CN101015684B - Recombinant erythropoietin liquor preparation - Google Patents
Recombinant erythropoietin liquor preparation Download PDFInfo
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- CN101015684B CN101015684B CN 200610012397 CN200610012397A CN101015684B CN 101015684 B CN101015684 B CN 101015684B CN 200610012397 CN200610012397 CN 200610012397 CN 200610012397 A CN200610012397 A CN 200610012397A CN 101015684 B CN101015684 B CN 101015684B
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Abstract
This invention relates to a solution preparation of recombinant human erythropoietin. The preparation is prepared from recombinant human erythropoietin, recombinant human serum albumin, sodium citrate, and sodium chloride buffer. It can be used for treating renal anemia, preoperative surgical red blood cell mobilization, and anemia due to chemotherapy of tumor.
Description
Technical field
The present invention relates to a kind of erythropoietin (EPO), particularly relate to the pharmaceutical solutions of a kind of recombinant human erythropoietin (rhEPO).
Background technology
Erythropoietin is the erythropoietic peptide hormone of a kind of adjusting, and 90% is produced by kidney.The recombinant human erythropoietin is all identical with the structure and the physiological action of natural erythropoietin, has been widely used in the anemia that erythrocyte is mobilized and chemotherapy of tumors causes of renal anemia, surgery peri-operation period etc. clinically.
The human albumin is a kind of blood products, extracts through methods such as cold induced proteins separation by healthy people's blood plasma, and the preparation of behind inactivation of viruses, making.The human albumin is because the characteristics of self, as rigid-framed structure, good stability, binding ability that bio-compatibility is good, strong, can play slow releasing function to other drug, be often used as medicine, especially the adjuvant of recombinant protein medicine, gathering, modification and the degraded of protein drug can be prevented, and the consumption of other stabilizing agent can be significantly reduced.What recombinant human erythropoietin's formulation products in the market generally adopted is to be the pharmaceutical formulation of adjuvant with human albumin.For example, recombinant human erythropoietin's injection (trade name: Epogen of U.S. Amgen (Amgen)
), (trade name: the Ji arteries and veins is glad for recombinant human erythropoietin's injection of North China Pharmacuetical Jintan Biotechnology Co., Ltd
) all adopt blood source albumin as stabilizing agent.
Because blood source albumin raw material sources are in human blood, though The World Health Organization (WHO), food and drug administration (FDA), human drug legislation specification requirement international coordination meeting (ICH) and state food and drug administration (SFDA) etc. to blood products and raw materials for production thereof, all there are strict requirements for process, but, exist potential viral pollution problem (as hepatitis virus, HIV (human immunodeficiency virus) etc.) inevitably as blood products.In addition, also there is periodically source of goods problem of shortage in blood source albumin.Therefore, research worker is being sought the stabilizing agent that can replace the human albumin always.
Summary of the invention
Compare with blood source albumin (pHSA), the chemical homogeneous degree of recombinant human serum albumin (rHSA) is higher.From the albumin of blood plasma purification preparation at the very big (Day of differences between batches aspect glycosylation, fat content and the bonded micromolecule aglucon, et al., " Nonenzymatically Glucosylated Albumin ", The Journalof Biological Chemistry, vol.254, No.3, pp.595-597, Feb.10,1979.), then there are not these problems in rHSA.Simultaneously, among the rHSA dimer and oligomer content far below the aggressiveness content among the pHSA.So recombinant human serum albumin is the more more clear and definite protein of homogeneous, composition of chemical constituent, aspect the physiologically active that keeps protein drug, has the incomparable advantage of blood source albumin.
The present invention utilizes the stabilizing agent of recombinant human serum albumin as the recombinant human erythropoietin, has solved pathogen contamination and periodicity source of goods problem of shortage that blood source albumin may bring.The present invention is that adjuvant prepares recombinant erythropoietin liquor preparation with blood source albumin and recombinant human serum albumin.(37 ℃, 25 ℃, 2-8 ℃) were placed 3-24 month under different ambient temperatures, compare with pHSA, with rHSA is that the rhEPO that stabilizing agent prepares has all shown higher activity in vivo and in vitro, and rHSA has kept purity and the stability higher than pHSA in the rhEPO pharmaceutical solutions.
Description of drawings
Fig. 1: rhEPO places 3 months external activity testing result under 37 ℃ of environment;
Fig. 2: rhEPO places 3 months activity in vivo testing result under 37 ℃ of environment;
Fig. 3: rhEPO places 15 months external activity testing result under 25 ℃ of environment;
Fig. 4: rhEPO places 15 months activity in vivo testing result under 25 ℃ of environment;
Fig. 5: rhEPO places 24 months external activity testing result under 2-8 ℃ of environment;
Fig. 6: rhEPO places 24 months activity in vivo testing result under 2-8 ℃ of environment;
Among Fig. 1-Fig. 6,
2.5mg/ml rHSA
4.0mg/ml rHSA
2.5mg/ml pHSA.
The specific embodiment
Embodiment
RhEPO stock solution: provide by North China Pharmacuetical Jintan Biotechnology Co., Ltd
RHSA: provide by North China Pharmacuetical Group New Drug Research ﹠ Development Co., Ltd
PHSA: available from Shanghai Laishi Blood Products Co.,Ltd
Enzyme-linked immunoassay instrument: microplate reader (Molecular Device, USA) (model: VERSAmax)
High-pressure liquid phase: Waters HPLC high-pressure liquid phase system (996 photodiode array detector)
Embodiment 1: contain the preparation of the 3000IU/ml rhEPO of rHSA
Prescription:
The constituent concentration consumption
rhEPO 3000IU/ml 165ml
rHSA 2.5mg/ml 137.5ml
Sodium citrate, chlorination sodium citrate 20mmol/L, sodium chloride
10697.5ml
Sodium buffer 100mmol/L (transferring pH to 6.9)
10000 components
Get rhEPO stock solution (2.0 * 10
5IU/ml) 165ml adds rHSA (200mg/ml) 137.5ml, adding citric acid sodium, sodium chloride buffer 10697.5ml.Fully behind the mixing, be aseptically filled in the container of sterilization, be the semi-finished product of rhEPO.
Then with the semi-finished product solution fill to the 3ml cillin bottle, every bottle adds 1.1ml.Jump a queue, roll lid, labeling at last, make the rhEPO injection that 3000IU/ props up.
Embodiment 2: contain the preparation of the 1500IU/ml rhEPO of rHSA
Prescription:
The component content consumption
rhEPO 1500IU/ml 82.5ml
rHSA 2.5mg/ml 137.5ml
Sodium citrate, chlorination sodium citrate 20mmol/L, sodium chloride
10780ml
Sodium buffer 100mmol/L (transferring pH to 6.9)
10000 components
Preparation method is with embodiment 1
Embodiment 3: contain the preparation of the 6000IU/ml rhEPO of rHSA
Prescription:
The component content consumption
rhEPO 6000IU/ml 330ml
rHSA 4.0mg/ml 220ml
Sodium citrate, chlorination sodium citrate 20mmol/L, sodium chloride
10450ml
Sodium buffer 100mmol/L (transferring pH to 6.9)
10000 components
Preparation method is with embodiment 1
Embodiment 4: contain the preparation of the 3000IU/ml rhEPO of pHSA
Prescription:
The component content consumption
rhEPO 3000IU/ml 165ml
pHSA 2.5mg/ml 137.5ml
Sodium citrate, chlorination sodium citrate 20mmol/L, sodium chloride
10697.5ml
Sodium buffer 100mmol/L (transferring pH to 6.9)
10000 components
Get rhEPO stock solution (2.0 * 10
5IU/ml) 165ml adds pHSA (200mg/ml) 137.5ml, adding citric acid sodium, sodium chloride buffer 10697.5ml.Fully behind the mixing, be aseptically filled in the container of sterilization, be the semi-finished product of rhEPO.
Then with the semi-finished product solution fill to the 3ml cillin bottle, every bottle adds 1.1ml.Jump a queue, roll lid, labeling at last, make the rhEPO injection that 3000IU/ props up.
Experimental example
Experimental example 1:rhEPO is active to be detected
In order to prove conclusively the steady quality of biological product in the storage life, at least three batches of preproductions should be deposited in and carry out study on the stability under the different condition, general storage condition comprises 2～8 ℃, room temperature, 37 ℃ (" biotech drug research and development and quality control ", the Wang Junzhi chief editor, Science Press, 2002, the 173rd page).Sample was placed respectively 3 months, 15 months and 24 months under the environmental condition of 37 ℃, 25 ℃, 2-8 ℃.Detect interior, the external activity (" Chinese biological goods rules " version in 2000, the recombinant human erythropoietin makes and vertification regulation, the 399-408 page or leaf) of body of each batch rhEPO, investigate the influence of rHSA the rhEPO biologic activity.
Pharmaceutical solutions according to embodiment 1, and the rhEPO of the 3000IU/ml specification of rHSA content 4.0mg/ml, pHSA content 2.5mg/ml (with reference to embodiment 1 preparation), the active testing result in three batches inside and outsides averaged be figure (seeing Figure of description 1-6).The result shows that rHSA and rhEPO compatibility are good.Under the condition of identical storage temperature and holding time, compare with the pHSA of 2.5mg/ml, rHSA with 2.5mg/ml and 4.0mg/ml makes adjuvant, can more effectively keep the effect of inside and outside activity, the especially rHSA2.5mg/ml of rhEPO obviously to be better than pHSA 2.5mg/ml.
Experimental example 2:rHSA Detection of Stability
The rhEPO of the 3000IU/ml specification of embodiment 1 and embodiment 4 preparations, be positioned over respectively in the environment of 37 ℃, 25 ℃, 2-8 ℃, regularly with molecular sieve high pressure liquid chromatography (HPLC) (SEC-HPLC, Superdex200HR10/30) rHSA and the pHSA purity in the detection rhEPO pharmaceutical solutions, the stability of rHSA and pHSA in the investigation preparation.The result shows that rHSA can keep the purity higher than pHSA in the rhEPO pharmaceutical solutions, have good stable.Testing result sees Table 1.
The Detection of Stability method:
Instrument: Waters HPLC system (996 photodiode array detector)
Chromatographic column: Superdex200 HR10/30 (Amersham Biosciences, Sweden)
Mobile phase: 100mmol/L PB, pH7.0
Flow velocity: 0.5ml/min
Applied sample amount: 50 μ l
Detect wavelength: 280nm
HSA purity (%) in the table 1rhEPO pharmaceutical solutions
The inventor has also carried out same experiment by experimental example 1 and experimental example 2 to the rhEPO pharmaceutical solutions of 1500IU/ml, the 6000IU/ml of embodiment 2 and embodiment 3 preparations.Experimental result unanimity, recombinant human serum albumin keep in the rhEPO pharmaceutical solutions than the higher purity of blood source albumin (containing the albuminous rhEPO in blood source with reference to what embodiment 4 can prepare 1500IU/ml, 6000IU/ml equally), and stability is also better.
Claims (5)
1. recombinant erythropoietin liquor preparation is made up of recombinant human erythropoietin and recombinant human serum albumin and sodium citrate, sodium chloride buffer.
2. the pharmaceutical solutions of claim 1, wherein recombinant human serum albumin content is 2.5-4.0mg/ml.
3. the pharmaceutical solutions of claim 2, wherein recombinant human serum albumin content is 2.5mg/ml.
4. the pharmaceutical solutions of claim 2, wherein recombinant human serum albumin content is 4.0mg/ml.
5. the arbitrary described pharmaceutical solutions of claim 1 to 4 is injection.
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| CN 200610012397 CN101015684B (en) | 2006-02-10 | 2006-02-10 | Recombinant erythropoietin liquor preparation |
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|---|---|---|---|
| CN 200610012397 CN101015684B (en) | 2006-02-10 | 2006-02-10 | Recombinant erythropoietin liquor preparation |
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| CN101015684B true CN101015684B (en) | 2011-08-24 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4879272A (en) * | 1984-10-09 | 1989-11-07 | Chugai Seiyaku Kabushiki Kaisha | Method and composition for preventing the adsorption of a medicine |
| CN1354669A (en) * | 1999-04-09 | 2002-06-19 | 奥索-麦克尼尔药品公司 | Pharmaceutical compositions of erythropoietin |
| CN1429116A (en) * | 2000-05-15 | 2003-07-09 | 霍夫曼-拉罗奇有限公司 | Pharmaceutical composition |
| EP1475104A1 (en) * | 2003-01-14 | 2004-11-10 | Nipro Corporation | Stabilized proteinic preparation |
-
2006
- 2006-02-10 CN CN 200610012397 patent/CN101015684B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4879272A (en) * | 1984-10-09 | 1989-11-07 | Chugai Seiyaku Kabushiki Kaisha | Method and composition for preventing the adsorption of a medicine |
| CN1354669A (en) * | 1999-04-09 | 2002-06-19 | 奥索-麦克尼尔药品公司 | Pharmaceutical compositions of erythropoietin |
| CN1429116A (en) * | 2000-05-15 | 2003-07-09 | 霍夫曼-拉罗奇有限公司 | Pharmaceutical composition |
| EP1475104A1 (en) * | 2003-01-14 | 2004-11-10 | Nipro Corporation | Stabilized proteinic preparation |
Non-Patent Citations (1)
| Title |
|---|
| 侯绪凤等.重组人红细胞生成素新制剂及其稳定性研究.《微生物学杂志》.2003,第23卷(第01期),15-18. * |
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Owner name: NORTH-CHINA PHARMACEUTICAL JINTAN BIOTECHNOLOGY CO Free format text: FORMER OWNER: NCPC NEW DRUG RESEARCH AND DEVELOPMENT CO., LTD. Effective date: 20150728 |
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Effective date of registration: 20150728 Address after: 050035 No. 106 Tianshan Avenue, Shijiazhuang hi tech Development Zone, Hebei, China Patentee after: North-China Pharmaceutical Jintan Biotechnology Co., Ltd. Address before: 050015 Heping East Road, Hebei, Shijiazhuang, No. 388 Patentee before: Ncpc New Drug Research And Development Co., Ltd. |