CN1247257C - Stable erythropoietin of recombined human red blood cell - Google Patents
Stable erythropoietin of recombined human red blood cell Download PDFInfo
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- CN1247257C CN1247257C CN 03100653 CN03100653A CN1247257C CN 1247257 C CN1247257 C CN 1247257C CN 03100653 CN03100653 CN 03100653 CN 03100653 A CN03100653 A CN 03100653A CN 1247257 C CN1247257 C CN 1247257C
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- erythropoietin
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Abstract
The present invention relates to a stable recombined human erythropoietin preparation, particularly to an injection, which has the essential technical scheme that the present invention contains a certain amount of the following components: recombined human erythropoietin (rHuEPO) as an active component, mannitol as a protein protectant, sodium chloride which is physiologically soluble, maintains a certain pH value and is used as an isotonic agent of buffer solution, and water as the rest. The present invention is characterized in that recombined human erythropoietin (rHuEPO) can be prepared into a soluble and stably stored injection used for treating symptoms of renal anemia, erythrocyte mobilization of a surgery preoperative period, anemia caused by tumor chemotherapy, etc., and has the characteristics of safe and convenient use, economy, no side effect, etc.
Description
Technical field
The present invention relates to a kind of genetic engineering recombiant protein (recombinant human erythropoietin rHuEPO) preparation that does not contain the human albumin and preparation method thereof.
Background technology
(Erythropoietin is a kind of glycoprotein EPO) to erythropoietin, and molecular weight is about 30～39KD.Difference according to its sugared type structure can be divided into two kinds of α, β.Its physiological action is to stimulate hemoglobin or erythrocytic formation in the bone marrow, is mainly generated and entered bone marrow by circulation blood by kidney to play a role.Recombinant human erythropoietin rHuEPO has structure and the physiological action identical with natural erythropoietin EPO, has been widely used in the anemia that erythrocyte is mobilized, chemotherapy of tumors causes of renal anemia, surgery peri-operation period etc. clinically.
EPO working concentration in vivo is very low, and human body is very responsive for the minor variations of EPO level, must be based upon an exogenous hormone that can guarantee this trace accurately on the basis of the preparation of administration so depend on the treatment of rHuEPO.But known rHuEPO is in aqueous solution and unstable (seeing patent US 4,806,524), so the stability that adds one or more protective agents increase rHuEPO in rHuEPO solution prevents that on its inwall that is attracted to container be necessary.
Known in gene recombinant protein quasi drugs preparation, the human albumin is a kind of common and effective stabilizer (seeing patent US 4,703,008).But, inevitably exist potential haematogenous pollution problem (as infecting hepatitis virus, HIV (human immunodeficiency virus) etc.) because the human albumin is a blood products.Therefore research worker is by having adopted the kinds of protect agent to replace the human albumin to the research of rHuEPO.For example patent US 4,879, the lecithin of mentioning in 272, Gentran 40, methylcellulose, Polyethylene Glycol etc.But patent US 4,992, and 419 studies show that with such reagent and can not reach long term stabilization, and promptly the effectiveness of rHuEPO reduces greatly in mouse experiment, and can cause immunogenic response when injecting these reagent; In addition, from patent US 4,992,419 and patent US 5,376,632, the stabilizing agent that some is applicable to lyophilizing rHuEPO preparation be can recognize, carbamide, each seed amino acid and cyclodextrin comprised.But adopt freeze-dried formulation inconvenience for ejection preparation, increased the chance of dissolved step and operation and pollution impurity.In addition,, not only increased cost, and made quality be not easy to control, the stability between the different batches is affected owing to increased freeze dried operation in process of production.
Therefore, problem is to locate a kind of be easy to mix, the rHuEPO preparation that can stablize storage, can guarantee its accurate administration, can prevent that again it is adsorbed on peace bottle and the injector wall; Can not only keep its secular biological activity and stability, and production technology is simple, production cost is low.
Summary of the invention
The purpose of this invention is to provide a kind of genetic engineering recombiant protein (recombinant human erythropoietin, rHuEPO) preparation and preparation method thereof that mix, stable storage that does not contain the human albumin.Repeatedly testing discovery according to us and add mannitol can prevent effectively that rHuEPO is adsorbed in rHuEPO solution, increase the stability of rHuEPO and active, is protective agent so the present invention adopts mannitol, and production technology is simple, and is safe and reliable.
The object of the present invention is achieved like this: the invention provides a kind of recombinant erythropoietin liquor preparation that is mainly used in the treatment renal anemia, said preparation contains protective agent mannitol.Pharmaceutical solutions of the present invention is the aqueous solution for injection agent, can contain following component:
(1) is recombinant human erythropoietin's protein 10 0～20000 iu/milliliter of active component;
Buffer 5～the 100mM that (2) can mix on the physiology, pH4.0～8.5;
(3) isoosmotic adjusting agent sodium chloride 0.05～1.0%;
(4) be protective agent mannitol 1.0～20.0%;
(5) water.
The buffer that mixes on the selected physiology of the present invention can adopt citrate buffer or adopt phosphate buffer or adopt glycine buffer or adopt acetate buffer or carbonate buffer solution, working concentration is 5～100mM, pH4.0～8.5.Be used for the requirement of required pH value of stable prod and drug solubility.
The selected citrate buffer of the present invention can also be got rid of the proteinic destruction of trace heavy metal ion pair in the injection as the chela mediating recipe except keeping the suitable pH value of rHuEPO, the working concentration scope is 5～100mM.
Sodium chloride of the present invention has guaranteed the osmotic pressure that is consistent in injection and the body, and concentration is 0.05～1.0%.
The present invention adopts mannitol to cook protective agent, stops proteinic condensing to increase proteinic dissolubility, and concentration is between 1.0～20.0%.
Also can contain metal ion chela mediating recipe in the preparation of the present invention as the disodium salt disodium, reaction has remarkable catalytic action to trace metal ion to autoxidation, can add the generation that metal ion chela mediating recipe suppresses the autoxidation reaction.
Also can contain the L-histidine hydrochloride in the preparation of the present invention, there are certain biocompatibility in it and protein, and protein is had the certain protection effect, simultaneously, as antioxidant, can reduce the oxidative degradation of rHuEPO in the long preservation process.
Above-described percentage composition is a weight percentage.
Characteristics of the present invention are: use method provided by the invention, can produce a kind of do not contain the human albumin mix, storage period is long and the genetic engineering recombinant human erythropoietin rHuEPO of stable storage.The present invention also has characteristics such as preparation method is simple, production cost is low, effect is good.
The specific embodiment
Below will the present invention will be further described by embodiment, but following example only is the present invention's example wherein, the interest field of not representing the present invention and being limited.
Embodiment 1.
The rHuEPO of 1000 200 units of preparation:
Component content
Citrate buffer solution 20mM
20% mannitol 4%
Sodium chloride 0.585%
Recombinant human erythropoietin's 200 ius
Surplus is a water.
Measure 700 milliliters of ultra-pure waters, accurately take by weighing citrate dihydrate trisodium 5.80 grams, citric acid monohydrate 0.062 gram, sodium chloride 5.85 grams then, be dissolved in 700 milliliters of ultra-pure waters, fully after the dissolving, make A liquid, keep solution temperature, measure the solution pH value between 6.9～7.0 at 20～25 ℃.
Accurately measure 200 milliliter of 20% mannitol and be dissolved in the A liquid, fully mixing keeps solution temperature at 20～25 ℃.Add 2 * 10 in the mixed solution
5The rHuEPO albumen of unit is settled to 1000 milliliters with ultra-pure water.The finished product preparation of preparation rHuEPO.
Finished product preparation is aseptically filled in the container of sterilization.Packing then, every bottle adds 1 milliliter of finished product preparation, and the nitrogen that passes to aseptic filtration in whole process for preparation rolls the rHuEPO injection that lid is made 200 units at last to get rid of air in the bottle.
Embodiment 2.
The rHuEPO of 1000 1000 units of preparation:
Component content
Phosphate buffer 1 0mM
20% mannitol 2%
Sodium chloride 0.584%
Recombinant human erythropoietin's 1000 ius
Surplus is a water.
Measure 700 milliliters of ultra-pure waters, accurately take by weighing sodium hydrogen phosphate 2.51 grams, sodium dihydrogen phosphate 0.47 gram, sodium chloride 5.84 grams, be dissolved in 700 milliliters of ultra-pure waters, fully after the dissolving, make A liquid, keep solution temperature, measure the solution pH value between 6.9～7.0 at 20～25 ℃.
Accurately measure 100 milliliter of 20% mannitol and be dissolved in the A liquid, fully mixing keeps solution temperature at 20～25 ℃.Add 1 * 10 in the mixed solution
6The rHuEPO albumen of unit is settled to 1000 milliliters with ultra-pure water.The finished product preparation of preparation rHuEPO.
Finished product preparation is aseptically filled in the container of sterilization.Packing then, every bottle adds 1 milliliter of finished product preparation, and the nitrogen that passes to aseptic filtration in whole process for preparation rolls the rHuEPO injection that lid is made 1000 units at last to get rid of air in the bottle.
Embodiment 3.
The rHuEPO of 1000 4000 units of preparation:
Component content
Glycine buffer 50mM
20% mannitol 8%
Sodium chloride 0.9%
Disodium salt disodium 0.01%
Recombinant human erythropoietin's 4000 ius
Surplus is a water.
Measure 500 milliliters of ultra-pure waters, accurately take by weighing glycine 3.75 grams, sodium chloride 9.0 grams, be dissolved in 500 milliliters of ultra-pure waters, fully after the dissolving, make A liquid, keep solution temperature, measure the solution pH value between 6.9～7.0 at 20～25 ℃.
Take by weighing disodium salt disodium 0.10 gram, add in the A liquid, fully mixed dissolution keeps solution temperature at 20～25 ℃, and this is a B liquid.
Accurately measure 400 milliliter of 20% mannitol and be dissolved in the B liquid, fully mixing keeps solution temperature at 20～25 ℃.Add 4 * 10 in the mixed solution
6The rHuEPO albumen of unit is settled to 1000 milliliters with ultra-pure water.The finished product preparation of preparation rHuEPO.
Finished product preparation is aseptically filled in the container of sterilization.Packing then, every bottle adds 1 milliliter of finished product preparation, and the nitrogen that passes to aseptic filtration in whole process for preparation rolls the rHuEPO injection that lid is made 4000 units at last to get rid of air in the bottle.
Embodiment 4.
The rHuEPO of 1000 8000 units of preparation:
Component content
Phosphate buffer 1 0mM
20% mannitol 4%
Sodium chloride 0.584%
L-histidine hydrochloride 0.1%
Recombinant human erythropoietin's 8000 ius
Surplus is a water.
Measure 700 milliliters of ultra-pure waters, accurately take by weighing sodium hydrogen phosphate 2.51 grams, sodium dihydrogen phosphate 0.47 gram, sodium chloride 5.84 grams, be dissolved in 700 milliliters of ultra-pure waters, fully after the dissolving, make A liquid, keep solution temperature, measure the solution pH value between 6.9～7.0 at 20～25 ℃.
Take by weighing L-histidine hydrochloride 1 gram, add in the A liquid, abundant mixed dissolution, this is a B liquid.
Accurately measure 200 milliliter of 20% mannitol and be dissolved in the B liquid, fully mixing keeps solution temperature at 20～25 ℃.Add 8 * 10 in the mixed solution
6The rHuEPO albumen of unit is settled to 1000 milliliters with ultra-pure water.The finished product preparation of preparation rHuEPO.
Finished product preparation is aseptically filled in the container of sterilization.Packing then, every bottle adds 1 milliliter of finished product preparation, and the nitrogen that passes to aseptic filtration in whole process for preparation rolls the rHuEPO injection that lid is made 8000 units at last to get rid of air in the bottle.
Embodiment 5.
The rHuEPO of 1000 10000 units of preparation:
Component content
Citrate buffer solution 20mM
20% mannitol 2%
Sodium chloride 0.585%
L-histidine hydrochloride 0.1%
Disodium salt disodium 0.01%
Recombinant human erythropoietin's 10000 ius
Surplus is a water.
Measure 700 milliliters of ultra-pure waters, accurately take by weighing citrate dihydrate trisodium 5.80 grams, citric acid monohydrate 0.062 gram, sodium chloride 5.85 grams, be dissolved in 700 milliliters of ultra-pure waters, fully after the dissolving, make A liquid, keep solution temperature, measure the solution pH value between 6.9～7.0 at 20～25 ℃.
Accurately take by weighing disodium salt disodium 0.1 gram, add in the A liquid, abundant mixed dissolution, this is a B liquid.
Accurately take by weighing L-histidine hydrochloride 1 gram, add in the B liquid, abundant mixed dissolution, this is a C liquid.
Accurately measure 100 milliliter of 20% mannitol and be dissolved in the C liquid, fully mixing keeps solution temperature at 20～25 ℃.Add 1 * 10 in the mixed solution
7The rHuEPO albumen of unit is settled to 1000 milliliters with ultra-pure water.The finished product preparation of preparation rHuEPO.
Finished product preparation is aseptically filled in the container of sterilization.Packing then, every bottle adds 1 milliliter of finished product preparation, and the nitrogen that passes to aseptic filtration in whole process for preparation rolls the rHuEPO injection that lid is made 10000 units at last to get rid of air in the bottle.
In order to measure the bin stability of the different finished product preparation of preparing, carried out the preparation stored controlled trial.Adopt recombinant human erythropoietin (rHuEPO) semi-finished product of same batch (to meet " Chinese biological goods vertification regulation ", purity is greater than 98%), carry out the test preparation of different size preparation, wherein matched group is made into the preparation that contains 0.25% human albumin.Be divided into two groups according to different temperature conditions: 37 ℃ of groups and 4 ℃ of groups, the observation period was respectively 3 months and 2 years, regularly detected outward appearance, pH value, purity and the activity in vivo of rHuEPO in the preparation.The result shows, under 37 ℃ of conditions, included human albumin's matched group in three months all to significantly decrease with the activity that does not contain human albumin's test group, but both do not have evident difference.Under 4 ℃ of conditions, including human albumin's matched group in 2 years does not all have decline with the activity that does not contain human albumin's test group, and both do not have evident difference (the results are shown in following table).
Preserve trimestral experimental result for 37 ℃
| Group | PH value | Purity | Active (IU/ml) | Outward appearance |
| Embodiment 1 | 6.92 | 96.4％ | 137 | Qualified |
| Contrast | 6.98 | 96.8％ | 152 | |
| Embodiment 2 | 7.00 | 95.6％ | 640 | |
| Contrast | 6.94 | 96.1％ | 634 | |
| Embodiment 3 | 7.01 | 96.0％ | 2418 | |
| Contrast | 6.98 | 96.4％ | 2720 | |
| Embodiment 4 | 6.95 | 95.0％ | 4482 | |
| Contrast | 7.00 | 96.2％ | 4640 | |
| Embodiment 5 | 6.90 | 95.5％ | 5506 | |
| Contrast | 6.92 | 96.0％ | 5524 |
4 ℃ of experimental results of preserving 2 years
| Group | PH value | Purity | Active (IU/ml) | Outward appearance |
| Embodiment 1 | 7.00 | 98.4％ | 212 | Qualified |
| Contrast | 6.96 | 98.8％ | 208 | |
| Embodiment 2 | 6.94 | 99.2％ | 986 | |
| Contrast | 6.98 | 99.0％ | 1020 | |
| Embodiment 3 | 6.97 | 99.0％ | 3890 | |
| Contrast | 7.00 | 99.2％ | 3865 | |
| Embodiment 4 | 6.92 | 98.5％ | 7921 | |
| Contrast | 6.89 | 99.2％ | 7963 | |
| Embodiment 5 | 6.95 | 98.9％ | 10040 | |
| Contrast | 6.97 | 99.0％ | 10120 |
The result shows, use method provided by the invention, produce a kind of genetic engineering recombinant human erythropoietin rHuEPO that mix, stable storage that does not contain the human albumin, can keep stable 4 ℃ of activity of preserving 2 years recombinant human erythropoietin rHuEPO.
Claims (7)
1, a kind of erythropoietin liquor preparation is characterized in that, it is composed as follows:
(1) erythropoietin, 100～20000 iu/milliliters;
Buffer solution 5～the 100mM that (2) can mix on the physiology, pH4.0～8.5;
(3) sodium chloride, 0.05～1.0%;
(4) mannitol, 1.0～20.0%;
(5) metal ion chela mediating recipe, 0.001～0.2%;
(6) L-histidine hydrochloride, 0.01～1.5 ‰;
(7) water.
2, the preparation of claim 1, buffer solution wherein are selected from phosphate buffered solution, citric acid solution, glycine buffer solution, carbonate buffer solution, acetate buffer solution.
3, the preparation of claim 1, described metal ion chela mediating recipe is the disodium salt disodium.
4, the preparation of claim 1, erythropoietin wherein are that gene recombinaton erythropoietin content is 200～10000 iu/milliliters.
5, the preparation method of the preparation of claim 1 is characterized in that, mannitol, erythropoietin, metal ion chela mediating recipe, L-histidine hydrochloride are mixed with the buffer solution that contains sodium chloride.
6, the preparation of claim 1 is an injection.
7, the application of the preparation of claim 1 in the medicine that the preparation promoting erythrocyte generates.
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| CN 03100653 CN1247257C (en) | 2003-01-20 | 2003-01-20 | Stable erythropoietin of recombined human red blood cell |
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| CN 03100653 CN1247257C (en) | 2003-01-20 | 2003-01-20 | Stable erythropoietin of recombined human red blood cell |
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| CN1247257C true CN1247257C (en) | 2006-03-29 |
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Owner name: SHENYANG SANSHENG PHARMACEUTICAL CO. Free format text: FORMER NAME OR ADDRESS: SANSHENG PHARMACY CO., LTD., SHENYANG CITY |
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Address after: 1 No. 3, ten, road 110141, Shenyang economic and Technological Development Zone, Liaoning, China Patentee after: SHENYANG SUNSHINE PHARMACEUTICAL Co.,Ltd. Address before: 1 No. 3, ten, road 110141, Shenyang economic and Technological Development Zone, Liaoning, China Patentee before: Shenyang Sunshine Pharmaceutical Co.,Ltd. |
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