CN85101930A - The preparation method of stable type gamma interferon preparation - Google Patents

Abstract

Glucosan or hetastarch are added in the aqueous solution of human body gamma interferon, carry out freezing, if desired, can also be under reduced pressure further dry this frozen soln, can prepare a kind of stable human body gamma interferon goods.

Description

The preparation method of stable type gamma interferon preparation

The present invention relates to a kind of IFN-goods.The human interferon has three kinds, α, β and γ type.α and β type are more stable, and the clinical parenterai administration that is mainly used in still is in deeply among the systematically clinical research at present.IFN-(after this being called for short IFN-γ) is very unstable, and its aqueous solution will obtain being applicable to very difficulty of clinical stable article storing, actively in the freezing or freezing dry process descending easily, has an a series of difficult problem to need to solve.The IFN-anti-tumor activity is the strongest, in case stable dosage form has been arranged, clinically will extensive use.

According to the present invention, the IFN-γ that uses can be any deutero-human body IFN-, natural or make with recombinant, the concentrate of for example natural generation human body IFN-γ, that is the IFN-γ (rIFN-γ) of native IFN-y (rIFN-γ) and recombinant, i.e. the material that contains human body IFN-γ that the microorganism that can produce human body IFN-γ that is obtained by gene transformation by artificial culture is prepared is (referring to European Patent Publication NO 0089676; Nucleic Acids Research 10,2487-2501,1982; Nature, 295,503-508,1982; Nucleic Acids Research 10,3605～3615,1982).Specifically, aforementioned r-IFN-γ comprises the polypeptide (its amino acid sequence is seen Fig. 1) be made up of 146 aminoacid and various (broken) fragment of polypeptide, as aminoterminal room fragment and c-terminus room fragment, amino room refers to that the amino end of polypeptide lacks the aminoacid of 4 of less thaies, these fragments also all are on the 131st position that aminoacid is later on the polypeptide, and are perhaps splitted on the position in aminoterminal room.In addition, r-IFN-γ also include polypeptide (with) the cysteine nubbin that belongs to polypeptide that replaced by serine or threonine.

Wherein the most suitable use contains 146 amino acid whose polypeptide and lacks C S-T

R-C

S (takes off (C S-T R-C S) aminoterminal room fragment IFN-γ).

It is the most favourable that use contains the high aqueous solution of human body IFN-γ concentration, and high concentration can be accomplished with gene recombinant method.

The specific activity of human body IFN-γ preferably will reach 1 * 10 ⁵To 1 * 10 ⁷Iu/milligram (after this being abbreviated as IU/mg), the activity of aqueous solution should reach 1 * 10 ²To 1 * 10 ⁷IU/ml is preferably 1 * 10 most ⁴To 1 * 10 ⁷IU/ml.

Glucosan and hetastarch, the available product of available markets.But goods of the present invention are used for clinical grade, should be identical with the grade of blood plasma substitute with the non-intestinal of preparation fully.The mean molecule quantity of glucosan is 10,000～100,000, is preferably 40,000～70,000; The hetastarch mean molecule quantity is 10,000～200,000, is preferably 20,000～60,000 or 200,000 most.

Glucosan and hetastarch concentration should be preferably 3～50mg/ml most in the IFN-γ aqueous solution more than 1mg/ml.

Except that glucosan and hetastarch, also can be added to human serum Bai Danbai (HSA) in the goods, addition is preferably 2～20mg/ml IFN-γ aqueous solution most.

According to the present invention, when in the humanIFN-, containing the residual thing of cysteine, these goods also can add goes back the original shape sulphur compound, as glutathion (having gone back original shape), thioctic acid, cysteine, sulfo-diethylene glycol, thioethanolamine, single thioglycerol, dithiothreitol dithio and the sulfo-alkanoic acid of 1 to 7 carbon atom is arranged, wherein the most suitable with the amino sulfur of bran (going back original shape).If when allowing coexistence, this class also the most suitable consumption of original shape sulphur compound is not less than 0.1mg/ml, is preferably 0.5～10mg/ml IFN-γ solution.

Also can add one or more aminoacid used as stabilizers, as glycine, glutamic acid, alanine, physiologically acceptable salt class and derivant, monosaccharide such as glucose, fructose, mannose, galactose and disaccharidase class, as sucrose, maltose and lactose etc., these all have the further effect that improves stability.Certainly, these materials also can have combination with selecting.

In the aforementioned stable agent, the sucrose effect is better, and incorporation should be 10～50mg/mlIFN-γ aqueous solution.

These goods also can further add surfactant, as Tween-20, and buffer agent, isotonic agent (isotonizing agent) etc.

According to the present invention, human body IFN-γ goods are frozen type or lyophilization type, can adopt following method production, for example, glucosan or hetastarch be added to contain human body IFN-γ, and concentration is 1 * 10 ²To 1 * 10 ⁷In the aqueous solution of IU/ml, concentration reaches more than the 1mg/ml, is preferably 3～50mg/ml, also can add human plasma Bai Danbai (HSA), sucrose and other agent.IFN-γ aqueous solution also can contain the above reduction-sulfurization compound of 0.1mg/ml, be preferably 0.5～10mg/ml, and/or the trace surfactant, perhaps being added to again in this solution going back original shape sulphur compound and/or surfactant, the stabilizing agent situation is the same as described above.

According to the present invention, can make freezing human body IFN-γ goods with the method for freezing this aqueous solution, be as cold as-80 ℃ to-30 ℃ usually, frozen product should be stored among-80 ℃ to-10 ℃ environment.This refrigerated products of drying under reduced pressure can make lyophilization IFN-γ goods again, and drying pressure is no more than 1 millimetres of mercury, within temperature cryogenic temperature to the 30 in the early stage ℃ scope.The refrigerated products that also can thaw in the ampoule of packing into, carries out freezingly and dry under decompression, makes freeze-dried product.

According to the present invention, cryodesiccated human body IFN-γ goods also have a series of other steps as ejection preparation when producing, such as aseptic filtration, and sterilization fill and lyophilization etc.

Freezing and lyophilization human body IFN-γ goods are useful, because its active reduction is very little in lyophilization and storage afterwards.

Freeze-dried product is the stable human body IFN-γ solid that contains, and is suitable for parenterai administration especially.Freeze-dried product faces with wanting elder generation dissolve in injection distilled water, normal saline or the injectable dextrose monohydrate solution ampoule of 1～100ml before as injection medicament.Adopt appropriate carriers, excipient or diluent, this goods also can be used as eye, ear, nose medicine preparation.

Freezing or lyophilization human body IFN-γ goods of the present invention are stable, low toxicity, can be used for various purposes as existing human body IFN-γ goods.

The activity of IFN-γ refers to the activity that suppresses the viral growth breeding, with iu (IU) or unit (U) expression, measures as follows:

IU: select a kind of IFN-γ international standard sample of having determined unit of activity, with standard sample with treat that test sample carries out in the human body amniotic membrane is derived FL cell line the inhibition test to Sindbis virus induction cytopathic effect respectively, calculate the vigor of testing sample then according to the ratio of gained data.

U: select a kind of IFN-γ international standard sample of having determined unit of activity and a kind of leukocyte thick IFN-γ sample of deriving, carry out in the human body amniotic membrane is derived FL cell line inhibition test to oral cavity herpes virus (VSV) inducing cell pathological changes effect, gained data relatively can be determined the derive vigor of thick IFN-γ of leukocyte; The IFN-γ vigor of testing sample, then by in the deutero-WISH cell of human body amniotic membrane system to the inhibition test of VSV inducing cell pathological changes effect, measure its antiviral activity, and be standard with thick IFN-γ, calculate according to gained vigor ratio and determine.Always carry out parallel test in the mensuration with standard I FN-γ sample.

White content of solution dawn is equivalent to 1mg and calculates according to hypothesis E:280nm=1.0.Pass between iu that obtains like this (IU) and the unit (U) is

IU

1/4 U

Brief Description Of Drawings

Fig. 1 illustrates to contain amino acid whose order among 146 amino acid whose human body IFN-γ.

Fig. 2, the description references 4(ⅰ that gives an example) structure of the cellular content PLC2 in.

Below will describe content of the present invention in detail for example, but this is not the finger limitation of the present invention.

Used human body IFN-γ for example, except that specializing all with the reference 2(II of giving an example) in the method introduced make.For example in 6, human body IFN-γ uses by making with reference to 3 methods of introducing for example, takes off (C in 8,9 and 10 for example S-T

R-C S) IFN-γ makes with the methods with reference to 5 introductions for example.

(giving an example 1)

60mg glucosan (mean molecule quantity 70,000) and 0.5ml injection distilled water are added to the human body IFN-γ (2 * 10 that 1ml contains the 3mg glutathion ⁵U/ml) in the aqueous solution, carry out aseptic filtration again, get aqueous solution 1.5ml, put into ampoule, in-30 ℃ of freezing or lyophilizations down; Lyophilization product reuse injection distilled water recovers ortho states, assay determination IFN-γ vigor.

Usually when carrying out lyophilization, get IFN-γ solution in addition, add 50mg D-mannitol, and do not add glucosan, in contrast sample.This also will be with identical method lyophilization to solution in the same old way.Vigor with human body IFN-γ before the lyophilization is a benchmark, and the residue vigor of control sample is 61%, and freeze-dried product residue vigor of the present invention is 94%.

(giving an example 2)

Get 1ml human body IFN-γ (7 * 10 ⁵U/ml) aqueous solution includes the 3mg glutathion, adds 30mg hetastarch (mean molecule quantity 200,000) and 0.5ml injection distilled water, carries out aseptic filtration, gets aqueous solution 1.5ml, puts into regulating the spleen and stomach, in-30 ℃ of freezing or lyophilizations down.Add the injection distilled water in the lyophilization product again, recover ortho states, measure its IFN-γ vigor respectively.With the vigor before the freezing or lyophilization is benchmark, and the refrigerated products residue vigor under-30 ℃ is 111%, and the freeze-dried product vigor is 117%.This has shown the stability of goods.At (refrigerated products) under-30 ℃ or after (freeze-dried product) under 40 ℃ stored for two weeks, calculate according to the initial stage vigor, the two residue vigor is respectively 121% and 107%.

(giving an example 3)

Press 2 method for example, other adds the 10mg Sodium Glutamate, stores for two weeks down in-30 ℃, and freeze-dried product residue vigor is 83%, and refrigerated products is 105%; After storing for two weeks under 40 ℃, the residue vigor of freeze-dried product is in a ratio of 105% with the vigor of the goods at the initial stage of storage.In view of this, product is stable.

(giving an example 4)

Press 1 method for example: the glucosan amount reduces to 30mg, and other adds 5mg human plasma Bai Danbai (HSA), is 78% at-30 ℃ of freeze-dried product residue vigor of storing for two weeks down, and refrigerated products is 90%; After being stored in 40 ℃ of following two weeks, the residue vigor of freeze-dried product is 112% of the storage initial stage, and this illustrates that also product is stable.

(giving an example 5)

According to the IFN-γ solution of 2 method preparation for example, other is with sucrose 51mg, lyophilization, and reuse injection distilled water recovers ortho states, measures its human body IFN-γ vigor, is as the criterion by the vigor of the human body IFN-γ solution before the lyophilization, and its residue vigor is 99%.

(giving an example 6)

Get 1ml by with reference to humanIFN-(the IFN-γ 1.6 * 10 of 3 method preparation for example ⁶IU/ml), include glutathion 3mg, add 30mg hetastarch (mean molecule quantity 200,000) and 0.5ml injection distilled water, carry out aseptic filtration, filtrate 1.5ml in the ampoule of packing into, carries out lyophilization.The lyophilization product recovers ortho states with distilled water, measures human body IFN-γ vigor.Vigor with aqueous solution before the lyophilization is a benchmark, and the residue vigor is 88%.

(giving an example 7)

Get 1ml with taking off N ₂The humanIFN-of distilled water for injection preparation, concentration 3.0 * 10 ⁵IU/ml is with hetastarch (30mg) aqueous solution of 1.5ml, lyophilization in ampoule.Lyophilization thing reuse injection distilled water recovers liquid, measures its vigor, and the residue vigor is 74% before the lyophilization.

(giving an example 8)

Get 1ml by taking off-(C made from reference to 5 method for example S-T

R-C

S) IFN-γ solution (2.5 * 10 ⁶IU/ml), adding 30mg mean molecule quantity is 70,000 glucosan and 0.5ml injection distilled water, carries out aseptic filtration, filtrate 1.5ml, lyophilization in ampoule.The lyophilization product recovers liquid with distilled water, measures the residue vigor, is 92%.

(giving an example 9)

According to 8 method for example, other adds the 30mg hetastarch, replaces the 30mg glucosan, and the residue vigor that obtains goods is 95%.

(giving an example 10)

Press 9 method for example, append the 10mg Sodium Glutamate.The residue percentage vigor that obtains goods is 104%.

(with reference to giving an example 1)

Bacterial strain RRI(pRK 248 CIts, pRC 231/IFI-900), be loaded with human body IFN-γ and represent gene, be placed in the M-9 glucose medium and under 30 ℃, cultivate, reach 3～4 * 10 until cell concentration ⁸Individual cell/ml, adding glucose and tyrosine to concentration respectively is 1.0% and 0.5%; After inducing 1 hour under 42 ℃, centrifugalize culture, the cell of collection are after freezing, and storage is got up.(consulting example 8 Japanese unexamined patent bulletin № 189197/1983)

(with reference to giving an example 2)

Get 1000g by the frozen cell that obtains with reference to 1 method of giving an example, add Tris-hcl buffer (PH7.0) 3000ml of 100mM, contain 7M guanidine hydrochloride and 2mM phenol ethyl sulfonyl fluorides in the Tris-hcl buffer.Stir this mixture under 4 ℃, centrifugalize (17000rpm) 30 minutes gets peace and quiet transparent supernatant, and the buffer that reuse is called for short PBS dilutes 70 times.The PBS buffer contains 137mM sodium chloride, 27mM potassium chloride, 8mM sodium hydrogen phosphate and 147mM potassium dihydrogen phosphate.Reuse Sharples seperator (10,000rpm) remove precipitation, with Perico membrane filter (Millipore Corp; Blocking molecular weight 10,000) concentrated supernatant (220l) is to 15l, places 40 ℃ to spend the night; Concentrated solution is removed precipitation once more with the Sharples centrifuge again, and the supernatant that obtains is introduced the filter post (Ab(Mo γ 2-11.1) that antibody is housed with flow velocity 1000ml/hr; 5 * 30cm) (consulting Specification Japanese patent application NO 176091/1983 filed on Sept.22,1983); After this following solution is passed through this antibody column successively: 2500ml PBS solution, 5000ml contains phosphate (10mM) buffer of 1M sodium chloride and 0.1% Tween 20, and 2500ml PBS and 2500ml contain 0.5M guanidine hydrochloride (20mM) phosphate buffer (PH7.0).At last, with a kind of solution flushing in back antibody column, obtaining 500ml has the fraction that washes out that suppresses virus activity.

(II) gets the above-mentioned fraction that washes out of 420ml, adds glutathion (having gone back original shape), makes it concentration and reaches 10mM, is incorporated into Sephacryl S-200(pharmacy again) filter post (in 9 * 100cm).This filter post needs to compensate with the 25mM acetate buffer in advance.Acetate buffer (PH6.0) contains 1mM editic acid salt, 150mM sodium chloride, 10mM glutathion (being reduced shape) and 2M guanidine hydrochloride.The buffer flushing filter post that last reuse is same is collected monomer and is washed out fraction (450ml).Handling IFN-γ (0.410mg/ml) specific activity that obtains like this is 3.4 * 10 ⁶IU/mg.

(with reference to giving an example 3)

Get 450ml and wash out fraction by the monomer I FN-γ that contains with reference to 2 preparations for example, add acetate (25mM) buffer (PH6.0) 25ml that contains 10mM glutathion (having gone back original shape), 150mM sodium chloride, 0.5M guanidine hydrochloride and 0.01%Tween 20, promptly get the low concentration solution of white content 0.05mg/ml of dawn after the stirring.Again this solution is filtered post (14 * 100cm) by Sephadex G-25, with aforesaid acetate buffer (PH6.0) flushing filter post, that must not have that guanidine hydrochloride contains IFN-γ washes out fraction 3180ml, white content 55.8mg/ml of dawn, white concentration 0.049mg/ml of solution dawn, the white response rate 84.8% of dawn, specific activity 3.5 * 10 ⁶The IU/mg(dawn is white).Sephadex G-25 filter post need compensate with the acetate buffer of aforementioned component in advance.

By on the solution that obtains in 4 ℃ down after aging 48 hours, use Diaflo-PM10, diameter 43mm(Amicon ultra-filtration thin film) ultrafiltration membrane be concentrated into 159ml, concentrated solution is peace and quiet transparent, white concentration 0.92mg/ml of dawn, white response rate 93.9%(146.3mg of dawn), IFN-γ specific activity 6.8 * 10 ⁶IU/mg.

(with reference to giving an example 4)

Production is taken off-(C

S-T

R-C S) IFN-γ

(ⅰ) the conversion body produces:

The representative cellular content PRC23/IFI-900 (referring to example 7) of IFN-γ digests with restriction endonuclease Nde I and Nco I, isolates Nde I-Nco I dna fragmentation (A) of a 710bp, and this fragment (A) contains IFN-γ gene territory.In addition, cellular content is limited enzyme Bgl II again respectively and Eco R I digests, and isolates a 265bp dna fragmentation (B) again, this fragment section (B) contain λ Pe promote in.Segment A, B and with the synthetic synthetic in vain oligonucleotide that starts codon of dawn that contains of chemical method

AATTCATGCAGGATCCA

GTACGTCCTAGGTAT

Use the T4-DNA ligase, as junction point, make each other to couple together again with Nde I and Eco R I viscosity end.The dna fragmentation that obtains like this, the effect and the cellular content pRC 23/IFI-900 of process Nco I and Bgl II are connected, and have constituted one and have represented cellular content pLC2, and password is C

S-T

The IFN-γ polypeptide (see figure 2) in r-Cys room.This cellular content.According to the method for humans such as Cohn, be used to transmit Escherichia Coli RRI(pRK 248 cIts) (P.N.A.S.(USA), 69,2110,1972), obtain a kind of conversion body, Escherichia Coli(=E.Coli) PRI(pLC2, pRK 248 cIts).

(ⅱ) the conversion body is cultivated

Bacterial strain E.Coli RRI(pLC2, pRK 248 cIts), be loaded with the cellular content that constitutes by (ⅰ), under 35 ℃, in the 50ml liquid medium, shake cultivation, contain white Peptone of 1% antibacterial pancreas dawn in this liquid medium, 0.5% yeast extract, 0.5% sodium chloride and 7 μ g/ml tetracyclines.Then culture fluid is moved in the M9 culture medium of 2.5l, grew up 4 hours down in 35 ℃, grew up 3 hours down in 42 ℃, absorb cell with centrifugal separation at last, and it is stored in the .M-9 culture medium contains 0.5% tyrosine, 0.5% glucose and 7 μ g/ml tetracyclines in-80 ℃ of environment.

(ⅲ) purge process

Get the frozen cell that 7.1g makes by (ⅱ) joint method, being suspended in 22ml contains in Tris-hydrogen chloride (0.1M) buffer of 7M guanidine hydrochloride and 2mM phenolic group methanesulfonyl fluoride, after stirring 1 hour (4 ℃), with 10, the speed centrifugalize of 000 * g 30 minutes, get the 24ml supernatant, reuse 300ml PBS dilution, and be incorporated into antibody column (Mo γ 2-11.1, column capacity 15ml), flow velocity ImI/min washes post with sodium phosphate (20mM) buffer that 60ml contains 0.5 M guanidine hydrochloride then, reuse 45ml contains the sodium phosphate buffer flushing of 2M guanidine hydrochloride, obtaining 25ml has the sick active fraction of inhibition, and this fraction is passed through Sephacryl S-200(pharmacy) post (2.6 * 94cm, column capacity 500ml), the ammonium acetate buffer of reuse 25mM (PH6.0) flushing gets the fraction that 40ml has antiviral activity.Ammonium acetate buffer contains the 1mM editic acid, 0.15M sodium chloride, 10mM cysteine and 2M guanidine hydrochloride; Sephacryl S-200(pharmacy) needs with these ammonium acetate buffer compensation deals before post uses.

Obtained (C at last

S-T

R-C

S) the IFN-γ polypeptide in room takes off (C

S-T

R-C

S) IFN-γ, heavy 7.0mg, specific activity 2.7 * 10 ⁶IU/mg.

(with reference to giving an example 5)

Get 2.2ml by with reference to 4(ⅱ for example) method takes off-(C containing of obtaining

S-T

R-C

S) IFN-γ washes out fraction, white content of dawn is 0.331mg/ml, acetate (25mM) buffer that contains 150mM sodium chloride and 2M guanidine hydrochloride that adds 8 times of volumes, dilute (PH6.0), stir, (2.6 * 15cm), usefulness contains the acetate buffer flushing of 150mM sodium chloride to the low concentration solution that obtains, and obtains not having (the C that takes off of guanidine hydrochloride by Sephadex G25 post again

S-T What r-Cys) contain IFN-γ washes out fraction 30ml.White concentration 0.022mg/ml of this fraction dawn, peace and quiet transparent, after under 4 ℃ aging 24 hours, use Diaflo-PMIO, diameter 25mm ultrafiltration membrane (Amicon ultrafiltration membrane) is concentrated into 0.68ml, and then filters with (0.2 μ m) filter, gets the as clear as crystal supernatant of 0.68ml, white concentration 0.67mg/ml of its dawn, the white response rate 63% of dawn.Sephadex G-25 post compensates processing with the acetate buffer that contains 150nM NaCl in advance.

Each symbolic significance is as follows among Fig. 1

The Ala alanine

The Asn asparagine

The Asp aspartic acid

The Cys cysteine

The Gln glutamine

Glu glutamic acid

The His histidine

The Ile isoleucine

The Leu leucine

Lys lysine

Met dawn propylhomoserin

The Phe phenylalanine

The Pro proline

The Ser serine

Ihr tyrosine

The Val valine

The Arg arginine

The Gly glycine

Claims (22)

1, the preparation method of human body gamma interferon preparation, be characterized in human body gamma interferon solution adding glucosan and (or) hetastarch, then that resulting solution is freezing to produce frozen preparation.If necessary, also can be under reduced pressure with the frozen preparation drying, to produce freeze-dried preparation.

2,, be characterized in that the human body gamma interferon is a recombinant human body gamma interferon according to the method for claim 1.

3,, be characterized in that recombinant human body gamma interferon is the aqueous solution that derives from the human body recombinant gamma interferon of high concentration according to the method for claim 2.

4, according to the method for claim 2, the specific activity that is characterized in the human body gamma interferon of recombinant is 1 * 10 ⁵To 1 * 10 ⁷Iu/milligram.

5,, be characterized in that human body gamma interferon preparation is that concentration is 1 * 10 according to the method for claim 1 ²To 1 * 10 ⁷The aqueous solution of iu/milliliter.

6,, be characterized in containing in the preparation glucosan according to the method for claim 1.

7, according to the method for claim 6, the mean molecule quantity that is characterized in glucosan is 10000 to 100,000.

8,, be characterized in containing in the preparation hetastarch according to the method for claim 1.

9, according to the method for claim 8, the mean molecule quantity that is characterized in hetastarch is 10,000 to 200,000.

10, according to the method for claim 1, be characterized in glucosan and (or) hetastarch is that concentration is the aqueous solution of 3～50 mg/ml.

11, according to the method for claim 1, be characterized in, also contain the human serum albumin in the preparation.

12,, be characterized in also containing in the preparation disaccharidase according to the method for claim 1.

13,, be characterized in that said disaccharidase is sucrose according to the method for claim 12.

14,, be characterized in also containing in the preparation aminoacid according to the method for claim 1.

15,, be characterized in also containing in the preparation reduction-sulfurization compound according to the method for claim 1.

According to the method for claim 15, be characterized in that 16, these characteristics are that this reduced sulphur is glutathion (reduced form).

17,, be characterized in that the reduction-sulfurization compound is that concentration is the aqueous solution of 0.5～100 mg/ml according to the method for claim 15.

18,, be characterized in that preparation is a frozen form according to the method for claim 1.

19,, be characterized in that preparation is a freeze-dried according to the method for claim 1.

20,, be characterized in that refrigerating process is to carry out in-80 ℃ to-30 ℃ temperature range according to the method for claim 1.

21, according to the method for claim 1, be characterized in that dry run under reduced pressure carries out, its pressure is no more than 0.1 millimetres of mercury.

22, a kind of stable method of human body gamma interferon that makes, be characterized in glucosan or hetastarch, or the mixture of the two joins in the aqueous solution of human body gamma interferon, then that resulting solution is freezing, and if necessary, also prepared frozen soln under reduced pressure can be carried out drying.

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