CN101134020A - Method for producing recombination human body keratinized cell growth factor -2 freeze-dried powder for injection - Google Patents
Method for producing recombination human body keratinized cell growth factor -2 freeze-dried powder for injection Download PDFInfo
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- CN101134020A CN101134020A CNA2006100306915A CN200610030691A CN101134020A CN 101134020 A CN101134020 A CN 101134020A CN A2006100306915 A CNA2006100306915 A CN A2006100306915A CN 200610030691 A CN200610030691 A CN 200610030691A CN 101134020 A CN101134020 A CN 101134020A
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Abstract
The present invention provides a kind of human horny cell growth factor-2(KGF-2) freeze-dried injection and its production method. Said production method includes the procedures of adding auxiliary material and freeze-drying treatment. The adoption of said method can prepare human horny cell growth factor-2 freeze-dried injection with high activity and high stability.
Description
Technical field
The present invention relates to the preparation production field.More specifically, the invention provides a kind of human body keratinized cell growth factor-2 (KGF-2) lyophilized injectable powder, and corresponding production method, comprise relevant adjuvant screening and freeze-dry process.
Background technology
1996, (Am J Physiol Gastrointest Liver Physiol 2000Nov such as Yamasaki; 279 (5)) from the embryo of Mus, increase and keratinocyte growth factor (Keratinocyte GrowthFactor, KGF) has highly homologous a kind of brand-new cytokine, be called body keratinized cell growth factor-2 (KGF-2), be called again fibroblast growth factor-10 (Fibroblast Growth Factor-10, FGF-10).Afterwards, (J Burn Care Rehabil 2000 Jan-Feb such as Emoto; 21 (1Pt 1): 5-9) be cloned into people's KGF-2cDNA, its encoded protein is made up of 208 aminoacid, and the N end has 39 amino acid whose hydrophobic signal peptides, 169 aminoacid of maturation protein (40-208aa), theoretical molecular is 19.3kDa, and heparin is had stronger affinity interaction.
KGF-2 can be specific be incorporated into tyrosine kinase receptor FGFR2IIIb (KGFR), the FGFR1 on target cell surface and the triggering signal conduction, thus the performance physiological action.External activity studies show that, when 0.1-5nM (2-100ng/mL) concentration, cutin epithelial cell Bal/MK had significant short splitting action, NIH3T3 does not have effect to fibroblast, and when being higher than the concentration of 5nM NIH3T3 is had significant promotion division and growth effect equally.Studies show that the expression of KGF-2mRNA mainly is among embryo's growth course, and at specific adult tissue such as lung, prostate.KGF-2 is the formation of initial appendage bud and the cytokine that promotes the key of its growth in embryo development procedure.KGF-2mRNA high level expression in the process of wound healing, animal test results shows that KGF-2 plays an important role in wound healing.
(J Pathol 1999 Aug such as Han; 188 (4): 431-8) utilize the scale-model investigation of indometacin inducing mouse small intestinal ulcer to show, the KGF-2 intravenous injection significantly reduces acute and chronic damage of intestines.It produces by the prostaglandin that stimulates epithelium recovery and protectiveness and suppresses the small intestinal inflammation; The patient that burn etc. needs large area skin to transplant faces serious metabolic problems and infects lethal danger, (Pharmacol Exp Ther 1999Jul such as Smith; 290 (1): 464-71) with the skin transplantation test model of nude mouse, examination KGF-2 forms effect of kinetics to the skin epithelium, shows that KGF-2 has very significantly facilitation to the healing of cutify; (J Surg Res 1999 Feb such as Xia; 81 (2): the 238-42) rabbit ear corium ulcer model that utilizes ischemia to damage, examination KGF-2 is to wound healing and to the influence of cicatrization, the result shows that KGF-2 significantly promotes epidermal growth, the lifting epithelium forms, strengthen the formation (KGF does not have this effect) of granulation tissue in addition, than cytokines such as KGF-1, TGF-beta, KGF-2 the most effectively and hardly causes tangible cicatrix; Miceli etc. utilize the inductive Mus ulcer of dextran sulfate sodium salt model, find that KGF-2 almost completely recovers the normal cell structure on intestinal surface, has shown its clinical treatment meaning to inflammatory bowel.Jimenez etc. show by the scale-model investigation of mouse back skin cutting wound, and it is significant that KGF-2 quickens traumatic wound healing to initial sum.
Domestic development and use to KGF-2 at present are not seen in report as yet, only are confined to a spot of basic clinical experimental study.Abroad to its utilize then be in flourish among.U.S. FDA approved U.S. HUMANGEMNOME SCIENCES, company (HGS) carry out the clinical trial II phase to rhKGF-2 (Repifermin) and study.
Repifermin is HUMAN GEMNOME SCIENCES, the trade name of the reorganization KGF-2 of Inc (HGS) exploitation, begin two I phase experimental studies that KGF-2 is used for wound healing in February, 1998, one is the local application of skin surface, and another is the systemic administration by injection.The purpose of test is to estimate safety, pharmacokinetics and the appropriate dosage of KGF-2.Result of the test shows that under selected dosage, system applies KGF-2 is safe, and the healthy volunteer can be well tolerable, almost has no side effect.
The relevant mucositis II clinical trial phase of in January, 2000 starting treatment treatment of cancer (high dose chemotherapy before the bone marrow transplantation), the II clinical trial phase of begin treatment venous ulcer in early time.Purpose is safety and the effectiveness of evaluation system application KGF-2, and in JIUYUE, 2000 have been announced the IIa clinical trial phase result of treatment venous ulcer, and 94 patients have been investigated in test, and its venous ulcer area is from 3-30cm
2, continued 3-36 month.Local application's treatment twice weekly, heals fully up to 12 weeks or wound, because venous ulcer may need many month ability to heal fully, added up partly patient's number of healing in the test.
The IIa result of the test shows that the KGF-2 therapeutic effect is remarkable, and patient can be well tolerable, is free from side effects substantially, and HGS determines more massive IIb test, with the effectiveness that venous ulcer fully healed of the patient of investigation more than 600 with further evaluation KGF-2.Other clinical trial is in progress.
We have selected the dosage form, adjuvant, the production technology that are fit to, by controlling crucial process conditions, make the product that makes keep very high active and stable.Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low.Be fit to industrialization production.
Summary of the invention
Purpose of the present invention just provides a kind of human body keratinized cell growth factor-2 (KGF-2) lyophilized injectable powder.
Another object of the present invention just provides the preparation process of the human body keratinized cell growth factor-2 of optimization.
In a first aspect of the present invention, just provided a kind of human body keratinized cell growth factor-2 (KGF-2) lyophilized injectable powder, it is by human body keratinized cell growth factor-2, sucrose, mannitol, NaCl, EDTA and citric acid one sodium citrate buffer system lyophilizing form.
In a second aspect of the present invention, the preparation process of human body keratinized cell growth factor-2 is provided, the method comprising the steps of:
A. human body keratinized cell growth factor-2 is mixed with adjuvant;
B. add suitable buffer solution;
C. under appropriate condition, lyophilizing;
D. by suitable specification packing;
In another preference, described adjuvant is 7% sucrose, 7% mannitol, 0.02M NaCl, 1mMEDTA.
In another preference, described buffer solution is 40mM citric acid one sodium citrate, pH6.2.
In another preference of the present invention, human body keratinized cell growth factor-2 obtains product moisture＜3% through after the lyophilizing, and stability can reach 2 years, and the back clarity of redissolving is good.
Description of drawings
Fig. 1 .KGF-2 sample freeze-drying curve, this figure have write down temperature and vacuum value and actual value that KGF-2 sample freeze-drying process is set.
The specific embodiment
The inventor is extensive studies by going deep into, by screening and optimization to factors such as adjuvant, buffer solution, freeze-dry process, produce the freeze-dried powder product of human body keratinized cell growth factor-2, obtain high stability and active lyophilized injectable powder, realize the human body keratinized cell growth factor-2 large-scale production, finished the present invention on this basis.
The present invention sums up according to the inquiry of related data arrangement, by to the screening of adjuvant, buffer solution and freeze-dry process with grope, is index optimization technology with parameters such as outward appearance, dissolubility, moisture, activity change, freeze-drying curves, method summary such as following table.
Table 1 method summary
| Step | Test name | Get a little | Working foundation | The parameter that obtains | The single test time |
| Test 1 | Supplementary product kind | The 3-5 kind, concentration 5-15% | Document, solubility property | Outward appearance, dissolubility, moisture, activity change, freeze-drying curve | The 1st week |
| Test 2 | Adjuvant concentration | The 1-2 kind, the concentration refinement | Test 1 | Outward appearance, dissolubility, moisture, activity change, freeze-drying curve | The 2nd week |
| Test 3 | Lyophilisation condition | The 1-2 kind | Test 2 | Outward appearance, dissolubility, moisture, activity change, freeze-drying curve | The 3rd week |
| Test 4 | The preliminarily stabilised test data | The 1-2 kind | Test 3 | Outward appearance, dissolubility, moisture, activity change, freeze-drying curve | The beginning of the 4th week, about 2 months |
| Test 4 | Pilot scale is amplified | 1 kind | Test 3 | Outward appearance, dissolubility, moisture, activity change, freeze-drying curve | |
| Test 5 | Pilot scale is optimized | 1 kind | Outward appearance, dissolubility, moisture, activity change, freeze-drying curve, industrialization value |
In another example of the present invention, carried out the screening of buffer solution and pH thereof.Guaranteed the active and stable of sample.
In an example of the present invention, carried out the screening of supplementary product kind and concentration thereof, guaranteed that further human body keratinized cell growth factor-2 is active and stable.
By above method, the technology that is optimized, the human body keratinized cell growth factor-2 powder ampoule agent for injection that makes by this technology.The preliminarily stabilised test shows that this injectable powder is stable.
Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, is fit to industrialization production.
The invention has the advantages that:
(1) adjuvant of selecting for use and buffering solution are easy to get, and cheapness is fit to industrialization production.
(2) freeze-dry process after the optimization is stable, and is simple to operate, the efficient height, and cost is low, is fit to industrialization production.
(3) owing to adopt freeze-dry process, by controlling crucial manufacturing condition, moisture＜3%, solubility is good, makes stability can reach 2 years.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The experiment of embodiment 1.KGF-2 freeze-dried powder preliminarily stabilised
A. adjuvant: mannitol and sucrose
Adjuvant concentration is: mannitol concentration: 5%, 7.5%, 10%;
Sucrose concentration: 5%, 6%, 7.5%.
The accelerated stability experimental result:
Clarity: under lamp, detect by an unaided eye kgf-2 at room temperature 2 days all samples white point or flocculent undissolved substance all appear, a large amount of white points and flocculent undissolved substance then appearred after 3 days.Sample white point or flocculent undissolved substance are less in 4 ℃.White point is not seen in freeze dried sample redissolution back perusal under common daylight lamp, but under specific lamp, observe, 1, No. 2 visible opalescence of sample, 6, No. 7 the sample dissolution degree is low, the sample that adds Tween80 has the saloid exchange phenomenon of class when just having dissolved, liquid sensation thickness but after the earthquake this phenomenon disappear.No special evident difference between other samples.Therefore the clarity of 3, No. 4 samples is best.
Moisture: in water content detection, when just offeing for sale, the sample structure pine solute that removes No. 7 is low again, and moisture reaches 8%, and all below 2.2%, moisture is qualified for other samples.Recheck moisture except 3, No. 4 samples after 30 days below 3%, all the other all greater than the moisture of 3, No. 4 samples after 3%, 60 day still below 3%.
Active: No. 2 activity is best, but its toxic dose and effective dose are very near, and No. 3 activity are then apparently higher than other several samples.
Embodiment 2.KGF-2 freeze-dried powder pilot scale stability test
Detect at moisture content, 5% concentration sucrose dressing is qualified always.Outward appearance detects sucrose and compares mannitol, and sample does not have Powderedly in the time of 40 ℃, and dressing three temperature profile of sample when being mannitol are all qualified; The clarity detection is along with mannitol concentration constantly increases, and dissolubility reduces, so clarity is defective.Under two kinds of dressing conditions, mannitol has dimer as dressing, and sucrose only has dimer as dressing 37 ℃ of April, does not occur under other conditions.
The accelerated stability experiment shows: comprehensive clarity, activity, moisture, stable several aspects, determine that the adjuvant that KGF-2 adds is 0.02MNaCl, 7% mannitol and 7% sucrose.Test agent its stability in 2 years respectively detects index all in normal range in three batches, still determine that the effect duration of KGF-2 is 2 years
Claims (6)
1. a human body keratinized cell growth factor-2 (KGF-2) lyophilized injectable powder is characterized in that, it is by human body keratinized cell growth factor-2, and proper supplementary material and suitable buffer system lyophilizing form.
2. lyophilized injectable powder as claimed in claim 1 is characterized in that, described adjuvant is: 5-15% sucrose, 5-15% mannitol, 0.01-0.05M NaCl, 0.1-10mM EDTA.Preferable, 7% sucrose, 7% mannitol, 0.02MNaCl, 1mMEDTA.
3. lyophilized injectable powder as claimed in claim 1 is characterized in that, described buffer system is the 10-100mM citric acid-sodium citrate, pH5-7.Preferable, 40mM citric acid-sodium citrate, pH6.2.
4. the formulation process method of a human body keratinized cell growth factor-2 is characterized in that, the method comprising the steps of:
A. human body keratinized cell growth factor-2 is mixed with adjuvant;
B. add suitable buffer solution;
C. under appropriate condition, lyophilizing;
D. by suitable specification packing;
5. method as claimed in claim 4 is characterized in that, the described adjuvant of step (a) is 5-15% sucrose, 5-15% mannitol, 0.01-0.05M NaCl, 0.1-10mMEDTA.Preferable, 7% sucrose, 7% mannitol, 0.02M NaCl, 1mMEDTA.
6. method as claimed in claim 4 is characterized in that, the described buffer solution of step (b) is the 10-100mM citric acid-sodium citrate, pH5-7.Preferable, buffer is the 40mM citric acid-sodium citrate, pH6.2.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106377760A (en) * | 2016-10-26 | 2017-02-08 | 李天学 | Application of preparation containing KGF-2 (keratinocyte growth factor-2) to relieving of diabetic ulcer |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106377760A (en) * | 2016-10-26 | 2017-02-08 | 李天学 | Application of preparation containing KGF-2 (keratinocyte growth factor-2) to relieving of diabetic ulcer |
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