CN1522159A - Interferon formulations - Google Patents

Abstract

The invention relates to interferon compositions, such as pharmaceutical interferon compositions and methods of their preparation. In particular it relates to stabilized compositions comprising an interferon polypeptide and a sulfoalkyl ether cyclodextrin derivative.

Description

Interferon formulations

Technical field

The present invention relates to Inteferon compositions such as medicinal Inteferon compositions and preparation method thereof.Specifically it relates to the stable composition that contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant (sulfoalkyl ethercyclodextrin derivative).

Background technology

Interferon is an important cytokine, it is characterized in that having antiviral anti-hypertrophy, and immunoregulatory activity.These activity have formed and have comprised hepatitis, the basis of observed clinical efficacy in the numerous disease of various cancers and multiple sclerosis.I type interferon comprises interferon-ALPHA, β, and τ and ω, and interferon gamma is unique known member in the dissimilar II type.The summary of interferon is referring to Aggarwall and Gutterman, and at Human Cytokines, I rolls up, and Blackwell Science is among the Inc.1996.

Interferon beta and variant thereof and conjugate are described in WO01/15736 and PCT/DK02/00128, and this paper quotes its content for your guidance.

Interferon gamma is a kind of cytokine of being produced by T-lymphocyte and natural killer cell and exists as the homodimer of two non-covalent bonded polypeptide subunits.Its precursor forms that each monomeric mature form comprises 143 amino acid residues (representing with SEQ ID NO:2) and comprises signal sequence contains 166 amino acid residues (representing with SEQ ID NO:3).

Each subunit 25 and 97 has two potential N-glycosylation sites (Aggarwal etc., Human Cytokines, Blackwell Scientific Publications, 1992) in the position.According to degree of glycosylation, the molecular weight of the interferon gamma of dimeric forms be 34-50kDa (Farrar etc., Ann.Rev.Immunol, 1993,11:571-611).

The primary sequence of wild type human interferon is by (Nature 298:859-863 such as Gray, 1982), (EMBO J.1:953-958 for Taya etc., 1982), (J.Biol.Chem.259:6790-6797,1984) such as Devos etc. (Nucleic Acids Res.10:2487-2501,1982) and Rinderknecht, with at EP 77670, report among EP 89676 and the EP 110044.

Interferon gamma variant and conjugate thereof are described in WO01/36001, and this paper quotes its content for your guidance.

The experiment three dimensional structure of the wild type human interferon gamma of measuring by the X-radiocrystallography is by Ealick etc., Science 252:698-702 (1991) report, and they have reported the C-α vestige of interferon gamma homodimer.Walter etc., Nature 376:230-235 (1995) discloses the structure with the compound interferon gamma homodimer of bimolecular interferon gamma receptor soluble form.Yet the coordinate of this structure can not openly obtain.Thiel etc., Structure 8:927-936 (2000) show that the structure with the compound interferon gamma homodimer of bimolecular interferon gamma receptor soluble form has structurally and with the interferon gamma homodimer interactional the 3rd acceptor molecule do not take place.

As medicinal compound, use recombinant human interferon gamma to obtain some success, especially to some viral infection and tumor.Recombinant human interferon gamma is applicable to usually by parenteral, preferably by subcutaneous, injects.

Be mixed with the gathering that problem is an interferon polypeptides that medicinal product interrelates and recognizes with interferon.Attempted by using various stabilizing agents to solve this problem, for example, at WO98/28007, described in WO99/15193 and the WO01/24814.Commercial available interferon beta product is stablized by end user's serum albumin.

The invention summary

According to the present invention, the new Pharmaceutical composition that contains interferon is provided, said composition comprises the sulfoalkyl ether cyclodextrin derivant as stabilizing agent.

Therefore, first aspect of the present invention relates to the stable Pharmaceutical composition that contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.

Interferon activity attempted to show in term " interferon polypeptides ", for example by Aggarwel and Gutterman, and the active polypeptide that ibid limits.

Interferon polypeptides generally is selected from by interferon-ALPHA, interferon beta, and interferon ω, interferon-tau is in the group that interferon ε and interferon gamma are formed.

Another aspect of the present invention relates to first product container that contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.

Another aspect of the present invention relates to the method for the stability that increases the interferon polypeptides that is mixed with Pharmaceutical composition, and described method is included in and mixes sulfoalkyl ether cyclodextrin derivant and optional a kind of buffer agent in the described compositions.

Another aspect of the present invention relates to the method for mammal being carried out interferon therapy, and this method comprises the compositions that contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant of effective dose on the administering therapeutic.

Another aspect of the present invention relates to the Pharmaceutical composition that contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.

Another aspect of the present invention relates to the compositions that contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant and is used for the treatment of purposes in disease or the disorderly medicine in preparation.

When interferon polypeptides is an interferon-ALPHA, or interferon beta, or when its variant or conjugate, the invention provides the viral infection that is used for the treatment of most of types, cancer or tumor or tumor vessel form, Chrohn ' s disease, ulcerative colitis, Guillain-Barr é syndrome, glioma, primary pulmonary fibre modification, abnormal cell growth, perhaps be used for any suitable animal, preferred mammal, and particularly human immunoregulatory compositions and method.For example, compositions of the present invention can be used for treating osteosarcoma, the matrix cells cancer, ovarian cancer, neck abnormal development, neck cancer, the larynx papillomatosis, Mycophyta mycosis, glioma, acute myeloid leukemia, multiple myeloma, Hokdkin disease, melanoma, breast carcinoma, nonsmall-cell lung cancer, malignant melanoma (accessory drugs, late period, and preventive), carcinoid tumor, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, Kaposi sarcoma, chronic lymphocytic leukemia, renal cell carcinoma, recurrent bladder surface cancer, colorectal carcinoma, hairy cell leukemia, and viral infection, for example human papillomavirus, viral hepatitis, genital herpes, herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis, cytomegaloviral pneumonia, rhinovirus chronic and refractory hepatitis, chronic stadium HCV (I type), chronic stadium HCV (II type) and chronic viral hepatitis B.Specifically, polypeptide of the present invention or compositions can be used for the treatment of multiple sclerosis (MS), for example 4 types the MS of Gong Rening is (optimum, recurrence alleviates type MS (RRMS), carrying out property of essential MS (PPMS) and carrying out property of secondary type MS (SPMS)) in any and be used for monosymptom MS, cancer or tumor, hepatitis, for example hepatitis B and hepatitis C, the perhaps treatment of herpes infection (the optional treatment with IL-10 of a kind of treatment in back combines).

When interferon polypeptides was interferon gamma or its variant or conjugate, compositions of the present invention can be used for treating arbitrary medical science indication of describing among the WO01/36001, particularly between matter lung disease, the most specifically primary pulmonary fibre modification.Existing suggestion is used for the treatment of a matter lung disease (IPF) with interferon gamma, and for this purpose, interferon gamma can be used in combination with meticortelone.Except IPF, granuloma disease, some mycobacterial infections, renal carcinoma, osteopetrosis, scleroderma, hepatitis B, hepatitis C, the plain γ treatment of septic shock and rheumatoid arthritis available interference.

On the other hand, the present invention relates to contain the test kit of with good grounds compositions of the present invention.

Detailed Description Of The Invention

Term " is stablized " and is attempted to represent that said composition compares the bin stability with increase with the compositions that does not contain the sulfoalkyl ether cyclodextrin derivant.For example, in the liquid formulations of before storing with liquid formulations storage itself or with freezing state and using, melting, reformulate the dried forms of liquid form before using afterwards, for example lyophilizing, in spray drying or the air drying form,, be intended for use in the solid form of lung or nasal administration for example, and/or, prepare the bin stability that any other form (for example microsphere or the like) that is used for special drug-supplying system is observed increase for example.The bin stability of this increase is general to be measured according to the biological activity of comparing increase under identical storage requirement with reference composition.The bin stability of this increase attempts to comprise physics and/or chemical stability.

Term " biological activity " is attempted to represent by biological activity in the external and/or body of any suitable test determination.For example, biological activity can be according to the methods known in the art relevant with the purpose interferon polypeptides with antiviral activity, antiproliferative activity, and immunoregulatory activity, receptors bind/activating activities etc. are measured.

Have been found that the sulfoalkyl ether cyclodextrin derivant has great stabilizing active to form accumulative interferon polypeptides at lay up period.Therefore, the present invention has found to be used to stablize the concrete purposes of this interferon polypeptides.In addition, can be stabilized in lay up period owing to other reason according to the present invention, for example at lay up period because chemistry or other mechanical degradation and the interferon polypeptides of loss of activity.

The physics that term " aggregation formation " is attempted to represent to cause to form between the interferon polypeptides of oligomer interacts.It is unwelcome that aggregation forms, because in most of the cases it causes reducing or even loss of biological activity and/or increase immunogenicity.When compositions of the present invention was fluid composition, aggregation can keep solvable or be precipitated out from solution with the form of large-scale visible aggregation.When said composition is dried forms, during its preparation, can forms aggregation and cause obtaining inferior preparation.This aggregation forms and can measure by visual inspection, perhaps measures with any suitable spectrophotometer.

Interferon polypeptides

The present invention generally is applicable to all types of interferon, comprises from the isolating interferon of natural origin (for example, human leukocyte or fibroblast) the naturally occurring or variant interferon that reorganization produces, and the interferon of chemosynthesis.For example, interferon polypeptides can be, but to be not limited to be I type interferon or II type interferon, for example be selected from by interferon-ALPHA, and interferon beta, interferon ω, interferon-tau is in the group that interferon ε and interferon gamma are formed.

Interferon polypeptides can have the aminoacid sequence (wild type interferon) of natural discovery or can be the variant of this wild type interferon.

More particularly, interferon polypeptides can be to contain one or more amino acid modifiedly, that is, disappearance is inserted or replaced, and shows the variant of the wild type interferon of interferon activity.This modification can the locus specificity mode (for example, by using direct mutagenesis) or with at random or half random fashion, for example, by using at random or localized random mutagenesis, the mutation that for example limits in WO01/04287 is perhaps by using the orthogenesis technology, for example, press Stemmer, Bio/Technology 13:549-553 (1995), US 5,605, and 793, US 5,830, and 721, US 5,811, and described technology such as 238 are carried out.In general, this variant comprises maximum 15 amino acid whose modifications, and for example 1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15 modifications.Most preferably, this interferon polypeptides is human interferon (aminoacid sequence with naturally occurring human interferon) or its variant.

Heterozygosis (with the compound or chimeric implication) molecule by the covalently bound formation of one or more interferon polypeptides and one or more non-polypeptide half point attempted to represent in term " conjugate " (perhaps interchangeable " coupling polypeptide ").Term is covalently bound to be meant between interferon polypeptides and non-polypeptide half point are mutually directly covalently boundly, and perhaps by such as bridge joint, at interval, or one or more connection half point one or more interleave half point covalently bound indirectly between mutually.Preferably, link coupled interferon polypeptides is soluble under relevant concentration and condition, that is, be soluble in the physiology's liquid such as blood.The example of link coupled interferon polypeptides comprises the interferon polypeptides of glycosylation and/or PEGization.Term " non-link coupled polypeptide " can be used for the polypeptide portion of link coupled interferon polypeptides.

Plan to allow to exist other difference except this specific amino acids is distinguished with the interrelate term " difference " that uses or " different " of specific modification.Therefore, except importing specific modification disclosed herein, if desired, this interferon polypeptides can comprise other modification.For example, they can be included in the one or more extra residues of the terminal interpolation of N-, for example N-terminal add a Met residue and/the one or more C-terminal residues of truncate and " conserved amino acid replacement ", promptly have similar features, for example, p1 amino acid, acidic amino acid, polar amino acid, basic amino acid, the replacement of carrying out in the aminoacid group of hydrophobic amino acid and ArAA.Specifically, the conservative example that replaces can be selected from the listed group of following table.

????1	Alanine (A) glycine (G) serine (S) threonine (T)
		????2	Aspartic acid (D) glutamic acid (E)
????3	Agedoite (N) glutamine (Q)
		????4	Arginine (R) histidine (H) lysine (K)
????5	Isoleucine (I) leucine (L) methionine (M) valine (V)
		????6	Phenylalanine (F) tyrosine (Y) tryptophan (W)

Term " random mutagenesis " be meant for tried mutational site in the nucleic acid be at random and be at random method of mutagenesis for the sudden change that imports, for example, chemomorphosis, ultraviolet or gamma-radiation are by going down to posterity of repair-deficiency cell etc.Term " local mutation " is used for representing that method of mutagenesis preferentially takes place at predetermined portions that is tried nucleic acid or subsequence.In the context of the invention, " direct mutagenesis " is meant the change at one or more predetermined nucleotide positions, is purpose with the one or more amino acid residues that change in the amino acid sequence coded usually.Direct mutagenesis designs according to the one-level of polypeptide to be finished or the analysis of three grades of (for example model) structures usually.

Term " linking group " attempt to represent can with the link coupled amino acid residue group of relevant non-polypeptide half point such as multimeric molecule or saccharide half point.Non-polypeptide half point of useful linking group and coupling thereof is conspicuous from following table.

Linking group	Aminoacid	The example of non-polypeptide half point	Associated methods/activatory PEG	List of references
					-NH 2	The N-end, Lys	Polymer, for example PEG	mPEG-SPA TresylatedmPEG	Shearwater Inc.Delgado etc., critical reviews in Therapeutic Drug Carrier Systems 9 (3,4): 249-304 (1992)
-COOH	The C-end, Asp, Glu	Polymer, for example PEG saccharide half point	The external coupling of mPEG-Hz	Shearwater?Inc
					-SH	Cys	Polymer, for example PEG saccharide half point	The external coupling of PEG-vinyl sulfone(Remzaol PEG-maleimide	Shearwater Inc Delgado etc., critical reviews in Therapeutic Drug Carrier Systems 9 (3,4): 249-304 (1992)
-OH	Ser，Thr，OH-， Lys	Saccharide half point	The glycosylation that O-connects in the body
					-CONH 2	Asn is as the part of N-glycosylation site	Saccharide half point	Glycosylation in the body
Aromatic residues	Phe，Tyr，Trp	Saccharide half point	External coupling
					-CONH 2	Gln	Saccharide half point	External coupling	Yan and Wold, Biochemistry, on July 31st, 1984; 23 (16): 3759-65
Aldehyde ketone	The saccharide of oxidation	Polymer, PEG for example, PEG-hydrazides	PEGization	Andresz etc., 1978, Makromol.Chem.179:301; WO92/16555, WO00/23 114
					Guanidine radicals	Arg	Saccharide half point	External coupling	Lundblad and Noyes, Chimical Reagents for Protein Modification, CRC Press Inc. Boca Raton, FI
Imidazole ring	His	Saccharide half point	External coupling	For guanidine

For N-glycosylation in the body, term " linking group " with unconventional mode be used to represent to form the N-glycosylation site amino acid residue (have sequence N-X '-S/T/C-X "; wherein X ' is the arbitrary amino acid residue except that proline; X " be and arbitrary amino acid residue that X ' is identical or different and preferably be different from proline, N is an agedoite, and S/T/C is a serine, one of threonine or cysteine, preferably serine or threonine, and most preferably be threonine).Although the asparagine residue of N-glycosylation site is to connect the residue of saccharide half point during the glycosylation, unless there is other amino acid residue of N-glycosylation site, otherwise can not finish this connection.Therefore, when this non-polypeptide half point is that saccharide half point and this connection are when finishing by the N-glycosylation, the change of term " amino acid residue that contains the linking group of non-polypeptide half point " and parent's amino acid sequence of polypeptide interrelates and is interpreted as constituting one of the N-glycosylation site when using, two or all amino acid residues change by this way, are about to functional N-glycosylation site and import in the aminoacid sequence or remove from described sequence.

Obviously, when interferon polypeptides is when having the conjugate form of non-polypeptide half point of one or more connections, remove and/or the amino acid residue that imports the linking group that contains non-polypeptide half point is first-selected.By removing and/or import the amino acid residue of the linking group that contains non-polypeptide half point, can optimize with the number of link coupled non-polypeptide half point of interferon polypeptides and distribution (for example, guarantee the best distribution of non-polypeptide half point on the interferon polypeptides surface, thereby, for example, the effectively epi-position of shielding polypeptide and other surface portion and not obvious its function that reduces), as in WO01/15736 and the detailed explanation in PCT/DK02/00128.For example, by importing linking group, can improve or change the content of the bonded specific amino acid residue of relevant non-polypeptide half point, thereby realization is more effective, more special and/or coupling widely.By removing one or more linking groups, can avoid in part of polypeptide coupling with non-polypeptide half point, wherein this coupling is for for example, is positioned at or is disadvantageous (because in the coupling in this site owing to damaged interferon activity inactivation or the reduction that receptor identification causes the conjugate of gained) near the amino acid residue in this polypeptide functional site.In addition, removing near a linking group of another linking group is favourable to avoid the heterogeneity coupling with this group.

Should understand that the amino acid residue that contains the sub-linking group of non-polypeptide half point that removes or import can be according to the characteristic of non-polypeptide half point, and in most of the cases, select according to the coupling method that uses.For example, when this non-polypeptide half point is multimeric molecule such as Polyethylene Glycol or polyalkylene oxide derived molecules, can be as the optional free lysine of the amino acid residue that linking group works, cysteine, aspartic acid is in the group that glutamic acid and arginine are formed.When this non-polypeptide half point is the saccharide half point period of the day from 11 p.m. to 1 a.m, linking group is glycosylation site, preferably a N-glycosylation site in the body.

The amino acid residue that imports or remove generally is positioned at the surperficial exposure position of interferon polypeptides, preferably in the position that is occupied by such amino acid residue, promptly this amino acid residue has and surpasses its side chain of 25% and contact with solvent, preferably contacts with solvent above its side chain of 50%.And it is with to remove the linking group that occupies the position that is positioned at receptor binding site in the interferon polypeptides relevant.In addition, it is relevant with the position or its epi-position that the linking group importing are arranged in interferon polypeptides.This position can for example use the method described in the interferon beta WO01/15736 as an example to identify according to the analysis of the three dimensional structure of interferon polypeptides or by arbitrary other suitable method.

Cause surpassing the replacement that position that amino acid residue that 25% side chain is exposed to the surface occupies imports another N-glycosylation site and comprising being exposed to interferon beta polypeptides surface and being had:

S2N+N4S/T, L6S/T, L5N+G7S/T, F8N+Q10S/T, L9N+R11S/T, R11N, R11N+S13T, S12N+N14S/T, F15N+C17S/T, Q16N+Q18S/T, Q18N+L20S/T, K19N+L21S/T, W22N+L24S/T, Q23N+H25S/T, G26N+L28S/T, R27N+E29S/T, L28S+Y30S/T, Y30N+L32S/T, L32N+D34S/T, K33N+R35S/T, R35N+N37S/T, M36N+F38S/T, D39S/T, D39N+P41S/T, E42N+I44S/T, Q43N+K45S/T, K45N+L47S/T, Q46N+Q48S/T, L47N+Q49T/S, Q48N+F50S/T, Q49N+Q51S/T, Q51N+E53S/T, K52N+D54S/T, L57N+I59S/T, Q64N+I66S/T, A68N+F70S/T, R71N+D73S/T, Q72N, Q72N+S74T, D73N, D73N+S75T, S75N+T77S, S75N, S76N+G78S/T, E81N+I83S/T, T82N+V84S/T, E85N+L87S/T, L88S/T, A89N+V91S/T, Y92S/T, Y92N+Q94S/T, H93N+I95S/T, L98S/T, H97N+K99S/T, K99N+V101S/T, T100N+L102S/T, E103N+K105S/T, E104N+L106S/T, K105N+E107S/T, E107N+E109S/T, K108N+D110S/T, E109N+F111S/T, D110N+T112S, D110N, F111N+R113S/T, R113N+K115S/T, G114N+L116S/T, K115N+M117S/T, L116N, L116N+S118T, S119N+H212S/T, L120N+L122S/T, H121N+K123S/T, K123N+Y125S/T, R124N+Y126S/T, G127N+I129S/T, R128N+L130S/T, L130N+Y132S/T, H131N+L133S/T, K134N+K136S/T, A135N+E137S/T, K136N+Y138S/T, E137N, Y138N+H140S/T, H140N+A142S/T, V148N+I150S/T, R152N+F154S/T, Y155N+I157S/T, L160S/T, R159N+T161S, R159N, G162N+L164S/T, and Y163N+R165S/T, this replacement is represented with respect to the aminoacid sequence of the wild type human interferon beta shown in the SEQ ID NO 1.

Cause surpassing the replacement that the position that is exposed to the interferon beta polypeptides surface that 50% side chain is exposed to the surface imports another N-glycosylation site and comprising having:

L6S/T, L5N+G7S/T, F8N+Q10S/T, L9N+R11S/T, S12N+N14S/T, F15N+C17S/T, Q16N+Q18S/T, K19N+L21S/T, W22N+L24S/T, Q23N+H25S/T, G26N+L28S/T, R27N+E29S/T, Y30N+L32S/T, K33N+R35S/T, R35N+N37S/T, M36N+F38S/T, D39S/T, D39N+P41S/T, E42N+I44S/T, Q46N+Q48S/T, Q48N+F50S/T, Q49N+Q51S/T, Q51N+E53S/T, K52N+D54S/T, L57N+I59S/T, R71N+D73S/T, D73N, D73N+S75T, S75N+T77S, S75N, S76N+G78S/T, E81N+I83S/T, T82N+V84S/T, E85N+L87S/T, A89N+V91S/T, Y92S/T, Y92N+Q94S/T, H93N+I95S/T, T100N+L102S/T, E103N+K105S/T, E104N+L106S/T, E107N+E109S/T, K108N+D110S/T, D110N+T112S, D110N, F111N+R113S/T, R113N+K115S/T, L116N, L116N+S118T, K123N+Y125S/T, R124N+Y126S/T, G127N+I129S/T, H131N+L133S/T, K134N+K136S/T, A135N+E137S/T, E137N, V148N+I150S/T, and Y155N+I157S/T, this replacement is represented with respect to the aminoacid sequence of the wild type human interferon beta shown in the SEQ ID NO 1.

In above-mentioned replacement, preferably in 141-terminal amino acid residues, particularly in 116-terminal amino acid residues, has the replacement of the N residue of importing.

The replacement that causes importing a N-glycosylation site by a unique amino acid whose replacement comprises: L6S/T, R11N, D39S/T, Q72N, D73N, S75N, L88S/T, Y92S/T, L98S/T, D110N, L116N, E137N, R159N and L160S/T, this replacement is represented with respect to the aminoacid sequence of the wild type human interferon beta shown in the SEQ ID NO 1.Wherein, the preferred replacement, be selected from by L6S/T R11N, D39S/T, Q72N, D73N, S75N, L88S/T, Y92S/T, L98S/T in the group that D110N and L116N form, more preferably is selected from by L6S/T, D39S/T, D73N, S75N, L88S/T, D110N is in the group that L116N and E137N form; And most preferably be selected from by L6S/T, D39S/T, D73N, S75N, L88S/T is in the group that D110N and L116N form.

Preferably, the amino acid residue of the linking group that contains non-polypeptide half point of importing produces new N-glycosylation site or new PEGization site.

In addition, when interferon polypeptides comprises glycosylation site, can optimize the utilization in this site.Can realize this point by modifying near the amino acid residue of described glycosylation site, this modification belongs to and causes increasing glycosylated type.In general, glycosylation site is the N-glycosylation site in the body, but it also can be the O-glycosylation site.

In the context of the invention, term " glycosylation of increase " attempts to represent that the carbohydrate molecule level that connects improves, and generally the utilization raising (or better) owing to one or more glycosylation sites obtains.Known in the art any appropriate method of the carbohydrate structure by being used to analyze connection can be measured the glycosylation of increase.

Term in the context of the invention " the N-degree of glycosylation increases in the body " or " increase of N-degree of glycosylation " attempt to represent that the carbohydrate molecule level that connects improves, and generally the utilization raising (or better) owing to one or more N-glycosylation sites obtains.For interferon gamma, that know is (Hooker etc., 1998, J.Interferon and Cytokine Res.18,287-295 and Sarenva etc., 1995, Biochem J., 308,9-14) when expressing in Chinese hamster ovary celI, the wild type human interferon gamma have only about 50% interferon gamma polypeptide to utilize two glycosylation sites, about 40% has utilized a glycosylation site (1N), and about 10% by glycosylation (ON).By any suitable method known in the art, for example, can measure the increase of N-degree of glycosylation in the body by SDS-PAGE.

Term " shows the interferon gamma activity " attempts to represent that this interferon gamma polypeptide has one or more functions of natural human interferon gamma or recombinant human interferon gamma, comprises the ability that combines with interferon gamma receptor and cause the signal of transduceing when the human interferon gamma measured in the outer or body of transductant is in conjunction with its receptor (external or body in biological activity).(Proc.Natl.Acad.Sci.USA 85:4837-4841,1988) such as Aguet etc. (Cell 55:273-280,1988) and Calderon have described this interferon gamma receptor.Detecting the active suitable test of interferon gamma is the test of disclosed herein being entitled as " one-level test ".

" interferon gamma polypeptide " is to show the active polypeptide of interferon gamma, and when suitable, this paper is used for the interferon gamma polypeptide with monomer or dimeric forms.For example, when the concrete replacement of expression, they are generally represented with respect to the interferon gamma polypeptide monomer.Should understand term " interferon gamma polypeptide " also comprise the wild type interferon gamma polypeptide the terminal truncate of C-and variant form.The object lesson of this variant comprises having such as S99T, the terminal clipped form of the variant of the modification of E38N+S40T and C-thereof.More many cases of suitable modification provides hereinafter.

In general, the variant form of interferon gamma polypeptide is compared (perhaps its clipped form, compare with the human interferon gamma of corresponding truncate) if desired with human interferon gamma has the difference of 1-15 amino acid residue (for example to have 1,2,3,4,5,6,7,8,9,10,11,12,13, the difference of 14 or 15 amino acid residues), 1-10 amino acid residue for example arranged, the difference of 1-5 amino acid residue or 1-3 amino acid residue.

As mentioned above, known activity that can not eliminate the interferon gamma of this polypeptide from 1-15 amino acid residue of C-terminal deletion.Therefore, term " interferon gamma polypeptide " also comprises such interferon gamma polypeptide (having people's wild-type sequence or its variant), and wherein the interferon gamma polypeptide of 1-15 is 1-15 amino acid residue of the terminal truncate of C-.A concrete example comprises the interferon gamma polypeptide of 3 amino acid residues of the terminal truncate of C-.

The mature form of the wild type human interferon gamma of aminoacid sequence shown in the SEQ ID NO:2 attempted to represent to have in term " human interferon gamma ".

The mature form with wild type human interferon gamma of aminoacid sequence shown in the SEQ ID NO:2 that produces with recombination form attempted to comprise in term " recombinant human interferon gamma ".

Term " Actimmune used herein ^" be meant 140 amino acid whose forms (open) of the interferon gamma that the escherichia coli antibacterial by the fermentation genetic modification obtains with SEQ ID NO:4.Actimmune ^Out of Memory can Www.actimmune.com.Last acquisition.

Amino acid residue " be positioned at " glycosylation site close and normally be positioned at the glycosylation site that connects with respect to saccharide amino acid residue-4 ,-3 ,-2 ,-1 ,+1 ,+2 ,+3 or+4.This position can be selected from-1 ,+1, or+3, particularly+1 or+3.Therefore, the amino acid residue that is positioned near N-glycosylation site (have sequence N-X '-S/T/C-X ") is positioned at-4 ,-3 with respect to the N residue ;-2 ;-1, at X ' or X " position (amino acid residue of Dao Ruing preferably is different from proline in this case), or with respect to X " residue+1.Amino acid modified generally is to replace, and replaces to use to compare arbitrary other amino acid residue that causes the interferon polypeptides glycosylation to increase with the polypeptide of unmodified and carry out.This other amino acid residue can be tested by test and error pattern and be measured (that is, be replaced to arbitrary other amino acid residue by the amino acid residue with relevant position, and measure the gained glycosylation of gained variant).

When modify with respect to the N-residue+2 the time, should understand that it is possible having only a limited number of modification, because in order to keep/import N-glycosylation site in the body, the amino acid residue of described position can be Ser, one of Thr or Cys.In an especially preferred embodiment, with respect to N-glycosylation site in the body+modification of 2 amino acid residue is the replacement that described amino acid residue is replaced by the Thr residue.On the other hand, if described amino acid residue has been the Thr residue, generally preferred or needn't carry out any replacement in this position.When modifying X ', X ' should not be Pro and preferably not be Trp, Asp, Glu and Leu.If modify X ', the amino acid residue that needs to import is preferably selected from by Phe Asn, Gln, Tyr, Val, Ala, Met, Ile, Lys, Gly, Arg, Thr, His, in the group that Cys and Ser form, more preferably Ala, Met, Ile, Lys, Gly, Arg, Thr, His, Cys and Ser, particularly Ala or Ser.When modify with respect to the N-residue+during 3 positions, the amino acid residue that needs to import is preferably selected from by His, Asp, Ala, Met, Asn, Thr, Arg, in the group that Ser and Cys form, more preferably Thr, Arg, Ser and Cys.If X ' residue is the Ser residue, this modification is suitable especially.

In addition, can use such as neutral amino acid residue, for example Gly by for example, Val, Ala, Leu, Ile, Tyr, Phe, His, Trp, Ser, another amino acid residue of Thr or Met, preferably with Ser or Thr replace can remove interferon polypeptides free cysteine (promptly, do not form the cysteine residues of the part of cysteine bridged bond), for example, press US 4,959,314 or EP 192,811 described in.

Interferon polypeptides can be with non-polypeptide half point, and for example, (when interferon polypeptides during by glycosylation) carries out derivatization such as the multimeric molecule of Polyethylene Glycol or saccharide half point.

Term " non-polypeptide half point " attempt to represent can with the link coupled molecule of the linking group of polypeptide of the present invention.The preferred example of this molecule comprises multimeric molecule, saccharide half point, lipophilic compound, or organic derivating agent.When in the context of conjugate of the present invention, using, should understand that non-polypeptide half point is connected with the polypeptide portion of conjugate by the linking group of this polypeptide.

Term " multimeric molecule " is defined as by the covalently bound molecule that forms of two or more monomers, and wherein neither one is an amino acid residue in the monomer, unless this polymer is human albumin or another high abundance plasma proteins.Term " polymer " is used interchangeably with term " multimeric molecule ".This term is attempted to comprise through external glycosylation,, relates to the optional cross-linking agent that uses under the normal condition with the covalently bound external synthetic glycosylation of carrying out to the linking group of polypeptide of carbohydrate molecule, the carbohydrate molecule of connection that is.Be called " saccharide half point " by carbohydrate molecule (as the following further describing) this paper that connects such as glycosylation in N-or the glycosylated body of O-.Except specially showing in the conjugate situation, mention that at every turn " non-polypeptide half point " that use among contained or the present invention in the conjugate should be meant one or more non-polypeptide half point such as multimeric molecule or saccharide half point in the conjugate such as the number of non-polypeptide half point of multimeric molecule or saccharide half point.

When interferon polypeptides comprised the amino acid residue of linking group of containing of importing non-polypeptide half point, this non-polypeptide half point preferably was connected with this amino acid residue.

Aspect first, the present invention relates to contain the stabilized composition of interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.

Aspect second, the present invention relates to contain the stable drug composition of interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.

In one embodiment, this sulfoalkyl ether cyclodextrin derivant is with from 1mg/ml to 150mg/ml, and for example the concentration from 5mg/ml to 100mg/ml exists.

In one embodiment, this interferon polypeptides comprises the amino acid residue that contains the sub-linking group of non-polypeptide half point at least one importing and/or that at least one removes.In another embodiment, this interferon polypeptides comprise at least one importing with at least one amino acid residue that contains the sub-linking group of non-polypeptide half point that removes.In another embodiment, this interferon polypeptides comprises the amino acid residue of the sub-linking group of non-polypeptide half point of containing of at least one importing.In another embodiment, this interferon polypeptides comprises the amino acid residue that contains the sub-linking group of non-polypeptide half point that at least one removes.

In another embodiment, this non-polypeptide half point comprises the multimeric molecule such as Polyethylene Glycol, or saccharide half point.In a specific embodiments, this multimeric molecule comprises Polyethylene Glycol.In another embodiment, this non-polypeptide half point comprises saccharide half point, particularly at mammalian cell, expresses saccharide half point that interferon polypeptides obtains in the preferred Chinese hamster ovary celI.

In another embodiment, this interferon polypeptides comprises the glycosylation site of at least one importing, with at least one saccharide half point that is connected with the glycosylation site that imports.In another embodiment, this interferon polypeptides comprises the glycosylation site of at least one importing, with at least one saccharide half point and multimeric molecule such as Polyethylene Glycol that is connected with the glycosylation site that imports.In an especially preferred embodiment, this interferon polypeptides comprises 3 saccharide half point, and each saccharide half point links to each other with the N-glycosylation site.

In a preferred embodiment, interferon polypeptides is by glycosylation and/or PEGization.In another embodiment, interferon polypeptides is by glycosylation and PEGization.In another embodiment, interferon polypeptides is by glycosylation.In another embodiment, interferon polypeptides is by PEGization.When interferon polypeptides during by glycosylation, preferably by the N-glycosylation.When interferon polypeptides during by glycosylation, it contains 1-5 saccharide half point usually, for example 1-3 saccharide half point.In another embodiment, interferon polypeptides is by the N-glycosylation, and comprises 1-5 saccharide half point, for example 1-3 saccharide half point.When interferon polypeptides during by PEGization, it contains 1-5 Polyethylene Glycol (PEG) molecule usually.In another embodiment, interferon polypeptides comprises 1-5 PEG molecule, for example 1,2 or 3 PEG molecule.In another embodiment, each PEG molecule has the molecular weight of about 5kDa (kilodalton) to 100kDa.In another embodiment, each PEG molecule has the molecular weight of about 10kDa to 40kDa.In another embodiment, each PEG molecule has the molecular weight of about 12kDa.In another embodiment, each PEG molecule has the molecular weight of about 20kDa.Preferred interferon polypeptides comprises 1-3 PEG molecule that has about 12kDa molecular weight respectively, or has 1 PEG molecule of about 20kDa molecular weight.Suitable PEG molecule can obtain and can be selected from SS-PEG, NPC-PEG, aldehyde radical-PEG from Shearwater Polymers company and Enzon company, mPEG-SPA, mPEG-SCM, mPEG-BTC, SC-PEG, (US 5 for tresylated mPEG, 880,255), or oxygen carbonyl-oxygen-N-dicarboximide-PEG (US 5,122,614).

In a preferred embodiment, interferon polypeptides is an interferon beta polypeptides, for example, wild type human interferon beta or its variant, optional and non-polypeptide half point is connected.

For example, interferon beta polypeptides can be the variant of wild type human interferon beta, and wherein another amino acid residue of 17 cysteine residues disappearance or quilt such as above-mentioned neutral amino acid residue replaces in the position.For example, this interferon beta polypeptides comprises the C17S sudden change, replaces to use with respect to the aminoacid sequence of the wild type human interferon beta shown in the SEQ ID NO 1 and represents.

In preferred embodiments, this interferon beta polypeptides is the arbitrary polypeptide described in the WO01/15736.In another embodiment preferred, interferon beta polypeptides is at the arbitrary polypeptide described in the PCT/DK02/00128.Preferred variant for example is included in, S2, and Q49, Q51, or the F111 position has the variant of the glycosylation site of at least one importing; With for example, K19, K33, K45 or K123 position have the variant at least one PEGization of removing site.

In another embodiment, interferon beta polypeptides is the conjugate that comprises with covalently bound at least one first non-polypeptide half point of interferon beta polypeptides, and this amino acid sequence of polypeptide and the difference of wild type human interferon beta are to have imported at least one and have removed at least one to contain the amino acid residue of the sub-linking group of the described first non-polypeptide half point.

In another embodiment, interferon beta polypeptides is the conjugate that comprises with bonded at least one first non-polypeptide half point of at least one lysine residue of interferon beta polypeptides, and this amino acid sequence of polypeptide and the difference of wild type human interferon beta are to have imported at least one and/or have removed at least one lysine residue.

In another embodiment, interferon beta polypeptides is the conjugate that comprises with bonded at least one first non-polypeptide half point of at least one cysteine residues of interferon beta polypeptides, and this amino acid sequence of polypeptide difference is to have imported at least one cysteine residues in the position that the amino acid residue that the wild type human interferon beta is exposed by the surface occupies.

In another embodiment, interferon beta polypeptides is to comprise to have the conjugate of acidic-group as at least one first non-polypeptide half point of linking group, this half point combines with at least one aspartic acid or the glutaminic acid residue of interferon beta polypeptides, and this amino acid sequence of polypeptide and the difference of wild type human interferon beta are to have imported at least one and/or have removed at least one aspartic acid or glutaminic acid residue.

In another embodiment, first non-polypeptide half point comprises the multimeric molecule such as Polyethylene Glycol, or saccharide half point.

In another embodiment, interferon beta polypeptides is the conjugate that comprises with covalently bound at least one multimeric molecule of interferon beta polypeptides and at least one saccharide half point, and this amino acid sequence of polypeptide is distinguished with the wild type human interferon beta and is

A) imported at least one and/or removed at least one linking group that contains this multimeric molecule amino acid residue and

B) imported at least one and/or removed at least one and contained the amino acid residue of the linking group of this saccharide half point,

Prerequisite is that the linking group when this multimeric molecule is a cysteine residues, and this saccharide half point is the saccharide half point period of the day from 11 p.m. to 1 a.m that N-connects, and does not insert cysteine residues in the mode of destroying the N-glycosylation site.

In another embodiment, interferon beta polypeptides is the conjugate that comprises interferon beta polypeptides, the difference of this amino acid sequence of polypeptide and wild type human interferon beta is to have imported at least one glycosylation site, and this conjugate also contains saccharide half point of at least one un-PEGization that is connected with the glycosylation site that imports.

In another embodiment, interferon beta polypeptides is the conjugate that comprises interferon beta polypeptides, the difference of this amino acid sequence of polypeptide and wild type human interferon beta is to have imported at least one glycosylation site, and this conjugate also contains at least one saccharide half point that is connected with the glycosylation site that imports.

Therefore, one concrete aspect, the present invention relates to the compositions of stabilisation, comprising:

A) contain the conjugate of interferon beta polypeptides, the difference of this amino acid sequence of polypeptide and wild type human interferon beta is to have imported at least one glycosylation site, this conjugate also contain at least one saccharide half point of being connected with the glycosylation site that imports and

B) sulfoalkyl ether cyclodextrin derivant.

In another embodiment, interferon beta polypeptides is the conjugate that comprises interferon beta polypeptides, and the difference of this amino acid sequence of polypeptide and wild type human interferon beta is that the position that occupies by the amino acid residue that is exposed by the surface at the wild type human interferon beta imports or the mode of removing the one or more amino acid residues that constitute a glycosylation site part imports or has removed a glycosylation site.

In another embodiment, interferon beta polypeptides is the conjugate that comprises interferon beta polypeptides, and the difference of this amino acid sequence of polypeptide and wild type human interferon beta is that the mode that the position that occupies by the amino acid residue that is exposed by the surface at the wild type human interferon beta imports the one or more amino acid residues that constitute a glycosylation site part has imported a glycosylation site.

In also having another embodiment, interferon beta polypeptides is the conjugate that comprises with covalently bound saccharide half point of interferon beta polypeptides, and the difference of this amino acid sequence of polypeptide and wild type human interferon beta is to have removed at least one glycosylation site.

In also having another embodiment, interferon beta polypeptides is the glycosylation variant that contains parent's interferon beta polypeptides of glycosylation site at least one individuality, and the amino acid residue of wherein having modified the described parent's polypeptide that is positioned at close described glycosylation site is to obtain to compare with the glycosylation of parent's interferon beta polypeptides the variant polypeptide of glycosylation increase.

In another embodiment, interferon beta polypeptides comprises the aminoacid replacement K19R+K45R+K123R that is selected from by in the following group of forming; K19Q+K45R+K123R; K19R+K45Q+K123R; K19R+K45R+K123Q; K19Q+K45Q+K123R; K19R+K45Q+K123Q; K19Q+K45R+K123Q; K19Q+K45Q+K123Q; K45R+K123R; K45Q+K123R; K45Q+K123Q; K45R+K123Q; K19R+K123R; K19Q+K123R; K19R+K123Q; K19Q+K123Q; K19R+K45R; K19Q+K45R; K19R+K45Q; K19Q+K45Q; K52R+K134R; K99R+K136R; K33R+K105R+K136R; K52R+K108R+K134R; K99R+K115R+K136R; K19R+K33R+K45R+K123R; K19R+K45R+K52R+K123R; K19R+K33R+K45R+K52R+K123R; K19R+K45R+K52R+K99R+K123R; K19R+K45R+Q49N+Q51T+F111N+R113T+K123R; K19R+K45R+Q49N+Q51T+F111N+R113T; K19R+K45R+Q49N+Q51T+K123R; S2N+N4T/S; L9N+R11T/S; R11N; S12N+N14T/S; F15N+C17S/T; Q16N+Q18T/S; K19N+L21T/S; Q23N+H25T/S; G26N+L28T/S; R27N+E29T/S; L28N+Y30T/S; D39T/S; K45N+L47T/S; Q46N+Q48T/S; Q48N+F50T/S; Q49N+Q51T/S; Q51N+E53T/S; R71N+D73T/S; Q72N; D73N; S75N; S76N+G78T/S; L88T/S; Y92T/S; N93N+I95T/S; L98T/S; E103N+K105T/S; E104N+L106T/S; E107N+E109T/S; K108N+D110T/S; D110N; F111N+R113T/S; L116N; S2N+N4T; L9N+R11T; 49N+Q51T; F111N+R113T; R71N+D73T; 49N+Q51T; F111N+R113T; R71N+D73T; Q49N+Q51T+F111N+R113T; Q49N+Q51T+R71N+D73T+F111N+R113T; S2N+N4T+F111N+R113T; S2N+N4T+Q49N+Q51T; S2N+N4T+Q49N+Q51T+F111N+R113T; S2N+N4T+L9N+R11T+Q49N+Q51T; S2N+N4T+L9N+R11T+F111N+R113T; S2N+N4T+L9N+R11T+Q49N+Q51T+F111N+R113T; L9N+R11T+Q49N+Q51T; L9N+R11T+Q49N+Q51T+F111N+R113T; L9N+R11T+F111N+R113T; R27K+R159K; R27K+K45R+R159K; R27K+Q49K+E85K+A89K; R27K+K45R+Q49K+E85K+A89K; R27K+D39K+Q49K+E85K+A89K; R27K+D39K+K45R+Q49K+E85K+A89K; N4K+R27K+D39K+Q49K+E85K+A89K; N4K+R27K+D39K+K45R+Q49K+E85K+A89K; R27K+K123R+R159K; R27K+K45R+K123R+] R159K; R27K+Q49K+E85K+A89K+K123R; R27K+K45R+Q49K+E85K+A89K+K123R; R27K+D39K+Q49K+E85K+A89K+K123R; R27K+D39K+K45R+Q49K+E85K+A89K+K123R; N4K+R27K+D39K+Q49K+E85K+A89K+K123R; N4K+R27K+D39K+K45R+Q49K+E85K+A89K+K123R; K19R+K45R+F111K+K123R; K19R+K45R+Q49K+F111K+K123R; K19R+K45R+Q49K+K123R; K19R+K45R+F111K; K19R+K45R+Q49K+F111K; K19R+Q49K+K123R; K19R+Q49K+F111K+K123R; K45Q+F111K+K123Q; K45R+Q49K+K123R; Or K45R+Q49K+F111K+K123R; This replacement is represented with respect to the aminoacid sequence of the wild type human interferon beta shown in the SEQ ID NO 1.Each concrete variant is considered to an embodiment of interferon beta variant.

The interested concrete interferon beta variant of the present invention comprises the aminoacid replacement Q49N+Q51T that is selected from by in the following group of forming; Q49N+Q51T+F111N+R113T; F111N+R113T; C17S+Q49N+Q51T+L980+F111N+R113T; S2N+N4T+C17S+Q51N+E53T; S2N+N4T+C17S+Q51N+E53T+F111N+R113T; C17S+Q49N+Q51T+F111N+R113T; C17S+Q49N+Q51T+D110F+F111N+R113T; C17S+Q48F+Q49N+Q51T+D110F+F111N+R113T; C17S+Q48Y+Q49N+Q51T+D110Y+F111N+R113T; K19R+K45R+K123R; K19R+K45R+Q49N+Q51T+F111N+R113T+K123R; C17S+K19R+K45R+Q49N+Q51T+F111N+R113T+K123R; C17S+K19R+K45R+Q49N+Q51T+F111N+R113T+K123R; C17S+K19R+Q49N+Q51T+F111N+R113T+K123R; C17S+K19R+K45R+Q49N+Q51T+D110F+F111N+R113T+K123R; C17S+K19R+Q49N+Q51T+D110F+F111N+R113T+K123R; S2N+N4T+C17S+K19R+K45R+Q51N+E53T+K123R; C17S+K19R+K45R+Q48F+Q49N+Q51T+D110F+F111N+R113T+K123R; S2N+N4T+C17S+K19R+K45R+Q51N+E53T+D110F+F111N+R113T+K123R; C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T; C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T+K123R; C17S+K19R+K33R+K45R+Q49N+Q51T+F111N+R113T; And C17S+K19R+K33R+K45R+Q49N+Q51T+F111N+R113T+K123R; This replacement is represented with respect to the aminoacid sequence of the wild type human interferon beta shown in the SEQ ID NO 1.Each concrete variant is considered to an embodiment of interferon beta variant.For example, an embodiment is C17S+Q49N+Q51T+D110F+F111N+R113T; Another embodiment is C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T.The sudden change embodiment preferred is that the replacement wherein represented only is the replacement with respect to the wild type human interferon beta shown in the SEQ ID NO 1 in the above-mentioned concrete variant.

These variants can be the forms of conjugate, and it also comprises one or more non-polypeptide half point.For example, this variant is by glycosylation and/or PEGization.When interferon beta polypeptides during by glycosylation, it is preferably by the N-glycosylation.When interferon beta polypeptides during by glycosylation, it contains 1-5 saccharide half point usually, for example 1-3 saccharide half point.In another embodiment, interferon beta polypeptides is by the N-glycosylation, and contains 1-5 saccharide half point, for example 1-3 saccharide half point.

When interferon beta polypeptides contained the glycosylation site of at least one importing, preferably this polypeptide also contained at least one saccharide half point that is connected with the glycosylation site that imports.Specifically, this interferon beta polypeptides contains 2-5 saccharide half point, for example 2-3 saccharide half point.In another embodiment, interferon beta polypeptides is by the N-glycosylation, and comprises 2-5 saccharide half point, for example 2-3 saccharide half point.In an especially preferred embodiment, interferon beta polypeptides comprises 3 saccharide half point, and each saccharide half point is connected with a N-glycosylation site.

When interferon beta polypeptides during by PEGization, it comprises 1-5 Polyethylene Glycol (PEG) molecule usually.In another embodiment, interferon beta polypeptides comprises 1-5 PEG molecule, for example 1,2 or 3 PEG molecule.In another embodiment, each PEG molecule has the molecular weight of about 5kDa (kilodalton) to 100kDa.In another embodiment, each PEG molecule has the molecular weight of about 10kDa to 40kDa.In another embodiment, each PEG molecule has the molecular weight of about 12kDa.In another embodiment, each PEG molecule has the molecular weight of about 20kDa.Preferred interferon beta polypeptides comprises 1-3 PEG molecule that has about 12kDa molecular weight respectively, or 1 PEG molecule with about 20kDa molecular weight.More preferably interferon beta polypeptides contains saccharide half point and 1 PEG molecule with about 20Kda molecular weight that at least one is connected with the glycosylation site that imports.Most preferably interferon beta polypeptides contains 3 saccharide half point and 1 the PEG molecule with about 20Kda molecular weight that is connected with 3 N-glycosylation sites.

Interferon polypeptides can be according to methods known in the art production.Preferably, interferon polypeptides is by expressing the generation (at WO01/15736, describing in detail among PCT/DK02/00128 and the WO01/36001) of recombinating from the glycosylation host cell.Expression host cell can be selected from fungus (filamentous fungi or yeast), insecticide, mammalian cell, transgenic plant cells or transgenic animal.In addition, when the nucleotide sequence of polypeptide portion that in gene therapy, uses coding conjugate of the present invention or polypeptide of the present invention, can in human body, finish glycosylation.In one embodiment, host cell is a mammalian cell, Chinese hamster ovary celI for example, BHK or HEK cell, for example HEK293, or insect cell, SF9 cell for example, perhaps yeast cells, Saccharomyces cerevisiae (Saccharomyces cerevisiae) for example, Pichiia pastoris or any other suitable glycosylation host.The example of suitable mammalian host cell comprises Chinese hamster ovary (CHO) cell line, (for example, CHO-K1; ATCC CCL-61), grivet cell line (COS) (for example COS1 (ATCC CRL-1650), COS7 (ATCC CRL-1651)); Mouse cell (for example NS/O), hamster kidney childhood (BHK) cell line (for example ATCC CRL-1632 or ATCC CCL-10) and people's cell (for example HEK 293 (ATCC CRL-1573)), and the plant cell in the tissue culture.Optional is that saccharide half point that is connected with polypeptide by glycosylation in the body can for example use Neose by using glycosyl transferase, Horsham, PA, the glycoAdvance that USA sells ^TMTechnology is further modified.Therefore, for example can increase the sialylated of the interior glycosylation of expressing cho cell and body glycosylated polypeptides afterwards.

Can be according to methods known in the art purifying alpha-interferon polypeptide with the interferon goods that obtain enough purity so that as medicine.In general, this purification process comprises ultrafiltration, diafiltration, cation-exchange chromatography (for example, from the S-Sepharose of Pharmacia), hydrophobic interaction chromatography, hydroxylapatite chromatography method, and/or the separation on the Sephacryl post.

In another embodiment, interferon polypeptides is an interferon gamma polypeptide, and for example, wild type human interferon gamma, or its variant are chosen wantonly and sub combination of non-polypeptide half point, for example at arbitrary variant or the conjugate described in the WO 01/36001.

In another embodiment of the present invention, this interferon gamma polypeptide is selected from the interferon gamma variant, for example hereinafter the disclosed arbitrary variant of " interferon gamma variant " part.

The interferon gamma variant

Interferon gamma variant with N-glycosylation site of optimization

Have been found that by replacing the serine residue that is positioned at 99 of human interferon gammas and can increase the glycosylation that is positioned at 97 naturally occurring N-glycosylation sites of human interferon gamma with threonine residues, can obtain to increase fully, the perhaps composition of complete glycosylated interferon gamma polypeptide basically.For example, have been found that by replacing S99T, about 90% the interferon gamma polypeptide that exists in the culture medium of results has utilized two N-glycosylation sites, and the human interferon gamma polypeptide that the reorganization that exists in the culture medium of results produces has only about 60% by glycosylation fully.

Therefore, in utmost point advantageous embodiment, interferon gamma polypeptide comprises and replaces S99T.

In optimizing 97 body, the required above-mentioned S99T sudden change of N-glycosylation site, also can optimize glycosylation site (referring to the part that is entitled as " the interferon gamma variant that non-polypeptide half point is saccharide half point ") in other body that has imported in the sequence.In general, glycosylation site is the N-glycosylation site in the body, but also comprises the O-glycosylation site under relevant situation.This optimization can particularly be modified near the position of N-glycosylation site in the body by being positioned near glycosylation site, preferably replaces and finishes.The object lesson that can import the suitable position of N-glycosylation site in the body is open in WO01/36001.

Therefore, for N-glycosylation in the naturally occurring body, expection is by being selected from by E93, K94, L95, T96, Y98, V100 and T101 are (promptly in the position-4 with respect to N97,-3 ,-2 ,-1, + 1 ,+3 or+4) position in the group formed carries out can further optimizing 97 N-glycosylation site such as the modification that replaces.Comprise Y98F at 98 object lessons that replace, Y98N, Y98Q, Y98V, Y98A, Y98M, Y98I, Y98K, Y98G, Y98R, Y98T, Y98H, Y98C and Y98S, preferred Y98A, Y98M, Y98I, Y98K, Y98G, Y98R, Y98T, Y98H, Y98C and Y98S, particularly Y98S.Comprise V100H at 100 object lessons that replace, V100D, V100A, V100M, V100N, V100T, V100R, V100S, or V100C, particularly V100T, V100R, V100S or V100C.

Equally, for N-glycosylation site in 25 body, expection is by being selected from by D21 V22, A23, D24, G26, L28 and F29 are (promptly in the position-4 with respect to N25 ,-3,-2 ,-1 ,+1 ,+3 or+4) a position in the group formed carries out can further optimizing this site such as the modification that replaces.Comprise G26F at 26 object lessons that replace, G26N, G26Y, G26Q, G26V, G26A, G26M, G26I, G26K, G26R, G26T, G26H, G26C and G26S, preferred G26A, G26M, G26I, G26K, G26R, G26T, G26H, G26C and G26S, more preferably G26A and G26S, particularly G26A.Comprise G28H at 28 object lessons that replace, G28D, G28A, G28M, G28N, G28T, G28R, G28S, or G28S, particularly G28A, G28T, G28R, G28S or G28C.

Obviously, with optimize 97 glycosylations interrelate described arbitrary modification can with optimize 25 glycosylations described arbitrary modification that interrelates and combine.

The interferon gamma variant that contains the sub-linking group of non-polypeptide half point

The modification of serum half life when the interested modification of another type that can import comprises AUC when being used to increase subcutaneous administration and/or intravenous administration.

In an interested embodiment, interferon gamma polypeptide comprises the amino acid residue that contains non-polypeptide linking group at least one importing and/or that at least one removes.

For fear of the 26S Proteasome Structure and Function that destroys interferon gamma polypeptide too much, generally be no more than 15 according to the sum of this embodiment amino acid residue to be finished.Usually this aminoacid sequence is compared and is contained 1-10 with SEQ ID NO:2 (or the terminal clipped form of its C-), and for example 1-5, for example 1-3 modification.Preferably, this modification is to replace.

Except removing and/or import this amino acid residue, this polypeptide can comprise other to be modified, for example with import and/or remove the irrelevant replacement of amino acid residue that contains the sub-linking group of non-polypeptide half point.The example of this modification comprises that conserved amino acid replaces and/or imports Cys-Tyr-Cys or Met at the N-end.

Can be used in conjunction with and the exact number of the linking group that in the interferon gamma polypeptide of dimeric forms, exists depend on need be by in conjunction with the effect that reaches.The effect that need to obtain for example depends on, bonded nature and extent (for example, the characteristic of non-polypeptide half point, need or may with the number of bonded non-polypeptide half point of this polypeptide, they should bonded positions or should avoid bonded position, etc.).

Should understand that the amino acid residue of the linking group that contains non-polypeptide half point that can remove or import can select according to the characteristic of selected non-polypeptide half point subdivision, and in most of the cases, select according to used associated methods.For example, when non-polypeptide half point is multimeric molecule such as Polyethylene Glycol or polyalkylene oxide derived molecules, can be as the optional free cysteine of the amino acid residue that linking group works, lysine, aspartic acid is in the group that glutamic acid and arginine are formed.Specifically, preferred cysteine.When this non-polypeptide half point is the saccharide half point period of the day from 11 p.m. to 1 a.m, linking group for example is, glycosylation site, preferably N-glycosylation site in the body.

No matter the linking group of non-polypeptide half point be need in interferon gamma polypeptide, import or remove, the position of polypeptide to be finished can be selected as follows easily:

This optimum seeking site is positioned at the surface of interferon gamma polypeptide, and more preferably had during with the three dimensional structure of its dimeric forms or model determination and surpass the amino acid residue that its side chain of 25% contacts with solvent and occupy according to interferon gamma, preferably surpass its side chain of 50% and contact with solvent, this structure or model option further contain one or two interferon gamma receptor molecule.List in this paper embodiment A this position.

In addition, interested may be to modify the one or more amino acid residues that are positioned at interferon gamma ring zone, because be positioned at most of amino acid residues in these ring zones be exposed to the surface and with functional site at a distance of enough far feasible can importings such as multimeric molecule, particularly non-polypeptide half point of PEG molecule, and/or N-glycosylation site and can not damage the function of this polypeptide.Can identify this ring zone by checking optional three dimensional structure with the compound human interferon gamma of its receptor.The amino acid residue that constitutes described ring zone is residue N16-K37 (" an A-B ring "), F60-S65 (" B-C ring "), N83-S84 (" C-D ring ") and Y98-L103 (" D-E ring ").

In addition, in interferon gamma polypeptide, preferably removed the linking group of the receptor binding site that is positioned at interferon gamma, preferably the amino acid residue that contains this group by replacement removes.The amino acid residue that constitutes the interferon gamma receptor binding site is Q1, D2, Y4, V5, E9, K12, G18, H19, S20, D21, V22, A23, D24, N25, G26, T27, L30, K34, K37, K108, H111, E112, I114, Q115, A118, E119 (also referring to this paper Embodiment B).

In order to measure the best distribution of linking group, according to the distance of three dimensional structure calculating between the amino acid residue on interferon gamma polypeptide surface of interferon gamma dimer polypeptide.More particularly, mensuration contains the distance between the CB of amino acid residue of this linking group, the perhaps functional group of a residue (NZ of lysine, the CG of aspartic acid, the CD of glutamic acid, the SG of cysteine) and contain distance between the CB of another amino acid residue of linking group.For glycine, use CA replaced C B.In interferon gamma polypeptide part, for fear of or reduce the heterogeneity combination, arbitrary described distance preferably surpasses 8 , particularly surpasses 10 .

As mentioned above, under physiological condition, interferon gamma exists with the dimer polypeptide.This polypeptide generally is homodimer form (for example by associating by two interferon gamma polypeptide preparations of preparation described herein).Yet if desired, interferon gamma polypeptide can single stranded form provide, and wherein two interferon gamma polypeptide monomers connect by peptide bond or peptide linker.The advantage that provides interferon gamma polypeptide to have with single stranded form is that the interferon gamma polypeptide of two components can be different, and this for example helps, and makes the asymmetric mutation of this polypeptide become possibility.For example, can remove the PEGization site from a monomeric receptor binding site, but in another monomer, keep.Therefore, after the PEGization, a monomer has a complete receptor binding site, and another monomer is by PEGization fully (and so provide the molecular weight of obvious increase).

Non-polypeptide half point is the interferon gamma variant of saccharide half point

In preferred embodiments, interferon gamma polypeptide contains glycosylation site at least one importing and/or that at least one removes, and promptly non-polypeptide half point is saccharide half point.Preferably, glycosylation site is a glycosylation site in the body, and promptly non-polypeptide half point is saccharide half point, for example O-saccharide half point that connect or that N-connects, preferably saccharide half point of N-connection.

In particularly preferred embodiments, interferon gamma polypeptide contains the glycosylation site of at least one importing, particularly N-glycosylation site in the body of Dao Ruing.Preferably, the glycosylation site of this importing imports by replacing.

For example, the N-glycosylation site can import in the position of the interferon gamma polypeptide that contains the amino acid residue that is exposed to the surface in the body.The amino acid residue that preferred described surface exposes has at least 25% side chain and is exposed to the surface, and particularly its side chain of at least 50% is exposed to the surface.Can be about the details of measuring this position referring to this paper embodiment A.

The N-glycosylation site imports by this way, and promptly the N-residue in described site is positioned at described position.Equally, can import the O-glycosylation site and be positioned at described position so that constitute the S or the T residue in this site.Should understand that when the importing of N-glycosylation site in term " at least 25% (or 50%) of its side chain is exposed to the surface " and the body interrelated use, this term was meant the surperficial accessibility at the amino acid side chain of the actual position that connects of saccharide half point.In many cases must with respect to the actual asparagicacid residue that connects of saccharide half point+2 import a serine or threonine residues, and allow embedding to import these positions of serine or threonine residues, promptly have the surface that its side chain that is no more than 25% (or 50%) is exposed to this molecule.

In addition, in order to ensure effective glycosylation, N residue or the S of O-glycosylation site or the 118N-terminal amino acid residue that the T residue is positioned at interferon gamma polypeptide of glycosylation site, particularly N-glycosylation site more preferably are positioned at the 97N-terminal amino acid residue in the preferred body.Even more preferably, only need the position (that is, producing required any other amino acid residue in functional glycosyl site position in this polypeptide Already in) of a sudden change to import glycosylation site in the body producing this site.

For example, cause being exposed to the replacement that position that the amino acid residue on surface (having in the structure of acceptor molecule) occupies imports another N-glycosylation site and comprising being exposed to interferon gamma polypeptide surface and being had at least 25% side chain: Q1N+P3S/T, P3N+V5S/T, K6N+A8S/T, E9N+L11S/T, K12S/T, K13N+F15S/T, Y14N+N16S/T, G18S/T, G18N, G18N+S20T, H19N+D21S/T, D21N+A23S/T, G26N+L28S/T, G31N+L33S/T, K34N+W36S/T, K37S/T, K37N+E39S/T, E38N, E38N+S40T, E39N+D41S/T, S40N+R42S/T, K55N+F57S/T, K58N+F60S/T, K61S/T, K61N+D63S/T, D62N+Q64S/T, D63N, D63N+S65T, Q64N+I66S/T, S65N+Q67S/T, Q67N, Q67N+S69T, K68N+V70S/T, E71N+I73S/T, T72N+K74S/T, K74N+D76S/T, E75N+M77S/T, K80S/T, V79N+F81S/T, K80N+F82S/T, N85S/T, S84N+K86S/T, K87S/T, K86N+K88S/T, K87N+R89S/T, D90N+F92S/T, E93N+L95S/T, K94N, K94N+T96S, T101N+L103S/T, D102N+N104S/T, L103N+V105S/T, Q106S/T, E119N, E119N+S121T, P122N+A124S/T, A123N+K125S/T, A124N, A124N+T126S, K125N+G127S/T, T126N+K128S/T, G127N+R129S/T, K128N+K130S/T, R129N+R131S/T and K130N.S/T represents to be replaced to serine or threonine residues, preferred threonine residues.

Cause comprising being exposed to interferon gamma polypeptide surface and having the replacement that position that at least 50% side chain is exposed to surface (having in the structure of acceptor molecule) imports another N-glycosylation site: P3N+V5S/T, K6N+A8S/T, K12S/T, K13N+F15S/T, G18S/T, D21N+A23S/T, G26N+L28S/T, G31N+L33S/T, K34N+W36S/T, K37N+E39S/T, E38N, E38N+S40S/T, E39N+D41S/T, K55N+F57S/T, K58N+F60S/T, K61S/T, D62N+Q64S/T, Q64N+I66S/T, S65N+Q67S/T, K68N+V70S/T, E71N+I73S/T, E75N+M77S/T, N85S/T, S84N+K86S/T, K86N+K88S/T, K87N+R89S/T, K94N, K94N+T96S, T101N+L103S/T, D102N+N104S/T, L103N+V105S/T, Q106S/T, P122N+A124S/T, A123N+K125S/T, A124N, A124N+T126S, K125N+G127S/T, T126N+K128S/T, G127N+R129S/T, K128N+K130S/T, R129N+R131S/T, K130N and K130N+S132T.S/T represents to be replaced to serine or threonine residues, preferred threonine residues.

Importing a N-glycosylation site only needs the replacement of an aminoacid replacement to comprise K12S/T, G18S/T, G18N, K37S/T, E38N, M45N, I49N, K61S/T, D63N, Q67N, V70N, K80S/T, F82N, N85S/T, K87S/T, K94N, Q106S/T, E119N, A124N, K130N and R140N, particularly K12S/T, G18N, G18S/T, K37S/T, E38N, K61S/T, D63N, Q67N, K80S/T, N85S/T, K94N, Q106S/T, A124N and K130N (in not having the structure of acceptor molecule, having the position that is exposed to the surface above its side chain of 25%), or more preferably G18N, E38N, D63N, Q67N, K94N, A124N and K130N (in not having the structure of acceptor molecule, having the position that is exposed to the surface above its side chain of 50%).

Usually, the preferred N-glycosylation site (except under special circumstances, referring to the part that is entitled as " the interferon gamma variant that receptor affinity descends ") that do not import in the zone that constitutes receptor binding site.Therefore, the Q1N+P3S/T that generally should not suddenly change, E9N+L11S/T, G18N, G18N+S20T, H19N+D21S/T, D21N+A23S/T, G26N+L28S/T, K34N+W36S/T, K37N+E39S/T, E119N and E119N+S121T are unless need to reduce receptor affinity.

Particularly preferred interferon gamma polypeptide comprises at least one selection by K12S, K12T, G18S, G18T, E38N, E38N+S40T, K61S, K61T, N85S, N85T, K94N, the replacement in the group that Q106S and Q106T form, more preferably be selected from by K12T G18T, E38N+S40T, K61T, N85T in the group that K94N and Q106T form, even more preferably is selected from by K12T G18T, E38N+S40T, in the group that K61T and N85T form, E38N+S40T particularly.

Should understand that the replacement of above determining preferably combines with the S99T sudden change.Therefore, interferon gamma polypeptide very preferably comprises and is selected from by K12S+S99T K12T+S99T, G18S+S99T, G18T+S99T, E38N+S99T, E38N+S40T+S99T, K61S+S99T, K61T+S99T, N85S+S99T, N85T+S99T, K94N+S99T, replacement in the group that S99T+Q106S and S99T+Q106T form, more preferably be selected from by K12T+S99T G18T+S99T, E38N+S40T+S99T, K61T+S99T, N85T+S99T in the group that K94N+S99T and S99T+Q106T form, even more preferably is selected from by K12T+S99T, G18T+S99T, E38N+S40T+S99T, in the group that K61T+S99T and N85T+S99T form, E38N+S40T+S99T particularly.

Should understand arbitrary above-mentioned modification can with the disclosed arbitrary modification of part that is entitled as " the interferon gamma variant of having optimized the N-glycosylation site ", combine particularly with replacement S99T, and with the disclosed arbitrary modification of part that is entitled as " non-polypeptide half point is with the interferon gamma variant of cysteine as the molecule of linking group ".

Non-polypeptide half point is with the interferon gamma variant of cysteine as the molecule of linking group

In another embodiment preferred, interferon gamma polypeptide comprises the cysteine residues of at least one importing.Preferably, import cysteine residues by replacing.

For example, can import cysteine residues in the position of the interferon gamma polypeptide that contains the amino acid residue that is exposed to the surface.The amino acid residue that preferred described surface exposes has at least 25% side chain and is exposed to the surface, and particularly its side chain of at least 50% is exposed to the surface.Can be about the details of measuring this position referring to this paper embodiment A.

For example, cause comprising being exposed to interferon gamma polypeptide surface and being had the replacement that at least 25% side chain is exposed to the cysteine residues of position importing that the amino acid residue on surface (having in the structure of acceptor molecule) occupies: Q1C, D2C, P3C, K6C, E9C, N10C, K13C, Y14C, N16C, G18C, H19C, D21C, N25C, G26C, G31C, K34C, N35C, K37C, E38C, E39C, S40C, K55C, K58C, N59C, K61C, D62C, D63C, Q64C, S65C, Q67C, K68C, E71C, T72C, K74C, E75C, N78C, V79C, K80C, N83C, S84C, N85C, K86C, K87C, D90C, E93C, K94C, T101C, D102C, L103C, N104C and E119C.

Cause comprising being exposed to interferon gamma polypeptide surface and being had the replacement that at least 50% side chain is exposed to the cysteine residues of position importing that the amino acid residue on surface (having in the structure of acceptor molecule) occupies: P3C, K6C, N10C, K13C, N16C, D21C, N25C, G26C, G31C, K34C, K37C, E38C, E39C, K55C, K58C, N59C, D62C, Q64C, S65C, K68C, E71C, E75C, N83C, S84C, K86C, K87C, K94C, T101C, D102C, L103C and N104C.

Usually, it is preferred that (except under special circumstances, referring to the part that is entitled as " the interferon gamma variant that receptor affinity descends ") imports cysteine residues (and subsequently these cysteine residues being connected with non-polypeptide half point) not in the zone that constitutes receptor binding site.Therefore, the Q1C that generally should not suddenly change, E9C, G18C, H19C, D21C, G26C, K34C, K37C and E119C are unless need to reduce receptor affinity.

Most preferably, by being selected from by N10C, N16C, E38C, N59C, N83C, K94C, the replacement in the group that N104C and A124C form imports described cysteine residues.

Preferably, the interferon gamma polypeptide of arbitrary above-mentioned modification also contains and replaces S99T.In above-mentioned replacement, preferred especially following replacement N10C+S99T, N16C+S99T, E38C+S99T, N59C+S99T, N83C+S99T, K94C+S99T, N104C+S99T and A124C+S99T.

The cysteine residues that should understand importing preferably with non-polypeptide half point, for example PEG or more preferably mPEG combination.The interferon gamma variant combines by using arbitrary conventional method to carry out with the activation multimeric molecule, for example, by following list of references described (wherein also having described the appropriate method of activation multimeric molecule): Harris and Zalipsky, eds., Poly (ethylene glycol) Chemistry and BiologicalApplications, AZC, Washington; R.F.Taylor, (1991), " Protein immobilisation.Fundamental and applications ", Marcel Dekker, N. Y.; S.S.Wong, (1992), " Chemistry of Protein Conjugation and Crosslinking ", CRC Press, BocaRaton; G.T.Hermanson etc., (1993), " Immobilized Affinity Ligand Techniques ", Academic Press, N.Y.).

The preferred especially and polymeric object lesson of the link coupled activated PEG of cysteine residues comprises following linear PEGs: vinyl sulfone(Remzaol-PEG (VS-PEG), optimal ethylene sulfone-mPEG (VS-mPEG); Maleimide-PEG (MAL-PEG), preferred maleimide-mPEG (MAL-mPEG) and positive pyridine radicals-disulphide-PEG (OPSS-PEG), preferred positive pyridine radicals-disulphide-mPEG (OPSS-mPEG).In general, this PEG or mPEG polymer have about 5kDa, approximately 10kD, the approximately size of 12kDa or about 20kDa.For with cysteine residues PEGization, before PEGization, use usually such as the Reducing agent of dithiothreitol, DTT (DTT) and handle the interferon gamma variant.Use any conventional method subsequently, for example remove this Reducing agent by desalination.PEG reaches 16 hours with combining generally of cysteine residues in 4 ℃ to 25 ℃ temperature range in the suitable buffer of pH6-9.

Should understand arbitrary above-mentioned modification can with the disclosed arbitrary modification of part that is entitled as " the interferon gamma variant of having optimized the N-glycosylation site ", combine particularly with replacement S99T, and with the disclosed arbitrary modification of part that is entitled as " the interferon gamma variant that non-polypeptide half point is saccharide half point ".

First non-polypeptide half point is that saccharide half point and second non-polypeptide half point are to do with cysteine Interferon gamma variant for the molecule of linking group

In another particularly preferred embodiment, interferon gamma polypeptide comprises the N-glycosylation site of at least one importing and the cysteine residues of at least one importing.Preferably import cysteine residues and/or N-glycosylation site by replacing.Prepare this polypeptide by being chosen in the residue described in two aforementioned parts describing the correct position that imports N-glycosylation site and cysteine residues respectively.Therefore, in an interested embodiment, interferon gamma polypeptide comprises and is selected from by K12T+N16C, K12T+E38C, K12T+N59C, K12T+N83C, K12T+K94C, K12T+N104C, K12T+A124C, G18T+N10C, G18T+E38C, G18T+N59C, G18T+N83C, G18T+K94C, G18T+N104C, G18T+A124C, E38N+S40T+N10C, E38N+S40T+N16C, E38N+S40T+N59C, E38N+S40T+N83C, E38N+S40T+K94C, E38N+S40T+N104C, E38N+S40T+A124C, K61T+N10C, K61T+N16C, K61T+E38C, K61T+N83C, K61T+K94C, K61T+N104C, K61T+A124C, N85T+N10C, N85T+N16C, N85T+E38C, N85T+N59C, N85T+K94C, N85T+N104C, N85T+A124C, K94N+N10C, K94N+N16C, K94N+E38C, K94N+N59C, K94N+N83C, K94N+N104C, K94N+A124C, Q106T+N10C, Q106T+N16C, Q106T+E38C, Q106T+N59C, Q106T+N83C, replacement in the group that Q106T+K94C and Q106T+A124C form more preferably is selected from by E38N+S40T+N10C E38N+S40T+N16C, E38N+S40T+N59C, E38N+S40T+N83C, E38N+S40T+K94C is in the group that E38N+S40T+N104C and E38N+S40T+A124C form.

Preferably, the interferon gamma polypeptide of arbitrary above-mentioned modification also comprises and replaces S99T, be that interferon gamma polypeptide comprises and is selected from by K12T+N16C+S99T, K12T+E38C+S99T, K12T+N59C+S99T, K12T+N83C+S99T, K12T+K94C+S99T, K12T+N104C+S99T, K12T+A124C+S99T, G18T+N10C+S99T, G18T+E38C+S99T, G18T+N59C+S99T, G18T+N83C+S99T, G18T+K94C+S99T, G18T+N104C+S99T, G18T+A124C+S99T, E38N+S40T+N10C+S99T, E38N+S40T+N16C+S99T, E38N+S40T+N59C+S99T, E38N+S40T+N83C+S99T, E38N+S40T+K94C+S99T, E38N+S40T+N104C+S99T, E38N+S40T+A124C+S99T, K61T+N10C+S99T, K61T+N16C+S99T, K61T+E38C+S99T, K61T+N83C+S99T, K61T+K94C+S99T, K61T+N104C+S99T, K61T+A124C+S99T, N85T+N10C+S99T, N85T+N16C+S99T, N85T+E38C+S99T, N85T+N59C+S99T, N85T+K94C+S99T, N85T+N104C+S99T, N85T+A124C+S99T, K94N+N10C+S99T, K94N+N16C+S99T, K94N+E38C+S99T, K94N+N59C+S99T, K94N+N83C+S99T, K94N+N104C+S99T, K94N+A124C+S99T, Q106T+N10C+S99T, Q106T+N16C+S99T, Q106T+E38C+S99T, Q106T+N59C+S99T, Q106T+N83C+S99T, replacement in the group that Q106T+K94C+S99T and Q106T+A124C+S99T form more preferably is selected from by E38N+S40T+N10C+S99T E38N+S40T+N16C+S99T, E38N+S40T+N59C+S99T, E38N+S40T+N83C+S99T, E38N+S40T+K94C+S99T is in the group that E38N+S40T+N104C+S99T and E38N+S40T+A124C+S99T form.

Should understand, the cysteine residues of importing preferably with non-polypeptide half point, for example PEG or more preferably mPEG combination.Combining and can finish in any suitable manner between the polypeptide variants that contains cysteine and the multimeric molecule, for example described by the part that is entitled as " non-polypeptide half point is with the interferon gamma variant of cysteine as the molecule of linking group ".

Should understand arbitrary above-mentioned modification can with the part disclosed arbitrary modification that is entitled as " the interferon gamma variant of having optimized N-glycosylation site in the body ", particularly with replace S99T and combine.

The interferon gamma variant that receptor affinity reduces

A kind of mode that increases interferon gamma polypeptide serum half life is to reduce receptor-mediated internalization and therefore reduce receptor-mediated removing.Receptor-mediated internalization depends on the affinity of interferon gamma dimer to the interferon gamma receptor complex, so the plain γ polypeptide of expected interference can make internalization to the affinity reduction of interferon gamma receptor complex, and the degree of therefore removing is lower.

Undertaken one or more by receptor binding domain and modify, particularly replace the affinity that can reduce interferon gamma dimer and its receptor complex at interferon gamma polypeptide.The amino acid residue that constitutes receptor binding domain limits in this paper Embodiment B.It is that conserved amino acid replaces that the class that can carry out replaces.In another embodiment, the modification of carrying out causes importing N-glycosylation site.

Therefore, in another interested embodiment, interferon gamma polypeptide contains at least one modification at receptor binding site (limiting by this paper).In particular, interferon gamma polypeptide contains at least one modification that produces N-glycosylation site in the body at described receptor binding domain, the preferred replacement.For example, this replaces optional free Q1N+P3S/T, D2N+Y4S/T, Y4N+K6S/T, V5N+E7S/T, E9N+L11S/T, K12N+Y14S/T, G18N, G18N+S20T, H19N+D21S/T, S20N+V22S/T, D21N+A23S/T, V22N+D24S/T, D24N+G26S/T, G26N+L28S/T, L30N+I32S/T, K34N+W36S/T, K37N+E39S/T, K108N+I110S/T, H111N+L113S/T, E112N+I114S/T, I114N+V116S/T, Q115N+M117S/T, A118N+L120S/T, in the group that E119N and E119N+S121T form, be preferably selected from by Q1N+P3S/T D2N+Y4S/T, E9N+L11S/T, K12N+Y14S/T, G18N, G18N+S20T, H19N+D21S/T, S20N+V22S/T, D21N+A23S/T, K34N+W36S/T, K37N+E39S/T, H111N+L113S/T, Q115N+M117S/T, A118N+L120S/T (imports the N-glycosylation site at least 25% position that is exposed to the amino acid residue on surface of containing its side chain) in the group that E119N and E119N+S121T form, more preferably be selected from by Q1N+P3S/T, D2N+Y4S/T, E9N+L11S/T, G18N, G18N+S20T, H19N+D21S/T, S20N+V22S/T, D21N+A23S/T, K34N+W36S/T, K37N+E39S/T, Q115N+M117S/T, A118N+L120S/T, (import the N-glycosylation site) in the group that E119N and E119N+S121T form at least 50% position that is exposed to the amino acid residue on surface of containing its side chain, even more preferably be selected from by Q1N+P3T D2N+Y4T, E9N+L11T, G18N+S20T, H19N+D21T, S20N+V22T, D21N+A23T, K34N+W36T, K37N+E39I, Q115N+M117I is in the group that A118N+L120T and E119N+S121T form, most preferably be selected from by G18N+S20T, H19N+D21T, in the group that D21N+A23T and E119N+S121T form, D21N+A23T particularly.

Expect that this variant compares the receptor affinity that shows reduction with human interferon gamma or Actimmune .This receptor affinity can be measured and is known to those skilled in the art by any suitable test.An example measuring the suitable test of receptors bind affinity is Michiels etc., the described BIAcore  test of Int.J.Biochem.Cell Biol.30:505-516 (1998).

In general, for example, when detecting in " one-level test " as herein described, the interferon gamma polypeptide that receptor affinity reduces shows the interferon gamma activity of reduction.For example, interferon gamma polypeptide can show the interferon gamma activity of the 1-95% of Actimunne  or human interferon gamma, 1-75% for example, 1-50% for example, for example the Actimunne  of 1-20% or 1-10% or with the interferon gamma activity of the human interferon gamma of its glycosylation form.

Obviously, the arbitrary above-mentioned modification that causes the receptors bind affinity to reduce can combine with arbitrary other modification disclosed herein, particularly be entitled as " the interferon gamma variant of optimizing the N-glycosylation site ", " the interferon gamma variant that contains the sub-linking group of non-polypeptide half point ", " non-polypeptide half point is the interferon gamma variant of saccharide half point ", the modification that " non-polypeptide half point is with the interferon gamma variant of cysteine as the molecule of linking group " and " first non-polypeptide half point is that saccharide half point and second non-polypeptide half point are with the interferon gamma variant of cysteine as the molecule of linking group " part is mentioned, for example be selected from by E38N, S40T, modification, particularly E38N+S40T+S99T in the group that S99T and combination thereof are formed.

In another embodiment, interferon gamma polypeptide contains the glycosylation site of at least one importing, with at least one saccharide half point that is connected with the glycosylation site that imports.In another embodiment, interferon gamma polypeptide comprises the glycosylation site and at least one saccharide half point and multimeric molecule such as Polyethylene Glycol that is connected with the glycosylation site that imports of at least one importing.In particularly preferred embodiments, interferon gamma polypeptide contains 3 saccharide half point, and each saccharide half point is connected with a N-glycosylation site.

Truncate analysis to interferon gamma polypeptide

Can measure the terminal truncate of the C-of interferon gamma polypeptide purification of samples in many ways.

A kind of mode of illustrating the terminal truncate of interferon gamma polypeptide C-depends on the accurate mass measurement by mass spectrography.Unfortunately, the glycosylation of interferon gamma is inhomogenous, the therefore feasible extremely difficult accurate mass of directly measuring glycoprotein.Therefore, generally use the enzymatic deglycosylation of varying level to combine with mass spectrography.

In one approach, use whole polysaccharide parts of endoglycosidase PNGase F cracking interferon gamma polypeptide, then use ESI mass spectrography or MALDI-TOF mass spectrography to carry out accurate mass measurement.The known amino acid sequence of comparative experiments quality and interferon gamma makes the site of measuring the terminal truncate of C-become possibility.

In another method, the only sialic acid of the polysaccharide of cracking interferon gamma polypeptide part rather than all polysaccharide.In some cases, it is enough that the inhomogeneity of sample is reduced to such level, promptly can infer the site that the terminal truncate of C-after using ESI mass spectrography or MALDI-TOF mass spectrography to carry out accurate mass measurement under this level.

A kind of more traditional mode of illustrating the terminal truncate of interferon gamma polypeptide C-is to adopt peptide mapping connexus spectrometry and chemical amino acid sequencing.Briefly, interferon gamma polypeptide then uses RP-HPLC to carry out peptide and separates with known specific protease (Asp-N protease) degraded.Use ESI mass spectrography or off-line to use the MALDI-TOF mass spectrography that the fractionated composition is carried out quality analysis then on the line.Relatively the known amino acid sequence of peptide quality of Huo Deing and interferon gamma makes the possible site of measuring the terminal truncate of C-become possibility.Be confirmed by amino acid sequencing then.

The sulfoalkyl ether cyclodextrin derivant

In compositions of the present invention, the sulfoalkyl ether cyclodextrin derivant is at US 5,874,418, US5, and 376,645 and US 5,134, the arbitrary derivant described in 127, this paper quotes its content for your guidance.The sulfoalkyl ether cyclodextrin derivant also is described in WO91/11172, and this paper quotes its content for your guidance.In one embodiment of the invention, sulfoalkyl ether cyclodextrin is the chemical compound with formula (I):

Wherein

N is 4,5 or 6,

R ₁, R ₂, R ₃, R ₄, R ₅, R ₆, R ₇, R ₈, and R ₉Be independently respectively ,-O-or-O-(C ₂-C ₆Alkylidene)-SO ₃-group, wherein R ₁And R ₂In at least one is-O-(C independently ₂-C ₆Alkylidene)-SO ₃-group, and

S ₁, S ₂, S ₃, S ₄, S ₅, S ₆, S ₇, S ₈And S ₉Be respectively pharmaceutically useful cation independently.

In another embodiment of the chemical compound of formula (I), n is 5.

In another embodiment of the chemical compound of formula (I), n is 6.

In another embodiment of the chemical compound of formula (I), R ₁And R ₂In at least one is-O-(CH ₂) _m-SO ₃, and m is 2,3,4,5 or 6.

In another embodiment of the chemical compound of formula (I), R ₁And R ₂Be independently selected from-OCH ₂CH ₂CH ₂SO ₃-or-OCH ₂CH ₂CH ₂CH ₂SO ₃-.

In another embodiment of the chemical compound of formula (I), R ₄, R ₆And R ₈In at least one is-O-(C independently ₂-C ₆Alkylidene)-SO ₃-; And R ₅, R ₇And R ₉All be-O-.

In another embodiment of the chemical compound of formula (I), S ₁, S ₂, S ₃, S ₄, S ₅, S ₆, S ₇, S ₈And S ₉Be respectively pharmaceutically useful cation independently, this cation is selected from H ⁺, alkali metal (Li for example ⁺, Na ⁺, K ⁺), alkaline-earth metal (for example, Ca ^{+ 2}, Mg ^{+ 2}), ammonium ion and such as (C ₁-C ₆) alkylamine, piperidines, pyrazine, (C ₁-C ₆) alkanolamine and (C ₄-C ₈) the cationic amine cation of cycloalkanes hydramine.

In another embodiment of the chemical compound of formula (I), S ₁, S ₂, S ₃, S ₄, S ₅, S ₆, S ₇, S ₈And S ₉Be independently selected from alkali metal cation, alkaline earth metal cation, quartemary ammonium cation, trivalent ammonium cation and bivalence ammonium cation.

In another embodiment, R ₄, R ₆And R ₈In at least one is-O-(C independently ₂-C ₆Alkylidene)-SO ₃-; And R ₅, R ₇And R ₉All be-O-.

Term used herein " alkylidene " and " alkyl " are (for example at-O-(C ₂-C ₆Alkylidene)-SO ₃-group or in alkylamine) comprise line style respectively, ring-type, and branch, the divalent alkyl group and the monovalent alkyl group of saturated and unsaturated (that is, contain one two keys).Term herein " alkanol " comprises line style equally, ring-type, and branch, and the triacontanol group of saturated and unsaturated alkyl composition, wherein hydroxyl can be positioned at arbitrary position of alkyl half point.Term " cyclic alkanol " comprises the cyclic alcohol that does not replace or replace (for example, with methyl or ethyl).

(Kansas 66213, US) for Cydex, Overland Park for the salt that at present preferred sulfoalkyl ether cyclodextrin derivant is a beta cyclodextrin sulfo group butyl ether (particularly with its sodium salt of the obtainable SBE7-of the being also referred to as β-CD of Captisol ).

Other embodiment of the present composition

In general, the pH scope of compositions of the present invention is 3-8,4-8 for example, 5-8,6-8,7-8,4-7,4-6, or 4-5.For interferon beta, preferred pH scope is 4-8, preferred 5-8, or alternative 4-7.In another embodiment, this pH scope is 5-6, for example about 5.5.Generally can obtain this pH by the buffer agent that uses the appropriate amount that further describes hereinafter.

In addition, need said composition and blood almost to wait and ooze, that is, have the Morie osmolarity of about 240-360mOsmol/kg, 280-320mOsmol/kg for example, particularly about 300mOsmol/kg.Generally can obtain this Morie osmolarity by the tonicity agent that uses the appropriate amount that hereinafter further describes.

Therefore, aspect generalized, the present invention relates to contain the stabilized composition of interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.This interferon polypeptides generally be selected from above with embodiment in mention any one.The preferred embodiment of interferon polypeptides is interferon beta and interferon gamma, particularly interferon beta variant and interferon gamma variant, the variant of glycosylation and/or PEGization for example, detailed description as mentioned.

Interferon polypeptides generally exists with about 1-100MIU/ agent with the concentration of about 1-100MIU/ml or in solid articles in flowing product.About 1-10MIU/ml in the optional comfortable flowing product of this concentration, 1-20MIU/ml, 1-30MIU/ml, 1-40MIU/ml, 1-50MIU/ml, 1-60MIU/ml, 1-70MIU/ml, 1-80MIU/ml, 1-90MIU/ml, 10-20MIU/ml, 20-30MIU/ml, 30-40MIU/ml, 40-50MIU/ml, 50-60MIU/ml, 60-70MIU/ml, 70-80MIU/ml, 80-90MIU/ml, 90-100MIU/ml, 5-95MIU/ml, 15-85MIU/ml, 25-75MIU/ml, 35-65MIU/ml, and 45-55MIU/ml.In addition, the about 1-10MIU/ agent in the optional comfortable solid articles of this concentration, 1-20MIU/ agent, the 1-30MIU/ agent, 1-40MIU/ agent, 1-50MIU/ agent, the 1-60MIU/ agent, 1-70MIU/ agent, 1-80MIU/ agent, the 1-90MIU/ agent, 10-20MIU/ agent, 20-30MIU/ agent, the 30-40MIU/ agent, the 40-50MIU/ agent, 50-60MIU/ agent, 60-70MIU/ agent, the 70-80MIU/ agent, the 80-90MIU/ agent, 90-100MIU/ agent, 5-95MIU/ agent, the 15-85MIU/ agent, 25-75MIU/ agent, 35-65MIU/ agent and 45-55MIU/ agent.

The sulfoalkyl ether cyclodextrin derivant generally exists with the concentration of about 1-150mg/ml.This concentration is optional from about 1-10mg/ml, 1-20mglml, 1-30mg/ml, 1-40mg/ml, 1-50mg/ml, 1-60mg/ml, 1-70mg/ml, 1-80mg/ml, 1-90mg/ml, 1-100mg/ml, 1-110mg/ml, 1-120mg/ml, 1-130mg/ml, 1-140mg/ml, 10-20mg/ml, 20-30mg/ml, 30-40mg/ml, 40-50mg/ml, 50-60mg/ml, 60-70mg/ml, 70-80mg/ml, 80-90mg/ml, 90-100mg/ml, 100-110mg/ml, 110-120mg/ml, 120-130mg/ml, 130-140mg/ml, 140-150mg/ml, 5-100mg/ml, 5-95mg/ml, 5-90mg/ml, 5-85mg/ml, 5-80mg/ml, 5-75mg/ml, 5-70mg/ml, 5-65mg/ml, 5-60mg/ml, 5-55mg/ml, 5-50mg/ml, 5-45mg/ml, 5-40mg/ml, 5-35mg/ml, 5-30mg/ml, 5-25mg/ml, 5-20mg/ml, 5-15mg/ml, and 5-10mg/ml.

In one embodiment, the pH scope of compositions of the present invention is 4-8, and Morie osmolarity is about 240-360mOsmol/kg, 280-320mOsmol/kg for example, particularly about 300mOsmol/kg.

One concrete aspect, compositions of the present invention relates to the liquid solution that contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.

Another concrete aspect, compositions of the present invention relates to the aqueous solution that contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.

The sulfoalkyl ether cyclodextrin derivant generally exists with the concentration of about 1-150mg/ml.Yet arbitrary above-mentioned concentration range is all thought one embodiment of the invention.

Interferon polypeptides generally in liquid solution the concentration with about 1-100MIU/ml exist.Yet arbitrary above-mentioned concentration range is all thought one embodiment of the invention.

In one embodiment, liquid solution or aqueous solution are isoosmotic and have the Morie osmolarity of about 240-360mOsmol/kg.In another embodiment, liquid solution or aqueous solution be isoosmotic and have the Morie osmolarity of about 240-360mOsmol/kg, and have the pH scope of 4-8,5-8 for example, 6-8,7-8,4-7,4-6, or 4-5.In also having another embodiment, liquid solution or aqueous solution also contain the tonicity agent of the Morie osmolarity that about 240-360mOsmol/kg is provided.This tonicity agent can be any suitable tonicity agent, for example hereinafter arbitrary tonicity agent of mentioning of " parenteral goods " part.In also having another embodiment, this liquid solution or aqueous solution also contain the buffer agent that exists with the concentration up to 100mM.The optional arbitrary concentration range mentioned from " parenteral goods " hereinafter part of the concentration of buffer agent.This buffer agent can be any suitable reducing, for example hereinafter arbitrary buffer agent of mentioning of " parenteral goods " part.

As mentioned above, interferon polypeptides generally be selected from above with embodiment in arbitrary polypeptide of mentioning.The preferred embodiment of interferon polypeptides is interferon beta and interferon gamma, particularly arbitrary interferon beta variant and interferon gamma variant, the variant of for example arbitrary glycosylation and/or PEGization, detailed description as mentioned.Other embodiment preferred of interferon polypeptides is people's wild type interferon beta and people's wild type interferon gamma, and for example glycosylation and/or PEG chemoattractant molecule for example with polymer, for example comprise the link coupled glycosylated interferon β-1a of polymer of Polyethylene Glycol.

Preferably, compositions of the present invention is that wherein interferon polypeptides was stored for 1 week under 37 ℃ temperature at least, preferably keeps its bioactive compositions (measuring in accelerated stability test) during at least 2,3 or 4 weeks basically.Wherein interferon polypeptides keeps its biological activity basically and is meant that to keep it bioactive at least 80%, preferably its bioactive at least 90%.

In addition, preferred interferon polypeptides was stored for 4 weeks under 25 ℃ temperature at least in compositions of the present invention, for example kept its biological activity basically during at least 5,6,7,8,9,10,11 or 12 weeks.In another was selected, compositions of the present invention was that wherein interferon polypeptides keeps its bioactive compositions basically during storing at least 1 week under 37 ℃ the temperature and during storing at least 4 weeks under 25 ℃ the temperature.Biological activity to be measured is, for example antiviral activity.

Antiviral activity can be measured with methods known in the art, for example, and by using in the algoscopy described in WO01/15736 (interferon beta) and the WO01/36001 (interferon gamma).

The algoscopy of WO01/15736 is as follows:

Use A549 cell (CCL 185, U.S. tissue culture preservation center) and encephalomyo-carditis (EMC) virus (VR-129B, U.S. tissue culture preservation center) to carry out the antiviral bioassay.Be seeded in 96 hole tissue culture plate with the concentration of 10,000 cells/well cell and cultivating in the air at 5%CO2 under 37 ℃.In containing hyclone and antibiotic 100 μ l DMEM culture medium altogether, add polypeptide of the present invention or conjugate with concentration range (being generally 100-0.0001IU/mL) from about 100-0.0001IU/mL.Take out culture medium after 24 hours and in each hole, add the 0.1mL fresh culture that contains EMC virus.After 24 hours, cause that in the cell culture of noiseless element-β the concentration of 100% cell death adds EMC virus.After 24 hours, use the WST-1 algoscopy to measure the antiviral effect of this polypeptide or conjugate.In the 0.1mL culture, add 0.01mL WST-1 (WST-1 hyperplasia agent, Roche Diagnostics GmbH, Mannheim, Germany) and under 37 ℃ at 5%CO ₂Air in cultivated 1/2-2 hour.Mitochondrial dehydrogenase cracking tetrazolium WST-1 causes forming and can carry out the quantitative first moon for (formazan) by measure absorbance under 450nm in living cells.

Calculate output with respect to known standard with U/ml, this standard is included on the identical flat board and analyzes under the same conditions.

Algoscopy among the WO01/36001 is as follows:

The interferon gamma receptor that disclosed in the past on interferon gamma and the HeLa cell interacts and the activation this receptor.Thereby, contain interferon irritant reaction element (ISRE) but promoter on activated transcription.Therefore to screen the agonist of interferon receptors be possible to the ISRE (ISRE-luc) that is positioned over the coupling luciferase reporter gene in the HeLa cell by use.

With ISRE-Luc and pCDNA 3.1/hygro cotransfection HeLa cell, by in containing the DMEM culture medium of HYG, selecting to produce transforming focus (cell clone).The luciferase activity of screening cell clone under the condition that has or do not exist interferon gamma.Those clones that show the ceiling rate of the luciferase activity that stimulates and do not stimulate are used for further test.

In order to screen mutant, in 96 hole culture plates, inoculate 15,000 cells/well and overnight incubation in the DMEM culture medium.In cell, added mutant and known standard with various concentration in second day.Dull and stereotyped under 37 ℃ at 5%CO ₂Air in cultivated 6 hours, in each hole, add LucLite substrate (Packard Bioscience, Groningen The Netherlands) subsequently.Seal plate is also upward measured luminous in SPC (single photon counting) mode at TopCount luminometer (Packard).Each flat board contains the hole of useful interferon gamma cultivation as stimulating contrast, and other hole is contained normal culture medium and contrasted as stimulating.Ratio between the luciferase activity that stimulate and that do not stimulate is as the internal standard of error between mutant activity and experiment and experiment.

Pharmaceutical composition of the present invention can be a various forms, comprises liquid, gel, dried frozen aquatic products, lung dispersant, perhaps any other suitable form, for example compression solid.Term " aqueous " attempted to comprise in term in the context of the invention " liquid ".

In another embodiment of the present invention, said composition is dried forms or liquid formulations.In another embodiment of the present invention, said composition is a dried forms.Said composition is a liquid form in another embodiment of the present invention.In another embodiment of the present invention, said composition is an aqueous solution.Said composition is an aqueous suspensions in another embodiment of the present invention.

Preferred form depends on the concrete indication of being treated and apparent to one skilled in the art.For example, lyophilizing solution can be used for further increasing stability.

Pharmaceutical composition of the present invention can pass through the oral cavity, intravenous, and in the brain, intramuscular, intraperitoneal, intradermal, subcutaneous, intranasal, in the lung, or, for example use drug-supplying system to carry out administration such as PowderJect or ProLease technology with any other acceptable manner.Preferred administering mode depends on the concrete indication of being treated and apparent to one skilled in the art.

The compositions of the particular type that is suitable for preparation has been described hereinafter.Should understand that the property quality and quantity of the various additives of use depends on the type and the route of administration of interferon polypeptides and preparation.In general, compositions of the present invention comprises buffer agent, tonicity agent, preservative agent, wetting agent, viscosity increasing agent, and/or one or more stabilizing agents except the sulfoalkyl ether cyclodextrin derivant.Should understand that this stabilizing agent must not have adverse effect to the stablizing effect of sulfoalkyl ether cyclodextrin derivant.Other component of said composition further describes hereinafter.

In addition, compositions of the present invention can comprise human blood-pure albumen or be used for stablizing said composition and/or minimize and store other human body protein of absorption of the container of said composition.Yet in a specific embodiments, said composition does not conform to human serum albumin or other human body protein basically, because may not need to exist this protein from check viewpoint.

Compositions of the present invention can prepare with the conventional method of preparation Pharmaceutical composition.For example, by before mixing interferon polypeptides, being pre-mixed stable and buffer agent and any other additive can prepare said composition.The void volume that purifies partially filled product container with the purifying aqueous preparation of nitrogen and/or nitrogen prepares said composition in one embodiment.In another embodiment, need not this nitrogen purify and prepare said composition.

The amount that is present in the interferon polypeptides in the compositions depends on interferon polypeptides, the characteristic of preparation and route of administration.For example, when said composition is that interferon beta polypeptides exists with the amount corresponding to 1-100MIU/ml when containing the compositions of interferon beta, be generally 1-50MIU/ml (when being mixed with liquid formulations) or 1-100MIU/ agent (when being mixed with solid formulation).

The parenteral goods

An example of Pharmaceutical composition is the solution that is designed for parenteral.Although in many cases, the medical solution preparation provides with the liquid form that is suitable for using immediately, this parenteral preparation also can be freezing or lyophilized form provide.In the previous case, said composition must used preceding thawing.A kind of form in back is usually used in the stability of reactive compound contained in the enhancing composition under various storage requirements, because those skilled in the art will recognize that freeze-dried products is generally more stable than its liquid homologue.This freeze-dried products is being rebuild by adding one or more suitable pharmaceutically useful diluent such as Injectable sterile water or sterile physiological saline solution with preceding.

For the parenteral goods, can be by mixing interferon polypeptides and one or more pharmaceutically useful carriers suitably with required purity, excipient or stabilizing agent (they all are called " excipient ") that this area is commonly used, buffer agent for example, stabilizing agent, preservative agent, tonicity agent, nonionic detergent, antioxidant and/or other various additives prepare them and are used for storing as lyophilizing preparation or aqueous solution.

Buffer agent exists with such concentration, and promptly this concentration guarantees that pH maintains desired level, for example near the level of physiological level.This buffer agent has suitable concn up to 100mM for various buffer type.For most buffering agents, this concentration generally exists with the scope of 1-100mM, 1-90mM for example, 1-80mM, 1-70mM, 1-60mM, 1-50mM, 1-40mM, 1-30mM, 1-20mM, 1-10mM, 5-15mM, 15-25mM, 25-35mM, 35-45mM, 45-55mM, 55-65mM, 65-75mM, 75-85mM, 85-95mM.Suitable concentration can be by determination of technical staff.Buffer agent generally is a weak acid, the solution of weak base or anion salt that should acid.Being used for suitable buffer agent of the present invention comprises organic and mineral acid and salt thereof, for example citrate buffer agent (for example, sodium dihydrogen citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-sodium dihydrogen citrate mixture, Deng), the succinate buffer agent (for example, succinic acid-succinic acid one sodium mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, Deng), the tartrate buffer agent (for example, tartaric acid-sodium tartrate mixture, tartaric acid-Soluble tartar. mixture, tartaric acid-sodium hydroxide mixture, Deng), Fumaric acid salt buffer agent (for example, Fumaric acid-Fumaric acid one sodium mixture, Fumaric acid-Fumaric acid disodium mixture, Fumaric acid one sodium-Fumaric acid disodium mixture, Deng), maleate buffer agent (for example, Monosodium maleate), the gluconic acid salt buffer agent (for example, gluconic acid-gluconic acid sodium salt mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, Deng), oxalates buffer agent (for example, oxalic acid-Disodium oxalate. mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, Deng), lactate buffer agent (for example, lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffer (for example, acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.).Other probability is a phosphate buffer, carbonate buffer agent (for example, sodium carbonate), histidine buffer and glutamate, Glu buffer agent.Add preservative agent stoping growth of microorganism, and generally add with the amount of about 0.2%-1% (w/v).Be used for suitable preservative agent of the present invention and comprise phenol, benzyl alcohol, metacresol, alkyl paraben, for example methyl parahydroxybenzoate or propyl p-hydroxybenzoate, zephiran halogenide (for example, zephiran chlorine, bromine or iodine), catechol, resorcinol, 3-amylalcohol and its suitable mixture.

Therefore, in another embodiment, compositions of the present invention comprises the buffer agent such as above-mentioned any or its mixture.

In another embodiment, compositions of the present invention also contains preservative agent and/or viscosity increasing agent.

In another embodiment, compositions of the present invention does not contain preservative agent.

In another embodiment, compositions of the present invention comprises and is selected from citrate buffer agent, the succinate buffer agent, the tartrate buffer agent, Fumaric acid salt buffer agent, maleate buffer agent, the gluconic acid salt buffer agent, the oxalates buffer agent, phosphate buffer, carbonate buffer agent, histidine buffer, the glutamate, Glu buffer agent, a kind of buffer agent of lactate buffer agent and acetate buffer.

In another embodiment, compositions of the present invention comprises the buffer agent that exists with the concentration up to 100mM, 1mM to 100mM for example, 1-90mM, 1-80mM, 1-70mM, 1-60mM, 1-50mM, 1-40mM, 1-30mM, 1-20mM, 1-10mM, 5-15mM, 15-25mM, 25-35mM, 35-45mM, 45-55mM, 55-65mM, 65-75mM, 75-85mM, or 85-95mM.

Add tonicity agent and comprise the polyhydroxy sugar alcohol with isotonicity and this tonicity agent of guaranteeing fluid composition, preferred trihydroxy or more senior sugar alcohol, glycerol for example, erithritol, 1,2,3,4,5-pentanepentol, xylitol, sorbitol and mannitol (mannitol generally exists with the concentration up to 50mg/ml), or NaCl (NaCl generally exists with the concentration up to 9mg/ml).Consider the relative quantity of other composition, polyhydroxy-alcohol with account for weight 0.1% and 25% between amount exist, be generally 1% to 5%.

Stabilizing agent is meant various excipient, but by function from extender to the solubilising therapeutic agent or help prevent degeneration or adhere to the additive of chamber wall and change.Typical stabilizing agent can be polyhydroxy sugar alcohol (above enumerate); Such as arginine, lysine, glycine, glutamine, agedoite, histidine, alanine, ornithine, the L-leucine, 2-phenylalanine, glutamic acid, the aminoacid of threonine etc., organic saccharide or sugar alcohol, lactose for example, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol etc. comprise the cyclic alcohol such as inositol; Polyethylene Glycol; The aminoacid polymer; Such as carbamide, glutathion, thioctic acid, sodium mercaptoacetate, thioglycerol, the sulfur-bearing Reducing agent of α-monothioglycerol and sodium thiosulfate; Low molecular weight polypeptide (residue promptly＜10); Such as the human serum albumin, bovine serum albumin, the protein of gelatin or immunoglobulin; Hydrophilic polymer such as polyvinylpyrrolidone; Such as xylose, mannose, the monosaccharide of fructose and glucose; Such as lactose, the disaccharide of maltose and sucrose; Such as the trisaccharide of Raffinose with such as the polysaccharide of glucosan.Stabilizing agent generally exists with the scope based on from 0.1 to 10,000 part of weight of activated protein weight.

Can exist non-ionic surface active agent or detergent (being also referred to as " wetting agent ") to assemble to help solubilising therapeutic agent and the stirring induction type that prevents therapeutical peptide, it also allows this preparation to be exposed to shear surface tension force and does not cause the polypeptide degeneration.Suitable ionic surfactant pack is drawn together Polysorbate (20,80 etc.), polyoxamers (184,188 etc.), Pluronic ^Polyhydric alcohol, polyethylene glycol oxide anhydro sorbitol monoether (Tween ^-20 (Tween-20 generally exists with the concentration up to 2mg/ml), Tween ^-80 (Tween-80 generally exists with the concentration up to 2mg/ml), etc.).

In a preferred embodiment, when comprising interferon beta polypeptides, compositions do not add surfactant such as non-ionic surface active agent.

Other various excipient comprise extender or filler (for example, starch), and chelating agen (for example, EDTA), antioxidant (for example, ascorbic acid, methionine, vitamin E) and cosolvent.At colloidal drug delivery system (for example, liposome, the albumin microsphere spheroid, micro emulsion, nano-particle and Nano capsule) or in slow release goods (referring to next part), this active component also can be embedded in by for example, in coascervation technology or the microcapsule by the interfacial polymerization preparation, hydroxy methocel for example is in gelatin or poly--(methyl methacrylate) microcapsule.This technology is at Remington ' s PharmaceuticalSciences, by E.W.Martin, the 18th edition, A.R.Gennaro, Ed., Mack PublishingCompany[1990]; Open among the Pharmaceutical Formulation Development of Peptides andProteins.

The parenteral preparation that is used for vivo medicine-feeding must be aseptic.For example, can easily realize this point by the aseptic filtration membrane filtration.

Slow release goods (being used for parenteral or other purposes)

The suitable example of slow release goods comprises solid-state hydrophobic polymer and/or the polymeric semi permeability substrate of hydrophilic that contains interferon polypeptides, and this substrate has such as thin film, and is shaft-like, the suitable form of microcapsule or microsphere.The example of sustained-release matrix comprises polyester, hydrogel (for example, poly-(2-ethoxy-methacrylate) or poly-(vinyl alcohol)), polyactide, L-glutamic acid and ethyl-L-glutamic acid copolymer, nondegradable ethylene vinyl acetate, glucosan, starch is such as ProLease ^Technology or Lupron Depot ^The degradable lactic acid-ethanol copolymer of (the injectable microsphere of forming by lactic acid-ethanol copolymer and leuprolide acetic acid) and poly--D-(-)-3-hydroxybutyric acid.Although discharging molecule such as the poly physical ability of ethylene vinyl acetate and lactic acid-ethanol such as reaching or surpassing in 100 days long-time, some hydrogel discharges protein in the time bar of weak point.

Lung or nasal administration preparation

This preparation can be liquid form or solid formulation, uses as the dispersion form or with this form.

All relative assemblies that are used for administration and/or generation dispersion need use the preparation that is suitable for disperseing this interferon polypeptides.In general, each preparation has specificity and can comprise use conventional diluent except being used for the treatment of, the suitable propellant material that adjuvant and/or carrier are outer for the type of device that adopts.Each preparation generally also has specificity for the interferon polypeptides as the dispersion administration.In addition, also comprise the use liposome, microcapsule or microsphere, inclusion complexs, or the carrier of other type.Description now can be used to implement interferon polypeptides preparation of the present invention in the lung dispersal device of common type.

The aerosol apparatus interferon formulations

Be suitable for generally comprising with for example with the interferon polypeptides preparation that sprays or hyperacoustic aerosol apparatus uses, about 0.01 to the every mL solution of 25mg interferon polypeptides, preferably approximately 0.1 to 10mg/mL concentration interferon polypeptides soluble in water.This preparation also can comprise buffer agent and tonicity agent, for example is used for the saccharide that protein stabilization and osmotic pressure are regulated, and/or 0.1 human serum albumin who arrives in the 10mg/ml concentration range.The example of spendable buffer agent has sodium acetate, citrate and glycine.Preferably, buffer agent has and is suitable for pH value of solution is adjusted to component and molar concentration in 3 to 9 scopes.In general, the buffer agent molar concentration from 1mM to 50mM is applicable to this purpose.The example of spendable saccharide is a lactose, maltose, and mannitol, sorbitol, trehalose and xylose, content range account for 1% to 10% of preparation weight usually.

The aerosol apparatus preparation also can contain the proteinic spatial induction type gathering of surfactant to reduce or to prevent to be caused by solution spray when forming aerosol.Can use various conventional surfactants, for example polyoxyethylene fatty acid esters and alcohol and polyethylene glycol oxide fatty acid esters of sorbitan.Content range generally preparation weight 0.001% and 4% between the change.The particularly preferred surfactant that is used for the object of the invention is a polyethylene glycol oxide anhydro sorbitol monooleate.

The concrete preparation and the method for suitable dispersion that produces liquid particles be at WO 9420069, US5915378, and US 5960792, and US 5957124, US 5934272, and US 5915378, and US 5855564, describe among US 5826570 and the US 5522385, they are quoted for your guidance in this article.

3 object lessons that are suitable for the commercially available aerosol apparatus of the present invention's practice are by Mallinckrodt, Inc., St.Louis, Mo. the Ultravent aerosol apparatus of Sheng Chaning is by Marquest MedicalProducts, Englewood, the Acorn II aerosol apparatus that Colorado produces, with by AradigmCorporation, the AERx lung drug-supplying system that Hayward, Califomia produce.

Be used to indicate the inhaler of dosage and the dry preparation of powder inhalator

The interferon formulations that uses with the inhaler device of indicating dosage generally comprises relevant shape, the meticulous powder that separates of surface and size.This powder can by lyophilizing and if desired, the abrasive solid preparation be produced subsequently, also can contain the stabilizing agent such as human serum albumin (HSA).In general, add the HAS that surpasses 0.5% (w/w).In addition, if desired, can in goods, add one or more sugar or sugar alcohol.Its example comprises lactose, maltose, mannose, sorbitol, sorbose, trehalose, xylitol, and xylose.Amount in the adding preparation is from changing about 0.01 to 100% (w/w), preferably from about 1 to 50%.This preparation of lyophilizing and grind to form required granular size then.

Then by means of surfactant with the particle suspending of suitable size in propellant.Propellant can be to be used for the arbitrary conventional substances of this purpose, chlorofluorocarbon for example, HCFC (hydrochlorofluorocarbon), hydrogen fluorohydrocarbon (hydrofluorocarbon), or Hydrocarbon, comprise trifluorochloromethane, dichlorodifluoromethane, dichloro-tetrafluoro ethanol, with 1,1,1,2-tetrafluoroethane, or its combination.Suitable surfactant comprises sorbitan trioleate and soybean lecithin.Oleic acid is useful as surfactants also.Then this mixture is loaded in the doser.Be applicable to and of the present inventionly commercially availablely indicate that the example of the inhaler of dosage is by Glaxo Inc., Research Triangle Park, the Ventolin that N.C produces indicates the inhaler of dosage.

This interferon formulations that is used for powder inhalator comprises the meticulous dried powder that separates that contains conjugate and also can comprise extender, lactose for example, sorbitol, sucrose, or mannitol, its content helps powder to scatter from this device, for example accounts for 50% to 90% of preparation weight.Particles of powder should have the pulmonary air dynamics.Generally correspond to granule, preferably between 0.5 to 5 micron, most preferably between 1.5 to 3.5 microns with the about 1g/cm2 density that is no more than 10 microns mid-diameters.

An example according to the suitable powder inhalator of the instruction of this paper is by Fisons Corp., Bedford, the Spinhaler powder inhalator that Mass produces.

The powder that is used for these devices can pass through US 5997848, and US 5993783, and US 5985248, and US 5976574, and US 5922354, and disclosed method produces and/or administration among US 5785049 and the US 55654007.

The machinery that is used for dispersion administration of the present invention

The Pharmaceutical composition that contains interferon polypeptides can be by being designed for the various machinery administrations of the lung administration for the treatment of product, this device includes, but are not limited to aerosol apparatus, indicates the inhaler of dosage, and powder inhalator, all these those skilled in the art are afamiliar with.

Some object lessons that are applicable to the commercially available device that the present invention puts into practice are by Mallinckrodt, Inc., St.Louis, the Ultravent aerosol apparatus that Missouri produces; By MarquestMedical Products, Englewood, the Acorn II aerosol apparatus that Colorado produces; By Glaxo Inc., Research Triangle Park, the Ventolin that North Carolina produces indicates the inhaler of dosage; By Fisons Corp., Bedford, the Spinhaler powder inhalator that Massachusetts produces; InhaleTherapeutic Systems, Inc., San Carlos, " static mist (standing cloud) " device of California; Alkermes, Cambridge, the AIR inhaler that Massachusetts produces; With by AradigmCorporation, the AERx lung drug-supplying system that Hayward, Califomia produce.

The present invention also provides the major product that contains present composition container.This container can be the container that is suitable for any kind of described compositions, for example the container of being made by rustless steel or glass.This container can be a syringe, for example Yu Zhuan syringe.

In addition, the invention provides and be used for test kit and the operation instruction of parenteral administration according to liquid interferon compositions of the present invention.This test kit can comprise Inteferon compositions as unique medical active composition, perhaps can be included in one or more other medical active compositions in same container or the different vessels.

On the other hand, the present invention relates to increase the method for the stability of the interferon polypeptides that is mixed with Pharmaceutical composition, described method is included in and mixes sulfoalkyl ether cyclodextrin derivant and a kind of buffer agent in the described compositions.

When interferon polypeptides lay up period show that aggregation forms and be enough to reduce amount that the interferon polypeptides aggregation forms when mixing the sulfoalkyl ether cyclodextrin derivant this method suitable especially.This interferon polypeptides arbitrary polypeptide preferably as herein described.The sulfoalkyl ether cyclodextrin derivant is preferably at US5, and 376,645, US 5,874, and 418 or US 5,134, arbitrary derivant described in 127, the particularly salt of beta cyclodextrin sulfo group butyl ether for example can be used as the sodium salt that Captisol  obtains.

On the other hand, the present invention relates to mammal is carried out the method for interferon therapy, this method comprise effective dose on the administering therapeutic according to compositions of the present invention.In addition, the present invention relates to compositions of the present invention treatment in the disease purposes and relate to the purposes of compositions of the present invention in the medicine of production for treating disease.Clearly when compositions of the present invention was used as the medicine of treatment disease or disorder, it was a kind of Pharmaceutical composition.

Pharmaceutical composition of the present invention can combine administration with other therapeutic agent.The part that these therapeutic agents can be used as same Pharmaceutical composition mix or can with this interferon polypeptides simultaneously or according to any other acceptable treatment plan administration respectively.In addition, Pharmaceutical composition of the present invention can be used as the auxiliary of other therapies.

Should understand that medicable disease depends on the type of the interferon polypeptides that exists in the compositions.

When interferon polypeptides is an interferon-ALPHA, or interferon beta, or when its variant or conjugate, the invention provides the viral infection that is used for the treatment of most of types, cancer or tumor or tumor vessel form, Chrohn ' s disease, ulcerative colitis, Guillain-Barr é syndrome, glioma, primary pulmonary fibre modification, abnormal cell growth, perhaps be used for any suitable animal, preferred mammal, and particularly human immunoregulatory compositions and method.For example, compositions of the present invention can be used for treating osteosarcoma, the matrix cells cancer, ovarian cancer, neck abnormal development, neck cancer, the larynx papillomatosis, Mycophyta mycosis, glioma, acute myeloid leukemia, multiple myeloma, Hokdkin disease, melanoma, breast carcinoma, nonsmall-cell lung cancer, malignant melanoma (accessory drugs, late period, and preventive), carcinoid tumor, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, Kaposi sarcoma, chronic lymphocytic leukemia, renal cell carcinoma, recurrent bladder surface cancer, colorectal carcinoma, hairy cell leukemia, and viral infection, for example human papillomavirus, viral hepatitis, genital herpes, herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis, cytomegaloviral pneumonia, rhinovirus chronic and refractory hepatitis, chronic stadium HCV (I type), chronic stadium HCV (II type) and chronic viral hepatitis B.

At this on the one hand, said composition can be used for the CML monotherapy or combines with cytosine arabinoside, be used for B-cell lymphoma monotherapy or combine with therapy based on amycin, be used for follicular lymphoma therapy assisting as CHOP-class therapy, be used for the hepatitis C monotherapy or combine with ribavirin, be used for the multiple myeloma monotherapy or and VBMCP, BCNU or VBMCP+HiCy combination, perhaps be used for the renal carcinoma monotherapy or and vincaleucoblastine, floxuridine, 5-fluorouracil or IL-10 combination.

Specifically, polypeptide of the present invention or compositions can be used for the treatment of multiple sclerosis (MS), for example 4 types the MS of Gong Rening is (optimum, recurrence alleviates type MS (RRMS), carrying out property of essential MS (PPMS) and carrying out property of secondary type MS (SPMS)) in any and be used for monosymptom MS, cancer or tumor, hepatitis, for example hepatitis B and hepatitis C, the perhaps treatment of herpes infection (the optional treatment with IL-10 of a kind of treatment in back combines).

Therefore, the invention still further relates to the compositions that contains interferon beta polypeptides and sulfoalkyl ether cyclodextrin derivant and be used for the treatment of viral infection in preparation, cancer or tumor or tumor vessel form, Chrohn ' s disease, ulcerative colitis, Guillain-Barr é syndrome, glioma, primary pulmonary fibre modification, abnormal cell growth, perhaps be used for any suitable animal, preferred mammal, and the purposes in the particularly human immunoregulatory medicine.More particularly, the compositions of the present invention that contains interferon beta polypeptides and sulfoalkyl ether cyclodextrin derivant can be used for treating osteosarcoma, the matrix cells cancer, ovarian cancer, neck abnormal development, neck cancer, the larynx papillomatosis, Mycophyta mycosis, glioma, acute myeloid leukemia, multiple myeloma, Hokdkin disease, melanoma, breast carcinoma, nonsmall-cell lung cancer, malignant melanoma (accessory drugs, late period, and preventive), carcinoid tumor, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, Kaposi sarcoma, chronic lymphocytic leukemia, renal cell carcinoma, recurrent bladder surface cancer, colorectal carcinoma, hairy cell leukemia, and viral infection, human papillomavirus for example, viral hepatitis, genital herpes, herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis, cytomegaloviral pneumonia, rhinovirus chronic and refractory hepatitis, chronic stadium HCV (I type), chronic stadium HCV (II type), chronic viral hepatitis B, chronic or acute hepatitis C, or herpes infection, multiple sclerosis (MS), for example 4 types the MS of Gong Rening is (optimum, recurrence alleviates type MS (RRMS), former carrying out property MS (PPMS) and carrying out property of secondary MS (SPMS)) in any be used for monosymptom MS.

One concrete aspect, the compositions that the present invention relates to contain interferon beta polypeptides and sulfoalkyl ether cyclodextrin derivant is used for the treatment of in preparation and is selected from osteosarcoma, the matrix cells cancer, ovarian cancer, neck abnormal development, neck cancer, the larynx papillomatosis, the Mycophyta mycosis, glioma, acute myeloid leukemia, multiple myeloma, Hokdkin disease, melanoma, breast carcinoma, nonsmall-cell lung cancer, malignant melanoma (accessory drugs, late period, and preventive), carcinoid tumor, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, Kaposi sarcoma, chronic lymphocytic leukemia, renal cell carcinoma, recurrent bladder surface cancer, purposes in colorectal carcinoma and the hairy cell leukemia in the medicine of any cancer, wherein interferon beta polypeptides contains with respect to the wild type human interferon beta shown in the SEQ ID NO 1 and replaces C17S+Q49N+Q51T+D110F+F111N+R113T, and have 3 saccharide half point that are connected with 3 N-glycosylation sites and 1 PEG molecule (object lesson of this interferon beta polypeptides for example is, carries out the interferon beta variant of the embodiment 3 of PEGization with 20kDa-PEG) with about 20kDa molecular weight.Each above-mentioned cancer is thought one embodiment of the invention.This cancer is a melanoma in a preferred embodiment.In another embodiment preferred, this cancer is a breast carcinoma.In another embodiment preferred, this cancer is a nonsmall-cell lung cancer.In another embodiment preferred, this cancer is a malignant melanoma.

Therefore, the invention still further relates to the compositions that contains interferon-ALPHA polypeptide and sulfoalkyl ether cyclodextrin derivant and be used for the treatment of viral infection in preparation, cancer or tumor or tumor vessel form, Chrohn ' s disease, ulcerative colitis, Guillain-Barr é syndrome, glioma, primary pulmonary fibre modification, abnormal cell growth, perhaps be used for any suitable animal, preferred mammal, and the purposes in the particularly human immunoregulatory medicine.More particularly, the compositions of the present invention that contains interferon-ALPHA polypeptide and sulfoalkyl ether cyclodextrin derivant can be used for treating osteosarcoma, the matrix cells cancer, ovarian cancer, neck abnormal development, neck cancer, the larynx papillomatosis, Mycophyta mycosis, glioma, acute myeloid leukemia, multiple myeloma, Hokdkin disease, melanoma, breast carcinoma, nonsmall-cell lung cancer, malignant melanoma (accessory drugs, late period, and preventive), carcinoid tumor, B-cell lymphoma, T-cell lymphoma, follicular lymphoma, Kaposi sarcoma, chronic lymphocytic leukemia, renal cell carcinoma, recurrent bladder surface cancer, colorectal carcinoma, hairy cell leukemia, and viral infection, human papillomavirus for example, viral hepatitis, genital herpes, herpes zoster, herpetic keratitis, herpes simplex, viral encephalitis, cytomegaloviral pneumonia, rhinovirus chronic and refractory hepatitis, chronic stadium HCV (I type), chronic stadium HCV (II type), chronic viral hepatitis B, chronic or acute hepatitis C, or herpes infection, multiple sclerosis (MS), for example 4 types the MS of Gong Rening is (optimum, recurrence alleviates type MS (RRMS), former carrying out property MS (PPMS) and carrying out property of secondary MS (SPMS)) in any be used for monosymptom MS.

In addition, when said composition contains interferon beta polypeptides, the present invention relates to the mammiferous method of a kind of treatment, this animal has anti-such as Avonex ^TMOr Rebif ^Interferon beta 1a or such as Betaseron ^The circulating antibody of 1b, this method comprises uses that the reaction that contains with described antibody reduces or the compositions of the present invention of nonreactive interferon beta polypeptides.This chemical compound is with the effective dose administration.This mammal is preferably human.Need the mammal of treatment can suffer from above listed arbitrary disease that interferon beta is useful therapeutic agent.Specifically, this aspect of the present invention is for treatment multiple sclerosis (above listed arbitrary type), and hepatitis or cancer are favourable.Term " circulating antibody " is attempted to be illustrated in the mammal with (Rebif, Betaseron Avonex) treat the antibody, particularly neutralizing antibody that reacts and form with arbitrary commercially available interferon beta goods.

When interferon polypeptides was interferon gamma or its variant or conjugate, compositions of the present invention can be used for treating arbitrary medical science indication of describing among the WO01/36001, particularly between matter lung disease, the most specifically primary pulmonary fibre modification.Existing suggestion is used for the treatment of a matter lung disease (being also referred to as a matter pnemnofibrosis (IPF)) ((N.Engl.J.Med.341:1264-1269 such as Ziesche with interferon gamma, 1999 and Chest110:Suppl:25S, 1996) and EP 795332), for this purpose, interferon gamma can be used in combination with meticortelone.Except IPF, granuloma disease (Bolinger etc., Clinical Pharmacy, 1992,11:834-850), some mycobacterial infections (N.Engl.J.Med.330:1348-1355,1994), renal carcinoma (J.Urol.152:841-845,1994), osteopetrosis (N.Engl.J.Med.332:1594-1599,1995), scleroderma (J.Rheumatol.23:654-658,1996), hepatitis B (Hepatogastroenterology 45:2282-2294,1998), hepatitis C (Int.Hepatol.Communic.6:264-273,1997), the plain γ treatment of septic shock (Nature Medicine 3:678-681,1997) and rheumatoid arthritis available interference.

Therefore, the invention still further relates to the compositions that contains interferon gamma polypeptide and sulfoalkyl ether cyclodextrin derivant and be used for the treatment of matter lung disease, the most specifically a primary pulmonary fibre modification in preparation, between matter lung disease (being also referred to as a matter pnemnofibrosis (IPF)), the granuloma disease, mycobacterial infections, renal carcinoma, osteopetrosis, scleroderma, hepatitis B, hepatitis C, purposes in the medicine of septic shock and rheumatoid arthritis.

Embodiment

The material and the method for preparation interferon gamma

Material

CHO-K1 cell (can obtain (ATCC#CCL-61)) from American type culture collection.

HeLa cell (can obtain (ATCC#CCL-2)) from American type culture collection.

ISRE-Luc is from Stratagene, and La Jolla USA obtains.

PCDNA 3.1/hygro is from Invitrogen, and Carlsbad USA obtains.

Restriction Enzyme and polymerase can be from New England Biolabs Inc., Beverly, and USA obtains.

The DMEM culture medium: the Eagle culture medium (DMEM) of Dulbecco ' s improvement, 10% hyclone and HYG can be from Life Technologies A/S, Copenhagen, Denmark obtains.

The LucLite substrate can be from Packard Bioscience, Groningen, and The Netherlands obtains.

The TopCount luminometer can be from Packard Bioscience, Groningen, and The Netherlands obtains.

The anti-human interferon gamma antibodies of biotin labeled polyclone BAF285 can be from R; D Systems Inc., Minneapolis, USA obtains.

The bonded Succ-PEG-DSPE P0397 of horseradish peroxidase can be from DAKO, Copenhagen, and Denmark obtains.

TMB trace reagent can be from KEM-EN-TEC, Copenhagen, and Denmark obtains.

Method

Interferon algoscopy summary

The interferon gamma receptor that disclosed in the past on interferon gamma and the HeLa cell interacts and the activation this receptor.Thereby, contain interferon irritant reaction element (ISRE) but promoter on activated transcription.Therefore to screen the agonist of interferon receptors be possible to the ISRE (ISRE-luc) that is positioned over the coupling luciferase reporter gene in the HeLa cell by use.

The one-level algoscopy

With ISRE-Luc and pCDNA 3.1/hygro cotransfection HeLa cell, by in containing the DMEM culture medium of HYG, selecting to produce transforming focus (cell clone).The luciferase activity of screening cell clone under the condition that has or do not exist interferon gamma.Those clones that show the ceiling rate of the luciferase activity that stimulates and do not stimulate are used for further test.

In order to screen polypeptide, in 96 hole culture plates, inoculate 15,000 cells/well and overnight incubation in the DMEM culture medium.In cell, added polypeptide and known standard with various concentration in second day.Dull and stereotyped under 37 ℃ at 5%CO ₂Air in cultivated 6 hours, in each hole, add LucLite substrate (Packard Bioscience, Groningen, The Netherlands) subsequently.Seal plate is also upward measured luminous in SPC (single photon counting) mode at TopCount luminometer (Packard).

Each flat board contains the hole of useful interferon gamma cultivation as stimulating contrast, and other hole of containing normal culture medium contrasts as stimulating.Ratio between the luciferase activity that stimulate and that do not stimulate is as the internal standard of error between interferon gamma activity and experiment and experiment.

Identify the amino acid residue that the surface exposes

Structure

The experiment three dimensional structure of the human interferon gamma of measuring by the X-radiocrystallography is by Ealick etc., Science 252:698-702 (1991) report, and they have reported the C-α vestige of interferon gamma homodimer.Walter etc., Nature 376:230-235 (1995) has reported the structure with the compound interferon gamma homodimer of bimolecular interferon gamma receptor soluble form.Yet the coordinate of this structure can not openly obtain.Thiel etc., the structure of Structure 8:927-936 (2000) report and the compound interferon gamma homodimer of bimolecular interferon gamma receptor soluble form has structurally and with the interferon gamma homodimer interactional the 3rd acceptor molecule does not take place.

Accessible surface area (ASA)

The program that uses a computer Access (B.Lee and F.M.Richards, J.Mol.Biol.55:379-400 (1971)) version 2 (Copyright (c) 1983 Yale University) calculates the accessible surface area (ASA) of each atom in structure.This method is general to be used the probe of 1.4 sizes and accessible surface area (ASA) is defined as the area that probe central authorities form.Before the calculating, from set of coordinates (coordinate set), remove all hydrones, hydrogen atom and other atom not directly related with this protein.

The part A SA of side chain

Come the part A SA of calculation side chain atom divided by the summation of atom ASA in this side chain by the value of this residue type side chain atom ASA in the ALA-x-ALA tripeptides that extends with representative.Referring to Hubbard, Campbell ﹠amp; Thomton (1991) J.Mol.Biol.220,507-530.For example, the CA atom is thought the pendant moiety of glycine residue rather than remaining residue.Following table is used as the standard 100%ASA of side chain:

Ala?????69.23 ²????????????Leu?????140.76 ²

Arg?????200.35 ²???????????Lys?????162.50 ²

Asn?????106.25 ²???????????Met?????156.08 ²

Asp?????102.06 ²???????????Phe?????163.90 ²

Cys?????96.69 ²????????????Pro?????119.65 ²

Gln?????140.58 ²???????????Ser?????78.16 ²

Glu?????134.61 ²???????????Thr?????101.67 ²

Gly?????32.28 ²????????????Trp?????210.89 ²

His?????147.00 ²???????????Tyr?????176.61 ²

Ile?????137.91 ²???????????Val?????114.14 ²

The residue that does not detect in structure is defined as has 100% exposure, because think that they are present in the elastic region.

Measure interatomic distance:

Use the molecule graphics software, routine InsightII v.98.0, MSI INC measures interatomic distance.

Measure receptor binding site:

Receptor binding site is defined as all residues that its accessible surface area changes when comprising receptors bind.It can be measured by at least two kinds of ASA algorithms; A kind of at the isolating ligands in the ligand/receptor complex, a kind of at complete ligand/receptor complex.

The aminoacid that embodiment A-the mensuration surface exposes

The X-ray structure that uses belongs to Thiel etc., have structurally not and the interferon gamma homodimer of Structure 8:927-936 (2000) report interactional the 3rd interferon gamma receptor molecule take place with the compound interferon gamma homodimer of bimolecular interferon gamma receptor soluble form.This structure is made up of the interferon gamma homodimer, and wherein two molecular markers are A and B.In order to make up purpose, before the interferon gamma sequence, exist another methionine of being labeled as M0 and this sequence 10 residues of the terminal truncate of C-(Q133 is last residue in the polypeptide that is making up).In all result of calculations of this embodiment, from structure, remove M0.Two monomeric structures of interferon gamma have extremely weak electron density and only are models up to the residue of residue T126 after residue 120.Therefore, from structure, remove residue S121-T126 before the calculating in the present embodiment.The 3rd acceptor molecule and the interferon gamma homodimer that are labeled as two the receptor segments of C and D and interferon gamma homodimer generation direct interaction and are labeled as E do not come in contact and are not included in these result of calculations.

The surface exposes:

The homodimer of the molecule A that do not comprise M0 and S121-T126 in two molecules and B is carried out part A SA to calculate following residue and has and surpass its side chain of 25% and be exposed at least one monomeric surface: Q1, D2, P3, K6, E9, N10, K12, K13, Y14, N16, G18, H19, S20, D21, A23, D24, N25, G26, T27, G31, K34, N35, K37, E38, E39, S40, K55, K58, N59, K61, D62, D63, Q64, S65, Q67, K68, E71, T72, K74, E75, N78, V79, K80, N83, S84, N85, K86, K87, D90, E93, K94, N97, S99, T101, D102, L103, N104, H111, Q115, A118 and E119.

Its side chain that following residue has above 50% is exposed at least one monomeric surface: Q1, D2, P3, K6, E9, N10, K13, N16, G18, H19, S20, D21, A23, D24, N25, G26, T27, G31, K34, K37, E38, E39, K55, K58, N59, D62, Q64, S65, K68, E71, E75, N83, S84, K86, K87, K94, N97, S99, T101, D102, L103, N104, Q115, A118, E119.

To not comprising M0 and S121-T126 in two molecules and comprising the molecule A of acceptor molecule C and D and the homodimer of B carries out part A SA and calculates following residue and have and surpass its side chain of 25% and be exposed at least one monomeric surface: Q1, D2, P3, K6, E9, N10, K13, Y14, N16, G18, H19, D21, N25, G26, G31, K34, N35, K37, E38, E39, S40, K55, K58, N59, K61, D62, D63, Q64, S65, Q67, K68, E71, T72, K74, E75, N78, V79, K80, N83, S84, N85, K86, K87, D90, E93, K94, N97, S99, T101, D102, L103, N104, E119.

Its side chain that following residue has above 50% is exposed at least one monomeric surface: P3, K6, N10, K13, N16, D21, N25, G26, G31, K34, K37, E38, E39, K55, K58, N59, D62, Q64, S65, K68, E71, E75, N83, S84, K86, K87, K94, N97, S99, T101, D102, L103 and N104.

The result who lists for twice relatively lists and has removed K12, S20, A23 the result from surpassing 25% side chain ASA when drawing receptors bind, D24, T27, H111, Q115 and A118, and list and removed Q1, D2 the result from surpassing 50% side chain ASA during receptors bind, E9, G18, H19, S20, A23, D24, T27, Q115, A118 and E119.

Undeterminate residue exposes processing fully by the surface in this structure, i.e. residue S121, P122, A123, A124, K125, T126, G127, K128, R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140, A141, S142, Q143.These residues also are configured for importing the independent target (perhaps can think and for example, have the group that belongs to the surperficial amino acid residue that exposes above 25% or above 50% side chain that exposes) of linking group.

Embodiment B-mensuration receptor binding site

Calculate the following residue of interferon gamma polypeptide so that isolating dimeric result of calculation is compared the ASA:Q1 that has reduction at least one monomer of complex, D2, Y4, V5, E9 by the above-mentioned ASA that carries out, K12, G18, H19, S20, D21, V22, A23, D24, N25, G26, T27, L30, K34, K37, K108, H111, E112, I114, Q115, A118, E119.

Embodiment C-be designed for is expressed having of interferon gamma and is optimized the codon that is used for Chinese hamster ovary celI The expressed sequence box of selecting

Modification comprises the DNA sequence of full-length cDNA that coding has the ripe human interferon gamma of its natural signals peptide, and promptly GenBank accession number X13274 expresses at the Chinese hamster ovary celI camber so that help.Select to be partial to the codon that human codon commonly used is modified the human interferon gamma nucleotide sequence by being formed on codon.Subsequently, be substituted in some nucleotide in this sequence so that import the recognition site of DNA restriction endonuclease with other nucleotide.The design primer is so that synthetic this gene.

Use Platinum Pfx-polymerase kit (Life Technologies) and three step of standard PCR loop parameter primer to be assembled into synthetic gene by a step PCR.Use identical condition to have the sequence shown in the SEQ ID NO:5 by gene and this gene of this assembling of pcr amplification.Synthetic gene is cloned between the BamHI and the 3 ' XbaI that holds that into pcDNA3.1/hygro (InVitrogen) 5 ' holds, produce pIGY-22.

Embodiment D-direct mutagenesis

Produce the glycosylation variant

In order in interferon gamma, to import sudden change, design oligonucleotides by this way, i.e. the change that produces of PCR-can import in the expression plasmid (pIGY-22) by traditional two-step pcr.

Use two carrier primers and specific mutations primer: ADJ013:5 '-GATGGCTGGCAACTAGAAG-3 ' (SEQ ID NO:6), (antisense downstream carrier primer) and ADJ014:5 '-TGTACGGTGGGAGGTCTAT-3 ' (SEQ ID NO:7), (adopted upstream carrier primer is arranged).

Use ADJ013 and ADJ014 as the carrier primer, ADJ093 (5 '-GTTCAGGTCTGTCACGGTGTAATTGGTCAGCTT-3 ') (SEQ ID NO:8), and ADJ094 (5 '-AAGCTGACCAATTACACCGT GACAGACCTGAAC-3 ') (SEQID NO:9) produces S99T variant as template by traditional two-step pcr as mutant primer and pIGY-22.Use BamHI and XbaI that 447 bp PCR product sub-clones are advanced pcDNA3.1/Hygro (InVitrogen), produce plasmid pIGY-48.

By using Lipofectaim2000 (Life Technologies) CHO K1 cell to be advanced in the pIGY-48 transfection as transfection agents.Gather in the crops culture medium after 24 hours and measure the interferon gamma activity.Use one-level algoscopy as herein described, obtained following activity: 5.1 * 10 ⁶AU/ml.

Use ADJ013 and ADJ014 as the carrier primer, ADJ091 (5 '-CATGATCTTCCGATCGGTCTCGTTCTTCCAATT-3 ') (SEQ ID NO:10), and ADJ092 (5 '-AATTGGAAGAACGAGACCGATCGG AAGATCATG-3 ') (SEQID NO:11), as mutant primer, and pIGY-48 produces the E38N+S40T+S99T variant as template by traditional two-step pcr.Use BamHI and xbaI that 447 bp PCR product sub-clones are advanced pcDNA3.1/Hygro (InVitrogen), produce plasmid pIGY-54.

By using Lipofectaim2000 (Life Technologies) CHO K1 cell to be advanced in the pIGY-54 transfection as transfection agents.Gather in the crops culture medium after 24 hours and measure the interferon gamma activity.Use one-level algoscopy as herein described, obtained 1.3 * 10 ⁷The activity of AU/ml.

Use above-mentioned similar standard technique, prepared many total length interferon gamma glycosylation variants.

Produce the interferon gamma variant of the terminal truncate of C-

Use pIGY-22, pIGY-48 and pIGY-54 make template and are created in the interferon gamma variant that contains the terminal truncate of C-of a termination codon near the downstream of Leu135 codon by a step PCR, then use BamHI and xbaI that PCR product sub-clone is advanced pcDNA3.1/Hygro (InVitrogen).The primer that is used to make up these variants is: ADJ014 (seeing above the upstream) and: 5 ' GAGTCTAGATTACAGC ATCTGGCTTCTCTT-3 ' (downstream).The gained plasmid is called pIGY-72 (the wild type interferon gamma of truncate behind the Leu135), pIGY-73 (the S99T variant of truncate behind the Leu135) and pIGY-74 (E38N+S40T+S99T of truncate behind the Leu135).

Generation contains the interferon gamma variant of cysteine

Use Stratagene ' s QuikChange ^TMXL direct mutagenesis test kit produces the interferon gamma variant that contains cysteine residues according to manufacturers instruction.Use pIGY-48 to make 7 interferon gamma variant: N10C+S99T, N16C+S99T, E38C+S99T, N59C+S99T, N83C+S99T, K94C+S99T and S99T+N104C that template produces the cysteine that respectively contains an importing.Equally, use pIGY-54 to make 6 interferon gamma variant: N10C+E38N+S40T+S99T that template produces the cysteine that respectively contains an importing, N16C+E38N+S40T+S99T, E38N+S40T+N59C+S99T, E38N+S40T+N83C+S99T, E38N+S40T+K94C+S99T and E38N+S40T+S99T+N104C.

Embodiment E-PEGization contains the variant of cysteine

The deoxidation before use of all buffer.Protein concentration is estimated by measuring A280.

Use OPSS coupling chemical agent carry out PEGization

7.2ml at the 5mM sodium succinate, 4% mannitol, 0.01%Tween 20, and the interferon gamma variant N16C+S99T (total length) of the 1.3mg/ml among the pH6.0 by reducing with incubation under the 300 μ l 0.5M DTT room temperatures in 30 minutes.Upward (pH8.1) the middle post of crossing is to the desalination of interferon gamma variant for 50mM sodium phosphate, 1mM EDTA in buffer A at NAP25 solvent resistant column (Pharmacia) by 3 aliquots with 2.5ml.Each aliquot eluting in 3.5ml.

MPEG-OPSS (10KDa) is dissolved in the concentration that reaches 2mg/ml in the buffer A and also at room temperature shook incubation gently 60 minutes in the interferon gamma variant that adds reduction and desalination with equal-volume.

Use Vivaspin20 post (VivaScience) that the reactant mixture of 11ml is concentrated to 1-6ml and use and remove remaining mPEG by gel filtration with the equilibrated Sephacryl S-100 of buffer A post (Pharmacia).

Use Vivaspin 6 posts (VivaScience) that the 5mM sodium succinate is advanced in the interferon gamma variant diafiltration of PEGization, 4% mannitol is among the pH6.0 and add Tween 20 to 0.01%.The interferon gamma variant of the PEGization of purification is measured as in one-level algoscopy as herein described has 1.3 * 10 ⁶The ratio work (the interferon gamma variant of corresponding not PEGization is than 15% of work) of AU/mg.

Use MAL coupling chemical agent carry out PEGization

1.6ml at the 5mM sodium succinate, 4% mannitol, 0.01%Tween 20, and the interferon gamma variant N59C+S99T (total length) of the 1.5mg/ml among the pH6.0 by reducing with incubation under the 64 μ l 0.5M DTT room temperatures in 30 minutes.NAP25 solvent resistant column (Pharmacia) go up buffer A (the 50mM sodium phosphate, 1mM EDTA, pH8.1) in to the desalination of interferon gamma variant.Interferon gamma variant eluting in 3.5ml.

MPEG-MAL (5kDa) is dissolved in the concentration that reaches 0.5mg/ml in the buffer A and also at room temperature shook incubation gently 120 minutes in the interferon gamma variant that adds reduction and desalination with equal-volume.

Add ammonium sulfate to the concentration of 0.9M and to the interferon gamma variant use of PEGization buffer B (the 20mM sodium phosphate, 0.9M ammonium sulfate, pH6.6) in equilibrated 1ml Resource ^TMPhenyl post (Pharmacia).(buffer B with 5 times of column volumes before the 20mM sodium phosphate, the bonded PEGization interferon gamma of line style gradient elution variant pH6.6) is washed pillar with from 0 to 50% buffer C in 30 times of column volumes.The interferon gamma variant of PEGization is at about 0.6M ammonium sulfate place eluting.

Merge the fraction of the interferon gamma variant contain PEGization and use Vivaspin 6 posts (VivaScience) diafiltration to advance the 5mM sodium succinate, 4% mannitol is among the pH6.0 and add Tween 20 to 0.01%.The interferon gamma variant of the PEGization of purification is measured as in one-level algoscopy as herein described has 2.4 * 10 ⁶The ratio work (the interferon gamma variant of corresponding not PEGization is than 15% of work) of AU/mg.

Embodiment F is expressed interferon gamma polypeptide in mammalian cell

For the transient expression interferon gamma, with cell at the culture medium (Dulbecco ' s MEM/Nut.-mix F-12 (Ham) L-glutamine that contains 1: 10 hyclone (BioWhittakerCat#02-701F) and 1: 100 penicillin and streptomycin (BioWhittaker Cat#17-602E), 15mM Hepes, pyridoxol-HCl (Life Technologies Cat#31330-038)) growing to 95% in converges.Use Lipofectamine2000 (Life Technologies) plasmid transfection of the plain γ of coded interference to be advanced cell according to manufacturers instruction.After the transfection 24 hours, collect culture medium and measure the interferon gamma activity.In addition, for the relative number of the quantitative glycosylation site that utilizes, use the culture medium of results to carry out the Western trace.

Produce the stable clone of expressing interferon gamma by then in the culture medium that contains the 0.36mg/ml hygromycin, cultivating this cell with the plasmid transfection CHO K1 cell of the plain γ of coded interference.The separating stable cells transfected is also passed through the limiting dilution sub-clone.Identify the clone who produces the plain γ of high levels of interference by ELISA.

Embodiment G-large-scale production

The stabilized cell of expressing interferon gamma or variant ties up to 1700cm2 and shakes bottle (Coming, Dulbecco ' s MEM/Nut.-mix F-12 (Ham) L-glutamine #431200), 15mM Hepes, pyridoxol-HCl (Life Technologies Cat#31330-038), 1: 10 hyclone (BioWhittaker Cat#02-701F) grows in 1: 100 penicillin and the streptomycin (BioWhittaker Cat#17-602E) and converges.Then culture medium is replaced with and contains L-glutamine (BioWhittaker Cat #12-724Q) and added 1: 500 EX-CYTE VLE (Serological Proteins Inc.#81-129) and the 300ml UltraCHO of 1: 100 penicillin and streptomycin (BioWhittaker Cat #17-602E).Grow after 48 hours, change culture medium with the fresh UltraCHO that contains identical additive.After the regrowth 48 hours, with having added 1: 100 ITS-A (Gibco/BRL#51300-044), Dulbecco ' s MEM/Nut.-mix F-12 (Ham) the L-glutamine of 1: 500 EX-CYTE VLE (SerologicalProteins Inc.#81-129) and 1: 100 penicillin and streptomycin (BioWhittaker Cat#17-602E), pyridoxol-HCl (LifeTechnologies Cat#21041-025) changes culture medium.Subsequently, every 24 hours, the results culture medium also used the 300ml serum-free medium that contains identical additive to change.The culture medium of collecting is filtered to remove cell by 0.22 μ m filter.

Embodiment H-purification

Use the ultrafiltration of Millipore TFF system to preceding microporous filter (the 0.22 μ m) filtrate of about 1/15 volume.In same system, use 10mM Tris, this concentrate of pH7.6 diafiltration.Add the concentration of ammonium sulfate, stir the back and use the GS3 rotor in the Sorvall centrifuge, to remove precipitation in centrifugal 25 minutes with 8000rpm to 1.7M.

To in advance at 10mM Tris, 1.7M ammonium sulfate is among the pH7.6 on equilibrated 25mlPhenyl High Performance (Pharmacia) post with sample on the supernatant.Behind the last sample with the 10mM Tris of 3 times of column volumes, 1.7M ammonium sulfate, pH7.6 washs pillar, then to reach 100%10mMTris in 10 times of column volumes, the interferon gamma variant of the linear gradient elution of bound of pH7.6.The interferon gamma variant of fractionated effluent and eluting.Merging is rich in the fraction of interferon gamma variant and is used the molecular weight cutoff value is 10, and 10mM Tris advances by diafiltration in the Vivaflow200 system (VivaScience) of 000Da, exchange buffering liquid among the pH9.0.

Then with sample on the interferon gamma variant in advance at 10mM Tris, among the pH9.0 on equilibrated 18mlQ-sepharose Fast Flow (Pharmacia) post.Behind the last sample in 15 times of column volumes with 10mM Tris from 0-100%, 0.5M NaCl, with the 10mM Tris of 3 times of column volumes, pH9.0 washs pillar before the bonded interferon gamma variant of the gradient elution of pH9.0.The interferon gamma variant of fractionated effluent and eluting.Merging is rich in the fraction of interferon gamma variant and is used the molecular weight cutoff value is 10, and the Vivaspin20 of 000Da (VivaScience) post becomes 10mM sodium phosphate, pH7.0 by diafiltration with buffer exchange.

Then, with sample on the interferon gamma variant at the 10mM sodium phosphate, among the pH7.0 on the 8ml CHT of the pre-balance pottery hydroxyapatite column (Biorad).Behind the last sample in 30 times of column volumes with from the 0-60%500mM sodium phosphate, with the 10mM sodium phosphate of 5 times of column volumes, pH7.0 washs pillar before the bonded interferon gamma variant of the gradient elution of pH7.0.The interferon gamma variant of fractionated effluent and eluting.Merge and be rich in the fraction of interferon gamma variant and use Vivaspin20 post (VivaScience) that buffer exchange is become the 5mM sodium succinate, 4% mannitol, pH6.0 also add the concentration of Tween 20 to 0.01% subsequently.Aseptic filtration interferon gamma variant also is stored in-80 ℃.

In addition, the interferon gamma variant can carry out purification according to following purification scheme:

Use the ultrafiltration of Millipore TFF system to preceding microporous filter (the 0.22 μ m) filtrate of about 1/15 volume.Use 10mM Tris in same system, this concentrate of pH7.6 diafiltration is transferred to pH 9.0 and remove precipitation by microporous filter afterwards.

With sample on this sample at 10mM Tris, among the pH9.0 on Q-sepharose FastFlow (Pharmacia) post of pre-balance.Behind the last sample in 15 times of column volumes with 10mM Tris from 0-100%, 0.5M NaCl, with the 10mM Tris of 3 times of column volumes, pH9.0 washs pillar before the bonded interferon gamma variant of the gradient elution of pH9.0.The interferon gamma variant of fractionated effluent and eluting.Merge the fraction that is rich in the interferon gamma variant, and with pH regulator to 7.6.Add ammonium sulfate to 1.5M and after stirring by the centrifugal precipitation of removing.

Then with sample on the interferon gamma variant in advance at 10mM Tris, 1.5M ammonium sulfate is among the pH7.6 on the equilibrated phenyl sepharose high-efficiency column (Pharmacia).

Behind the last sample with the 10mM Tris of 3 times of column volumes, 1.5M ammonium sulfate, pH7.6 washs pillar, then to reach 100%10mM Tris in 10 times of column volumes, the interferon gamma variant of the linear gradient elution of bound of pH7.6.The interferon gamma variant of fractionated effluent and eluting.Merge and be rich in the fraction of interferon gamma variant and ammonium sulfate is adjusted to 1.7M.

Then with sample on the interferon gamma variant at the 10mM sodium phosphate, 1.7M ammonium sulfate is among the pH7.6 on the butyl-agarose post of pre-balance.Use the 10mM sodium phosphate behind the last sample, using the 10mM sodium phosphate before the interferon gamma variant of elution of bound in the step of pH6.5,1.7M ammonium sulfate, pH7.6 washs pillar.The interferon gamma variant of fractionated effluent and eluting.

Merge the fraction be rich in the interferon gamma variant then and go up sample at the 10mM sodium phosphate, among the pH6.5 on the hydroxyapatite column of pre-balance.Behind the last sample in 30 times of column volumes with from the 0-100%500mM sodium phosphate, with the 10mM sodium phosphate of 5 times of column volumes, pH6.5 washs pillar before the interferon gamma variant of the linear gradient elution of bound of pH6.5.The interferon gamma variant of fractionated effluent and eluting.

Merging contains the fraction of interferon gamma variant and buffer exchange is become to contain the 5mM sodium succinate, 4% mannitol, the buffer of pH6.0.The concentration that adds Tween 20 to 0.01% subsequently.Aseptic filtration interferon gamma variant also is stored in-80 ℃.

The preparation of interferon polypeptides

Embodiment 1

The preparation of interferon beta (IFN-/3) variant Q49N+Q51T+F111N+R113T

This variant is pressed the embodiment 7 of WO01/15736 and 8 described structures and expression and with following two-step method purification:

Centrifugal from shaking bottle culture medium of results and filtering by 0.22um filter membrane (PVDF).Filtering culture medium is being equipped with in Vivaflow 200 systems that cutoff value is 10000 polyether sulfone (PES) film diafiltration and is going up sample to using the 50mM sodium acetate, and 50mM sodium chloride is on the equilibrated S-Sepharose post of pH5.5 (Pharmacia).Be attached to the interferon variant 50mM sodium acetate on the pillar, 0.5M sodium chloride, pH5.5 eluting.Sodium chloride concentration in the eluate of S-Sepharose post is adjusted to 1.0M and with sample on the sample to using the 50mM sodium acetate, 1.0M sodium chloride is on the equilibrated phenyl of pH5.5-agarose high-efficiency column (Pharmacia).Use Milli Q water washing pillar behind the last sample.The interferon beta variant is used from Milli Q water to 60% ethylene glycol, the 50mM sodium acetate, and the gradient of pH5.5 is with 30 times of column volume eluting.Collection contains the fraction of complete glycosylated interferon beta variant.In being equipped with the Vivaspin 20ml concentrator that cutoff value is 10000 PES film, the buffer exchange in the goods is become 50mM sodium phosphate, pH7.0.

The IFN-β variant of purification is mixed with contains that initial concentration is the variant of 10MIU/ml and contains 35mg/ml mannitol and the compositions of 2mg/mlTween 80 in 50mM sodium phosphate buffer (being adjusted to pH7.0 at last).A kind of compositions (compositions A) is not added Captisol  (can obtain from CyDex Inc.).Another compositions (compositions B) contains 10mg/ml Captisol .

Compositions was stored 18 days down at-80 ℃ and 35 ℃ respectively with the aliquot (without nitrogen or argon purification) in 0.5ml Eppendorf pipe of 50 μ l.Use the antivirus test described in the WO01/15736 to measure antiviral activity.

The result is expressed as " in the average percentage activity of 35 ℃ of samples of storing down, as the active function (analyzing on the same day) of the sample of storing down at-80 ℃ ".

Store natural law " 35 ℃ with-80 ℃ under active % "

Compositions

A?????????B

10??????????????????24????????144

18??????????????????27????????78

Embodiment 2

The differential scanning calorimetry that contains the preparation of IFN-β variant Q49N+Q51T+F111+R113T

The proteinic sample of analyzing embodiment 1 by differential scanning calorimetry (DSC) is proteinicly separated folding (or degeneration) and particularly is determined at the proteinic folding temperature (Tm) of separating in each operation to study.

The parent material that is used for dsc analysis is the protein solution of 50mM sodium acetate buffer (being adjusted to pH5.5 at last).Prepared a series of solution with the final protein concentration that adds various excipient generation 0.4mg/mL.Use the blank of injection water, because focus only is to measure the variation of Tm value as DSC.Before carrying out dsc analysis, it is described by using the vacuum time enough to give all solution degasification to press MicroCal Inc..

By using this proteinic behavior of DSC device (VP-DSC type) assessment of MicroCal Inc.The temperature of described solution is increased to about 120 ℃ with the speed of 1.5 ℃ of per minutes gradually from ambient temperature (25 ℃).Along with incident takes place twice in the increase of temperature.Incident is to separate folding reaction (heat absorption) for the first time, and observes peak value upwards in scanning.Incident is precipitation (exothermic reaction) for the second time, and observes downward peak value in scanning.

The Tween 80 that in starting soln, adds 2.4mg/ml; 5mg/ml sodium chloride or 40mg/mlCaptisol  cause the Tm-value of Δ Tm to change, and are respectively :-0.7; + 1.2 or+7.2 ℃.Wherein Δ Tm is defined as:

ΔTm＝(Tm ₂-Tm ₁)

" Tm wherein ₁" relate to the DSC scanning and the " Tm of the starting soln that does not add other excipient ₂" relate to and added 2.4mg/ml Tween 80; The difference DSC scanning of the solution of 5mg/ml sodium chloride or 40mg/ml Captisol .

These data clearly prove the protein that adds in the Captisol  stabilizing solution; Thereby supported

Discovery among the embodiment 1.

Embodiment 3

The production of [C17S+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant, purification and PEGization

A CHO K1 sub-clone (5/G-10) inoculation that will produce [C17S+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant is advanced 6 200ml that shake bottle and has been replenished the DMEM/F-12 culture medium (LifeTechnologies of 10%FBS and penicillin/streptomycin (P/S); Cat.#31330) in, each shakes bottle and has 1700cm ²Unfolded surface (Coming, USA).Change culture medium after 2 days.Converge after 2 days latter two shaking flask inoculations nearly 100%, culture medium is replaced with has replenished 1/500 EX-CYTE (Serologicals Proteins; Cat.#81129N) and the 300ml serum-free UltraCHO culture medium (BioWhittaker of P/S; Cat.#12-724).The raising of the growth cell concentration of cell in this culture medium, higher than accessible amount in containing the culture medium of serum.Upgrade culture medium after 2 days.After 2 days, culture medium is replaced with the production culture medium: replenished 1/100 ITSA (Life Technologies; Cat.#51300-044) [ITSA represents to be used for insulin (1.0g/L)-transferrin (0.55g/L)-selenium (0.67mg/L) additive of adhere-wall culture thing], the DMEM/F-12 culture medium (LifeTechnologies of 1/500 EC-CYTE and P/S; Cat.#21041).The culture medium that merging is gathered in the crops from shake bottle is taken out media samples afterwards and is used for IFN-'beta ' activity mensuration.In 21 days, gather in the crops 1.8l culture medium and freezing every day at-80 ℃.

Centrifugal from shaking bottle culture medium of results and filtering by 0.22 μ m filter membrane (PVDF).Filtering culture medium be equipped with in the Vivaflow200 system that cutoff value is 10000 poly (ether sulfone) film diafiltration and on sample to S-Sepharose post (Pharmacia).

Use the 50mM sodium acetate, 50mM sodium chloride, pH5.5 balance S.Sepharose post, and use the 50mM sodium acetate, 0.5M sodium chloride, pH5.5 eluting interferon variant.Sodium chloride concentration in the eluate is adjusted to 1.0M.

Will be from sample in the eluate of S-Sepharose post to using the 50mM sodium acetate, 1.0M sodium chloride is on the equilibrated phenyl of pH5.5-agarose high-efficiency column (Pharmacia).Use the 50mM sodium acetate behind the last sample, 50mM sodium chloride, pH5.5 washs pillar.IFN-β variant sodium acetate from 50mM, 50mM sodium chloride, pH5.5 be to 60% ethylene glycol, the 50mM sodium acetate, and the gradient of pH5.5 is with 30 times of column volume eluting.Collection also merges the fraction that contains complete glycosylated IFN-β variant.

By with eluate by using the 50mM sodium acetate, 50mM sodium chloride, the equilibrated S-Sepharose post of pH5.5 removes the ethylene glycol in phenyl-agarose eluate.Ethylene glycol flows through and the interferon variant is attached on the pillar.Use the 20mM sodium acetate behind the last sample, pH5.5 washing pillar is also used 100mM sodium phosphate, pH7.5 eluting interferon variant.

Phosphate concn in the eluate is adjusted to the 15mM sodium phosphate buffer, pH7.2, and go up sample to using the 15mM sodium phosphate, the equilibrated hydroxyapatite column of pH7.2 (CHT I, Ceramichydroxyapatite, Type I, Biorad) on.The glycosylation form is passed through pillar fully, and the not enough form of glycosylation uses a site more than needed to combine with pillar, uses from 15mM to the 200mM sodium phosphate, and the line style sodium phosphate gradient of pH6.8 is with 20 times of column volume eluting.

The purity of identifying complete glycosylated variant [C17S+Q49N+Q51T+D110F+F111N+R113T] IFN-β according to SDS-PAGE is higher than 95%.

Behind the purification, variant carry out PEGization.The fresh stock solution that in 96% ethanol, prepares 10mg/mlSCM-PEG (the succinimido ester of carboxy methylation PEG, from Shearwater, Alabama, 12k or 20k) before each experiment.

0.1mg/ml at the 20mM sodium phosphate, the protein solution SCM-PEG among the pH7.0,20K carry out PEGization, and PEG is than possible PEGization site, i.e. the terminal superfluous 0.75 times molal quantity of lysine and N-.Incubation is after 30 minutes, by adding superfluous 20mM glycine, pH8.0 cessation reaction under the room temperature.Reactant mixture contains single, 2 and the mixture of the material of PEGization not.Use cation-exchange chromatography or size exclusion chromatography or both combinations to separate single PEGization material and other kind.With the pH regulator in the PEGization solution to pH2.7 and with sample on the sample to using the 20mM sodium citrate, on the equilibrated SP-Sepharose HR of pH2.7 (Pharmacia) post.With the 50mM sodium acetate that contains 1M sodium chloride from eluting PEGization protein on the pillar and go up sample to using the 100mM sodium acetate, 200mM sodium chloride, the equilibrated size-exclusion column Sephacryl S-100 of pH5.5 is on ((16/60) Pharmacia).Merge the fraction of the material that contains single PEGization and further evaluation.

In another experiment, 0.16mg/ml is at the 20mM sodium phosphate, the protein solution SCM-PEG among the pH7.0, and 12K carry out PEGization, and PEG is than possible PEGization site, i.e. the molal quantity of 2 times of lysine and N-end surpluses.Incubation is after 30 minutes, by adding superfluous 20mM glycine, pH8.0 cessation reaction under the room temperature.Reactant mixture contains single, and 2, the mixture of 3 PEGization materials and underivatized material.Use cation-exchange chromatography or size exclusion chromatography or both protein in conjunction with separation PEGization material and unmodified.With the pH regulator in the PEGization solution to pH2.7 and with sample on the sample to using the 20mM sodium citrate, on the equilibrated SP-Sepharose HR of pH2.7 (Pharmacia) post.With the 50mM sodium acetate that contains 1M sodium chloride from eluting PEGization protein on the pillar and go up sample to using the 100mM sodium acetate, 200mM sodium chloride, the equilibrated size-exclusion column SephacrylS-100 of pH5.5 is on ((16/60) Pharmacia).Merging contains single, and the fraction of 2 and 3 the proteinic mixture of PEGization is also further identified.

Embodiment 4

The differential scanning calorimetry that contains the preparation of [17S+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant

The sample (embodiment 3 preparations) of analyzing IFN-β glycosylation variant by differential scanning calorimetry (DSC) is proteinicly separated folding (or degeneration) and particularly is determined at the proteinic folding temperature (Tm) of separating in each operation to study.

The parent material that is used for dsc analysis is the protein solution of 20mM sodium phosphate (being adjusted to β H7.1 at last).Prepare solution with the final protein concentration that adds various excipient generation 0.4mg/mL.Use the blank of injection water, because focus only is to measure the variation of Tm value as DSC.Before carrying out dsc analysis, it is described by using the vacuum time enough to give all solution degasification to press MicroCal Inc..

By using this proteinic behavior of DSC device (VP-DSC type) assessment of MicroCal Inc.The temperature of described solution is increased to about 120 ℃ with the speed of 1.5 ℃ of per minutes gradually from ambient temperature (25 ℃).Along with being increased in of temperature observed in the scanning and separated folding reaction (heat absorption) peak value for making progress.

Relatively the DSC operation of the solution that adds " 0.2M mannitol " or " 0.2M mannitol+35mg/ml Captisol  " has been disclosed about 6.4 ℃ of the sample Tm-value increase that contains Captisol .These data have confirmed that clearly adding Captisol  can be stabilized in the protein in the solution.

Embodiment 5

Contain preparation with [C17S+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant of 12kDa PEGization

The IFN-β glycosylation variant with 12kDa PEGization of purification (embodiment 3 preparations) is mixed with down is listed in following buffer: 10mM sodium acetate buffer (being adjusted to pH5.5 at last) and contain 45mg/ml mannitol and 2mg/ml Tween 80 (buffer I), or 50mM sodium phosphate buffer (being adjusted to pH7.0 at last) and contain 30mg/ml mannitol and 2mg/ml Tween 80 (buffer II) in contain the compositions that initial concentration is the variant of about 5MIU/ml.For buffer system I: a kind of compositions (compositions A) is not added Captisol .Another compositions (compositions B) contains 10mg/ml Captisol .For buffer system II: a kind of compositions (compositions C) is not added Captisol .Another compositions (compositions D) contains 10mg/ml Captisol .

Compositions is down stored different time length (without nitrogen or argon purify) at-80 ℃ with the aliquot of 50 μ l in 0.5ml Eppendorf pipe.At least the sample of 0.4mL is stored (purifying with nitrogen before the sealing) down at 5,25 and 35 ℃ in silication vial (I type glass).

Use the antivirus test described in the WO01/15736 to measure antiviral activity.

The result of antiviral activity test is shown " in the average percentage activity to the sample of storing under the fixed temperature, as the active function (analyzing on the same day) of the sample of storing down at-80 ℃ " at the following table invading the exterior:

Storage temperature (℃)	?????????????5				?????????????25				????????????????35
													Compositions	??A	??B	??C	??D	??A	??B	??C	??D	??A	??B	??C	??D
Store natural law
													??4	??-	???-	??-	??-	??-	??-	??-	??-	??13 ??2	??83	??33	??35
??21	??-	???-	??-	??-	??-	??-	??-	??-	??0	??22	??5	??0
													??32	??-	???-	??-	??-	??-	??-	??-	??-	??0	??10	??3	??1
??38	??114	??173	??86	??73	??48	??90	??29	??16	??-	??-	??-	??-
													??67	??64	??107	??-	??-	??0	??20	??-	??-	??-	??-	??-	??-
??80	??45	??69	??-	??-	??-	??9	??-	??-	??-	??-	??-	??-

If NB. put the sample of not analyzing said composition, then be expressed as "-" in preset time.

These data prove that clearly the biological activity that adding Captisol  can delay under some pH value in pharmaceutically useful buffer system loses.

In addition, the visual observation to the various compositionss of storing in bottle has disclosed the precipitation that the compositions that contains Captisol  prevented or delayed the IFN-β variant of PEGization.For example, compositions A and B store at 35 ℃ and were observed " highly muddy " and " settled solution " in 34 days respectively.

Embodiment 6

Contain preparation with [C17S+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant of 20kDa PEGization

The IFN-β variant with 20kDa PEGization of purification (embodiment 3 preparations) is mixed with and contains the following compositions that initial concentration is the variant of 11-14MIU/ml in following buffer: 10mM sodium acetate buffer (being adjusted to pH5.5 at last) and contain 45mg/ml mannitol and 2mg/ml Tween 80 and 6.7mg/ml Captisol  (compositions A), or 50mM sodium phosphate buffer (being adjusted to pH7.0 at last) and contain 30mg/ml mannitol and 2mg/ml Tween 80 and 10mg/ml Captisol  (compositions B).

Compositions is down stored different time length (without nitrogen or argon purify) at-80 ℃ with the aliquot of 50 μ l in 0.5ml Eppendorf pipe.At least the sample of 0.4mL is stored (purifying with nitrogen before the sealing) down at 5,25 and 35 ℃ in silication vial (I type glass).

Use the antivirus test described in the WO01/15736 to measure antiviral activity.

The result of antiviral activity test is shown " in the average percentage activity to the sample of storing under the fixed temperature, as the active function (analyzing on the same day) of the sample of storing down at-80 ℃ " at the following table invading the exterior:

Storage temperature (℃)	??????????5		?????????25		??????????35
							Compositions	????A	????B	????A	????B	????A	????B
Store natural law
							????4	????-	????-	????-	????-	????137	????116
????21	????-	????-	????-	????-	????19	????1
							????32	????-	????-	????-	????-	????6	????1
????38	????144	????114	????105	????24	????-	????-
							????67	????101	????103	????69	????-	????-	????-
????80	????117	????101	????52	????1	????-	????-
							????108	????130	????89	????-	????-	????-	????-

If NB. put the sample of not analyzing said composition, then be expressed as "-" in preset time.

These data are clearly supported the discovery of embodiment 5, show that wherein adding Captisol  can delay or even prevent that the biological activity under some pH value from losing in pharmaceutically useful buffer system.

Embodiment 7

The production of [C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant, purification and PEGization

A CHO K1 sub-clone (5/G-10) that will produce [C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant is produced in the bottle and by the used scheme purification of embodiment 3 by described the shaking at 6 of embodiment 3.The purity of identifying complete glycosylated variant [C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T] IFN-β according to SDS-PAGE is higher than 95%.

Behind the purification, variant carry out PEGization.The fresh stock solution that in ethanol, prepares SCM-PEG (the succinimido ester of carboxy methylation PEG, from Shearwater, Alabama, 12kD or 20kD) before each experiment.

0.1mg/ml at the 20mM sodium phosphate, the protein solution SCM-PEG among the pH7.0,20K carry out PEGization, and PEG is than possible PEGization site, i.e. the terminal superfluous 3 times molal quantity of lysine and N-.Incubation is after 30 minutes, by adding superfluous 20mM glycine, pH8.0 cessation reaction under the room temperature.Reactant mixture contains single, 2 and the mixture of the material of PEGization not.Use cation-exchange chromatography or size exclusion chromatography or both combinations to separate single PEGization material and other kind.With the pH regulator in the PEGization solution to pH2.7 and with sample on the sample to using the 20mM sodium citrate, on the equilibrated SP-Sepharose HR of pH2.7 (Pharmacia) post.With the 50mM sodium acetate that contains 1M sodium chloride from eluting PEGization protein on the pillar and go up sample to using the 100mM sodium acetate, 200mM sodium chloride, the equilibrated size-exclusion column Sephacryl S-100 of pH5.5 is on ((16/60) Pharmacia).Merge the fraction of the material that contains single PEGization and further evaluation.

In another experiment, 0.1mg/ml is at the 20mM sodium phosphate, and the protein solution among the pH7.0 is with (10mg/ml) SCM-PEg, and 12K carry out PEGization, and PEG is than possible PEGization site, i.e. the molal quantity of 5 times of lysine and N-end surpluses.Incubation is after 30 minutes, by adding superfluous 20mM glycine, pH8.0 cessation reaction under the room temperature.Reactant mixture contains single, and 2, the mixture of 3 PEGization materials and underivatized material.Use cation-exchange chromatography or size exclusion chromatography or both protein in conjunction with separation PEGization material and unmodified.With the pH regulator in the PEGization solution to pH2.7 and with sample on the sample to using the 20mM sodium citrate, on the equilibrated SP-Sepharose HR of pH2.7 (Pharmacia) post.With the 50mM sodium acetate that contains 1M sodium chloride from eluting PEGization protein on the pillar and go up sample to using the 100mM sodium acetate, 200mM sodium chloride, the equilibrated size-exclusion column Sephacryl S-100 of pH5.5 is on ((16/60) Pharmacia).Merging contains single, and the fraction of 2 and 3 the proteinic mixture of PEGization is also further identified.

Embodiment 8

Contain preparation with [C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant of 20kDa PEGization

The IFN-β variant with 20kDa PEGization (embodiment 7 preparations) of purification is mixed with and contains the following compositions that initial concentration is the variant of 5-10MIU/ml in following buffer: 10mM sodium acetate buffer (being adjusted to pH5.0 at last ,～buffer A); The 10mM sodium acetate buffer (is adjusted to pH5.5 at last,～buffer B), 10mM sodium succinate buffer (is adjusted to pH5.5 at last,～buffer C), 10mM sodium succinate buffer (is adjusted to pH6.0 at last,～buffer D) and 10mM sodium citrate buffer solution (being adjusted to pH6.0 at last ,～buffer E).The following 3 kinds of excipient Tween 80 (do not have, 0.2 and 2.0mg/ml) that in each of 5 kinds of described buffer systems, add various combinations, Captisol  (10 and 50mg/ml) and mannitol (17 and 39mg/ml).In addition, for buffer system C and E, also studied no Tween 80 or Captisol  but only added the combination of mannitol (be respectively 34 and 32mg/ml).

The amount of regulating mannitol is suitable for parenteral to guarantee isosmotic solution.

Compositions is down stored different time length (without nitrogen or argon purify) at-80 ℃ and 5 ℃ with the aliquot of 25 μ l in 0.5ml Eppendorf pipe.At least the sample of 0.4ml is stored (purifying with nitrogen before the sealing) down at 25 ℃ in silication vial (I type glass).

Use the antivirus test described in the WO01/15736 to measure antiviral activity.

The result of antiviral activity test is shown " in the average percentage activity to the sample of storing under the fixed temperature, as the active function (analyzing on the same day) of the sample of storing down at-80 ℃ " at the following table invading the exterior.

7 different preparations that all contain 10mM sodium succinate buffer and mannitol (being adjusted to pH5.5 at last ,～buffer C) are stored down at-80 ℃ and 25 ℃ before antiviral is analyzed.Do not conform to stability that the preparation of Tween 80 and Captisol  keeps late 20 days than other preparation.

????Captisol ????(mg/ml)	???????????Tween?80(mg/ml)
				????0	????0.2	????2
	????0	????40 *
????10				????64	????101	????116
	????50	????117	????109				????65

25 ℃ of average percentage activity of storing 94-95 days buffer C composition sample down, as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

^*) 25 ℃ of average percentage activity of storing this buffer C composition sample of 74 days down, as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

????Captisol ????(mg/ml)	??????????Tween?80(mg/ml)
				????0	????0.2	????2
	????0	????17 *
????10				????120	????72	????41
	????50	????96	????91				????116

25 ℃ of average percentage activity of storing 160 days buffer C composition sample down, as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

^*) 25 ℃ of average percentage activity of storing this buffer C composition sample of 140 days down, as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

7 different preparations that all contain 10mM sodium citrate buffer solution and mannitol (being adjusted to pH6.0 at last ,～buffer E) are stored down at-80 ℃ and 25 ℃ before antiviral is analyzed.Do not contain stability that the preparation of Tween 80 and Captisol  keeps late 20 days than other preparation.

????Captisol ????(mg/ml)	??????????Tween?80(mg/ml)
				????0	????0.2	????2
	????0	????68 *
????10				????86	????81	????61
	????50	????103	????117				????111

25 ℃ of average percentage activity of storing 94-95 days buffer E composition sample down, as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

^*) 25 ℃ of average percentage activity of storing this buffer E composition sample of 75 days down, as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

????Captisol ????(mg/ml)	?????????Tween?80(mg/ml)
				????0	????0.2	????2
	????0	????25 *
????10				????83	????45	????8
	????50	????122	????80				????45

25 ℃ of average percentage activity of storing 164 days buffer E composition sample down, as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

^*) 25 ℃ of average percentage activity of storing this buffer E composition sample of 144 days down, as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

Consider that the compositions that does not contain Captisol  compares the time much shorter that remaining compositions is stored, therefore these data clearly illustrate that add in pharmaceutically useful buffer system that Captisol  can delay or even prevent biological activity loss under some pH value, even store the longer time at elevated temperatures.

Embodiment 9

The stability of IFN-β variant of selected purification that comprises the concrete variant of embodiment 1-8 in conjunction with following parameters research:

A) " various concentration " is selected from 1-50MIU/ml.

B) buffer type is selected from sodium acetate, sodium succinate, and sodium citrate, Monosodium maleate, sodium carbonate, sodium tartrate, sodium lactate and its mixture, every kind of buffer type has the suitable concn up to 100mM.

C) the pH scope is selected from pH4.0-7.0.

D) concentration of Captisol  is selected from 5-100mg/ml.

E) other type and amount about excipient is selected from Tween 20 (up to 2mg/ml), Tween80 (up to 2mg/ml), mannitol (up to 50mg/ml), sodium chloride (up to 9mg/ml).

F) be adapted to pass through the main container of the given product of parenteral administration.

Embodiment 10

Contain preparation with [C17S+Q49N+Q51T+D110F+F111N+R113T] IFN-β glycosylation variant of 20kDa PEGization

Before the preparation, use the IFN-β variant with 20kDa PEGization (embodiment 3 preparations) of the solution equilibria purification of forming by the 10mM sodium acetate buffer that contains 25mg/ml mannitol (finally being adjusted to pH5.5).This material is mixed with in following buffer, contains the following compositions that initial concentration is the variant of 100 μ g/ml: the mannitol that contains following combination, sodium chloride (NaCl), the 10mM sodium acetate buffer of Tween 80 and Captisol  (being adjusted to pH5.5 at last).

Preparation	????Tween?80 ????(mg/ml)	????Captisol ????(mg/ml)	Mannitol (mg/ml)	????NaCl ????(mg/ml)
					????M01	????0	????0	????48	????0
????M02	????0.05	????0	????48	????0
					????M03	????0.02	????0	????48	????0
????M04	????0.05	????10	????45	????0
					????M05	????0.20	????10	????45	????0
????M06	????0	????25	????38	????0
					????M07	????0.05	????25	????38	????0
????M08	????0.20	????25	????38	????0
					????M09	????0	????50	????28	????0
????M10	????0.05	????50	????28	????0
					????M11	????0	????100	????9.3	????0
????M12	????0.05	????100	????9.3	????0
					????NM01	????0	????0	????9.3	????6.8
????NM02	????0.05	????0	????9.3	????6.8
					????NM03	????0.20	????0	????9.3	????6.8
????NM04	????0.05	????10	????9.3	????6.2
					????NM05	????0.20	????10	????9.3	????6.2
????NM06	????0	????25	????9.3	????5.1
					????NM07	????0.05	????50	????9.3	????5.1
????NM08	????0.20	????25	????9.3	????5.1
					????NM09	????0	????50	????9.3	????3.4
????NM10	????0.05	????50	????9.3	????3.4

Compositions is by filtration sterilization, in the sterile chamber of packing under aseptic condition and store different time length.The aliquot of 20tl packed into store down in the 0.5ml Eppendorf pipe and at-80 ℃.At least the aliquot of 0.3ml is packed into and is stored down in the silication vial (I type glass) and at 5,25 and 35 ℃.

Use the antivirus test described in the WO01/15736 to measure antiviral activity.

The result who stores antiviral activity test after about 1 month is shown " in the average percentage activity to the sample of storing under the fixed temperature, as the active function of storing down at-80 ℃ of sample (analyzing on the same day) " at the following table invading the exterior:

????Captisol ????(mg/ml)	??????????Tween?80(mg/ml)
				????0	????0.05	????0.2
	????0	????76	????65				????56
????10					????123	????110
	????25	????124	????111				????113
????50				????101	????109
	????100	????155	????128

The average percentage activity of the sample of the preparation compositions M01 to M012 that store under 25 ℃ is as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

????Captisol ????(mg/ml)	???????????Tween?80(mg/ml)
				????0	????0.05	????0.2
	????0	????38	????16				????7
????10					????71	????68
	????25	????109	????90				????73
????50				????87	????93
	????100	????115	????100

The average percentage activity of the sample of the preparation compositions M01 to M012 that store under 35 ℃ is as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

These data are clearly supported the discovery among the embodiment 5, show that wherein adding Captisol  can delay or even prevent that the biological activity under some pH value from losing in pharmaceutically useful buffer system.

Compare with embodiment 6, use the Tween 80 of obvious much less in the present embodiment, soluble observed stability raising when described polypeptide temperature raises.

Embodiment 11

The preparation that contains wild type IFN-β (IFN-β)

By being the bulk article of final step by the method purification IFN-β that forms by the gel filtration of Superdex 75 posts with embodiment 3 described variant differences.Use this material of solution equilibria of forming by the 50mM sodium acetate (being adjusted to pH5.5) that contains 0.1M sodium chloride and 0.2M mannitol then.

This material is mixed with in following buffer, contains the following compositions that initial concentration is the IFN-β of about 5MIU/ml: contain 28mg/ml mannitol, 1.3mg/ml sodium chloride and 2mg/ml Tween80 and do not have Captisol  (preparation A) or the 50mM sodium acetate buffer (being adjusted to pH5.5 at last) of 10mg/ml Captisol  (preparation B).

Pack in Eppendorf pipe with the aliquot of 50 μ l said composition and, store the different time length down for-20 and 5 ℃ at-80 ℃.

Use the antivirus test described in the WO01/15736 to measure antiviral activity.

The result who stores antiviral activity test after about 354 days is shown " in the average percentage activity to the sample of storing under the fixed temperature, as the active function of storing down at-80 ℃ of sample (analyzing on the same day) " at the following table invading the exterior:

Storage temperature	Preparation A	Preparation B
			????-20℃	????32	????35
????5℃	????9	????31

The average percentage activity of the sample of 354 days preparation compositions A of storage and B under-20 ℃ and 5 ℃ is as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

Embodiment 12

The preparation that contains wild type IFN-γ (IFN-γ)

By the described a large amount of preparations of carrying out IFN-γ of embodiment H in " material and the method for preparation interferon gamma " part.These goods are mixed with in following buffer, contain the following compositions that initial concentration is the IFN-γ of 0.5mg/ml: contain 40mg/ml mannitol and 0.01% Tween 20 and do not have Captisol  (preparation A) or the 5mM sodium succinate buffer (being adjusted to pH6.0 at last) of 50mg/ml Captisol  (preparation B).

Compositions is by filtration sterilization, in the sterile chamber of packing under aseptic condition and store different time length.The aliquot of 20 μ l packed into store down in the 0.5ml Eppendorf pipe and at-80 ℃.At least the aliquot of 0.15ml is packed in the silication vial (I type glass) and 5,25, is stored down for 35 and 40 ℃.

Use the luciferase assay method described in " material and the method for preparation interferon gamma " part to measure active.

Store that the result of luciferase assay is shown at the following table invading the exterior after 8 days " in average percentage activity, as the active functions of storing down at-80 ℃ of sample (analyzing on the same day) " to the sample of storing under the fixed temperature:

Storage temperature	Preparation A	Preparation B
			????25℃	????63	????118
????35℃	????44	????95
			????40℃	????39	????83

The average percentage activity of the sample of 8 days preparation compositions A of storage and B under 25,35 and 40 ℃ is as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

Embodiment 13

The preparation that contains [E38N+S40T+S99T] IFN-γ glycosylation variant

The described preparation of embodiment D IFN-γ variant by " material and the method for preparation interferon gamma " part description.This material of the described purification of embodiment H by " material and the method for preparation interferon gamma " part description.The material of this purification is mixed with in following buffer, contains the following compositions that initial concentration is the variant of 0.5mg/ml: contain 40mg/ml mannitol and 0.01% Tween 20; And the 5mM sodium succinate buffer (being adjusted to pH6.0 at last) that does not have Captisol  (preparation A) or 50mg/ml Captisol  (preparation B).

Compositions is by filtration sterilization, in the sterile chamber of packing under aseptic condition and store different time length.The aliquot of 20 μ l packed into store down in the 0.5ml Eppendorf pipe and at-80 ℃.At least the aliquot of 0.15ml is packed into and is stored down in the silication vial (I type glass) and at 5 and 25 ℃.

Use the luciferase assay method described in " material and the method for preparation interferon gamma " part to measure active.

The result that luciferase activity is measured is shown " in the average percentage activity to the sample of storing under the fixed temperature, as the active function of storing down at-80 ℃ of sample (analyzing on the same day) " at the following table invading the exterior:

Store natural law	Preparation A		Preparation B
					Store down at 5 ℃	Store down at 25 ℃	Store down at 5 ℃	Store down at 25 ℃
	??6		??5						??35
??14					?47	??0	?72	??9
	??28	?13		?44

The average percentage activity of preparation compositions A that store under 5 and 25 ℃ and the sample of B is as the active function of storing down at-80 ℃ of sample (analyzing on the same day).

Sequence table

＜110〉Maxygen Inc. (Maxygen ApS)

Mark Scegen Pty Ltd. (Maxygen Holdings Ltd.)

＜120〉interferon formulations

<130>0232wo410

<140>

<141>

<150>DK?PA?2001?01040

<151>2001-06-29

<150>DK?PA?2001?01277

<151>2001-08-30

<150>DK?PA?2002?00257

<151>2002-02-19

<160>11

<170>FastSEQ?for?Windows?Version?4.0

<210>1

<211>166

<212>PRT

＜213〉people (Homo sapiens)

<400>1

Met?Ser?Tyr?Asn?Leu?Leu?Gly?Phe?Leu?Gln?Arg?Ser?Ser?Asn?Phe?Gln

1???????????????5??????????????????10??????????????????15

Cys?Gln?Lys?Leu?Leu?Trp?Gln?Leu?Asn?Gly?Arg?Leu?G1u?Tyr?Cys?Leu

20??????????????????25??????????????????30

Lys?Asp?Arg?Met?Asn?Phe?Asp?Ile?Pro?Glu?Glu?Ile?Lys?Gln?Leu?Gln

35??????????????????40??????????????????45

Gln?Phe?Gln?Lys?Glu?Asp?Ala?Ala?Leu?Thr?Ile?Tyr?Glu?Met?Leu?Gln

50??????????????????55??????????????????60

Asn?Ile?Phe?Ala?I1e?Phe?Arg?G1n?Asp?Ser?Ser?Ser?Thr?G1y?Trp?Asn

65??????????????????70??????????????????75??????????????????80

Glu?Thr?Ile?Val?Glu?Asn?Leu?Leu?Ala?Asn?Val?Tyr?His?Gln?Ile?Asn

85??????????????????90??????????????????95

His?Leu?Lys?Thr?Val?Leu?Glu?Glu?Lys?Leu?Glu?Lys?Glu?Asp?Phe?Thr

100?????????????????105?????????????????110

Arg?Gly?Lys?Leu?Met?Ser?Ser?Leu?His?Leu?Lys?Arg?Tyr?Tyr?Gly?Arg

115?????????????????120?????????????????125

Ile?Leu?His?Tyr?Leu?Lys?Ala?Lys?Glu?Tyr?Ser?His?Cys?Ala?Trp?Thr

130?????????????????135?????????????????140

Ile?Val?Arg?Val?Glu?Ile?Leu?Arg?Asn?Phe?Tyr?Phe?Ile?Asn?Arg?Leu

145?????????????????150?????????????????155?????????????????160

Thr?Gly?Tyr?Leu?Arg?Asn

165

<210>2

<211>143

<212>PRT

＜213〉people (Homo sapiens)

<400>2

Gln?Asp?Pro?Tyr?Val?Lys?Glu?Ala?Glu?Asn?Leu?Lys?Lys?Tyr?Phe?Asn

1???????????????5??????????????????10??????????????????15

Ala?Gly?His?Ser?Asp?Val?Ala?Asp?Asn?Gly?Thr?Leu?Phe?Leu?Gly?Ile

20??????????????????25??????????????????30

Leu?Lys?Asn?Trp?Lys?Glu?Glu?Ser?Asp?Arg?Lys?Ile?Met?Gln?Ser?Gln

35??????????????????40??????????????????45

Ile?Val?Ser?Phe?Tyr?Phe?Lys?Leu?Phe?Lys?Asn?Phe?Lys?Asp?Asp?Gln

50??????????????????55??????????????????60

Ser?Ile?Gln?Lys?Ser?Val?Glu?Thr?Ile?Lys?Glu?Asp?Met?Asn?Val?Lys

65??????????????????70??????????????????75??????????????????80

Phe?Phe?Asn?Ser?Asn?Lys?Lys?Lys?Arg?Asp?Asp?Phe?Glu?Lys?Leu?Thr

85??????????????????90??????????????????95

Asn?Tyr?Ser?Val?Thr?Asp?Leu?Asn?Val?Gln?Arg?Lys?Ala?Ile?His?Glu

100?????????????????105?????????????????110

Leu?Ile?Gln?Val?Met?Ala?Glu?Leu?Ser?Pro?Ala?Ala?Lys?Thr?Gly?Lys

115?????????????????120?????????????????125

Arg?Lys?Arg?Ser?Gln?Met?Leu?Phe?Arg?Gly?Arg?Arg?Ala?Ser?Gln

130?????????????????135?????????????????140

<210>3

<211>166

<212>PRT

＜213〉people (Homo sapiens)

<400>3

Met?Lys?Tyr?Thr?Ser?Tyr?Ile?Leu?Ala?Phe?Gln?Leu?Cys?Ile?Val?Leu

1???????????????5??????????????????10??????????????????15

Gly?Ser?Leu?Gly?Cys?Tyr?Cys?Gln?Asp?Pro?Tyr?Val?Lys?Glu?Ala?Glu

20??????????????????25??????????????????30

Asn?Leu?Lys?Lys?Tyr?Phe?Asn?Ala?Gly?His?Ser?Asp?Val?Ala?Asp?Asn

35??????????????????40??????????????????45

Gly?Thr?Leu?Phe?Leu?Gly?Ile?Leu?Lys?Asn?Trp?Lys?Glu?Glu?Ser?Asp

50??????????????????55??????????????????60

Arg?Lys?Ile?Met?Gln?Ser?Gln?Ile?Val?Ser?Phe?Tyr?Phe?Lys?Leu?Phe

65??????????????????70??????????????????75??????????????????80

Lys?Asn?Phe?Lys?Asp?Asp?Gln?Ser?Ile?Gln?Lys?Ser?Val?Glu?Thr?Ile

85??????????????????90??????????????????95

Lys?Glu?Asp?Met?Asn?Val?Lys?Phe?Phe?Asn?Ser?Asn?Lys?Lys?Lys?Arg

100?????????????????105?????????????????110

Asp?Asp?Phe?Glu?Lys?Leu?Thr?Asn?Tyr?Ser?Val?Thr?Asp?Leu?Asn?Val

115?????????????????120?????????????????125

Gln?Arg?Lys?Ala?Ile?His?Glu?Leu?Ile?Gln?Val?Met?Ala?Glu?Leu?Ser

130?????????????????135?????????????????140

Pro?Ala?Ala?Lys?Thr?Gly?Lys?Arg?Lys?Arg?Ser?Gln?Met?Leu?Phe?Arg

145?????????????????150?????????????????155?????????????????160

Gly?Arg?Arg?Ala?Ser?Gln

165

<210>4

<211>140

<212>PRT

＜213〉artificial sequence

<220>

<223>Actimmune

<400>4

Met?Gln?Asp?Pro?Tyr?Val?Lys?Glu?Ala?Glu?Asn?Leu?Lys?Lys?Tyr?Phe

1???????????????5??????????????????10??????????????????15

Asn?Ala?Gly?His?Ser?Asp?Val?Ala?Asp?Asn?Gly?Thr?Leu?Phe?Leu?Gly

20??????????????????25??????????????????30

Ile?Leu?Lys?Asn?Trp?Lys?Glu?Glu?Ser?Asp?Arg?Lys?Ile?Met?Gln?Ser

35??????????????????40??????????????????45

Gln?Ile?Val?Ser?Phe?Tyr?Phe?Lys?Leu?Phe?Lys?Ash?Phe?Lys?Asp?Asp

50??????????????????55??????????????????60

Gln?Ser?Ile?Gln?Lys?Ser?Val?Glu?Thr?Ile?Lys?Glu?Asp?Met?Asn?Val

65??????????????????70??????????????????75??????????????????80

Lys?Phe?Phe?Asn?Ser?Asn?Lys?Lys?Lys?Arg?Asp?Asp?Phe?Glu?Lys?Leu

85??????????????????90??????????????????95

Thr?Asn?Tyr?Ser?Val?Thr?Asp?Leu?Asn?Val?Gln?Arg?Lys?Ala?Ile?His

100?????????????????105?????????????????110

Glu?Leu?Ile?Gln?Val?Met?Ala?Glu?Leu?Ser?Pro?Ala?Ala?Lys?Thr?Gly

115?????????????????120?????????????????125

Lys?Arg?Lys?Arg?Ser?Gln?Met?Leu?Phe?Arg?Gly?Arg

130?????????????????135?????????????????140

<210>5

<211>498

<212>DNA

＜213〉artificial sequence

<220>

＜223〉expression cassette that the expression of interferon gamma in Chinese hamster ovary celI optimized

<400>5

atgaagtaca?caagctatat?cctggccttt?cagctgtgca?tcgtgctggg?ctccctgggc?60

tgctattgcc?aggaccctta?cgtgaaggag?gccgagaacc?tgaagaagta?ctttaacgcc?120

ggccacagcg?atgtggccga?caatggcaca?ctgtttctgg?gcatcctgaa?gaattggaag?180

gaggagagcg?atcggaagat?catgcagtcc?cagatcgtgt?ccttctattt?caagctgttt?240

aagaatttca?aggacgatca?gtccatccag?aagtccgtgg?agaccatcaa?ggaggacatg?300

aacgtgaagt?ttttcaatag?caataagaag?aagagagacg?atttcgagaa?gctgaccaat?360

tactccgtga?cagacctgaa?cgtgcagaga?aaggccatcc?acgagctgat?ccaggtgatg?420

gccgagctgt?cccccgccgc?caagaccggc?aagagaaaga?gaagccagat?gctgttcaga?480

ggcagacggg?ccagccag???????????????????????????????????????????????498

<210>6

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>6

gatggctggc?aactagaag???????????????????????????????????????????????19

<210>7

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>7

tgtacggtgg?gaggtctat?????????????????????????????????????????19

<210>8

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>8

gttcaggtct?gtcacggtgt?aattggtcag?ctt?????????????????????????33

<210>9

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>9

aagctgacca?attacaccgt?gacagacctg?aac??????????????????????????33

<210>10

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>10

catgatcttc?cgatcggtct?cgttcttcca?att??????????????????????????33

<210>11

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>11

aattggaaga?acgagaccga?tcggaagatc?atg??????????????????????????33

Claims (49)

1. the compositions of a stabilisation, it contains interferon polypeptides and sulfoalkyl ether cyclodextrin derivant.

2. according to the compositions of claim 1, wherein this interferon polypeptides is to show the polypeptide that aggregation forms and/or biological activity loses at lay up period.

3. according to the compositions of claim 1 or 2, wherein this interferon polypeptides is people's wild type interferon polypeptides or its variant.

4. according to the compositions of claim 3, wherein this interferon polypeptides is to be selected from interferon-ALPHA, interferon beta, interferon ω, interferon-tau, people's wild type interferon polypeptides of interferon ε and interferon gamma, or its variant.

5. according to each compositions of claim 1-4, wherein this interferon polypeptides is by the sub-derivatization of non-polypeptide half point.

6. according to the compositions of claim 5, wherein this interferon polypeptides is by glycosylation and/or PEGization.

7. according to each compositions of claim 1-6, wherein this interferon polypeptides is the wild type human interferon beta.

8. according to each compositions of claim 5-6, wherein this interferon polypeptides is the conjugate that contains interferon beta polypeptides, the difference of the aminoacid sequence of this interferon beta polypeptides and wild type human interferon beta is to have imported at least one glycosylation site, and this conjugate also contains at least one saccharide half point that is connected with the glycosylation site that imports.

9. compositions according to Claim 8, wherein the glycosylation site of at least one importing is to be in the N-glycosylation site that is selected from following position: S2N+N4S/T, L6S/T, L5N+G7S/T, F8N+Q10S/T, L9N+R11S/T, R11N, R11N+S13T, S12N+N14S/T, F15N+C17S/T, Q16N+Q18S/T, Q18N+L20S/T, K19N+L21S/T, W22N+L24S/T, Q23N+H25S/T, G26N+L28S/T, R27N+E29S/T, L28S+Y30S/T, Y30N+L32S/T, L32N+D34S/T, K33N+R35S/T, R35N+N37S/T, M36N+F38S/T, D39S/T, D39N+P41S/T, E42N+I44S/T, Q43N+K45S/T, K45N+L47S/T, Q46N+Q48S/T, L47N+Q49T/S, Q48N+F50S/T, Q49N+Q51S/T, Q51N+E53S/T, K52N+D54S/T, L57N+I59S/T, Q64N+I66S/T, A68N+F70S/T, R71N+D73S/T, Q72N, Q72N+S74T, D73N, D73N+S75T, S75N+T77S, S75N, S76N+G78S/T, E81N+I83S/T, T82N+V84S/T, E85N+L87S/T, L88S/T, A89N+V91S/T, Y92S/T, Y92N+Q94S/T, H93N+I95S/T, L98S/T, H97N+K99S/T, K99N+V101S/T, T100N+L102S/T, E103N+K105S/T, E104N+L106S/T, K105N+E107S/T, E107N+E109S/T, K108N+D110S/T, E109N+F111S/T, D110N+T112S, D110N, F111N+R113S/T, R113N+K115S/T, G114N+L116S/T, K115N+M117S/T, L116N, L116N+S118T, S119N+H212S/T, L120N+L122S/T, H121N+K123S/T, K123N+Y125S/T, R124N+Y126S/T, G127N+I129S/T, R128N+L130S/T, L130N+Y132S/T, H131N+L133S/T, K134N+K136S/T, A135N+E137S/T, K136N+Y138S/T, E137N, Y138N+H140S/T, H140N+A142S/T, V148N+I150S/T, R152N+F154S/T, Y155N+I157S/T, L160S/T, R159N+T161S, R159N, G162N+L164S/T, and Y163N+R165S/T, described replacement is represented with respect to the aminoacid sequence of the wild type human interferon beta shown in the SEQ ID NO 1.

10. according to each compositions of claim 1-9, wherein this interferon polypeptides is the interferon beta polypeptides that also contains the C17S sudden change, and described replacement is represented with respect to the aminoacid sequence of the wild type human interferon beta shown in the SEQ ID NO 1.

11. according to claim 1-3,5,6, each compositions among the 8-9, wherein this interferon beta polypeptides is selected from

Q49N+Q51T；

F111N+R113T；

Q49N+Q51T+F111N+R113T；

C17S+Q49N+Q51T+L98P+F111N+R113T；

S2N+N4T+C17S+Q51N+E53T；

S2N+N4T+C17S+Q51N+E53T+F111N+R113T；

C17S+Q49N+Q51T+F111N+R113T；

C17S+Q49N+Q51T+D110F+F111N+R113T；

C17S+Q48F+Q49N+Q51T+D110F+F111N+R113T；

C17S+Q48Y+Q49N+Q51T+D110Y+F111N+R113T；

K19R+K45R+K123R；

K19R+K45R+Q49N+Q51T+F111N+R113T+K123R；

C17S+K19R+K45R+Q49N+Q51T+F111N+R113T+K123R；

C17S+K19R+K45R+Q49N+Q51T+F111N+R113T+K123R；

C17S+K19R+Q49N+Q51T+F111N+R113T+K123R；

C17S+K19R+K45R+Q49N+Q51T+D110F+F111N+R113T+K123R；

C17S+K19R+Q49N+Q51T+D110F+F111N+R113T+K123R；

S2N+N4T+C17S+K19R+K45R+Q51N+E53T+K123R；

C17S+K19R+K45R+Q48F+Q49N+Q51T+D110F+F111N+R113T+K123R；

S2N+N4T+C17S+K19R+K45R+Q51N+E53T+D110F+F111N+R113T+K123R；

C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T；

C17S+K19R+K33R+K45R+Q49N+Q51T+D110F+F111N+R113T+K123R；

C17S+K19R+K33R+K45R+Q49N+Q51T+F111N+R113T; With

C17S+K19R+K33R+K45R+Q49N+Q51T+F111N+R113T+K123R；

Described replacement is represented with respect to the aminoacid sequence of wild type human interferon beta shown in the SEQ ID NO 1.

12. according to each compositions of claim 1-6, wherein this interferon polypeptides is an interferon gamma polypeptide.

13. according to the compositions of claim 12, wherein this interferon gamma polypeptide is wild type human interferon gamma or its variant.

14. according to the compositions of claim 13, wherein this interferon gamma polypeptide is the variant that replaces S99T that contains of people's wild type interferon gamma, this replacement is represented with respect to the aminoacid sequence of the wild type human interferon gamma shown in the SEQ ID NO 2.

15. according to the compositions of claim 14, wherein this variant also contains and replaces E38N+S40T, this replacement is represented with respect to the aminoacid sequence of the wild type human interferon gamma shown in the SEQ ID NO 2.

16. according to each compositions of claim 12-15, wherein this interferon gamma polypeptide in the terminal truncate of C-1-15 amino acid residue.

17. according to each compositions of claim 1-16, this sulfoalkyl ether cyclodextrin derivant chemical compound that is formula (I) wherein:

Wherein

N is 4,5 or 6,

R ₁, R ₂, R ₃, R ₄, R ₅, R ₆, R ₇, R ₈, and R ₉Be independently respectively ,-O-or-O-(C ₂-C ₆Alkylidene)-SO ₃-group, wherein R ₁And R ₂In at least one is-O-(C independently ₂-C ₆Alkylidene)-SO ₃-group, and

S ₁, S ₂, S ₃, S ₄, S ₅, S ₆, S ₇, S ₈And S ₉Be respectively pharmaceutically useful cation independently.

18. according to the compositions of claim 17, wherein this sulfoalkyl ether cyclodextrin is a beta cyclodextrin sulfo group butyl ether, for example its salt form, for example its sodium salt, preferably Captisol .

19. according to each compositions of claim 1-18, wherein said sulfoalkyl ether cyclodextrin derivant exists with the concentration from 1mg/ml to 150mg/ml.

20. according to each compositions of claim 1-19, wherein said interferon polypeptides is with corresponding to 1-100MIU/ml, the liquid formulations of 1-50MIU/ml for example, and perhaps 1-100MIU/ agent, for example the amount of the solid formulation of 1-50MIU/ agent exists.

21. according to each compositions of claim 1-20, the pH scope of wherein said compositions is 4-8, preferred pH5-8, or alternative 4-7.

22. according to each compositions of claim 1-21, it also contains a kind of buffer agent.

23. according to the compositions of claim 22, wherein said buffer agent exists with the concentration up to 100mM.

24. according to each compositions of claim 1-23, wherein said compositions is a kind of liquid isosmotic solution and has the Morie osmolarity of about 240-360mOsmol/kg.

25. according to each compositions of claim 1-24, it also contains a kind of tonicity agent.

26. according to each compositions of claim 1-23, it is dried forms or liquid formulations, for example aqueous solution or suspension.

27. according to each compositions of claim 1-26, it is a liquid solution, for example aqueous solution.

28. according to the compositions of claim 26, it is refrigerated liquid formulations, spray drying, perhaps cryodesiccated preparation form.

29. require each compositions according to aforesaid right, it is suitable for parenteral, nose or lung administration.

30. according to the compositions of claim 29, it is suitable for intravenous, intramuscular or subcutaneous administration.

31. according to each compositions of claim 1-30, it also comprises another kind of excipient.

32. according to each compositions of claim 1-31, it also comprises the gathering that can reduce interferon polypeptides and/or second kind of stabilizing agent of chemical degradation.

33. according to each compositions of claim 1-32, it also contains a kind of preservative agent and/or viscosifier.

34. according to each compositions of claim 1-32, it does not contain preservative agent.

35. according to each compositions of claim 1-34, it also contains HSA.

36. according to each compositions of claim 1-34, it does not contain HSA.

37. according to each compositions of claim 1-36, it also contains a kind of surfactant, for example non-ionic surface active agent.

38. according to each compositions of claim 1-36, it does not contain surfactant, particularly non-ionic surface active agent.

39. according to each compositions of claim 1-38, wherein this interferon polypeptides a) is being stored at least 1 week and/or b under 37 ℃ the temperature) keep its antiviral activity basically during storing at least 4 weeks under 25 ℃ the temperature.

40. according to each compositions of claim 7-39, wherein this interferon polypeptides also comprises multimeric molecule, for example Polyethylene Glycol.

41. according to the compositions of claim 40, wherein this multimeric molecule comprises a PEG molecule.

42. contain each the major product container of compositions of with good grounds claim 1-41.

43. according to the container of claim 42, it is the syringe of prepackage.

44. an enhancing is mixed with the method for stability of the interferon polypeptides of Pharmaceutical composition, described method comprises mixes the sulfoalkyl ether cyclodextrin derivant in described compositions, and optionally comprises a kind of buffer agent.

45. according to the method for claim 44, wherein this interferon polypeptides shows aggregation formation at lay up period, and the incorporation of this sulfoalkyl ether cyclodextrin derivant is enough to reduce the aggregation formation of interferon polypeptides.

46. according to the method for claim 44 or 45, wherein said composition is by each qualification of claim 1-41.

47. mammal is carried out the method for interferon therapy, this method comprise effective dose on the administering therapeutic according to each compositions of claim 1-41.

48. according to each compositions of claim 1-41 as the purposes of medicine.

49. be used for the treatment of purposes in disease or the disorderly medicine in preparation according to each compositions of claim 1-41.