CN101591387A - PEG-IFN omega conjugate - Google Patents
PEG-IFN omega conjugate Download PDFInfo
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- CN101591387A CN101591387A CNA200810110734XA CN200810110734A CN101591387A CN 101591387 A CN101591387 A CN 101591387A CN A200810110734X A CNA200810110734X A CN A200810110734XA CN 200810110734 A CN200810110734 A CN 200810110734A CN 101591387 A CN101591387 A CN 101591387A
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- human interferon
- interferon omega
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Abstract
The invention discloses PEG-IFN omega conjugate and preparation technology thereof, compare the human interferon omega of unmodified, in human body, has the longer transformation period, can obviously reduce the medication number of times, reduce toxic side effect, be particularly suitable for treating chronic viral infection, be with a wide range of applications clinically Interferon, rabbit ω sensitivity.
Description
Technical field
Relate to the medicine for treating viral infections preparation of a kind of treatment, especially a kind ofly can obviously reduce the medication number of times Interferon, rabbit ω sensitivity, the Pegylation recombinant human interferon alpha 2 conjugates with long-acting with and preparation technology.
Background technology
Interferon, rabbit is naturally occurring small protein of a class and glycoprotein, and they produce the reaction of virus infection and other antigenic stimulation by most of karyocytes and secrete.Interferon, rabbit makes cell can resist virus infection and pair cell shows effect widely.They are by bringing into play its cytoactive with the specific membranes receptors bind of cell surface.
Chronic hepatitis mainly refers to chronic hepatitis B and chronic hepatitis C, and two kinds of hepatitis are all very common in China, especially chronic hepatitis B.If do not carry out antiviral therapy effectively, the patient of part chronic hepatitis develops into liver cirrhosis and liver cancer the most at last.The pharmacoeconomics analysis revealed, the treatment liver cirrhosis that causes of chronic hepatitis or the cost of liver cancer are higher than the needed cost of antiviral therapy of commitment far away.
Interferon, rabbit a is the choice drug of treatment chronic hepatitis B or chronic hepatitis C, can suppress or remove virus effectively, and curative effect is lasting, stops the generation of liver cirrhosis or liver cancer.But common interferon therapy has self unsurmountable shortcoming, and the transformation period is short, has only 4 hours.So Interferon, rabbit has to every other day just inject once, uses very inconvenient.In addition, the host of injection of interferon can produce the antibody at Interferon, rabbit.This reaction can cause the symptom that is similar to influenza, and this symptom is used as the side effect of Interferon, rabbit and reports; This reaction simultaneously also can cause the destruction to Interferon, rabbit, thereby more Large dosage interferon is to reach result of treatment.
In order to solve the deficiency of common Interferon, rabbit on clinical treatment, how tame biopharmaceutical company is devoted to develop long lasting Interferon, rabbit.Its means are by carrying out polyethyleneglycol modified to common Interferon, rabbit.
(Poly ethlene glycol is a kind of water-soluble high-molecular substance of nontoxic, non-immunogenicity pEG) to polyoxyethylene glycol, can close the mode modifying protein by covalency spare bond.Polyethyleneglycol modified pharmaceutical grade protein not only can reduce immunogenicity and toxicity but also can obviously prolong plasma half-life, improves the stability of pharmaceutical grade protein.
Clinically, the Peg-Intron of widespread use comprise the exploitation of Schering Plough company glycol interferon alpha-2b (trade(brand)name, wear happy can, PegIntron); (trade(brand)name, Pai Luoxin PegasyS), are widely used in chronic type b and therapy for hepatitis C by uniting with ribavirin to the glycol interferon alpha-2a of Roche Holding Ag's exploitation.
The invention provides a kind of peg molecule and combine, and recombinant human interferon omega molecule is in conjunction with the preparation method of the Pegylation recombinant human interferon omega medicine of a peg molecule with the N-terminal of recombinant human interferon omega.
Summary of the invention
Primary technical task to be solved by this invention provides and a kind ofly helps suitability for industrialized production and can obviously reduce the medication number of times, be used for the Pegylation recombinant human interferon alpha 2 medicine of chronic hepatitis clinical treatment.
For achieving the above object, the present invention is by the following technical solutions:
A kind of Pegylation recombinant human interferon omega medicine is characterized in that: Pegylation recombinant human interferon alpha 2 medicine is to be connected with covalent linkage with recombinant human interferon omega by polyoxyethylene glycol.
Described Pegylation recombinant human interferon alpha 2 medicine is that the N-terminal by polyoxyethylene glycol and recombinant human interferon omega is connected by covalent linkage.
Each peg molecule connects a recombinant human interferon omega molecule.
Described polyoxyethylene glycol is mono methoxy polyethylene glycol (Monomethoxy polyethyleneglycol, mPEG) activated molecule, comprise mono methoxy polyethylene glycol butyraldehyde, mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD) and Y type polyglycol ethanal, its molecular weight is between 5000-40000; Mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD) most preferably.
The ratio (mol ratio) of polyoxyethylene glycol and recombinant human interferon alpha 2 generation covalent attachment reaction is about 0.25～20, and preferred proportion is between 2.5～10.
Another technical task to be solved by this invention provides a kind of above-mentioned Pegylation recombinant human interferon alpha 2 medicine production technology.
Comprise following process:
1) provides the buffer system of the salt concn of pH 4.0～9.0,100mmol/L;
2) in the buffer system that provides, add an amount of recombinant human interferon omega, polyoxyethylene glycol and NaBH3CN successively;
3) said mixture reacted 12～72 hours at 4～37 ℃;
4) product that obtains of step 3) reaction carries out SDS-PAGE and analyzes, and determines the degree of modifying;
5) dialyse or diluted reaction mixture with the acetate buffer solution of 20mM pH5.0, last CM cationic exchange coloum, with the acetate buffer solution gradient elution of the 20mM pH5.0 that contains 0-1M NaCl, 280nm monitors elution peak;
6) SDS-PAGE analyzes elution peak, collects mainly to contain the single-point modification elution peak that a peg molecule connects a recombinant human interferon alpha 2 molecule;
7) single-point is modified elution peak and is carried out concentration with ultra-filtration centrifuge tube after, lyophilize.
Compared with prior art, outstanding substantive distinguishing features that the present invention possessed and significant technical progress.
Mainly show following 3 aspects:
The advantage of Pegylation recombinant human interferon alpha 2 provided by the present invention-ω medicine provides in vivo the blood levels of longer time, has significantly long-lastingly, reduces frequency injection and makes the patient acceptant;
Recombinant human interferon alpha 2-ω of the present invention adopts engineered method, uses the patent carrier of this research department, use Pichia anomala expression, and through fermenting, obtain behind the purifying, purity is greater than 98%, and keeps higher biological activity;
The present invention adopts the modifying method high to the N-terminal selectivity of recombinant human interferon alpha 2-ω, the N-terminal that is polyethylene active aldehyde and recombinant human interferon omega is connected by covalent, the homogeneity of product is greatly enhanced, easy purifying, the activity of purified product can keep preferably.
Summary of drawings
Fig. 1 is that recombinant human interferon alpha 2-ω and polyoxyethylene glycol (PEG) are at 25 ℃, 12 hours SDS-PAGE of reaction figure under the condition of pH5.0, M represents the low molecular weight protein (LMWP) standard among the figure, 1,2,3 represent that respectively the molar ratio of recombinant human interferon alpha 2-ω and mono methoxy polyethylene glycol propionic aldehyde-20K is 1: 2.5,1: 5,1: 10, the recombinant human interferon alpha 2-ω after 5 expressions, 1 molecule is polyethyleneglycol modified.
Fig. 2 is that recombinant human interferon alpha 2-ω and polyoxyethylene glycol (PEG) are at 25 ℃, 24 hours SDS-PAGE of reaction figure under the condition of pH5.0, M represents the low molecular weight protein (LMWP) standard among the figure, 1,2,3 represent that respectively the molar ratio of recombinant human interferon alpha 2-ω and mono methoxy polyethylene glycol propionic aldehyde-20K is 1: 2.5,1: 5,1: 10, the recombinant human interferon alpha 2-ω of 4 expression unmodifieds, the recombinant human interferon alpha 2-ω after 5 expressions, 1 molecule is polyethyleneglycol modified.
Fig. 3 is that recombinant human interferon alpha 2-ω and polyoxyethylene glycol (PEG) are at 25 ℃, 48 hours SDS-PAGE of reaction figure under the condition of pH5.0, M represents the lower molecular weight standard among the figure, 1,2,3 represent that respectively the molar ratio of recombinant human interferon alpha 2-ω and mono methoxy polyethylene glycol propionic aldehyde-20K is 1: 2.5,1: 5,1: 10, the recombinant human interferon alpha 2-ω of 4 expression unmodifieds, the recombinant human interferon alpha 2-ω after 5 expressions, 1 molecule is polyethyleneglycol modified.
Fig. 4 is that recombinant human interferon alpha 2-ω and polyoxyethylene glycol (PEG) are at 25 ℃, 60 hours SDS-PAGE of reaction figure under the condition of pH5.0, M represents the lower molecular weight standard among the figure, recombinant human interferon alpha 2-the ω of 1 expression unmodified, 2,3,4 represent that respectively the molar ratio of recombinant human interferon alpha 2-ω and mono methoxy polyethylene glycol propionic aldehyde-20K is 1: 2.5,1: 5,1: 10, the recombinant human interferon alpha 2-ω after 5 expressions, 1 molecule is polyethyleneglycol modified.
Fig. 5 is that subcutaneous injection PEG-rhIFN-ω and rhIFN-ω are at the intravital Cot curve of rabbit;
The purity of the recombinant human interferon omega of Shi Yonging is greater than 98% in the present embodiment, and maintains higher biological activity;
Recombinant human interferon alpha 2 is dissolved in the acetate buffer (pH5.0) of 100mM, in the ratio adding of recombinant human interferon omega and polyoxyethylene glycol (PEG) 1: 2.5 (mol ratio);
Add the mono methoxy polyethylene glycol propionic aldehyde (mPEG-propionaldehyde, mPEG-ALD) stirring and dissolving, add again NaBH4CN (with the mol ratio of Interferon, rabbit be 20: 1), room temperature reaction 12-60 hour.
The purity of the recombinant human interferon omega of Shi Yonging is greater than 98% in the present embodiment, and maintains higher biological activity;
Recombinant human interferon alpha 2 is dissolved in the acetate buffer (pH5.0) of 100mM, in the ratio adding of recombinant human interferon omega and polyoxyethylene glycol (PEG) 1: 5 (mol ratio);
Add the mono methoxy polyethylene glycol propionic aldehyde (mPEG-propionaldehyde, mPEG-ALD) stirring and dissolving, add again NaBH4CN (with the mol ratio of Interferon, rabbit be 20: 1), room temperature reaction 12-60 hour.
The purity of the recombinant human interferon omega of Shi Yonging is greater than 98% in the present embodiment, and maintains higher biological activity;
Recombinant human interferon alpha 2 is dissolved in the acetate buffer (pH5.0) of 100mM, in the ratio adding of recombinant human interferon omega and polyoxyethylene glycol (PEG) 1: 10 (mol ratio);
Add the mono methoxy polyethylene glycol propionic aldehyde (mPEG-propionaldehyde, mPEG-ALD) stirring and dissolving, add again NaBH4CN (with the mol ratio of Interferon, rabbit be 20: 1), room temperature reaction 12-60 hour.
The purity of the recombinant human interferon omega of Shi Yonging is greater than 98% in the present embodiment, and maintains higher biological activity.
Recombinant human interferon omega is dissolved in the phosphate buffered saline buffer (pH7.2) of 20mM, add recombinant human interferon omega and the mono methoxy polyethylene glycol propionic aldehyde (mPEG-propionaldehyde that contracts successively in the ratio of recombinant human interferon omega and polyoxyethylene glycol (PEG) 1: 2.5 (mol ratio), mPEG-ALD), stirring and dissolving, add again NaBH4CN (with the mol ratio of Interferon, rabbit be 20: 1), room temperature reaction 24-72 hour.
The purity of the recombinant human interferon omega of Shi Yonging is greater than 98% in the present embodiment, and maintains higher biological activity.
Recombinant human interferon omega is dissolved in the phosphate buffered saline buffer (pH7.2) of 20mM, add recombinant human interferon omega and the mono methoxy polyethylene glycol propionic aldehyde (mPEG-propionaldehyde that contracts successively in the ratio of recombinant human interferon omega and polyoxyethylene glycol (PEG) 1: 5 (mol ratio), mPEG-ALD), stirring and dissolving, add again NaBH4CN (with the mol ratio of Interferon, rabbit be 20: 1), room temperature reaction 24-72 hour.
Embodiment 6
The purity of the recombinant human interferon omega of Shi Yonging is greater than 98% in the present embodiment, and maintains higher biological activity.
Recombinant human interferon omega is dissolved in the phosphate buffered saline buffer (pH7.2) of 20mM, add recombinant human interferon omega and the mono methoxy polyethylene glycol propionic aldehyde (mPEG-propionaldehyde that contracts successively in the ratio of recombinant human interferon omega and polyoxyethylene glycol (PEG) 1: 10 (mol ratio), mPEG-ALD), stirring and dissolving, add again NaBH4CN (with the mol ratio of Interferon, rabbit be 20: 1), room temperature reaction 24-72 hour.
The separation purifying technique of embodiment 7 Pegylation recombinant human interferon omegas
After the acetate buffer solution dialysis of product that the reaction of recombinant human interferon alpha 2 and polyoxyethylene glycol is obtained to 20mM pH5.0, last CM cationic exchange coloum, the absorption back acetate buffer solution gradient elution of the 20mM PH5.0 that contains 0-1M NaCl, 280nm monitors elution peak, collect and mainly contain the Pegylation recombinant human interferon alpha 2 that a peg molecule connects a recombinant human interferon alpha 2 molecule, through after the concentration, the purity of resulting Pegylation recombinant human interferon alpha 2 medicine can be product greater than 98% after degerming after filtration, lyophilize.
The retention time experiment of embodiment 8 Pegylation recombinant human interferon alpha 2 medicines in rabbit blood.
This research is carried out in rabbit (the big ear of New Zealand is white), adopts subcutaneous injection Pegylation recombinant human interferon alpha 2, and with recombinant human interferon alpha 2 (rIFN-ω) in contrast, estimates its retention time in rabbit blood.
Respectively at 5min, 10min, 15min, 30min, 45min, 1h, 1.5h, 2h, 4h, 8h, 12h, 24h gets blood, adds the heparin sodium anti-freezing, and centrifuging and taking goes out blood plasma, and freezing preservation is to be measured.
The content of Pegylation recombinant human interferon alpha 2 and recombinant human interferon alpha 2 in the employing double antibodies sandwich method mensuration blood plasma, measure wavelength 490nm, with time is X-coordinate, and the OD value is an ordinate zou, draws mPEG-rhIFN-ω and the retention time curve of rhIFN-ω in rabbit anteserum.
This test-results shows that rhIFN-ω is after subcutaneous injection, and 10min-30min peaks in mouse blood, descends rapidly subsequently, and 4h is eliminated in blood substantially; And the Pegylation recombinant human interferon alpha 2, can continue to keep more than 24 hours after 45min peaks in rabbit blood through subcutaneous injection, still has the PEG-rhIFN-ω of higher concentration in 24 hours in the body.This explanation is compared with IFN-ω reference substance, and the Pegylation recombinant human interferon alpha 2 can prolong the retention time in blood significantly through the subcutaneous injection administration, the time that delay is eliminated, has long lasting effect.
Claims (12)
1, a kind of Pegylation human interferon omega conjugates, than unconjugated human interferon omega, has the transformation period in the longer organism, keep at least 10% antiviral biologic activity, wherein each human interferon omega (hIFN-ω) is with the polyoxyethylene glycol of covalent linkage connection a part, and molecular weight polyethylene glycol is between 5000～40000.
2, a kind of Pegylation human interferon omega conjugates according to claim 1, wherein human interferon omega is recombinated;
3, a kind of Pegylation human interferon omega conjugates according to claim 1, wherein human interferon omega is to be connected with polyoxyethylene glycol by N-terminal amino covalence key.
4, a kind of Pegylation human interferon omega conjugates according to claim 1, wherein human interferon omega is to be connected with polyoxyethylene glycol by lysine amino.
5, a kind of Pegylation human interferon omega conjugates according to claim 1, wherein human interferon omega is that sulfydryl by halfcystine is connected with polyoxyethylene glycol.
6, Pegylation human interferon omega conjugates according to claim 1 is characterized in that: each peg molecule connects a recombinant human interferon omega (hIFN-ω) molecule.
7, Pegylation human interferon omega conjugates according to claim 1, wherein PEG is linear;
8, Pegylation human interferon omega conjugates according to claim 1, wherein PEG is a ramose;
9, PEG-IFN omega conjugate according to claim 1, it is characterized in that: described polyoxyethylene glycol is mono methoxy polyethylene glycol (monomethoxy polyetnylene glycol, mPEG) activated molecule, comprise mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD), mono methoxy polyethylene glycol butyraldehyde, Y type polyglycol ethanal, mono methoxy polyethylene glycol succinate, mono methoxy polyethylene glycol maleimide, its molecular weight is between 5000～40000; Mono methoxy polyethylene glycol propionic aldehyde (mPEG-ALD) most preferably, molecular weight is 20000.
10, Pegylation human interferon omega conjugates according to claim 1, it is characterized in that: (mol ratio is 0.25～20 to the ratio of polyoxyethylene glycol and recombinant human interferon omega (rhIFN-ω) generation covalent attachment reaction, and preferred proportion is between 2.5～10.
11, Pegylation recombinant human interferon omega medicine production technology according to claim 1 comprises following process:
(1) provides the buffer system of the salt concn of pH4.0～9.0,10～100mmol/L;
(2) recombinant human interferon omega and polyethylene glycol aldehyde reacted 12～72 hours in the system that (1) step provides, and temperature is controlled at 4～37 ℃;
(3) the product process cation-exchange chromatography separation and purification that obtains through step (2) reaction, collect the Pegylation recombinant human interferon omega (PEG-rhIFN-ω) that a peg molecule connects a recombinant human interferon omega molecule, through filtration sterilization, after the lyophilize promptly.
12, Pegylation recombinant human interferon omega medicine production technology according to claim 6, it is characterized in that: (the preferred pH value of buffer system of covalent attachment reaction takes place between 4.5～5.5 in (hIFN-ω) for polyoxyethylene glycol and recombinant human interferon omega, preferred temperature is at 20～25 ℃, and the preferred time is 24～48 hours.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA200810110734XA CN101591387A (en) | 2008-05-28 | 2008-05-28 | PEG-IFN omega conjugate |
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| CNA200810110734XA CN101591387A (en) | 2008-05-28 | 2008-05-28 | PEG-IFN omega conjugate |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234331A (en) * | 2010-05-06 | 2011-11-09 | 华东师范大学 | Composition of Exendin-4 derivative and preparation method and application thereof |
| CN102617736A (en) * | 2010-07-20 | 2012-08-01 | “拜奥卡德”股份公司 | The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer |
| CN107118269A (en) * | 2017-06-05 | 2017-09-01 | 北京交通大学 | A kind of PEG method of modifying of the albumen of recombined human IL 24 |
-
2008
- 2008-05-28 CN CNA200810110734XA patent/CN101591387A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234331A (en) * | 2010-05-06 | 2011-11-09 | 华东师范大学 | Composition of Exendin-4 derivative and preparation method and application thereof |
| CN102617736A (en) * | 2010-07-20 | 2012-08-01 | “拜奥卡德”股份公司 | The new stable polyethylene glycol conjugate of interferon alpha, represented by one positional isomer |
| CN102617736B (en) * | 2010-07-20 | 2015-11-25 | “拜奥卡德”股份公司 | The stable interferon alpha polyoxyethylene glycol conjugate represented by a kind of positional isomers |
| CN107118269A (en) * | 2017-06-05 | 2017-09-01 | 北京交通大学 | A kind of PEG method of modifying of the albumen of recombined human IL 24 |
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Open date: 20091202 |