CN102327242B - Polyethylene glycol-integrated interferon variant lyophilized preparation - Google Patents

Abstract

The invention relates to polyethylene glycol-integrated interferon variant injection. Each 1ml of injection comprises 50 to 500 micrograms of polyethylene glycol-integrated interferon variant protein, a buffer system with the pH value of between 4.5 and 7.5 and a freeze-dried protective agent formed by a mannitol-alkaline amino acid mixed system. The polyethylene glycol-integrated interferon variant injection is stable and good in redissolution effect, and does not have the phenomenon of opalescence.

Description

A kind of polyethylene glycol-integrated interferon variant lyophilized preparation

Technical field

The present invention relates to a kind of polyethylene glycol-integrated interferon variant lyophilized preparation, belong to field of pharmaceutical preparations.

Background technology

As far back as nineteen fifty-seven, the people such as lssacs find that the cell of viral infection produces a kind of factor, can support antiviral infection, copying of viral interference, thus called after interferon (interferon, IFN).Interferon be by deactivation or live virus function after permissive cell, the one group of glycoprotein produced by permissive cell genome encoding.Its activity and antigenicity all depend on the protein in molecule, and have nothing to do with its glycosyl.According to its source and structure, IFN can be divided into IFN-α, IFN-β, IFN-γ, they are produced by leukocyte, fibroblast and activating T cell respectively.IFN-α is polygenes product, has more than ten to plant different subtype, but their biological activity is substantially identical.IFN, apart from outside antivirus action, also has antitumor, immunomodulating, control cell proliferation and causes the effects such as heating.The interferon used clinically is at present obtained by gene recombination technology mostly.

IFN is that chronic viral hepatitis is through the effective first-line treatment medicine of clinical verification.IFN α-2a and the IFN α-2b of what Clinical practice was more is natural structure.The two all belongs to fugitive interferon, and the half-life is short, and (4 ~ 16 h), and serum-concentration is very low after injection 24 h.Its pharmacokinetics process influence in vivo 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 serum levels fluctuates widely and easily makes virus levels bounce, and produces drug resistance, reduces therapeutic effect.Meanwhile, it is large to there is toxicity in fugitive interferon, and people's life-time service toleration is poor, and immunogenicity waits by force not enough.Therefore, fugitive interferon is modified, extend its half-life in vivo, maintain the stable of blood drug level in body, the effectiveness improving its treatment viral hepatitis will be contributed to, and alleviate toxic and side effects.

For improving the activity of natural interferon, Amgen company of the U.S. has synthesized a kind of non-natural interferon (commodity are called Infergen for Consensus interferon, integrated interferon) first.Infergen is non-natural recombinantα-interferon of a kind of machine as calculated optimum organization, and its sequence is formed by the optimum organization of 20 kinds of natural alpha-interferon subtype sequences, and its antiviral activity improves 5 ~ 10 times than natural interferon.1997, FDA ratified the treatment that Infergen is applied to hepatitis C, and result of study display curative effect is obviously better than plain interferon.US Patent No. 4695623, US4897471, US5372808 disclose the interferon activity that Interferon Alfacon-1 has wide spectrum, have the activity of stronger antiviral and antitumor and activation 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 cysteine need form disulfide bond with 99 cysteine simultaneously, effectively can not carry out N-terminal modification.Although US Patent No. 005985265 A of Amgen company discloses the method terminal modified with the N of Infergen albumen, the N-terminal that the biological activity no matter retained after modification rate and modification is all starkly lower than other albumen modifies (recombinant human granulocyte colony stimulating factor G-CSF and recombinant human somatropin GH etc.).

On Infergen basis, according to homologous sequence supreme principle, inventor has devised 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 (or claiming recombinant consensus interferon variant protein) into according to the nomenclature principle of interferon.Be made up of 171 aminoacid, 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 Anti-viral activity in vitro improves 50% simultaneously.This new structure and recombinate preparation and the application in antiviral property disease, obtained Chinese invention patent mandate, the patent No.: ZL01102915.3.

But, the same with other oroteins, use integrated interferon variant usually also can be subject to the restriction of some shortcomings as medicine, comprise immunogenicity and the half-life short, its immunogenicity causes the formation of neutralizing antibody and the loss of clinical therapeutic efficacy, and the half-life is short, means and needs patient's frequent drug administration to maintain effective treatment concentration of albumen.

Polyethylene Glycol (polyetnylene glycol, PEG) is the polymer 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 solublity 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) katre, Advanced Drug Delivery Systems, 10:91 (1993)).

But although interferon-polymer conjugate is effective clinically, such conjugate widely using in clinical practice, needing can at the preparation produced and store in elongated segment phase during being distributed to medical institutions.Drying keeps one of material unlikely putrid and deteriorated method, but some drying meanss carry out at more than 0 DEG C or higher temperature.Theory and the biological property of dry rear product can change a lot, and lyophilization occurs in less than 0 DEG C, refers to the process being removed moisture or other solvent by distillation from the material freezed.Distillation refers to solvent, such as water, and as dry ice, without liquid state, from the solid-state process directly becoming gaseous state, the product that lyophilization obtains is called lyophilized products, and this process is called lyophilizing.In freezing dry process, the structure of sample can not be destroyed, because solid constituent is support by the black ice on its position, distilling at ice is that it can leave hole in the surplus materials of drying, so just remains the biological and chemical structure of product and the integrity of activity thereof.

At present, freeze-drying is usually used in the preservation of many bioactive materials (comprising polypeptide, protein etc.), but lyophilization also has himself limitation, and as in freezing and removal process, albumen overrich, may cause the instability of product.Therefore; except the desamidation that can occur at lyophilised state and liquid state and oxidation reaction; lyophilization may cause the rising being cross-linked (generating covalency oligomer) and natural polymer ratio, in order to strengthen the stability of bioactive materials, often needs some protective agents.

At present, the protective agent of the biological product of most of injection protide selects human albumin usually, and human albumin is expensive, causes this freeze-drying prods cost high, and human albumin has and takes viruliferous danger in addition.In order to address this problem; many people are just being devoted to the research of protide lyophilized formulations; attempt replacing human albumin as freeze drying protectant using other material; as selected dextran as the freeze drying protectant of recombinant interferon in Chinese patent CN1260171A; also useful mannitol is as freeze drying protectant; with mannitol as freeze drying protectant time; often occur that solubility is poor, affect the problem of albumen effect; and alone dextran; for some albumen; be unfavorable for that it is preserved for a long time, preparation stability is relatively poor.

Known aminoacid under certain conditions can as the protective agent of lyophilized protein product; it is reported; sodium glutamate and lysinate; to a kind of albumen; the freeze denaturation of lactic acid dehydrogenase has antifreeze effect (Seguro; et al.; Cryobiology 27:70-79 (1990)) etc. the molecule of other kind; comprise monosaccharide and disaccharide, such as lactose and trehalose, and polymer such as PVP; also report is had as the protective agent of lyophilized protein; but for these disaccharide (lactose and trehalose), it is expensive, when doing cryoprotective agent with it, cost is higher.But due to the difference of the physicochemical property of different proteins own, to arbitrary given protein product, their effectiveness is unpredictable.

At present, the Polyethylene Glycol integrated interferon variant preparation of listing does not still have, therefore, need to provide a kind of stable, can for the clinical easy to use and preparation of proper storage.

Summary of the invention

Research worker of the present invention, when preparing Polyethylene Glycol integrated interferon variant injection, finds, when using mannitol as lyophilizing cryoprotective agent; there is opalescence after redissolving in freeze-drying prods, and water redissolves in liquid; loss of proteins is comparatively large, and stability is relatively also poor, is unfavorable for long-term preservation.

The object of the present invention is to provide a kind of stable polyethylene glycol-integrated interferon variant lyophilized preparation, and lyophilized formulations solubility of the present invention is good, and performance is more stable, is suitable for long-term preservation.

Polyethylene glycol-integrated interferon variant lyophilized preparation of the present invention; comprise the polyethylene glycol-integrated interferon variant of every milliliter 50 to 500 microgram; and comprise maintenance pH at the buffer system of 4.5-7.5, a certain amount of cryoprotective agent; wherein; described integrated interferon variant introduces GlySerGlyGlyGly five amino acid sequence in N-terminal design, and at the Lys of integrated interferon mature amino acid sequence ¹²¹sport Arg ¹²¹, Glu ¹⁶⁶sport Asp ¹⁶⁶.

In the present invention, described maintenance pH value can be acetate salt buffer system, carbonate buffer system, citrate buffer system or phosphate buffer in the buffer system of 4.5-7.5.

In the present invention, preferred buffer system is phosphate buffer, and preferred pH value range is 6.5-7.5.

Cryoprotective agent described in the present invention is mannitol-amino acid system, and described aminoacid is basic amino acid, example hydrochloric acid arginine or lysine hydrochloride, and mannitol and amino acid whose ratio are 4:1 ~ 2:1.

Research worker of the present invention finds; by mannitol and basic amino acid; such as arginine hydrochloride or lysine hydrochloride; after certain proportion mixing; as the cryoprotective agent of Polyethylene Glycol integrated interferon variant freeze-drying prods; in the Polyethylene Glycol integrated interferon variant lyophilized preparation preparing gained; after the freeze-drying prods redissolution of gained; there will not be opalescence, and, after water redissolves; albumen (Polyethylene Glycol integrated interferon variant) content loss is few; preserve after 18 months for 4 DEG C, the activity preservation rate of product is high, is more suitable for long-term preservation.

Polyethylene Glycol integrated interferon variant lyophilized preparation of the present invention, also can comprise stabilizing agent further, and described stabilizing agent is Polysorbate, such as polyoxyethylene sorbitan monoleate, and consumption is 0.05-0.20mg/ml.In lyophilized formulations, after adding a certain amount of stabilizing agent, preparation stability can correspondingly be improved.

As polyethylene glycol-integrated interferon variant compound protein, Polyethylene Glycol used, it can be straight chain Polyethylene Glycol, also can be branched chair polymacrogol, be preferably straight chain Polyethylene Glycol, more specifically, the Polyethylene Glycol of coupling integrated interferon variant of the present invention is mono methoxy polyethylene glycol (mPEG) aldehyde, as being mono methoxy polyethylene glycol acetaldehyde, mono methoxy polyethylene glycol propionic aldehyde, mono methoxy polyethylene glycol butyraldehyde, to realize the pointed decoration to integrated interferon N-terminal, it is 5000-50000 that the molecular weight ranges of mPEG used can be selected from, be preferably the scope of 10000-20000, as being mPEG ₁₂₀₀₀, mPEG ₂₀₀₀₀.

In the present invention, when as a kind of lyophilizing stock solution, after with water preparation, it contains following component:

The polyethylene glycol-integrated interferon variant of 50 to 500 micrograms, wherein said integrated interferon variant introduces GlySerGlyGlyGly five amino acid sequence in N-terminal design, and at the Lys of integrated interferon mature amino acid sequence ¹²¹sport Arg ¹²¹, Glu ¹⁶⁶sport Asp ¹⁶⁶;

PH is kept to be the NaH of 6.5-7.5 ₂pO ₄.2H ₂o-Na ₂hPO ₄.12H ₂o buffer system;

The mannitol of 10-40mg/ml;

The lysine hydrochloride of 10-20mg/ml or arginine hydrochloride, and,

The polyoxyethylene sorbitan monoleate of 0.05-0.15mg/ml;

Wherein, the weight ratio of mannitol and arginine hydrochloride or lysine hydrochloride is 4:1 ~ 2:1;

Water for injection.

In injection, polyethylene glycol-integrated interferon is the mono methoxy polyethylene glycol-integrated interferon variant carrying out single modification on N-terminal GlySerGlyGlyGly.

Modify the Polyethylene Glycol of integrated interferon, for molecular weight is the mPEG of 12kDa or 20kDa.

After liquid carries out lyophilizing before by the stable lyophilizing of gained, can obtain being more conducive to storage is lyophilized powder needle-like injection.

In one embodiment of the present of invention; have studied mannitol-basic amino acid (such as arginine hydrochloride or lysine hydrochloride) the cryoprotective agent system of different proportion; with single use mannitol as cryoprotective agent; it is when redissolving; redissolution Contrast on effect; result shows, after adding a certain amount of basic amino acid, its redissolution successful is better than alone mannitol.

In another embodiment of the invention; to the freeze-drying prods of gained of the present invention; preserve under 4 DEG C of conditions after 24 months; protein active retention rate and single Polyethylene Glycol integrated interferon variant content contrast; in stability; after adding a certain amount of basic amino acid, also obviously to be better than alone mannitol as cryoprotective agent.

Embodiment

The present invention is illustrated further by following embodiment, but the restriction that any embodiment or its combination not should be understood to scope of the present invention or embodiment.Scope of the present invention is defined by the appended claims, and in conjunction with the general general knowledge of this description and this area, those of ordinary skill in the art can clearly understand claims limited range.Under prerequisite without departing from the spirit and scope of the present invention, those skilled in the art can carry out any amendment or change to technical scheme of the present invention, and this amendment and change are also contained in scope of the present invention.

Embodiment 1 prepares mPEG _20kDa-IFN-SA

It is that 100mM, pH6.0 include 20mMNaCNBH that IFN-SA solution (3.5mg/ml) is dissolved in sodium phosphate concentration ₃solution in, after 4 DEG C of coolings, fully mixing, adds the methoxy poly (ethylene glycol) aldehyde (mPEG-propionic aldehyde, mean molecule quantity is 20kDa) exceeding 4 times of moles.

In course of reaction, the degree of modification reversed-phase HPLC of protein is monitored, and after 10 hours, analysed by reverse phase HPLC result shows, the protein transduction that the N-end of 80% has untight α amino becomes mPEG-IFN-SA derivant.

At 10 hours points, reactant mixture dilute with water 5 times, the purification ion-exchange chromatography of mPEG-IFN-SA derivant has come, its post used is Hiload 16/10 S Sepharose HP post, balanced with the sodium-acetate buffer pH5.5 of 20mM, reactant mixture loads in post with the flow velocity of 1ml/min, unreacted mPEG aldehyde is with 3 times of same buffer to column volume eluting in addition, then under 4 DEG C of conditions, carry out the eluting that protein-polymer connects molecule, linear elution 420min is carried out with the 20mM sodium-acetate buffer of 0%-75%, pH of buffer is 5.5, containing 1M NaCl, merge stream part of mPEG-IFN-SA derivant, refilter after concentrated.

Embodiment 2 prepares the lyophilizing stock solution that following proportioning is made, and is lyophilized into finished product

Na ₂HPO ₄.12H ₂O，2.58mg/ml，NaH ₂PO ₄.2H ₂O，0.44mg/ml；

(1) lysine hydrochloride of the mannitol+10mg/ml of 40mg/ml;

(2) arginine hydrochloride of the mannitol+12mg/ml of 35mg/ml;

(3) lysine hydrochloride of the mannitol+15mg/ml of 30mg/ml;

(4) mannitol of 40mg/ml;

In proportion; get mannitol-lysine hydrochloride or the mannitol-arginine hydrochloride of mannitol or different proportion; take pH as the phosphate buffered saline of 6.5-7.5, filtration sterilization, preparation is containing the mannitol of different proportion or the mPEG20 of right mannitol-aminoacid cryoprotective agent _kDa-IFN-SA solution, containing mPEG in every ml soln _20kDa-IFN-SA 50-160 μ g/ml, fully mixes, is packed as 1ml/ bottle, carry out lyophilization, lyophilization condition is-40 DEG C of precoolings 4 hours, then vacuum drying 20 hours under-40 DEG C to-7 DEG C conditions, then be warming up to 10 DEG C gradually to keep 3 hours, prepare lyophilized formulations, preserve.

Lyophilizing sample, dissolves with water for injection, detects visible foreign matters and occurs the phenomenon of opalescence, and measure the amount of wherein Polyethylene Glycol integrated interferon variant by HPLC method, the results are shown in Table 1 under lamp inspection instrument.

As can be seen from upper table result; when with mannitol as lyophilizing cryoprotective agent time; make Polyethylene Glycol integrated interferon variant lyophilizing finished product; after redissolving with water; there will be degradation problem under opalescence, visible foreign matters and effective ingredient dissolution rate; and when adding a certain amount of basic amino acid in mannitol; such as lysine hydrochloride or arginine hydrochloride; after the lyophilizing finished product made redissolves with water again; opalescence disappears; and visible foreign matters detects and conforms with the regulations, and the effective ingredient dissolution rate after redissolving also obviously will be improved.

Embodiment 3 mPEG _20kDa-IFN-SA lyophilized formulations

Preparation method and process:

By the accurate prepared and diluted liquid of following diluent formula, for diluting stock solution after preparation, vessel used need through apyrogeneity process, and overall process aseptic condition operates.Diluent formula: mannitol 38.6g, lysine hydrochloride 10.2 g, Na ₂hPO ₄12H ₂o 2.58 g, NaH ₂pO ₄2H ₂o 0.44 g, water for injection is accurately settled to 1 L, and diluent pH is to about 7.2

Get the Polyethylene Glycol restructuring integrated interferon variant stock solution of assay approval, accurate its volume of amount, be 0.16mg/ml with diluted to protein concentration, aseptic filtration, obtains semi-finished product.

2 ml cillin bottles and plug are carried out aseptic apyrogeneity process, in the aseptic apyrogeneity process such as pipeline and subpackage syringe needle of the subpackage machine that semi-finished product directly contact, subpackage semi-finished product under aseptic condition, every bottled amount 1ml semi-finished product solution, lyophilization, lyophilization condition is-40 DEG C of precoolings 4 hours, then vacuum drying 20 hours under-40 DEG C to-7 DEG C conditions, then be warming up to 10 DEG C gradually to keep 3 hours, obtain finished product.

Embodiment 4 mPEG _20kDa-IFN-SA lyophilized formulations

Preparation technology is with embodiment 3

Embodiment 5

Preparation technology is with embodiment 3

Embodiment 6

Preparation technology is with embodiment 3

Sample is dissolved with water again, Simultaneously test mPEG20 _kDa-IFN-SA biological activity (IU/ml), single mPEG _20kDathe purity of-IFN-SA, the results are shown in Table 1, and table 1 is that different protective agent is to mPEG20 _kDathe stability that-IFN-SA preserves at 4 DEG C.

Under 4 DEG C of conditions, place 24 months, sample is dissolved with water again, Simultaneously test mPEG20 _kDa-IFN-SA biological activity (IU/ml), single mPEG _20kDathe purity of-IFN-SA, the results are shown in Table 2, and measure its activity decrease and single polyethyleneglycol content situation of change, potency labeling amount is 4.0 × 10 ⁶iU/ bottle, the results are shown in Table 2.

The calibrating of table 2 Pegylation restructuring integrated interferon variant injection

As can be seen from above-mentioned standard, three preparation-obtained mPEG of Example formulations being added with basic amino acid _20kDa-IFN-SA lyophilized formulations, deposits 24 months at 4 DEG C, and its interferon activity retention rate is high; Further, after depositing 24 months, redissolve with water, visible foreign matters also still conforms with the regulations, agalactia optical phenomenon, mPEG _20kDa-IFN-SA content reduces not obvious, and only uses mannitol as freezing drying protective agent, and they are after 24 months, and opalescence phenomenon of redissolving is obvious, and after redissolving, effective ingredient stripping quantity is low, and lamp inspection visible foreign matters is more, against regulation.

Claims (9)

1. a Polyethylene Glycol integrated interferon variant lyophilized preparation; comprise the polyethylene glycol-integrated interferon variant of every milliliter 50 to 500 microgram; and comprise the buffer system of maintenance pH at 4.5-7.5, the mixture of freeze drying protectant; wherein said integrated interferon variant introduces GlySerGlyGlyGly five amino acid sequence in N-terminal design, and at the Lys of integrated interferon mature amino acid sequence ¹²¹sport Arg ¹²¹, Glu ¹⁶⁶sport Asp ¹⁶⁶; Described freeze drying protectant is mannitol-basic amino acid mixed system, and the mass ratio of described mannitol-basic amino acid is 4:1-1:1.

2. lyophilized formulations according to claim 1, wherein said basic amino acid is arginine or lysine, or arginine hydrochloride or lysine hydrochloride.

3. lyophilized formulations according to claim 1, wherein said maintenance pH is phosphate buffer, acetate salt buffer system or citrate buffer system in the buffer system of 4.5-7.5.

4. lyophilized formulations according to claim 3, the pH value of wherein said buffer system is 6.5-7.5.

5. the lyophilized formulations according to a claim any in claim 1-4, wherein said preparation also comprises stabilizing agent further.

6. lyophilized formulations according to claim 5, wherein said stabilizing agent polyoxyethylene sorbitan monoleate.

7. lyophilized formulations according to claim 6, wherein said Polysorbate is polyoxyethylene sorbitan monoleate, and the consumption of described polyoxyethylene sorbitan monoleate is 0.05-0.2mg/ml.

8. a Polyethylene Glycol integrated interferon lyophilized formulations, before being mixed with lyophilizing with water during liquid, it contains following component:

The polyethylene glycol-integrated interferon variant of 80 to 200 micrograms, wherein said integrated interferon variant introduces GlySerGlyGlyGly five amino acid sequence in N-terminal design, and at the Lys of integrated interferon mature amino acid sequence ¹²¹sport Arg ¹²¹, Glu ¹⁶⁶sport Asp ¹⁶⁶;

PH is kept to be the NaH of 6.5-7.5 ₂pO ₄.2H ₂o-Na ₂hPO ₄.12H ₂o buffer system;

The mannitol of 10-40mg/ml;

The arginine hydrochloride of 10-20mg/ml or lysine hydrochloride;

Water for injection.

9. lyophilized formulations according to claim 8, the polyoxyethylene sorbitan monoleate of wherein said preparation also further containing 0.05-0.15mg/ml.