CN103694335A - Polyethylene glycol omega-interferon with long connection bridge and preparation process thereof - Google Patents
Polyethylene glycol omega-interferon with long connection bridge and preparation process thereof Download PDFInfo
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Abstract
The invention relates to the field of biological medicines and specifically relates to polyethylene glycol (PEG) omega-interferon with a long connection bridge and a preparation method thereof. The method comprises: (1) 2-imino thiacyclopentane reacts with a primary amine group of the omega-interferon, sulfydryl is introduced, the sulfydryl on the surface of the omega-interferon reacts with PEG-maleimide to realize PEG-modified omega-interferon; (2), a longer connection bridge exists between the PEG and the omega-interferon, so that space shielding effect of the PEG on the omega-interferon can be predicated to be lowered, and the biological activity of the omega-interferon can be improved; (3), compared with un-modified omega-interferon, the polyethylene glycol omega-interferon has a longer circulation half-life period in vivo, can be used for obviously reducing drug-use times and lowering the toxic and side effect, is especially suitable for treating a patient resistant to the alpha-interferon, and has wide application prospect in clinical.
Description
Technical field
The present invention relates to biomedicine field, particularly, the present invention relates to a kind of polyoxyethylene glycol (PEG) with long cross structure and change ω-Interferon, rabbit, with and preparation technology.
Background technology
Interferon, rabbit (interferon, IFN) be body by virus infection after, a kind of glycoprotein being produced by Immune responses of the antivirus by white corpuscle, has the biological activitys such as antiviral, antitumor and immunomodulatory.Since 1986, Interferon, rabbit becomes firstly to be ratified by U.S. FDA, and for clinical cytokine.Interferon, rabbit is divided into I type and II type the earliest.In I type, there are again two kinds of compositions, can be divided into α and β two classes.The ε finding subsequently, κ, ω, δ-IFN all belongs to interferon type Ⅰ.II type is defined as γ interferoid.ω-Interferon, rabbit is the interferon type Ⅰ that the people such as Austrian scientist Rudelf Hamptan found in 1985.GuntherR.Adolf etc. find that in 1987 ω-Interferon, rabbit is close with alpha-interferon function, but antigenicity is different with immunogenicity.Tiefenthaler etc. were discovery in 1997, and ω-Interferon, rabbit and α-2a-Interferon, rabbit in vitro inhibition tumor cell are active, and the proliferation of bone marrow cells that especially suppresses chronic cellulous leukemia of bone marrow (CML) has closely similar effect.The people such as Hagelstein finds, Human IFN-ω has similar to alpha-interferon, gamma-interferon and tumour necrosis factor antiviral copying and the effect such as inhibition of cell proliferation.Because ω-Interferon, rabbit has stronger antiviral copying, the effect such as inhibition of cell proliferation and immunomodulatory, ω-Interferon, rabbit has broad application prospects in the treatment of kinds of tumors, virus disease and to the patient that alpha-interferon has a resistance, and its clinical application launches gradually.
Interferon, rabbit is the choice drug for the treatment of chronic hepatitis B or chronic hepatitis C, can effectively suppress or remove virus, and can stop the generation of liver cirrhosis or liver cancer.Because hepatitis B virus (HBV) and hepatitis C virus (HCV) copy very soon, during interferon therapy, need to maintain the viral inhibition of constant Plasma Concentration competence exertion maximum.Interferon, rabbit is generally every other day and is administered once, and after administration, Plasma Concentration reaches rapidly peak value.The intermittent phase of 24 hours and administration after medication, the Plasma Concentration of Interferon, rabbit is down to very low, and virus restarts to copy, " peak-paddy " effect of Here it is Interferon, rabbit.This is mainly because interferon molecule amount is little, in vivo easily by proteasome degradation and glomerular filtration, causes Interferon, rabbit circulating half-life in vivo short.In addition, also there is anaphylaxis and a series of toxic side effect such as thrombopenia, irregular pulse, influenza-like symptom, urticaria, maculopapule in Interferon, rabbit in clinical application simultaneously.These problems produce immune response by extrinsic protein and cause in human body, have seriously restricted its clinical application.
Therefore, Chinese scholars has competitively been carried out the research of long-acting interferon.Wherein, polyoxyethylene glycol (PEG) modification is to prepare at present one of long-acting protein and the most successful strategy of polypeptide drugs.PEG be one with-CH
2cH
2o-is the high polymer of foundation structure, is commonly used to carry out the modification of protein.PEG modifies the pharmaceutical grade protein shielding effect of having living space, and can effectively reduce the Degradation of proteolytic ferment, reduces its antigenicity and immunogenicity, extends circulating half-life, improves stability and solvability simultaneously.PEG is modified in the long-acting dosage form exploitation of Interferon, rabbit and is successfully applied, as the α 2a-Interferon, rabbit (Pegasys of PEGization, Roche company) and the α 2b-Interferon, rabbit (PEG-Intron of PEGization, Schering company) etc., by combine the treatment that is widely used in hepatitis with ribavirin.The random α 2a-Interferon, rabbit of modifying of ramiform PEG that Pegasys is 40kDa by molecular weight.The circulating half-life of Pegasys is 96 hours, can keep 7% of interferon alpha 2a antiviral activity.The random α 2b-Interferon, rabbit of modifying of line style PEG that PEG-Intron is 12kDa by molecular weight.The circulating half-life of PEG-Intron is 45 hours, can keep 28% of α 2b-antiviral activity of interferon.At present, ω-Interferon, rabbit that PEG modifies not yet goes on the market.
Although PEG modifies the circulating half-life that can extend ω-Interferon, rabbit, improve the pharmaceutical properties of ω-Interferon, rabbit, PEG modifies ω-Interferon, rabbit and also has some problems at present.For example, because PEG is chain high molecular polymer, inevitably can cover to a certain extent the avtive spot on ω-Interferon, rabbit surface, thereby reduce the biological activity of ω-Interferon, rabbit.Therefore, need to when extending ω-Interferon, rabbit circulating half-life, reduce the spatial masking effect of PEG to ω-Interferon, rabbit, improve the biological activity of ω-Interferon, rabbit.2-imino-thiacyclopentane (2-iminothiolane) is ring-type thioimines ester cpds, can react with the primary amine groups of ω-Interferon, rabbit and introduce sulfydryl.The sulfydryl on ω-interferon molecule surface reacts with PEG-maleimide, can realize PEG and modify ω-Interferon, rabbit (Fig. 1).Owing to existing longer cross structure between PEG and ω-Interferon, rabbit, expectation can reduce the spatial masking effect of PEG to ω-Interferon, rabbit, improves the biological activity of ω-Interferon, rabbit.The present invention adopts the PEG of the auxiliary ω-Interferon, rabbit of 2-imino-thiacyclopentane to modify.Interferon medicine on sale is based on alpha-interferon and beta-interferon mostly in the market, not yet occurs the PEG modified product of ω-Interferon, rabbit.
Summary of the invention
For the problems referred to above, invention has herein proposed a kind of method that PEG modifies ω-Interferon, rabbit.With 2-imino-thiacyclopentane, react with the primary amine groups of ω-Interferon, rabbit, the PEG-maleimide that the sulfydryl of introducing is 20kDa and 40kDa with molecular weight respectively reacts, and can realize PEG and modify ω-Interferon, rabbit.By separation and purification, the PEG that obtains molecular weight homogeneous modifies ω-Interferon, rabbit, and each ω-interferon molecule is in conjunction with 1 PEG molecule.This modifying method expection can reduce the loss of ω-interferon biological activity.
The present invention relates to prepare the method that PEG modifies ω-Interferon, rabbit, comprise the following steps:
(1) determine the preparation condition of IFN-mal-P20K: ω-interferon solution is replaced in the phosphoric acid buffer (pH7.0-7.4) of 20mM.Adjusting ω-Interferon, rabbit concentration is 2.2 mg/ml (0.1mM), by ω-Interferon, rabbit: 2-imino-thiacyclopentane: different mol ratio routine, in 4 ℃, react respectively 2-24 hour.Subsequently, the super filter tube that is 10kDa with molecular weight cut-off is centrifugal removes unreacted 2-imino-thiacyclopentane.The PEG-maleimide (20kDa) that adds different mol ratio example, reacts respectively 2-24 hour in 4 ℃, completes the chemically modified of PEG to ω-Interferon, rabbit.Wherein, to take the product that mol ratio is 1:1 be object product, i.e. IFN-mal-P20K to the PEG that ω-Interferon, rabbit and molecular weight are 20kDa.With Superdex200 gel-filtration column (1cm * 30cm, U.S. GE Healthcare company), detect the PEG modified outcome of preparing under differential responses condition, elutriant is 20mM phosphoric acid buffer (pH7.4), and flow velocity is 0.5 ml/min.By detecting the productive rate of IFN-mal-P20K, determine optimum reaction conditions.
(2) determine the preparation condition of IFN-mal-P40K: ω-interferon solution is replaced in the phosphoric acid buffer (pH7.0-7.4) of 20mM.Adjusting ω-Interferon, rabbit concentration is 2.2 mg/ml (0.1mM), by ω-Interferon, rabbit: 2-imino-thiacyclopentane: different mol ratio routine, in 4 ℃, react respectively 2-24 hour.Subsequently, the super filter tube that is 10kDa with molecular weight cut-off is centrifugal removes unreacted 2-imino-thiacyclopentane.The PEG-maleimide (40kDa) that adds different mol ratio example, reacts respectively 2-24 hour in 4 ℃, completes the chemically modified of PEG to ω-Interferon, rabbit.Wherein, to take the product that mol ratio is 1:1 be object product, i.e. IFN-mal-P40K to the PEG that ω-Interferon, rabbit and molecular weight are 40kDa.With Superdex200 gel-filtration column (1cm * 30cm, U.S. GE Healthcare company), detect the PEG modified outcome of preparing under differential responses condition, elutriant is 20mM phosphoric acid buffer (pH7.4), and flow velocity is 0.5 ml/min.By detecting the productive rate of IFN-mal-P40K, determine optimum reaction conditions.
(3) first step 1 and the resulting reaction mixture of step 2 are carried out to purifying by SP Sepharose HP post (1.6cm * 25cm, U.S. GE Healthcare company).SP Sepharose HP post carries out balance by 20mM sodium acetate-acetate buffer (pH5.0).After loading, continue to carry out abundant wash-out with this damping fluid, flow velocity is 1.0 ml/min, can remove unreacted PEG-maleimide.With the 20mM sodium acetate-acetate buffer (pH5.0) that contains 0.5M sodium-chlor, continue wash-out, flow velocity is 1.0 ml/min, collects elution peak.Elution peak continues to carry out purifying by Superdex200 post (2.6cm * 60cm, U.S. GEHealthcare company).Superdex200 post, by phosphoric acid buffer (pH7.4) balance of 20mM, continues after loading to carry out wash-out with this damping fluid, and flow velocity is 3.0 ml/min.Collection, corresponding to the mono-modified product of PEG of ω-Interferon, rabbit, is carried out Product Identification by SDS-PAGE and gel filtration chromatography after concentrating.
(4) evaluation of PEG decorating site: ω-Interferon, rabbit and PEG modified outcome are replaced to the NH containing 2.0M urea
4hCO
3in (0.05M, pH8.3) solution, protein concentration is adjusted into 1.0 mg/ml.Trypsinase and albumen be take to the ratio that mass ratio is 1:100, hatch 10 hours at 37 ℃, enzymolysis product is used reversed-phase HPLC post (0.46cm * 25cm, Japanese Proteonavi company) carry out the separated of endonuclease bamhi and identify.
(5) PEG modifies the antiviral activity of ω-Interferon, rabbit: by measuring the cytopathic effect of stomatitis herpesvirus to WISH cell, can assess the antiviral activity of ω-Interferon, rabbit.Measure ω-Interferon, rabbit to viral-induced cytolytic suppression efficiency, can calculate the antiviral activity that PEG modifies ω-Interferon, rabbit.
(6) PEG modifies the pharmacokinetics of ω-Interferon, rabbit: choose 15 of 2.3-2.8 kilogram of male New Zealand large ear rabbits, be divided at random 3 groups.Wherein, the 1st group is ω-Interferon, rabbit group, and the 2nd group is that molecular weight is ω-Interferon, rabbit (IFN-mal-P20K) group that 20kDa PEG modifies, and the 3rd group is that molecular weight is ω-Interferon, rabbit (IFN-mal-P40K) group that 40kDa PEG modifies.Injected dose is 20 microgram ω-Interferon, rabbit/kilogram white rabbit.After subcutaneous injection, in the 0th, 0.1,0.5,1,2,4,8,12,24,48,72,96,120,144,168,192,216,288 and 312 hour, carry out ear vein and get blood, centrifugal collection blood plasma.With the ω-Interferon, rabbit content in Human IFN-ω ELISA kit measurement blood plasma.
The invention has the advantages that, the mono-modified productive rate in modified outcome is higher, and two modifications and the many modified outcomes that generate are less, are conducive to the separation and purification of the mono-modified product of PEG.
Accompanying drawing explanation
The preparation feedback formula of Fig. 1 PEGization ω-Interferon, rabbit.
Fig. 2 molecular weight is the gel filtration chromatography figure of PEGization ω-Interferon, rabbit of 20kDa.That use is Superdex200 gel-filtration column (1cm * 30cm), and eluent flow rate is 0.5 ml/min, and elution buffer is 20mM phosphoric acid buffer (pH7.4).Sample 1 is ω-Interferon, rabbit, and sample 2-6 is ω-Interferon, rabbit: 2-imino-thiacyclopentane: PEG-maleimide (20kDa) mol ratio is the PEG modified outcome of preparing under the ratio of 1:1:1,1:1:2,1:2:2,1:2:4 and 1:4:4.
Fig. 3 molecular weight is the gel filtration chromatography figure of PEGization ω-Interferon, rabbit of 40kDa.That use is Superdex200 gel-filtration column (1cm * 30cm), and eluent flow rate is 0.5 ml/min, and elution buffer is 20mM phosphoric acid buffer (pH7.4).Sample 1-5 is ω-Interferon, rabbit: 2-imino-thiacyclopentane: PEG-maleimide (40kDa) mol ratio is the PEG modified outcome of preparing under the ratio of 1:1:1,1:1:2,1:2:2,1:2:4 and 1:4:4.
Fig. 4 SDS-PAGE analyzes PEGization ω-Interferon, rabbit.The 1st swimming lane is standard protein, and the 2nd swimming lane is ω-Interferon, rabbit, and the 3rd swimming lane is IFN-mal-P20K, and the 4th swimming lane is IFN-mal-P40K.
Fig. 5 analysed by gel filtration chromatography PEGization ω-Interferon, rabbit.That use is Superdex200 gel-filtration column (1cm * 30cm), and eluent flow rate is 0.5 ml/min, and elution buffer is 20mM phosphoric acid buffer (pH7.4).
The decorating site of Fig. 6 IFN-mal-P20K is identified.Using reversed-phase HPLC post (0.46cm * 25cm, Japanese Proteonavi company) to carry out enzyme, to cut peptide section separated.
The antiviral activity of Fig. 7 PEGization ω-Interferon, rabbit.By measuring ω-Interferon, rabbit to viral-induced cytolytic suppression efficiency, calculate the antiviral activity of PEGization ω-Interferon, rabbit.
The concentration curve of ω-Interferon, rabbit in Fig. 8 New Zealand large ear rabbit blood plasma.New Zealand's large ear rabbit is through subcutaneous injection PEG modified outcome, and in blood plasma, the content of ω-Interferon, rabbit is measured by ELISA quantification kit.
Embodiment
Determining of embodiment 1IFN-mal-P20K preparation condition
ω-Interferon, rabbit is replaced in 50mM NaAc-HAc damping fluid (pH5.5), and adjusting ω-Interferon, rabbit concentration is that 2.2mg/mL(is 0.1mM).By ω-Interferon, rabbit: 2-imino-thiacyclopentane: PEG-maleimide (20kDa) mol ratio is 1:1:1,1:1:2,1:2:2,1:2:4 and 1:4:4, in 4 ℃ of reactions spend the night (～16 hours) respectively.With Superdex200 gel-filtration column (1cm * 30cm), modified outcome is detected, elutriant is 20mM phosphoric acid buffer (pH7.4).The result of the amine-modified ω-Interferon, rabbit of 20kDa PEG-maleimide as shown in Figure 2, is weighed productive rate with peak area (2 peak) and the total peak area ratio size of IFN-mal-P20K.Along with the raising of 2-imino-thiacyclopentane and 20kDa PEG-maleimide reaction ratio, the productive rate of IFN-mal-P20K also improves thereupon, and the peak area of ω-Interferon, rabbit reduces (3 peak).But, along with the increase of reaction ratio, there is the ω-Interferon, rabbit (1 peak) of many modifications, and increased along with the increase of reaction ratio.Although can access higher IFN-mal-P20K when mol ratio is 1:4:4, consider that IFN-mal-P20K not easily separated with ω-Interferon, rabbit of modifying more, therefore selecting mol ratio is that the reaction ratio of 1:2:4 is prepared IFN-mal-P20K.
Determining of embodiment 2IFN-mal-P40K preparation condition
ω-Interferon, rabbit is replaced in 50mM NaAc-HAc damping fluid (pH5.5), and adjusting ω-Interferon, rabbit concentration is that 2.2mg/mL(is 0.1mM).By ω-Interferon, rabbit: 2-imino-thiacyclopentane: PEG-maleimide (40kDa) mol ratio is 1:1:1,1:1:2,1:2:2,1:2:4 and 1:4:4, in 4 ℃ of reactions spend the night (～16 hours) respectively.With Superdex200 gel-filtration column (1cm * 30cm), modified outcome is detected, elutriant is 20mM phosphoric acid buffer (pH7.4).The result of the amine-modified ω-Interferon, rabbit of 40kDa PEG-maleimide as shown in Figure 3, is weighed productive rate with peak area (2 peak) and the total peak area ratio size of IFN-mal-P40K.Along with the raising of 2-imino-thiacyclopentane and 40kDa PEG-maleimide reaction ratio, the productive rate of IFN-mal-P40K also improves thereupon, and the peak area of ω-Interferon, rabbit reduces (3 peak).But, along with the increase of reaction ratio, there is the ω-Interferon, rabbit (1 peak) of many modifications, and increased along with the increase of reaction ratio.Although can access higher IFN-mal-P40K when mol ratio is 1:2:4 and 1:4:4, consider that IFN-mal-P40K not easily separated with ω-Interferon, rabbit of modifying more, therefore selecting mol ratio is that the reaction ratio of 1:2:2 is prepared IFN-mal-P40K.
The evaluation of embodiment 3IFN-mal-P20K and IFN-mal-P40K
By SDS-PAGE and gel filtration chromatography, identify IFN-mal-P20K and IFN-mal-P40K.As shown in Figure 4, IFN-mal-P20K and IFN-mal-P40K all show a band on SDS-PAGE electrophorogram, and this shows that these two kinds of PEG modified outcomes have higher purity.The rate of migration of IFN-mal-P40K electrophoresis band will be slower than IFN-mal-P20K, and IFN-mal-P20K will be slower than ω-Interferon, rabbit.This shows that PEG modifies the molecular weight that can increase ω-Interferon, rabbit, and the molecular weight of IFN-mal-P40K will be higher than IFN-mal-P20K.As shown in Figure 5, after buffer solution elution, there is 1 single elution peak at Superdex200 gel-filtration column (1.0cm * 30cm) respectively in IFN-mal-P20K and IFN-mal-P40K.Wherein, the appearance time of IFN-mal-P20K will be faster than IFN-mal-P40K, and will be slower than ω-Interferon, rabbit.This shows that IFN-mal-P20K and IFN-mal-P40K have higher purity, and molecular weight is greater than ω-Interferon, rabbit.
The evaluation of embodiment 4PEG decorating site
By ω-Interferon, rabbit and IFN-mal-P20K through the NH containing 2.0M urea
4hCO
3in the damping fluid of (0.05M, pH8.3), fully dialysis, then regulates concentration to 1.0 mg/ml.Getting trypsinase, is the liquid storage of 1.0 mg/ml with ultrapure water configuration concentration.The ratio that is 1:100 according to the mass ratio of trypsinase and albumen, adds respectively trypsinase liquid storage, in 37 ℃, hatches 10 hours.Use reversed-phase HPLC post (Proteonavi, 0.46cm * 25cm) to carry out enzyme and cut the separation of peptide section.Reversed-phase HPLC post is used 95% solution A (containing the ultrapure water of 0.1% trifluoroacetic acid) and 5% the abundant balance of solution B (containing the acetonitrile of 0.1% trifluoroacetic acid), and flow velocity is 0.5 ml/min.Adopt linear elution mode to rise to 50% solution B in 100 minutes from 5% solution B, detect effluent liquid in the absorption value of 214 nanometers.
As shown in Figure 6, trypsinase can be cut into different peptide sections from IFN-mal-P20K by ω-Interferon, rabbit to result.Wherein with T11(75-123) peptide section is in contrast.Compare with the ω-Interferon, rabbit of unmodified, the T1 of IFN-mal-P20K (1-14) peptide section, T4 (26-33) peptide section, T14 (131-134) peptide section and the corresponding peak height of T17+18 (138-152) peptide section all decrease.This shows that chemically modified has occurred for PEG molecule and these peptide sections, causes the corresponding peak height of these peptide sections to reduce.In addition, other endonuclease bamhi is compared without noticeable change with ω-Interferon, rabbit.Due to 2-imino-thiacyclopentane can with the leucine (Leu of N-end
1) alpha-amino group and the epsilon-amino reaction of Methionin (Lys), thereby the decorating site of PEG concentrates on Leu
1, the Methionin (Lys of the 33rd
33), the Methionin (Lys of the 134th
134) and the Methionin (Lys of the 152nd
152).
The antiviral activity of embodiment 5IFN-mal-P20K and IFN-mal-P40K
By measuring the cytopathic effect of stomatitis herpesvirus to WISH cell, can assess the antiviral activity of ω-Interferon, rabbit.As shown in Figure 7, compare with the ω-Interferon, rabbit of unmodified, IFN-mal-P20K and IFN-mal-P40K can keep respectively 25.4% and 10.2% antiviral activity.This explanation PEG modifies the antiviral activity that can reduce ω-Interferon, rabbit, and PEG molecular weight is higher, and antiviral activity declines more.On the other hand, the antiviral activity that IFN-mal-P40K retains will be higher than Pegasys(7%).Wherein, IFN-mal-P40K is the ω-Interferon, rabbit that combines the ramiform PEG that 1 molecular weight is 40kDa, and Pegasys is the α 2a-Interferon, rabbit that combines the ramiform PEG that 1 molecular weight is 40kDa.This shows to extend the cross structure of PEG and ω-Interferon, rabbit, can effectively improve the antiviral activity that PEG modifies ω-Interferon, rabbit.
The Pharmacokinetic Evaluation of embodiment 6IFN-mal-P20K and IFN-mal-P40K
Choose 15 of 2.3-2.8 kilogram of male New Zealand large ear rabbits, be divided at random 3 groups.Wherein, the 1st group is ω-Interferon, rabbit group, and the 2nd group is IFN-mal-P20K group, and the 3rd group is IFN-mal-P40K group.After subcutaneous injection sample, in different time, get blood, measure subsequently the ω-Interferon, rabbit concentration in blood plasma.As shown in Figure 7, ω-Interferon, rabbit is removed fast from blood plasma, its circulating half-life (T
1/2) be only 1.54 hours.Compare with ω-Interferon, rabbit, IFN-mal-P20K and IFN-mal-P40K are difficult for being eliminated, and its circulating half-life is respectively 41.71 hours and 130.4 hours.Compare with ω-Interferon, rabbit, the bioavailability of IFN-mal-P20K and IFN-mal-P40K (CL) has increased respectively 27.1 times and 84.7 times.ω-Interferon, rabbit can be absorbed fast by body, retention time peak value (T
max) be 0.5 hour, fast-descending subsequently.By contrast, IFN-mal-P20K and IFN-mal-P40K absorption rate are slow, and retention time peak value is respectively 4.0 hours and 8.0 hours.Compare the peak plasma concentration (C of IFN-mal-P20K and IFN-mal-P40K with ω-Interferon, rabbit
max) increased respectively 1.23 times and 1.36 times; The time dependent area under curve of drug level (AUC) of IFN-mal-P20K and IFN-mal-P40K has increased respectively 16.3 times and 29.8 times.These data show, PEG modifies the pharmacokinetic properties that can significantly strengthen ω-Interferon, rabbit; And PEG molecular weight is larger, the pharmacokinetic properties increase of ω-Interferon, rabbit is more.
Claims (8)
1. polyoxyethylene glycol (PEG) is changed ω-Interferon, rabbit, it is characterized in that existing longer cross structure between PEG and ω-Interferon, rabbit, can reduce the spatial masking effect of PEG to ω-Interferon, rabbit.
2. Pegylation ω-Interferon, rabbit according to claim 1, its preparation process is by 2-imino-thiacyclopentane (2-iminothiolane), to be reacted with the primary amine groups of ω-Interferon, rabbit to introduce sulfydryl, the sulfydryl of ω-Interferon, rabbit and PEG-maleimide covalent attachment.
3. Pegylation ω-Interferon, rabbit according to claim 1, is characterized in that each ω-interferon molecule is only in conjunction with 1 PEG molecule.
4. Pegylation ω-Interferon, rabbit according to claim 1, is characterized in that PEG is mono methoxy polyethylene glycol (mPEG).
5. Pegylation ω-Interferon, rabbit according to claim 1, is characterized in that the molecular weight of PEG-maleimide is between 5kDa～40kDa, and preferred PEG molecular weight is 20kDa.
6. Pegylation ω-Interferon, rabbit according to claim 2, the mol ratio that it is characterized in that 2-imino-thiacyclopentane and ω-ifn response is between 1-20, preferred mol ratio is between 2-4.
7. Pegylation ω-Interferon, rabbit according to claim 2, the mol ratio that it is characterized in that PEG-maleimide and ω-Interferon, rabbit is between 1-20, preferred mol ratio is between 2-4.
8. ω-Interferon, rabbit that PEG according to claim 1 modifies, for the preparation of antiviral and antitumor drug.
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