared by 1kg of human placenta is about 100 mug (Bradford method), which is far higher than the existing report; and the low-speed centrifugation and clarification process is favorable for industrial production and preparation, the cost is reduced, the defects that the conventional ammonium sulfate fractional precipitation method is difficult to operate in industrial preparation and high-concentration ammonium sulfate pollutes the environment are effectively avoided, and the method has good application prospect in industrial large-scale preparation.
Drawings
FIG. 1 is a flow chart of a process for preparing human placenta-derived bFGF of the present invention.
FIG. 2 is an SDS-PAGE electrophoresis of human placental bFGF of the present invention.
FIG. 3 is an isoelectric focusing (IEF) electrophoretogram of human placenta-derived bFGF of the present invention.
FIG. 4 is a Balb/c 3T3 cell proliferation curve of human placental bFGF of the present invention.
FIG. 5 is a diagram showing a test of skin irritation of albino rabbit with human placenta-derived bFGF of the present invention.
FIG. 6 is a graph comparing the effect of the volunteers of the present invention before and after experiencing bFGF to repair lacrimal ducts.
Detailed Description
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described below by combining the specific drawings.
Human placenta source
The raw material human placenta is a healthy human placenta which is collected by strict aseptic operation and is used for producing biological products, and in order to ensure the quality and the safety of the human placenta, the collected contents comprise the blood plasma of a puerpera, the blood plasma of the placenta, an informed contract signed by the puerpera and a complete placenta blood and placenta collection registration form. The collection hospital is more than second class A and the like, and has primary screening detection qualities of HBV, HCV, TP, HIV and the like. According to the requirements of 'Chinese pharmacopoeia' 2020 edition and 'quality control standard of main raw and auxiliary materials of Chinese biological products', the collected material placenta is strictly screened, and the collected placenta is subjected to the inspection of HBsAg, syphilis, HIV-1/HIV-2 antibody and HCV antibody, etc. one by adopting a kit approved by the national drug administration, so that the placenta prescription of the total negative person can be put into use.
TABLE 1 Experimental instrumentation
Second, preparation method
As shown in fig. 1, a method for preparing basic fibroblast growth factor from human placenta in a large scale comprises the following steps:
step (1), cleaning: selecting 20 placenta (10kg) meeting the requirement, thawing, and cleaning with normal saline;
step (2), homogenizing W/V1: 5 adding 0.15M ammonium sulfate, homogenizing for 1.5 min/time and 3 times by a tissue homogenizer; homogenizing the homogenate for 20 minutes by using a pre-dispersing machine;
step (3), acid extraction;
step (4), centrifuging: centrifuging by a cloth bag centrifuge, wherein the centrifugal force is 500 g-1000 g, and removing homogenate extraction residues;
and (5) clarifying and filtering: diluting the feed liquid after the cloth bag centrifugation to the conductivity lower than 10mS/cm, adding chitosan to 0.01-0.05%, adjusting the pH value to 6.5-7.5, standing for 10-12 h, siphoning the supernatant, and further performing tangential flow filtration or filter core direct flow filtration by using hollow fibers of 0.45 mu m;
step (6), concentrating, replacing and buffering: ultrafiltering and concentrating the filtrate by a 10KD ultrafiltration membrane by 5-20 times, and washing and filtering the buffer solution of pH6.0100mM PB for buffer replacement;
and (7) carrying out CMFF chromatography and heparin affinity chromatography to obtain the product.
The acid extraction in the step (3) is to adjust the pH of the homogenate to be 4.5, stir and extract for 2 h:
the chromatography medium used in the cation exchange chromatography in the step (7) is CMFF, 500ml, and the chromatography conditions comprise equilibrium Buffer: pH6.0100mM PB, elution Buffer I: pH6.0100mM PB +0.15M NaCl, elution Buffer II: pH6.0100mM PB +0.6M NaCl.
The heparin affinity chromatography condition in the step (7) is that 50ml (pall) of affinity resin is applied, AKTA AVANT150 full-automatic intelligent protein purification system is used for operation, and the chromatography condition comprises balance Buffer: pH7.010mM Tris-HCl +0.6M NaCl, elution Buffer I: pH7.010mM Tris-HCl +1.1M NaCl, elution Buffer II pH7.010mM Tris-HCl +2M NaCl.
Third, experimental results
Collecting heparin target elution peak, measuring total protein concentration (Bradford method), and performing aseptic detection, endotoxin detection and the like to be qualified. Meanwhile, the molecular weight of the target protein is determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the isoelectric point (pI) is determined by isoelectric focusing, and then a cell proliferation promotion experiment, an animal experiment and a volunteer experience experiment are carried out. The results are as follows:
as shown in FIG. 2, SDS-PAGE analysis of purified human placenta bFGF showed 17.355kD molecular weight, and thin-layer scanning of purity showed 95% purity.
As shown in FIG. 3, isoelectric focusing (IEF) determined the isoelectric point (pI) of bFGF samples purified from human placenta, and showed only one distinct basic band in the region of pI 9.3-10.0, with a pI of about 9.7.
As shown in FIG. 4, the biological activity of human placenta-derived bFGF was measured by cell proliferation method (BALB/c 3T3 cells), and the experimental data were processed by four-parameter regression calculation method, and the results showed that bFGF purified from human placenta (dark color) and commercial recombinant bFGF (light color) both promoted the proliferative growth of fibroblasts at the same dilution factor and the proliferation curve was consistent, and that bFGF derived from human placenta (dark color) had higher biological activity than commercial recombinant bFGF sample (light color) by ED50Calculating human placenta bFGF biological activity greater than 10000U/ml and specific activity greater than 1 x 106U/mg。
Animal safety test
(1) Mouse abnormal toxicity test according to the method of appendix 1141 of the four parts of the 'Chinese pharmacopoeia' 2020 edition, 5 mice were each intraperitoneally injected with 0.5ml of human placenta-derived hbFGF sample, and after 7 days of observation, no mice died and the body weight increased normally. And the test requirements are met.
(2) Abnormal toxicity test of guinea pigs 2 guinea pigs were injected with 5.0ml of hbFGF sample, respectively, according to the method of appendix 1141 of the fourth part of the "Chinese pharmacopoeia" 2020 edition, and after 7 days, there was no death, no abnormal reaction, and normal weight gain. And the test requirements are met.
(3) Intradermal reaction test as shown in fig. 5, part 10 is evaluated according to medical device biology: stimulation is associated with the requirement of the delayed type hypersensitivity test (GB/T16886.10-2005/ISO 10993-10:2002) that the potential of this sample to produce a stimulatory response was evaluated by intradermal injection of human placental purified bFGF and the results of the test showed: after injecting test sample hbFGF and blank control (normal saline) into albino rabbits, 24h, 48h and 72h were observed, and no erythema or edema was observed at the injection site (sample, control). The score was scored and calculated according to the score criteria in the evaluation results and the difference between the mean scores of the test sample and the control was less than 1.0, indicating that the sample was not potentially stimulatory.
As shown in fig. 6, 10 volunteers, male and female, were recruited, and subjected to microneedle injection to experience the restoration of lacrimal canals by bFGF, which has a certain restoration effect on lacrimal canals.
bFGF prepared from human placenta can be used in multiple fields, and can be used for treating fresh wound surface repair after cosmetic plastic surgery (eyebrow tattooing, lip bleaching, nose humping, eyebrow washing, and eye line cutting); quick repair of fresh wound surfaces of burns, scratches, scalds, falling injuries and the like.
In conclusion, the alkaline fibroblast growth factor prepared by the method has high yield and stable quality, and has good effect of promoting the growth of the fibroblast; the method is simple and feasible, is suitable for large-scale production, and has good application prospect.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (4)
1. A method for preparing basic fibroblast growth factor from human placenta in large scale is characterized in that: the method comprises the following steps:
step (1), cleaning: selecting a placenta meeting the requirement, unfreezing, and cleaning with normal saline;
step (2), homogenizing: W/V1: 5 adding 0.15M ammonium sulfate, homogenizing for 1.5 min/time and 3 times by a tissue homogenizer; homogenizing the homogenate for 20 minutes by using a pre-dispersing machine;
step (3), acid extraction;
step (4), centrifuging: the method is characterized in that the method comprises the steps of centrifuging at a low speed and removing homogenate extraction residues by a centrifugal force of 500-1000 g;
and (5) diluting, clarifying and filtering: diluting the feed liquid after low-speed centrifugation to the conductivity of less than 10mS/cm, adding chitosan to the feed liquid to the concentration of 0.01-0.05%, adjusting the pH value to 6.5-7.5, standing for 10-12 h, siphoning the supernatant, and further performing tangential flow filtration or filter core direct flow filtration on the supernatant by using hollow fibers of 0.45 mu m;
step (6), concentrating, replacing and buffering: the method comprises the steps of performing ultrafiltration concentration on a filtered filtrate by a 10KD ultrafiltration membrane by 5-20 times, and then performing washing filtration on a buffer solution of PB (phosphate buffer) with the pH value of 6.0100mM for buffer replacement;
step (7), chromatography: cation exchange chromatography and heparin affinity chromatography.
2. The method of claim 1, wherein the human placenta is used for large scale production of basic fibroblast growth factor, wherein the human placenta is selected from the group consisting of: the acid extraction in the step (3) refers to: adjusting the pH value of the homogenate to 4.5, and stirring and extracting for 2-5 h.
3. The method of claim 1, wherein the human placenta is used for large scale production of basic fibroblast growth factor, wherein the human placenta is selected from the group consisting of: the cation exchange chromatography conditions in step (7) include equilibrium Buffer: pH6.0100mM PB, elution Buffer I: pH6.0100mM PB +0.15M NaCl, elution Buffer II: pH6.0100mM PB +0.6M NaCl.
4. The method of claim 1, wherein the human placenta is used for large scale production of basic fibroblast growth factor, wherein the human placenta is selected from the group consisting of: the heparin affinity chromatography conditions in the step (7) comprise balance Buffer: pH7.010mM Tris-HCl +0.6M NaCl, elution Buffer I: pH7.010mM Tris-HCl +1.1M NaCl, elution Buffer II pH7.010mM Tris-HCl +2M NaCl.
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| CN202110538517.6A Pending CN113956343A (en) | 2021-05-18 | 2021-05-18 | Method for large-scale preparation of basic fibroblast growth factor from human placenta |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4785079A (en) * | 1984-11-09 | 1988-11-15 | The Salk Institute For Biological Studies | Isolation of fibroblast growth factor |
| US4902782A (en) * | 1986-12-10 | 1990-02-20 | The Salk Institute For Biological Studies | Isolation of fibroblast growth factor |
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- 2021-05-18 CN CN202110538517.6A patent/CN113956343A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4785079A (en) * | 1984-11-09 | 1988-11-15 | The Salk Institute For Biological Studies | Isolation of fibroblast growth factor |
| US4902782A (en) * | 1986-12-10 | 1990-02-20 | The Salk Institute For Biological Studies | Isolation of fibroblast growth factor |
Non-Patent Citations (1)
| Title |
|---|
| 威尔逊等编著，屈伸等译: "实用生物化学原理和技术", 中国医药科技出版社, pages: 273 * |
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