CN101381412B - Polymer/recombinant human erythropoietin couple - Google Patents
Polymer/recombinant human erythropoietin couple Download PDFInfo
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Abstract
The invention discloses a polymer/recombinant human erythropoietin conjugate, wherein the polymer is a derivative of a hydrophilic polymer selected from polyethylene glycol, polypropylene glycol, polyvinyl alcohol, polypropylene morpholine and copolymers of the polyethylene glycol, the polypropylene glycol, the polyvinyl alcohol and the polypropylene morpholine, and the molecular weight of P is between 18,000 and 50, 000 daltons. The invention also discloses a medicine compound containing the conjugate, an application of the conjugate and a preparation method.
Description
Technical field
The present invention relates to a kind of conjugate of recombinant human erythropoietin; Particularly relate to a kind of polymkeric substance/recombinant human erythropoietin couple, contain medicine and this conjugate application aspect the preparation medicine of this conjugate, the invention still further relates to the preparation method of polymkeric substance/recombinant human erythropoietin couple.
Background technology
Erythropoiesis (Erythropoiesis) is meant the generation of erythrocyte, to compensate erythrocytic loss.It is a kind of controlled physiological mechanism that red blood corpuscle generates, and it provides enough erythrocytes to be used to organize oxygen supply.People's erythropoietin in the human body (Erythropoietin is called for short EPO) is by a kind of hormonelike material of kidney and hepatic secretion, can promote erythropoiesis.Natural EPO stimulates the division and the differentiation of directed red corpuscle progenitor cell in the marrow, and through bringing into play its BA (Krantz, BS (1991) Blood 77:419) on the acceptor that is attached to the red corpuscle precursor.
People's erythropoietin is through using recombinant DNA technology to be produced (Egrie, JC, Strickland; TW; Lane, J et al. (1986) Immunobiol.72:213-224), it is that Chinese hamster ovary histocyte (Chinese hamster ovary celI) and expressed products are therein inserted in people EPO gene clone.EPO is a kind of glycoprotein hormones, and the EPO that exists in the blood plasma is made up of 165 amino acid, and degree of glycosylation is very high.The molecular weight that does not have the EPO polypeptied chain of sugar moieties is 18,236Da.In complete EPO molecule, carbohydrate group accounts for the about 40% of molecular weight, this carbohydrate group at proteic glycosylation site place with protein glycosylation (Sasaki; H, Bothner, B; Dell, A andFukuda, M (1987) J.Biol.Chem.262:12059).
Because people's erythropoietin is extremely important in erythropoiesis, therefore this hormone can be used to low or lack in the treatment of blood disorder that erythropoiesis is a characteristic.Clinically, EPO can be used for treating anaemia (Eschbach, JW, Egri, JC, Downing, MR et al. (1987) the NEJM 316:73-78 among the chronic renal failure patient (CRF); Eschbach, JW, Abdulhadi, MH, Browne, JK et al. (1989) Ann.Intern.Med.111:992; Egrie, JC, Eschbach, JW, McGuire, T, Adamson, JW (1988) Kidney Intl.33:262; Lim, VS, Degowin, RL; Zavala, Det al. (1989) Ann.Intern.Med.110:108-114) and AIDS and the treatment (Dana, RP, the Rudnick that accept the cancer patients of chemotherapy; SA, Abels, RI In:MB; Garnick, ed.Erythropoietin in Clinical Applications-An International Perspective.New York, N.Y.:Marcel Dekker; 1990:P.301-324).
At present, existing on the market a lot of commercially available epo protein preparations, but because plasma half-life is very short and be prone in vivo be degraded, the bioavailability of EPO is greatly limited.
The Pegylation technology is a kind of effective ways that therapeutic protein transports in vivo that improve.Pegylation has changed the pharmacokinetic properties of medicine, and then pharmacodynamics is exerted an influence.Pegylation can reduce the removing of kidney to medicine, and the purpose that makes some drugs behind subcutaneous administration, can keep more stable absorption and reach restricted distribution.The change of these pharmacokinetics can bring and continue and stable plasma drug level, and then can improve the clinical efficacy relevant with Plasma Concentration.In addition, Pegylation can also reduce because the untoward reaction that the peak-the paddy Plasma Concentration is brought that frequent drug administration causes can also reduce proteic immunogenicity simultaneously.Reduce the spatial obstacle of medicine behind the Pegylation, thereby avoided immune identification, reduced the immunogenicity of pharmaceutical grade protein.
At present existing bibliographical information Pegylation alpha-interferon; Find through different Pegylation alpha-interferons carefully being assessed the back; Different pegylated medicaments exists difference on pharmacokinetics; Different polyoxyethylene glycol (PEG) conjugates have different effect characteristics, bring the difference of relevant pharmacodynamics thus.Because therefore the size of Pegylation part, conformation and can produce critical influence to these characteristics with the combining site of medicine, must carry out the Pegylation design of individuation to different proteins one by one, just possibly reach the result of treatment of hope.The length of every kind of polyoxyethylene glycol (PEG) molecule and shape are very important in decision aspect the influence of pharmacokinetics and pharmacodynamic properties.And, be necessary for the target treatment and select suitable or best PEG molecule with protein molecule because protein has nothing in common with each other.
Summary of the invention
The objective of the invention is the deficiency to prior art, provide a kind of and can effectively promote erythropoiesis, greatly improve conjugate, the pharmaceutical composition that comprises this conjugate and this conjugate application aspect the preparation medicine of the recombinant human erythropoietin of RT in vivo.
Another object of the present invention is to provide the preparation method of said conjugate.
For realizing above-mentioned purpose, the present invention has adopted following technical scheme:
The invention discloses a kind of polymkeric substance/recombinant human erythropoietin couple, said conjugate has following general formula:
Wherein, T is selected from following carbohydrate target agent: glucose, seminose, semi-lactosi, lactose, fructose or its oligosaccharides and verivate;
P is selected from following hydrophilic polymer: polyoxyethylene glycol, W 166, Z 150PH, Vestolen PP 7052 morpholine or their multipolymer, and the molecular weight of P is 18,000～50,000 dalton;
EPO is the recombinant human erythropoietin.
The binding site of said conjugate is the ε amino of lysine side-chain on the EPO aminoacid sequence or the imino-on terminal Histidine α amino of N-or the imidazolyl.
Preferably, P is a polyoxyethylene glycol, and the molecular weight of P is 30,000～40,000 dalton.
In the preferred embodiment of the present invention, said conjugate has with the structure shown in the following formula (II):
Wherein P is a polyoxyethylene glycol, and the molecular weight of P is 35,000 ± 3500 dalton.
In the concrete embodiment of the present invention, said EPO is the recombinant human erythropoietin that utilizes Chinese hamster ovary line to prepare through recombinant DNA technology.
Of the present invention preferred embodiment in, said EPO adopts the method that comprises following steps to prepare: make up and include the Chinese hamster ovary line of recombinant human epo's gene and carry out cell cultures, purifying epo protein from cell culture.
The invention also discloses the pharmaceutical composition that comprises above-mentioned polymkeric substance/recombinant human erythropoietin couple.
The present invention further discloses above-mentioned polymkeric substance/recombinant human erythropoietin couple and be used for treating the application of the medicine of erythropoiesis deficiency disorders in preparation.
The invention also discloses a kind of preparation method with polymkeric substance/recombinant human erythropoietin couple of the structure shown in the formula (II), said method comprises that the polyethyleneglycol derivative that will have structure shown in the following formula (III) mixes with the recombinant human erythropoietin,
Wherein, P is a polyoxyethylene glycol, and the molecular weight of P is 35,000 ± 3500 dalton;
Reaction times is at least 2 hours, and pH value in reaction is 7.0～7.5, and the mol ratio of said polyethyleneglycol derivative and recombinant human erythropoietin is 20～25: 1.
Preferably, the reaction times is 2.5 hours, and pH value in reaction is 7.2, and the mol ratio of said polyethyleneglycol derivative and recombinant human erythropoietin is 25: 1, and temperature of reaction is a room temperature.
Owing to adopted above technical scheme, the beneficial effect that the present invention is possessed is:
The present invention has the conjugate of single substituted type EPO of ad hoc structure through preparation; The single substituted type Pegylation EPO (PEG-EPO) that particularly has ad hoc structure through preparation; Can effectively promote red corpuscle growth greatly to have improved EPO RT in vivo, make the pharmaceutical prepn that these conjugates (like PEG-EPO) are prepared into; Under the prerequisite that guarantees result of treatment; Its medicine frequency reduces greatly, can be reduced to per at least two weeks injection once like the subcutaneous injection frequency, even preferred every injection all around once.
After polyethyleneglycol derivative is modified epo protein, molecular weight of albumen and sterically hindered increase, heat and ph stability are improved; And single Pegylation EPO molecule that replaces of the present invention has strict tissue distribution, only can pass through kidney and liver by metabolism, and the transformation period prolongs, and can reduce medication dose; Around the epo protein of Pegylation, can form one deck special " shell ", can reduce immunoreation and toxic side effect, reduce the vivo degradation effect, keep original BA simultaneously again.
Description of drawings
The GFC detected result figure of the PEG-EPO product that Fig. 1 makes for the embodiment of the invention 1.
The SDS-PAGE detected result figure of the PEG-EPO product that Fig. 2 prepares for the embodiment of the invention 1.
Fig. 3 is the reticulocyte percentage ratio graphic representation of the embodiment of the invention 3.
Fig. 4 is the RET figure of the embodiment of the invention 3.
Fig. 5 is the RBC figure of the embodiment of the invention 3.
Fig. 6 is the HCT figure (manual capillary vessel method) of the embodiment of the invention 4.
Fig. 7 is the HCT figure (sysmex) of the embodiment of the invention 4.
Fig. 8 is the HGB figure (sysmex) of the embodiment of the invention 4.
Fig. 9 is the RET figure (sysmex) of the embodiment of the invention 4.
Figure 10 is the RBC figure (sysmex) of the embodiment of the invention 4.
Embodiment
The present invention can effectively promote the red corpuscle growth on the one hand through preparation polymkeric substance/recombinant human erythropoietin couple, and the conjugate for preparing on the other hand can be kept the long metabolism time in vivo.Especially, the present invention is through selecting specific polymkeric substance for use, the conjugate that obtains after itself and recombinant human erythropoietin the are combined recombinant human erythropoietin couple of the prior art of comparing, and the survival time can further prolong in vivo, therefore makes medicine frequency become lower.
Polymkeric substance/recombinant human erythropoietin couple of the present invention is meant the conjugate with following structure:
Wherein, T is selected from following carbohydrate target agent: glucose, seminose, semi-lactosi, lactose, fructose or its oligosaccharides and verivate;
P is selected from following hydrophilic polymer: polyoxyethylene glycol, W 166, Z 150PH, Vestolen PP 7052 morpholine or their multipolymer, and the molecular weight of P is 18,000～50,000 dalton;
EPO is the recombinant human erythropoietin.In the preferred embodiment of the present invention, the EPO stoste that EPO preferably produces with Shenzhen Sciprogen Biology Medicine Co., Ltd.The preparation of this EPO stoste mainly is divided into steps such as cell strain structure, cell cultures and protein purification.
The EPO that Shenzhen Sciprogen Biology Medicine Co., Ltd produced is a kind of recombinant human protein; Through in Chinese hamster ovary cell (Chinese hamster ovary cell, CHO) clone, prepare (EP-B 0148605 and EP-P 0209539) through recombinant DNA technology.The clone and the expression method of EPO molecule are well known to those skilled in the art, and are disclosed in patent EP-B 0148605, EP-P0209539, and the content of each document is added as reference at this.
Cell cultures is the working cardial cell storehouse taking-up from liquid nitrogen is irritated that a bottle is derived from the Chinese hamster ovary celI system that produces EPO, is transferred in the square vase, and is cultured in the substratum of the buffered with bicarbonate in the moistening CO2gas incubator.Detect cell density after the some amount with increasing in cell transfer to the glass rolling bottle; After initial vegetative period; Cell culture is diluted with fresh substratum, reach initial cell density after, be inoculated in the cell cultures jar; Perfusion is cultivated, and the mode that stream adds is gathered in the crops and contained the cell culture of expressing EPO.The cell culture processes of EPO molecule is well known to those skilled in the art; The method of in serum free medium, expressing and preparing EPO is described in: WO96/35718 and european patent application NO.513738, the serum-free fermentation process that contains the recombinaant CHO cell of EPO gene in addition is described in EP-A 0513738, EP-A 0267678.
Protein purification is to be purified into EPO with methods such as blue-sepharose chromatography, DEAE ion exchange chromatography, reversed-phase HPLC, molecular sieves from containing the cell culture of expressing EPO successively.Those skilled in the art know preparation and the purification process of EPO; In EP-A 0267678, described a kind of ion exchange chromatography on the S agarose, a kind of preparation type reversed-phase HPLC and a kind of gel permeation chromatography on the C8 post is used for the EPO that serum-free culture prepares and after dialysis, carries out purifying.At this on the one hand, the gel permeation chromatography step can be flowed the ion exchange chromatography replacement on the S agarose soon.Also can before ion exchange chromatography, carry out the dyestuff chromatography on blue Trisacryl post.
In the preferred embodiment of the present invention, polymkeric substance/recombinant human erythropoietin couple of the present invention preferably has following structure:
And P is polyoxyethylene glycol (PEG), and the molecular weight of P is 35,000 ± 3500 dalton.。
Above-mentioned preferred polymkeric substance/recombinant human erythropoietin couple can be called as single Pegylation EPO molecule (PEG-EPO) that replaces.
In the preferred embodiment of the present invention, through the polyethyleneglycol derivative of selecting ad hoc structure for use EPO is carried out Pegylation, and then obtain single Pegylation EPO molecule (PEG-EPO) that replaces.The EPO molecule of comparing independent, these single Pegylation EPO molecules that replace have the residence time in the long body.With the pharmaceutical prepn that these PEG-EPO make, hypodermic frequency should be at least per two weeks injection once, even every injection all around once.Prepared single replacement Pegylation EPO (like mouse or other animal models) in vivo can effectively promote erythropoiesis, and keeps the long metabolism time, to reach the purpose that reduces medicine frequency.
PEG and verivate have following characteristic: water-soluble very strong, low toxicity and the immunoreation that can not cause are easy in the body remove.After polyethyleneglycol derivative is modified epo protein, molecular weight of albumen and sterically hindered increase, heat and ph stability are improved; And single Pegylation EPO molecule that replaces of the present invention has strict tissue distribution, only can pass through kidney and liver by metabolism, and the transformation period prolongs, and can reduce medication dose; Around the epo protein of Pegylation, can form one deck special " shell ", can reduce immunoreation and toxic side effect, reduce the vivo degradation effect, keep original BA simultaneously again.
PEG-EPO of the present invention obtains through the polyethyleneglycol derivative and the EPO prepared in reaction that will have formula (III) structure.
Wherein, P is a polyoxyethylene glycol, and the molecular weight of P is 30,000～40,000 dalton, further is preferably 35,000 ± 3500.
In the present invention; This polyethyleneglycol derivative is called GLUC-NHS-35K; It is to be that starting raw material prepares with the polyoxyethylene glycol, and its preparation method can be with reference to the patent No. by being put down in writing in 02818455.6 the Chinese invention patent, and the mode that this patent is quoted is in full incorporated the present invention into.The sample that adopts GLUC-NHS-35K and EPO prepared in reaction to obtain is called G-PEG-EPO or PEG-EPO in the present invention.
The reaction conditions for preparing G-PEG-EPO of the present invention is at room temperature, and the pH value is reaction in 7.0～7.5 o'clock at least 2 hours, and the mol ratio of said polyethyleneglycol derivative and recombinant human erythropoietin is 20～25: 1.In the preferred embodiment of the present invention, reaction conditions in room temperature under both mixed back oscillatory reaction 2.5 hour, and the mol ratio of said polyethyleneglycol derivative and recombinant human erythropoietin is 25: 1 for being at pH at 7.2 o'clock.
Another aspect of the present invention relates to the pharmaceutical composition that comprises polymkeric substance/recombinant human erythropoietin couple.Polymkeric substance/recombinant human erythropoietin couple of the present invention can the pure compound form or the appropriate drug compsn carry out administration, the reagent that can adopt any acceptable administering mode or be used for similar applications carries out.
The form of medication that adopts can be selected in through port, the nose, rectum, transdermal or drug administration by injection mode, and its form is solid, semisolid, lyophilized powder or liquid preparation form administration, the preferred presented in unit dosage form that adopts the simple administration that is applicable to exact dosage desired.Pharmaceutical composition of the present invention can comprise conventional pharmaceutical carrier or vehicle and as the polymkeric substance/recombinant human erythropoietin couple of the present invention of activeconstituents, in addition, also can comprise other medicament, carrier, assistant agent.
The preferred route of administration of the present invention is a drug administration by injection, adopts conventional per daily dose scheme, and this scheme can be adjusted according to the severity of disease.Conjugate of the present invention or its pharmacy acceptable salt also can be mixed with the injection agent; For example use about 0.5 to about 50% activeconstituents to be scattered in the medicinal adjuvant that can adopt the liquid form administration; Instance is water, salt solution, dextrose hydrate, glycerine, ethanol etc., thereby forms solution or suspensoid.
Through concrete embodiment the present invention is done further detailed description below.
Embodiment 1
The preparation of G-PEG-EPO and detection
Get 2mL EPO stoste (EPO content 5mg, Shenzhen Sciprogen Biology Medicine Co., Ltd, lot number :), add the 20mM phosphoric acid buffer of 3mL pH7.2, the final concentration of EPO is 1mg/mL.With the accurate weighing 109.4mg of Sartorius balance GLUC-NHS-35K, add the phosphoric acid buffer dissolving of 1094 μ L 2mM and pH3.0.GLUC-NHS-35K solution is all added in the EPO solution, and the pH that test paper is measured EPO solution is 7.2.Reaction mixture is placed on the vibrator, room temperature oscillatory reaction 2.5 hours.After reaction finishes, can pass through anion-exchange chromatography purification reaction product:
Ion exchange column: 2 * 5mL DEAE FF
Mobile phase A 1:50mM Bistris pH8.0
Mobile phase A 3:50mM Bistris pH8.0,1M NaCl.
Flow velocity: 10.0mL/min
Sample size: reaction mixture (EPO content 5mg) adds goes up appearance after deionized water is diluted to 100mL.
Collect component: 27-50
After reaction finished, product was stored in refrigerator, gets 5 μ L products simultaneously, added 25 μ L water dilution carrying out GFC and detected.
GFC detects chromatographic column: Waters UltrahydrogelTM 500,7.8 * 300mm, PartNo.WAT011530
Flow velocity: 0.5mL/min
Column temperature: room temperature
30 minutes working times
UV detector wavelength: 280mn
Sample size: 20 μ L
The GFC detected result is as shown in Figure 1, and the result shows does not have two PEG to replace EPO and free EPO in the reaction product of GLUC-NHS-35K and EPO.
Further adopt SDS-PAGE to detect degree of purity of production, the result is as shown in Figure 2.The result shows does not have two PEG to replace EPO and free EPO in the reaction product of GLUC-NHS-35K and EPO.
The external activity of G-PEG-EPO detects
The external activity of the G-PEG-EPO that employing ELISA double antibody sandwich method detection embodiment 1 makes is 4481IU/mL, and external is 8821IU/mg than living.And simultaneously, the external activity that detects used EPO stoste is 531480IU/mL, and external is 1.6 * 10 than work
5IU/mg.
The most of epitope position electricity that presentation of results embodiment 1 makes EPO among the G-PEG-EPO is modified the G-PEG that connects and is covered, and the EPO external activity that causes adopting the ELISA double antibody sandwich method to detect is about original 1/20.
G-PEG-EPO activity in vivo determination test
Purpose: whether measure G-PEG-EPO effectively with whether long-acting
Method: reticulocyte method (RET) and hematocrit (HCT)
Laboratory animal: the female BALB/C mice of SPF level (body weight 18～20g, female)
1, reticulocyte method (RET) test:
1.1 reticulocyte method (RET) test is divided into groups
| Test group 1 | |
|
| The blank group | EPO stoste group | The G-PEG-EPO sample sets |
The G-PEG-EPO of used G-PEG-EPO for preparing among the embodiment 1.
1.2 blood sampling time point
Injection back the 1st day to the 15th day totally 15 days
1.3 experimental animal number
Blank group treated animal number is 10,
EPO stoste treated animal number is 10,
G-PEG-EPO sample sets number of animals is 10,
Totally 30
1.4 injection and sampling
Blank group subcutaneous injection saline water, EPO stoste group and G-PEG-EPO sample sets are respectively with 0.08ug/ dosage subcutaneous injection only.Get mouse tail passages through which vital energy circulates blood every day after medication, presses the reticulocyte percentage ratio of each mouse of conventional determining.
1.5 the result is shown in Fig. 3～5.
Normal erythrocyte mouse bioassay is well known in the art, measures with mouse reticulocyte parallel method.Can find out that from shown in Fig. 3～5 through to behind the sample of every injected in mice with dosage, G-PEG-EPO has the reticulocyte and the red corpuscle of obvious increase, show that G-PEG-EPO has the transformation period in higher activity in vivo and the body that obviously prolongs.，
2, hematocrit (HCT) test
2.1 hematocrit (HCT) test is divided into groups
| Test group | ID (the every mouse of ug/) | Frequency injection weekly | ID (the every mouse of ug/) weekly |
| Blank group a | Equal-volume saline water | 1 | 0.000 |
| Blank group b | Equal- |
3 | 0.000 |
| EPO stoste group a | 0.080(12IU) | 1 | 0.080 |
| EPO stoste group b | 0.027(4IU) | 3 | 0.080 |
| G-PEG-EPO organizes a | 0.080 | 1 | 0.080 |
| G-PEG-EPO organizes b | 0.027 | 3 | 0.080 |
2.2 blood sampling time point
End around the
2.3 experimental animal number
Blank group a number of animals is 10,
Blank group b number of animals is 10,
It is 10 that EPO stoste group a injects number of animals once in a week,
EPO stoste group b time injection number of animals on every Wendesdays is 10,
It is 10 that G-PEG-EPO group a injects number of animals once in a week,
G-PEG-EPO group b time injection number of animals on every Wendesdays is 10,
Totally 60
2.4 injection and sampling
In 4 weeks of subcutaneous injection medication, kapillary eye socket venous blood collection is pressed the hematocrit (HCT) of each sample of centrifugal determination or with automatic blood analyzer analysis (full-automatic blood cell analyser XT2000iV of Shanghai sysmex company or XT1800IV)
2.5 the result is shown in following table and Fig. 6～10.
| Test group | ID (the every mouse of ug/) | Frequency injection weekly | ID (the every mouse of ug/) weekly | HCT (％) | Increase |
| Blank group a | 0.5ml saline water | 1 | 0.000 | 45.3 | 0.0 |
| Blank group b | 0.5 |
3 | 0.000 | 45.4 | 0.0 |
| EPO stoste group a | 0.080(12IU) | 1 | 0.080 | 48.0 | 2.7 |
| EPO stoste group b | 0.027(4IU) | 3 | 0.080 | 51.9 | 6.5 |
| G-PEG-EPO organizes a | 0.080 | 1 | 0.080 | 48.1 | 2.8 |
| G-PEG-EPO organizes b | 0.027 | 3 | 0.080 | 51.2 | 5.8 |
We have selected to treat in order to evaluation rhEPO clinically index-hematocrit value, reticulocyte count, red blood cell count(RBC), the oxyphorase of anaemia effect, reflect the action effect of G-PEG-rhEPO and rhEPO.Can find out from shown in Fig. 4～10; Through to behind the sample of every injected in mice with dosage; The result of treatment of G-PEG-rhEPO obviously is superior to rhEPO; The recombinant human erythropoietin couple for preparing in the prior art of having delivered of comparing, the survival time further prolongs in vivo, makes medicine frequency become lower.
Above content is to combine concrete preferred implementation to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.For the those of ordinary skill of technical field under the present invention, under the prerequisite that does not break away from the present invention's design, can also make some simple deduction or replace, all should be regarded as belonging to protection scope of the present invention.
Claims (1)
1. the preparation method of polymkeric substance/recombinant human erythropoietin couple, it comprises,
Get 2mL EPO stoste, wherein EPO content is 5mg, adds the 20mM phosphoric acid buffer of 3ml pH7.2;
With the accurate weighing 109.4mg of Sartorius balance GLUC-NHS-35K, add the phosphoric acid buffer dissolving of 1094 μ L 2mM and pH3.0;
GLUC-NHS-35K solution is all added in the EPO solution, and reaction mixture is placed on the vibrator, and room temperature is placed oscillatory reaction 2.5 hours; With
After reaction finishes, through anion-exchange chromatography purification reaction product,
Wherein, GLUC-NHS-35K has structure shown in the following formula (III):
Wherein, P is a polyoxyethylene glycol, and the molecular weight of P is 35,000 dalton.
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| CN200710123683XA CN101381412B (en) | 2007-09-30 | 2007-09-30 | Polymer/recombinant human erythropoietin couple |
| HK09106687.6A HK1127615A1 (en) | 2007-09-30 | 2009-07-22 | Polymer/ recombinant human erythropoietin conjugate |
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| CN102453087B (en) * | 2010-10-22 | 2013-12-25 | 广东赛保尔生物医药技术有限公司 | Method for purification and preparation of mono-substituted PEG-EPO (polyethylene glycol-erythropoietin) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1359392A (en) * | 1999-07-02 | 2002-07-17 | 霍夫曼-拉罗奇有限公司 | Coupled compound of erythropoietin with glycol |
| CN1235939C (en) * | 2002-05-14 | 2006-01-11 | 北京键凯科技有限公司 | Target hydrophili polymer and its combined object with interferon and medicinal composition conteining said combined object |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1359392A (en) * | 1999-07-02 | 2002-07-17 | 霍夫曼-拉罗奇有限公司 | Coupled compound of erythropoietin with glycol |
| CN1235939C (en) * | 2002-05-14 | 2006-01-11 | 北京键凯科技有限公司 | Target hydrophili polymer and its combined object with interferon and medicinal composition conteining said combined object |
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