CN102453089B - Preparation and application of recombinant consensus interferon mutant polyethylene glycol conjugate - Google Patents
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Abstract
The invention relates to a recombinant consensus interferon mutant polyethylene glycol conjugate, and a preparation method and applications thereof, and is characterized in that a recombinant consensus interferon mutant is chemically modified at a single locus at an amino terminal by an aldehyde group-activated polyethylene glycol molecule with a molecular weight of 10 KD-40 KD. The modified recombinant consensus interferon mutant polyethylene glycol conjugate has the characteristics of prolonged in-vivo half life, reduced immunogenicity, and long-term antiviral activity in vivo. The invention also discloses a preparation method of the recombinant consensus interferon mutant polyethylene glycol conjugate, and provides applications of its combined medicaments in the treatment and prevention of viral infection or hyperplastic diseases, or the regulation of immune functions.
Description
Technical field
The invention belongs to biotechnology and pharmacy field, particularly relate to polyethylene coupling compound of a kind of integrated interferon variant of recombinating and its preparation method and application.The present invention is that denomination of invention is the extension of " human alpha interferon derivative of antiviral activity " (patent No. is ZL01102915.3) patent of invention.
Background technology
As far back as nineteen fifty-seven, the people such as lssacs find that the cell of virus infection produces a kind of factor, can support antiviral infection, the copying of viral interference, thereby called after Interferon, rabbit (interferon, IFN).Interferon, rabbit be by deactivation or live virus function after permissive cell, the one group of glycoprotein being produced by permissive cell genome encoding.Its activity and antigenicity all depend on the protein in molecule, and irrelevant with its glycosyl.According to its source and structure, IEN can be divided into IFN-α, IFN-β, IFN-γ, they are produced by white corpuscle, fibroblast and activating T cell respectively.IFN-α is polygene product, has more than ten to plant different subtype, but their biological activity is basic identical.IFN is apart from outside antivirus action, in addition antitumor, immunomodulatory, control cell proliferation and cause the effects such as heating.The Interferon, rabbit using clinically at present obtains by gene recombination technology mostly.
IFN is that chronic viral hepatitis is through the effective first-line treatment medicine of clinical verification.That clinical use is more is IFN α-2a and the IFN α-2b of natural structure.The two all belongs to fugitive Interferon, rabbit, and the transformation period short (4～16h), serum-concentration is very low after injection 24h.Its pharmacokinetics process influence in vivo the late result of its antiviral therapy.According to current 3～6MU/kg, the administrated method of 3 times weekly, Interferon level presents typically " peak-paddy curve " in vivo.Interferon, rabbit serum level fluctuates widely and easily makes virus levels knock-on, produces resistance, reduces result for the treatment of.Meanwhile, fugitive Interferon, rabbit exists toxic side effects large, and people's life-time service tolerance is poor, and immunogenicity waits by force not enough.Therefore, fugitive Interferon, rabbit is modified, extended its transformation period in vivo, maintain the stable of the interior Plasma Concentration of body, will contribute to improve the validity of its treatment viral hepatitis, and alleviate toxic side effect.
Polyoxyethylene glycol (polyetnylene glycol, PEG) is the polymkeric substance of a kind of safety non-toxic, non-activity, is usually used in pharmaceutical grade protein and modifies.Albumen resistance to enzymolysis after modification, water-soluble increase, Increased Plasma Half-life, immunogenicity and toxicity reduce (Inada, et al., J.Bioact.And Compatible Polymers, 5:343 (1990) .Delgalo, et al., Critical Reviews in Therapeutic Drug Carrier systems, 9:249 (1992) kate, Advanced Drug Delivery Systems, 10:91 (1993)).The Chinese patent of Huffman-La Luoqi company: ZL93116479.6 and ZL97105433.9 disclose the preparation of PEG-inerferon conjugates, the Chinese patent 200580032850.2 of Schering Corp discloses the preparation of the preparation of stable Peg-Intron.At present, the human alpha interferon (PEGASYS of Huffman-La Luoqi and the PEGINTRON of Schering Corp) that existing two kinds of PEG modify is for the treatment of chronic the third type and hepatitis B.After both Pegylations, kidney clearance rate obviously reduces, and Increased Plasma Half-life (30～16h), immunogenicity reduce, and only need weekly administration 1 time.Aspect virus, be obviously better than fugitive Interferon, rabbit at removing HCV/HBV.The recent reactivity for the treatment of hepatitis C HCV-RNA can reach 69%, and sustained reaction rate reaches 39%, and conventional Interferon, rabbit is only 7%.Treatment hepatitis B HBeAg negative conversion rate can reach 37%; comprehensive response rate (be HBeAg turns out cloudy, HBV DNA suppress and ALT normalizing) is 35%; 61% patient follows HBVDNA to turn out cloudy (< 400copies/mL), and 7% has occurred that HBsAg turns out cloudy.And three indexs are only (15%, 25%, 12%) after conventional interferon therapy hepatitis B.Patient (ReddyKR, etal.Hepatology, 2001,33:433-438.Jessner W, et al.J Viral Hepat.2003,10:37-42.Shiffman ML, et al.Hepatology, 2004 of turning out cloudy without HBsAg; 40 (4, suppl 1): 314A.Davis GL, et al.Hepatology, 2003; 38:645-52.Janssen HL, et al.Lancet, 2005; 365:123-129.Cooksley WG et al..J Viral Hepat, 2003; 10:298-305.).
Recombinant human alpha interferon 2a (PEGASYS) decorating site that PEG modifies is Methionin, and recombinant human a interferon alpha-2 b (PEG-Intron) decorating site that PEG modifies is Histidine.Bibliographical information (Miller DL, et al.Science, 1982,215:689-690.Radhakrishnan R, et al.Structure, 1996; 4 (12): 1453-1463.Karpusas M, et al.Proc.Natl.Acad.sci.USA 1997,94:11813-11818), the receptor binding domain of IFN-α mainly comprises AB loop (Arg22, Leu26, Phe27, Leu30, Lys31, Arg33, His34), helixB (Ser68), helixC (Thr79, Lys83, Tyr85, Tyr89), helix D (Arg120, Lys121, Gln124, Lys131, and helixE (Arg144, Glu146) Glu132).The Histidine that the Methionin that PEGASYS modifies and PEG-Intron modify, all in above-mentioned receptor binding domain, is unfavorable for the combination of Interferon, rabbit and its acceptor.In addition, all non-specific single pointed decorations of end product after modifying by the two PEG, if PEGASYS is to modify Lys131 as main mixture (having 7 decorating sites), PEG-Intron modifies the mixture (having 4 decorating sites) that His34 is master.
The PEG in single site modifies the homogeneity that can simultaneously realize molecular weight and structure, improves biological activity and the interior security of body after modifying.Existing several different methods can realize the single site-specific sex modification of PEG to Interferon, rabbit.(1) WO2006/004959 disclose one utilize in IFN α-1b aminoacid sequence method and the product thereof of 86 free halfcystines (Cys86) specificity coupling maleinamide PEG (PEG maleimide); (2) in WO2008/074230, disclose by IFN α the 85th or 86 for amino acid mutation is Cys, recycling maleinamide PEG carries out specificity pointed decoration.(3) also can select containing holding the PEG of amino specific binding activity group to carry out N-terminal modification (as Chinese patent CN1229388C, CN100478359C and CN101381409A) to Interferon, rabbit with N.
The modification PEG of N-terminal, away from Interferon Receptors land, should be desirable decorating site from structural analysis.But the 1st of natural interferon alpha N end is halfcystine, and itself and 99 halfcystines form a pair of disulfide linkage (this disulfide linkage is very crucial to biological activity).Adopt the single-minded site PEG of interferon-alpha N-terminal is modified, modification rate is very low and large on biological activity impact.
For improving the activity of natural interferon, Amgen company of the U.S. has synthesized a kind of non-natural Interferon, rabbit (commodity are called Infergen for Consensus interferon, integrated interferon) first.Infergen is non-natural recombinantα-interferon of a kind of machine as calculated optimum combination, and its sequence is combined by 20 kinds of natural alpha-interferon hypotype sequence optimisation, and its antiviral activity has improved 5～10 times than natural interferon.1997, FDA approval Infergen was applied to the treatment of hepatitis C, and result of study shows that curative effect is obviously better than common Interferon, rabbit.U.S. Pat 4695623, US4897471, US5372808 disclose Interferon alfacon-1 and have had the interferon activity of wide spectrum, have stronger antiviral and antitumor and activate the activity of NK cell.But Infergen is identical with natural interferon alpha, there is the shortcomings such as poor stability, Half-life in vivo is short, immunogenicity is strong.N holds primary halfcystine to form disulfide linkage with 99 halfcystines simultaneously, can not effectively carry out N-terminal modification.Although the U.S. Pat 005985265A of Amgen company discloses the method terminal modified with the N of Infergen albumen, the biological activity no matter retaining at modification rate with after modifying is all starkly lower than the N-terminal of other albumen and modifies (recombinant methionyl human G-CSF G-CSF and recombinant human somatropin GH etc.).
Terminal modified for realizing the N of Interferon, rabbit, simultaneously according to homologous sequence supreme principle, the inventor has designed brand-new integrated interferon sequence, called after integrated interferon variant (super antiviral interferon, be called for short IFN-SA), now change restructuring integrated interferon variant into according to the nomenclature mo of Interferon, rabbit.Be made up of 171 amino acid, introduce Gly Ser Gly Gly Gly five amino acid sequence in N-terminal specialized designs, the single-minded site PEG that can realize N-terminal modifies.This structure is different from Infergen structure, and extracorporeal antivirus effect activity has improved 50% simultaneously.This novel texture and restructuring preparation and the application in antiviral property disease, obtained Chinese invention patent mandate, the patent No.: ZL01102915.3.
The invention provides the preparation method and application of a kind of restructuring integrated interferon variant (IFN-SA) polyethylene glycol conjugation thing (being called for short PEG-IFN-SA).It is modifier that the present invention preferably can hold the 20KD polyoxyethylene glycol (PEG-ALD) of the propionic aldehyde group of amino specific binding with N, select pH 4.5～6 conditions to modify, successfully obtained Pegylation restructuring integrated interferon variant conjugate (PEG-IFN-SA).This preparation method N holds single site chemical modification object, effectively improved this protein stability, extended the transformation period, reduced immunogenicity and toxic side effects.The medicine that is expected to become novel long-acting interferon prevention and treats the diseases such as viral infection disease, proliferative disease, enhancing immunologic function.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of restructuring integrated interferon variant (IFN-SA) the polyethylene glycol conjugation thing for the chemically modified of N end fixed point.This conjugate has the features such as good stability, long half time, immunogenicity and toxic side effects are low, can be used for preparing the medicine of Prevention and treatment viral infection disease, proliferative disease, enhancing immunologic function and other diseases.
The polyethylene glycol conjugation thing of the IFN-SA of a kind of chemically modified the present invention relates to.
IFN-SA represents the integrated interferon variant of recombinating, and it is characterized in that, the aminoacid sequence of described IFN-SA is shown in SEQ ID No:1:
Gly Ser Gly Gly Gly Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn
1 5 10 15
Arg Arg Ala Leu Ile Leu Leu Ala Gln Met Arg Arg Ile Ser Pro Phe
20 25 30
Ser Cys Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
35 40 45
Asp Gly Asn Gln Phe Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu
50 55 60
Met Ile Gln Gln Thr Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala
65 70 75 80
Ala Trp Asp Glu Ser Leu Leu Glu Lys Phe Tyr Thr Glu Leu Tyr Gln
85 90 95
Gln Leu Asn Asp Leu Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu
100 105 110
Glu Thr Pro Leu Met Asn Val Asp Ser Ile Leu Ala Val Arg Lys Tyr
115 120 125
Phe Gln Arg Ile Thr Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys
130 135 140
Ala Trp Glu Val Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser
145 150 155 160
Thr Asn Leu Gln Glu Arg Leu Arg Arg Lys Asp
165 170
IFN-SA polyethylene glycol conjugation thing, is characterized in that polyoxyethylene glycol is to be connected with the amino covalence of the α of restructuring integrated interferon variant N-terminal glycine with amido linkage.
Hold the polyoxyethylene glycol being connected it is characterized in that with the N of IFN-SA, described polyoxyethylene glycol is the mono methoxy polyethylene glycol molecule of aldehyde radical activation.
The mono methoxy polyethylene glycol molecule of aldehyde radical activation, is characterized in that, includes but not limited to mono methoxy PVOH propionic aldehyde (mPEG-ALD), mono methoxy polyethylene glycol butyraldehyde, mono methoxy polyethylene glycol acetaldehyde, mono methoxy polyethylene glycol valeral.Preferably mono methoxy polyethylene glycol propionic aldehyde.Peg molecule, according to coupling degree difference, can be that molecular weight is arbitrary molecule of 10KDa～40KDa, the peg molecule that wherein preferred molecular weight is 20KD.Peg molecule can be straight chain type or branching type, can be strand, two strands or multichain.Preferably straight chain type strand peg molecule.
The present invention also provides the preparation method of IFN-SA polyethylene glycol conjugation thing.Affiliated art person can select the required consumption of structure type, molecular weight and reaction of polyoxyethylene glycol according to general knowledge.The preparation method who provides by the application, according to structure and the character of protein molecular after modifying, selects modification condition: modify potential of hydrogen, reaction system, the mol ratio of albumen and polyoxyethylene glycol etc.
MPEG fixed point N-terminal take the preferred 20KDa of the present invention is modified IFN-SA as example, and the preparation method of conjugate is described.
The preparation of IFN-SA, is specifically shown in patent ZL01102915.3.
Well known in the art, under boron sodium cyanide existence condition, PEG meeting and primary amine generation reduction amination with aldehyde radical.Aldehyde radical is different with other close electroactive group, it only and amido react.Although the reactivity ratio NHS activity of aldehyde radical is low, it has reaction conditions gentleness, is easy to make PEG to be connected with the surface of protein or other material.Therefore,, under low pH, mPEG-aldehyde can carry out selective reaction to the N end of protein.This reaction conditions is in pH value 4.0～7.0, and under the buffer system of 10～100mmol/L salt concn, temperature is at 4～25 ℃, and the reaction times is 1～72 hour, reaction mol ratio PEG: albumen was at 1: 2～1: 10.Generally, the end modified product homogeneity of N-obtaining under selection low temperature, low pH, reaction times suitable condition is strong, and reaction conversion rate is higher.
The preparation process of conjugate described in patent of the present invention: the mPEG-ButyrALD of the phosphate buffered saline buffer of IFN-SA and 20KD is reacted adding under 20mM boron sodium cyanide condition, then adopt ordinary method to carry out separation and purification, filtration sterilization, obtains the conjugate of IFN-SA of the present invention.The preferred pH4.0-6.0 of phosphate buffered saline buffer used, concentration is 50-100mmol/L, the preferred 4-10 ℃ of temperature, preferably 24～48 hours reaction times, reaction mol ratio PEG: IFN-SA is 1: 2～1: 5, and modification rate reaches more than 50%.Selection and the modifying method of modification condition describe in detail in example.
The purification process of conjugate of the present invention is: IFN-SA and polyethylene glycol polymer are through reacted sample, with upper SPSepharose F.F. after 5 × DDW dialysis, through 10mM acetic acid-sodium acetate (pH5.5), after balance liquid balance, with the contrary degree of the salt ion wash-out of 1MNacl, penetrate with elution samples and detect purity with electrophoresis, RP-HPLC respectively.Containing polyethylene glycol conjugation thing component, again through Superdex75 molecular sieve (or SephacrylS-200), 10mM PB, (pH7.4) separates and removes high molecular polymer under condition.Structure and mass analysis method comprise mass spectrum, quality peptide figure, RP-HPLC, Gel-HPLC and SDS-PAGE.
The peg molecule (ALD-mPEG20K) of the propionic aldehyde activation of employing 20KD molecular weight is as modifier, through the pilot process test of at least 10 batches, every batch can obtain the IFN-SA polyethylene glycol conjugation thing (code name: PEG20-IFN-SA) that more than 1 gram purity is greater than 95%, modification rate approximately 50%, the terminal modified thing of non-N is lower than 3%, antiviral specific activity>=1.2 × 10
7iU/mg.Pharmacokinetics shows that at cynomolgus monkey Half-life in vivo be 34.5 hours, is obviously longer than the IFN-SA transformation period of 3 hours.Cynomolgus monkey long poison in June shows, immunogenicity is significantly lower than IFN-SA.Under 4 ℃ of conditions of aqueous solution state, can stablize 24 months.Therefore, IFN-SA polyethylene glycol conjugation thing of the present invention possesses feature long-acting, stable, reduced immunogenicity.
Separately, according to above-mentioned preparation method, select the mono methoxy polyethylene glycol butyraldehyde of 10KD and the N of IFN-SA end amino to carry out coupling and obtain PEG10-IFN-SA.Select the PEG-NHS of 40KD and the N of IFN-SA end amino to carry out coupling and obtain PEG40-IFN-SA.The extracorporeal antivirus effect activity of PEG10-IFN-SA is>=7.0 × 10
7u/mg, the transformation period is 15.3 hours.The extracorporeal antivirus effect activity of PEG40-IFN-S A is>=4.0 × 10
6u/mg, the transformation period is 42.29 hours.Show the increase along with coupling PEG molecular weight, Increased Plasma Half-life, but outer antiviral activity significantly declines.The present invention's discovery, straight chain type PEG modifies the modification that the impact of IFN-SA external activity is significantly less than to branched chain type PEG.Therefore, IFN-SA is modified in the preferred 20KD straight chain type of the present invention strand PEG coupling.
The PEG-IFN-SA being prepared by aforesaid method, is prepared into injection liquid or freeze-dried powder, the paste of external application type or the buccal tablets of emulsion or sprays and oral type or capsule, tablet by corresponding preparation technique.
Another feature of the polyethylene glycol conjugation thing of IFN-SA is, this conjugate can be used for the drug regimen of clinical treatment, the arbitrary conjugate that contains effective dose and medicinal diluent, adjuvant and carrier.
The effective constituent that PEG-IFN-SA molecule involved in the present invention is clinical medicinal preparations, said preparation includes but not limited to: thinner, stablizer, sanitas, solvating agent, emulsifying agent, adjuvant and carrier etc.1) diluent: the damping fluids such as phosphoric acid salt, acetate, Citrate trianion; 2) pH value and ionic strength; 3) stain remover and chaotropic agent: sorbyl alcohol, Tween-20, Tween-80; 4) filling agent: sorbyl alcohol, glucose, sucrose, N.F,USP MANNITOL, glycerine.The preferred 10mM PB of PEG-IFN-SA preparation (pH7.2), sorbyl alcohol, Tween-80.The effective dose of effective constituent refers to effective treatment or the preventive dose of considering the factor such as apparent molecular weight and patient body weight, age.Its effective dose of the PEG-IFN-SA the present invention relates to is 0.5ug～9ug/kg/ time.
The clinical medicinal preparations administering mode of PEG-IFN-SA enteron aisle external administration, includes but not limited to subcutaneous, muscle, vein, abdominal injection, suction, hypogloeeis or transdermal administration.The administration cycle is 3～14 days/time.
The present invention also discloses IFN-SA and polyethylene glycol conjugation thing for preventing and treat the method for viral infection disease, proliferative disease, enhancing immunologic function and other diseases.
In one embodiment, described virus sense is characterised in that and includes, without being limited to hepatites virus infections, human papillomavirus's infection, and herpes simplex viral infections, human immunodeficiency virus infection, ebv infection, SARS infect and influenza infection.Described hepatites virus infections is characterised in that and includes, without being limited to hepatitis C virus, hepatitis b virus infected, hepatitis A virus, hepatitis D virus, hepatitis E virus.Preferably hepatitis C, hepatitis b virus infected.Preferably hepatitis C, hepatitis b virus infected.
In another embodiment, described proliferative disease is characterised in that and includes, without being limited to malignant tumour, and pernicious swollen being characterised in that includes, without being limited to chronic marrow originality leukemia, melanoma, kidney, liver cancer.
Also provide drug regimen drug combination mode to include, without being limited to ribavirin, lamivudine.The anti-hepatitis c virus broad-spectrum antiviral class medicine synthetic with suppressing viral DNA and RNA uses as ribavirin combines; The antiviral class medicine of anti-hepatitis B virus and ucleosides is as lamivudine combination utilization; Treatment malignant tumour and the combination of other chemotherapeutics are used.
So far the present invention is described in detail, by reference to following example, can the present invention is had more clearly and be understood, described example be only intended to limit the invention for the purpose of illustration and not.
Accompanying drawing explanation
The mol ratio of accompanying drawing 1:IFN-SA and PEG affects SDS-PAGE electrophorogram to modification reaction, and wherein 1: be 1: 2 ratio, 2: be 1: 3
SDS-PAGE electrophorogram before and after accompanying drawing 2:IFN-SA modifies.Wherein 1: for modifying front IFN-SA, 2: be to modify after product, 3: be the PEG20-IFN-SA after purifying, M: molecular weight of albumen standard.
Ratio, 3: be 1: 4 ratio, 4: be 1: 5 ratio, 5: be 1: 6 ratio, M is molecular weight of albumen standard.
Accompanying drawing 3: the SDS-PAGE electrophorogram of the IFN-SA that different molecular weight PEG modifies.Wherein M is marker, and 1 is PEG10K-IFN-SA, and 2 is PEG20K-IFN-SA, and 3 is PEG40K-IFN-SA.In figure the IFN-SA albumen of three kinds of PEGization because of mobility slack-off bigger than normal than theoretical value.
The RP-HPLC color atlas of accompanying drawing 4:PEG20-IFN-SA.In figure, PEG20-IFN-SA is single absorption peak, and purity is 98.7%.
After the tryptic digestion of accompanying drawing 5:PEG20-IFN-SA and IFN-SA, go back proper mass peptide figure analysis color atlas.Wherein scheming A is the peptide figure of IFN-SA, and figure B is PEG20-IFN-SA peptide figure.The two only has the difference of two peptide Duan Feng, and example 3 is shown in variance analysis explanation.
Fig. 6: PEG20-IFN-SA is the rear interior resisting virus biological activity temporal evolution graphic representation of subcutaneous injection various dose (10ug/kg, 30ug/kg and 90ug/kg) in food crab.Wherein ● be 10ug/kg, zero is 30ug/kg, ▲ be 90ug/kg.X-coordinate be the time (hour), ordinate zou is antiviral activity unit in every milliliter of blood (IU/ml).
Embodiment
The modification condition of example 1, restructuring integrated interferon variant Pegylation conjugate (PEG20-IFN-SA)
IFN-SA solution (concentration is 10mg/mL, cushions PB, pH7.0 into 100mM) is prepared by Chongqing Fujin Biological Medicine Co. Ltd, and (purity > 95%, relative molecular mass is 20 × 10 to mPEG-ButyrALD-20KD
3) purchased from Jiankai Science and Technology Co., Ltd., Beijing, sodium cyanoborohydride (CH3BrNa) solution is purchased from Sigma company, SDS-PAGE related reagent and solution and various analytical pure are the raw chemical product in Shanghai.Temperature constant magnetic stirring instrument (Jiangsu Zhong great instrument plant), electrophoresis apparatus, SP Sepharose F.F., Superdex75 molecular sieve, AKTA explore chromatographic system, SDS-PAGE electrophoresis system are Pharmacia company product.
1, the impact of pH value in reaction on modified outcome
IFN-SA solution uses respectively 100mmol/L PB (pH4.0, pH5.0 and pH5.5) damping fluid to be made into 2mg/mL, and respectively getting portion, to add 1mol/LCH3BNNa mother liquor to final concentration be 20mmol/L.Within 1: 4 in molar ratio, (IFN-SA:mPEG-ButyrALD-20KD) weighing m PEG-ButyrALD-20KD adds in IFN-SA reaction soln, 100r/min stirring reaction 24h under 4 ℃ of conditions.SDS-PAGE electrophoresis is carried out in sampling respectively, determines the modification rate of PEG20-IFN-SA.
Experimental result shows: pH value is under 4.0,5.0,5.5 condition, and PEG1-IFN-SA proportion is respectively 18%, 31%, 40%.The modification rate that pH4.0 is described is minimum, and under the condition of pH5.5, modification rate is the highest.
2, the impact of the modification ratio of IFN-SA and PEG on modified outcome
IFN-SA solution is made into 2mg/mL with 100mmol/LPB pH5.5 damping fluid, and filling into 1mol/L CH3BNNa mother liquor to final concentration is 20mmol/L.By IFN-SA and mPEG-ButyrALD-20KD mol ratio be A (1: 2), B (1: 3), C (1: 4), D (1: 5), E (1: 6) respectively weighing m PEG-ButyrALD-20KD add in IFN-SA reaction soln, 100r/min stirring reaction 24hr under 4 ℃ of conditions, SDS-PAGE electrophoresis is carried out in sampling respectively, determines the modification rate of PEG20-IFN-SA.
Experimental result shows (Fig. 1): in the time that IFN-SA and mPEG-ButyrALD-20KD mol ratio are A (1: 2), B (1: 3), C (1: 4), D (1: 5), E (1: 6), PEGI-IFN-SA proportion is respectively 18%, 27%, 39%, 40%, 41%.When the mol ratio of PEG modifier reaches after 1: 4, can not further improve modification rate, reaction can cause the polymer that PEG modifies to increase.
3, temperature of reaction and the time impact on modified outcome
IFN-SA solution is made into 2mg/mL with 100mmol/L PB pH5.5 damping fluid, fill into 1mol/L CH3BNNa to final concentration be 20mmol/L.Get IFN-SA and mPEG-ButyrALD-20KD mass ratio 1: 4, respectively at 4 ℃ of reaction 48hr, 25 ℃ of reaction 24hr different time points, SDS-PAGE electrophoresis is carried out in sampling respectively, determines the modification rate of PEG20-IFN-SA.Experimental result shows: under 4 ℃ of conditions, by extending the reaction times, reach equally the modification rate of 25 ℃ of reactions of reaction.But 4 ℃ of conditions are modified the activity and the Stability Analysis of Structures that are more conducive to IFN-SA.
4, the impact of IFN-SA concentration on modified outcome
IFN-SA solution is made into as 1.0mg/mL with 100mmol/L PB pH5.5 damping fluid, 2.0mg/mL, 3.0mg/mL, 4.0mg/mL,, adds mPEG-ButyrALD-20KD in IFN-SA and the mPEG-ButyrALD-20KD mass ratio ratio of 1: 4 by totally 4 parts, 4 ℃, 100r/min stirring reaction 24hr, SDS-PAGE electrophoretic analysis is carried out in sampling respectively, determines the modification rate of PEG20-IFN-SA.
Experimental result shows, IFN-SA concentration is at 1.0mg/mL, 2.0mg/mL, and 3.0mg/mL, when 4.0mg/mL, PEG20-IFN-SA proportion is respectively 28%, 40%, and 45%, 48%.But while raising along with protein concentration, the modification of PEG20-IFN-SA increases to some extent, it while reaching 3.0～4.0mg/mL, is suitable concn.
The separation and purification of example 2, restructuring integrated interferon variant Pegylation conjugate (PEG20-IFN-SA)
Sample P EG-IFN-SA after modifying, by 5 × DDW dialysed overnight, SP Sepharose F.F chromatography column on sample after dialysis.SP SepharoseF.F loading after BufferA (10mM acetic acid-sodium acetate pH5.5 balance) balance for chromatography column, use respectively wash-out 1 (10mM acetic acid-sodium acetate 0.15M NaCl pH5.5), wash-out 2 (10mM acetic acid-sodium acetate 0.2M NaCl pH5.5), wash-out 3 (10mM acetic acid-sodium acetate 0.25M NaCl pH5.5), wash-out 4 (10mM acetic acid-sodium acetate 0.35M NaCl pH5.5), wash-out 5 (10mM acetic acid-sodium acetate 0.45MNaCl pH5.5) carries out wash-out, collect and penetrate peak and each elution peak respectively, and SDS-PAGE electrophoretic analysis is carried out respectively in sampling.Collect the PEG-IFN-SA sample after SPSepharose F.F chromatography purification, upper Superdex 75 molecular sieves.Superdex 75 gel molecular sieve 10mmol/LPB, pH7.4 balance, balance is rear loading well, then uses 10mmol/L PB, and pH7.4 constant gradient separates, and collects subsequently containing PEG20-IFN-SA main peak.SDS-PAGE electrophoretic analysis (seeing Fig. 2) is carried out in sampling respectively.
Experimental result shows, can effectively separate with free IFN-SA with the IFN-SA that modifies 2 PEG modifying 1 PEG with SuperdexG75 post through SP Sepharose.
Example 3,10KD and 40 polyethyleneglycol modified restructuring integrated interferon variants
Select respectively the mono methoxy polyethylene glycol butyraldehyde of 10KD and the branched chain mono methoxy polyethylene glycol propionic aldehyde of 40KD (being Beijing Jian Kai biotech firm product), according to the method for example 1 and example 2, IFN-SA is carried out to N and hold single site-specific sex modification, through separating and purifying acquisition PEG10-IFN-SA and PEG40-IFN-SA (seeing Fig. 3).
The physico-chemical property of example 4, restructuring integrated interferon variant Pegylation conjugate (PEG20-IFN-SA)
1, purity
The analytical column of analysis mode RP-HPLC is C18 (Waters Symmestry), mobile phase A liquid (0.1% trifluoroacetic acid, 5% acetonitrile), Mobile phase B liquid (0.1% trifluoroacetic acid, 95% acetonitrile).Applied sample amount 20 μ l, flow velocity is 1mL/min, gradient elution 70min (A liquid is from 100% to 30%, B liquid from 0 to 70%), detection wavelength is 214nm.Experimental result shows (Fig. 4), and PEG20-IFN-SA purity reaches more than 95%.
2, PEG20-IFN-SA decorating site is determined
Be SGG GCD LPQ THS LGN through Shanghai Inst. of Life Science, CAS Proteomic analysis center with the N terminal amino acid sequence of amino acid sequence analysis instrument mensuration PEG20-IFN-SA.First Gly of N-terminal before unmodified fails to measure, and proves that this site modified by PEG.
3, PEG20-IFN-SA decorating site was all once analyzed
It is amido modified that this product mPEG-ALD is modified to single-minded N-terminal, but under the existence of base boron sodium cyanide, may be modified at because modifying the different generating portion mPEG-ALD of condition the ε-NH of Methionin
2on residue.For proving that in PEG20-IFN-SA, PEG is only modified at N-terminal, by selecting the trypsin Sigma company that specificity is strong, protein groups classes and grades in school) specificity enzymolysis Methionin and arginic C end peptide bond, due to the impact of sterically hindered effect, the lysine sites after PEG modifies can not be cut by trypsinase identification and enzyme.IFN-SA quality peptide figure before and after relatively modifying, the N-terminal PEG that can obtain in this sample was all once modifying.
To after IFN-SA and bis-kinds of sample freeze-drying desalinations of PEG20-IFN-SA, use 1%NH
4hCO
3be dissolved into 2mg/ml, each sample is got 0.9ml, fills into 50uL bovine trypsin (mass ratio 1: 50), and 37 ℃ of reactions are after 24 hours, the 0.1MoL/L DTT reduction that fills into again 0.1ml is processed (37 ℃, 1hr) through the two quality peptide figure of RP-HPLC and mass spectroscopy.
From Fig. 5 A and Fig. 5 B relatively: in unmodified IFN-SA sample peptide figure, 13.5min peptide Duan Feng disappears in PEG20-IFN-SA, and in PEG20-IFN-SA peptide figure, occurs the peptide Duan Feng that 37.9min is new, and all the other peptide section peak shapes and peak area are in full accord.The 13.5min peptide section disappearing at PEG20-IFN-SA is 1655 through mass spectroscopy molecular weight, i.e. the first peptide section of IFN-SA (sequence is GSGGGCDLPQTHSLGNR), and this peptide section that the 37.9min peptide section occurring is PEGization.Relatively both quality peptide figure, have no the fragment that all the other are modified by PEG.Prove, IFN-SA modifies and is only positioned at N-terminal through the PEG of this patent method.
Example 5, the polyethyleneglycol modified IFN-SA Bioactivity of different molecular weight are measured
Adopt Wish/VSV cytopathic-effect inhibition assay to measure extracorporeal antivirus effect activity.After the national standard of humanα-interferon's Determination of biological activity (middle inspection institute) redissolves, be diluted to 1000IU/mL with mensuration nutrient solution.In 96 porocyte culture plates, do 4 times of gradient series dilutions, totally 8 extent of dilution, each extent of dilution does 2 holes.By PEG10-IFN-SA, PEG20-IFN-SA and PEG40-IFN-SA are diluted to 0.1 μ g/mL with mensuration nutrient solution respectively, be diluted to IFN-SA dilution be 1.0ng/mL, record pre-extension rate with mensuration nutrient solution.In 96 porocyte culture plates, then do 4 times of gradient series dilutions, totally 8 extent of dilution, each extent of dilution does 2 holes.By the antiviral external activity of active standard substance calculation sample.
PEG10-IFN-SA extracorporeal antivirus effect activity has retained the activity of IFN-SA approximately 8.01%, and PEGG20-IFN-SA has retained 1.24%, PEG40-IFN-SA and retained 0.43%.Adopt the active standard substance of international recombinant human integrated interferon in NIBSC source to do reference, four its extracorporeal antivirus effect activity all improve 50% left and right.
Example 6, IFN-SA and the comparative studies of PEG20-IFN-SA pharmacokinetics
12 of cynomolgus monkeys, male and female half and half, be divided into 2 groups, single gives the IFN-SA of 10 μ g/kg or the PEG20-IFN-SA of 50 μ g/kg respectively, Plasma Concentration after IFN-SA radioimmunity (RIA) kit measurement subcutaneous injection IFN-SA or the PEG20-IFN-SA of the development of employing our company, experimental data is intended the moving parameter of joint account medicine with 3P97 medicine dynamic program.Laboratory animal is from forelimb elbow anticoagulant heparin venous blood sampling, each 1.5ml.The blood time point of getting of IFN-SA is 0h 30min, 1hr, 2h, 4h, 8h, 12h, 16h and 24h.That PEG-IFN-SA is 0h 1hr, 2hr, 4,6,12,24,48,72,96,120,144 and 168h.Blood in the centrifugal 10min of 4000rpm, is isolated serum after room temperature is placed 30min, and-20 ℃ of preservations are used for the mensuration of Plasma Concentration.
Through comparision of goodness of fit, the medicine generation of the IFN-SA of unmodified in, meets a chamber and eliminates model, average out to peak time T
peak(measured value) is 5.33 ± 1.63h, on average eliminates the transformation period (t
1/2e) be 3.06 ± 1.19h; PEG20-IFN-SA average out to peak time T
peak(measured value) is 13.33 ± 2.07h; Average transformation period (the t that eliminates
1/2e) be 34.43 ± 4.77h.Than the Increased Plasma Half-life of IFN-SA more than 10 times.
Example 7, PEG20-IFN-SA in vivo bioactivity are measured
18 of cynomolgus monkeys, are divided into 3 groups at random, and 6 every group, male and female half and half.Respectively after subcutaneous shot PEG-IFN-SA 10 μ g/kg, 30 μ g/kg, 90 μ g/kg in 0,3,12,24,48,72,96,120,144hr is totally 9 time points, gets blood 1.0ml, and 4 ℃ of 3000rpm get after 10min is centrifugal and are no less than 0.4ml serum sample in-70 ℃ of preservations.Adopt Wish/VSV cytopathic-effect inhibition assay to measure interior resisting virus biological activity, when each, phase point serum sample was pressed three times of dilutions, totally 6 extent of dilution from 1: 45.Measurement result is calculated the interior resisting virus activity (IU/ml) of corresponding time point with active standard substance.
Result as shown in Figure 6,10,30 and the corresponding time point of tri-dosage of 90ug/kg within the scope of 3～144hr, body internal interference element antiviral activity is dose-response relationship.Three's activity in vivo all extends in time and reduces gradually, and high reactivity maintains between 12～24hr.After 30ug/kg dosage injection, more than 12hr occurs that high in-vivo activity reaches 15000IU/ml, within 24 hours, be 11000IU/ml, 48hr is 6500IU/ml, and 72hr is 4000IU/ml, and 96hr is 2500IU/ml, within 144 hours, is 500IU/ml.
Example 8, PEG20-IFN-SA and the comparative studies of IFN-SA immunogenicity
IFN-SA establishes 1.0 μ g/kg/d groups, 5 μ g/kg/d groups, 25 μ g/kg/d group subcutaneous injections, continuous 4 weeks.PEG-IFN-SA establishes 10.0 μ g/kg/ week groups, 30.0 μ g/kg/ week groups, 90.0 μ g/kg/ week group subcutaneous injections, continuous 4 weeks.Every group of 6 cynomolgus monkeys, different time points blood sampling detects the antibody titers of anti-IFN-SA, and the geometric mean of calculating antibody titre (being the mean value of the antilogarithm of antibody titers inverse).
Result shows: PEG20-IFN-SA has significantly reduced the interior immunogenicity of body of IFN-SA.
Example 9, the promoter action of PEG20-IFN-SA to cynomolgus monkey immunologic function
If PEG20-IFN-SA is 10 μ g/kg, basic, normal, high three the dosage groups of 30 μ g/kg, 90 μ g/kg, subcutaneous injection, weekly.Within after administration 30,58,175,180 days, carrying out peripheral lymphocyte proliferation test and NK cell killing activity detects.
Result demonstration, administration is after 2 months, and three dosage groups are compared with control group, and ConA induction peripheral lymphocyte proliferation ability and NK cell killing activity detect and all significantly increase (P < 0.05).Prompting, PEG-IFN-SA has the effect that strengthens immunologic function.
The activity of example 10, PEG20-IFN-SA In Vitro Anti hepatitis B virus
Select the 2.2.15 clone of hepatitis B virus (HBV) DNA clone transfected with human liver cancer cell (HepG2), observe PEG20-IFN-SA and integrated interferon (trade(brand)name Infergen or IFN (one) Con
1) impact on HBsAg and HBeAg secretion, HBV DNA copy number in vitro.If without the administration group of medicine cell control group and different pharmaceutical concentration, the final concentration of PEG20-IFN-SA is 1.0 × 10
6to 0.0032 × 10
6five times of gradient dilutions of IU/ml, change a not good liquor in every four days, then dosing again.Sample and cell act on after 9 days altogether, get the DNA copy number of supernatant mensuration HBsAg, HBeAg concentration and HBV, and remaining cell adds MTT to detect cell proliferation.By obtaining cytotoxic T C
50with the IC that suppresses HBsAg, HBeAg
50, calculating corresponding therapeutic index (TI) is TC
50/ IC
50.As TI is larger, show that the effect of anti-HBV is more remarkable.Adopt quantitative fluorescent PCR to measure the copy number of HBV-DNA, in upper cleer and peaceful born of the same parents, the maximum reduced rate of HBV DNA copy number PEG20-IFN-SA compared with control group is respectively 94% and 83%, and Infergen is 84% and 76%.
Result shows: PEG20-IFN-SA has the activity of stronger In Vitro Anti hepatitis B virus.
Example 11, the external restraining effect to tumor cell line of PEG20-IFN-SA
Select four strain tumor cell lines: hepatocellular carcinoma (HepG2), mammary cancer (MCF-7), acute promyelocytic leukemia (HL60), kidney (ACHN).If without the administration group of medicine cell control group and different pharmaceutical concentration, PEG20-IFN-SA final concentration is 4.0 × 10
5to 0.001 × 10
5iU/ml, four times of gradient dilutions, totally 8 concentration gradients, act on MTT after 48 hours and detect cell proliferation situation.
Experimental result shows: PEG20-IFN-SA all has obvious restraining effect to four strain tumour cells, and prompting PEG20-IFN-SA has potential therapeutic action to hepatocellular carcinoma, mammary cancer, acute promyelocytic leukemia, kidney.
Example 12, the effect of PEG20-IFN-SA anti-tumor in vivo
Select kidney (ACHN) cell strain, go down to posterity while growing to 80% fusion, be digested to single cell suspension, with 6.0 × 10
5individual/200ul, subcutaneous injection is subcutaneous at the back of female nude mice, observes and grows after knurl piece until subcutaneous for 4～5 days, with digital caliper detection tumor size, is divided at random model group and PEG-IFN-SA treatment group, 10 every group.PEG20-IFN-SA is by 10
6iU/kg, subcutaneous injection, 2 times weekly, the physiological saline of model group injection same volume.After the 5th administration 24 hours, put to death nude mice, peeling operation lump, digital caliper detects tumor size.Result shows, after PEG20-IFN-SA administration, tumor size reduces 33% left and right than model group, has the effect of certain anti-tumor in vivo growth.
Claims (1)
1. the polyethylene glycol conjugation thing preparation method of restructuring integrated interferon variant, described restructuring integrated interferon variant is referred to as IFN-SA, its aminoacid sequence is SEQ ID No:1, the polyethylene glycol conjugation thing of described restructuring integrated interferon variant is referred to as PEG20-IFN-SA, it is characterized in that, comprise the steps:
(1) IFN-SA solution is made into 2mg/mL with the PB damping fluid of 100mmol/L pH5.5, adding 1mol/LCH3BNNa mother liquor to final concentration is 20mmol/L, by the mol ratio of IFN-SA:mPEG-ButyrALD-20KD, 1: 4 weighing m PEG-ButyrALD-20KD adds in IFN-SA reaction soln, 100r/min stirring reaction 24h under 4 ℃ of conditions, SDS-PAGE electrophoresis is carried out in sampling, determines the modification rate of PEG20-IFN-SA;
(2) modify rear sample P EG20-IFN-SA, by 5 × DDW dialysed overnight, after dialysis, on sample, use the SP Sepharose F.F chromatography column after BufferA balance, use respectively wash-out 1, wash-out 2, wash-out 3, wash-out 4, wash-out 5 carries out wash-out, collect and penetrate peak and each elution peak respectively, and SDS-PAGE electrophoretic analysis is carried out respectively in sampling, wherein, the formula of described BufferA is acetic acid-sodium acetate of 10mM pH5.5, the formula of described wash-out 1 is 10mM acetic acid-sodium acetate and the 0.15M NaCl of pH5.5, the formula of described wash-out 2 is 10mM acetic acid-sodium acetate and the 0.2M NaCl of pH5.5, the formula of described wash-out 3 is 10mM acetic acid-sodium acetate and the 0.25M NaCl of pH5.5, the formula of described wash-out 4 is 10mM acetic acid-sodium acetate and the 0.35M NaCl of pH5.5, the formula of described wash-out 5 is 10mM acetic acid-sodium acetate and the 0.45MNaCl of pH5.5, (3) collect the PEG20-IFN-SA sample after SPSepharose F.F chromatography purification, upper Superdex75 molecular sieve, the PB balance of 10mmol/L pH7.4 for described molecular sieve, balance is rear loading well, separate with the PB constant gradient of 10mmol/L pH7.4 again, collect subsequently containing PEG20-IFN-SA main peak, SDS-PAGE electrophoretic analysis is carried out in sampling respectively.
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| CN103936847B (en) * | 2013-12-24 | 2014-12-10 | 北京凯因科技股份有限公司 | Polyethylene glycol-SA (PEG-SA) and medicinal composition thereof |
| WO2015096595A1 (en) * | 2013-12-24 | 2015-07-02 | 北京凯因科技股份有限公司 | Pegylated interferon derivative and pharmaceutical composition thereof |
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| CN101514229A (en) * | 2009-04-03 | 2009-08-26 | 海南四环心脑血管药物研究院有限公司 | Human interferon alpha derivative and polyethylene glycol modified substance thereof |
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| CN1313343A (en) * | 1994-10-12 | 2001-09-19 | 安姆根有限公司 | N-terminal modified protein compositions and methods therefor |
| CN1365983A (en) * | 2001-01-19 | 2002-08-28 | 重庆富进生物医药有限公司 | Human alpha interferon derivative with superstrong antiviral activity |
| CN1929857A (en) * | 2004-02-04 | 2007-03-14 | 法玛西雅公司 | N-terminally monopegylated human growth hormone conjugates, process for their preparation, and methods of use thereof |
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