CN103041370A - Drug composition containing recombinant human erythropoietin and preparation method of drug composition - Google Patents
Drug composition containing recombinant human erythropoietin and preparation method of drug composition Download PDFInfo
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- CN103041370A CN103041370A CN2013100092795A CN201310009279A CN103041370A CN 103041370 A CN103041370 A CN 103041370A CN 2013100092795 A CN2013100092795 A CN 2013100092795A CN 201310009279 A CN201310009279 A CN 201310009279A CN 103041370 A CN103041370 A CN 103041370A
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- human erythropoietin
- mannitol
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Abstract
The invention provides a drug composition containing recombinant human erythropoietin and a preparation method of the drug composition. The drug composition comprises the following components: mannitol, a disodium hydrogen phosphate-citric acid buffer, recombinant human albumin and an effective amount of recombinant human erythropoietin. According to the drug composition and the preparation method, the recombinant human albumin and mannitol serve as protective agents; the stability of the drug composition can be effectively improved; the drug composition can be effectively prevented from being absorbed on a wall of a syringe or a container; in addition, inactivation of protein at a room temperature can be effectively slowed down at a pH value and under specific buffer conditions; and therefore, an injection of the drug composition is easier to store and transport.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of pharmaceutical composition that contains recombinant human erythropoietin and preparation method thereof.
Background technology
Erythropoietin (Erythropoietin, EPO) be called for short erythropoietin, it is the glycoprotein for molecular weight 6-7 ten thousand, be a kind of cytokine that promotes the differentiation of erythroid hematopoiesis precursor, breeding, it can impel undifferentiated differentiation of stem cells protoerythrocyte lineage stem cells (erythropoietin responsive cell).To further again maturation be hemocytoblast, reticulocyte, hemoglobin synthetic and flow into peripheral vessel etc. facilitation is all arranged.Generally when anemia and hypoxia, according to the needs of bodily tissue to oxygen, the quantity delivered of erythropoietin will increase, but its content is then very low when the kidney anemia.Clinically, erythropoietin is mainly used in increasing erythrocytic number, is used for anemia, histodialysis, premature infant, is used in cancerology and hematology aspect.
Nineteen eighty-three is successfully separated by Lin etc. and has cloned the EPO gene, utilized technique for gene engineering to begin to produce in enormous quantities the recombined human promoting erythrocyte in 1985 and generate (rHuEPO) and begin for clinical experiment, FDA (Food and Drug Adminstration) (FDA) was applied to the clinical treatment of anemia in official approval rHuEPO in 1989.
RHuEPO is poor stability in aqueous solution, so rHuEPO needs to add protective agent increasing its stability in solution, and preventer is attracted on the inwall of container.CN1247257C discloses a kind of sufficient human forcing erythrogenin preparation, and it comprises the reagent such as mannitol, L-Histidine hydrochlorate, metal ion chelation agent, pH value buffer, sodium chloride.CN100502943C discloses a kind of drug injection of recombinant human erythropoietin, it comprise pH value buffer, polyoxyethylene sorbitan monoleate and polysorbate 20, sodium chloride and etc. composition.These two pieces of documents think that there are the problems such as potential blood source pollution and drug allergy in end user's blood albumin.Yet, use metal ion chelation agent may cause the problems such as low blood calcium, use polyoxyethylene sorbitan monoleate then can cause colloidal ion to form, some rHuEPO molecular aggregatess are to the surface of these colloidal ions, but enter excitating organism immune system behind the body, finally cause antibody to produce (Schellekens etc.), thereby cause antibody-mediated pure red cell aplasia (PRCA).
Studies show that rHuEPO is subject to preserve and transportation in the impact of excess Temperature, the cold chain degeneration may be the key that induce antibody produces in its structure.In addition, the inactivation that in transportation, very easily causes rHuEPO.Also there is not so far effective solution.
Summary of the invention
The object of the invention is to the problem for Recombinant Human Erythropoietin less stable in the prior art, provide a kind of stability better to contain the pharmaceutical composition of erythropoietin.
For achieving the above object, the present invention adopts following technical scheme:
A kind of pharmaceutical composition that contains Recombinant Human Erythropoietin, it comprises following component:
Mannitol 0.5～10% (W/V);
Sodium hydrogen phosphate-citrate buffer 10～100mM, pH5.5～7.5;
Recombinant human serum albumin 0.001～0.1%;
The effective dose Recombinant Human Erythropoietin.
Preferably, pharmaceutical composition of the present invention comprises following component:
Mannitol 5% (W/V);
Sodium dihydrogen phosphate-citrate buffer 25mM, pH6.5;
Recombinant human serum albumin 0.01%;
The effective dose Recombinant Human Erythropoietin.
The Recombinant Human Erythropoietin of above-mentioned effective dose can be the concentration of directly using clinically, and perhaps its concentrate for example can be 200～30000IU/ml, preferred 500～20000IU/ml.
Pharmaceutical composition of the present invention can be aqueous injection, also can make lyophilized powder, is preferably aqueous injection.
The present invention also provides the method for preparing aforementioned pharmaceutical compositions, and it is that sodium hydrogen phosphate-citrate buffer, mannitol, recombinant human serum albumin, Recombinant Human Erythropoietin are added the water for injection dissolving, and standardize solution.The step that also comprises degerming and fill behind the described standardize solution.The method of described degerming preferably adopts the filtration sterilization method, uses successively the membrane filtration degerming of 0.45 μ m and 0.22 μ m.When fill, in bottle, fill nitrogen, and lid is rolled in tamponade.
The present invention adopts recombinant human serum albumin and mannitol as protective agent; can its stability of Effective Raise; and effectively stop it in syringe or chamber wall absorption; simultaneously under pH value of the present invention and specific buffer condition; can effectively slow down the at normal temperatures inactivation of protein, thus so that the easier storage and transport of injection of the present invention.Ironically, use separately mannitol or human albumin to can not show a candle to the preservation effect of injection of the present invention as its preservation effect at normal temperatures of stabilizer.
The recombinant human serum albumin that human albumin among the present invention uses, thereby the risk problem that does not exist the blood source to pollute, and the human albumin's consumption among the present invention is extremely low, only be ten thousand of human albumin's injection/, thereby greatly reduced the risk of part population to human serum albumin.
The specific embodiment
Following examples are used for further specifying the present invention, but should not be construed as limitation of the present invention.Under the prerequisite that does not deviate from the present invention's spirit and essence, modification or replacement to the present invention does all belong to category of the present invention.
Embodiment 1
Prescription: pharmaceutical composition of the present invention comprises the composition of following ratio:
Mannitol 5% (W/V);
Sodium hydrogen phosphate-citrate buffer 25mM, pH6.5;
Recombinant human serum albumin 0.01% (W/V);
Recombinant Human Erythropoietin 5000IU/ml;
Surplus is water.
Preparation method:
Sodium hydrogen phosphate-citrate buffer (pH6.5) of configuration 500mL50mM adds 50g mannitol, and stirring and dissolving adds the 0.1g recombinant human serum albumin, and 5 * 10
6The IU Recombinant Human Erythropoietin is settled to 1000ml with water for injection.
Use successively the membrane filtration of 0.45 μ m and 0.22 μ m, and packing, every bottle of 1ml fills with nitrogen, and the recombinant human erythropoietin preparation that lid namely gets 5000IU/ml is rolled in tamponade.
Embodiment 2
Prescription: pharmaceutical composition of the present invention comprises the composition of following ratio:
Mannitol 0.5% (W/V);
Sodium hydrogen phosphate-citrate buffer 10mM, pH5.5;
Recombinant human serum albumin 0.001% (W/V);
Recombinant Human Erythropoietin 200IU/ml;
Surplus is water.
Preparation method:
Sodium hydrogen phosphate-citrate buffer (pH5.5) of configuration 500mL20mM adds 5g mannitol, and stirring and dissolving adds the 0.01g recombinant human serum albumin, and 2 * 10
5The IU Recombinant Human Erythropoietin is settled to 1000ml with water for injection.
Use successively the membrane filtration of 0.45 μ m and 0.22 μ m, and packing, every bottle of 1ml fills with nitrogen, and the recombinant human erythropoietin preparation that lid namely gets 200IU/ml is rolled in tamponade.
Embodiment 3
Prescription: pharmaceutical composition of the present invention comprises the composition of following ratio:
Mannitol 10% (W/V);
Sodium hydrogen phosphate-citrate buffer 100mM, pH7.5;
Recombinant human serum albumin 0.1% (W/V);
Recombinant Human Erythropoietin 30000IU/ml;
Surplus is water.
Preparation method:
Sodium hydrogen phosphate-citrate buffer (pH7.5) of configuration 500mL200mM adds 100g mannitol, and stirring and dissolving adds the 0.1g recombinant human serum albumin, and 3 * 10
7The IU Recombinant Human Erythropoietin is settled to 1000ml with water for injection.
Use successively the membrane filtration of 0.45 μ m and 0.22 μ m, and packing, every bottle of 1ml fills with nitrogen, and the recombinant human erythropoietin preparation that lid namely gets 30000IU/ml is rolled in tamponade.
Embodiment 4 safety testings
Materials and methods
Experimental group: experimental group 1～3 is respectively the recombinant human erythropoietin preparation of embodiment 1～3 configuration;
Matched group: matched group 1 is the method configuration according to embodiment 1, but does not contain recombinant human serum albumin;
Blank group: water for injection.
Subjects: 100 of healthy guinea pigs, male and female half and half, body weight 350～420g
Test method
Cavia porcellus is divided into 5 groups at random, 20 every group, male and female half and half, every dosage injection according to 200IU, blank group injecting normal saline 1ml, every other day injection is once injected 3 times altogether, observes 1 hour after the per injection.
Result of the test
The result shows that unusual or anaphylaxis all do not appear in experimental group and matched group, do not have significant difference between experimental group and the matched group.
Embodiment 5 stability tests
Experimental subject
Experimental group 1～3 is respectively the recombinant human erythropoietin preparation that embodiment 1～3 disposes;
Matched group 1 according to the method configuration of embodiment 1, but does not contain recombinant human serum albumin;
Matched group 2: according to the method configuration of embodiment 1, but do not contain mannitol.
More than each the group material all adopt same batch.
Test method
Experimental group and matched group were placed 3 months under 37 ℃, detected outward appearance, pH value and the activity in vivo of preparation.Other gets experimental group and matched group was deposited under 4 ℃ 2 years, regularly detects outward appearance, pH value and the activity in vivo of preparation.
Experimental result
By above-mentioned test method, test, 37 ℃ lower, and to place 3 months testing results as shown in table 1, and the result who deposits under 4 ℃ is shown in table 2 and 3.
Table 137 ℃ lower testing result of placing 3 months
| Numbering | Sample | Solution appearance | PH value | Active (IU/ml) |
| 1 | Experimental group 1 | Colourless clear liquid | 6.5 | 4762 |
| 2 | Experimental group 2 | Colourless clear liquid | 5.6 | 182 |
| 3 | Experimental group 3 | Colourless clear liquid | 7.3 | 27561 |
| 4 | Matched group 1 | Colourless clear liquid | 6.6 | 3417 |
| 5 | Matched group 2 | Colourless clear liquid | 6.5 | 3364 |
As can be seen from Table 1, compare Recombinant Human Erythropoietin injection of the present invention with matched group and can effectively prevent at normal temperatures inactivation, can place the longer time at normal temperatures, thereby be conducive to its transportation and preservation, significantly be better than matched group.
Described sample is placed 1 year indices testing result:
Table 2 sample indices testing result (placing after 1 year)
| Numbering | Sample | Solution appearance | PH value | Active (IU/ml) |
| 1 | Experimental group 1 | Colourless clear liquid | 6.5 | 4968 |
| 2 | Experimental group 2 | Colourless clear liquid | 5.6 | 201 |
| 3 | Experimental group 3 | Colourless clear liquid | 7.5 | 29348 |
| 4 | Matched group 1 | Colourless clear liquid | 6.5 | 4907 |
| 5 | Matched group 2 | Colourless clear liquid | 6.5 | 4926 |
Described sample is placed 2 years indices testing results:
Table 3 sample indices testing result (placing after 2 years)
| Numbering | Sample | Solution appearance | PH value | Active (IU/ml) |
| 1 | Experimental group 1 | Colourless clear liquid | 6.5 | 4953 |
| 2 | Experimental group 2 | Colourless clear liquid | 5.6 | 196 |
| 3 | Experimental group 3 | Colourless clear liquid | 7.4 | 29219 |
| 4 | Matched group 1 | Colourless clear liquid | 6.6 | 4892 |
| 5 | Matched group 2 | Colourless clear liquid | 6.5 | 4907 |
Can be found out by above long-time stability experimental result: recombinant erythropoietin injection provided by the invention is placed the rear stability height for a long time, clarity activity in vivo etc. indices changes all not obvious, and pH value does not change along with the prolongation of storage time yet.
Although above used general explanation, the specific embodiment and experiment, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (8)
1. pharmaceutical composition that contains Recombinant Human Erythropoietin, it comprises following component:
Mannitol 0.5～10% (W/V);
Sodium hydrogen phosphate-citrate buffer 10～100mM, pH5.5～7.5;
Recombinant human serum albumin 0.001～0.1%;
The effective dose Recombinant Human Erythropoietin.
2. pharmaceutical composition according to claim 1 is characterized in that it comprises following component:
Mannitol 5% (W/V);
Sodium dihydrogen phosphate-citrate buffer 25mM, pH6.5;
Recombinant human serum albumin 0.01%;
The effective dose Recombinant Human Erythropoietin.
3. pharmaceutical composition according to claim 1 and 2 is characterized in that, the content of described Recombinant Human Erythropoietin is 200～30000IU/ml.
4. pharmaceutical composition according to claim 3 is characterized in that, the content of described Recombinant Human Erythropoietin is 500～20000IU/ml.
5. prepare the method for each described pharmaceutical composition of claim 1～4, it is characterized in that, sodium hydrogen phosphate-citrate buffer, mannitol, recombinant human serum albumin, Recombinant Human Erythropoietin are added the water for injection dissolving, and standardize solution.
6. method according to claim 5 is characterized in that, also comprises the step of degerming and fill behind the standardize solution.
7. method according to claim 6 is characterized in that, the method for described degerming is to use successively the membrane filtration degerming of 0.45 μ m and 0.22 μ m.
8. method according to claim 6 is characterized in that, fill nitrogen during fill in bottle, and lid is rolled in tamponade.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1519021A (en) * | 2003-01-20 | 2004-08-11 | 沈阳三生制药股份有限公司 | Stable erythropoietin of recombined human red blood cell |
| CN1832754A (en) * | 2003-08-06 | 2006-09-13 | 希杰公司 | Formulation of albumin-free erythropoietin |
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- 2013-01-11 CN CN2013100092795A patent/CN103041370A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1519021A (en) * | 2003-01-20 | 2004-08-11 | 沈阳三生制药股份有限公司 | Stable erythropoietin of recombined human red blood cell |
| CN1832754A (en) * | 2003-08-06 | 2006-09-13 | 希杰公司 | Formulation of albumin-free erythropoietin |
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Application publication date: 20130417 |